US20040023325A1 - Altered OspA of Borrelia burgdorferi - Google Patents

Altered OspA of Borrelia burgdorferi Download PDF

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US20040023325A1
US20040023325A1 US10/369,339 US36933903A US2004023325A1 US 20040023325 A1 US20040023325 A1 US 20040023325A1 US 36933903 A US36933903 A US 36933903A US 2004023325 A1 US2004023325 A1 US 2004023325A1
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ospa
thr
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Benjamin Luft
John Dunn
Shohei Koide
Catherine Lawson
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Research Foundation of State University of New York
Brookhaven Science Associates LLC
University of Rochester
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Research Foundation of State University of New York
Brookhaven Science Associates LLC
University of Rochester
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Publication of US20040023325A1 publication Critical patent/US20040023325A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF ROCHESTER
Priority to US12/313,443 priority patent/US8680236B2/en
Priority to US13/944,078 priority patent/US8992936B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Lyme disease (Lyme borreliosis) is the most common tick-borne infectious disease in North America and Europe, and has been found in Russia, Japan, China and Australia. Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring during the course of infection.
  • the causative bacterial agent of this disease is the spirochete Borrelia burgdorferi , which was first isolated and cultivated in 1982 (Burgdorferi, W. A. et al., Science 216: 1317-1319 (1982); Steere, A. R. et al., N. Engl. J. Med ., 308:733-740 (1983)).
  • B. burgdorferi sensu stricto B. burgdorferi or B.b.s.s .
  • B. afzelii B. garinii
  • B. burgdorferi has an outer membrane whose major protein constituents are the outer surface proteins A and B (OspA and OspB).
  • OspA is a basic lipoprotein of approximately 31 kd, which is encoded on a large linear plasmid along with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and J. L. Benach, Microbiol. Rev ., 55:21 (1991)).
  • the immune response to these outer surface proteins tends to occur late in the disease, if at all (Craft, J. E. et al., J. Clin Invest . 78:934-939 (1986); Dattwyler, R. J. and B. J.
  • Lyme Disease is treated with a range of antibiotics, e.g., tetracyclines, penicillin and cephalosporins.
  • antibiotics e.g., tetracyclines, penicillin and cephalosporins.
  • Treatment is often delayed due to improper diagnosis with the deleterious effect that the infection proceeds to a chronic condition, where treatment with antibiotics is often not useful.
  • One of the factors contributing to delayed treatment is the lack of effective diagnostic tools.
  • Vaccines against Lyme borreliosis have been attempted.
  • a vaccine that consists of recombinant OspA may require frequent booster immunizations.
  • An additional concern of OspA-based vaccines is the recent identification of a putative autoreactive OspA domain with a high degree of similarity to a region of human leukocyte function-associated antigen-1 (hLFA-1) (Gross, D. M. et al., Science , 281: 703-706 (1998)).
  • modified OspA proteins having decreased cross-reactivity to hLFA-1 in order to reduce potential side effects of an OspA vaccine.
  • Development of OspA proteins with decreased hLFA-1 cross-reactivity that maintain or have increased immunoreactivity to more than one member of the Borrelia complex would also be desirable.
  • the conformations of these modified proteins must be sufficiently stable to retain certain OspA structural features that are required to elicit a protective immune response. OspA proteins with these features would allow for improvements in diagnosis and/or vaccination against all, or most, of the Borrelia that cause Lyme Disease.
  • the present invention is drawn to altered forms of OspA from Borrelia burgdorferi that have increased conformational stability while maintaining at least some of the antigenicity of wild type OspA.
  • the altered OspA polypeptide has decreased cross-reactivity with hLFA-1, as compared to the corresponding unaltered OspA polypeptide.
  • the altered OspA polypeptides can comprise almost all or only a portion of the native OspA polypeptide.
  • the altered OspA polypeptide can be part of a cocktail which includes one or more other proteins, such as, for example, other Borrelia burgdorferi polypeptides including OspA, OspB, OspC, OspD, p93 and p41.
  • the altered OspA polypeptide can be part of a chimeric protein, such as those described in U.S. Pat. No. 6,248,562, the entire teachings of which are incorporated herein by reference.
  • the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 139 to about residue 273, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 changed to methionine, residue 160 changed to tyrosine, residue 189 changed to methionine and combinations thereof.
  • the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 131 to about residue 273 or from about residue 17 to about residue 273.
  • the OspA polypeptides of the present invention can comprise longer or shorter fragments of OspA protein.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7 (OspA from B31).
  • the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least two alterations selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine and residue 170 changed to lysine.
  • the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from a sensu stricto strain of Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine, residue 170 changed to lysine and combinations thereof.
  • the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 150 to about residue 180 or from about residue 17 to about residue 273.
  • the OspA polypeptides of the present invention can comprise longer or shorter fragments of OspA protein.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • polypeptides of the present invention include polypeptides selected from the group consisting of: SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114 and 116.
  • the present invention is also drawn to polynucleotides encoding the amino acid sequences described herein, such as polynucleotides encoding OspA polypeptides from Borrelia burgdorferi from about residue 131 to about residue 273, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine and combinations thereof.
  • the polynucleotide encoding OspA polypeptides of the present invention can encode longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide encodes an amino acid sequence of an OspA polypeptide from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 165 encoding phenylalanine, codon 166 encoding threonine, codon 170 encoding lysine and combinations thereof.
  • the polynucleotides which encode OspA polypeptides of the present invention can encode longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotides of the present invention include a polynucleotide selected from the group consisting of: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115.
  • the present invention is also drawn to a method of generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the method comprises selecting a polynucleotide encoding a Borrelia burgdorferi OspA polypeptide that includes at least one of residues 139, 160 and 189, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide is altered such that at least one of the following alterations is present: residue 139 is changed to methionine, residue 160 is changed to tyrosine and residue 189 is changed to methionine or a combination thereof. In one embodiment, both the alteration at residue 160 and the alteration at 189 is made. In another embodiment, the alterations at all three residues are made.
  • the altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the present invention is drawn to a method of generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with an hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the method comprises selecting a polynucleotide encoding an OspA polypeptide from Borrelia burgdorferi that includes at least one of residues 165, 166 and 170, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide is altered such that at least one alteration from the following list is present: residue 165 is changed to phenylalanine, residue 166 is changed to threonine and residue 170 is changed to lysine or combination thereof.
  • the altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the present invention is also drawn to an expression vector which comprises an isolated DNA encoding an altered Borrelia OspA protein.
  • the present invention also encompasses a host cell which comprises a recombinant nucleic acid that encodes an altered OspA protein as described herein.
  • the present invention is also drawn to a method of delivering the altered Borrelia OspA polypeptides described herein.
  • the method comprises administering the altered OspA polypeptide in a physiologically-acceptable carrier to an individual.
  • the individual develops at least some immune response to the protein.
  • the individual generates a humoral immune response, wherein antibodies that recognize at least a portion of said polypeptide are produced by the individual.
  • the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope.
  • the present invention is also drawn to a method of delivering a nucleic acid which encodes an altered OspA polypeptide described herein.
  • the method comprises administering the nucleic acid in a physiologically-acceptable carrier to an individual.
  • the altered OspA polypeptide is at least transiently expressed and the individual develops at least some immune response, preferably an immunoprotective response to the altered OspA protein encoded by the nucleic acid.
  • the individual generates a humoral immune response, wherein antibodies that recognize at least a portion of the altered OspA polypeptide produced from the nucleic acid are produced by the individual.
  • the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope.
  • the invention also encompasses methods of using the proteins described herein in diagnostic assays.
  • the method can be used to detect the presence of OspA specific antibodies in a host sample of interest.
  • the method comprises contacting a host sample of interest with the altered protein, under conditions wherein antibodies, if present in the host sample, bind to the altered protein, forming antigen-antibody complexes.
  • the antigen-antibody complexes are then detected using standard methods known in the art.
  • the present invention is also drawn to a diagnostic kit comprising the altered polypeptides described herein.
  • the kit comprises an altered Borrelia burgdorferi OspA protein as described herein.
  • the kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspA altered protein and antibodies that are present in the user-supplied host sample.
  • OspA proteins, or fragments thereof, having either increased conformational stability while maintaining at least some antigenicity, or having reduced cross-reactivity to hLFA-1 are available for use in research, vaccines and/or diagnostic assays.
  • nucleic acids encoding OspA polypeptides having reduced cross-reactivity with hLFA-1 are available for research and vaccines.
  • the altered OspA polypeptides of the present invention are expected to allow for improved vaccines having fewer side effects.
  • FIG. 1 summarizes peptides and antigenic domains localized by proteolytic and chemical fragmentation of OspA.
  • FIG. 2 is a comparison of the antigenic domains depicted in FIG. 1, for OspA in nine strains of B. burgdorferi.
  • FIG. 3 is a graph depicting a plot of weighted polymorphism versus amino acid position among 14 OspA variants.
  • the marked peaks are: a) amino acids 132-145; b) amino acids 163-177; c) amino acids 208-221.
  • the upper dotted line at 1.520 is the same, except that the first 29 amino acids at the monomorphic-terminus have been removed from the original analysis.
  • FIG. 4 depicts the amino acid alignment of residues 200 through 220 for OspAs from strains B31 and K48 as well as for the site-directed mutants 613, 625, 640, 613/625, and 613/640. Arrow indicates Trp216. Amino acid changes are underlined.
  • FIG. 5 depicts a phylogenetic tree for strains of Borrelia described in Table I.
  • FIGS. 6A and 6B depict the nucleic acid sequence of OspA-B31 (SEQ ID NO: 6), and the encoded protein sequence (SEQ ID NO: 7).
  • FIGGS. 7A, 7B and 7 C depict the nucleic acid sequence of OspA-K48 (SEQ ID NO: 8), and the encoded protein sequence (SEQ ID NO: 9).
  • FIGGS. 8A, 8B and 8 C depict the nucleic acid sequence of OspA-PGau (SEQ ID NO: 10), and the encoded protein sequence (SEQ ID NO: 11).
  • FIGS. 9A and 9B depict the nucleic acid sequence of an OspA gene (SEQ ID NO: 127 and its encoded protein sequence (SEQ ID NO: 128).
  • FIGS. 10A, 10B and 10 C depict the nucleic acid sequence of the OspA-K48/OspA-PGau chimer (SEQ ID NO: 28) and the encoded chimeric protein sequence (SEQ ID NO: 29).
  • FIGS. 11A, 11B and 11 C depict the nucleic acid sequence of the OspA-B31/OspA-PGau chimer (SEQ ID NO: 30) and the encoded chimeric protein sequence (SEQ ID NO: 31).
  • FIGS. 12A and 12B depict the nucleic acid sequence of the OspA-B31/OspA-K48 chimer (SEQ ID NO: 32) and the encoded chimeric protein sequence (SEQ ID NO: 33).
  • FIGS. 13A, 13B and 13 C depict the nucleic acid sequence of the OspA-B31/OspA-25015 chimer (SEQ ID NO: 34) and the encoded chimeric protein sequence (SEQ ID NO: 35).
  • FIGS. 14A, 14B and 14 C depict the nucleic acid sequence of the OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 36) and the encoded chimeric protein sequence (SEQ ID NO: 37).
  • FIGS. 15A, 15B and 15 C depict the nucleic acid sequence of the OspA-B31/OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 38) and the encoded chimeric protein sequence (SEQ ID NO: 39).
  • FIGS. 16A, 16B and 16 C depict the nucleic acid sequence of the OspA-B31/OspB-B31 chimer (SEQ ID NO: 40) and the encoded chimeric protein sequence (SEQ ID NO: 41).
  • FIGS. 17A, 17B, 17 C, 17 D, 17 E, 17 F, 17 G, 17 H, 17 I, 17 J, 17 K, 17 L, 17 M, 17 N, 17 O, and 17 P depict an alignment of the nucleic acid sequences for OspA-B31 (SEQ ID NO: 6), OspA-pKa1 (SEQ ID NO: 42), OspA-N40 (SEQ ID NO: 43), OspA-ZS7 (SEQ ID NO: 44), OspA-25015 (SEQ ID NO: 12), OspA-pTrob (SEQ ID NO: 45), OspA-K48 (SEQ ID NO: 8), OspA-Hei (SEQ ID NO: 46), OspA-DK29 (SEQ ID NO: 21), OspA-Ip90 (SEQ ID NO: 22), OspA-pBo (SEQ ID NO: 23), OspA-Ip3 (SEQ ID NO: 24
  • FIGS. 18A and 18B depict the nucleic acid sequence of the OspA-Tro/OspA-Bo chimer (SEQ ID NO: 47) and the encoded chimeric protein sequence (SEQ ID NO: 48).
  • FIGS. 19A and 19B depict the nucleic acid sequence of the OspA-PGau/OspA-Bo chimer (SEQ ID NO: 49) and the encoded chimeric protein sequence (SEQ ID NO: 50).
  • FIGS. 20A and 20B depict the nucleic acid sequence of the OspA-B31/OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 53) and the encoded chimeric protein sequence (SEQ ID NO: 54).
  • FIG. 21A and 21B depict the nucleic acid sequence of the OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 51) and the encoded chimeric protein sequence (SEQ ID NO: 52).
  • FIG. 22 is a bar graph showing the reactivity (as measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis) against the indicated OspA or OspC antigens (legend) from the strain B31 ( Borrelia burgdorferi sensu stricto ).
  • FIG. 23 is a bar graph showing the reactivity (as measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis) against the indicated OspA or OspC antigens (legend) from the strain B31 ( Borrelia burgdorferi sensu stricto ).
  • a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response was specific for the C-terminal region of OspA.
  • FIG. 24 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspA/Bo, lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA antigens (legend) from strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and Pgau ( Borrelia afzelii ).
  • FIG. 25 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspAP/Bo, lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA (legend) from strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and Pgau ( Borrelia afzelii ). In all cases, a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response is specific for the C-terminal region of OspA.
  • B31 Borrelia burgdorferi sensu stricto
  • K48 Borrelia garinii
  • Pgau Borrelia afzelii
  • FIG. 26 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or lip OspC-B31)(X-axis) against the indicated OspC antigen (legend) from the strain B31 ( Borrelia burgdorferi sensu stricto ).
  • FIG. 27 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or Lip OspA K/T)(X-axis) against the indicated OspA antigens (legend) from strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and Pgau ( Borrelia afzelii ).
  • FIG. 28 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P, OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspA antigens (legend) from strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and Pgau ( Borrelia afzelii ).
  • FIG. 29 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P, OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspA (legend) from strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and Pgau ( Borrelia afzelii ). In all cases, a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response is specific for the C-terminal region of OspA.
  • B31 Borrelia burgdorferi sensu stricto
  • K48 Borrelia garinii
  • Pgau Borrelia afzelii
  • FIGS. 30A, 30B and 30 C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 55) and the encoded chimeric protein sequence (SEQ ID NO: 56).
  • FIGS. 31A, 31B and 31 C depict the nucleic acid sequence of the OspC-B31 (bp 55-624)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 57) and the encoded chimeric protein sequence (SEQ ID NO: 58).
  • FIGS. 32A, 32B and 32 C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 59) and the encoded chimeric protein sequence (SEQ ID NO: 60).
  • FIGS. 33A, 33B, and 33 C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 61) and the encoded chimeric protein sequence (SEQ ID NO: 62).
  • FIGS. 34A, 34B and 34 C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 63) and the encoded chimeric protein sequence (SEQ ID NO: 64).
  • FIGS. 35A, 35B and 35 C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 65) and the encoded chimeric protein sequence (SEQ ID NO: 66).
  • FIGS. 36A, 36B and 36 C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 67) and the encoded chimeric protein sequence (SEQ ID NO: 68).
  • FIGS. 37A, 37B and 37 C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 69) and the encoded chimeric protein sequence (SEQ ID NO: 70).
  • FIGS. 38A, 38B and 38 C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 71) and the encoded chimeric protein sequence (SEQ ID NO: 72).
  • FIGS. 39A, 39B, 39 C depict the nucleic acid sequence of the OspC-C12 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-822) chimer (SEQ ID NO: 73) and the encoded chimeric protein sequence (SEQ ID NO: 74).
  • FIGS. 40A, 40B and 40 C depict the nucleic acid sequence of the OspC-Pko (bp 55-639)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-651)/OspA-K48 (bp 652-825) chimer (SEQ ID NO: 75) and the encoded chimeric protein sequence (SEQ ID NO: 76).
  • FIGS. 41A, 41B and 41 C depict the nucleic acid sequence of the OspC-Tro (bp 55-624)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 77) and the encoded chimeric protein sequence (SEQ ID NO: 78).
  • FIGS. 42A and 42B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 394-820) chimer (SEQ ID NO: 79) and the encoded chimeric protein sequence (SEQ ID NO: 80).
  • FIGS. 43A and 43B depict the nucleic acid sequence of the OspC-B31 (bp 55-631)/OspA-B31 (bp 394-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 81) and the encoded chimeric protein sequence (SEQ ID NO: 82).
  • FIGS. 44A and 44B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 394-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 83) and the encoded chimeric protein sequence (SEQ ID NO: 84).
  • FIGS. 45A and 45B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-K48 (bp 394-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 85) and the encoded chimeric protein sequence (SEQ ID NO: 86).
  • FIGS. 46A, 46B and 46 C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 87) and the encoded chimeric protein sequence (SEQ ID NO: 88).
  • FIGS. 47A, 47B, and 47 C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 89) and the encoded chimeric protein sequence (SEQ ID NO: 90).
  • FIGS. 48A and 48B depict the nucleic acid and encoded protein sequence of an R139M altered OspA (SEQ ID NO: 95 and 96).
  • FIGS. 49A and 49B depict the nucleic acid and encoded protein sequence of an E160Y altered OspA (SEQ ID NO: 97 and 98).
  • FIGGS. 50A and 50B depict the nucleic acid and encoded protein sequence of an R139M, E160Y altered OspA (SEQ ID NO: 99 and 100).
  • FIGS. 51A and 51B depict the nucleic acid and encoded protein sequence of an E160Y altered OspA (SEQ ID NO: 101 and 102).
  • FIGS. 52A and 52B depict the nucleic acid and encoded protein sequence of an R139M, E160Y, K189M altered OspA (SEQ ID NO: 103 and 104).
  • FIGS. 53A and 53B depict the nucleic acid and encoded protein sequence of an Y165F altered OspA (SEQ ID NO: 105 and 106).
  • FIGS. 54A and 54B depict the nucleic acid and encoded protein sequence of an Y165F, V166T altered OspA (SEQ ID NO: 107 and 108).
  • FIGS. 55A and 55B depict the nucleic acid and encoded protein sequence of a V166T altered OspA (SEQ ID NO: 109 and 110).
  • FIGS. 56A and 56B depict the nucleic acid and encoded protein sequence of a V166T, T170K altered OspA (SEQ ID NO: 111 and 112).
  • FIGS. 57A and 57B depict the nucleic acid and encoded protein sequence of an Y165F, V166T, T170K altered OspA (SEQ ID NO: 113 and 114).
  • FIGS. 58A and 58B depict the nucleic acid and encoded protein sequence of an R139M, E160Y, K189M, Y165F, V166T, T170K altered OspA (SEQ ID NO: 115 and 116).
  • the present invention is drawn to altered forms of OspA from Borrelia burgdorferi that have increased conformational stability while maintaining antigenicity, as indicated, for example, by the ability to be bound by the LA-2 monoclonal antibody.
  • the altered OspA polypeptides also have decreased cross-reactivity with hLFA-1.
  • the altered OspA polypeptides can comprise all (with the exception of the alterations described herein) or a portion, such as the C-terminal portion, of a wild type OspA polypeptide.
  • OspA protein such as truncated versions of OspA
  • some forms of the OspA protein do not elicit a strong immunoprotective response when administered to an animal even when the OspA polypeptide has the immunoprotective LA-2 epitope sequence.
  • the structure for recombinant OspA has been determined to 1.95 ⁇ resolution in a binary complex with the Fab fragment of the nonprotective mouse mAb184.1, which is reactive with the OspA-terminus (Li et al., Proc. Natl. Acad. Sci. U.S.A . 94:3584-3589 (1997)).
  • the OspA polypeptide folds into 21 consecutive antiparallel ⁇ -strands followed by a C-terminal ⁇ -helix.
  • the structure is conveniently described as two discrete folded domains, an N-terminal sandwich domain and a C-terminal barrel domain, connected by a long central ⁇ -sheet.
  • One set of altered polypeptides described herein is designed to remove buried charges and/or salt bridges in the OspA C-terminal portion and replace them with residues that promote hydrophobic interactions.
  • the altered OspA polypeptides of the present invention comprise altered OspA protein or polypeptides from Borrelia burgdorferi from about residue 139 to about residue 273, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 changed to methionine, residue 160 changed to tyrosine, residue 189 changed to methionine and combinations thereof.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the altered OspA polypeptide has a tyrosine at residue position 160 and a methionine at residue position 189.
  • the altered OspA polypeptide has a methionine at residue position 139, a tyrosine at residue position 160 and a methionine at residue position 189.
  • the altered OspA polypeptides have both increased conformational stability and reduced cross-reactivity to the hLFA-1 protein.
  • the altered OspA sequence can be from any Lyme borreliosis strain of Borrelia burgdorferi , such as strains from Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii .
  • Strains of Borrelia burgdorferi are well known to those of skill in the art.
  • strains of Borrelia burgdorferi sensu stricto include B31
  • strains of Borrelia afzelii include Pgau and Pko
  • strains of Borrelia garinii include K48.
  • the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least two alterations selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine and residue 170 changed to lysine, wherein the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from a sensu stricto strain of Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine, residue 170 changed to lysine and combinations thereof.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the altered OspA polypeptide has a phenylalanine at residue position 165 and a threonine at residue position 166.
  • the altered OspA polypeptide has a phenylalanine at residue position 165, a threonine at residue position 166 and a lysine at residue position 170.
  • the altered OspA polypeptide includes all of the alterations described herein.
  • the altered OspA polypeptide has a methionine at residue position 139, a tyrosine at residue position 160, a methionine at residue position 189, a phenylalanine at residue position 165, a threonine at residue position 166 and a lysine at residue position 170.
  • This invention also pertains to polypeptides comprising SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, or 116.
  • the altered OspA polypeptides of the invention can be partially or substantially purified (e.g., purified to homogeneity), and/or substantially free of other proteins.
  • polynucleotide refers to a nucleotide multimer or oligomer which is composed of deoxyribonucleotides or ribonucleotides, or a combination thereof, having from a few, e.g., 2-20, to many, e.g., 20 to several thousand or more, nucleotides.
  • polynucleotides include nucleic acids of any length and further encompass both naturally-occurring and synthetic oligonucleotides and polynucleotides.
  • the polynucleotides of the present invention include polynucleotides encoding OspA polypeptides from Borrelia burgdorferi from about residue 139 to about residue 189, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine and combinations thereof.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide encoding the altered OspA sequence can be from any Lyme borreliosis strain of Borrelia burgdorferi.
  • the polynucleotide encodes an amino acid sequence of an OspA polypeptide from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 165 encoding phenylalanine, codon 166 encoding threonine, codon 170 encoding lysine and combinations thereof.
  • the numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • polynucleotides of the present invention include polynucleotides selected from the group consisting of: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115.
  • the altered OspA polypeptides of the present invention can be derived from OspA molecules comprising fragments, derivatives, analogs, variants and mutants of the OspA protein (modified OspA) or can be fragmented, derivatized, or otherwise altered after having the alterations described herein inserted. These modified OspA molecules possess OspA antigenic activity.
  • the present invention is also drawn to a method of generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the method comprises selecting a polynucleotide encoding a Borrelia burgdorferi OspA polypeptide that includes residues 139, 160 and 189, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide is altered such that residue 139 is methionine, residue 160 is tyrosine or residue 189 is methionine or combination thereof.
  • the altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • Methods of altering a polynucleotide are described below and in the Exemplification and are well known to those of skill in the art.
  • Methods of expressing the altered polypeptides of the invention are also described below and in the Exemplification and are well known to those of skill in the art.
  • One way to eliminate the cross-reactive sequence is to replace the ⁇ -13 region of OspA-B31 (YVLEGTLTA (SEQ ID NO: 129)) with an analogous region from a strain that does not possess the same sequence, such as that from a B. Afzelii strain, e.g., Pgau or Pko (U.S. Patent Application entitled “Recombinant Constructs of Borrelia burgdorferi ” by Lucas et al., filed on Aug. 7, 2001, the entire teachings of which are incorporated herein by reference).
  • the present invention is drawn to a method of generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • the method comprises selecting a polynucleotide encoding an OspA polypeptide from Borrelia burgdorferi that includes residues 165, 166 and 170, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
  • the polynucleotide is altered such that residue 165 is changed to phenylalanine, residue 166 is changed to threonine or residue 170 is changed to lysine or combination thereof.
  • the altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • an altered OspA polypeptide having reduced cross-reactivity to hLFA-1 while retaining the ability to be bound by LA-2 is generated.
  • residue 130 is methionine
  • residue 160 is tyrosine
  • residue 165 is phenylalanine
  • residue 166 is threonine
  • residue 170 is lysine
  • residue 189 is methionine.
  • Polynucleotides encoding Borrelia burgdorferi OspA polypeptides can be selected as described herein.
  • the altered OspA polypeptide includes the minimal sequence that includes the positions of the alterations.
  • the altered polypeptide can comprise OspA from about residue 139 to about residue 189, wherein the numbering corresponds to SEQ ID NO: 7.
  • the altered polypeptide can comprise OspA from about residue 165 to about residue 170, wherein the numbering corresponds to SEQ ID NO: 7.
  • the altered OspA polypeptides of the present invention also include larger fragments of OspA.
  • the altered OspA polypeptides include, but are not limited to, altered OspA polypeptides which comprise OspA from about residue 160 to about residue 170, OspA from about residue 150 to 180, OspA from about residue 131 to 273 or OspA from about residue 17 to 273.
  • altered OspA polypeptides which comprise OspA from about residue 160 to about residue 170, OspA from about residue 150 to 180, OspA from about residue 131 to 273 or OspA from about residue 17 to 273.
  • the OspA sequence used to generate the altered OspA polypeptide can itself be a chimeric OspA polypeptide, having two or more segments derived from OspA proteins from different genospecies or strains of Borrelia.
  • the size of the altered OspA polypeptide can vary depending on the method used to generate the altered polypeptide and/or the purpose for which it is generated, and such altered OspA chimeric polypeptides can include fragments of OspA.
  • the altered polypeptide can be part of a larger polypeptide, including additional OspA sequences on the N-terminus, the C-terminus or both termini.
  • a fragment of OspA protein can encompass polypeptides that are only a part of the full-length OspA protein. Such OspA fragments typically include at least one of the altered residues described herein and possess at least some of the antigenicity of wild type OspA. OspA fragments can be produced by amino and/or carboxyl terminal deletions, as well as internal deletions. Fragments can also be produced by enzymatic digestion. Such modified OspA molecules can be tested for antigenic activity as described herein or using methods known in the art.
  • the altered OspA polypeptide can be part of a cocktail with one or more other proteins, such as other Borrelia burgdorferi polypeptides, including but not limited to, OspA, OspB, OspC, OspD, p93 and p41.
  • the altered OspA polypeptide can be part of a larger molecule, such as a chimeric polypeptide, for example, as described in U.S. Pat. No. 6,248,562 and U.S. Patent Application entitled “Recombinant Constructs of Borrelia burgdorferi ”, by Lucas et al, filed on Aug. 7, 2001.
  • Such larger polypeptides can include amino acid sequences from other proteins including but not limited to, other Borrelia burgdorferi proteins and/or other proteins useful in generating fusion proteins for vaccine and/or immunodiagnostic methods.
  • Additional components for example, labels (a radioisotope, an epitope label (tag)(e.g., a hemagglutinin (HA) epitope, a hexahistidine tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme label, a fluorescent group, a chemiluminescent group) can be incorporated into the altered OspA polypeptides of the invention to assist in the isolation and/or purification of the polypeptide.
  • labels a radioisotope, an epitope label (tag)(e.g., a hemagglutinin (HA) epitope, a hexahistidine tag), an affinity label (e.g., biotin, avidin),
  • a hexahistidine tag would permit ready purification by nickel chromatography.
  • These and other components can also be incorporated in the altered OspA polypeptides of the invention in order to extend the half life of the polypeptides. Methods of incorporating such components into the polypeptides of the invention are well known to those of skill in the art.
  • the altered OspA polypeptide of the invention is a chimeric polypeptide.
  • the altered OspA polypeptide comprises the following: a) an amino acid sequence of a first OspA polypeptide from about residue 1 to about residue 164 from a first strain of Borrelia burgdorferi ; b) an amino acid sequence of a second OspA polypeptide from about residue 165 to about residue 179 from a second strain of Borrelia burgdorferi , wherein said second strain is a different strain from said first strain; c) an amino acid sequence of a third OspA polypeptide from about residue 180 to about residue 216 from a third strain of Borrelia burgdorferi , wherein said third strain is a different strain from said second strain; d) an amino acid sequence of a fourth OspA polypeptide from about residue 217 to about residue 273 from a fourth strain of Borrelia burgdorferi , wherein
  • Polypeptides described herein can be isolated from naturally-occurring sources, chemically synthesized or recombinantly produced.
  • the altered OspA polypeptides of the present invention can be derived from naturally-occurring OspA molecules or from nucleic acids which encode such molecules.
  • the OspA polypeptides of the present invention can comprise fragments, derivatives, analogs, variants and mutants of an OspA protein (modified OspA) and/or can be fragmented, derivatized or otherwise altered after having the alterations described herein inserted (also referred to as modified OspA).
  • modified OspA molecules possess at least some OspA antigenic activity.
  • the amino acid sequence of the altered OspA polypeptides of the invention can be that of a naturally-occurring protein or can comprise additional modifications.
  • additional modifications include conservative and/or non-conservative amino acid substitutions, additions of one or more amino acids, and/or deletions of one or more amino acids.
  • additional modifications should also preserve at least some activity of the encoded protein or polypeptide.
  • the further modified polypeptide or protein should have similar or improved conformational stability, similar or improved immunoprotective activity or reduced cross-reactivity to hLFA-1, as compared to the corresponding altered OspA polypeptide (i.e., the OspA polypeptide comprising one or more of the alterations described herein but not comprising the further modification(s)).
  • the further modification(s) preferably preserve the three-dimensional configuration of an antibody binding site of the native protein such as the LA-2 binding site.
  • the presence or absence of biological activity or activities can be determined by various functional assays as described herein or using methods that are known in the art, e.g., recognition using an ELISA assay, elicitation of an immune response (e.g., an immunoprotective response) in an animal.
  • Appropriate amino acid alterations that fall within the scope of the invention can be made on the basis of several criteria, including hydrophobicity, basic or acidic character, charge, polarity, size, the presence or absence of a functional group (e.g., —SH or a glycosylation site), and aromatic character, provided that the resulting molecule has at least one of the alterations described herein and maintains increased conformational stability and/or reduced cross-reactivity to hLFA-1. Assignment of various amino acids to similar groups based on the properties above will be readily apparent to the skilled artisan; further appropriate amino acid changes can also be found in Bowie ( Science , 247:1306-1310(1990)).
  • “Variants” and “mutants” of OspA can be produced using in vitro and/or in vivo techniques well-known to those of skill in the art, for example, site-specific mutagenesis, and oligonucleotide mutagenesis. Manipulations of the OspA polypeptide sequence can be made at the protein level as well. Chemical modifications can be carried out using known techniques including but not limited to, specific chemical cleavage using cyanogen bromide, trypsin and/or papain. OspA can also be structurally modified and/or denatured, for example, using heat. In general, mutations can be conservative or non-conservative amino acid substitutions, amino acid insertions or amino acid deletions.
  • a nucleic acid (e.g., DNA) encoding a modified OspA polypeptide can be prepared by site-directed mutagenesis of the nucleic acid (e.g., DNA) that encodes a wild type OspA.
  • Site-directed (site-specific) mutagenesis allows the production of OspA variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation (e.g., alteration, deletion, insertion), as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the desired mutation.
  • a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementary residues on both sides of the mutation of the sequence being altered.
  • site-specific mutagenesis is well known in the art, as exemplified by publications such as Edelman et al., DNA , 2:183, 1983.
  • a site-specific mutagenesis technique can employ a phage vector that exists in both a single-stranded and double-stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA , A.
  • phage vectors are commercially available and their use is well known to those skilled in the art.
  • a versatile and efficient procedure for the construction of oligonucleotide directed site-specific mutations in DNA fragments using M13-derived vectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res ., 10:6487-6500, 1982.
  • plasmid vectors that contain a single-stranded phage origin of replication can be employed to obtain single-stranded DNA (see for example, Veira et al., Meth Enzymol ., 153:3, (1987)).
  • nucleotide substitutions can be introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it using PCR procedures known in the art.
  • site-specific mutagenesis can be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc Natl Acad Sci USA ., 75:5765, 1978.
  • This primer can then be annealed with the single-stranded protein sequence-containing vector, and subjected to DNA polymerizing enzymes, e.g., E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand.
  • heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector can then be used to transform appropriate host cells such as JM 101 cells, and clones can be selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region can be removed and placed in an appropriate expression vector for protein production.
  • the PCR technique can be used in creating amino acid sequence variants of OspA.
  • primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • one of the primers can be designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer is preferably identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA.
  • sequence of the second primer be located within 500 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced.
  • PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the end position of the mutation specified by the primer.
  • the DNA fragments produced bearing the desired mutation can be used to replace the corresponding region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more) part ligation.
  • the starting material can be the plasmid (or vector) comprising the OspA DNA to be mutated.
  • the codon(s) within the OspA to be mutated are identified.
  • the plasmid is cut at these sites to linearize it.
  • a double stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • the plasmid now contains the mutated OspA DNA sequence, and can be subcloned and/or expressed to produce the modified OspA polypeptide or protein.
  • the OspA encoding nucleic acid molecules (e.g., polynucleotides) of the present invention have at least one of the alterations described herein and generally hybridize under high stringency hybridization conditions to a polynucleotide-encoding OspA nucleic acid or fragment thereof from a sensu stricto strain of Borrelia burgdorferi , e.g., SEQ ID NO: 7.
  • the OspA-encoding nucleic acid molecules (e.g., polynucleotides) of the present invention hybridize under high stringency hybridization conditions to a polynucleotide encoding OspA or fragment thereof from Borrelia afzelii , e.g., SEQ ID NO: 10.
  • the OspA-encoding nucleic acid molecules (e.g., polynucleotides) of the present invention hybridize under high stringency hybridization conditions to a polynucleotide encoding OspA or fragment thereof from Borrelia garinii , e.g., SEQ ID NO: 8.
  • the polynucleotides and polypeptides of the present invention include modified versions of OspA as described herein.
  • stringent hybridization conditions include a sodium ion concentration of no more than 1 M and a temperature of at least 25° C.
  • 5 ⁇ SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30° C., or equivalent conditions are suitable for specific hybridization.
  • Equivalent conditions can be determined by varying one or more of the parameters, as is known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
  • Hybridizable nucleic acid molecules are useful as probes and primers for diagnostic applications.
  • the invention pertains to nucleic acid molecules which have a substantial identity with the nucleic acid molecules encoding the altered OspA polypeptides described herein wherein the nucleic acid encodes for one or more of the alterations described herein; particularly preferred are nucleic acid molecules which have at least about 90%, more preferably at least about 95% and most preferably at least about 98% identity with nucleic acid molecules described herein, wherein the nucleic acid encodes for at least one of the alterations described herein. Sequence identity can be determined using publically or commercially available sequence alignment algorithms, using, for example, default parameters.
  • DNA molecules which comprise a sequence which is different from the naturally-occurring nucleic acid molecule but which, due to the degeneracy of the genetic code, encode the same protein or polypeptide are encompassed by the present invention.
  • the invention also encompasses variations of the nucleic acid molecules of the invention, such as those encoding portions, analogues or derivatives of the encoded protein or polypeptide.
  • Such variations can be naturally-occurring, such as in the case of allelic variation, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes, so long as the nucleic acid molecule encodes at least one of the alterations described herein and the encoded protein has either increased conformational stability and/or decreased cross-reactivity to hLFA-1 (as compared to the corresponding unaltered protein) that is conferred by the alterations described herein.
  • Intended variations include, but are not limited to, addition, deletion and/or substitution of one or more nucleotides which can result in conservative or non-conservative amino acid changes, including additions and deletions.
  • nucleotide or amino acid variations are silent; that is, they do not alter one or more characteristics or activity of the encoded altered OspA protein or polypeptide.
  • activities of the encoded protein or polypeptide include, but are not limited to, binding function, antigenic function and conformational stability.
  • the invention also provides expression vectors containing a nucleic acid sequence described herein, operably linked to at least one regulatory sequence.
  • Many such vectors are commercially available, and other suitable vectors can be readily prepared by the skilled artisan.
  • “Operably linked” is intended to mean that the nucleic acid molecule is linked to a regulatory sequence in a manner which allows expression of the nucleic acid sequence. Regulatory sequences are art-recognized and are selected to produce the encoded polypeptide or protein. Accordingly, the term “regulatory sequence” includes promoters, enhancers, and other expression control elements which are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • the native regulatory sequences or regulatory sequences native to the transformed host cell can be employed.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
  • the polypeptides of the present invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells or both (see, for example, Broach, et al., Experimental Manipulation of Gene Expression , ed. M. Inouye (Academic Press, 1983) p. 83 ; Molecular Cloning: A Laboratory Manual , 2nd Ed., ed.
  • expression constructs will contain one or more selectable markers, including but not limited to, the gene that encodes dihydrofolate reductase and the genes that confer resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin or streptomycin.
  • Prokaryotic and eukaryotic host cells transfected by the described vectors are also provided by this invention.
  • cells which can be transfected with the vectors of the present invention include, but are not limited to, bacterial cells such as E. coli (e.g., E. coli K12, BL21, DH5 ⁇ strains), Streptomyces, Pseudomonas, Serratia marcescens and Salmonella typhimurium , insect cells (baculovirus) including Drosophila, fungal cells, such as yeast cells, plant cells and mammalian cells, such as thymocytes, Chinese hamster ovary cells (CHO) and COS cells.
  • E. coli e.g., E. coli K12, BL21, DH5 ⁇ strains
  • Streptomyces e.g., Pseudomonas, Serratia marcescens and Salmonella typhimurium
  • insect cells baculovirus
  • a nucleic acid molecule comprising, for example SEQ ID NO: 6 with at least one of the specific alterations described herein, or a nucleic acid molecule which encodes, for example SEQ ID NO: 7 with at least one of the specific alterations described herein, can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes.
  • Ligating the nucleic acid molecule (e.g., polynucleotide) into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect, plant or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well known proteins. Similar procedures, or modifications thereof, can be employed to prepare recombinant proteins according to the present invention by microbial means or tissue-culture technology. Accordingly, the invention pertains to the production of encoded proteins or polypeptides by recombinant technology.
  • proteins and polypeptides of the present invention can be isolated or purified (e.g., to homogeneity) from recombinant cell culture by a variety of processes. These include, but are not limited to, anion or cation exchange chromatography, ethanol precipitation, affinity chromatography and high performance liquid chromatography (HPLC). The particular method used will depend upon the properties of the polypeptide and the selection of the host cell; appropriate methods will be readily apparent to those skilled in the art.
  • the present invention also pertains to pharmaceutical compositions comprising polypeptides and other compounds described herein.
  • a polypeptide or protein of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition.
  • the particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to well known procedures, and will depend on the ultimate pharmaceutical formulation desired.
  • Methods of introduction of exogenous polypeptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal.
  • Other suitable methods of introduction can also include gene therapy, rechargeable or biodegradable devices and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • the altered OspA proteins described herein can be produced so that they are highly soluble, hyper-produced in E. coli , and non-lipidated.
  • the altered OspA proteins can be designed to begin or end in a suitable affinity tag (e.g., a His-tag) to facilitate purification.
  • a suitable affinity tag e.g., a His-tag
  • the recombinant proteins described herein have been constructed to maintain high levels of antigenicity and improved conformational stability.
  • the altered OspA proteins of the current invention are advantageous in that they retain at least some specific reactivity to monoclonal and/or polyclonal antibodies against wild-type Borrelia proteins, are immunogenic, and inhibit the growth or induce lysis of Borrelia in vitro.
  • the proteins are particularly useful in immunodiagnostic assays.
  • proteins of the present invention can be used as reagents in assays to detect the presence of antibodies to native Borrelia in potentially infected individuals. These proteins can also be used as immunodiagnostic reagents, such as in dot blots, Western blots, enzyme-linked immunosorbent assays, or agglutination assays.
  • the altered OspA proteins of the present invention can be produced by known techniques, such as by recombinant methodology, polymerase chain reaction, or mutagenesis.
  • proteins of the current invention are useful as vaccine immunogens against Borrelia infection.
  • One or more of the altered proteins can be combined with a physiologically acceptable carrier and administered to a vertebrate animal through standard methods (e.g., intravenously or intramuscularly, for example).
  • the altered forms of the OspA proteins described herein were bioengineered such that at least one immunoprotective domain of the protein was maintained.
  • antigenic refers to the ability of a compound to bind products of an immune response, such as antibodies, T-cell receptors or both. Such responses can be measured using standard antibody detection assays, such as ELISA or standard T-cell activation assays.
  • the altered forms of OspA described herein elicit an immunoprotective response, for example, by eliciting antibodies that recognize the LA-2 epitope.
  • nucleic acids that encode the polypeptides that comprise the altered OspA protein can include extra nucleotides or fewer nucleotides in order to simplify the construction of the gene encoding the chimeric polypeptide, e.g., to allow for the use of convenient restriction endonuclease sites, or to allow the ligation of the gene fragments such that a contiguous coding region is created.
  • the altered OspA polypeptides of the present invention can be lipidated or non-lipidated.
  • mice can be immunized with OspA polypeptides or proteins containing the polypeptide sequences in aluminum hydroxide. Mice are then bled and tested for antibody responses against OspA derived from various strains of Borrelia. In additional experiments, these immunized mice can be challenged with ticks infected with Borrelia burgdorferi and transmission of infection can be assessed as described in the Exemplification which use OspA, OspC and OspC/OspA chimeric molecules. The results of such a tick challenge reveals whether the animal has developed a protective immune response. For example, an immunized animal that does not seroconvert in response to subsequent tick challenge has likely generated an immunoprotective response to the immunization.
  • the immunogenic compositions of the present invention can be used to immunize animals including humans. Immunization is understood to elicit specific immunogenic responses, preferably immunoprotective responses, as described above. As described herein, an immunogenic response includes responses that result in at least some level of immune response in the treated animal, where the animal was previously treated with a composition comprising at least one altered OspA polypeptide of the present invention.
  • Immunity as described herein, is understood to mean the ability of the treated animal to resist infection, to resist systemic infection, or to overcome infection such as systemic infection more easily or more quickly when compared to non-immunized or non-treated individuals. Immunity can also include an improved ability of the treated individual to sustain an infection with reduced or no clinical symptoms of systemic infection.
  • the individual may be treated with the altered OspA proteins of the present invention either proactively, e.g., once a year or, alternatively, after sustaining a tick bite.
  • the altered OspA protein of the present invention together with suitable excipients and/or adjuvants, is administered to an animal such that the animal develops an immune response to the OspA polypeptide of the composition.
  • the pharmaceutical composition can also be administered with other components suitable for in vitro and/or in vivo use. These additional components include buffers, carrier proteins, adjuvants, preservatives and combinations thereof.
  • the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope.
  • the present invention is also drawn to a physiological composition comprising an altered OspA protein.
  • the composition is useful to administer to an animal in order to generate an immune response or in the diagnostic methods described herein.
  • the composition of the present invention can include suitable adjuvants, well known in the art, to enhance immunogenicity, potency or half-life of the chimeric proteins in the treated animal.
  • adjuvants and their use are well known in the art (see for example PCT Publication WO 96/40290, the entire teachings of which are incorporated herein by reference).
  • the composition can be prepared by known methods of preparing vaccines.
  • the altered OspA polypeptides described herein can be isolated and/or purified using known techniques, such as by size exclusion chromatography, ion exchange chromatography, affinity chromatography, preparative electrophoresis, selective precipitation or combinations thereof.
  • the prepared proteins can be mixed with suitable other reagents as described above, wherein the protein is at a suitable concentration.
  • the dosage of the protein will vary and depends upon the age, weight and/or physical condition of the animal to be treated.
  • the optimal dosage can be determined by routine optimization techniques, using suitable animal models.
  • composition to be used as a vaccine can be administered by any suitable technique.
  • administration is by injection, e.g., subcutaneous, intramuscular, intravenous, or intra peritoneal injection.
  • the composition is administered to mucosa, e.g., by exposing nasal mucosa to nose drops containing the proteins of chimeric proteins of the present invention.
  • the immunogenic composition is administered by oral administration.
  • the chimeric proteins are administered by DNA immunization using nucleic acids encoding an altered OspA polypeptide.
  • the present invention is also drawn to a diagnostic kit comprising the altered OspA polypeptides described herein.
  • the kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspA protein and antibodies that are present in an sample, e.g., a user-supplied host sample.
  • the present invention is also drawn to methods of detecting an immune response to Lyme Disease-causing Borrelia in a host sample.
  • the method comprises contacting a host sample with an OspA altered protein, such that anti-OspA antibodies, if present in said sample, bind to said OspA protein.
  • the quantity of antibodies that have bound said OspA protein are measured, thereby detecting an immune response to Lyme disease-causing Borrelia.
  • This example details a method for the purification of large amounts of native outer surface protein A (OspA) to homogeneity, and describes mapping of the antigenic specificities of several anti-OspA MAbs.
  • OspA was purified to homogeneity by exploiting its resistance to trypsin digestion. Intrinsic labeling with 14 C-palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal cysteine of the molecule.
  • OspA Although both OspA and OspB are sensitive to proteinase K digestion, in contrast to OspB, OspA is resistant to cleavage by trypsin (Dunn, J. et al., Prot. Exp. Purif ., 1:159-168 (1990); Barbour, A. G. et al., Infect. Immun ., 45:94-100 (1984)).
  • trypsin Unn, J. et al., Prot. Exp. Purif ., 1:159-168 (1990); Barbour, A. G. et al., Infect. Immun ., 45:94-100 (1984)
  • the relative insensitivity to trypsin is surprising in view of the fact that OspA has a high (16% for B31) lysine content, and may relate to the relative configuration of OspA and B in the outer membrane.
  • the suspension was rotated overnight at 4° C., and the insoluble fraction removed as a pellet after centrifugation at 10,000 ⁇ g for 15 minutes at 4° C.
  • the supernatant (soluble fraction) was incubated at 37° C. for 15 minutes and centrifuged at room temperature at 1000 ⁇ g for 15 minutes to separate the aqueous and detergent phases.
  • the aqueous phase was decanted, and ice cold PBS added to the lower Triton phase, mixed, warmed to 37° C., and again centrifuged at 1000 ⁇ g for 15 minutes. Washing was repeated twice more.
  • detergent was removed from the preparation using a spin column of Bio-beads SM2 (BioRad, Melville, N.Y.) as described (Holloway, P. W., Anal. Biochem ., 53:304-308 (1973)).
  • Ion exchange chromatography was carried out as described by Dunn et al., (Dunn et al., Prot. Exp. Purif ., 1:159-168 (1990)) with minor modifications. Crude OspA was dissolved in buffer A (1% Triton X-100, 10 mM phosphate buffer (pH 5.0)) and loaded onto a SP Sepharose resin (Pharmacia, Piscataway, N.J.), pre-equilibrated with buffer A at 25° C. After washing the column with 10 bed-volumes of buffer A, the bound OspA was eluted with buffer B (1% Triton X-100, 10 mM phosphate buffer (pH 8.0)).
  • OspA fractions were detected by protein assay using the BCA method (Pierce, Rockford, Ill.), or as radioactivity when intrinsically labeled material was fractionated.
  • Triton X-100 was removed using a spin column of Bio-beads SM2.
  • This method purifies OspA from an outer surface membrane preparation.
  • OspA and B were the major components of the soluble fraction obtained after Triton partitioning of strain B31.
  • Triton extraction was carried out after trypsin-treatment, the OspB band is not seen.
  • Further purification of OspA-B31 on a SP Sepharose column resulted in a single band by SDS-PAGE. The yield following removal of detergent was approximately 2 mg per liter of culture.
  • This method of purification of OspA, as described herein for strain B31 can be used for other isolates of Borrelia as well. For strains such as strain K48, which lack OspB, trypsin treatment can be omitted.
  • OspA and B contain a signal sequence (L-X-Y-C) similar to the consensus described for lipoproteins of E. coli , and it has been predicted that the lipidation site of OspA and B should be the amino-terminal cysteine (Brandt, M. E. et al., Infect. Immun , 58:983-991 (1990)). The results presented herein support this prediction.
  • Strains K48, PGau and DK29 were supplied by R. Johnson, University of Minnesota; Pko and pTrob were provided by B. Wilske and V. Preac-Mursic of the Pettenkhofer Institute, Kunststoff, Germany; and Ip3 and Ip90 were supplied by L. Mayer of the Center for Disease Control, Atlanta, Ga.
  • the North American strains included strain 25015, provided by J. Anderson of the Connecticut Department of Agriculture; and strain B31 (ATCC 35210).
  • MAbs monoclonal antibodies
  • Prediction of the various cleavage sites was achieved by knowledge of the primary amino acid sequence derived from the full nucleotide sequences of OspA, many of which are currently available (see Table II, below). Cleavage sites can also be predicted based on the peptide sequence of OspA, which can be determined by standard techniques after isolation and purification of OspA by the method described above. Cleavage of several OspA isolates was conducted to determine the localization of monoclonal antibody binding of the proteins.
  • Proteins and peptides generated by cleavage were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U. K., Nature ( London ) 227:680-685 (1970)), and electroblotted onto immobilon Polyvinylidine Difluoride (PVDF) membranes (Ploskal, M. G. et al., Biotechniques , 4:272-283 (1986)). They were detected by amido black staining or by immunostaining with murine MAbs, followed by alkaline phosphatase-conjugated goat anti-mouse IgG. Specific binding was detected using a 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) developer system (KPL Inc., Gathersburg, Md.).
  • BCIP 5-bromo-4-chloro-3-indolylphosphate
  • NBT nitroblue
  • HA1 and HA2 were fragmented with hydroxylamine-HCl (HA) into two peptides, designated HA1 and HA2 (data not shown).
  • the HA1 band migrated at 27 kd and retained its radioactivity, indicating that the peptide included the lipidation site at the N-terminus of the molecule (data not shown). From the predicted cleavage point, HA1 should correspond to residues 1 to 251 of OspA-B31.
  • HA2 had a MW of 21.6 kd by SDS-PAGE, with amino-terminal sequence analysis showing it to begin at Gly72, i.e.
  • HA cleaved OspA-K48 into three peptides, designated HA1, HA2, and HA3 with apparent MWs of 22 kd, 16 kd and 12 kd, respectively.
  • Amino-terminal sequencing showed HA1 to start at Gly72, and HA3 at Gly142.
  • HA2 was found to have a blocked amino-terminus, as was observed for the full-length OspA protein.
  • HA1, 2 and 3 of OspA-K48 were predicted to be residues 72-274, 1 to 141 and 142 to 274, respectively.
  • N-Chlorosuccinimide cleaves tryptophan (W), which is at residue 216 of OspA-B31 or residue 217 of OspA-K48 (data not shown).
  • NCS cleaved OspA-B31 into 2 fragments, NCS1, with MW of 23 kd, residues 1-216 of the protein, and NCS2 with a MW of 6.2 kd, residues 217 to 273 (data not shown).
  • K48 OspA was divided into 2 pieces, NCS1 residues 1-217, and NCS2 residues 218 to 274 (data not shown).
  • Cleavage of OspA by cyanogen bromide (CNBr) occurs at the carboxy side of methionine, residue 39.
  • the major fragment, CNBr1 has a MW of 25.7 kd, residues 39-274 by amino-terminal amino acid sequence analysis (data not shown).
  • CNBr2 (about 4 kd) could not be visualized by amido black staining; instead, lightly stained bands of about 20 kd MW were seen. These bands reacted with anti-OspA MAbs, and most likely were degradation products due to cleavage by formic acid.
  • MAb 12 specific for OspA-B31, bound to both HA1 and HA2 of OspA-B31.
  • cleavage of OspA-B31 by NCS at residue Trp216 created fragments which did not react with MAb12, suggesting that the relevant domain is near or is structurally dependent upon the integrity of this residue (data not shown).
  • MAb 13 bound only to OspA-K48, and to peptides containing the amino-terminus of that molecule (e.g. HA2; NCS1). It did not bind to CNBr1 residues 39 to 274.
  • the domain recognized by MAb13 is in the amino-terminal end of OspA-K48, near Met38.
  • MAb15 reacts with the OspA of both the B31 and K48 strains, and to peptides containing the N-terminus of OspA, such as HA1 of OspA-B31 and NCS1, but not to peptides HA2 of OspA-B31 and HA1 of OspA-K48 (data not shown). Both peptides include residue 72 to the C-terminus of the molecules. MAb15 bound to CNBr1 of OspA-K48, indicating the domain for this antibody to be residues 39 to 72, specifically near Gly72 (data not shown).
  • MAb83 binds to OspA-K48, and to peptides containing the C-terminal portion of the molecule, such as HA1. They do not bind to HA2 of OspA-K48, most likely because the C-terminus of HA2 of OspA-K48 ends at 141. Similar to MAb12 and OspA-B31, binding of MAbs 83 and CIII.78 is eliminated by cleavage of OspA at the tryptophan residue. Thus binding of MAbs 12, 83 and CIII.78 to OspA depends on the structural integrity of the Trp 216 residue, which appears to be critical for antigenicity. Also apparent is that, although these MAbs bind to a common antigenic domain, the precise epitopes which they recognize are distinct from one another given the varying degrees of cross-reactivity to these MAbs among strains.
  • Site-directed mutagenesis was performed to convert residues within the 204-219 domain of the recombinant B31 OspA to the analogous residues of a European OspA variant, K48.
  • a European OspA variant K48.
  • Three oligonucleotides were generated, each containing nucleotide changes which would incorporate K48 amino acids at their analogous positions in the B31 OspA protein.
  • the oligonucleotides used to create the site-directed mutants were:
  • 5′-CTTAATGACTCTGACACTAGTGC-3′ (#613, which converts threonine at position 204 to serine, and serine at 206 to threonine (Thr204-Ser, Thr206-Ser)) (SEQ ID NO: 1); 5′-GCTACTAAAAAAACCGGGAAATGGAATTCA-3′ (#625, which converts alanine at 214 to glycine, and alanine at 215 to lysine (Ala214-Gly, Ala215-Lys)) (SEQ ID NO: 2); and
  • Site-directed mutagenesis was carried out by performing mutagenesis with pairs of the above oligonucleotides. Three site-directed mutants were created, each with two changes: OspA 613 (Thr204-Ser, Thr206-Ser), OspA 625 (Ala214-Gly, Ala215-Lys), and 640 (Asn217-Asp, Gly219-Lys).
  • OspA 613/625 Thr204-Ser, Thr206-Ser, Ala214-Gly, Ala215-Lys
  • OspA 613/640 Thr204-Ser, Thr206-Ser, Asn217-Asp, Gly219-Lys
  • the uncleaved material binds MAb 105; however, the 23.2 kd fragment is unreactive.
  • Similar Western blots with a TrpE-OspA fusion protein containing the carboxy-terminal portion of the OspA protein demonstrated that the small 6.2 kd piece also fails to bind MAb 105 (Schubach, W. H. et al., Infect. and Immun ., 59:1911 (1991)).
  • MAb H5332 is cross-reactive among all of the known phylogenetic groups, whereas MAb H3TS and MAb 105 seem to be specific to the B31 strain to which they were raised. Like MAb 105, the reactivities of H5332 and H3TS to OspA are abrogated by fragmentation of the protein at Trp216. MAb 336 was raised to whole spirochetes of the strain PGau. It cross-reacts to OspA from group 1 (the group to which B31 belongs) but not to group 2 (of which K48 is a member). Previous studies using fusion proteins and chemical cleavage have indicated that this antibody recognizes a domain of OspA in the region between residues 217 and 273. All of these MAbs agglutinate the B31 spirochete.
  • MAbs were used for Western Blot analysis of the site-directed OspA mutants induced in E. coli using the T7 expression system (Dunn, J. J. et al., Protein Expression and Purification , 1:159 (1990)).
  • E. coli cells carrying pET9c plasmids having a site-directed OspA mutant insert were induced at mid-log phase growth with IPTG for four hours at 37° C.
  • Cell lysates were made by boiling an aliquot of the induced cultures in SDS gel loading dye, and this material was then loaded onto a 12% SDS gel (BioRad mini-Protean II), and subjected to electrophoresis.
  • the proteins were then transferred to Imobilon-P membranes (Millipore) 70V, 2 hour at 4° C. using the BioRad mini transfer system. Western analysis was carried out as described by Schubach et al., ( Infect. Immun ., 59:1911 (1991)).
  • the 613/625 mutant failed to bind MAbs 105 and H5332, while the other mutants retained their ability to bind these MAbs. This is important in light of the data using fusion proteins that indicate that MAb 105 behaves more like MAb H3TS in terms of its serotype specificity and binding to OspA (Wilske, B. et al., Med. Microbiol. Immunol ., 181:191 (1992)).
  • the 613/625 protein has, in addition to the differences at residues Thr204 and Ser206, changes immediately amino-terminal to Trp216 (Ala214-Gly and Ala215-Lys). The abrogation of reactivity of MAbs 105 and H5332 to this protein indicated that the epitopes of OspA which bind these monoclonal antibodies are comprised of residues on the amino-terminal side of Trp216.
  • the altered OspA polypeptides of the current invention can be part of a cocktail with other proteins or can be joined to other proteins to form a chimeric protein.
  • the other polypeptides of the cocktail or chimeric can be derived from any Borrelia.
  • Representative proteins include OspA, OspB, OspC, OspD, p12, p39, p41 (fla), p66, and p93.
  • Nucleic acid sequences encoding several Borrelia proteins are available (see Table II for examples); alternatively, nucleic acid sequences encoding Borrelia proteins can be isolated and characterized using methods such as those described below.
  • Nucleic acid sequences encoding full length, lipidated proteins from known Borrelia strains were isolated using the polymerase chain reaction (PCR) as described below.
  • nucleic acid sequences were generated which encoded truncated proteins (proteins in which the lipidation signal has been removed, such as by eliminating the nucleic acid sequence encoding the first 18 amino acids, resulting in non-lipidated proteins).
  • Other proteins were generated which encoded polypeptides of a particular gene (i.e., encoding a segment of the protein which has a different number of amino acids than the protein does in nature).
  • primers can be generated from known nucleic acid sequences encoding Borrelia proteins and used to isolate other genes encoding Borrelia proteins.
  • Primers can be designed to amplify all of a gene, as well as to amplify a nucleic acid sequence encoding truncated protein sequences, such as described below for OspC, or nucleic acid sequences encoding a polypeptide derived from a Borrelia protein.
  • Primers can also be designed to incorporate unique restriction enzyme cleavage sites into the amplified nucleic acid sequences. Sequence analysis of the amplified nucleic acid sequences can then be performed using standard techniques.
  • Borrelia OspA sequences were isolated in the following manner: 100 ⁇ l reaction mixtures containing 50 mM KCl, 10 mM TRIS-HCl (pH 8,3), 1.5 mM MgCl 2 , 200 ⁇ M each NTP, 2.5 units of TaqI DNA polymerase (Amplitaq, Perkin-Elmer/Cetus) and 100 pmol each of the 5′ and 3′ primers (described below) were used. Amplification was performed in a Perkin-Elmer/Cetus thermal cycler as described (Schubach, W. H. et al., Infect. Immun ., 59:1811-1915 (1991)).
  • the amplicon was visualized on an agarose gel by ethidium bromide staining. Twenty nanograms of the chloroform-extracted PCR product were cloned directly into the PC-TA vector (Invitrogen) by following the manufacturer's instructions. Recombinant colonies containing the amplified fragment were selected, the plasmids were prepared, and the nucleic acid sequence of each OspA was determined by the dideoxy chain-termination technique using the Sequenase kit (United States Biochemical). Directed sequencing was performed with M13 primers followed by OspA-specific primers derived from sequences, previously obtained with M13 primers.
  • the 5′ and 3′ primers for cloning can be based upon any known OspA sequences. For example, the following primers based upon the OspA nucleic acid sequence from strain B31 were used:
  • OspA genes isolated in this manner include those for strains B31, K48, PGau, and 25015; the nucleic acid sequences are depicted in the sequence listing as SEQ ID NO: 6 (OspA-B31), SEQ ID NO: 8 (OspA-K48), SEQ ID NO: 10 (OspA-PGau), and SEQ ID NO: 12 (OspA-25015). An alignment of these and other OspA nucleic acid sequences is shown in FIG. 17.
  • amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO: 7 (OspA-B31), SEQ ID NO: 9 (OspA-K48), SEQ ID NO: 11 (OspA-PGau), and SEQ ID NO: 13 (OspA-25015).
  • nucleic acid sequences described above can be incorporated into expression plasmids, using standard techniques, and transfected into compatible host cells in order to express the proteins encoded by the nucleic acid sequences.
  • expression of the p12 gene and the isolation of p12 protein is set forth.
  • Amplification of the p12 nucleic acid sequence was conducted with primers that included a NdeI restriction site into the nucleic acid sequence.
  • the PCR product was extracted with phenol/chloroform and precipitated with ethanol.
  • the precipitated product was digested and ligated into an expression plasmid as follows: 15 ⁇ l (approximately 1 ⁇ g) of PCR DNA was combined with 2 ⁇ l 10 ⁇ restriction buffer for NdeI (Gibco/BRL), 1 ⁇ l NdeI (Gibco/BRL), and 2 ⁇ l distilled water, and incubated overnight at 37° C.
  • This mixture was subsequently combined with 3 ⁇ l 10 ⁇ buffer (buffer 3, New England BioLabs), 1 ⁇ l BamHI (NEB), and 6 ⁇ l distilled water, and incubated at 37° for two hours.
  • the resultant material was purified by preparative gel electrophoresis using low melting point agarose, and the band was visualized under long wave ultraviolet light and excised from the gel.
  • the gel slice was treated with Gelase using conditions recommended by the manufacturer (Epicentre Technologies).
  • the resulting DNA pellet was resuspended in 25-50 ⁇ l of 10 mM TRIS-CL (pH 8.0) and 1 mM EDTA (TE). An aliquot of this material was ligated into the pET9c expression vector (Dunn, J. J. et al., Protein Expression and Purification , 1:159 (1990)).
  • DH5-alpha is used as a “storage strain” for T7 expression clones, because it is RecA deficient, so that recombination and concatenation are not problematic, and because it lacks the T7 RNA polyierase gene necessary to express the cloned gene.
  • This strain allows for cloning of potentially toxic gene products while minimizing the chance of deletion and/or rearrangement of the desired genes.
  • Other cell lines having similar properties may also be used.
  • Kanamycin resistant colonies were single-colony purified on nutrient agar plates supplemented with kanamycin at 50 ⁇ g/ml. A colony from each isolate was inoculated into 3-5 ml of liquid medium containing 50 ⁇ g/ml kanamycin, and incubated at 37° C. without agitation. Plasmid DNA was obtained from 1 ml of each isolate using a hot alkaline lysis procedure (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)).
  • Plasmid DNA was digested with EcoRI and BglII in the following manner: 15 ⁇ l plasmid DNA was combined with 2 ⁇ l 10 ⁇ buffer 3 (NEB), 1 ⁇ l EcoRI (NEB), 1 ⁇ l BglII (NEB) and 1 ⁇ l distilled water, and incubated for two hours at 37° C. The entire reaction mixture was subjected to electrophoresis on an analytical agarose gel. Plasmids carrying the p12 insert were identified by the presence of a band corresponding to 925 base-pairs (full length p12) or 875 base-pairs (nonlipidated p12).
  • One or two plasmid DNAs from the full length and nonlipidated p12 clones in pET9c were used to transform BL21 DE3 pLysS to kanamycin resistance as described by Studier et al., ( Methods in Enzymology , Goeddel, D. (Ed.), Academic Press, 185: 60-89 (1990)).
  • One or two transformants of the full length and nonlipidated clones were single-colony purified on nutrient plates containing 25 ⁇ g/ml chloramphenicol (to maintain pLysS) and 50 ⁇ g/ml kanamycin at 37° C.
  • the induced cells were pelleted by centrifugation and resuspended in 25 ml of 20 mM NaPO 4 (pH 7.7). A small aliquot was removed for analysis by gel electrophoresis. Expressing clones produced proteins which migrated at the 12 kd position.
  • a crude cell lysate was prepared from the culture as described for recombinant OspA by Dunn, J. J. et al., ( Protein Expression and Purification , 1:159 (1990)). The crude lysate was first passed over a Q-sepharose column (Pharmacia) which had been pre-equilibrated in Buffer A: 10 mM NaPO 4 (pH 7.7), 10 mM NaCl, 0.5 mM PMSF.
  • the column was washed with 10 mM NaPO 4 , 50 mM NaCl and 0.5 mM PMSF and then p12 was eluted in 10 mM NaPO 4 , 0.5 mM PMSF with a NaCl gradient from 50-400 mM. p 12 eluted approximately halfway through the gradient between 100 and 200 mM NaCl.
  • the peak fractions were pooled and dialyzed against 10 mM NaPO4 (pH 7.7), 10 mM NaCl, 0.5 mM PMSF.
  • the protein was then concentrated and applied to a Sephadex G50 gel filtration column of approximately 50 ml bed volume (Pharmacia), in 10 mM NaPO 4 , 200 mM NaCl, 0.5 mM PMSF.
  • p12 would typically elute shortly after the excluded volume marker. Peak fractions were determined by running small aliquots of all fractions on a gel. The p12 peak was pooled and stored in small aliquots at ⁇ 20° C.
  • the megaprimer method of site directed mutagenesis and a modification were used to generate chimeric nucleic acid sequences (Sarkar and Sommer, Biotechniques , 8(4): 404-407 (1990); Aiyar, A. and J. Leis, Biotechniques , 14(3): 366-369 (1993)).
  • a 5′ primer for the first genomic template and a 3′ fusion oligo are used to amplify the desired region.
  • the fusion primer consists of a 3′ end of the first template (DNA that encodes the amino-proximal polypeptide of the fusion protein), coupled to a 5′ end of the second template (DNA that encodes the carboxy-proximal polypeptide of the fusion protein).
  • the PCR amplifications are performed using Taq DNA polymerase, 10 ⁇ PCR buffer, and MgCl 2 (Promega Corp., Madison, Wis.), and Ultrapure dNTPs (Pharmacia, Piscataway, N.J.).
  • One ⁇ g of genomic template 1, 5 ⁇ l of 10 ⁇ M 5′ oligo and 5 ⁇ l of 10 ⁇ M fusion oligo are combined with the following reagents at indicated final concentrations: 10 ⁇ Buffer-Mg FREE (1 ⁇ ), MgCl 2 (2 mM), dNTP mix (200 ⁇ M each dNTP), Taq DNA polymerase (2.5 units), water to bring final volume to 100 ⁇ l.
  • a Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.) is used to amplify under the following conditions: 35 cycles at 95° C. for one minute, 55° C. for two minutes, and 72° for three minutes. This procedure results in a “megaprimer”.
  • the resulting megaprimer is run on a 1 ⁇ TAE, 4% low-melt agarose gel.
  • the megaprimer band is cut from the gel and purified using the Promega Magic PCR Preps DNA purification system.
  • Purified megaprimer is then used in a second PCR step.
  • One ⁇ g of genomic template 2, approximately 0.5 ⁇ g of the megaprimer, and 5 ⁇ l of 10 ⁇ M 3′ oligo are added to a cocktail of 10 ⁇ buffer, MgCl 2 , dNTPs and Taq at the same final concentrations as noted above, and brought to 100 ⁇ l with water.
  • PCR conditions are the same as above.
  • the fusion product resulting from this amplification is also purified using the Promega Magic PCR Preps DNA purification system.
  • the fusion product is then ligated into TA vector and transformed into E. coli using the Invitrogen (San Diego, Calif.) TA Cloning Kit.
  • Approximately 50 ng of PCR fusion product is ligated to 50 ng of pCRII vector with 1 ⁇ Ligation Buffer, 4 units of T4 ligase, and brought to 10 ⁇ l with water.
  • This ligated product mixture is incubated at 12° C. overnight (approximately 14 hours).
  • Two ⁇ l of the ligation product mixture is added to 50 ⁇ l competent INC F′ cells and 2 ⁇ l beta mercaptoethanol. The cells are then incubated for 30 minutes, followed by heat shock treatment at 42° C.
  • the individual overnight cultures are prepared using Promega's Magic Miniprep DNA purification system. A small amount of the resulting DNA is cut using a restriction digest as a check. DNA sequencing is then performed to check the sequence of the fusion nucleic acid sequence, using the United States Biochemical (Cleveland, Ohio) Sequenase Version 2.0 DNA sequencing kit. Three to five ⁇ g of plasmid DNA is used per reaction. 2 ⁇ l 2M NaOH/2mM EDTA are added to the DNA, and the volume is brought to 20 ⁇ l with water. The mixture is then incubated at room temperature for five minutes. 7 ⁇ l water, 3 ⁇ l 3M NaAc, 75 ⁇ l ethanol are added.
  • the resultant mixture is mixed by vortex and incubated for ten minutes at ⁇ 70° C., and then subjected to microcentrifugation. After microcentrifugating for ten minutes, the supernatant is aspirated off, and the pellet is dried in the speed vac for 30 second. 6 ⁇ l water, 2 ⁇ l annealing buffer, and 2 ⁇ l of 10 ⁇ M of the appropriate oligo is then added. This mixture is incubated for 10 minutes at 37° C. and then allowed to stand at room temperature for 10 minutes. Subsequently, 5.5 ⁇ l of label cocktail (described above) is added to each sample of the mixture, which are incubated at room temperature for an additional five minutes. 3.5 ⁇ l labeled DNA is then added to each sample which is then incubated for five minutes at 37° C. 4 ⁇ l stop solution is added to each well. The DNA is denatured at 95° for two minutes, and then placed on ice.
  • Clones with the desired fusion nucleic acid sequences are then recloned in frame in the pET expression system in the lipidated (full length) and non-lipidated (truncated, i.e., without first 17 amino acids) forms.
  • the product is amplified using restriction sites contained in the PCR primers.
  • the vector and product are cut with the same enzymes and ligated together with T4 ligase.
  • the resultant plasmid is transformed into competent E. coli using standard transformation techniques. Colonies are screened as described earlier and positive clones are transformed into expression cells, such as E. coli BL21, for protein expression with IPTG for induction.
  • the expressed protein in its bacterial culture lysate form and/or purified form is then injected in mice for antibody production.
  • the mice are bled, and the sera collected for agglutination, in vitro growth inhibition, and complement—dependent and —independent lysis tests.
  • OspA A specific example of chimeric OspA is as follows. Other OspA chimeras can be made using the same method with suitable primers.
  • a chimer of OspA from strain K48 (OspA-K48) and OspA from strain PGau (OspA-PGau) was generated using the method described above.
  • This chimeric nucleic acid sequence included bp 1-654 from OspA-K48, followed by bp 655-820 from OspA-PGau.
  • the chimeric nucleic acid sequence is presented as SEQ ID NO: 28; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO: 29.
  • chimeric nucleic acid sequences described above, as well as chimeric nucleic acid sequences produced by the methods described above, are used to produce chimeric proteins encoded by the nucleic acid sequences.
  • Standard methods such as those described above in Example 3, concerning the expression of proteins from Borrelia genes, can be used to express the proteins in a compatible host organism.
  • the chimeric proteins can then be isolated and purified using standard techniques.
  • Nucleic acid encoding altered versions of OspA can be used to generate OspA chimeras.
  • nucleic acid encoding OspA chimeras can be used to generate the altered OspA polypeptides of the present invention.
  • the chimeric protein is soluble, it can be purified on a Sepharose column.
  • Insoluble proteins can be solubilized in guanidine and purified on a Ni 2+ column; alternatively, they can be solubilized in 10 mM NaPO 4 with 0.1-1% TRIXON X 114, and subsequently purified over an S column (Pharmacia). Lipidated proteins were generally purified by the latter method. Solubility was determined by separating both soluble and insoluble fractions of cell lysate on a 12% PAGE gel, and checking for the localization of the protein by Coomassie staining, or by Western blotting with monoclonal antibodies directed to an antigenic polypeptide of the chimeric protein.
  • a large number of chimeric nucleic acid sequences encoding proteins comprising at least a first and a second polypeptide from Borrelia burgdorferi were generated. These chimeric nucleic acid sequences were produced such that the encoded chimeric protein comprised a Borrelia burgdorferi OspC polypeptide upstream of (or N-terminal to) a Borrelia burgdorferi OspA polypeptide. The chimeric nucleic acid sequences were also produced such that the nucleic acid encoding one polypeptide was in the same reading frame as the nucleic acid sequence encoding the next polypeptide in the chimeric protein.
  • the general cloning strategy used to construct the chimeric nucleic acid sequences was as follows.
  • the desired fragment of OspC was amplified using a 5′ primer containing a restriction site suitable for cloning the resultant product into a vector of interest and a 3′ primer containing a restriction site suitable for ligating the OspC fragment to the OspA fragment.
  • the OspC product was cloned into a suitable vector.
  • the desired OspA fragment was amplified using a 5′ primer containing a restriction site for ligating the resultant OspA fragment to the OspC fragment and a 3′ primer containing a restriction site suitable for cloning the resultant OspA product into the vector with the OspC product.
  • the use of a restriction site to allow ligation of the OspC and OspA fragments results in the insertion of 0 to about 3 amino acids between the OspC and OspA fragments.
  • OspC portions of the chimeric nucleic acids desired fragments of OspC genes from various strains or genospecies were PCR amplified using a 5′ primer containing an NdeI site and a 3′ primer containing a NcoI site and a BamHI site. The amplified OspC product was then cloned into the NdeI and BamHI sites of the T7 promoter driven expression vector, pET9c.
  • OspA portion of the chimeric nucleic acid desired fragments of OspA genes from a strain of interest or genospecies of interest were PCR amplified using a 5′ primer containing an NcoI site and a 3′ primer containing a BamHI site. This OspA portion could then be directly cloned into the NcoI and BamHI sites of the pET9c vector containing the desired OspC sequence, thereby producing the desired OspC-OspA construct.
  • OspC-OspA chimeric nucleic acids which contain nonlipidated OspC B31 were generated using the following primers: (5′OspC-NdeI): 5′-GT CAT ATG GCT TGT AAT AAT TCA GGG AAA GA-3′; and (SEQ ID NO:91) (3′OspC-NcoI): 5′-T TTC CAT GGA AGG TTT TTT TGG ACT TTC TG-3′. (SEQ ID NO:92)
  • OspC-OspA chimeric nucleic acids which contain nonlipidated OspA B31
  • the following primers were used: (5′OspA-NcoI:) 5′-TT TCC ATG GCC AAG CAA AAT GTT AGC AGC C-3′; and (SEQ ID NO:93) (3′OspA-BamHI): 5′-TAA GGA TCC TTA TTT TAA AGC GTT TTT-3′.
  • SEQ ID NO:94 5′OspA-NcoI: 5′-TT TCC ATG GCC AAG CAA AAT GTT AGC AGC C-3′
  • SEQ ID NO:93 3′OspA-BamHI
  • Lipidated versions of the OspC/OspA chimeras can be constructed by designing primers to amplify the a portion of the template that includes the lipidation signal sequence or by generating a nucleic acid construct with a suitable lipidation signal sequence.
  • the leader sequence comprising a lipidation signal can be, for example, from a gene encoding the OspA, OspB or OspC polypeptides.
  • the purified OspA, OspC or OspC/OspA chimeric proteins that were used to immunize mice and in the ELISA tests described below, were generated and purified by cloning either OspA, OspC or OspC/OspA chimeric nucleic acid sequences, in frame, into the pET expression plasmid.
  • the expression plasmid was then transfected into the compatible expression cell line Escherichia coli strain BL21 (pLysS) or strain B834 (DE3).
  • the BL21 or B834 cells were grown in 10 mL LB media (5 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract, 25 mg/L chloramphenicol and 50 mg/L ampicillin) at 37° C., with shaking. When the optical density at 600 ⁇ reached 0.3-0.4 units, recombinant protein expression was induced by adding IPTG (isopropyl B-D-thiogalactopyranoside) to a final concentration of 0.5 mM and the cells were grown for an additional three hours. The cultures were harvested by centrifugation at 3800 ⁇ g for five minutes. The cells were resuspended in 20 mM NaPO 4 , pH 7.7, and stored at ⁇ 20° C. overnight.
  • IPTG isopropyl B-D-thiogalactopyranoside
  • the crude extracts were incubated with DNase (2 ⁇ g/mL) in the presence of 2.5 mM MgCl 2 at room temperature for thirty minutes and then spun at 14,000 rpm (Eppendorf 5417C) for five minutes.
  • the partially purified OspC was then passed over a Ni 2+ metal affinity column (Chelating Sepharose Fast Flow 2.2 ⁇ 10 cm, Pharmacia) equilibrated with 20 mM NaPO 4 , pH 8.0, and 250 mM NaCl.
  • the column was washed using a decreasing pH gradient of 20 mM sodium acetic acid and 250 mM NaCl and the bound OspC eluted around pH 5.7.
  • the OspC fractions were then concentrated by ultrafiltration and stored at ⁇ 70° C.
  • mice either C3H-J or ICR, were immunized with 3 ⁇ g of lipidated OspC/OspA chimeric protein or 6 ⁇ g of non-lipidated OspC/OspA chimer in 100 mL of aluminum hydroxide adjuvant (concentration of 1.8 mg/mL) by (SC) subcutaneous injection.
  • SC aluminum hydroxide adjuvant
  • mice were immunized with 100 mL of aluminum hydroxide adjuvant only. All mice received a total of three injections which were given at two week intervals.
  • the coating procedure was as follows: 100 ⁇ l of a solution containing the appropriate OspA or OspC protein (made up at a concentration of 250 ng/ml in the following coating buffer: 100 mM Bis-Tris propane, pH 9.7) was added to each well of a microtiter plate which was incubated for one hour at 37° C. The antigen solution was removed from the wells, the plate was washed three times with phosphate buffered saline (PBS) pH 9.0, and 300 ⁇ l of blocking buffer solution was added (3% dry milk, 0.1% polyoxyethylenesorbitan (referred to herein as Tween 20TM), 0.02% NaN 3 in 100 nM Bis-Tris propane, pH 9.7).
  • PBS phosphate buffered saline
  • Tween 20TM polyoxyethylenesorbitan
  • TBS-Tween 20TM wash buffer (20 mM Tris-Cl, pH 7.5, 136 mM NaCl, 0.1% Tween 20TM and 0.02% NaN 3 ) and then were allowed to dry. The plates were then wrapped in plastic and stored at 4° C. until they were used.
  • goat anti-mouse antisera conjugated to alkaline phosphatase-specific for either IgM (Fc) or IgG (Fab), (Jackson Immuno Research Laboratories) was diluted 1:750 in sample dilution buffer (1% dry milk, 136 mM NaCl, 0.1% Tween 20TM, 0.02% NaN 3 in 20 mM Tris-Cl, pH7.5) and 100 ⁇ l of the diluted secondary antibody was added to each well.
  • sample dilution buffer 1% dry milk, 136 mM NaCl, 0.1% Tween 20TM, 0.02% NaN 3 in 20 mM Tris-Cl, pH7.5
  • mice immunized with a non-lipidated OspC/OspA chimeric protein (OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a. 18-273 from strain B31) (SEQ ID NO: 60) produced an immune response both to OspA and OspC that was comparable to the immune response generated to non-lipidated OspA (OspA—a.a. 18-273 from strain B31) and non-lipidated OspC (OspC—a.a. 19-211 from strain B31) control proteins (FIG. 22). As indicated in FIG.
  • mice were immunized with OspA, OspC or OspC2-OspA proteins and immune responses of the sera were measured against B31 OspA antigen (stippled bars) and B31 OspC antigen (solid bars).
  • mice immunized with the same non-lipidated OspC/OspA chimeric protein (OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a. 18-273 from strain B31) (SEQ ID NO: 60) produced an immune response to the C-terminal portion of OspA that was comparable to the immune response generated to the C-terminal portion of a non-lipidated OspA (OspA—a.a. 18-273 from strain B31) control protein (FIG. 23). As indicated in FIG.
  • mice were immunized with OspA, OspC or OspC2-OspA proteins and immune responses of the sera were measured against B31 OspA antigen.
  • the protective antibody response to B31 OspA antigen is indicated in the stippled bars.
  • mice were immunized with a non-lipidated OspC/OspA chimeric protein (OspC-OspAB/P—composed of OspC (a.a. 19-211 from strain B31)/OspA (a.a. 18-216 from strain B31)/OspA (a.a.
  • mice immunized with the non-lipidated OspC/OspA chimeric protein produced an immune response to OspA from each of the Borrelia burgdorferi strains B31 ( Borrelia burgdorferi sensu stricto ), K48 ( Borrelia garinii ) and PGau ( Borrelia afzelii ), that was equivalent or greater than the immune response generated to the two lipidated OspA control proteins (lipOspAP/Bo and lipOspAb/P) (FIG. 24).
  • the OspC/OspA chimeric proteins of the present invention are also able to generate immune responses against OspA proteins that are derived from strains that are not represented in the chimeric protein.
  • Mice immunized with the OspC/OspA chimeric proteins, OspCB31-OspAB31 (SEQ ID NO: 56) and OspC2-OspAB31 (SEQ ID NO: 60) are not only able to generate immune responses that recognize OspA derived from strain B31 ( Borrelia burgdorferi sensu stricto ), but also recognize OspA derived from strain K48 ( Borrelia garinii ) and strain PGau ( Borrelia afzelii ) (FIG.
  • mice were also immunized with the lipidated OspA chimeric protein, Lip OspA K/T (composed of OspA (a.a. 1-217 from strain K48)/OspA (a.a. 218-273 from strain Tro)) (FIG. 27).
  • FIG. 28 presents the ELISA results from mice immunized with either OspCB31-OspAB/P (SEQ ID NO: 66), OspCB31-OspABPBP (SEQ ID NO: 88) or OspCB31-OspAB31 (SEQ ID NO: 56).
  • mice either C3H-J or JCR, that had been immunized as described above, were also challenged with either laboratory-infected nympha or field nympha.
  • the immunized mice were placed in isolation cages and each mouse received 5-10 nymphs. All of the nymphs were collected at counted after 6 days.
  • the mice were bled and sera was tested using commercially-available Western blot strips to Borrelia burgdorferi sensu stricto strain B31 (MarDx strips) and/or Borrelia garinii (MRL strips). Eight weeks after challenge, the mice were bled, sera was tested again by Western blot and ear punch and bladder samples were cultured.
  • As a positive control mice which had been immunized with only aluminum hydroxide adjuvant, as described above, were subjected to the same challenge.
  • the mutations described herein are made using nucleic acids (e.g., polynucleotides) encoding OspA polypeptides or fragments from any strain of Lyme Disease-causing Borrelia, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii .
  • nucleic acids e.g., polynucleotides
  • BPBP Borrelia burgdorferi sensu stricto
  • Borrelia afzelii Borrelia garinii
  • the creation of the construct OspA BPBP M3 was as follows.
  • BPBP is a chimeric OspA polypeptide, wherein residues 1-164 are from OspA of B31, residues 165-179 are from OspA of Pko or PGau, residues 180-216 are from OspA of B31 and residues 217-273 are from OspA of Pko (wherein the numbering is as shown in SEQ ID NO: 7).
  • the first step in creating this construct was to make the construct BPBP M1.
  • the M1 mutation consists of a mutation of codon 139 (aga) of OspA from the amino acid arginine to the amino acid methionine (codon atg).
  • a PCR reaction was set up as described above containing a polynucleotide encoding BPBP as the template DNA and the following oligonucleotide primers:
  • the PCR reaction also contained reaction buffer, 10 ng of the BPBP DNA template, 125 ng of each oligonucleotide primer, and the dNTP mix.
  • the parameters for the PCR were as outlined in Table V. TABLE V Parameters for PCR Segment cycles temp time 1 1 95° C. 30 sec. 2 18 95° C. 30 sec. 3 50° C. 1 min. 4 68° C. 12 min.
  • BPBP MI was then used as template to make the plasmid BPBP M2, which in addition to the R139M mutation, also contains a mutation that changes the glutamic acid codon (gag) at position 160 to the codon (tat) which encodes for tyrosine.
  • the oligonucleotide primers used to generate this plasmid were:
  • the parameters for the PCR were as outlined in Table VII.
  • the final mutation, M3, which alters a lysine residue at position 189 to a methionine residue was performed using BPBP M2 as a DNA template.
  • the oligonucleotide primers used to generate this plasmid were:
  • J1, J2 and J3 mutants were made using the same protocol described for the generation of the M1, M2 and M3 mutants.
  • Such mutants can be made using nucleic acids (e.g., polynucleotides) encoding OspA polypeptides fragments from any strain of Lyme Disease-causing Borrelia, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii.
  • oligonucleotide primers which were used to generate the J1 mutation (which contains a Y165F mutation (codon tat to ttt) and a V166T mutation (codon gtt to act)) were as follows:
  • gag ttc ctt caa gag taa agc ctt tta aaa cct g 3′ (SEQ ID NO: 124)
  • oligonucleotide primers which were used to generate the J2 mutation (which contains a T170K mutation (codon act to aag)) were as follows:
  • the template containing the J1 mutations was used with the oligonucleotide primers for generating the J2 mutation (5′ B31 T-K (SEQ ID NO: 125) and 3′ B31 T-K (SEQ ID NO: 126)).

Abstract

Provided herein are OspA polypeptides from Lyme Disease-causing Borrelia having certain alteration(s). In one embodiment, the alteration(s) increase the conformational stability of the OspA polypeptide containing the alteration(s) while maintaining at least some of the antigenicity of the corresponding unaltered OspA polypeptide. In another embodiment, the altered OspA polypeptide has reduced cross-reactivity to hLFA-1, as compared to the corresponding unaltered OspA polypeptide.

Description

    RELATED APPLICATIONS
  • This application is a continuation of International Application No. PCT/US01/25852, which designated the United States, was published in English and was filed on Aug. 17, 2001. This application also claims the benefit of U.S. Provisional Application No. 60/226,484, filed on Aug. 18, 2000. The entire teachings of both of these applications are incorporated herein by reference.[0001]
    • GOVERNMENT SUPPORT
  • [0002] The invention was supported, in whole or in part, by Grant 2R01A137256-05A1 from the National Institute of Allergy and Infectious Diseases. The Government has certain rights in the invention.
    • BACKGROUND OF THE INVENTION
  • Lyme disease (Lyme borreliosis) is the most common tick-borne infectious disease in North America and Europe, and has been found in Russia, Japan, China and Australia. Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring during the course of infection. The causative bacterial agent of this disease is the spirochete [0003] Borrelia burgdorferi, which was first isolated and cultivated in 1982 (Burgdorferi, W. A. et al., Science 216: 1317-1319 (1982); Steere, A. R. et al., N. Engl. J. Med., 308:733-740 (1983)).
  • Three pathogenic genospecies of Borrelia, [0004] B. burgdorferi sensu stricto (B. burgdorferi or B.b.s.s.), B. afzelii and B. garinii have been described (Baranton, G., et al., Int. J. Syst. Bacteriol., 42:378-383 (1992)). These are members of a species complex, B. burgdorferi sensu lato, which consists of at least 10 different genospecies (Piken, R. N. et al., J. Invest. Dermatol., 110:211-214 (1998); Postic, D. et al., Int. J. Syst. Bacteriol., 44:743-752 (1994); Valsangiacomo, C. T. et al., Int. J. Syst. Bacteriol., 47:1-10 (1997)). The three genospecies, B. burgdorferi sensu stricto, B. afzelii and B. garinii, are all thought to be pathogenic and all are found in Europe.
  • [0005] B. burgdorferi has an outer membrane whose major protein constituents are the outer surface proteins A and B (OspA and OspB). OspA is a basic lipoprotein of approximately 31 kd, which is encoded on a large linear plasmid along with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and J. L. Benach, Microbiol. Rev., 55:21 (1991)). The immune response to these outer surface proteins tends to occur late in the disease, if at all (Craft, J. E. et al., J. Clin Invest. 78:934-939 (1986); Dattwyler, R. J. and B. J. Luft, Rheum. Clin. North Am., 15:727-734 (1989)). Furthermore, patients acutely and chronically infected with B. burgdorferi respond variably to the different antigens, including OspA, OspB, OspC, OspD, p39, p41 and p93.
  • Currently, Lyme Disease is treated with a range of antibiotics, e.g., tetracyclines, penicillin and cephalosporins. However, such treatment is not always successful in clearing the infection. Treatment is often delayed due to improper diagnosis with the deleterious effect that the infection proceeds to a chronic condition, where treatment with antibiotics is often not useful. One of the factors contributing to delayed treatment is the lack of effective diagnostic tools. [0006]
  • Vaccines against Lyme borreliosis have been attempted. However, a vaccine that consists of recombinant OspA may require frequent booster immunizations. An additional concern of OspA-based vaccines is the recent identification of a putative autoreactive OspA domain with a high degree of similarity to a region of human leukocyte function-associated antigen-1 (hLFA-1) (Gross, D. M. et al., [0007] Science, 281: 703-706 (1998)).
  • Therefore, it should be advantageous to develop modified OspA proteins having decreased cross-reactivity to hLFA-1 in order to reduce potential side effects of an OspA vaccine. Development of OspA proteins with decreased hLFA-1 cross-reactivity that maintain or have increased immunoreactivity to more than one member of the Borrelia complex would also be desirable. To be useful as vaccines, the conformations of these modified proteins must be sufficiently stable to retain certain OspA structural features that are required to elicit a protective immune response. OspA proteins with these features would allow for improvements in diagnosis and/or vaccination against all, or most, of the Borrelia that cause Lyme Disease. [0008]
  • Analysis of the immune status of OspA immunized individuals revealed that the overall quantitative response is not predictive of protection, but rather the reactivity with a specific epitope of the OspA lipoprotein directly correlates to protective immunity. The anti-OspA monoclonal antibody, LA-2 (Kramer et al., 1990) defines an epitope of the lipoprotein that is apparently necessary for protective immunity after OspA vaccination. For instance, passive immunization of mice with this antibody leads to protection against infection with the spirochete (Schaible et al., 1993). In addition, immunization of mice and canines with OspA resulting in significant titers of LA-2 equivalent serum antibody accurately predicts protection from tick transmission of infection (Golde, 1997). Insufficient levels of LA-2 equivalent antibody result in a lack of protection in the face of high serum antibody titers to OspA (Johnson et al., 1995). [0009]
    • SUMMARY OF THE INVENTION
  • The present invention is drawn to altered forms of OspA from [0010] Borrelia burgdorferi that have increased conformational stability while maintaining at least some of the antigenicity of wild type OspA. In some embodiments, the altered OspA polypeptide has decreased cross-reactivity with hLFA-1, as compared to the corresponding unaltered OspA polypeptide. The altered OspA polypeptides can comprise almost all or only a portion of the native OspA polypeptide. In some embodiments, the altered OspA polypeptide can be part of a cocktail which includes one or more other proteins, such as, for example, other Borrelia burgdorferi polypeptides including OspA, OspB, OspC, OspD, p93 and p41. In other embodiments, the altered OspA polypeptide can be part of a chimeric protein, such as those described in U.S. Pat. No. 6,248,562, the entire teachings of which are incorporated herein by reference.
  • In one embodiment, the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from [0011] Borrelia burgdorferi from about residue 139 to about residue 273, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 changed to methionine, residue 160 changed to tyrosine, residue 189 changed to methionine and combinations thereof. In other embodiments, the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 131 to about residue 273 or from about residue 17 to about residue 273. The OspA polypeptides of the present invention can comprise longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7 (OspA from B31).
  • In another embodiment, the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from [0012] Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least two alterations selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine and residue 170 changed to lysine. In yet another embodiment, the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from a sensu stricto strain of Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine, residue 170 changed to lysine and combinations thereof. In other embodiments, the altered OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 150 to about residue 180 or from about residue 17 to about residue 273. The OspA polypeptides of the present invention can comprise longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • The polypeptides of the present invention include polypeptides selected from the group consisting of: SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114 and 116. [0013]
  • The present invention is also drawn to polynucleotides encoding the amino acid sequences described herein, such as polynucleotides encoding OspA polypeptides from [0014] Borrelia burgdorferi from about residue 131 to about residue 273, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine and combinations thereof. The polynucleotide encoding OspA polypeptides of the present invention can encode longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • In another embodiment, the polynucleotide encodes an amino acid sequence of an OspA polypeptide from [0015] Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 165 encoding phenylalanine, codon 166 encoding threonine, codon 170 encoding lysine and combinations thereof. The polynucleotides which encode OspA polypeptides of the present invention can encode longer or shorter fragments of OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • The polynucleotides of the present invention include a polynucleotide selected from the group consisting of: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115. [0016]
  • The present invention is also drawn to a method of generating an altered [0017] Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide encoding a Borrelia burgdorferi OspA polypeptide that includes at least one of residues 139, 160 and 189, wherein the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is altered such that at least one of the following alterations is present: residue 139 is changed to methionine, residue 160 is changed to tyrosine and residue 189 is changed to methionine or a combination thereof. In one embodiment, both the alteration at residue 160 and the alteration at 189 is made. In another embodiment, the alterations at all three residues are made. The altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • In another embodiment, the present invention is drawn to a method of generating an altered [0018] Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with an hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide encoding an OspA polypeptide from Borrelia burgdorferi that includes at least one of residues 165, 166 and 170, wherein the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is altered such that at least one alteration from the following list is present: residue 165 is changed to phenylalanine, residue 166 is changed to threonine and residue 170 is changed to lysine or combination thereof. The altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
  • The present invention is also drawn to an expression vector which comprises an isolated DNA encoding an altered Borrelia OspA protein. The present invention also encompasses a host cell which comprises a recombinant nucleic acid that encodes an altered OspA protein as described herein. [0019]
  • The present invention is also drawn to a method of delivering the altered Borrelia OspA polypeptides described herein. In one embodiment, the method comprises administering the altered OspA polypeptide in a physiologically-acceptable carrier to an individual. As a result of the administration of the altered OspA protein, the individual develops at least some immune response to the protein. As an example, the individual generates a humoral immune response, wherein antibodies that recognize at least a portion of said polypeptide are produced by the individual. In a preferred embodiment, the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope. [0020]
  • The present invention is also drawn to a method of delivering a nucleic acid which encodes an altered OspA polypeptide described herein. In one embodiment, the method comprises administering the nucleic acid in a physiologically-acceptable carrier to an individual. As a result of the administration of the nucleic acid, the altered OspA polypeptide is at least transiently expressed and the individual develops at least some immune response, preferably an immunoprotective response to the altered OspA protein encoded by the nucleic acid. As an example, the individual generates a humoral immune response, wherein antibodies that recognize at least a portion of the altered OspA polypeptide produced from the nucleic acid are produced by the individual. In a preferred embodiment, the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope. [0021]
  • The invention also encompasses methods of using the proteins described herein in diagnostic assays. In one embodiment, the method can be used to detect the presence of OspA specific antibodies in a host sample of interest. The method comprises contacting a host sample of interest with the altered protein, under conditions wherein antibodies, if present in the host sample, bind to the altered protein, forming antigen-antibody complexes. The antigen-antibody complexes are then detected using standard methods known in the art. [0022]
  • The present invention is also drawn to a diagnostic kit comprising the altered polypeptides described herein. The kit comprises an altered [0023] Borrelia burgdorferi OspA protein as described herein. The kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspA altered protein and antibodies that are present in the user-supplied host sample.
  • As a result of the present invention, OspA proteins, or fragments thereof, having either increased conformational stability while maintaining at least some antigenicity, or having reduced cross-reactivity to hLFA-1, are available for use in research, vaccines and/or diagnostic assays. Furthermore, as a result of the present invention, nucleic acids encoding OspA polypeptides having reduced cross-reactivity with hLFA-1 are available for research and vaccines. The altered OspA polypeptides of the present invention are expected to allow for improved vaccines having fewer side effects. [0024]
  • For a better understanding of the present invention together with other and further objects, reference is made to the following description, taken together with the accompanying drawings.[0025]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [0026]
  • FIG. 1 summarizes peptides and antigenic domains localized by proteolytic and chemical fragmentation of OspA. [0027]
  • FIG. 2 is a comparison of the antigenic domains depicted in FIG. 1, for OspA in nine strains of [0028] B. burgdorferi.
  • FIG. 3 is a graph depicting a plot of weighted polymorphism versus amino acid position among 14 OspA variants. The marked peaks are: a) amino acids 132-145; b) amino acids 163-177; c) amino acids 208-221. The lower dotted line at polymorphism value 1.395 demarcates statistically significant excesses of polymorphism at p=0.05. The upper dotted line at 1.520 is the same, except that the first 29 amino acids at the monomorphic-terminus have been removed from the original analysis. [0029]
  • FIG. 4 depicts the amino acid alignment of [0030] residues 200 through 220 for OspAs from strains B31 and K48 as well as for the site-directed mutants 613, 625, 640, 613/625, and 613/640. Arrow indicates Trp216. Amino acid changes are underlined.
  • FIG. 5 depicts a phylogenetic tree for strains of Borrelia described in Table I. The strains are as follows: 1=B31; 2=Pka1; 3=ZS7; 4=N40; 5=25015; 6=K48; 7=DK29; 8=PHei; 9=Ip90; 10=PTrob; 11=ACAI; 12=PGau; 13=Ip3; 14=PBo; 15=Pko. [0031]
  • FIGS. 6A and 6B depict the nucleic acid sequence of OspA-B31 (SEQ ID NO: 6), and the encoded protein sequence (SEQ ID NO: 7). [0032]
  • FIGS. 7A, 7B and [0033] 7C depict the nucleic acid sequence of OspA-K48 (SEQ ID NO: 8), and the encoded protein sequence (SEQ ID NO: 9).
  • FIGS. 8A, 8B and [0034] 8C depict the nucleic acid sequence of OspA-PGau (SEQ ID NO: 10), and the encoded protein sequence (SEQ ID NO: 11).
  • FIGS. 9A and 9B depict the nucleic acid sequence of an OspA gene (SEQ ID NO: 127 and its encoded protein sequence (SEQ ID NO: 128). [0035]
  • FIGS. 10A, 10B and [0036] 10C depict the nucleic acid sequence of the OspA-K48/OspA-PGau chimer (SEQ ID NO: 28) and the encoded chimeric protein sequence (SEQ ID NO: 29).
  • FIGS. 11A, 11B and [0037] 11C depict the nucleic acid sequence of the OspA-B31/OspA-PGau chimer (SEQ ID NO: 30) and the encoded chimeric protein sequence (SEQ ID NO: 31).
  • FIGS. 12A and 12B depict the nucleic acid sequence of the OspA-B31/OspA-K48 chimer (SEQ ID NO: 32) and the encoded chimeric protein sequence (SEQ ID NO: 33). [0038]
  • FIGS. 13A, 13B and [0039] 13C depict the nucleic acid sequence of the OspA-B31/OspA-25015 chimer (SEQ ID NO: 34) and the encoded chimeric protein sequence (SEQ ID NO: 35).
  • FIGS. 14A, 14B and [0040] 14C depict the nucleic acid sequence of the OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 36) and the encoded chimeric protein sequence (SEQ ID NO: 37).
  • FIGS. 15A, 15B and [0041] 15C depict the nucleic acid sequence of the OspA-B31/OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 38) and the encoded chimeric protein sequence (SEQ ID NO: 39).
  • FIGS. 16A, 16B and [0042] 16C depict the nucleic acid sequence of the OspA-B31/OspB-B31 chimer (SEQ ID NO: 40) and the encoded chimeric protein sequence (SEQ ID NO: 41).
  • FIGS. 17A, 17B, [0043] 17C, 17D, 17E, 17F, 17G, 17H, 17I, 17J, 17K, 17L, 17M, 17N, 17O, and 17P, depict an alignment of the nucleic acid sequences for OspA-B31 (SEQ ID NO: 6), OspA-pKa1 (SEQ ID NO: 42), OspA-N40 (SEQ ID NO: 43), OspA-ZS7 (SEQ ID NO: 44), OspA-25015 (SEQ ID NO: 12), OspA-pTrob (SEQ ID NO: 45), OspA-K48 (SEQ ID NO: 8), OspA-Hei (SEQ ID NO: 46), OspA-DK29 (SEQ ID NO: 21), OspA-Ip90 (SEQ ID NO: 22), OspA-pBo (SEQ ID NO: 23), OspA-Ip3 (SEQ ID NO: 24), OspA-Pko (SEQ ID NO: 25), OspA-ACAI (SEQ ID NO: 26), and OspA-PGau (SEQ ID NO: 10). Nucleic acids which are identical to those in the lead nucleic acid sequence (here, OspA-B31) are represented by a period (.); differing nucleic acids are shown in lower case letters.
  • FIGS. 18A and 18B depict the nucleic acid sequence of the OspA-Tro/OspA-Bo chimer (SEQ ID NO: 47) and the encoded chimeric protein sequence (SEQ ID NO: 48). [0044]
  • FIGS. 19A and 19B depict the nucleic acid sequence of the OspA-PGau/OspA-Bo chimer (SEQ ID NO: 49) and the encoded chimeric protein sequence (SEQ ID NO: 50). [0045]
  • FIGS. 20A and 20B depict the nucleic acid sequence of the OspA-B31/OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 53) and the encoded chimeric protein sequence (SEQ ID NO: 54). [0046]
  • FIG. 21A and 21B depict the nucleic acid sequence of the OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 51) and the encoded chimeric protein sequence (SEQ ID NO: 52). [0047]
  • FIG. 22 is a bar graph showing the reactivity (as measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis) against the indicated OspA or OspC antigens (legend) from the strain B31 ([0048] Borrelia burgdorferi sensu stricto).
  • FIG. 23 is a bar graph showing the reactivity (as measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis) against the indicated OspA or OspC antigens (legend) from the strain B31 ([0049] Borrelia burgdorferi sensu stricto). For the ELISA results to the B31 OspA antigen, a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response was specific for the C-terminal region of OspA.
  • FIG. 24 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspA/Bo, lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA antigens (legend) from strains B31 ([0050] Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
  • FIG. 25 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspAP/Bo, lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA (legend) from strains B31 ([0051] Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii). In all cases, a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response is specific for the C-terminal region of OspA.
  • FIG. 26 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or lip OspC-B31)(X-axis) against the indicated OspC antigen (legend) from the strain B31 ([0052] Borrelia burgdorferi sensu stricto).
  • FIG. 27 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or Lip OspA K/T)(X-axis) against the indicated OspA antigens (legend) from strains B31 ([0053] Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
  • FIG. 28 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P, OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspA antigens (legend) from strains B31 ([0054] Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
  • FIG. 29 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P, OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspA (legend) from strains B31 ([0055] Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii). In all cases, a purified fragment of B31 OspA (amino acids 18-139) was added in excess to the sera so that the detected immune response is specific for the C-terminal region of OspA.
  • FIGS. 30A, 30B and [0056] 30C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 55) and the encoded chimeric protein sequence (SEQ ID NO: 56).
  • FIGS. 31A, 31B and [0057] 31C depict the nucleic acid sequence of the OspC-B31 (bp 55-624)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 57) and the encoded chimeric protein sequence (SEQ ID NO: 58).
  • FIGS. 32A, 32B and [0058] 32C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 59) and the encoded chimeric protein sequence (SEQ ID NO: 60).
  • FIGS. 33A, 33B, and [0059] 33C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 61) and the encoded chimeric protein sequence (SEQ ID NO: 62).
  • FIGS. 34A, 34B and [0060] 34C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 63) and the encoded chimeric protein sequence (SEQ ID NO: 64).
  • FIGS. 35A, 35B and [0061] 35C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 65) and the encoded chimeric protein sequence (SEQ ID NO: 66).
  • FIGS. 36A, 36B and [0062] 36C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 67) and the encoded chimeric protein sequence (SEQ ID NO: 68).
  • FIGS. 37A, 37B and [0063] 37C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 69) and the encoded chimeric protein sequence (SEQ ID NO: 70).
  • FIGS. 38A, 38B and [0064] 38C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 71) and the encoded chimeric protein sequence (SEQ ID NO: 72).
  • FIGS. 39A, 39B, [0065] 39C depict the nucleic acid sequence of the OspC-C12 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-822) chimer (SEQ ID NO: 73) and the encoded chimeric protein sequence (SEQ ID NO: 74).
  • FIGS. 40A, 40B and [0066] 40C depict the nucleic acid sequence of the OspC-Pko (bp 55-639)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-651)/OspA-K48 (bp 652-825) chimer (SEQ ID NO: 75) and the encoded chimeric protein sequence (SEQ ID NO: 76).
  • FIGS. 41A, 41B and [0067] 41C depict the nucleic acid sequence of the OspC-Tro (bp 55-624)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 538-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 77) and the encoded chimeric protein sequence (SEQ ID NO: 78).
  • FIGS. 42A and 42B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 394-820) chimer (SEQ ID NO: 79) and the encoded chimeric protein sequence (SEQ ID NO: 80). [0068]
  • FIGS. 43A and 43B depict the nucleic acid sequence of the OspC-B31 (bp 55-631)/OspA-B31 (bp 394-651)/OspA-K48 (bp 652-820) chimer (SEQ ID NO: 81) and the encoded chimeric protein sequence (SEQ ID NO: 82). [0069]
  • FIGS. 44A and 44B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 394-651)/OspA-Pko (bp 652-820) chimer (SEQ ID NO: 83) and the encoded chimeric protein sequence (SEQ ID NO: 84). [0070]
  • FIGS. 45A and 45B depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-K48 (bp 394-654)/OspA-Tro (bp 655-819) chimer (SEQ ID NO: 85) and the encoded chimeric protein sequence (SEQ ID NO: 86). [0071]
  • FIGS. 46A, 46B and [0072] 46C depict the nucleic acid sequence of the OspC-B31 (bp 55-633)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 87) and the encoded chimeric protein sequence (SEQ ID NO: 88).
  • FIGS. 47A, 47B, and [0073] 47C depict the nucleic acid sequence of the OspC-C2 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31 (bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 89) and the encoded chimeric protein sequence (SEQ ID NO: 90).
  • FIGS. 48A and 48B depict the nucleic acid and encoded protein sequence of an R139M altered OspA (SEQ ID NO: 95 and 96). [0074]
  • FIGS. 49A and 49B depict the nucleic acid and encoded protein sequence of an E160Y altered OspA (SEQ ID NO: 97 and 98). [0075]
  • FIGS. 50A and 50B depict the nucleic acid and encoded protein sequence of an R139M, E160Y altered OspA (SEQ ID NO: 99 and 100). [0076]
  • FIGS. 51A and 51B depict the nucleic acid and encoded protein sequence of an E160Y altered OspA (SEQ ID NO: 101 and 102). [0077]
  • FIGS. 52A and 52B depict the nucleic acid and encoded protein sequence of an R139M, E160Y, K189M altered OspA (SEQ ID NO: 103 and 104). [0078]
  • FIGS. 53A and 53B depict the nucleic acid and encoded protein sequence of an Y165F altered OspA (SEQ ID NO: 105 and 106). [0079]
  • FIGS. 54A and 54B depict the nucleic acid and encoded protein sequence of an Y165F, V166T altered OspA (SEQ ID NO: 107 and 108). [0080]
  • FIGS. 55A and 55B depict the nucleic acid and encoded protein sequence of a V166T altered OspA (SEQ ID NO: 109 and 110). [0081]
  • FIGS. 56A and 56B depict the nucleic acid and encoded protein sequence of a V166T, T170K altered OspA (SEQ ID NO: 111 and 112). [0082]
  • FIGS. 57A and 57B depict the nucleic acid and encoded protein sequence of an Y165F, V166T, T170K altered OspA (SEQ ID NO: 113 and 114). [0083]
  • FIGS. 58A and 58B depict the nucleic acid and encoded protein sequence of an R139M, E160Y, K189M, Y165F, V166T, T170K altered OspA (SEQ ID NO: 115 and 116).[0084]
    • DETAILED DESCRIPTION OF THE INVENTION
  • A description of preferred embodiments of the invention follows. [0085]
  • The present invention is drawn to altered forms of OspA from [0086] Borrelia burgdorferi that have increased conformational stability while maintaining antigenicity, as indicated, for example, by the ability to be bound by the LA-2 monoclonal antibody. In some embodiments, the altered OspA polypeptides also have decreased cross-reactivity with hLFA-1. The altered OspA polypeptides can comprise all (with the exception of the alterations described herein) or a portion, such as the C-terminal portion, of a wild type OspA polypeptide. Applicants have found that some forms of the OspA protein, such as truncated versions of OspA, do not elicit a strong immunoprotective response when administered to an animal even when the OspA polypeptide has the immunoprotective LA-2 epitope sequence.
  • The structure for recombinant OspA has been determined to 1.95 Å resolution in a binary complex with the Fab fragment of the nonprotective mouse mAb184.1, which is reactive with the OspA-terminus (Li et al., [0087] Proc. Natl. Acad. Sci. U.S.A. 94:3584-3589 (1997)). The OspA polypeptide folds into 21 consecutive antiparallel β-strands followed by a C-terminal α-helix. The structure is conveniently described as two discrete folded domains, an N-terminal sandwich domain and a C-terminal barrel domain, connected by a long central β-sheet. One set of altered polypeptides described herein is designed to remove buried charges and/or salt bridges in the OspA C-terminal portion and replace them with residues that promote hydrophobic interactions.
  • Accordingly, in one embodiment, the altered OspA polypeptides of the present invention comprise altered OspA protein or polypeptides from [0088] Borrelia burgdorferi from about residue 139 to about residue 273, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 changed to methionine, residue 160 changed to tyrosine, residue 189 changed to methionine and combinations thereof. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7. In one embodiment, the altered OspA polypeptide has a tyrosine at residue position 160 and a methionine at residue position 189. These alterations have been found to stabilize the conformation of the immunoprotective LA-2 epitope in LA-2-containing OspA polypeptides. In another embodiment, the altered OspA polypeptide has a methionine at residue position 139, a tyrosine at residue position 160 and a methionine at residue position 189. In other embodiments, the altered OspA polypeptides have both increased conformational stability and reduced cross-reactivity to the hLFA-1 protein.
  • For the alterations at [0089] positions 139, 160 and 189, the altered OspA sequence can be from any Lyme borreliosis strain of Borrelia burgdorferi, such as strains from Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Strains of Borrelia burgdorferi are well known to those of skill in the art. For example, strains of Borrelia burgdorferi sensu stricto include B31, strains of Borrelia afzelii include Pgau and Pko and strains of Borrelia garinii include K48.
  • In one embodiment, the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from [0090] Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least two alterations selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine and residue 170 changed to lysine, wherein the numbering of the residues corresponds to the numbering of SEQ ID NO: 7. In another embodiment, the OspA polypeptides of the present invention comprise an amino acid sequence of OspA protein from a sensu stricto strain of Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 changed to phenylalanine, residue 166 changed to threonine, residue 170 changed to lysine and combinations thereof. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7. In another embodiment, the altered OspA polypeptide has a phenylalanine at residue position 165 and a threonine at residue position 166. In still another embodiment, the altered OspA polypeptide has a phenylalanine at residue position 165, a threonine at residue position 166 and a lysine at residue position 170. In yet another embodiment, the altered OspA polypeptide includes all of the alterations described herein. In this embodiment, the altered OspA polypeptide has a methionine at residue position 139, a tyrosine at residue position 160, a methionine at residue position 189, a phenylalanine at residue position 165, a threonine at residue position 166 and a lysine at residue position 170.
  • This invention also pertains to polypeptides comprising SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, or 116. The altered OspA polypeptides of the invention can be partially or substantially purified (e.g., purified to homogeneity), and/or substantially free of other proteins. [0091]
  • The present invention is also drawn to polynucleotides encoding the amino acid sequences described herein. As defined herein, the term “polynucleotide” refers to a nucleotide multimer or oligomer which is composed of deoxyribonucleotides or ribonucleotides, or a combination thereof, having from a few, e.g., 2-20, to many, e.g., 20 to several thousand or more, nucleotides. As such, polynucleotides include nucleic acids of any length and further encompass both naturally-occurring and synthetic oligonucleotides and polynucleotides. [0092]
  • The polynucleotides of the present invention include polynucleotides encoding OspA polypeptides from [0093] Borrelia burgdorferi from about residue 139 to about residue 189, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine and combinations thereof. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7. As described above for the polypeptides, in the case of alterations at positions 139, 160 and 189, the polynucleotide encoding the altered OspA sequence can be from any Lyme borreliosis strain of Borrelia burgdorferi.
  • In another embodiment, the polynucleotide encodes an amino acid sequence of an OspA polypeptide from [0094] Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 165 encoding phenylalanine, codon 166 encoding threonine, codon 170 encoding lysine and combinations thereof. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
  • The polynucleotides of the present invention include polynucleotides selected from the group consisting of: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115. [0095]
  • The altered OspA polypeptides of the present invention can be derived from OspA molecules comprising fragments, derivatives, analogs, variants and mutants of the OspA protein (modified OspA) or can be fragmented, derivatized, or otherwise altered after having the alterations described herein inserted. These modified OspA molecules possess OspA antigenic activity. [0096]
  • The present invention is also drawn to a method of generating an altered [0097] Borrelia burgdorferi OspA polypeptide with increased conformational stability, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide encoding a Borrelia burgdorferi OspA polypeptide that includes residues 139, 160 and 189, wherein the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is altered such that residue 139 is methionine, residue 160 is tyrosine or residue 189 is methionine or combination thereof. The altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. Methods of altering a polynucleotide are described below and in the Exemplification and are well known to those of skill in the art. Methods of expressing the altered polypeptides of the invention are also described below and in the Exemplification and are well known to those of skill in the art.
  • Residues 165-173 on β-[0098] strand 13 of OspA have been implicated in induction of Lyme-related arthritis (Gross D. M. et al., Science 281:703-706 (1998)). This region has homology to residues 332-340 of hLFA-1, suggesting that this protein has a cross-reactive T cell epitope (YVLEGTLTA-B31 (SEQ ID NO: 129 and YVIEGTSKQ-hLFA-1 (SEQ ID NO: 130), respectively). Although B. burgdorferi sensu stricto is generally believed to be more arthritogenic that other Borrelia strains, a recent study of ospA alleles in synovial fluid of patients with Lyme arthritis indicates that B. garinii and B. afzelii may also cause arthritis (Eiffert, L. F. et al., Scand. J. Infect. Dis. 30:265-268 (1998)).
  • One way to eliminate the cross-reactive sequence is to replace the β-13 region of OspA-B31 (YVLEGTLTA (SEQ ID NO: 129)) with an analogous region from a strain that does not possess the same sequence, such as that from a [0099] B. Afzelii strain, e.g., Pgau or Pko (U.S. Patent Application entitled “Recombinant Constructs of Borrelia burgdorferi” by Luft et al., filed on Aug. 7, 2001, the entire teachings of which are incorporated herein by reference).
  • Accordingly, in another embodiment, the present invention is drawn to a method of generating an altered [0100] Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule, as compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide encoding an OspA polypeptide from Borrelia burgdorferi that includes residues 165, 166 and 170, wherein the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is altered such that residue 165 is changed to phenylalanine, residue 166 is changed to threonine or residue 170 is changed to lysine or combination thereof. The altered polynucleotide is expressed, thereby generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide. In another embodiment, an altered OspA polypeptide having reduced cross-reactivity to hLFA-1 while retaining the ability to be bound by LA-2 is generated. In that embodiment, for example, residue 130 is methionine, residue 160 is tyrosine, residue 165 is phenylalanine, residue 166 is threonine, residue 170 is lysine and residue 189 is methionine. Polynucleotides encoding Borrelia burgdorferi OspA polypeptides can be selected as described herein.
  • In one embodiment, the altered OspA polypeptide includes the minimal sequence that includes the positions of the alterations. For example, the altered polypeptide can comprise OspA from about residue 139 to about residue 189, wherein the numbering corresponds to SEQ ID NO: 7. In another embodiment, the altered polypeptide can comprise OspA from about residue 165 to about [0101] residue 170, wherein the numbering corresponds to SEQ ID NO: 7. The altered OspA polypeptides of the present invention also include larger fragments of OspA. For example, the altered OspA polypeptides include, but are not limited to, altered OspA polypeptides which comprise OspA from about residue 160 to about residue 170, OspA from about residue 150 to 180, OspA from about residue 131 to 273 or OspA from about residue 17 to 273. Methods of generating and expressing varying-sized fragments of OspA which incorporate one or more of the alterations described herein are described below and are well known to those of skill in the art.
  • As described herein, the OspA sequence used to generate the altered OspA polypeptide can itself be a chimeric OspA polypeptide, having two or more segments derived from OspA proteins from different genospecies or strains of Borrelia. The size of the altered OspA polypeptide can vary depending on the method used to generate the altered polypeptide and/or the purpose for which it is generated, and such altered OspA chimeric polypeptides can include fragments of OspA. The altered polypeptide can be part of a larger polypeptide, including additional OspA sequences on the N-terminus, the C-terminus or both termini. A fragment of OspA protein can encompass polypeptides that are only a part of the full-length OspA protein. Such OspA fragments typically include at least one of the altered residues described herein and possess at least some of the antigenicity of wild type OspA. OspA fragments can be produced by amino and/or carboxyl terminal deletions, as well as internal deletions. Fragments can also be produced by enzymatic digestion. Such modified OspA molecules can be tested for antigenic activity as described herein or using methods known in the art. [0102]
  • In some embodiments, the altered OspA polypeptide can be part of a cocktail with one or more other proteins, such as other [0103] Borrelia burgdorferi polypeptides, including but not limited to, OspA, OspB, OspC, OspD, p93 and p41. In other embodiments, the altered OspA polypeptide can be part of a larger molecule, such as a chimeric polypeptide, for example, as described in U.S. Pat. No. 6,248,562 and U.S. Patent Application entitled “Recombinant Constructs of Borrelia burgdorferi”, by Luft et al, filed on Aug. 7, 2001. Such larger polypeptides can include amino acid sequences from other proteins including but not limited to, other Borrelia burgdorferi proteins and/or other proteins useful in generating fusion proteins for vaccine and/or immunodiagnostic methods. Additional components, for example, labels (a radioisotope, an epitope label (tag)(e.g., a hemagglutinin (HA) epitope, a hexahistidine tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme label, a fluorescent group, a chemiluminescent group) can be incorporated into the altered OspA polypeptides of the invention to assist in the isolation and/or purification of the polypeptide. For example, a hexahistidine tag would permit ready purification by nickel chromatography. These and other components can also be incorporated in the altered OspA polypeptides of the invention in order to extend the half life of the polypeptides. Methods of incorporating such components into the polypeptides of the invention are well known to those of skill in the art.
  • In one embodiment, the altered OspA polypeptide of the invention is a chimeric polypeptide. In a particular embodiment, the altered OspA polypeptide comprises the following: a) an amino acid sequence of a first OspA polypeptide from about [0104] residue 1 to about residue 164 from a first strain of Borrelia burgdorferi; b) an amino acid sequence of a second OspA polypeptide from about residue 165 to about residue 179 from a second strain of Borrelia burgdorferi, wherein said second strain is a different strain from said first strain; c) an amino acid sequence of a third OspA polypeptide from about residue 180 to about residue 216 from a third strain of Borrelia burgdorferi, wherein said third strain is a different strain from said second strain; d) an amino acid sequence of a fourth OspA polypeptide from about residue 217 to about residue 273 from a fourth strain of Borrelia burgdorferi, wherein said fourth strain is a different strain from said third strain; wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 being methionine, residue 160 being tyrosine, residue 189 being methionine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
  • Polypeptides described herein can be isolated from naturally-occurring sources, chemically synthesized or recombinantly produced. The altered OspA polypeptides of the present invention can be derived from naturally-occurring OspA molecules or from nucleic acids which encode such molecules. The OspA polypeptides of the present invention can comprise fragments, derivatives, analogs, variants and mutants of an OspA protein (modified OspA) and/or can be fragmented, derivatized or otherwise altered after having the alterations described herein inserted (also referred to as modified OspA). Such modified OspA molecules possess at least some OspA antigenic activity. According to the invention, the amino acid sequence of the altered OspA polypeptides of the invention can be that of a naturally-occurring protein or can comprise additional modifications. Such additional modifications include conservative and/or non-conservative amino acid substitutions, additions of one or more amino acids, and/or deletions of one or more amino acids. Such additional modifications should also preserve at least some activity of the encoded protein or polypeptide. For example, the further modified polypeptide or protein should have similar or improved conformational stability, similar or improved immunoprotective activity or reduced cross-reactivity to hLFA-1, as compared to the corresponding altered OspA polypeptide (i.e., the OspA polypeptide comprising one or more of the alterations described herein but not comprising the further modification(s)). [0105]
  • For example, the further modification(s) preferably preserve the three-dimensional configuration of an antibody binding site of the native protein such as the LA-2 binding site. The presence or absence of biological activity or activities can be determined by various functional assays as described herein or using methods that are known in the art, e.g., recognition using an ELISA assay, elicitation of an immune response (e.g., an immunoprotective response) in an animal. Appropriate amino acid alterations that fall within the scope of the invention can be made on the basis of several criteria, including hydrophobicity, basic or acidic character, charge, polarity, size, the presence or absence of a functional group (e.g., —SH or a glycosylation site), and aromatic character, provided that the resulting molecule has at least one of the alterations described herein and maintains increased conformational stability and/or reduced cross-reactivity to hLFA-1. Assignment of various amino acids to similar groups based on the properties above will be readily apparent to the skilled artisan; further appropriate amino acid changes can also be found in Bowie ([0106] Science, 247:1306-1310(1990)).
  • “Variants” and “mutants” of OspA can be produced using in vitro and/or in vivo techniques well-known to those of skill in the art, for example, site-specific mutagenesis, and oligonucleotide mutagenesis. Manipulations of the OspA polypeptide sequence can be made at the protein level as well. Chemical modifications can be carried out using known techniques including but not limited to, specific chemical cleavage using cyanogen bromide, trypsin and/or papain. OspA can also be structurally modified and/or denatured, for example, using heat. In general, mutations can be conservative or non-conservative amino acid substitutions, amino acid insertions or amino acid deletions. [0107]
  • For example, a nucleic acid (e.g., DNA) encoding a modified OspA polypeptide can be prepared by site-directed mutagenesis of the nucleic acid (e.g., DNA) that encodes a wild type OspA. Site-directed (site-specific) mutagenesis allows the production of OspA variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation (e.g., alteration, deletion, insertion), as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the desired mutation. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementary residues on both sides of the mutation of the sequence being altered. In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as Edelman et al., [0108] DNA, 2:183, 1983. For example, a site-specific mutagenesis technique can employ a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam, 1981. This and other phage vectors are commercially available and their use is well known to those skilled in the art. A versatile and efficient procedure for the construction of oligonucleotide directed site-specific mutations in DNA fragments using M13-derived vectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res., 10:6487-6500, 1982. In addition, plasmid vectors that contain a single-stranded phage origin of replication can be employed to obtain single-stranded DNA (see for example, Veira et al., Meth Enzymol., 153:3, (1987)).
  • Alternatively, nucleotide substitutions can be introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it using PCR procedures known in the art. [0109]
  • In general, site-specific mutagenesis can be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., [0110] Proc Natl Acad Sci USA., 75:5765, 1978. This primer can then be annealed with the single-stranded protein sequence-containing vector, and subjected to DNA polymerizing enzymes, e.g., E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector can then be used to transform appropriate host cells such as JM 101 cells, and clones can be selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region can be removed and placed in an appropriate expression vector for protein production.
  • The PCR technique can be used in creating amino acid sequence variants of OspA. When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers can be designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer is preferably identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer be located within 500 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the end position of the mutation specified by the primer. [0111]
  • The DNA fragments produced bearing the desired mutation can be used to replace the corresponding region in the plasmid that served as PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more) part ligation. [0112]
  • An additional method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., [0113] Gene, 34:315, 1985. The starting material can be the plasmid (or vector) comprising the OspA DNA to be mutated. The codon(s) within the OspA to be mutated are identified. There must be unique restriction endonuclease sites on each side of the identified mutation site(s). If such restriction sites do not exist, they can be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the OspA DNA or they can be generated using PCR and the desired primers as described in the Exemplification. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. The plasmid now contains the mutated OspA DNA sequence, and can be subcloned and/or expressed to produce the modified OspA polypeptide or protein.
  • The OspA encoding nucleic acid molecules (e.g., polynucleotides) of the present invention have at least one of the alterations described herein and generally hybridize under high stringency hybridization conditions to a polynucleotide-encoding OspA nucleic acid or fragment thereof from a sensu stricto strain of [0114] Borrelia burgdorferi, e.g., SEQ ID NO: 7. In one embodiment, the OspA-encoding nucleic acid molecules (e.g., polynucleotides) of the present invention hybridize under high stringency hybridization conditions to a polynucleotide encoding OspA or fragment thereof from Borrelia afzelii, e.g., SEQ ID NO: 10. In another embodiment, the OspA-encoding nucleic acid molecules (e.g., polynucleotides) of the present invention hybridize under high stringency hybridization conditions to a polynucleotide encoding OspA or fragment thereof from Borrelia garinii, e.g., SEQ ID NO: 8. Thus, the polynucleotides and polypeptides of the present invention include modified versions of OspA as described herein.
  • Appropriate selective stringency conditions are known to those skilled in the art or can be found in standard texts such as [0115] Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, stringent hybridization conditions include a sodium ion concentration of no more than 1 M and a temperature of at least 25° C. In one embodiment, conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C., or equivalent conditions, are suitable for specific hybridization. Equivalent conditions can be determined by varying one or more of the parameters, as is known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used. Hybridizable nucleic acid molecules are useful as probes and primers for diagnostic applications.
  • Accordingly, the invention pertains to nucleic acid molecules which have a substantial identity with the nucleic acid molecules encoding the altered OspA polypeptides described herein wherein the nucleic acid encodes for one or more of the alterations described herein; particularly preferred are nucleic acid molecules which have at least about 90%, more preferably at least about 95% and most preferably at least about 98% identity with nucleic acid molecules described herein, wherein the nucleic acid encodes for at least one of the alterations described herein. Sequence identity can be determined using publically or commercially available sequence alignment algorithms, using, for example, default parameters. [0116]
  • Thus, DNA molecules which comprise a sequence which is different from the naturally-occurring nucleic acid molecule but which, due to the degeneracy of the genetic code, encode the same protein or polypeptide are encompassed by the present invention. The invention also encompasses variations of the nucleic acid molecules of the invention, such as those encoding portions, analogues or derivatives of the encoded protein or polypeptide. Such variations can be naturally-occurring, such as in the case of allelic variation, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes, so long as the nucleic acid molecule encodes at least one of the alterations described herein and the encoded protein has either increased conformational stability and/or decreased cross-reactivity to hLFA-1 (as compared to the corresponding unaltered protein) that is conferred by the alterations described herein. Intended variations include, but are not limited to, addition, deletion and/or substitution of one or more nucleotides which can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably, such nucleotide or amino acid variations are silent; that is, they do not alter one or more characteristics or activity of the encoded altered OspA protein or polypeptide. As used herein, activities of the encoded protein or polypeptide include, but are not limited to, binding function, antigenic function and conformational stability. [0117]
  • The invention also provides expression vectors containing a nucleic acid sequence described herein, operably linked to at least one regulatory sequence. Many such vectors are commercially available, and other suitable vectors can be readily prepared by the skilled artisan. “Operably linked” is intended to mean that the nucleic acid molecule is linked to a regulatory sequence in a manner which allows expression of the nucleic acid sequence. Regulatory sequences are art-recognized and are selected to produce the encoded polypeptide or protein. Accordingly, the term “regulatory sequence” includes promoters, enhancers, and other expression control elements which are described in Goeddel, [0118] Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). For example, the native regulatory sequences or regulatory sequences native to the transformed host cell can be employed. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. For instance, the polypeptides of the present invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells or both (see, for example, Broach, et al., Experimental Manipulation of Gene Expression, ed. M. Inouye (Academic Press, 1983) p. 83; Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. Sambrook et al., (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17). Typically, expression constructs will contain one or more selectable markers, including but not limited to, the gene that encodes dihydrofolate reductase and the genes that confer resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin or streptomycin.
  • Prokaryotic and eukaryotic host cells transfected by the described vectors are also provided by this invention. For instance, cells which can be transfected with the vectors of the present invention include, but are not limited to, bacterial cells such as [0119] E. coli (e.g., E. coli K12, BL21, DH5αstrains), Streptomyces, Pseudomonas, Serratia marcescens and Salmonella typhimurium, insect cells (baculovirus) including Drosophila, fungal cells, such as yeast cells, plant cells and mammalian cells, such as thymocytes, Chinese hamster ovary cells (CHO) and COS cells.
  • Thus, a nucleic acid molecule comprising, for example SEQ ID NO: 6 with at least one of the specific alterations described herein, or a nucleic acid molecule which encodes, for example SEQ ID NO: 7 with at least one of the specific alterations described herein, can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the nucleic acid molecule (e.g., polynucleotide) into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect, plant or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well known proteins. Similar procedures, or modifications thereof, can be employed to prepare recombinant proteins according to the present invention by microbial means or tissue-culture technology. Accordingly, the invention pertains to the production of encoded proteins or polypeptides by recombinant technology. [0120]
  • The proteins and polypeptides of the present invention can be isolated or purified (e.g., to homogeneity) from recombinant cell culture by a variety of processes. These include, but are not limited to, anion or cation exchange chromatography, ethanol precipitation, affinity chromatography and high performance liquid chromatography (HPLC). The particular method used will depend upon the properties of the polypeptide and the selection of the host cell; appropriate methods will be readily apparent to those skilled in the art. [0121]
  • The present invention also pertains to pharmaceutical compositions comprising polypeptides and other compounds described herein. For instance, a polypeptide or protein of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to well known procedures, and will depend on the ultimate pharmaceutical formulation desired. Methods of introduction of exogenous polypeptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include gene therapy, rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents. [0122]
  • The altered OspA proteins described herein can be produced so that they are highly soluble, hyper-produced in [0123] E. coli, and non-lipidated. In addition, the altered OspA proteins can be designed to begin or end in a suitable affinity tag (e.g., a His-tag) to facilitate purification. The recombinant proteins described herein have been constructed to maintain high levels of antigenicity and improved conformational stability.
  • The altered OspA proteins of the current invention are advantageous in that they retain at least some specific reactivity to monoclonal and/or polyclonal antibodies against wild-type Borrelia proteins, are immunogenic, and inhibit the growth or induce lysis of Borrelia in vitro. The proteins are particularly useful in immunodiagnostic assays. For example, proteins of the present invention can be used as reagents in assays to detect the presence of antibodies to native Borrelia in potentially infected individuals. These proteins can also be used as immunodiagnostic reagents, such as in dot blots, Western blots, enzyme-linked immunosorbent assays, or agglutination assays. The altered OspA proteins of the present invention can be produced by known techniques, such as by recombinant methodology, polymerase chain reaction, or mutagenesis. [0124]
  • Furthermore, the proteins of the current invention are useful as vaccine immunogens against Borrelia infection. One or more of the altered proteins can be combined with a physiologically acceptable carrier and administered to a vertebrate animal through standard methods (e.g., intravenously or intramuscularly, for example). [0125]
  • The altered forms of the OspA proteins described herein were bioengineered such that at least one immunoprotective domain of the protein was maintained. As described herein, antigenic refers to the ability of a compound to bind products of an immune response, such as antibodies, T-cell receptors or both. Such responses can be measured using standard antibody detection assays, such as ELISA or standard T-cell activation assays. In a preferred embodiment, the altered forms of OspA described herein elicit an immunoprotective response, for example, by eliciting antibodies that recognize the LA-2 epitope. [0126]
  • It is understood that the nucleic acids that encode the polypeptides that comprise the altered OspA protein can include extra nucleotides or fewer nucleotides in order to simplify the construction of the gene encoding the chimeric polypeptide, e.g., to allow for the use of convenient restriction endonuclease sites, or to allow the ligation of the gene fragments such that a contiguous coding region is created. Based on the guidance provided herein, one of ordinary skill in the art would readily be able to add or remove nucleotides from the termini of the gene fragments encoding the polypeptides of the OspA protein in order to generate the altered OspA proteins of the present invention with no experimentation or using only routine experimentation. The altered OspA polypeptides of the present invention can be lipidated or non-lipidated. [0127]
  • To test the antigenicity of the altered OspA polypeptides, mice can be immunized with OspA polypeptides or proteins containing the polypeptide sequences in aluminum hydroxide. Mice are then bled and tested for antibody responses against OspA derived from various strains of Borrelia. In additional experiments, these immunized mice can be challenged with ticks infected with [0128] Borrelia burgdorferi and transmission of infection can be assessed as described in the Exemplification which use OspA, OspC and OspC/OspA chimeric molecules. The results of such a tick challenge reveals whether the animal has developed a protective immune response. For example, an immunized animal that does not seroconvert in response to subsequent tick challenge has likely generated an immunoprotective response to the immunization.
  • The immunogenic compositions of the present invention can be used to immunize animals including humans. Immunization is understood to elicit specific immunogenic responses, preferably immunoprotective responses, as described above. As described herein, an immunogenic response includes responses that result in at least some level of immune response in the treated animal, where the animal was previously treated with a composition comprising at least one altered OspA polypeptide of the present invention. [0129]
  • Immunity, as described herein, is understood to mean the ability of the treated animal to resist infection, to resist systemic infection, or to overcome infection such as systemic infection more easily or more quickly when compared to non-immunized or non-treated individuals. Immunity can also include an improved ability of the treated individual to sustain an infection with reduced or no clinical symptoms of systemic infection. The individual may be treated with the altered OspA proteins of the present invention either proactively, e.g., once a year or, alternatively, after sustaining a tick bite. [0130]
  • In one embodiment, the altered OspA protein of the present invention, together with suitable excipients and/or adjuvants, is administered to an animal such that the animal develops an immune response to the OspA polypeptide of the composition. The pharmaceutical composition can also be administered with other components suitable for in vitro and/or in vivo use. These additional components include buffers, carrier proteins, adjuvants, preservatives and combinations thereof. In a preferred embodiment, the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope. [0131]
  • The present invention is also drawn to a physiological composition comprising an altered OspA protein. The composition is useful to administer to an animal in order to generate an immune response or in the diagnostic methods described herein. [0132]
  • For use as a vaccine, the composition of the present invention can include suitable adjuvants, well known in the art, to enhance immunogenicity, potency or half-life of the chimeric proteins in the treated animal. Adjuvants and their use are well known in the art (see for example PCT Publication WO 96/40290, the entire teachings of which are incorporated herein by reference). The composition can be prepared by known methods of preparing vaccines. For example, the altered OspA polypeptides described herein can be isolated and/or purified using known techniques, such as by size exclusion chromatography, ion exchange chromatography, affinity chromatography, preparative electrophoresis, selective precipitation or combinations thereof. The prepared proteins can be mixed with suitable other reagents as described above, wherein the protein is at a suitable concentration. The dosage of the protein will vary and depends upon the age, weight and/or physical condition of the animal to be treated. The optimal dosage can be determined by routine optimization techniques, using suitable animal models. [0133]
  • The composition to be used as a vaccine can be administered by any suitable technique. In one embodiment, administration is by injection, e.g., subcutaneous, intramuscular, intravenous, or intra peritoneal injection. In another embodiment, the composition is administered to mucosa, e.g., by exposing nasal mucosa to nose drops containing the proteins of chimeric proteins of the present invention. In another embodiment, the immunogenic composition is administered by oral administration. In another embodiment of the present invention the chimeric proteins are administered by DNA immunization using nucleic acids encoding an altered OspA polypeptide. [0134]
  • The present invention is also drawn to a diagnostic kit comprising the altered OspA polypeptides described herein. The kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspA protein and antibodies that are present in an sample, e.g., a user-supplied host sample. [0135]
  • The present invention is also drawn to methods of detecting an immune response to Lyme Disease-causing Borrelia in a host sample. The method comprises contacting a host sample with an OspA altered protein, such that anti-OspA antibodies, if present in said sample, bind to said OspA protein. The quantity of antibodies that have bound said OspA protein are measured, thereby detecting an immune response to Lyme disease-causing Borrelia. [0136]
    • EXEMPLIFICATION Example 1 Purification of Borrelia burgdorferi Outer Surface Protein A and Analysis of Antibody Binding Domains
  • This example details a method for the purification of large amounts of native outer surface protein A (OspA) to homogeneity, and describes mapping of the antigenic specificities of several anti-OspA MAbs. OspA was purified to homogeneity by exploiting its resistance to trypsin digestion. Intrinsic labeling with [0137] 14C-palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal cysteine of the molecule.
  • The reactivity of seven anti-OspA murine monoclonal antibodies to nine different Borrelia isolates was ascertained by Western blot analysis. The reactivity of the altered OspA polypeptides described herein was tested using similar methods. Intact, lipidated or non-lipidated OspA and altered OspA can also be tested using similar methods. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains (see FIG. 1). [0138] Domain 3, which included residues 190-220 of OspA, was reactive with protective antibodies known to agglutinate the organism in vitro, and included distinct specificities, some of which were not restricted to a genotype of B. burgdorferi.
  • A. Purification of Native OspA [0139]
  • Detergent solubilization of [0140] B. burgdorferi strips the outer surface proteins and yields partially-purified preparations containing both OspA and outer surface protein B (OspB) (Barbour, A. G. et al., Infect. Immun., 52(5):549-554 (1986); Coleman, J. L. et al., J. Infect. Dis., 155 (4):756-765 (1987); Cunningham, T. M. et al., Ann. NY Acad. Sci., 539:376-378 (1988); Brandt, M. E. et al., Infect. Immun., 58: 983-991 (1990); Sambri, V. and R. Cevenini, Microbiol., 14:307-314 (1991)). Although both OspA and OspB are sensitive to proteinase K digestion, in contrast to OspB, OspA is resistant to cleavage by trypsin (Dunn, J. et al., Prot. Exp. Purif., 1:159-168 (1990); Barbour, A. G. et al., Infect. Immun., 45:94-100 (1984)). The relative insensitivity to trypsin is surprising in view of the fact that OspA has a high (16% for B31) lysine content, and may relate to the relative configuration of OspA and B in the outer membrane.
  • Intrinsic Radiolabeling of Borrelia [0141]
  • Labeling for lipoproteins was performed as described by Brandt et al. (Brandt et al., [0142] Infect. Immun., 58:983-991 (1990)). 14C-palmitic acid (ICN, Irvine, Calif.) was added to the BSK II media to a final concentration of 0.5 μCi per milliliter (ml). Organisms were cultured at 34° C. in this medium until a density of 108 cells per ml was achieved.
  • Purification of OspA Protein from Borrelia Strain B31 [0143]
  • [0144] Borrelia burgdorferi, either 14C-palmitic acid-labeled or unlabeled, were harvested and washed as described (Brandt, M. E. et al., Infect. Immun., 58:983-991 (1990)). Whole organisms were trypsinized according to the protocol of Barbour et al., (Infect. Immun., 45:94-100 (1984)) with some modifications. The pellet was suspended in phosphate buffered saline (PBS, 10 mM, pH 7.2), containing 0.8% tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma, St. Louis, Mo.), the latter at a ratio of 1 μg per 108 cells. Reaction was carried out at 25° C. for 1 hour, following which the cells were centrifuged. The pellet was washed in PBS with 100 μg/ml phenylmethylsulfonyl fluoride (PMSF). Triton X-114 partitioning of the pellet was carried out as described by Brandt et al., (Brandt et al., Infect. Immun., 58:983-991 (1990)). Following trypsin treatment, cells were resuspended in ice-cold 2% (v/v) Triton X-114 in PBS at 109 cells per ml. The suspension was rotated overnight at 4° C., and the insoluble fraction removed as a pellet after centrifugation at 10,000×g for 15 minutes at 4° C. The supernatant (soluble fraction) was incubated at 37° C. for 15 minutes and centrifuged at room temperature at 1000×g for 15 minutes to separate the aqueous and detergent phases. The aqueous phase was decanted, and ice cold PBS added to the lower Triton phase, mixed, warmed to 37° C., and again centrifuged at 1000×g for 15 minutes. Washing was repeated twice more. Finally, detergent was removed from the preparation using a spin column of Bio-beads SM2 (BioRad, Melville, N.Y.) as described (Holloway, P. W., Anal. Biochem., 53:304-308 (1973)).
  • Ion exchange chromatography was carried out as described by Dunn et al., (Dunn et al., [0145] Prot. Exp. Purif., 1:159-168 (1990)) with minor modifications. Crude OspA was dissolved in buffer A (1% Triton X-100, 10 mM phosphate buffer (pH 5.0)) and loaded onto a SP Sepharose resin (Pharmacia, Piscataway, N.J.), pre-equilibrated with buffer A at 25° C. After washing the column with 10 bed-volumes of buffer A, the bound OspA was eluted with buffer B (1% Triton X-100, 10 mM phosphate buffer (pH 8.0)). OspA fractions were detected by protein assay using the BCA method (Pierce, Rockford, Ill.), or as radioactivity when intrinsically labeled material was fractionated. Triton X-100 was removed using a spin column of Bio-beads SM2.
  • This method purifies OspA from an outer surface membrane preparation. In the absence of trypsin-treatment, OspA and B were the major components of the soluble fraction obtained after Triton partitioning of strain B31. In contrast, when Triton extraction was carried out after trypsin-treatment, the OspB band is not seen. Further purification of OspA-B31 on a SP Sepharose column resulted in a single band by SDS-PAGE. The yield following removal of detergent was approximately 2 mg per liter of culture. This method of purification of OspA, as described herein for strain B31, can be used for other isolates of Borrelia as well. For strains such as strain K48, which lack OspB, trypsin treatment can be omitted. [0146]
  • Lipidation Site of OspA-B31 [0147]
  • [0148] 14C-palmitic acid labeled OspA from strain B31 was purified as described above and partially digested with endoproteinase Asp-N. Following digestion, a new band of lower molecular weight was apparent by SDS-PAGE, found by direct amino-terminal sequencing to begin at Asp25. This band had no trace of radioactivity by autoradiography. OspA and B contain a signal sequence (L-X-Y-C) similar to the consensus described for lipoproteins of E. coli, and it has been predicted that the lipidation site of OspA and B should be the amino-terminal cysteine (Brandt, M. E. et al., Infect. Immun, 58:983-991 (1990)). The results presented herein support this prediction.
  • B. Comparison of OspA Antibody Binding Regions in Nine Strains of [0149] Borrelia burgdorferi
  • The availability of the amino acid sequenced for OspA from a number of different isolates, combined with peptide mapping and Western blot analysis, permitted the identification of the antigenic domains recognized by monoclonal antibodies (MAbs) and allowed inference of the key amino acid residues responsible for specific antibody reactivity. [0150]
  • Strains of [0151] Borrelia burgdorferi
  • Nine strains of Borrelia, including seven European strains and two North American strains, were used in this study of antibody binding domains of several proteins. Information concerning the strains is summarized in Table I, below. [0152]
    TABLE I
    Representative Borrelia Strains
    Strain Location and Source Reference for Strain
    K48 Czechoslovakia, none
    Ixodes ricinus
    PGau Germany, human ACA Wilske, B. et al., J. Clin. Microbiol.
    32:340-350 (1993)
    DK29 Denmark, human EM Wilske, B. et al.
    Pko Germany, human EM Wilske, B. et al.
    PTrob Germany, human skin Wilske, B. et al.
    Ip3 Khabarovsk, Russia, Asbrink, E. et al., Acta Derm.
    I. persulcatus Venereol., 64:506-512 (1984)
    Ip90 Khabarovsk, Russia, Asbrink, E. et al.
    persulcatus
    25015 Milibrook, NY, Barbour, A. G. et al., Curr. Microbiol.,
    I. persulcatus 8:123-126 (1983)
    B31 Shelter Island, NY, Luft, B. J. et al., Infect. Immun, 60:
    I. scapularis 4309-4321 (1992); ATCC 35210
    PKa1 Germany, human CSF Wilske, B. et al.
    ZS7 Freiburg, Germany, Wallich, R. et al., Nucl. Acids Res,
    I. ricinus 17:8864 (1989)
    N40 Westchester Co., NY Fikrig, B. et al., Science, 250:553-556
    (1990)
    PHei Germany, human CSF Wilske, B. et al.
    ACAI Sweden, human ACA Luft, B. J. et al., FEMS Microbiol.
    Lett. 93:73-68 (1992)
    PBo Germany, human CSF Wilske, B. et al.
  • Strains K48, PGau and DK29 were supplied by R. Johnson, University of Minnesota; Pko and pTrob were provided by B. Wilske and V. Preac-Mursic of the Pettenkhofer Institute, Munich, Germany; and Ip3 and Ip90 were supplied by L. Mayer of the Center for Disease Control, Atlanta, Ga. The North American strains included [0153] strain 25015, provided by J. Anderson of the Connecticut Department of Agriculture; and strain B31 (ATCC 35210).
  • Monoclonal Antibodies [0154]
  • Seven monoclonal antibodies (MAbs) were utilized in this study. Five of the MAbs (12, 13, 15, 83 and 336) were produced from hybridomas cloned and subcloned as previously described (Schubach, W. H., et al., [0155] Infect. Immun., 59(6):1911-1915 (1991)). MAb H5332 (Barbour, A. G. et al., Infect. Immun., 41: 795-804 (1983)) was a gift from Drs. Alan Barbour, University of Texas, and MAb CIII.78 (Sears, J. E. et al., J. Immunol., 147(6):1995-2000 (1991)) was a gift from Richard A. Flavell, Yale University. MAbs 12 and 15 were raised against whole sonicated B3; MAb 336 was produced against whole PGau; and MAbs 13 and 83 were raised to a truncated form of OspA cloned from the K48 strain and expressed in E. coli using the T7 RNA polymerase system (McGrath, B. C. et al., Vaccines, Cold Spring Harbor Laboratory Press, Plainview, N.Y., pp. 365-370 (1993)). All MAbs were typed as being Immunoglobulin G (IgG).
  • Methods of Protein Cleavage, Western Blotting and Amino-Terminal Sequencing [0156]
  • Prediction of the various cleavage sites was achieved by knowledge of the primary amino acid sequence derived from the full nucleotide sequences of OspA, many of which are currently available (see Table II, below). Cleavage sites can also be predicted based on the peptide sequence of OspA, which can be determined by standard techniques after isolation and purification of OspA by the method described above. Cleavage of several OspA isolates was conducted to determine the localization of monoclonal antibody binding of the proteins. [0157]
  • Hydroxylamine-HCl (HA), N-chlorosuccinimide (NCS), and cyanogen bromide cleavage of OspA followed the methods described by Bornstein ([0158] Biochem. 9 (12):2408-2421 (1970)), Shechter et al., (Biochem., 15 (23):5071-5075 (1976)), and Gross (in Hirs, C. H. W. (ed): Methods in Enzymology, (N.Y. Acad. Press), 11:238-255 (1967)) respectively. Protease cleavage by endoproteinase, Asp-N (Boehringer Mannheim, Indianapolis, Ind.), was performed as described by Cleveland D. W. et al., (J. Biol. Chem., 252:1102-1106 (1977)). Ten micrograms of OspA were used for each reaction. The ratio of enzyme to OspA was approximately 1 to 10 (w/w).
  • Proteins and peptides generated by cleavage were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U. K., [0159] Nature (London) 227:680-685 (1970)), and electroblotted onto immobilon Polyvinylidine Difluoride (PVDF) membranes (Ploskal, M. G. et al., Biotechniques, 4:272-283 (1986)). They were detected by amido black staining or by immunostaining with murine MAbs, followed by alkaline phosphatase-conjugated goat anti-mouse IgG. Specific binding was detected using a 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) developer system (KPL Inc., Gathersburg, Md.).
  • In addition, amino-terminal amino acid sequence analysis was carried out on several cleavage products, as described by Luft et al., ([0160] Infect. Immun., 57:3637-3645 (1989)). Amido black stained bands were excised from PVDF blots and sequenced by Edman degradation using a Biosystems model 475A sequenator with model 120A PTH analyzer and model 900A control/data analyzer.
  • Cleavage Products of Outer Surface Protein A Isolates [0161]
  • Purified OspA-B31, labeled with [0162] 14C-palmitic acid, was fragmented with hydroxylamine-HCl (HA) into two peptides, designated HA1 and HA2 (data not shown). The HA1 band migrated at 27 kd and retained its radioactivity, indicating that the peptide included the lipidation site at the N-terminus of the molecule (data not shown). From the predicted cleavage point, HA1 should correspond to residues 1 to 251 of OspA-B31. HA2 had a MW of 21.6 kd by SDS-PAGE, with amino-terminal sequence analysis showing it to begin at Gly72, i.e. residues 72 to 273 of OspA-B31. By contrast, HA cleaved OspA-K48 into three peptides, designated HA1, HA2, and HA3 with apparent MWs of 22 kd, 16 kd and 12 kd, respectively. Amino-terminal sequencing showed HA1 to start at Gly72, and HA3 at Gly142. HA2 was found to have a blocked amino-terminus, as was observed for the full-length OspA protein. HA1, 2 and 3 of OspA-K48 were predicted to be residues 72-274, 1 to 141 and 142 to 274, respectively.
  • N-Chlorosuccinimide (NCS) cleaves tryptophan (W), which is at [0163] residue 216 of OspA-B31 or residue 217 of OspA-K48 (data not shown). NCS cleaved OspA-B31 into 2 fragments, NCS1, with MW of 23 kd, residues 1-216 of the protein, and NCS2 with a MW of 6.2 kd, residues 217 to 273 (data not shown). Similarly, K48 OspA was divided into 2 pieces, NCS1 residues 1-217, and NCS2 residues 218 to 274 (data not shown).
  • Cleavage of OspA by cyanogen bromide (CNBr) occurs at the carboxy side of methionine, [0164] residue 39. The major fragment, CNBr1, has a MW of 25.7 kd, residues 39-274 by amino-terminal amino acid sequence analysis (data not shown). CNBr2 (about 4 kd) could not be visualized by amido black staining; instead, lightly stained bands of about 20 kd MW were seen. These bands reacted with anti-OspA MAbs, and most likely were degradation products due to cleavage by formic acid.
  • Determination of Antibody Binding Domains for Anti-OspA Monoclonal Antibodies [0165]
  • The cleavage products of OspA-B31 and OspA-K48 were analyzed by Western blot to assess their ability to bind to the six different MAbs. Preliminary Western blot analysis of the cleavage products demonstrated that strains K48 and DK29 have similar patterns of reactivity, as do IP3, PGau and Pko. The OspA of strain PTrob was immunologically distinct from the others, being recognized only by [0166] MAb 336. MAb 12 recognized only the two North American strains, B31 and 25015. When the isolates were separated into genogroups, it was remarkable that all the MAbs, except MAb 12, crossed over to react with multiple genogroups.
  • [0167] MAb 12, specific for OspA-B31, bound to both HA1 and HA2 of OspA-B31. However, cleavage of OspA-B31 by NCS at residue Trp216 created fragments which did not react with MAb12, suggesting that the relevant domain is near or is structurally dependent upon the integrity of this residue (data not shown). MAb 13 bound only to OspA-K48, and to peptides containing the amino-terminus of that molecule (e.g. HA2; NCS1). It did not bind to CNBr1 residues 39 to 274. Thus the domain recognized by MAb13 is in the amino-terminal end of OspA-K48, near Met38.
  • MAb15 reacts with the OspA of both the B31 and K48 strains, and to peptides containing the N-terminus of OspA, such as HA1 of OspA-B31 and NCS1, but not to peptides HA2 of OspA-B31 and HA1 of OspA-K48 (data not shown). Both peptides include [0168] residue 72 to the C-terminus of the molecules. MAb15 bound to CNBr1 of OspA-K48, indicating the domain for this antibody to be residues 39 to 72, specifically near Gly72 (data not shown).
  • MAb83 binds to OspA-K48, and to peptides containing the C-terminal portion of the molecule, such as HA1. They do not bind to HA2 of OspA-K48, most likely because the C-terminus of HA2 of OspA-K48 ends at 141. Similar to MAb12 and OspA-B31, binding of [0169] MAbs 83 and CIII.78 is eliminated by cleavage of OspA at the tryptophan residue. Thus binding of MAbs 12, 83 and CIII.78 to OspA depends on the structural integrity of the Trp216 residue, which appears to be critical for antigenicity. Also apparent is that, although these MAbs bind to a common antigenic domain, the precise epitopes which they recognize are distinct from one another given the varying degrees of cross-reactivity to these MAbs among strains.
  • Although there is similar loss of binding activity of MAb336 with cleavage at Trp[0170] 216, this MAb does not bind to HA1 of OspA-B31, suggesting the domain for this antibody includes the carboxy-terminal end of the molecule, inclusive of residues 251 to 273. Low MW peptides, such as HA3 (10 kd) and NCS2 (6 kd), of OspA-K48 do not bind this MAb on Western blots. In order to confirm this observation, we tested binding of the 6 MAbs with a recombinant fusion construct p3A/EC that contains a trpE leader protein fused with residues 217 to 273 of OspA-B31 (Schubach, W. H. et al., Infect. Immun., 59(6):1911-1915 (1991)). Only MAb336 reacted with this construct (data not shown). Peptides and antigenic domains localized by fragmentation of OspA are summarized in FIG. 1.
    • Example 2 Site-Directed Mutagenesis within Hypervariable Domains A., (Residues 120-140), B., (Residues 150-180) and C., (Residues 200-216 or 217)
  • Site-directed mutagenesis was performed to convert residues within the 204-219 domain of the recombinant B31 OspA to the analogous residues of a European OspA variant, K48. In the region of OspA between residues 204 and 219, there are seven amino acid differences between OspA-B31 and OspA-K48. Three oligonucleotides were generated, each containing nucleotide changes which would incorporate K48 amino acids at their analogous positions in the B31 OspA protein. The oligonucleotides used to create the site-directed mutants were: [0171]
  • 5′-CTTAATGACTCTGACACTAGTGC-3′ (#613, which converts threonine at position 204 to serine, and serine at 206 to threonine (Thr204-Ser, Thr206-Ser)) (SEQ ID NO: 1); 5′-GCTACTAAAAAAACCGGGAAATGGAATTCA-3′ (#625, which converts alanine at 214 to glycine, and alanine at 215 to lysine (Ala214-Gly, Ala215-Lys)) (SEQ ID NO: 2); and [0172]
  • 5′-GCAGCTTGGGATTCAAAAACATCCACTTTAACA-3′ (#640, which converts asparagine at 217 to aspartate, and glycine at 219 to lysine (Asn217-Asp, Gly219-Lys)) (SEQ ID NO: 3). [0173]
  • Site-directed mutagenesis was carried out by performing mutagenesis with pairs of the above oligonucleotides. Three site-directed mutants were created, each with two changes: OspA 613 (Thr204-Ser, Thr206-Ser), OspA 625 (Ala214-Gly, Ala215-Lys), and 640 (Asn217-Asp, Gly219-Lys). There were also two proteins with four changes: [0174] OspA 613/625 (Thr204-Ser, Thr206-Ser, Ala214-Gly, Ala215-Lys) and OspA 613/640 (Thr204-Ser, Thr206-Ser, Asn217-Asp, Gly219-Lys).
  • Specificity of Antibody Binding to Epitopes of the Non-mutated Hypervariable Region [0175]
  • Monoclonal antibodies that agglutinate spirochetes, including several which are neutralizing in vitro, recognize epitopes that map to the hypervariable region around Trp216 (Barbour, A. G. et al., [0176] Infect. and Immun., 41:759 (1983); Schubach, W. H. et al., Infect. and Immun., 59:1911 (1991)). Western Blot analysis demonstrated that chemical cleavage of OspA from the B31 strain at Trp 216 abolishes reactivity of the protein with the agglutinating MAB 105, a monoclonal raised against B31 spirochetes. The reagent, n-chlorosuccinimide (NCS), cleaves OspA at the Trp 216, forming a 23.2 kd fragment and a 6.2 kd peptide which is not retained on the Imobilon-P membrane after transfer. The uncleaved material binds MAb 105; however, the 23.2 kd fragment is unreactive. Similar Western blots with a TrpE-OspA fusion protein containing the carboxy-terminal portion of the OspA protein demonstrated that the small 6.2 kd piece also fails to bind MAb 105 (Schubach, W. H. et al., Infect. and Immun., 59:1911 (1991)).
  • Monoclonal antibodies H5332 and H3TS (Barbour, A. G. et al., [0177] Infect. and Immun., 41:759 (1983)) have been shown by immunofluorescence to decorate the surface of fixed spirochetes (Wilske, B. et al., World J. Microbiol., 7:130 (1991)). These monoclonal antibodies also inhibit the growth of the organism in culture. Epitope mapping with fusion proteins has confirmed that the epitopes which bind these MAbs are conformationally determined and reside in the carboxy half of the protein. MAb H5332 is cross-reactive among all of the known phylogenetic groups, whereas MAb H3TS and MAb 105 seem to be specific to the B31 strain to which they were raised. Like MAb 105, the reactivities of H5332 and H3TS to OspA are abrogated by fragmentation of the protein at Trp216. MAb 336 was raised to whole spirochetes of the strain PGau. It cross-reacts to OspA from group 1 (the group to which B31 belongs) but not to group 2 (of which K48 is a member). Previous studies using fusion proteins and chemical cleavage have indicated that this antibody recognizes a domain of OspA in the region between residues 217 and 273. All of these MAbs agglutinate the B31 spirochete.
  • Western Blot Analysis of Antibody Binding to Mutated Hypervariable Regions [0178]
  • MAbs were used for Western Blot analysis of the site-directed OspA mutants induced in [0179] E. coli using the T7 expression system (Dunn, J. J. et al., Protein Expression and Purification, 1:159 (1990)). E. coli cells carrying pET9c plasmids having a site-directed OspA mutant insert were induced at mid-log phase growth with IPTG for four hours at 37° C. Cell lysates were made by boiling an aliquot of the induced cultures in SDS gel loading dye, and this material was then loaded onto a 12% SDS gel (BioRad mini-Protean II), and subjected to electrophoresis. The proteins were then transferred to Imobilon-P membranes (Millipore) 70V, 2 hour at 4° C. using the BioRad mini transfer system. Western analysis was carried out as described by Schubach et al., (Infect. Immun., 59:1911 (1991)).
  • Western Blot analysis indicated that only the 625 mutant (Ala214-Gly and Ala215-Lys) retained binding to the agglutinating monoclonal H3TS. However, the 613/625 mutant which has additional alterations to the amino terminus of Trp216 (Ser204-Thr and Thr206-Ser) did not bind this monoclonal. Both 640 and 613/640 OspAs which have the Asn217-Asp and Gly219-Lys changes on the carboxy-terminal side of Trp216 also failed to bind MAb H3TS. This indicated that the epitope of the B31 OspA which binds H3TS is comprised of amino acid side-chains on both sides of Trp216. [0180]
  • The 613/625 mutant failed to bind MAbs 105 and H5332, while the other mutants retained their ability to bind these MAbs. This is important in light of the data using fusion proteins that indicate that MAb 105 behaves more like MAb H3TS in terms of its serotype specificity and binding to OspA (Wilske, B. et al., [0181] Med. Microbiol. Immunol., 181:191 (1992)). The 613/625 protein has, in addition to the differences at residues Thr204 and Ser206, changes immediately amino-terminal to Trp216 (Ala214-Gly and Ala215-Lys). The abrogation of reactivity of MAbs 105 and H5332 to this protein indicated that the epitopes of OspA which bind these monoclonal antibodies are comprised of residues on the amino-terminal side of Trp216.
  • The two proteins carrying the Asn217-Asp and Gly219-Lys replacements on the carboxy-terminal side of Trp216 (OspAs 640 and 613/640) retained binding to MAbs 105 and H5332; however, they failed to react with [0182] MAb 336, a monoclonal which has been mapped with TrpE-OspA fusion proteins and by chemical cleavage to a more carboxy-terminal domain. This result may explain why MAb 336 failed to recognize the K48-type of OspA (Group 2).
  • It is clear that amino acids Ser204 and Thr206 play an important part in the agglutinating epitopes in the region of the B31 OspA flanking Trp216. Replacement of these two residues altered the epitopes of OspA that bind MAbs 105, H3TS and H5332. The ability of the 640 changes alone to abolish reactivity of [0183] MAb 336 indicated that Thr204 and Ser206 are not involved in direct interaction with MAb 336.
  • The results indicated that the epitopes of OspA which are available to MAbs that agglutinate spirochetes are comprised at least in part by amino acids in the immediate vicinity of Trp216. Since circular dichroism analysis indicated that the structures of B31 and K48 OspA differ very little within this domain, it is unlikely that the changes made by mutation have radically altered the overall structure of the OspA protein (France, L. L. et al., [0184] Biochem. Biophys. Acta, 1120:59 (1992); and France et al., Biochem. Biophys Acta, submitted (1993)). This hypothesis is supported by the finding that the recombinant, mutant OspAs exhibit the same high solubility and purification properties as the parent B31 protein (data not shown).
  • In summary, amino acid side-chains at Ser204 and Thr206 are important for many of the agglutinating epitopes. However, a limited set of conservative changes at these sites were not sufficient to abolish binding of all of the agglutinating MAbs. These results suggested that the agglutinating epitopes of OspA are distinct, yet may have some overlap. The results also supported the hypothesis that the surface-exposed epitope around Trp216 which is thought to be important for immune recognition and neutralization is a conformationally-determined and complex domain of OspA. [0185]
    • Example 3 Borrelia Strains and Proteins
  • A. Genes Encoding Borrelia Proteins [0186]
  • The altered OspA polypeptides of the current invention can be part of a cocktail with other proteins or can be joined to other proteins to form a chimeric protein. The other polypeptides of the cocktail or chimeric can be derived from any Borrelia. Representative proteins include OspA, OspB, OspC, OspD, p12, p39, p41 (fla), p66, and p93. Nucleic acid sequences encoding several Borrelia proteins are available (see Table II for examples); alternatively, nucleic acid sequences encoding Borrelia proteins can be isolated and characterized using methods such as those described below. [0187]
    TABLE II
    References for Nucleic Acid Sequences for Several Proteins of
    Various Borrelia Strains
    Strain OspA
    K48 X62624 (SID 8) 
    PGau X62387 (SID 10)
    DK29 X63412 (SID 21)
    Pko X65599 (SID 25)
    PTrob X65598 (SID 45)
    Ip3 X70365 (SID 24)
    Ip90 Kryuchechnikov, V. N. et al.,
    J. Microbiol. Epid.
    Immunobiol. 12:41-44
    (1988) (SID 22)
    25015 Fikrig, E. S. et al., J.
    Immunol. 7:2256-2260
    (1992) (SID 12)
    B31 Bergstrom, S. etal., Mol.
    Microbiol. 3:479-486
    (1989) (SID 6)
    PKa1 X69606 (SID 42)
    ZS7 Jonsson, M. et al., Infect.
    Immun. 60:1845-1853
    (1992) (SID 44)
    N40 Kryuchechnikov, V. N. et al.
    (SID 43)
    PHei X65600 (SID 46)
    ACAI Kryuchechnikov, V. N. et al.
    (SID 26)
    PBo X65605 (SID 23)
  • B. Isolation of Borrelia Genes [0188]
  • Nucleic acid sequences encoding full length, lipidated proteins from known Borrelia strains were isolated using the polymerase chain reaction (PCR) as described below. In addition, nucleic acid sequences were generated which encoded truncated proteins (proteins in which the lipidation signal has been removed, such as by eliminating the nucleic acid sequence encoding the first 18 amino acids, resulting in non-lipidated proteins). Other proteins were generated which encoded polypeptides of a particular gene (i.e., encoding a segment of the protein which has a different number of amino acids than the protein does in nature). Using similar methods as those described below, primers can be generated from known nucleic acid sequences encoding Borrelia proteins and used to isolate other genes encoding Borrelia proteins. Primers can be designed to amplify all of a gene, as well as to amplify a nucleic acid sequence encoding truncated protein sequences, such as described below for OspC, or nucleic acid sequences encoding a polypeptide derived from a Borrelia protein. Primers can also be designed to incorporate unique restriction enzyme cleavage sites into the amplified nucleic acid sequences. Sequence analysis of the amplified nucleic acid sequences can then be performed using standard techniques. [0189]
  • Cloning and Sequencing of OspA Genes and Relevant Nucleic Acid Sequences [0190]
  • Borrelia OspA sequences were isolated in the following manner: 100 μl reaction mixtures containing 50 mM KCl, 10 mM TRIS-HCl ([0191] pH 8,3), 1.5 mM MgCl2, 200 μM each NTP, 2.5 units of TaqI DNA polymerase (Amplitaq, Perkin-Elmer/Cetus) and 100 pmol each of the 5′ and 3′ primers (described below) were used. Amplification was performed in a Perkin-Elmer/Cetus thermal cycler as described (Schubach, W. H. et al., Infect. Immun., 59:1811-1915 (1991)). The amplicon was visualized on an agarose gel by ethidium bromide staining. Twenty nanograms of the chloroform-extracted PCR product were cloned directly into the PC-TA vector (Invitrogen) by following the manufacturer's instructions. Recombinant colonies containing the amplified fragment were selected, the plasmids were prepared, and the nucleic acid sequence of each OspA was determined by the dideoxy chain-termination technique using the Sequenase kit (United States Biochemical). Directed sequencing was performed with M13 primers followed by OspA-specific primers derived from sequences, previously obtained with M13 primers.
  • Because the 5′ and 3′ ends of the OspA gene are highly conserved (Fikrig, E. S. et al., [0192] J. Immunol., 7:2256-2260 (1992); Bergstrom, S. et al., Mol. Microbiol. 3:479-486 (1989); Zumstein, G. et al., Med. Microbiol. Immunol., 181:57-70 (1992)), the 5′ and 3′ primers for cloning can be based upon any known OspA sequences. For example, the following primers based upon the OspA nucleic acid sequence from strain B31 were used:
  • 5′-GGAGAATATATTATGAAA-3′ (−12 to+6) (SEQ ID NO: 4); and [0193]
  • 5′-CTCCTTATTTTAAAGCG-3′ (+826 to+809) (SEQ ID NO: 5). (Schubach, W. H. et al., [0194] Infect. Immun, 59:1811-1915 (1991)).
  • OspA genes isolated in this manner include those for strains B31, K48, PGau, and 25015; the nucleic acid sequences are depicted in the sequence listing as SEQ ID NO: 6 (OspA-B31), SEQ ID NO: 8 (OspA-K48), SEQ ID NO: 10 (OspA-PGau), and SEQ ID NO: 12 (OspA-25015). An alignment of these and other OspA nucleic acid sequences is shown in FIG. 17. The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO: 7 (OspA-B31), SEQ ID NO: 9 (OspA-K48), SEQ ID NO: 11 (OspA-PGau), and SEQ ID NO: 13 (OspA-25015). [0195]
  • The following primers were used to generate specific nucleic acid sequences of the OspA gene: [0196]
    5′-GTCTGCAAAAACCATGACAAG-3′ (plus strand primer #369); (SEQ ID NO:14)
    5′-GTCATCAACAGAAGAAAAATTC-3′ (plus strand primer #357); (SEQ ID NO:15)
    5′-CCGGATCCATATGAAAAAATATTTATTGGG-3′ (plus strand primer #607); (SEQ ID NO:16)
    5′-CCGGGATCCATATGGCTAAGCAAAATGTTAGC-3′ (plus strand primer #584); (SEQ ID NO:17)
    5′-GCGTTCAAGTACTCCAGA-3′ (minus strand primer #200); (SEQ ID NO:18)
    5′-GATATCTAGATCTTATTTTAAAGCGTT-3′ (minus strand primer #586); and (SEQ ID NO:19)
    5′-GGATCCGGTGACCTTTTAAAGCGTTTTTAAT-3′ (minus strand primer #1169). (SEQ ID NO:20)
  • C. Expression of Proteins From Borrelia Genes [0197]
  • The nucleic acid sequences described above can be incorporated into expression plasmids, using standard techniques, and transfected into compatible host cells in order to express the proteins encoded by the nucleic acid sequences. As an example, the expression of the p12 gene and the isolation of p12 protein is set forth. [0198]
  • Amplification of the p12 nucleic acid sequence was conducted with primers that included a NdeI restriction site into the nucleic acid sequence. The PCR product was extracted with phenol/chloroform and precipitated with ethanol. The precipitated product was digested and ligated into an expression plasmid as follows: 15 μl (approximately 1 μg) of PCR DNA was combined with 2 [0199] μl 10×restriction buffer for NdeI (Gibco/BRL), 1 μl NdeI (Gibco/BRL), and 2 μl distilled water, and incubated overnight at 37° C. This mixture was subsequently combined with 3 μl 10×buffer (buffer 3, New England BioLabs), 1 μl BamHI (NEB), and 6 μl distilled water, and incubated at 37° for two hours. The resultant material was purified by preparative gel electrophoresis using low melting point agarose, and the band was visualized under long wave ultraviolet light and excised from the gel. The gel slice was treated with Gelase using conditions recommended by the manufacturer (Epicentre Technologies). The resulting DNA pellet was resuspended in 25-50 μl of 10 mM TRIS-CL (pH 8.0) and 1 mM EDTA (TE). An aliquot of this material was ligated into the pET9c expression vector (Dunn, J. J. et al., Protein Expression and Purification, 1:159 (1990)).
  • To ligate the material into the pET9c expression vector, 20-50 ng of p12 nucleic acid sequences cut and purified as described above was combined with 5 [0200] μl 10 One-Phor-All (OPA) buffer (Pharmacia), 30-60 ng pET9c cut with NdeI and BamHI, 2.5 μl 20 mM ATP, 2 μl T4 DNA ligase (Pharmacia) diluted 1:5 in 1×OPA buffer, and sufficient distilled water to bring the final volume to 50 μl. The mixture was incubated at 12° C. overnight.
  • The resultant ligations were transformed into competent DH5-alpha cells and plated on nutrient agar plates containing 50 μg/ml kanamycin and incubated overnight at 37° C. DH5-alpha is used as a “storage strain” for T7 expression clones, because it is RecA deficient, so that recombination and concatenation are not problematic, and because it lacks the T7 RNA polyierase gene necessary to express the cloned gene. The use of this strain allows for cloning of potentially toxic gene products while minimizing the chance of deletion and/or rearrangement of the desired genes. Other cell lines having similar properties may also be used. [0201]
  • Kanamycin resistant colonies were single-colony purified on nutrient agar plates supplemented with kanamycin at 50 μg/ml. A colony from each isolate was inoculated into 3-5 ml of liquid medium containing 50 μg/ml kanamycin, and incubated at 37° C. without agitation. Plasmid DNA was obtained from 1 ml of each isolate using a hot alkaline lysis procedure (Maniatis, T. et al., [0202] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)).
  • Plasmid DNA was digested with EcoRI and BglII in the following manner: 15 μl plasmid DNA was combined with 2 [0203] μl 10×buffer 3 (NEB), 1 μl EcoRI (NEB), 1 μl BglII (NEB) and 1 μl distilled water, and incubated for two hours at 37° C. The entire reaction mixture was subjected to electrophoresis on an analytical agarose gel. Plasmids carrying the p12 insert were identified by the presence of a band corresponding to 925 base-pairs (full length p12) or 875 base-pairs (nonlipidated p12). One or two plasmid DNAs from the full length and nonlipidated p12 clones in pET9c were used to transform BL21 DE3 pLysS to kanamycin resistance as described by Studier et al., (Methods in Enzymology, Goeddel, D. (Ed.), Academic Press, 185: 60-89 (1990)). One or two transformants of the full length and nonlipidated clones were single-colony purified on nutrient plates containing 25 μg/ml chloramphenicol (to maintain pLysS) and 50 μg/ml kanamycin at 37° C. One colony of each isolate was inoculated into liquid medium supplemented with chloramphenicol and kanamycin and incubated overnight at 37° C. The overnight culture was subcultured the following morning into 500 ml of liquid broth with chloramphenicol (25 μg/ml) and kanamycin (50 μg/ml) and grown with aeration at 37° C. in an orbital air-shaker until the absorbance at 600 nm reached 0.4-0.7. Isopropyl-thio-galactoside (IPTG) was added to a final concentration of 0.5 mM, for induction, and the culture was incubated for 3-4 hours at 37° C. as before. The induced cells were pelleted by centrifugation and resuspended in 25 ml of 20 mM NaPO4 (pH 7.7). A small aliquot was removed for analysis by gel electrophoresis. Expressing clones produced proteins which migrated at the 12 kd position.
  • A crude cell lysate was prepared from the culture as described for recombinant OspA by Dunn, J. J. et al., ([0204] Protein Expression and Purification, 1:159 (1990)). The crude lysate was first passed over a Q-sepharose column (Pharmacia) which had been pre-equilibrated in Buffer A: 10 mM NaPO4 (pH 7.7), 10 mM NaCl, 0.5 mM PMSF. The column was washed with 10 mM NaPO4, 50 mM NaCl and 0.5 mM PMSF and then p12 was eluted in 10 mM NaPO4, 0.5 mM PMSF with a NaCl gradient from 50-400 mM. p 12 eluted approximately halfway through the gradient between 100 and 200 mM NaCl. The peak fractions were pooled and dialyzed against 10 mM NaPO4 (pH 7.7), 10 mM NaCl, 0.5 mM PMSF. The protein was then concentrated and applied to a Sephadex G50 gel filtration column of approximately 50 ml bed volume (Pharmacia), in 10 mM NaPO4, 200 mM NaCl, 0.5 mM PMSF. p12 would typically elute shortly after the excluded volume marker. Peak fractions were determined by running small aliquots of all fractions on a gel. The p12 peak was pooled and stored in small aliquots at −20° C.
    • Example 4 Generation of Chimeric Nucleic Acid Sequences and Chimeric Proteins
  • A. General Protocol for Creation of Chimeric Nucleic Acid Sequences [0205]
  • The megaprimer method of site directed mutagenesis and a modification were used to generate chimeric nucleic acid sequences (Sarkar and Sommer, [0206] Biotechniques, 8(4): 404-407 (1990); Aiyar, A. and J. Leis, Biotechniques, 14(3): 366-369 (1993)). A 5′ primer for the first genomic template and a 3′ fusion oligo are used to amplify the desired region. The fusion primer consists of a 3′ end of the first template (DNA that encodes the amino-proximal polypeptide of the fusion protein), coupled to a 5′ end of the second template (DNA that encodes the carboxy-proximal polypeptide of the fusion protein).
  • The PCR amplifications are performed using Taq DNA polymerase, 10×PCR buffer, and MgCl[0207] 2 (Promega Corp., Madison, Wis.), and Ultrapure dNTPs (Pharmacia, Piscataway, N.J.). One μg of genomic template 1, 5 μl of 10 μM 5′ oligo and 5 μl of 10 μM fusion oligo are combined with the following reagents at indicated final concentrations: 10×Buffer-Mg FREE (1×), MgCl2 (2 mM), dNTP mix (200 μM each dNTP), Taq DNA polymerase (2.5 units), water to bring final volume to 100 μl. A Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.) is used to amplify under the following conditions: 35 cycles at 95° C. for one minute, 55° C. for two minutes, and 72° for three minutes. This procedure results in a “megaprimer”.
  • The resulting megaprimer is run on a 1×TAE, 4% low-melt agarose gel. The megaprimer band is cut from the gel and purified using the Promega Magic PCR Preps DNA purification system. Purified megaprimer is then used in a second PCR step. One μg of [0208] genomic template 2, approximately 0.5 μg of the megaprimer, and 5 μl of 10 μM 3′ oligo are added to a cocktail of 10×buffer, MgCl2, dNTPs and Taq at the same final concentrations as noted above, and brought to 100 μl with water. PCR conditions are the same as above. The fusion product resulting from this amplification is also purified using the Promega Magic PCR Preps DNA purification system.
  • The fusion product is then ligated into TA vector and transformed into [0209] E. coli using the Invitrogen (San Diego, Calif.) TA Cloning Kit. Approximately 50 ng of PCR fusion product is ligated to 50 ng of pCRII vector with 1×Ligation Buffer, 4 units of T4 ligase, and brought to 10 μl with water. This ligated product mixture is incubated at 12° C. overnight (approximately 14 hours). Two μl of the ligation product mixture is added to 50 μl competent INC F′ cells and 2 μl beta mercaptoethanol. The cells are then incubated for 30 minutes, followed by heat shock treatment at 42° C. for 60 seconds, and an ice quenching for two minutes. 450 μl of warmed SOC media is then added to the cells, resulting in a transformed cell culture which is incubated at 37° C. for one hour with slight shaking. 50 μl of the transformed cell culture is plated on LB+50 μg/μl ampicillin plates and incubated overnight at 37° C. Single white colonies are picked and added to individual overnight cultures containing 3 ml LB with ampicillin (50 μg/μl).
  • The individual overnight cultures are prepared using Promega's Magic Miniprep DNA purification system. A small amount of the resulting DNA is cut using a restriction digest as a check. DNA sequencing is then performed to check the sequence of the fusion nucleic acid sequence, using the United States Biochemical (Cleveland, Ohio) Sequenase Version 2.0 DNA sequencing kit. Three to five μg of plasmid DNA is used per reaction. 2 μl 2M NaOH/2mM EDTA are added to the DNA, and the volume is brought to 20 μl with water. The mixture is then incubated at room temperature for five minutes. 7 μl water, 3 μl 3M NaAc, 75 μl ethanol are added. The resultant mixture is mixed by vortex and incubated for ten minutes at −70° C., and then subjected to microcentrifugation. After microcentrifugating for ten minutes, the supernatant is aspirated off, and the pellet is dried in the speed vac for 30 second. 6 μl water, 2 μl annealing buffer, and 2 μl of 10 μM of the appropriate oligo is then added. This mixture is incubated for 10 minutes at 37° C. and then allowed to stand at room temperature for 10 minutes. Subsequently, 5.5 μl of label cocktail (described above) is added to each sample of the mixture, which are incubated at room temperature for an additional five minutes. 3.5 μl labeled DNA is then added to each sample which is then incubated for five minutes at 37° C. 4 μl stop solution is added to each well. The DNA is denatured at 95° for two minutes, and then placed on ice. [0210]
  • Clones with the desired fusion nucleic acid sequences are then recloned in frame in the pET expression system in the lipidated (full length) and non-lipidated (truncated, i.e., without first 17 amino acids) forms. The product is amplified using restriction sites contained in the PCR primers. The vector and product are cut with the same enzymes and ligated together with T4 ligase. The resultant plasmid is transformed into competent [0211] E. coli using standard transformation techniques. Colonies are screened as described earlier and positive clones are transformed into expression cells, such as E. coli BL21, for protein expression with IPTG for induction. The expressed protein in its bacterial culture lysate form and/or purified form is then injected in mice for antibody production. The mice are bled, and the sera collected for agglutination, in vitro growth inhibition, and complement—dependent and —independent lysis tests.
  • A specific example of chimeric OspA is as follows. Other OspA chimeras can be made using the same method with suitable primers. [0212]
  • OspA-K48/OspA-PGau [0213]
  • A chimer of OspA from strain K48 (OspA-K48) and OspA from strain PGau (OspA-PGau) was generated using the method described above. This chimeric nucleic acid sequence included bp 1-654 from OspA-K48, followed by bp 655-820 from OspA-PGau. Primers used included: the amino-terminal sequence of OspA primer #607 (SEQ ID NO: 16); the fusion primer, 5′-AAAGTAGAAGTTTTTGAATCCCATTTTCCAGTTTTTTT-3′ (minus strand primer #668-654) (SEQ ID NO: 27); the carboxy-terminal sequence of OspA primer #586 (SEQ ID NO: 19); and the sequence primers #369 (SEQ ID NO: 14) and #357 (SEQ ID NO: 15). The chimeric nucleic acid sequence is presented as SEQ ID NO: 28; the chimeric protein encoded by this chimeric nucleic acid sequence is presented as SEQ ID NO: 29. [0214]
  • C. Purification of Proteins Generated by Chimeric Nucleic Acid Sequences [0215]
  • The chimeric nucleic acid sequences described above, as well as chimeric nucleic acid sequences produced by the methods described above, are used to produce chimeric proteins encoded by the nucleic acid sequences. Standard methods, such as those described above in Example 3, concerning the expression of proteins from Borrelia genes, can be used to express the proteins in a compatible host organism. The chimeric proteins can then be isolated and purified using standard techniques. [0216]
  • Nucleic acid encoding altered versions of OspA can be used to generate OspA chimeras. In addition, nucleic acid encoding OspA chimeras can be used to generate the altered OspA polypeptides of the present invention. [0217]
  • If the chimeric protein is soluble, it can be purified on a Sepharose column. Insoluble proteins can be solubilized in guanidine and purified on a Ni[0218] 2+ column; alternatively, they can be solubilized in 10 mM NaPO4 with 0.1-1% TRIXON X 114, and subsequently purified over an S column (Pharmacia). Lipidated proteins were generally purified by the latter method. Solubility was determined by separating both soluble and insoluble fractions of cell lysate on a 12% PAGE gel, and checking for the localization of the protein by Coomassie staining, or by Western blotting with monoclonal antibodies directed to an antigenic polypeptide of the chimeric protein.
    • Example 5 Generation of OspC/OspA Chimeric Nucleic Acids and Chimeric Proteins
  • A. General Protocol for Creation of Chimeric Nucleic Acid Sequences [0219]
  • A large number of chimeric nucleic acid sequences encoding proteins comprising at least a first and a second polypeptide from [0220] Borrelia burgdorferi were generated. These chimeric nucleic acid sequences were produced such that the encoded chimeric protein comprised a Borrelia burgdorferi OspC polypeptide upstream of (or N-terminal to) a Borrelia burgdorferi OspA polypeptide. The chimeric nucleic acid sequences were also produced such that the nucleic acid encoding one polypeptide was in the same reading frame as the nucleic acid sequence encoding the next polypeptide in the chimeric protein.
  • The general cloning strategy used to construct the chimeric nucleic acid sequences was as follows. The desired fragment of OspC was amplified using a 5′ primer containing a restriction site suitable for cloning the resultant product into a vector of interest and a 3′ primer containing a restriction site suitable for ligating the OspC fragment to the OspA fragment. The OspC product was cloned into a suitable vector. For-the OspA portion of the chimeric nucleic acid, the desired OspA fragment was amplified using a 5′ primer containing a restriction site for ligating the resultant OspA fragment to the OspC fragment and a 3′ primer containing a restriction site suitable for cloning the resultant OspA product into the vector with the OspC product. The use of a restriction site to allow ligation of the OspC and OspA fragments results in the insertion of 0 to about 3 amino acids between the OspC and OspA fragments. [0221]
  • A specific example of such a construction follows. It is understood that other suitable restriction sites could be used with no or only routine experimentation. The resultant OspC/OspA Chimer could have, therefore, the addition of 0 to about 3 amino acids or more between the OspC and OspA fragments, depending on the restriction site used. [0222]
  • For the OspC portions of the chimeric nucleic acids, desired fragments of OspC genes from various strains or genospecies were PCR amplified using a 5′ primer containing an NdeI site and a 3′ primer containing a NcoI site and a BamHI site. The amplified OspC product was then cloned into the NdeI and BamHI sites of the T7 promoter driven expression vector, pET9c. For the OspA portion of the chimeric nucleic acid, desired fragments of OspA genes from a strain of interest or genospecies of interest were PCR amplified using a 5′ primer containing an NcoI site and a 3′ primer containing a BamHI site. This OspA portion could then be directly cloned into the NcoI and BamHI sites of the pET9c vector containing the desired OspC sequence, thereby producing the desired OspC-OspA construct. By including the sequence for the NcoI restriction site in the primers, a nine nucleotide linker sequence encoding the amino acids Ser-Met-Ala was produced at the junction between the N-terminal OspC sequence and the C-terminal OspA sequence. The use of the NcoI restriction enzyme (CCATGG) in this cloning strategy was a suitable choice as Borrelia is an AT-rich organism which possesses only a few NcoI sites in its genome. One of ordinary skill in the art would know that different restriction sites wold be utilized and would know how to generate the chimeric OspC/A constructs for use in subsequent alterations described herein with no or only routine experimentation. [0223]
  • As an example, OspC-OspA chimeric nucleic acids which contain nonlipidated OspC B31 were generated using the following primers: [0224]
    (5′OspC-NdeI): 5′-GT CAT ATG GCT TGT AAT AAT TCA GGG AAA GA-3′; and (SEQ ID NO:91)
    (3′OspC-NcoI): 5′-T TTC CAT GGA AGG TTT TTT TGG ACT TTC TG-3′. (SEQ ID NO:92)
  • For OspC-OspA chimeric nucleic acids which contain nonlipidated OspA B31, the following primers were used: [0225]
    (5′OspA-NcoI:) 5′-TT TCC ATG GCC AAG CAA AAT GTT AGC AGC C-3′; and (SEQ ID NO:93)
    (3′OspA-BamHI): 5′-TAA GGA TCC TTA TTT TAA AGC GTT TTT-3′. (SEQ ID NO:94)
  • Lipidated versions of the OspC/OspA chimeras can be constructed by designing primers to amplify the a portion of the template that includes the lipidation signal sequence or by generating a nucleic acid construct with a suitable lipidation signal sequence. The leader sequence comprising a lipidation signal can be, for example, from a gene encoding the OspA, OspB or OspC polypeptides. [0226]
  • B. Protein Expression [0227]
  • As described in the previous examples, it is possible to express and purify Borrelia proteins or polypeptides, for example, OspA polypeptides, OspC polypeptides, chimeric OspC/OspA polypeptides and polypeptides comprising the OspA alterations described herein. This is accomplished by incorporating the desired nucleic acid sequence, which encodes the protein of choice, into an expression plasmid, for example, using standard techniques. This expression plasmid can then be transfected into a compatible host cell in order to express the desired protein. [0228]
  • For example, the purified OspA, OspC or OspC/OspA chimeric proteins that were used to immunize mice and in the ELISA tests described below, were generated and purified by cloning either OspA, OspC or OspC/OspA chimeric nucleic acid sequences, in frame, into the pET expression plasmid. The expression plasmid was then transfected into the compatible expression cell line [0229] Escherichia coli strain BL21 (pLysS) or strain B834 (DE3). The BL21 or B834 cells were grown in 10 mL LB media (5 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract, 25 mg/L chloramphenicol and 50 mg/L ampicillin) at 37° C., with shaking. When the optical density at 600λ reached 0.3-0.4 units, recombinant protein expression was induced by adding IPTG (isopropyl B-D-thiogalactopyranoside) to a final concentration of 0.5 mM and the cells were grown for an additional three hours. The cultures were harvested by centrifugation at 3800×g for five minutes. The cells were resuspended in 20 mM NaPO4, pH 7.7, and stored at −20° C. overnight. Once thawed, the crude extracts were incubated with DNase (2 μg/mL) in the presence of 2.5 mM MgCl2 at room temperature for thirty minutes and then spun at 14,000 rpm (Eppendorf 5417C) for five minutes.
  • To purify the OspC proteins described below, the crude extracts from the OspC-expressing cells were loaded onto an anion exchange column (Q Sepharose Fast Flow, 2.2×10 cm, Pharmacia) which had been pre-equilibrated with 20 mM Tris-Cl, pH 9.3. The column was washed in the same buffer (20 mM Tris-Cl, pH 9.3) which eluted the OspC protein. The wash fractions that contained OspC were concentrated using Amicon 10K and then were dialyzed with a solution containing 20 mM NaPO[0230] 4, pH 8.0, and 250 mM NaCl. The partially purified OspC was then passed over a Ni2+ metal affinity column (Chelating Sepharose Fast Flow 2.2×10 cm, Pharmacia) equilibrated with 20 mM NaPO4, pH 8.0, and 250 mM NaCl. The column was washed using a decreasing pH gradient of 20 mM sodium acetic acid and 250 mM NaCl and the bound OspC eluted around pH 5.7. The OspC fractions were then concentrated by ultrafiltration and stored at −70° C.
  • For purification of OspA proteins, the same procedure was followed, except that the dialysis step, after the Amicon 10K cutoff, was done in 20 mM NaPO[0231] 4, pH 6.0. The partially purified OspA was then applied to a cation exchange column (S Sepharose Fast Flow 2.2×10 cm, Pharmacia) equilibrated with 20 nM NaPO4, pH 6.0. The column was washed using an increasing NaCl gradient from 0 to 100 mM. The OspA-containing fractions were concentrated by ultrafiltration and stored at −70° C.
  • As previously indicated, both lipidated and non-lipidated (e.g., truncated, lacking the first 17 amino acids) forms of OspC, OspA and OspC/OspA chimeric proteins were generated. [0232]
  • The above-described techniques were also used to express proteins comprising the altered OspA polypeptides of the present invention. [0233]
  • C. Immunization of Mice and Serologic Characterization Using ELISA (Enzyme-Linked Immunosorbent Assay) [0234]
  • Immunization of Mice [0235]
  • Mice, either C3H-J or ICR, were immunized with 3 μg of lipidated OspC/OspA chimeric protein or 6 μg of non-lipidated OspC/OspA chimer in 100 mL of aluminum hydroxide adjuvant (concentration of 1.8 mg/mL) by (SC) subcutaneous injection. As a negative control, mice were immunized with 100 mL of aluminum hydroxide adjuvant only. All mice received a total of three injections which were given at two week intervals. One week after the final immunization, blood was drawn from each mouse (including negative control mice) and the serum was tested for IgG reactivity using the ELISA method described below, for the presence of anti-OspA antibodies to three different purified OspA proteins ([0236] Borrelia burgdorferi sensu stricto (B31), Borrelia garinii (K48) and Borrelia afzelii (PGau). The sera was tested at a dilution of 1:1000.
  • Mice were immunized with the chimeric proteins described in Table III. [0237]
    TABLE III
    Chimeric Proteins Used to Immunize Mice
    SEQ SEQ
    ID NO: ID NO:
    Description (nucleic (poly-
    Name (amino acid) acid) peptide) FIG. No:
    OspA OspA-B31(18-273)  6  7 22, 23
    OspC OspC-B31(19-211) * * 22, 23
    OspC2- OspC-C2(19-204)/ 59 60 22, 23
    OspA OspA-B31(18-273)
    1lipOspAP/ OspA-PGau(1-217)/ 49 50 24, 25
    Bo OspA-Bo(218-273)
    1lipOspAB/P OspA-B31(1-216)/ * * 24, 25
    OspA-Pko(217-273)
    OspC- OspC-B31(19-211)/ 65 66 24, 25,
    OspAB/P OspA-B31(18-216)/ 27, 28, 29
    OspA-Pko(217-273)
    OspCB31- OspC-B31(19-211)/ 55 56 26, 27,
    OspAB31 OspA-B31(18-273) 28, 29
    OspC2- OspC-C2(19-204)/ 59 60 26, 27
    OspAB31 OspA-B31(18-273)
    1lip OspA OspA-K48(1-217)/ * * 27
    K/T OspA-Tro(218-273)
    1lip OspC- OspC-B31(1-211) * * 26
    B31
    OspCB31- OspC-B31(19-211)/ 87 88 28, 29
    OspABPBP OspA-B31(30-150)/
    OspA-Pko(151-179)/
    OspA-B31(180-216)
    (190 N deletion)/
    OspA-Pko(217-273)
    B31/B31/Pko
  • Immobilization of Antigen Onto ELISA Plates [0238]
  • A solution of purified recombinant OspC or OspA protein from each of the [0239] Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii) was added to sodium phosphate buffer, pH 9.0, and was used to coat a commercial microwell plate (MaxiSorp®, Nunc). The coating procedure was as follows: 100 μl of a solution containing the appropriate OspA or OspC protein (made up at a concentration of 250 ng/ml in the following coating buffer: 100 mM Bis-Tris propane, pH 9.7) was added to each well of a microtiter plate which was incubated for one hour at 37° C. The antigen solution was removed from the wells, the plate was washed three times with phosphate buffered saline (PBS) pH 9.0, and 300 μl of blocking buffer solution was added (3% dry milk, 0.1% polyoxyethylenesorbitan (referred to herein as Tween 20™), 0.02% NaN3 in 100 nM Bis-Tris propane, pH 9.7). Following a one hour incubation at 37° C., the plates were washed four times with TBS-Tween 20™ wash buffer (20 mM Tris-Cl, pH 7.5, 136 mM NaCl, 0.1% Tween 20™ and 0.02% NaN3) and then were allowed to dry. The plates were then wrapped in plastic and stored at 4° C. until they were used.
  • ELISA (Enzyme-Linked Immunosorbent Assay) Tests [0240]
  • The standard procedure for the ELISA tests was as follows: mouse serum was diluted 1:1000 in sample dilution buffer (1% dry milk, 136 mM NaCl, 0.1[0241] % Tween 20™, 0.02% NaN3 in 20 mM Tris-Cl, pH7.5) and 100 μl of the diluted serum was added to the ELISA microtiter plate wells that had been coated with antigen as described above. Following incubation for 1 hour at 37° C., the samples were removed and the plates were washed four times in TBS-Tween™ (20 mM Tris-Cl, pH 7.5; 136 mM NaCl; 0.1% Tween 20™ and 0.02% NaN3). For the secondary antibody, goat anti-mouse antisera conjugated to alkaline phosphatase-specific for either IgM (Fc) or IgG (Fab), (Jackson Immuno Research Laboratories) was diluted 1:750 in sample dilution buffer (1% dry milk, 136 mM NaCl, 0.1% Tween 20™, 0.02% NaN3 in 20 mM Tris-Cl, pH7.5) and 100 μl of the diluted secondary antibody was added to each well. Following incubation for thirty minutes at 37° C., the plates were washed three times with TBS-Tween™ and 100 μl of Phosphatase Substrate solution (5 mg of p-nitrophenylphosphate tablets dissolved in 1×diethanolamine substrate buffer to yield a 2 mg/ml solution—Kirkegaard Perry Laboratory) was added to each well. The plates were incubated for thirty minutes at 37° C. and 100 μl of stop solution (5% EDTA) was added to each well. The absorbance at 405 nm was read on a microplate reader (Dynatech). A sample was considered positive if it produced an average absorbance greater than the mean of the negative controls plus three standard deviations.
  • Previous work has demonstrated that it is the carboxy-terminal region of OspA that contains the antigenic sites that provide the immunoprotective response. Thus, in addition to the ELISA test described above, a modified ELISA was performed (herein referred to as the Protective ELISA Test), wherein the purified N-terminal region of B31 OspA (amino acids 18-139) was used in excess to block any antibodies present in the mouse serum that had specificity to this N-terminal OspA region. These protective ELISA tests were performed as above, except that 80 μg/ml of a purified B31 OspA fragment (amino acids 18-139) was added to the diluted mouse serum prior to adding the sera to the antigen-coated ELISA microtiter plate wells. [0242]
  • Results of ELISA Tests [0243]
  • Using the above-described ELISA tests, it was demonstrated that mice immunized with a non-lipidated OspC/OspA chimeric protein (OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a. 18-273 from strain B31) (SEQ ID NO: 60) produced an immune response both to OspA and OspC that was comparable to the immune response generated to non-lipidated OspA (OspA—a.a. 18-273 from strain B31) and non-lipidated OspC (OspC—a.a. 19-211 from strain B31) control proteins (FIG. 22). As indicated in FIG. 22 and described above, mice were immunized with OspA, OspC or OspC2-OspA proteins and immune responses of the sera were measured against B31 OspA antigen (stippled bars) and B31 OspC antigen (solid bars). [0244]
  • Using the above-described Protective ELISA Test, it was also shown that mice immunized with the same non-lipidated OspC/OspA chimeric protein (OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a. 18-273 from strain B31) (SEQ ID NO: 60) produced an immune response to the C-terminal portion of OspA that was comparable to the immune response generated to the C-terminal portion of a non-lipidated OspA (OspA—a.a. 18-273 from strain B31) control protein (FIG. 23). As indicated in FIG. 23, mice were immunized with OspA, OspC or OspC2-OspA proteins and immune responses of the sera were measured against B31 OspA antigen. The protective antibody response to B31 OspA antigen is indicated in the stippled bars. [0245]
  • Thus, these results clearly demonstrate that non-lipidated chimeric OspC/OspA proteins are able to induce immune responses in mice that are comparable to the immune response generated against non-lipidated OspC and OspA control proteins. [0246]
  • It had been previously thought that the lipidation signals that are present on [0247] Borrelia burgdorferi outer surface proteins were required for immunogenicity and that OspC and OspA proteins that lacked this lipidation signal would be less or non-immunogenic. To test this idea, mice were immunized with a non-lipidated OspC/OspA chimeric protein (OspC-OspAB/P—composed of OspC (a.a. 19-211 from strain B31)/OspA (a.a. 18-216 from strain B31)/OspA (a.a. 217-273 from strain Pko)(SEQ ID NO: 66) as well as two lipidated OspA proteins, lipOspAP/Bo (composed of OspA (a.a. 1-217 from strain PGau)/OspA (a.a. 218-273 from strain Bo)) and lipOspAB/P (composed of OspA (a.a. 1-216 from strain B31)/OspA (a.a. 217-273 from strain Pko)) and were subjected ELISA tests. Mice immunized with the non-lipidated OspC/OspA chimeric protein (OspC-OspAB/P) produced an immune response to OspA from each of the Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii), that was equivalent or greater than the immune response generated to the two lipidated OspA control proteins (lipOspAP/Bo and lipOspAb/P) (FIG. 24).
  • Similar results to these were obtained using the Protective ELISA Test described above. Mice immunized with the non-lipidated OspC/OspA chimeric protein (OspC-OspAB/P) produced an immune response to the C-terminal region of OspA from each of the [0248] Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii), that was equivalent or greater than the immune response generated to the C-terminal region of OspA from the two lipidated OspA control proteins (lipOspAP/Bo and lipOspAb/P) (FIG. 25).
  • In addition to the comparisons between non-lipidated OspC/OspA chimeric proteins and lipidated OspA control proteins, experiments were also performed to compare non-lipidated OspC/OspA chimeric proteins with a lipidated OspC control protein (FIG. 26). Mice that were immunized with either the non-lipidated OspC/OspA chimeric protein OspCB31-OspAB31 (composed of OspC (a.a. 19-211 from strain B31)/OspA (a.a. 18-273 from strain B31) (SEQ ID NO: 56) or the non-lipidated OspC/OspA chimeric protein OspC2-OspAB31 (composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a. 18-273 from strain B31) (SEQ TD NO: 60) produced an immune response to OspC derived from the [0249] Borrelia burgdorferi strain B31 that was comparable to the immune response produced by a lipidated OspC control protein (lip OspC-B31−composed of OspC (a.a. 1-211 from strain B31)) (FIG. 26).
  • Thus, these results clearly demonstrate that non-lipidated chimeric OspC/OspA proteins are able to induce immune responses against OspA and OspC that are comparable to the immune response generated against OspA and OspC using lipidated OspA or OspC control proteins. The use of unlipidated forms of these proteins as vaccine immunogens or diagnostic antigens is highly desirable because the product yield is much greater and the proteins are much easier to purify. For these reasons, the production of these proteins is less expensive. [0250]
  • The OspC/OspA chimeric proteins of the present invention are also able to generate immune responses against OspA proteins that are derived from strains that are not represented in the chimeric protein. Mice immunized with the OspC/OspA chimeric proteins, OspCB31-OspAB31 (SEQ ID NO: 56) and OspC2-OspAB31 (SEQ ID NO: 60), are not only able to generate immune responses that recognize OspA derived from strain B31 ([0251] Borrelia burgdorferi sensu stricto), but also recognize OspA derived from strain K48 (Borrelia garinii) and strain PGau (Borrelia afzelii) (FIG. 27). For comparison, mice were also immunized with the lipidated OspA chimeric protein, Lip OspA K/T (composed of OspA (a.a. 1-217 from strain K48)/OspA (a.a. 218-273 from strain Tro)) (FIG. 27).
  • Additional antibody responses to OspA derived from strain B31 ([0252] Borrelia burgdorferi sensu stricto), strain K48 (Borrelia garinii) and strain PGau (Borrelia afzelii) are also presented for sera from mice immunized with other OspC/OspA chimeric proteins. Thus, FIG. 28 presents the ELISA results from mice immunized with either OspCB31-OspAB/P (SEQ ID NO: 66), OspCB31-OspABPBP (SEQ ID NO: 88) or OspCB31-OspAB31 (SEQ ID NO: 56). In each case, sera from the immunized mice was tested against OspA derived from each of strain B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii). In all cases, a strong immune response was generated (FIG. 28). As with the previously described OspC/OspA chimeric proteins, the three OspC/OspA chimeric proteins used to immunize the mice in FIG. 27 also elicited a strong immune response to the C-terminal region of OspA when examined using the Protective ELISA Test described above (FIG. 29).
  • The above-described techniques are also used to immunize mice and to serologically characterize the immune response against the proteins comprising the altered OspA polypeptides of the present invention. [0253]
  • Tick Challenge of Immunized Mice [0254]
  • Mice, either C3H-J or JCR, that had been immunized as described above, were also challenged with either laboratory-infected nympha or field nympha. The immunized mice were placed in isolation cages and each mouse received 5-10 nymphs. All of the nymphs were collected at counted after 6 days. Four week after challenge, the mice were bled and sera was tested using commercially-available Western blot strips to [0255] Borrelia burgdorferi sensu stricto strain B31 (MarDx strips) and/or Borrelia garinii (MRL strips). Eight weeks after challenge, the mice were bled, sera was tested again by Western blot and ear punch and bladder samples were cultured. As a positive control, mice which had been immunized with only aluminum hydroxide adjuvant, as described above, were subjected to the same challenge.
  • The results of the tick challenge studies (Table IV) demonstrate that while immunization with lipidated OspC protein was unable to protect the mice, as evidenced by a positive Western blot signal (in 4 out of 5 mice), immunization with two different OspC/OspA chimeric proteins (SEQ ID NO: 56 and SEQ ID NO: 62) did provide protection, as indicated by the absence of Western blot signal (in 0 out of 8 mice and 0 out of 3 mice) (Table IV). The sham positive control showed that the challenge by the ticks was successful in all cases, as evidenced by 100% positive signal in Western blots (Table IV). Results from the tick challenge experiments are shown in Table IV. [0256]
    TABLE IV
    Effect of Vaccination on Transmission of Borrelia from Ticks
    Seroconversion Seroconversion
    Vaccine (Western Blots) (Western Blots)
    Candidate Mouse Tick-nymph Vaccinated Sham
    OspC1- C3H-J Long Island 0+/8 8+/8
    OspAB31
    OspC2- C3H-J Long Island 0+/3 4+/4
    OspAB31
    Lip OspC12 ICR Long Island 4+/5 5+/5
  • The above-described techniques are also used to measure the ability of mice immunized with proteins comprising the altered OspA polypeptides of the present invention to resist or respond to transmission of Borrelia from ticks. [0257]
    • Example 6 Generation of OspA M1, M2, M3, J1, J2 and J3 constructs using site directed mutagenesis and PCR
  • All constructs were made using Stratagene's QuikChange site-directed mutagenesis kit. Site-directed mutagenesis was performed using Pfu Turbo DNA polymerase II and a thermal temperature cycler. Pfu Turbo DNA polymerase replicates both plasmid strands with high fidelity and without displacing the mutant oligonucleotide primers. The basic procedure used a supercoiled double-stranded DNA vector (pET 9c for all constructs) with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation(s). The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during the temperature cycling by the Pfu Turbo DNA polymerase. Incorporation of the oligonucleotide primers created a mutated plasmid which contained staggered nicks. Following temperature cycling, the linear product was treated with the restriction enzyme Dpn I, which is specific for methylated DNA. DNA isolated from most [0258] E. coli strains is dam methylated and therefore is susceptible to Dpn I digestion. Digestion with Dpn I therefore destroyed the original template DNA leaving only the nicked plasmid DNA (which was not methylated) containing the desired mutation(s). This nicked vector DNA (containing the desired mutation(s)) was then transformed into competent E. coli which were plated on antibiotic-containing plates. Colonies containing the plasmid (which encodes for antibiotic resistance in addition to the altered OspA polypeptide) were grown and plasmids were purified and sequenced to confirm that they possessed the desired mutation(s).
  • 1) M1, M2 and M3 Mutants [0259]
  • The mutations described herein are made using nucleic acids (e.g., polynucleotides) encoding OspA polypeptides or fragments from any strain of Lyme Disease-causing Borrelia, such as [0260] Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii. As an example, the generation of the M3 mutations in an OspA chimera, called “BPBP”, is described below. The creation of the construct OspA BPBP M3 was as follows. BPBP is a chimeric OspA polypeptide, wherein residues 1-164 are from OspA of B31, residues 165-179 are from OspA of Pko or PGau, residues 180-216 are from OspA of B31 and residues 217-273 are from OspA of Pko (wherein the numbering is as shown in SEQ ID NO: 7).
  • The first step in creating this construct was to make the construct BPBP M1. The M1 mutation consists of a mutation of codon 139 (aga) of OspA from the amino acid arginine to the amino acid methionine (codon atg). A PCR reaction was set up as described above containing a polynucleotide encoding BPBP as the template DNA and the following oligonucleotide primers: [0261]
  • a) 5′ R139M: [0262]
  • 5′ gaa aaa ata ata aca atg gca gac gga [0263] acc 3′ (SEQ ID NO: 117).
  • b) 3′ R139M: [0264]
  • 5′ ggt tcc gtc tgc cat tgt tat [0265] tat ttt ttc 3′ (SEQ ID NO: 118)
  • The PCR reaction also contained reaction buffer, 10 ng of the BPBP DNA template, 125 ng of each oligonucleotide primer, and the dNTP mix. The parameters for the PCR were as outlined in Table V. [0266]
    TABLE V
    Parameters for PCR
    Segment cycles temp time
    1  1 95° C. 30 sec.
    2 18 95° C. 30 sec.
    3 50° C.  1 min.
    4 68° C. 12 min.
  • Following PCR, the product was transformed into competent [0267] E. coli XL1-Blue cells. Colonies formed on the selective agar plates (containing kanamycin) were grown and plasmids were purified and sequenced to confirm that they possessed the desired mutation. The construct BPBP MI was then used as template to make the plasmid BPBP M2, which in addition to the R139M mutation, also contains a mutation that changes the glutamic acid codon (gag) at position 160 to the codon (tat) which encodes for tyrosine. The oligonucleotide primers used to generate this plasmid were:
  • a) 5′ E160Y [0268]
  • 5′ gga aaa gct aaa tat gtt tta [0269] aaa ggc 3′ (SEQ ID NO: 119)
  • b) 3′ E160Y [0270]
  • 5′ gcc ttt taa aac ata ttt [0271] age ttt tcc 3′ (SEQ ID NO: 120)
  • The parameters for the PCR were as outlined in Table VII. The final mutation, M3, which alters a lysine residue at position 189 to a methionine residue was performed using BPBP M2 as a DNA template. The oligonucleotide primers used to generate this plasmid were: [0272]
  • a) 5′ K189M [0273]
  • 5′ gtt act tta age atg aat att [0274] tca aaa tc 3′ (SEQ ID NO: 121)
  • b) 3′ K189M [0275]
  • 5′ ga ttt tga aat att cat get taa agt [0276] aac 3′ (SEQ ID NO: 122)
  • This final mutation yielded the desired construct, BPBP M3. [0277]
  • [0278] 2) J1, J2 and J3 Mutants
  • J1, J2 and J3 mutants were made using the same protocol described for the generation of the M1, M2 and M3 mutants. Such mutants can be made using nucleic acids (e.g., polynucleotides) encoding OspA polypeptides fragments from any strain of Lyme Disease-causing Borrelia, such as [0279] Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii.
  • The oligonucleotide primers which were used to generate the J1 mutation (which contains a Y165F mutation (codon tat to ttt) and a V166T mutation (codon gtt to act)) were as follows: [0280]
  • a) 5′ B31 YV-FT [0281]
  • 5′ gag gtt tta aaa ggc ttt act ctt gaa [0282] gga act c 3′ (SEQ ID NO: 123)
  • b) 3′ B31 YV-FT [0283]
  • 5′ gag ttc ctt caa gag taa agc ctt tta [0284] aaa cct g 3′ (SEQ ID NO: 124)
  • The oligonucleotide primers which were used to generate the J2 mutation (which contains a T170K mutation (codon act to aag)) were as follows: [0285]
  • a) 5′B31 T-K [0286]
  • 5′ tct tga agg aaa gct [0287] aac tgc tg 3′ (SEQ ID NO: 125)
  • b) 3′ B31 T-K [0288]
  • 5′ cag cag tta gct ttc [0289] ctt caa ga 3′ (SEQ ID NO: 126)
  • To generate the J3 mutant (which contains a Y165F mutation (codon tat to ttt), a V166T mutation (codon gtt to act) and a T170K mutation (codon act to aag)), the template containing the J1 mutations was used with the oligonucleotide primers for generating the J2 mutation (5′ B31 T-K (SEQ ID NO: 125) and 3′ B31 T-K (SEQ ID NO: 126)). [0290]
  • The altered OspA polypeptides described herein are expressed, used to immunize mice and characterized using ELISA as described in the Experiments above. [0291]
  • While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. [0292]
  • 1 130 1 23 DNA Artificial Sequence Oligonucleotide Primer 1 cttaatgact ctgacactag tgc 23 2 30 DNA Artificial Sequence Oligonucleotide Primer 2 gctactaaaa aaaccgggaa atggaattca 30 3 33 DNA Artificial Sequence Oligonucleotide Primer 3 gcagcttggg attcaaaaac atccacttta aca 33 4 18 DNA Artificial Sequence Oligonucleotide Primer 4 ggagaatata ttatgaaa 18 5 17 DNA Artificial Sequence Oligonucleotide Primer 5 ctccttattt taaagcg 17 6 822 DNA Borrelia burgdorferi CDS (1)...(822) 6 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 7 273 PRT Borrelia burgdorferi 7 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 8 825 DNA Borrelia burgdorferi CDS (1)...(825) 8 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta agc aag aac att 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 tta aaa tcc gga gaa ata aca gtt gca ctt gat gac tct gac act act 624 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 cag gct act aaa aaa act gga aaa tgg gat tca aaa act tcc act tta 672 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser Thr Leu 210 215 220 aca att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa 720 Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys 225 230 235 240 gaa gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta 768 Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 gaa ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct 816 Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala 260 265 270 tta aaa taa 825 Leu Lys * 9 274 PRT Borrelia burgdorferi 9 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser Thr Leu 210 215 220 Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys 225 230 235 240 Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala 260 265 270 Leu Lys 10 822 DNA Borrelia burgdorferi CDS (1)...(822) 10 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa att gca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 aaa tct gga gaa gta aca gtt gct ctt aat gac act aac act act cag 624 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 gct act aaa aaa act ggc gca tgg gat tca aaa act tct act tta aca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaa caa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 tac aca ata act gta aaa caa tac gac tcc gca ggt acc aat tta gaa 768 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 11 273 PRT Borrelia burgdorferi 11 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Lys 12 819 DNA Borrelia burgdorferi CDS (1)...(819) 12 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gct tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggc aag tac agt cta atg gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Met Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga aca tct gat aaa aac aat gga tct ggg gtg ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agc aaa gta aaa tta aca gtt tct gac gat cta agc aca 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Val Ser Asp Asp Leu Ser Thr 85 90 95 acc aca ctt gaa gtt tta aaa gaa gat ggc aaa aca tta gtg tca aaa 336 Thr Thr Leu Glu Val Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa aga act tct aaa gat aag tca tca aca gaa gaa aag ttc aat gaa 384 Lys Arg Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggc gaa tta gtt gaa aaa ata atg gca aga gca aac gga acc ata 432 Lys Gly Glu Leu Val Glu Lys Ile Met Ala Arg Ala Asn Gly Thr Ile 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 act tta aaa gaa tat gtt ctt gaa gga act cta act gct gaa aaa gca 528 Thr Leu Lys Glu Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Ala 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agt aag cac att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys His Ile Ser 180 185 190 aaa tct gga gaa gta aca gct gaa ctt aat gac act gac agt act caa 624 Lys Ser Gly Glu Val Thr Ala Glu Leu Asn Asp Thr Asp Ser Thr Gln 195 200 205 gct act aaa aaa act ggg aaa tgg gat gca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Gly Lys Trp Asp Ala Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac aac aaa aaa act aaa gcc ctt gta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 gac aca att aca tca caa aaa tac gac tca gca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aga 819 Arg 13 273 PRT Borrelia burgdorferi 13 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Met Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Val Ser Asp Asp Leu Ser Thr 85 90 95 Thr Thr Leu Glu Val Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Arg Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Leu Val Glu Lys Ile Met Ala Arg Ala Asn Gly Thr Ile 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Thr Leu Lys Glu Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Ala 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys His Ile Ser 180 185 190 Lys Ser Gly Glu Val Thr Ala Glu Leu Asn Asp Thr Asp Ser Thr Gln 195 200 205 Ala Thr Lys Lys Thr Gly Lys Trp Asp Ala Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Arg 14 21 DNA Artificial Sequence Oligonucleotide Primer 14 gtctgcaaaa accatgacaa g 21 15 22 DNA Artificial Sequence Oligonucleotide Primer 15 gtcatcaaca gaagaaaaat tc 22 16 30 DNA Artificial Sequence Oligonucleotide Primer 16 ccggatccat atgaaaaaat atttattggg 30 17 32 DNA Artificial Sequence Oligonucleotide Primer 17 ccgggatcca tatggctaag caaaatgtta gc 32 18 18 DNA Artificial Sequence Oligonucleotide Primer 18 gcgttcaagt actccaga 18 19 27 DNA Artificial Sequence Oligonucleotide Primer 19 gatatctaga tcttatttta aagcgtt 27 20 31 DNA Artificial Sequence Oligonucleotide Primer 20 ggatccggtg accttttaaa gcgtttttaa t 31 21 825 DNA Borrelia burgdorferi 21 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaa aaatagcgtt tcagtagatt tacctggtgg aatgacagtt 120 cttgtaagta aagaaaaaga caaagacggt aaatacagtc tagaggcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aacggttctg gaacacttga aggtgaaaaa 240 actgacaaaa gtaaagtaaa atcaacaatt gctgatgacc taagtcaaac taaatttgaa 300 attttcaaag aagatggcaa aacattagta tcaaaaaaag taacccttaa agacaagtca 360 tcaacagaag aaaaattcaa cggaaagggt gaaacatctg aaaaaacaat agtaagagca 420 aatggaacca gacttgaata cacagacata aaaagcgatg gatccggaaa agctaaagaa 480 gttttaaaag actttactct tgaaggaact ctagctgctg acggcaaaac aacattgaaa 540 gttacagaag gcactgttgt tttaagcaag aacattttaa aatccggaga aataacagct 600 gcacttgatg actctgacac tactcgggct actaaaaaaa ctggaaaatg ggattcaaag 660 acttccactt taacaattag tgtgaatagc caaaaaacca aaaaccttgt attcacaaaa 720 gaagacacaa taacagtaca aagatacgac tcagcaggca ccaatctaga aggcaaagca 780 gtcgaaatta caacacttaa agaacttaaa aacgctttaa aataa 825 22 824 DNA Borrelia burgdorferi 22 atgaaaaaat atttattggg aataggtcta atattagcat taatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaa aaatagcgtt tcagtagatt tacctggtgg aatgcaagtt 120 cttgtaagta aagaaaaaga caaagatggt aaatacagtc taatggcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aacggttctg gaacacttga aggtgaaaaa 240 actgacaaaa gtaaagcaaa attaacaatt gctgaggatc taagtaaaac cacatttgaa 300 atcttcaaag aagatggcaa aacattagta tcaaaaaaag taacccttaa agacaagtca 360 tcaacagaag aaaaattcaa cgcaaagggt gaagcatctg aaaaaacaat agtaagagca 420 aatggaacca gacttgaata cacagacata aaaagcgata aaaccggaaa agctaaagaa 480 gttttaaaag actttgctct tgaaggaact ctagctgctg acggcaaaac aacattaaaa 540 gttacagaag gcactgttgt tttaagcaaa cacatttcaa actctggaga aataacagtt 600 gagcttaatg actctgacac tactcaggct actaaaaaaa ctggaacatg ggattcaaag 660 acttccactt taacaattag tgtgaatagc cgaaaaacca aaaaccttgt attcacaaaa 720 gaagacacaa taacagtaca aaaatacgac tcagcaggca ccaatctaga aggcaaagca 780 gtcgaaatta caacgcttaa agaacttaaa gatgctttaa aata 824 23 821 DNA Borrelia burgdorferi 23 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtga gatgaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctg gggtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taggtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagcaaa aaaactacac aacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 24 821 DNA Borrelia burgdorferi 24 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtga gattaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctg gagtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taagtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagtaaa aaaactacac aacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 25 821 DNA Borrelia burgdorferi 25 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtga gatgaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctg gggtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taggtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagcaaa aaaactacac aacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 26 822 DNA Borrelia burgdorferi 26 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtga gatgaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctg gagtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taagtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagcaaa aaaactacac aacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttgaaat aa 822 27 38 DNA Artificial Sequence Oligonucleotide Primer 27 aaagtagaag tttttgaatc ccattttcca gttttttt 38 28 825 DNA Artificial Sequence Chimeric nucleic acid 28 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta agc aag aac att 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 tta aaa tcc gga gaa ata aca gtt gca ctt gat gac tct gac act act 624 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 cag gct act aaa aaa act gga aaa tgg gat tca aaa act tct act tta 672 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser Thr Leu 210 215 220 aca att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaa 720 Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys 225 230 235 240 caa tac aca ata act gta aaa caa tac gac tcc gca ggt acc aat tta 768 Gln Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 gaa ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct 816 Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 tta aaa taa 825 Leu Lys * 29 274 PRT Artificial Sequence Chimeric protein 29 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser Thr Leu 210 215 220 Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys 225 230 235 240 Gln Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 Leu Lys 30 822 DNA Artificial Sequence CDS (1)...(822) Chimeric nucleic acid 30 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa att gca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 aaa tct gga gaa gta aca gtt gct ctt aat gac act aac act act cag 624 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 gct act aaa aaa act ggc gca tgg gat tca aaa act tct act tta aca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaa caa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 tac aca ata act gta aaa caa tac gac tcc gca ggt acc aat tta gaa 768 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 31 273 PRT Artificial Sequence Chimeric protein 31 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Lys 32 822 DNA Artificial Sequence Chimeric nucleic acid 32 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 33 273 PRT Artificial Sequence Chimeric protein 33 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 34 819 DNA Artificial Sequence Chimeric nucleic acid 34 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat gca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac aac aaa aaa act aaa gcc ctt gta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 gac aca att aca tca caa aaa tac gac tca gca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aga 819 Arg 35 273 PRT Artificial Sequence Chimeric protein 35 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Arg 36 822 DNA Artificial Sequence Chimeric nucleic acid 36 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta agc aag att tca 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 37 273 PRT Artificial Sequence Chimeric protein 37 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 38 822 DNA Artificial Sequence Chimeric nucleic acid 38 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta agc aag att tca 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 39 273 PRT Artificial Sequence Chimeric protein 39 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 40 822 DNA Artificial Sequence Chimeric nucleic acid 40 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat gac agt act agc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Asp Ser Thr Ser Thr Leu Thr 210 215 220 att agt gct gac agc aaa aaa act aaa gat ttg gtg ttc tta aca gat 720 Ile Ser Ala Asp Ser Lys Lys Thr Lys Asp Leu Val Phe Leu Thr Asp 225 230 235 240 ggt aca att aca gta caa caa tac aac aca gct gga acc agc cta gaa 768 Gly Thr Ile Thr Val Gln Gln Tyr Asn Thr Ala Gly Thr Ser Leu Glu 245 250 255 gga tca gca agt gaa att aaa aat ctt tca gag ctt aaa aac gct tta 816 Gly Ser Ala Ser Glu Ile Lys Asn Leu Ser Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 41 273 PRT Artificial Sequence Chimeric protein 41 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Asp Ser Thr Ser Thr Leu Thr 210 215 220 Ile Ser Ala Asp Ser Lys Lys Thr Lys Asp Leu Val Phe Leu Thr Asp 225 230 235 240 Gly Thr Ile Thr Val Gln Gln Tyr Asn Thr Ala Gly Thr Ser Leu Glu 245 250 255 Gly Ser Ala Ser Glu Ile Lys Asn Leu Ser Glu Leu Lys Asn Ala Leu 260 265 270 Lys 42 822 DNA Borrelia burgdorferi 42 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgacgagaa aaacagcgtt tcagtagatt tgcctggtga aatgaaagtt 120 cttgtaagca aagaaaaaaa caaagacggc aagtacgatc taattgcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aatggatctg gagtacttga aggcgtaaaa 240 gctgacaaaa gtaaagtaaa attaacaatt tctgacgatc taggtcaaac cacacttgaa 300 gttttcaaag aagatggcaa aacactagta tcaaaaaaag taacttccaa agacaagtca 360 tcaacagaag aaaaattcaa tgaaaaaggt gaagtatctg aaaaaataat aacaagagca 420 gacggaacca gacttgaata cacaggaatt aaaagcgatg gatctggaaa agctaaagag 480 gttttaaaag gctatgttct tgaaggaact ctaactgctg aaaaaacaac attggtggtt 540 aaagaaggaa ctgttacttt aagcaaaaat atttcaaaat ctggggaagt ttcagttgaa 600 cttaatgaca ctgacagtag tgctgctact aaaaaaactg cagcttggaa ttcaggcact 660 tcaactttaa caattactgt aaacagtaaa aaaactaaag accttgtgtt tacaaaagaa 720 aacacaatta cagtacaaca atacgactca aatggcacca aattagaggg gtcagcagtt 780 gaaattacaa aacttgatga aattaaaaac gctttaaaat aa 822 43 822 DNA Borrelia burgdorferi 43 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgacgagaa aaacagcgtt tcagtagatt tgcctggtga aatgaacgtt 120 cttgtaagca aagaaaaaaa caaagacggc aagtacgatc taattgcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aatggatctg gagtacttga aggcgtaaaa 240 gctgacaaaa gtaaagtaaa attaacaatt tctgacgatc taggtcaaac cacacttgaa 300 gttttcaaag aagatggcaa aacactagta tcaaaaaaag taacttccaa agacaagtca 360 tcaacagaag aaaaattcaa tgaaaaaggt gaagtatctg aaaaaataat aacaagagca 420 gacggaacca gacttgaata cacagaaatt aaaagcgatg gatctggaaa agctaaagag 480 gttttaaaag gctatgttct tgaaggaact ctaactgctg aaaaaacaac attggtggtt 540 aaagaaggaa ctgttacttt aagcaaaaat atttcaaaat ctggggaagt ttcagttgaa 600 cttaatgaca ctgacagtag tgctgctact aaaaaaactg cagcttggaa ttcaggcact 660 tcaactttaa caattactgt aaacagtaaa aaaactaaag accttgtgtt tacaaaagaa 720 aacacaatta cagtacaaca atacgactca aatggcacca aattagaggg gtcagcagtt 780 gaaattacaa aacttgatga aattaaaaac gctttaaaat aa 822 44 822 DNA Borrelia burgdorferi 44 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgacgagaa aaacagcgtt tcagtagatt tgcctggtga aatgaacgtt 120 cttgtaagca aagaaaaaaa caaagacggc aagtacgatc taattgcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aatggatctg gagtacttga aggcgtaaaa 240 gctgacaaaa gtaaagtaaa attaacaatt tctgacgatc taggtcaaac cacacttgaa 300 gttttcaaag aagatggcaa aacactagta tcaaaaaaag taacttccaa agacaagtca 360 tcaacagaag aaaaattcaa tgaaaaaggt gaagtatctg aaaaaataat aacaagagca 420 gacggaacca gacttgaata cacagaaatt aaaagcgatg gatctggaaa agctaaagag 480 gttttaaaaa gctatgttct tgaaggaact ttaactgctg aaaaaacaac attggtggtt 540 aaagaaggaa ctgttacttt aagcaaaaat atttcaaaat ctggggaagt ttcagttgaa 600 cttaatgaca ctgacagtag tgctgctact aaaaaaactg cagcttggaa ttcaggcact 660 tcaactttaa caattactgt aaacagtaaa aaaactaaag accttgtgtt tacaaaagaa 720 aacacaatta cagtacaaca atacgactca aatggcacca aattagaggg gtcagcagtt 780 gaaattacaa aacttgatga aattaaaaac gctttaaaat aa 822 45 821 DNA Borrelia burgdorferi 45 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgatgagaa aaacagcgtt tcagtagatt tacctggtga aatgaaagtt 120 cttgtaagca aagaaaaaga caaagatggt aaatacagtc taatggcaac agtagacaag 180 ctagagctta aaggaacttc tgataaaagc aacggttctg gaacacttga aggtgaaaaa 240 tctgacaaaa gtaaagcaaa attaacaatt tctgaagatc taagtaaaac cacatttgaa 300 attttcaaag aagatggcaa aacattagta tcaaaaaaag taaattctaa agataagtca 360 tcaatagaag aaaaattcaa cgcaaaaggt gaattatctg aaaaaacaat actaagagca 420 aacggaacca ggcttgaata cacagaaata aaaagcgatg gaaccggaaa agctaaagaa 480 gctttaaaag actttgctct tgaaggaact ctagctgccg acaaaacaac attgaaagtt 540 acagaaggca ctgttgtttt aagcaaacac attccaaact ctggagaaat aacagttgag 600 cttaatgact ctaactctac tcaggctact aaaaaaactg gaaaatggga ttcaaatact 660 tccactttaa caattagtgt gaatagcaaa aaaactaaaa acattgtatt tacaaaagaa 720 gacacaataa cagtacaaaa atacgactca gcaggcacca atctagaagg caacgcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 46 821 DNA Borrelia burgdorferi 46 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaa aaatagcgtt tcagtagatt tacctggtgg aatgaaagtt 120 cttgtaagta aagaaaaaga caaagatggt aaatacagtc taatggcaac agtagaaaag 180 cttgagctta aaggaacttc tgataaaaac aacggttctg gaacacttga aggtgaaaaa 240 actgacaaaa gtaaagtaaa attaacaatt gctgaggatc taagtaaaac cacatttgaa 300 atcttcaaag aagatggcaa aacattagta tcgaaaaaag taacccttaa agacaagtca 360 tcaacagaag aaaaattcaa cgaaaagggt gaaatatctg aaaaaacaat agtaagagca 420 aatggaacca gacttgaata cacagacata aaaagcgata aaaccggaaa agctaaagaa 480 gttttaaaag actttactct tgaaggaact ctagctgctg acggcaaaac aacattgaaa 540 gttacagagg gcactgttac tttaagcaag aacatttcaa aatccggaga aataacagtt 600 gcacttgatg acactgactc tagcggcaat aaaaaatccg gaacatggga ttcaggtact 660 tctactttaa caattagtaa aaacagacaa aaaactaaac aacttgtatt cacaaaagaa 720 gacacaataa cagtacaaaa ctacgactca gcaggcacca atctagaagg caaagcagtc 780 gaaattacaa cacttaaaga acttaaaaac gctttaaaat a 821 47 825 DNA Artificial Sequence Chimeric nucleic acid 47 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta agc aag aac att 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 tta aaa tcc gga gaa ata aca gtt gca ctt gat gac tct gac act act 624 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 cag gct act aaa aaa act gga aaa tgg gat tca aat act tcc act tta 672 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu 210 215 220 aca att agt gtg aat agc aaa aaa act aaa aac att gta ttt aca aaa 720 Thr Ile Ser Val Asn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys 225 230 235 240 gaa gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta 768 Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 gaa ggc aac gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct 816 Glu Gly Asn Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 tta aaa tag 825 Leu Lys * 48 274 PRT Artificial Sequence Chimeric protein 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu 210 215 220 Thr Ile Ser Val Asn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys 225 230 235 240 Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 Glu Gly Asn Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 Leu Lys 49 822 DNA Artificial Sequence Chimeric nucleic acid 49 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa att gca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 aaa tct gga gaa gta aca gtt gct ctt aat gac act aac act act cag 624 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 gct act aaa aaa act ggc gca tgg gat tca aaa act tct act tta aca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaa caa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 gac aca ata act gta caa aaa tac gac tcc gca ggt acc aat tta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggt aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aaa tag 822 Lys * 50 273 PRT Artificial Sequence Chimeric protein 50 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Lys 51 822 DNA Artificial Sequence Chimeric nucleic acid 51 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa att tca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 52 273 PRT Artificial Sequence Chimeric protein 52 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 53 822 DNA Artificial Sequence Chimeric nucleic acid 53 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca aat gga acc aaa 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa att tca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 54 273 PRT Artificial Sequence Chimeric protein 54 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 55 1362 DNA Artificial Sequence Chimeric nucleic acid 55 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc ctt gac gag aaa aac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 gaa aaa aac aaa gac ggc aag tac gat cta att gca aca gta gac aag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aat gga tct gga gta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta aca att tct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 cta gta tca aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 gac agt agt gct gct act aaa aaa act gca gct tgg aat tca ggc act 1200 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr 385 390 395 400 tca act tta aca att act gta aac agt aaa aaa act aaa gac ctt gtg 1248 Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val 405 410 415 ttt aca aaa gaa aac aca att aca gta caa caa tac gac tca aat ggc 1296 Phe Thr Lys Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly 420 425 430 acc aaa tta gag ggg tca gca gtt gaa att aca aaa ctt gat gaa att 1344 Thr Lys Leu Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile 435 440 445 aaa aac gct tta aaa taa 1362 Lys Asn Ala Leu Lys * 450 56 453 PRT Artificial Sequence Chimeric protein 56 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr 385 390 395 400 Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val 405 410 415 Phe Thr Lys Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly 420 425 430 Thr Lys Leu Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile 435 440 445 Lys Asn Ala Leu Lys 450 57 1353 DNA Artificial Sequence Chimeric nucleic acid 57 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca gcc 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Ala 180 185 190 atg gcc aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca 624 Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser 195 200 205 gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac 672 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 210 215 220 aaa gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt 720 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 225 230 235 240 aaa gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta 768 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 245 250 255 aaa gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt 816 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 260 265 270 caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca 864 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 275 280 285 aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat 912 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 290 295 300 gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc 960 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 305 310 315 320 aga ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa 1008 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 325 330 335 gag gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa 1056 Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys 340 345 350 aca aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att 1104 Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 355 360 365 tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt 1152 Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 370 375 380 gct gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta 1200 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 385 390 395 400 aca att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa 1248 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys 405 410 415 gaa aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta 1296 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu 420 425 430 gag ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct 1344 Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala 435 440 445 tta aaa taa 1353 Leu Lys * 450 58 450 PRT Artificial Sequence Chimeric protein 58 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Ala 180 185 190 Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser 195 200 205 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 210 215 220 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 225 230 235 240 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 245 250 255 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 260 265 270 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 275 280 285 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 290 295 300 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 305 310 315 320 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 325 330 335 Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys 340 345 350 Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 355 360 365 Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 370 375 380 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 385 390 395 400 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys 405 410 415 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu 420 425 430 Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala 435 440 445 Leu Lys 450 59 1341 DNA Artificial Sequence Chimeric nucleic acid 59 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta gat ttg cct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggc aag 672 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 tac gat cta att gca aca gta gac aag ctt gag ctt aaa gga act tct 720 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa gct gac aaa 768 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 agt aaa gta aaa tta aca att tct gac gat cta ggt caa acc aca ctt 816 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa aaa gta act 864 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa aaa ggt gaa 912 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 gta tct gaa aaa ata ata aca aga gca gac gga acc aga ctt gaa tac 960 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 aca gga att aaa agc gat gga tct gga aaa gct aaa gag gtt tta aaa 1008 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca aca ttg gtg 1056 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 gtt aaa gaa gga act gtt act tta agc aaa aat att tca aaa tct ggg 1104 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct gct act aaa 1152 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 aaa act gca gct tgg aat tca ggc act tca act tta aca att act gta 1200 Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr Ile Thr Val 385 390 395 400 aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa aac aca att 1248 Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu Asn Thr Ile 405 410 415 aca gta caa caa tac gac tca aat ggc acc aaa tta gag ggg tca gca 1296 Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu Gly Ser Ala 420 425 430 gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta aaa taa 1341 Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu Lys * 435 440 445 60 446 PRT Artificial Sequence Chimeric protein 60 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr Ile Thr Val 385 390 395 400 Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu Asn Thr Ile 405 410 415 Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu Gly Ser Ala 420 425 430 Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu Lys 435 440 445 61 1362 DNA Artificial Sequence Chimeric nucleic acid 61 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc ctt gac gag aaa aac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 gaa aaa aac aaa gac ggc aag tac gat cta att gca aca gta gac aag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aat gga tct gga gta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta aca att tct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 cta gta tca aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 gac agt agt gct gct act aaa aaa act gca gct tgg aat tca aaa act 1200 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr 385 390 395 400 tcc act tta aca att agt gtg aat agc caa aaa acc aaa aac ctt gta 1248 Ser Thr Leu Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val 405 410 415 ttc aca aaa gaa gac aca ata aca gta caa aaa tac gac tca gca ggc 1296 Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly 420 425 430 acc aat cta gaa ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt 1344 Thr Asn Leu Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu 435 440 445 aaa aac gct tta aaa taa 1362 Lys Asn Ala Leu Lys * 450 62 453 PRT Artificial Sequence Chimeric protein 62 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr 385 390 395 400 Ser Thr Leu Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val 405 410 415 Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly 420 425 430 Thr Asn Leu Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu 435 440 445 Lys Asn Ala Leu Lys 450 63 1341 DNA Artificial Sequence Chimeric nucleic acid 63 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta gat ttg cct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggc aag 672 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 tac gat cta att gca aca gta gac aag ctt gag ctt aaa gga act tct 720 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa gct gac aaa 768 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 agt aaa gta aaa tta aca att tct gac gat cta ggt caa acc aca ctt 816 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa aaa gta act 864 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa aaa ggt gaa 912 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 gta tct gaa aaa ata ata aca aga gca gac gga acc aga ctt gaa tac 960 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 aca gga att aaa agc gat gga tct gga aaa gct aaa gag gtt tta aaa 1008 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca aca ttg gtg 1056 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 gtt aaa gaa gga act gtt act tta agc aaa aat att tca aaa tct ggg 1104 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct gct act aaa 1152 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 aaa act gca gct tgg aat tca aaa act tcc act tta aca att agt gtg 1200 Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa gac aca ata 1248 Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu Asp Thr Ile 405 410 415 aca gta caa aaa tac gac tca gca ggc acc aat cta gaa ggc aaa gca 1296 Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala 420 425 430 gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta aaa taa 1341 Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys * 435 440 445 64 446 PRT Artificial Sequence Chimeric protein 64 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu Asp Thr Ile 405 410 415 Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala 420 425 430 Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys 435 440 445 65 1362 DNA Artificial Sequence Chimeric nucleic acid 65 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg tca tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Ser Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg gat tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Asp Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc ctt gac gag aaa aac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 gaa aaa aac aaa gac ggc aag tac gat cta att gca aca gta gac aag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aat gga tct gga gta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta aca att tct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 cta gta tca aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 gac agt agt gct gct act aaa aaa act gca gct tgg aat tca aaa act 1200 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr 385 390 395 400 tct act tta aca att agt gtt aac agc aaa aaa act aca caa ctt gtg 1248 Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val 405 410 415 ttt act aaa caa gac aca ata act gta caa aaa tac gac tcc gca ggt 1296 Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly 420 425 430 acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt 1344 Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu 435 440 445 aaa aac gct tta aaa taa 1362 Lys Asn Ala Leu Lys * 450 66 453 PRT Artificial Sequence Chimeric protein 66 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Ser Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Asp Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360 365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370 375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr 385 390 395 400 Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val 405 410 415 Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly 420 425 430 Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu 435 440 445 Lys Asn Ala Leu Lys 450 67 1341 DNA Artificial Sequence Chimeric nucleic acid 67 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta gat ttg cct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggc aag 672 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 tac gat cta att gca aca gta gac aag ctt gag ctt aaa gga act tct 720 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa gct gac aaa 768 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 agt aaa gta aaa tta aca att tct gac gat cta ggt caa acc aca ctt 816 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa aaa gta act 864 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa aaa ggt gaa 912 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 gta tct gaa aaa ata ata aca aga gca gac gga acc aga ctt gaa tac 960 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 aca gga att aaa agc gat gga tct gga aaa gct aaa gag gtt tta aaa 1008 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca aca ttg gtg 1056 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 gtt aaa gaa gga act gtt act tta agc aaa aat att tca aaa tct ggg 1104 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct gct act aaa 1152 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 aaa act gca gct tgg aat tca aaa act tct act tta aca att agt gtt 1200 Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 aac agc aaa aaa act aca caa ctt gtg ttt act aaa caa tac aca ata 1248 Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln Tyr Thr Ile 405 410 415 act gta aaa caa tac gac tcc gca ggt acc aat tta gaa ggc aca gca 1296 Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala 420 425 430 gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta aaa taa 1341 Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys * 435 440 445 68 446 PRT Artificial Sequence Chimeric protein 68 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln Tyr Thr Ile 405 410 415 Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala 420 425 430 Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys 435 440 445 69 1365 DNA Artificial Sequence Chimeric nucleic acid 69 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa tat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa aca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc ctt gat gaa aaa aat 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat tta cct ggt gga atg aca gtt ctt gta agt aaa 672 Ser Val Ser Val Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys 210 215 220 gaa aaa gac aaa gac ggt aaa tac agt cta gag gca aca gta gac aag 720 Glu Lys Asp Lys Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aac ggt tct gga aca ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu 245 250 255 gaa ggt gaa aaa act gac aaa agt aaa gta aaa tta aca att gct gat 816 Glu Gly Glu Lys Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp 260 265 270 gac cta agt caa act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca 864 Asp Leu Ser Gln Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr 275 280 285 tta gta tca aaa aaa gta acc ctt aaa gac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aac gaa aag ggt gaa aca tct gaa aaa aca ata gta aga gca 960 Lys Phe Asn Glu Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala 305 310 315 320 aat gga acc aga ctt gaa tac aca gac ata aaa agc gat gga tcc gga 1008 Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gaa gtt tta aaa gac ttt act ctt gaa gga act cta gct 1056 Lys Ala Lys Glu Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala 340 345 350 gct gac ggc aaa aca aca ttg aaa gtt aca gaa ggc act gtt gtt tta 1104 Ala Asp Gly Lys Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu 355 360 365 agc aag aac att tta aaa tcc gga gaa ata aca gtt gca ctt gat gac 1152 Ser Lys Asn Ile Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp 370 375 380 tct gac act act cag gct act aaa aaa act gga aaa tgg gat tca aat 1200 Ser Asp Thr Thr Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Asn 385 390 395 400 act tcc act tta aca att agt gtg aat agc aaa aaa act aaa aac att 1248 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Lys Asn Ile 405 410 415 gta ttt aca aaa gaa gac aca ata aca gta caa aaa tac gac tca gca 1296 Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 420 425 430 ggc acc aat cta gaa ggc aac gca gtc gaa att aaa aca ctt gat gaa 1344 Gly Thr Asn Leu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu Asp Glu 435 440 445 ctt aaa aac gct tta aaa tag 1365 Leu Lys Asn Ala Leu Lys * 450 70 454 PRT Artificial Sequence Chimeric protein 70 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys 210 215 220 Glu Lys Asp Lys Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu 245 250 255 Glu Gly Glu Lys Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp 260 265 270 Asp Leu Ser Gln Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr 275 280 285 Leu Val Ser Lys Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys Phe Asn Glu Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala 305 310 315 320 Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala 340 345 350 Ala Asp Gly Lys Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu 355 360 365 Ser Lys Asn Ile Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp 370 375 380 Ser Asp Thr Thr Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Asn 385 390 395 400 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Lys Asn Ile 405 410 415 Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 420 425 430 Gly Thr Asn Leu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu Asp Glu 435 440 445 Leu Lys Asn Ala Leu Lys 450 71 1344 DNA Artificial Sequence Chimeric nucleic acid 71 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta gat tta cct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa gac ggt aaa 672 Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys Asp Gly Lys 210 215 220 tac agt cta gag gca aca gta gac aag ctt gag ctt aaa gga act tct 720 Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa act gac aaa 768 Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys Thr Asp Lys 245 250 255 agt aaa gta aaa tta aca att gct gat gac cta agt caa act aaa ttt 816 Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln Thr Lys Phe 260 265 270 gaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa aaa gta acc 864 Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 ctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa aag ggt gaa 912 Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 aca tct gaa aaa aca ata gta aga gca aat gga acc aga ctt gaa tac 960 Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg Leu Glu Tyr 305 310 315 320 aca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa gtt tta aaa 1008 Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 gac ttt act ctt gaa gga act cta gct gct gac ggc aaa aca aca ttg 1056 Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys Thr Thr Leu 340 345 350 aaa gtt aca gaa ggc act gtt gtt tta agc aag aac att tta aaa tcc 1104 Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile Leu Lys Ser 355 360 365 gga gaa ata aca gtt gca ctt gat gac tct gac act act cag gct act 1152 Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr Gln Ala Thr 370 375 380 aaa aaa act gga aaa tgg gat tca aat act tcc act tta aca att agt 1200 Lys Lys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu Thr Ile Ser 385 390 395 400 gtg aat agc aaa aaa act aaa aac att gta ttt aca aaa gaa gac aca 1248 Val Asn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys Glu Asp Thr 405 410 415 ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa ggc aac 1296 Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Asn 420 425 430 gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta aaa tag 1344 Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys * 435 440 445 72 447 PRT Artificial Sequence Chimeric protein 72 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys Asp Gly Lys 210 215 220 Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys Thr Asp Lys 245 250 255 Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln Thr Lys Phe 260 265 270 Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg Leu Glu Tyr 305 310 315 320 Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys Thr Thr Leu 340 345 350 Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile Leu Lys Ser 355 360 365 Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr Gln Ala Thr 370 375 380 Lys Lys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu Thr Ile Ser 385 390 395 400 Val Asn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys Glu Asp Thr 405 410 415 Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Asn 420 425 430 Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys 435 440 445 73 1305 DNA Artificial Sequence Chimeric nucleic acid 73 atg gct tgt aat aat tca gga aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att aca gaa tct aac gca gtt gtt ctg gct gtg aaa gaa att gaa act 144 Ile Thr Glu Ser Asn Ala Val Val Leu Ala Val Lys Glu Ile Glu Thr 35 40 45 ttg ctt gca tct ata gat gaa ctt gct act aaa gct att ggt aaa aaa 192 Leu Leu Ala Ser Ile Asp Glu Leu Ala Thr Lys Ala Ile Gly Lys Lys 50 55 60 ata caa caa aat ggt ggt tta gct gtc gaa gcg ggg cat aat gga aca 240 Ile Gln Gln Asn Gly Gly Leu Ala Val Glu Ala Gly His Asn Gly Thr 65 70 75 80 ttg tta gca ggt gct tat aca ata tca aaa cta ata aca caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Thr Ile Ser Lys Leu Ile Thr Gln Lys Leu 85 90 95 gat gga ttg aaa aat tca gaa aaa tta aag gaa aaa att gaa aat gct 336 Asp Gly Leu Lys Asn Ser Glu Lys Leu Lys Glu Lys Ile Glu Asn Ala 100 105 110 aag aaa tgt tct gaa gat ttt act aaa aaa cta gaa gga gaa cat gcg 384 Lys Lys Cys Ser Glu Asp Phe Thr Lys Lys Leu Glu Gly Glu His Ala 115 120 125 caa ctt gga att gaa aat gtt act gat gag aat gca aaa aaa gct att 432 Gln Leu Gly Ile Glu Asn Val Thr Asp Glu Asn Ala Lys Lys Ala Ile 130 135 140 tta ata aca gat gca gct aaa gat aag ggc gct gca gag ctt gaa aag 480 Leu Ile Thr Asp Ala Ala Lys Asp Lys Gly Ala Ala Glu Leu Glu Lys 145 150 155 160 cta ttt aaa gca gta gaa aac ttg gca aaa gca gct aaa gag atg ctt 528 Leu Phe Lys Ala Val Glu Asn Leu Ala Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agt cct att gtg cat ggc gtt tca 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Ile Val His Gly Val Ser 180 185 190 gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac 624 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 195 200 205 aaa gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt 672 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 210 215 220 aaa gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta 720 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 aaa gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt 768 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca 816 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat 864 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc 912 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 aga ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa 960 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 gag gtt tta aaa aaa ttt act ctt gaa gga aaa gta gct aat gat aaa 1008 Glu Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 gta aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag aac att 1056 Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 340 345 350 tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt 1104 Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 355 360 365 gct gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta 1152 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 370 375 380 aca att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa 1200 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys 385 390 395 400 gaa aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta 1248 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu 405 410 415 gag ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct 1296 Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala 420 425 430 tta aaa taa 1305 Leu Lys * 74 434 PRT Artificial Sequence Chimeric protein 74 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Glu Ser Asn Ala Val Val Leu Ala Val Lys Glu Ile Glu Thr 35 40 45 Leu Leu Ala Ser Ile Asp Glu Leu Ala Thr Lys Ala Ile Gly Lys Lys 50 55 60 Ile Gln Gln Asn Gly Gly Leu Ala Val Glu Ala Gly His Asn Gly Thr 65 70 75 80 Leu Leu Ala Gly Ala Tyr Thr Ile Ser Lys Leu Ile Thr Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Ser Glu Lys Leu Lys Glu Lys Ile Glu Asn Ala 100 105 110 Lys Lys Cys Ser Glu Asp Phe Thr Lys Lys Leu Glu Gly Glu His Ala 115 120 125 Gln Leu Gly Ile Glu Asn Val Thr Asp Glu Asn Ala Lys Lys Ala Ile 130 135 140 Leu Ile Thr Asp Ala Ala Lys Asp Lys Gly Ala Ala Glu Leu Glu Lys 145 150 155 160 Leu Phe Lys Ala Val Glu Asn Leu Ala Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Ile Val His Gly Val Ser 180 185 190 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 195 200 205 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 210 215 220 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 Glu Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 340 345 350 Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 355 360 365 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 370 375 380 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys 385 390 395 400 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu 405 410 415 Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala 420 425 430 Leu Lys 75 1332 DNA Artificial Sequence Chimeric nucleic acid 75 atg gct tgt agt aat tca ggg aaa ggt ggg gat tct gca tct act aat 48 Met Ala Cys Ser Asn Ser Gly Lys Gly Gly Asp Ser Ala Ser Thr Asn 1 5 10 15 cct gct gac gag tct gcg aaa ggg cct aat ctt aca gaa ata agc aaa 96 Pro Ala Asp Glu Ser Ala Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys 20 25 30 aaa att aca gat tct aat gca ttt gta ctt gct gtt aaa gaa gtt gag 144 Lys Ile Thr Asp Ser Asn Ala Phe Val Leu Ala Val Lys Glu Val Glu 35 40 45 act ttg gtt tta tct ata gat gaa ctt gct aag aaa gct att ggt caa 192 Thr Leu Val Leu Ser Ile Asp Glu Leu Ala Lys Lys Ala Ile Gly Gln 50 55 60 aaa ata gac aat aat aat ggt tta gct gct tta aat aat cag aat gga 240 Lys Ile Asp Asn Asn Asn Gly Leu Ala Ala Leu Asn Asn Gln Asn Gly 65 70 75 80 tcg ttg tta gca gga gcc tat gca ata tca acc cta ata aca gaa aaa 288 Ser Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Thr Glu Lys 85 90 95 ttg agt aaa ttg aaa aat tta gaa gaa tta aag aca gaa att gca aag 336 Leu Ser Lys Leu Lys Asn Leu Glu Glu Leu Lys Thr Glu Ile Ala Lys 100 105 110 gct aag aaa tgt tcc gaa gaa ttt act aat aaa cta aaa agt ggt cat 384 Ala Lys Lys Cys Ser Glu Glu Phe Thr Asn Lys Leu Lys Ser Gly His 115 120 125 gca gat ctt ggc aaa cag gat gct acc gat gat cat gca aaa gca gct 432 Ala Asp Leu Gly Lys Gln Asp Ala Thr Asp Asp His Ala Lys Ala Ala 130 135 140 att tta aaa aca cat gca act acc gat aaa ggt gct aaa gaa ttt aaa 480 Ile Leu Lys Thr His Ala Thr Thr Asp Lys Gly Ala Lys Glu Phe Lys 145 150 155 160 gat tta ttt gaa tca gta gaa ggt ttg tta aaa gca gct caa gta gca 528 Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala 165 170 175 cta act aat tca gtt aaa gaa ctt aca agt cct gtt gta gca gaa agt 576 Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser 180 185 190 cca aaa aaa cct tcc atg gcc gtt tca gta gat ttg cct ggt gaa atg 624 Pro Lys Lys Pro Ser Met Ala Val Ser Val Asp Leu Pro Gly Glu Met 195 200 205 aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggc aag tac gat cta 672 Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu 210 215 220 att gca aca gta gac aag ctt gag ctt aaa gga act tct gat aaa aac 720 Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn 225 230 235 240 aat gga tct gga gta ctt gaa ggc gta aaa gct gac aaa agt aaa gta 768 Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val 245 250 255 aaa tta aca att tct gac gat cta ggt caa acc aca ctt gaa gtt ttc 816 Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe 260 265 270 aaa gaa gat ggc aaa aca cta gta tca aaa aaa gta act tcc aaa gac 864 Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp 275 280 285 aag tca tca aca gaa gaa aaa ttc aat gaa aaa ggt gaa gta tct gaa 912 Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu 290 295 300 aaa ata ata aca aga gca gac gga acc aga ctt gaa tac aca gga att 960 Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile 305 310 315 320 aaa agc gat gga tct gga aaa gct aaa gag gtt tta aaa aaa ttt act 1008 Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys Lys Phe Thr 325 330 335 ctt gaa gga aaa gta gct aat gat aaa gta aca ttg gaa gta aaa gaa 1056 Leu Glu Gly Lys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu 340 345 350 gga acc gtt act tta agt aag aat att tca aaa tct ggg gaa gtt tca 1104 Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser 355 360 365 gtt gaa ctt aat gac act gac agt agt gct gct act aaa aaa act gca 1152 Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala 370 375 380 gct tgg aat tca aaa act tcc act tta aca att agt gtg aat agc caa 1200 Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Gln 385 390 395 400 aaa acc aaa aac ctt gta ttc aca aaa gaa gac aca ata aca gta caa 1248 Lys Thr Lys Asn Leu Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln 405 410 415 aaa tac gac tca gca ggc acc aat cta gaa ggc aaa gca gtc gaa att 1296 Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala Val Glu Ile 420 425 430 aca aca ctt aaa gaa ctt aaa aac gct tta aaa taa 1332 Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys * 435 440 76 443 PRT Artificial Sequence Chimeric protein 76 Met Ala Cys Ser Asn Ser Gly Lys Gly Gly Asp Ser Ala Ser Thr Asn 1 5 10 15 Pro Ala Asp Glu Ser Ala Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys 20 25 30 Lys Ile Thr Asp Ser Asn Ala Phe Val Leu Ala Val Lys Glu Val Glu 35 40 45 Thr Leu Val Leu Ser Ile Asp Glu Leu Ala Lys Lys Ala Ile Gly Gln 50 55 60 Lys Ile Asp Asn Asn Asn Gly Leu Ala Ala Leu Asn Asn Gln Asn Gly 65 70 75 80 Ser Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Thr Glu Lys 85 90 95 Leu Ser Lys Leu Lys Asn Leu Glu Glu Leu Lys Thr Glu Ile Ala Lys 100 105 110 Ala Lys Lys Cys Ser Glu Glu Phe Thr Asn Lys Leu Lys Ser Gly His 115 120 125 Ala Asp Leu Gly Lys Gln Asp Ala Thr Asp Asp His Ala Lys Ala Ala 130 135 140 Ile Leu Lys Thr His Ala Thr Thr Asp Lys Gly Ala Lys Glu Phe Lys 145 150 155 160 Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala 165 170 175 Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser 180 185 190 Pro Lys Lys Pro Ser Met Ala Val Ser Val Asp Leu Pro Gly Glu Met 195 200 205 Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu 210 215 220 Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn 225 230 235 240 Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val 245 250 255 Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe 260 265 270 Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp 275 280 285 Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu 290 295 300 Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile 305 310 315 320 Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys Lys Phe Thr 325 330 335 Leu Glu Gly Lys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu 340 345 350 Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser 355 360 365 Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala 370 375 380 Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Gln 385 390 395 400 Lys Thr Lys Asn Leu Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln 405 410 415 Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala Val Glu Ile 420 425 430 Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys 435 440 77 1317 DNA Artificial Sequence Chimeric nucleic acid 77 atg gct tgt aat aat tca ggt ggg gat tct gca tct act aat cct gat 48 Met Ala Cys Asn Asn Ser Gly Gly Asp Ser Ala Ser Thr Asn Pro Asp 1 5 10 15 gag tct gca aaa gga cct aat ctt acc gta ata agc aaa aaa att aca 96 Glu Ser Ala Lys Gly Pro Asn Leu Thr Val Ile Ser Lys Lys Ile Thr 20 25 30 gat tct aat gca ttt tta ctg gct gtg aaa gaa gtt gag gct ttg ctt 144 Asp Ser Asn Ala Phe Leu Leu Ala Val Lys Glu Val Glu Ala Leu Leu 35 40 45 tca tct ata gat gaa ctt tct aaa gct att ggt aaa aaa ata aaa aat 192 Ser Ser Ile Asp Glu Leu Ser Lys Ala Ile Gly Lys Lys Ile Lys Asn 50 55 60 gat ggt act tta gat aac gaa gca aat cga aac gaa tca ttg ata gca 240 Asp Gly Thr Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu Ile Ala 65 70 75 80 gga gct tat gaa ata tca aaa cta ata aca caa aaa tta agt gta ttg 288 Gly Ala Tyr Glu Ile Ser Lys Leu Ile Thr Gln Lys Leu Ser Val Leu 85 90 95 aat tca gaa gaa tta aag gaa aaa att aaa gag gct aag gat tgt tcc 336 Asn Ser Glu Glu Leu Lys Glu Lys Ile Lys Glu Ala Lys Asp Cys Ser 100 105 110 gaa aaa ttt act act aag cta aaa gat agt cat gca gag ctt ggt ata 384 Glu Lys Phe Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu Gly Ile 115 120 125 caa agc gtt cag gat gat aat gca aaa aaa gct att tta aaa aca cat 432 Gln Ser Val Gln Asp Asp Asn Ala Lys Lys Ala Ile Leu Lys Thr His 130 135 140 gga act aaa gac aag ggt gct aaa gaa ctt gaa gag tta ttt aaa tca 480 Gly Thr Lys Asp Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe Lys Ser 145 150 155 160 cta gaa agc ttg tca aaa gca gcg caa gca gca tta act aat tca gtt 528 Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ala Leu Thr Asn Ser Val 165 170 175 aaa gag ctt aca aat cct gtt gtg gca gaa agt cca aaa aaa cct tcc 576 Lys Glu Leu Thr Asn Pro Val Val Ala Glu Ser Pro Lys Lys Pro Ser 180 185 190 atg gcc gtt tca gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc 624 Met Ala Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser 195 200 205 aaa gaa aaa aac aaa gac ggc aag tac gat cta att gca aca gta gac 672 Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp 210 215 220 aag ctt gag ctt aaa gga act tct gat aaa aac aat gga tct gga gta 720 Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val 225 230 235 240 ctt gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta aca att tct 768 Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser 245 250 255 gac gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa 816 Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys 260 265 270 aca cta gta tca aaa aaa gta act tcc aaa gac aag tca tca aca gaa 864 Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu 275 280 285 gaa aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga 912 Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg 290 295 300 gca gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct 960 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 305 310 315 320 gga aaa gct aaa gag gtt tta aaa aaa ttt act ctt gaa gga aaa gta 1008 Gly Lys Ala Lys Glu Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val 325 330 335 gct aat gat aaa gta aca ttg gaa gta aaa gaa gga acc gtt act tta 1056 Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu 340 345 350 agt aag aac att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac 1104 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 355 360 365 act gac agt agt gct gct act aaa aaa act gca gct tgg aat tca aaa 1152 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 370 375 380 act tct act tta aca att agt gtt aac agc aaa aaa act aca caa ctt 1200 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu 385 390 395 400 gtg ttt act aaa caa gac aca ata act gta caa aaa tac gac tcc gca 1248 Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 405 410 415 ggt acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt gat gaa 1296 Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 420 425 430 ctt aaa aac gct tta aaa taa 1317 Leu Lys Asn Ala Leu Lys * 435 78 438 PRT Artificial Sequence Chimeric protein 78 Met Ala Cys Asn Asn Ser Gly Gly Asp Ser Ala Ser Thr Asn Pro Asp 1 5 10 15 Glu Ser Ala Lys Gly Pro Asn Leu Thr Val Ile Ser Lys Lys Ile Thr 20 25 30 Asp Ser Asn Ala Phe Leu Leu Ala Val Lys Glu Val Glu Ala Leu Leu 35 40 45 Ser Ser Ile Asp Glu Leu Ser Lys Ala Ile Gly Lys Lys Ile Lys Asn 50 55 60 Asp Gly Thr Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu Ile Ala 65 70 75 80 Gly Ala Tyr Glu Ile Ser Lys Leu Ile Thr Gln Lys Leu Ser Val Leu 85 90 95 Asn Ser Glu Glu Leu Lys Glu Lys Ile Lys Glu Ala Lys Asp Cys Ser 100 105 110 Glu Lys Phe Thr Thr Lys Leu Lys Asp Ser His Ala Glu Leu Gly Ile 115 120 125 Gln Ser Val Gln Asp Asp Asn Ala Lys Lys Ala Ile Leu Lys Thr His 130 135 140 Gly Thr Lys Asp Lys Gly Ala Lys Glu Leu Glu Glu Leu Phe Lys Ser 145 150 155 160 Leu Glu Ser Leu Ser Lys Ala Ala Gln Ala Ala Leu Thr Asn Ser Val 165 170 175 Lys Glu Leu Thr Asn Pro Val Val Ala Glu Ser Pro Lys Lys Pro Ser 180 185 190 Met Ala Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser 195 200 205 Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp 210 215 220 Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val 225 230 235 240 Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser 245 250 255 Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys 260 265 270 Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu 275 280 285 Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg 290 295 300 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 305 310 315 320 Gly Lys Ala Lys Glu Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val 325 330 335 Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu 340 345 350 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 355 360 365 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 370 375 380 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu 385 390 395 400 Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 405 410 415 Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 420 425 430 Leu Lys Asn Ala Leu Lys 435 79 1029 DNA Artificial Sequence Chimeric nucleic acid 79 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt tct gaa aaa ata ata aca aga 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 gca gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct 672 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 gga aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta 720 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 act gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta 768 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 agc aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac 816 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 act gac agt agt gct gct act aaa aaa act gca gct tgg aat tca ggc 864 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly 275 280 285 act tca act tta aca att act gta aac agt aaa aaa act aaa gac ctt 912 Thr Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu 290 295 300 gtg ttt aca aaa gaa aac aca att aca gta caa caa tac gac tca aat 960 Val Phe Thr Lys Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn 305 310 315 320 ggc acc aaa tta gag ggg tca gca gtt gaa att aca aaa ctt gat gaa 1008 Gly Thr Lys Leu Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu 325 330 335 att aaa aac gct tta aaa taa 1029 Ile Lys Asn Ala Leu Lys * 340 80 342 PRT Artificial Sequence Chimeric protein 80 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly 275 280 285 Thr Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu 290 295 300 Val Phe Thr Lys Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn 305 310 315 320 Gly Thr Lys Leu Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu 325 330 335 Ile Lys Asn Ala Leu Lys 340 82 342 PRT Artificial Sequence Chimeric protein 82 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 275 280 285 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu 290 295 300 Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320 Gly Thr Asn Leu Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu 325 330 335 Leu Lys Asn Ala Leu Lys 340 83 1029 DNA Artificial Sequence Chimeric nucleic acid 83 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt tct gaa aaa ata ata aca aga 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 gca gac gga acc aga ctt gaa tac aca gga att aaa agc gat gga tct 672 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 gga aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta 720 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 act gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta 768 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 agc aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac 816 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 act gac agt agt gct gct act aaa aaa act gca gct tgg aat tca aaa 864 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 275 280 285 act tct act tta aca att agt gtt aac agc aaa aaa act aca caa ctt 912 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu 290 295 300 gtg ttt act aaa caa gac aca ata act gta caa aaa tac gac tcc gca 960 Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320 ggt acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt gat gaa 1008 Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 325 330 335 ctt aaa aac gct tta aaa taa 1029 Leu Lys Asn Ala Leu Lys * 340 84 342 PRT Artificial Sequence Chimeric protein 84 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 275 280 285 Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu 290 295 300 Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320 Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 325 330 335 Leu Lys Asn Ala Leu Lys 340 85 1035 DNA Artificial Sequence Chimeric nucleic acid 85 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa tat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa aca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt aca tct gaa aaa aca ata gta 624 Lys Pro Ser Met Ala Lys Gln Asn Val Thr Ser Glu Lys Thr Ile Val 195 200 205 aga gca aat gga acc aga ctt gaa tac aca gac ata aaa agc gat gga 672 Arg Ala Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly 210 215 220 tcc gga aaa gct aaa gaa gtt tta aaa gac ttt act ctt gaa gga act 720 Ser Gly Lys Ala Lys Glu Val Leu Lys Asp Phe Thr Leu Glu Gly Thr 225 230 235 240 cta gct gct gac ggc aaa aca aca ttg aaa gtt aca gaa ggc act gtt 768 Leu Ala Ala Asp Gly Lys Thr Thr Leu Lys Val Thr Glu Gly Thr Val 245 250 255 gtt tta agc aag aac att tta aaa tcc gga gaa ata aca gtt gca ctt 816 Val Leu Ser Lys Asn Ile Leu Lys Ser Gly Glu Ile Thr Val Ala Leu 260 265 270 gat gac tct gac act act cag gct act aaa aaa act gga aaa tgg gat 864 Asp Asp Ser Asp Thr Thr Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp 275 280 285 tca aat act tcc act tta aca att agt gtg aat agc aaa aaa act aaa 912 Ser Asn Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Lys 290 295 300 aac att gta ttt aca aaa gaa gac aca ata aca gta caa aaa tac gac 960 Asn Ile Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp 305 310 315 320 tca gca ggc acc aat cta gaa ggc aac gca gtc gaa att aaa aca ctt 1008 Ser Ala Gly Thr Asn Leu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu 325 330 335 gat gaa ctt aaa aac gct tta aaa tag 1035 Asp Glu Leu Lys Asn Ala Leu Lys * 340 86 344 PRT Artificial Sequence Chimeric protein 86 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Thr Ser Glu Lys Thr Ile Val 195 200 205 Arg Ala Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly 210 215 220 Ser Gly Lys Ala Lys Glu Val Leu Lys Asp Phe Thr Leu Glu Gly Thr 225 230 235 240 Leu Ala Ala Asp Gly Lys Thr Thr Leu Lys Val Thr Glu Gly Thr Val 245 250 255 Val Leu Ser Lys Asn Ile Leu Lys Ser Gly Glu Ile Thr Val Ala Leu 260 265 270 Asp Asp Ser Asp Thr Thr Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp 275 280 285 Ser Asn Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Lys 290 295 300 Asn Ile Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp 305 310 315 320 Ser Ala Gly Thr Asn Leu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu 325 330 335 Asp Glu Leu Lys Asn Ala Leu Lys 340 87 1323 DNA Artificial Sequence Chimeric nucleic acid 87 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc gtt tca gta gat ttg cct ggt gaa atg aaa gtt 624 Lys Pro Ser Met Ala Val Ser Val Asp Leu Pro Gly Glu Met Lys Val 195 200 205 ctt gta agc aaa gaa aaa aac aaa gac ggc aag tac gat cta att gca 672 Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala 210 215 220 aca gta gac aag ctt gag ctt aaa gga act tct gat aaa aac aat gga 720 Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly 225 230 235 240 tct gga gta ctt gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta 768 Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu 245 250 255 aca att tct gac gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa 816 Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu 260 265 270 gat ggc aaa aca cta gta tca aaa aaa gta act tcc aaa gac aag tca 864 Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser 275 280 285 tca aca gaa gaa aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata 912 Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile 290 295 300 ata aca aga gca gac gga acc aga ctt gaa tac aca gga att aaa agc 960 Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser 305 310 315 320 gat gga tct gga aaa gct aaa gag gtt tta aaa ggc ttt act ctt gaa 1008 Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys Gly Phe Thr Leu Glu 325 330 335 gga aaa gta gct aat gat aaa gta aca ttg gaa gta aaa gaa gga acc 1056 Gly Lys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr 340 345 350 gtt act tta agt aag att tca aaa tct ggg gaa gtt tca gtt gaa ctt 1104 Val Thr Leu Ser Lys Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu 355 360 365 aat gac act gac agt agt gct gct act aaa aaa act gca gct tgg aat 1152 Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn 370 375 380 tca aaa act tct act tta aca att agt gtt aac agc aaa aaa act aca 1200 Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr 385 390 395 400 caa ctt gtg ttt act aaa caa gac aca ata act gta caa aaa tac gac 1248 Gln Leu Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp 405 410 415 tcc gca ggt acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt 1296 Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu 420 425 430 gat gaa ctt aaa aac gct tta aaa taa 1323 Asp Glu Leu Lys Asn Ala Leu Lys * 435 440 88 440 PRT Artificial Sequence Chimeric protein 88 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Val Ser Val Asp Leu Pro Gly Glu Met Lys Val 195 200 205 Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala 210 215 220 Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly 225 230 235 240 Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu 245 250 255 Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu 260 265 270 Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser 275 280 285 Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile 290 295 300 Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser 305 310 315 320 Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys Gly Phe Thr Leu Glu 325 330 335 Gly Lys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr 340 345 350 Val Thr Leu Ser Lys Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu 355 360 365 Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn 370 375 380 Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr 385 390 395 400 Gln Leu Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp 405 410 415 Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu 420 425 430 Asp Glu Leu Lys Asn Ala Leu Lys 435 440 89 1302 DNA Artificial Sequence Chimeric nucleic acid 89 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc gtt tca 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Val Ser 180 185 190 gta gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac 624 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 195 200 205 aaa gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt 672 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 210 215 220 aaa gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta 720 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 aaa gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt 768 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 caa acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca 816 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 aaa aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat 864 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 gaa aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc 912 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 aga ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa 960 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 gag gtt tta aaa ggc ttt act ctt gaa gga aaa gta gct aat gat aaa 1008 Glu Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 gta aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag att tca 1056 Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Ile Ser 340 345 350 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 1104 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 355 360 365 gct act aaa aaa act gca gct tgg aat tca aaa act tct act tta aca 1152 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 370 375 380 att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaa caa 1200 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 385 390 395 400 gac aca ata act gta caa aaa tac gac tcc gca ggt acc aat tta gaa 1248 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 405 410 415 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 1296 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 420 425 430 aaa taa 1302 Lys * 90 433 PRT Artificial Sequence Chimeric protein 90 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Val Ser 180 185 190 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn 195 200 205 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 210 215 220 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 Glu Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Ile Ser 340 345 350 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 355 360 365 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 370 375 380 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 385 390 395 400 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 405 410 415 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 420 425 430 Lys 91 31 DNA Artificial Sequence Oligonucleotide Primer 91 gtcatatggc ttgtaataat tcagggaaag a 31 92 30 DNA Artificial Sequence Oligonucleotide Primer 92 tttccatgga aggttttttt ggactttctg 30 93 30 DNA Artificial Sequence Oligonucleotide Primer 93 tttccatggc caagcaaaat gttagcagcc 30 94 27 DNA Artificial Sequence Oligonucleotide Primer 94 taaggatcct tattttaaag cgttttt 27 95 822 DNA Artificial Sequence Altered OspA Nucleic Acid 95 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 96 273 PRT Artificial Sequence Altered OspA protein 96 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 97 822 DNA Artificial Sequence Altered OspA Nucleic Acid 97 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 98 273 PRT Artificial Sequence Altered OspA protein 98 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 99 822 DNA Artificial Sequence Altered OspA Nucleic Acid 99 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 100 273 PRT Artificial Sequence Altered OspA Protein 100 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 101 822 DNA Artificial Sequence Altered OspA Nucleic Acid 101 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 102 273 PRT Artificial Sequence Altered OspA Protein 102 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 103 822 DNA Artificial Sequence Altered OspA Nucleic Acid 103 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 104 273 PRT Artificial Sequence Altered OspA Protein 104 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 105 822 DNA Artificial Sequence Altered OspA Nucleic Acid 105 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc ttt gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Phe Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 106 273 PRT Artificial Sequence Altered OspA protein 106 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Phe Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 107 822 DNA Artificial Sequence Altered OspA Nucleic Acid 107 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc ttt act ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Phe Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 108 273 PRT Artificial Sequence Altered OspA Protein 108 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Phe Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 109 822 DNA Artificial Sequence Altered OspA Nucleic Acid 109 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat act ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 110 273 PRT Artificial Sequence Altered OspA Protein 110 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 111 822 DNA Artificial Sequence Altered OspA Nucleic Acid 111 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat act ctt gaa gga aag cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 112 273 PRT Artificial Sequence Altered OspA Protein 112 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 113 822 DNA Artificial Sequence Altered OspA Nucleic Acid 113 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc ttt act ctt gaa gga aag cta act gct gaa aaa aca 528 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 114 273 PRT Artificial Sequence Altered OspA protein 114 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 115 822 DNA Artificial Sequence Altered OspA Nucleic Acid 115 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc ttt act ctt gaa gga aag cta act gct gaa aaa aca 528 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc atg aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Met Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 116 273 PRT Artificial Sequence Altered OspA protein 116 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Met Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 117 30 DNA Artificial Sequence Oligonucleotide Primer 117 gaaaaaataa taacaatggc agacggaacc 30 118 30 DNA Artificial Sequence Oligonucleotide Primer 118 ggttccgtct gccattgtta ttattttttc 30 119 27 DNA Artificial Sequence Oligonucleotide Primer 119 ggaaaagcta aatatgtttt aaaaggc 27 120 27 DNA Artificial Sequence Oligonucleotide Primer 120 gccttttaaa acatatttag cttttcc 27 121 29 DNA Artificial Sequence Oligonucleotide Primer 121 gttactttaa gcatgaatat ttcaaaatc 29 122 29 DNA Artificial Sequence Oligonucleotide Primer 122 gattttgaaa tattcatgct taaagtaac 29 123 34 DNA Artificial Sequence Oligonucleotide Primer 123 gaggttttaa aaggctttac tcttgaagga actc 34 124 34 DNA Artificial Sequence Oligonucleotide Primer 124 gagttccttc aagagtaaag ccttttaaaa cctg 34 125 23 DNA Artificial Sequence Oligonucleotide Primer 125 tcttgaagga aagctaactg ctg 23 126 23 DNA Artificial Sequence Oligonucleotide Primer 126 cagcagttag ctttccttca aga 23 127 819 DNA Borrelia burgdorferi CDS (1)...(819) 127 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat gca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac aac aaa aaa act aaa gcc ctt gta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 gac aca att aca tca caa aaa tac gac tca gca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aga 819 Arg 128 273 PRT Borrelia burgdorferi 128 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Arg 129 9 PRT Artificial Sequence Residue 165-173 of B31 OspA 129 Tyr Val Leu Glu Gly Thr Leu Thr Ala 1 5 130 9 PRT Artificial Sequence Residue 332-340 of hLFA-1 130 Tyr Val Ile Glu Gly Thr Ser Lys Gln 1 5

Claims (30)

What is claimed is:
1. A polypeptide, comprising an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 139 to about residue 273 of Borrelia burgdorferi OspA protein, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 being methionine, residue 160 being tyrosine, residue 189 being methionine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
2. The polypeptide of claim 1, wherein the polypeptide has increased conformational stability compared to the corresponding unaltered OspA polypeptide.
3. The polypeptide of claim 1, wherein residue 160 is tyrosine and residue 189 is methionine.
4. The polypeptide of claim 1, wherein residue 139 is methionine, residue 160 is tyrosine and residue 189 is methionine.
5. The polypeptide of claim 1, wherein the polypeptide comprises residues 131 through 273 of the Borrelia burgdorferi OspA protein.
6. The polypeptide of claim 1, wherein the polypeptide comprises residues 17 through 273 of the Borrelia burgdorferi OspA protein.
7. The polypeptide of claim 1, wherein the polypeptide is derived from an OspA protein of a sensu stricto strain of Borrelia burgdorferi.
8. A polypeptide, comprising an amino acid sequence of OspA protein from a sensu stricto strain of Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 being phenylalanine, residue 166 being threonine, residue 170 being lysine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
9. The polypeptide of claim 8, wherein cross-reactivity to an hLFA-1 molecule is reduced compared to the corresponding unaltered OspA polypeptide.
10. The polypeptide of claim 8, wherein residue 165 is phenylalanine and residue 166 is threonine.
11. The polypeptide of claim 8, wherein residue 165 is phenylalanine, residue 166 is threonine and residue 170 is lysine.
12. The polypeptide of claim 8, wherein residue 139 is methionine, residue 160 is tyrosine and residue 189 is methionine.
13. The polypeptide of claim 8, wherein the polypeptide comprises residues 150 through 180 of the Borrelia burgdorferi OspA protein.
14. The polypeptide of claim 8, wherein the polypeptide comprises residues 17 through 273 of the Borrelia burgdorferi OspA protein.
15. A polypeptide selected from the group consisting of: SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114 and 116.
16. A polynucleotide encoding an amino acid sequence of an OspA protein from Borrelia burgdorferi from about residue 139 to about residue 273, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
17. The polynucleotide of claim 16, wherein codon 160 encodes tyrosine and codon 189 encodes methionine.
18. The polynucleotide of claim 16, wherein codon 139 encodes methionine, codon 160 encodes tyrosine and codon 189 encodes methionine.
19. The polynucleotide of claim 16, wherein the encoded polypeptide comprises residues 131 through 273 of the Borrelia burgdorferi OspA protein.
20. The polynucleotide of claim 16, wherein the encoded polypeptide comprises residues 17 through 273 of the Borrelia burgdorferi OspA protein.
21. The polynucleotide of claim 16, wherein the encoded polypeptide corresponds to OspA protein of a sensu stricto strain of Borrelia burgdorferi.
22. A polynucleotide encoding an amino acid sequence of an OspA protein from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence encodes at least one alteration selected from the group consisting of: codon 165 encoding phenylalanine, codon 166 encoding threonine, codon 170 encoding lysine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
23. The polynucleotide of claim 22, wherein codon 165 encodes phenylalanine and codon 166 encodes threonine.
24. The polynucleotide of claim 22, wherein codon 165 encodes phenylalanine, codon 166 encodes threonine and codon 170 encodes lysine.
25. A polynucleotide selected from the group consisting of: SEQ ID NO: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115.
26. A method of generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide comprising;
a) selecting a polynucleotide encoding a Borrelia burgdorferi OspA polypeptide that includes residues 139, 160 and 189, wherein the numbering corresponds to the numbering of SEQ ID NO: 7;
b) altering the polynucleotide such that residue 139 is methionine, residue 160 is tyrosine and residue 189 is methionine; and
c) expressing said altered polynucleotide;
thereby generating an altered Borrelia burgdorferi OspA polypeptide with increased conformational stability compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
27. A method of generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with an hLFA-1 molecule, compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide, comprising;
a) selecting a polynucleotide encoding an OspA polypeptide from Borrelia burgdorferi that includes residues 165, 166 and 170, wherein the numbering corresponds to the numbering of SEQ ID NO: 7;
b) altering the polynucleotide such that residue 165 is phenylalanine, residue 166 is threonine and residue 170 is lysine; and
c) expressing said altered polynucleotide;
thereby generating an altered Borrelia burgdorferi OspA polypeptide with reduced cross-reactivity with the hLFA-1 molecule compared to the corresponding unaltered Borrelia burgdorferi OspA polypeptide.
28. Use of an altered OspA polypeptide for the manufacture of a medicament for immunizing a mammal, wherein the altered OspA polypeptide comprises:
a) an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 139 to about residue 273 of Borrelia burgdorferi OspA protein, wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 being methionine, residue 160 being tyrosine, residue 189 being methionine and combinations thereof, wherein the polypeptide has increased conformational stability compared to a corresponding wild type OspA polypeptide; and/or
b) an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least one alteration selected from the group consisting of: residue 165 being phenylalanine, residue 166 being threonine, residue 170 being lysine and combinations thereof, wherein the polypeptide has decreased cross-reactivity to hLFA-1 compared to a corresponding wild type OspA polypeptide;
wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
29. A polypeptide, comprising an amino acid sequence of OspA protein from Borrelia burgdorferi from about residue 160 to about residue 170, wherein the sequence includes at least two alterations selected from the group consisting of: residue 165 being phenylalanine, residue 166 being threonine and residue 170 being lysine, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
30. A chimeric polypeptide, comprising:
a) an amino acid sequence of a first OspA polypeptide from about residue 1 to about residue 164 from a first strain of Borrelia burgdorferi;
b) an amino acid sequence of a second OspA polypeptide from about residue 165 to about residue 179 from a second strain of Borrelia burgdorferi, wherein said second strain is a different strain from said first strain;
c) an amino acid sequence of a third OspA polypeptide from about residue 180 to about residue 216 from a third strain of Borrelia burgdorferi, wherein said third strain is a different strain from said second strain;
d) an amino acid sequence of a fourth OspA polypeptide from about residue 217 to about residue 273 from a fourth strain of Borrelia burgdorferi, wherein said fourth strain is a different strain from said third strain;
wherein the sequence includes at least one alteration selected from the group consisting of: residue 139 being methionine, residue 160 being tyrosine, residue 189 being methionine and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
US10/369,339 2000-08-18 2003-02-18 Altered OspA of Borrelia burgdorferi Abandoned US20040023325A1 (en)

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US6210676B1 (en) * 1998-07-31 2001-04-03 Gundersen Lutheran Medical Foundation Compositions and methods using the borreliacidal epitope(s) of borrelia burgdorferi outer surface protein C (OspC) for the diagnosis and prevention of lyme disease

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US20040033236A1 (en) * 1993-11-01 2004-02-19 Research Foundation Of State University Of New York Recombinant constructs of borrelia burgdorferi
US20050271682A1 (en) * 1993-11-01 2005-12-08 Dattwyler Raymond J Recombinant constructs of Borrelia burgdorferi
US7008625B2 (en) 1993-11-01 2006-03-07 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US20070020286A1 (en) * 1993-11-01 2007-01-25 Dattwyler Raymond J Recombinant constructs of Borrelia burgdorferi
US7179448B2 (en) 1993-11-01 2007-02-20 Research Foundation Of The State Of New York Recombinant constructs of Borrelia burgdorferi
US7605248B2 (en) 1993-11-01 2009-10-20 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US8992936B2 (en) 2000-08-18 2015-03-31 Research Foundation Of The State University Of New York Altered OspA of Borrelia burgdorferi
US20090326200A1 (en) * 2000-08-18 2009-12-31 Brookhaven Sciences Associates, Llc Altered ospa of borrelia burgdorferi
US8680236B2 (en) 2000-08-18 2014-03-25 Brookhaven Sciences Associates, Llc Altered OspA of borrelia burgdorferi
US20170107263A1 (en) * 2012-07-06 2017-04-20 Valneva Austria Gmbh Mutant fragments of ospa and methods and uses relating thereto
US8986704B2 (en) * 2012-07-06 2015-03-24 Valneva Austria Gmbh Mutant fragments of OspA and methods and uses relating thereto
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US20150250865A1 (en) * 2012-07-06 2015-09-10 Valneva Austria Gmbh MUTANT FRAGMENTS OF OspA AND METHODS AND USES RELATING THERETO
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US8992936B2 (en) 2015-03-31
US20090326200A1 (en) 2009-12-31
AU2001285049A1 (en) 2002-03-04
EP1311540A2 (en) 2003-05-21
WO2002016421A8 (en) 2002-07-11
DE60131982T2 (en) 2008-12-11
US8680236B2 (en) 2014-03-25
DE60131982D1 (en) 2008-01-31
PT1311540E (en) 2008-03-20
WO2002016421A3 (en) 2003-03-06
US20140030285A1 (en) 2014-01-30
DK1311540T3 (en) 2008-04-28
ES2298249T3 (en) 2008-05-16

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