FITZGERALD J ROSS (GB)
BEN ZAKOUR NOURI L (AU)
BANNOEHR JEANETTE (GB)
FITZGERALD J ROSS (GB)
BEN ZAKOUR NOURI L (AU)
| WO2002076498A2 | 2002-10-03 |
| US0578044A | 1897-03-02 | |||
| US5780448A | 1998-07-14 |
RODRIQUEZ ET AL.: "Vectors: a Survey of Molecular Cloning Vectors and their Uses", 1988, BUTTERSWORTH
"Current Protocols in Molecular Biology", 1989, JOHN WILEY & SONS
PH.EUR. MONOGRAPH, 2001, pages 0062
PH.EUR. MONOGRAPH, 1997, pages 0193
"PCR Primer: A Laboratory Manual", COLD SPRING HARBOUR LABORATORY PRESS
JOSEPH SAMBROOK; DAVID RUSSELL: "Molecular Cloning: A Laboratory Manual", COLD SPRING HARBOUR LABORATORY PRESS
OLENA MOROZOVAA; MARCO A. MARRA: "Applications of next-generation sequencing technologies in functional genomics", GENOMICS, vol. 92, no. 5, November 2008 (2008-11-01), pages 255 - 264, XP025535324, DOI: doi:10.1016/j.ygeno.2008.07.001
RONAGHI: "Pyrosequencing sheds light on DNA sequencing", GENOME RESEARCH, vol. 11, 2001, pages 3 - 11, XP000980886, DOI: doi:10.1101/gr.11.1.3
HARLOW; LANE: "Using Antibodies: A Laboratory Manual", 1999, CSHLP
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1988, CSHLP
BANNOEHR J; BEN ZAKOUR NL; WALLER AS; GUARDABASSI L; THODAY KL; VAN DEN BROEK AH; FITZGERALD JR.: "Population genetic structure of the Staphylococcus intermedius group: insights into agr diversification and the emergence of methicillin-resistant strains", J BACTERIOL., vol. 189, 2007, pages 8685 - 92
BEN ZAKOUR; N. L., GUINANE; C. M.; FITZGERALD, J. R.: "Pathogenomics of the staphylococci: insights into niche adaptation and the emergence of new virulent strains", FEMS MICROBIOL LETT, vol. 289, 2008, pages 1 - 12
CLARKE, S. R.; FOSTER, S. J.: "Surface adhesins of Staphylococcus aureus", ADV MICROB PHYSIOL, vol. 51, 2006, pages 187 - 224
CORRIGAN, R. M.; MIAJLOVIC, H.; FOSTER, T. J.: "Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells", BMC MICROBIOL, vol. 9, 2009, pages 22, XP021048189, DOI: doi:10.1186/1471-2180-9-22
CLARKE, S. R.; ANDRE, G.; WALSH, E. J.; DUFRENE, Y. F.; FOSTER, T. J.; FOSTER, S. J.: "Iron-regulated surface determinant protein A mediates adhesion of Staphylococcus aureus to human comeocyte envelope proteins", INFECT IMMUN, vol. 77, 2009, pages 2408 - 16
CURTIS, C.F.: "Masked, controlled study to investigate the efficacy of a Staphylococcus intermedius autogenous bacterin for the control of canine idiopathic recurrent superficial pyoderma", VET DERMATO, vol. 17, 2006, pages 163 - 8
FORSYTHE, P. J.; HILL, P. B.; THODAY, K. L.; BROWN, J.: "Use of computerized . image analysis to quantify staphylococcal adhesion to canine comeocytes: does breed and body site have any relevance to the pathogenesis of pyoderma?", VET DERMATOL, vol. 13, 2002, pages 29 - 36
FOSTER, T. J.; HOOK, M.: "Surface protein adhesins of Staphylococcus aureus", TRENDS MICROBIOL, vol. 6, 1998, pages 484 - 8, XP001038138, DOI: doi:10.1016/S0966-842X(98)01400-0
GANESH, V. K.; RIVERA, J. J.; SMEDS, E.; KO, Y. P.; BOWDEN, M. G.; WANN, E. R.; GURUSIDDAPPA, S.; FITZGERALD, J. R.; HOOK, M.: "A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics", PLOS PATHOG, vol. 4, 2008, XP007915427, DOI: doi:10.1371/journal.ppat.1000226
GUARDABASSI, L.; SCHWARZ, S.; LLOYD, D. H.: "Pet animals as reservoirs of antimicrobial-resistant bacteria", JANTIMICROB CHEMOLHER, vol. 54, 2004, pages 321 - 32
HALL, A. E.; DOMANSKI, P. J.; PATEL, P. R.; VERNACHIO, J. H.; SYRIBEYS, P. J.; GOROVITS, E. L.; JOHNSON, M. A.; ROSS, J. M.; HUTCH: "Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A", INFECT IMMUN, vol. 71, 2003, pages 6864 - 70, XP009029021, DOI: doi:10.1128/IAI.71.12.6864-6870.2003
HILL, P.B. ET AL.: "Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice", VET REC, vol. 158, 2006, pages 533 - 9
JOSEFSSON, E.; HARTFORD, O.; O'BRIEN, L.; PATTI, J. M.; FOSTER, T.: "Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant", J INFECT DIS, vol. 184, 2001, pages 1572 - 80, XP009028976, DOI: doi:10.1086/324430
LINDSAY, J. A.; MOORE, C. E.; DAY, N. P.; PEACOCK, S. J.; WITNEY, A. A.; STABLER, R. A.; HUSAIN, S. E.; BUTCHER, P. D.; HINDS, J.: "Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes", J BACTERIOL, vol. 188, 2006, pages 669 - 76
MAZMANIAN, S.K. ET AL.: "Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall", SCIENCE, vol. 285, 1999, pages 760 - 3, XP002288783, DOI: doi:10.1126/science.285.5428.760
NANRA, J. S.; TIMOFEYEVA, Y.; BUITRAGO, S. M.; SELLMAN, B. R.; DILTS, D. A.; FINK, P.; NUNEZ, L.; HAGEN, M.; MATSUKA, Y. V.; MININ: "Heterogeneous in vivo expression of clumping factor A and capsular polysaccharide by Staphylococcus aureus: implications for vaccine design", VACCINE, vol. 27, 2009, pages 3276 - 80, XP026122449, DOI: doi:10.1016/j.vaccine.2009.01.062
OTTO, M.: "Targeted immunotherapy for staphylococcal infections : focus on anti-MSCRAMM antibodies", BIODRUGS, vol. 22, 2008, pages 27 - 36, XP009132068
PATTI, J. M.: "A humanized monoclonal antibody targeting Staphylococcus aureus", VACCINE, vol. 22, no. 1, 2004, pages 39 - 43
PIZZA, M. ET AL.: "Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing", SCIENCE, vol. 287, 2008, pages 1816 - 1820, XP002414564, DOI: doi:10.1126/science.287.5459.1816
| Claims 1. A Staphylococcus pseudintermedius protein or nucleic acid comprising a sequence at least 65% homologous or identical to (i) the nucleic acid of SEQ ID NO: 5 or a fragment thereof or (ii) the amino acid sequence of SEQ ID NO: 6 or an antigenic fragment thereof, for use in raising an immune response in a subject. 2. A vaccine for use in treating, preventing or controlling a disease caused or contributed to by Staphylococcus pseudintermedius in a subject, said vaccine comprising nucleic acid sequence or peptide/protein having a sequence at least 65% homologous or identical to the nucleic acid of SEQ ID NO: 5 or amino acid sequence of SEQ ID NOS: 6 or an antigenic fragment thereof. 3. A Staphylococcus pseudintermedius protein or nucleic acid comprising a sequence at least 65% homologous or identical to a nucleic acid or amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4 and 7-36; and fragments of any of SEQ ID NOS: 1-4 and 7-36, for use in raising an immune response in a subject. 4. A vaccine for use in treating, preventing or controlling disease caused or contributed to by Staphylococcus pseudintermedius in a subject, said vaccine comprising one or more amino acid or nucleic acid sequence(s) at least 65% homologous or identical to a nucleic acid or amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4 and 7-36; and fragments of any of SEQ ID NOS: 1-4 and 7-36. 5. The Staphylococcus pseudintermedius protein or nucleic acid for use of claim 1 or vaccine of claim 2, further comprising one or more protein or nucleic acid sequence(s) at least 65% homologous or identical to an amino acid or nucleic acid sequence selected from the group consisting of SEQ ID NOS 1-4 and 7-36; and fragments of any of SEQ ID NOS: 1-4 and 7-36. 6. A method of raising an immune response to Staphylococcus pseudintermedius in a subject, said method comprising immunising said subject with, or administering said subject a vaccine according to claim 2, 4 or 5 7. The protein/nucleic acid for use of claims 1 , 3 and 5, vaccine of claim 2, 4 or 5 or method of claim 6, wherein the subject is a canine. 8. The vaccine of claims 2, 4, 5 or 6, wherein the disease caused or contributed to by Staphylococcus pseudintermedius is canine pyoderma. 9. The vaccine of claim 2, 4, 5 or 8, wherein the vaccine further comprises one or more adjuvant(s) and/ or antigens for use in treating or preventing another diseases and/or condition. 10. An isolated and/or substantially purified or recombinant Staphylococcus pseudintermedius nucleic acid or protein sequence encoding a cell-wall anchored (CWA) or microbial surface components recognising adhesive matrix molecule (MSCRAMM) wherein said nucleic acid sequence comprises a nucleic acid at least 65% homologous or identical to the nucleic acid sequence of SEQ ID NO: 5 or fragments, portions, mutants, derivatives and/or homologoues/orthologues thereof and said protein sequence comprises an amino acid sequence at least 65% homologous or identical to an amino acid sequence of SEQ ID NO: 6 or fragments, portions, mutants, derivatives and/or homologoues/orthologues thereof. 1 1. An isolated and/or substantially purified or recombinant Staphylococcus pseudintermedius nucleic acid or protein sequence encoding a cell-wall anchored (CWA) or microbial surface components recognising adhesive matrix molecule (MSCRAMM) wherein said nucleic acid or protein sequence comprises a nucleic acid or amino acid sequence at least 65% homologous or identical to a nucleic acid or amino acid sequences selected from the group consisting of SEQ ID NOS: 1 -36 or fragments, portions, mutants, derivatives and/or homologoues/orthologues thereof. 12. A nucleic acid comprising a replicable expression vector and nucleic acid according to claims 10 or 1 1. 13. A host cell transformed with the nucleic acid of claim 12. 14. A process for the production of recombinant S. pseudintermedius CWA or MSCRAMM proteins, said process comprising the steps of: (a) transforming a host cell with the nucleic acid of claim 12; (b) culturing the cells obtained in (a) under conditions in which expression of a peptide/protein encoded by the nucleic acid occurs; and (c) isolating the expressed peptide/protein from the cell culture or/and a culture supernatant derived therefrom. 15. An antibody that selectively and/or specifically binds to, or exhibits affinity for the S. pseudintermedius CWA or MSCRAMM proteins encoded by the nucleic acid of claim 10 or 1 1 and/or proteins recombinantly produced by the process of claim 14. 16. A method of treating, preventing or controlling a Staphylococcus pseudintermedius infection or disease in a canine, said method comprising administering to the canine one or more of the nucleic acid(s)/protein(s) provided by claims 10 or 1 1. 17. A compound for use in treating an infection or disease caused or contributed to by Staphylococcus pseudintermedius, wherein said compound antagonises (i) the function, expression and/or activity of a protein and/or nucleic acid encoded by one or more of the sequences provided by claim 1 or (ii) the interaction between a protein and/or nucleic acid having the sequence of claim 10 or 1 1 and a native ligand. 18. The compound for use of claim 17, wherein the compound is a small organic molecule, antibody, peptide or carbohydrate 19. The compound for use of claim 17 or 14, wherein the compound comprises an antibody according to claim 5 or a polypeptide or polynucleotide sequence comprising a fragment, portion, mutant, derivative and/or homologoue/orthologue of one or more of the sequences given as SEQ ID NOS: 5/6 and/or 1-4, 7-36. 20. A method of diagnosing infections, diseases and/or conditions caused or contributed to by S. pseudintermedius, said methods comprising the steps of identifying in a sample a level of a protein, peptide or nucleic acid (for example a gene) having a sequence homologous or identical to a sequence provided by SEQ ID NOS: 5/6 and/or 1- 4, 7-36 or a fragment, portion, mutant, derivative and/or homologoue/orthologue thereof, wherein detection of a level of a protein, peptide or nucleic acid (for example a gene) having a sequence homologous or identical to a sequence provided by SEQ ID NOS: 5/6 or 1-4, 7-36 in sample, indicates that the sample may have been provided by a subject suffering from a S. pseudintermedius infection, disease and/or condition. 21. The method of claim 20, wherein the sample comprises canine skin cells. 22. The method of claims 20 or 21 , wherein the detection of a level of a protein, peptide or nucleic acid comprises the use of a molecular detection technique and/or an immunological detection technique. 23. A kit for diagnosing, detecting and/or evaluating possible S. pseudintermedius infections, diseases and/or conditions, said kit comprising one or more components selected from the group consisting of: (i) substrates having S. pseudintermedius proteins or agents capable of binding 5. pseudintermedius proteins, bound thereto; (ii) antibodies which exhibit specificity and/or selectivity for one or more S. pseudintermedius proteins; and (iii) oligonucleotides/primers for detecting/amplifying/probing samples for S. pseudintermedius protein encoding sequences. 24. The kit of claim 19, wherein the S. pseudintermedius proteins or S. pseudintermedius protein encoding sequences comprise sequences corresponding to (or homogous/identical to) to SEQ ID NOS: 5/6 and/or 1-4, 7-36 and/or fragments, portions, mutants, derivatives and/or homologoues/orthologues thereof. |
FIELD OF THE INVENTION
The present invention provides novel staphylococcal cell wall associated proteins, genes encoding the same and vaccines for use in treating/preventing Staphylococcal infections.
BACKGROUND OF THE INVENTION
Skin diseases are a major cause of morbidity in dogs and an important animal welfare issue (Hill et al, 2006). In particular, superficial bacterial folliculitis (pyoderma) caused by Staphylococcus pseudintermedius (formerly known as Staphylococcus intermedius) is one of the most common diseases seen in small animal veterinary practice, worldwide (Hill et al, 2006). Superficial pyoderma is characterized by the formation of follicular pustules and is often associated with pruritus, alopecia, erythema and swelling. This may develop into deep pyoderma which typically includes pain, crusting, odor, and exudation of blood and pus. The disease often occurs as a secondary infection in dogs with atopic dermatitis (AD) resulting from a type I hypersensitivity reaction (IgE antibody-associated) to environmental allergens (Hill et al, 2006). Treatment of canine pyoderma is often difficult without resorting to aggressive, medium-term administration of systemic antibacterial agents to prevent relapse of infection, and such therapy predisposes to the development of bacterial resistance that may be transferred to bacteria infecting humans (Guardabassi et al, 2004). Worringly, methicillin-resistant S. pseudintermedius has recently emerged as a major problem in veterinary clinics worldwide (Bannoehr et al, 2007). Although rare, several episodes of life-threatening infections of humans by S. pseudintermedius have been reported with the typical route of transmission being through dog bite wounds (Bannoehr et al, 2007). Previously, crude vaccine preparations based on Staphylococcus aureus phage lysate or S. pseudintermedius autogenous bacterin have shown promise as adjunctive therapies for treatment of pyoderma (Curtis et al, 2006), and a rationally-designed effective vaccine would be a highly desireable means to reducing or eliminating the suffering associated with the disease.
Accordingly, the present invention aims to obviate one or more of the problems associated with the prior art. SUMMARY OF THE INVENTION
The present invention is based upon the identification of novel gene sequences encoding proteins potentially useful in the treatment and/or prevention of canine disorders. In particular, the proteins encoded by the genes described herein, may find application in the treatment and/or prevention of diseases caused or contributed to by the bacterial pathogen Staphylococcus pseudintermedius.
The inventors have identified a number of Staphylococcus pseudintermedius genes encoding proteins which may broadly be classed as members of the cell-wall anchored (CWA) family of proteins. In certain embodiments, these CWA proteins may be further grouped as surface proteins known as Microbial Surface Components Recognising Adhesive Matrix Molecules (MSCRAMM). It should be understood that while a number microbial organisms may be known to express MSCRAMM type proteins, the term "MSCRAMM" describes the phenotypic function of a wide range of diverse surface-associated proteins of Gram-positive bacteria. As such, while MSCRAMM proteins may all possess cell-wall anchor motifs and signal sequences for cell wall transportation, the proteins belonging to this group may otherwise be structurally diverse. Furthermore, bacterial species within a particular genus, for example the genus Staphylococcus, may possess unique MSCRAMM profiles.
In view of the above, the present invention relates to a group of surface expressed proteins derived from Staphylococcus pseudintermedius that may be referred to either as CWA or MSCRAMM proteins.
As such, a first aspect of this invention provides an isolated and/or substantially purified Staphylococcus pseudintermedius CWA or MSCRAMM nucleic acid or protein sequence comprising a nucleic acid or amino acid sequence homologous or identical to any one of the nucleic acid or amino acid sequences provided as SEQ ID NOS: 1 -36 below.
SEQ ID NO: 1
atggaaaacaaaaacttttttagtattcgtaaactatctattggtgtaggttcttgc tta atcgcgagttctttacttgtaaacacgccaagttttgctgaagaaacagataatgcgaac attaatgacgcacaacaaaacgccttttatgaaattttacatttgccaaacttaactgaa gagcaacaaaatggattcatccaaagtcttaaagatgatccaagtgtgagcaacgacatt ttagtagaagctaagaaattaaatgacactcaagctaaacctgattacagtgaagcacaa caaaatgcattttatgaaattttacatttgtcaaacttaactgaagagcaacaaaatgga ttcatccaaagtcttaaagatgatccaagtgtgagcaacgacattttagtagaagctaag aagttaaatgacactcaagctaaacctgattacagtgaagcacaacaaaatgcattttat gaaattttacatttgtcaaacttaactgaagagcaacaaaatgggttcatccaaagcctt aaagatgatccaagtgtaagtaaagaaattttagcagaagctaagaagttaaatgatagt caagcacctaaagttgataaagctaaaaaaactgacaaagctgaagcgaaagcagatgat aaagctaaaggtgaagaagccaaaaaatctgaagacaaaaaagatagcaaagcagataag gcaaaatcgaaaaacgctacacatgttgttaaacctggtgaaactttagataatattgct aaagatcatcatacaactgttgataaaattgctaaagataacaaaataaaagataaaaat gtgattaaactaggtcaaaaacttgttgttgataaacaaaaagcaactcaaggaaaacaa gaagctgtagcgaaaaatgaagtgaaggctttacctaatactggtgaaaatgatgatatc gcattattcagcacaacagttgcgggtggcgtaagtatcgctttaggttcattattatta ggaagaaacagaaaaactagctaa
The protein sequence translated from SEQ ID NO 1 is designated SEQ ID NO: 2 and is shown below:
SEQ ID NO: 2
MENKNFFSIRKLSIGVGSCLIASSLLVNTPSFAEETDNANINDAQQNAFYEILHLPNLT
EEQQNGFIQSLKDDPSVSNDILVEAKKLNDTQAKPDYSEAQQNAFYEILHLSNLTEE
QQNGFIQSLKDDPSVSNDILVEAKKLNDTQAKPDYSEAQQNAFYEILHLSNLTEEQQ
NGFIQSLKDDPSVSKEILAEAKKLNDSQAPKVDKAKKTDKAEAKADDKAKGEEAKKS
EDKKDSKADKAKSKNATHWKPGETLDNIAKDHHTTVDKIAKDNKIKDKNVIKLGQKL
WDKQKATQGKQEAVAKNEVKALPNTGENDDIALFSTTVAGGVSIALGSLLLGRNRK
TS
SEQ ID NO: 3
atggaaaacaaaaactttttcagcattcgtaaattatcaattggggtgggttcatgt tta at cgcgagctctttacttgtgaatacaccaagttt cgcagaagaaggagataataacgca gaagcgcaacaaaacgctttct ctgaggtagtaaaatt acctaaccttagcgaagaacaa cgtaatggtttcattcaaagccttaaagatgatccaagtacaagt caagatgtgcttaat gaagctaaaaaattaaatgatagtcaagagggatctcaacctgctcctgattacagtgat gaacaacaaaatgcattttatgaaattttacaccttccaaacttaactgaagaacaacgc aatggctatattcaaagtcttaaagatgacccaagtgtaagcgctaatattcttgttgaa gctaaaaatatgaatgttaaccaaacacctacacaacctgcgccaagtttcgatgaagcg caacaaaatgcattctatgagattgtaaacttaccaaatcttactgaagagcaacgtaac ggtttcatccaaagccttaaagacgatccaagtgtaagtaaagatatccttgttgaagct aaaaagttaaatgacagccaagcaaaacctgattacagtgaagcgcaacaaaatgcattt tatgaaattttacaccttccaaacttaactgaagaacaacgtaacggtttcatccaaagc cttaaagacgatccgagtgtaagtagtgatattcttgctgaagctaagaaattaaatgac agccaagcgcctaaagaagacaacaacgtaaaagacaataattcaggtgaaaacaaagct gaagacaaaggcaacaaagaaaacaaagctgaagataaaggcagcaaagaagacaaagct gaagataaaggcagcaaagaagacaaagctgaagataaaggcagcaaagaagacaaagct
gaagataaaggcagcaaagaagacaaagctgaagataaaggcagcatagaagataaa gct
aaagacaaagacaacaaagaaggcaaagctgcagacaaaggtatggacaaagcgaaa gat
gcaatgcatgtcgttcaacctggtgaaacagtagaaaaaattgctaaagctaataac aca
actgtagaacaaatcgctaaagataatcatttagaagataaaaacatgattttacca ggt
caaaaacttgttgttgacaaccaaaaagcaatgaaagacagccaagaagctaaagca aac
cacgaaatgaaagctttacctgaaacaggtgaagaaaacgatatggcattattcggt aca
tcacttacaggtggtcttagcttagcattaggtttatacatcttaggacgtggcaga aaa
acaaactaa
The protein sequence translated from SEQ ID NO 3 is designated SEQ ID NO: 4 and is shown below:
SEQ ID NO: 4
MENKNFFSIRKLSIGVGSCLIASSLLVNTPSFAEEGDNNAEAQQNAFSEVV LPNLSEEQRNGFIQSLKDDPS TSQDVLNEAKKLNDSQEGSQPAPDYSDEQQNAFYEILHLPNLTEEQRNGYIQSLKDDPSV SANILVEAKNMNV NQTPTQPAPSFDEAQQNAFYEIVNLPNLTEEQRNGFIQSLKDDPSVSKDILVEAKKLNDS QAKPDYSEAQQNA FYEILHLPNLTEEQRNGFIQSLKDDPSVSSDILAEAKKLNDSQAPKEDNNVKDNNSGENK AEDKGNKEN AED KGSKEDKAEDKGSKEDKAEDKGSKEDKAEDKGSKEDKAED GSIEDKAKDKDNKEGKAADKGMDKAKDAMHVV QPGETVEKIAKANNTTVEQIAKDNHLEDKNMILPGQKLVVDNQKAM DSQEAKANHEMKALPETGEENDMALF GTSLTGGLSLALGLYILGRGRKTN
SEQ ID NO: 5
gtgtacaaaaatgaagaagaaaagcattcaataagaaagttatctataggagccgca tct
gtcattgttgggggactcatgtatggtgttttgggaaatgatgaagctcaagcgaat gaa
gatgtcactgaaacaactgggagaaattcagtgacaacgcaagcttctgagcaacat ttg
caagtggaagcagtacctcaagaaggcaataatgtaaatgtatcctctgtaaaagta cct
acgaatacggcaacgcaagcacaagaagatgt tgcaagtgtatccgatgt taaagcacat
gctgatgatgcattacaagtacaagaaagtagtcatactgatggtgtttcttcagaa ttc
aagcaggagacagcttatgcgaatcctcaaacagctgagacagttaaacctaatagt gaa
gcagtgcatcagtctgaatacgaggataagcaaaaacccgtatcatctagccgcaaa gaa
gatgagactatgcttcagcagcaacaagttgaagccaaaaatgttgtgagtgcggag gaa
gtgtctaaagaagaaaatactcaagtgatgcaatcccctcaagacgttgaacaacat gta
ggtggtaaagatatctctaatgaggttgtagtggataggagtgatatcaaaggattt aac
agcgaaactactattcgacctcatcagggacaaggtgg aggttgaattatcaattaaag
tttcctagcaatgtaaagccaggcgatcagtttactataaaattatctgacaatatc aat
acacatggtgtttctgttgaaagaaccgcaccgagaatcatggctaaaaatactgaa ggt
gcgacggatgtaat gctgaaggtctagtgttggaagatggtaaaaccatcgtatataca
tttaaagactatgtaaatggcaagcaaaatttgactgctgagttatcagtgagctat ttc
gtaagtccggaaaaagtcttgactactgggacacaaacattcacgacgatgatcggt aat
cattcaacgcaatccaatattgacgtttattatgataatagtcattatgtagatgga cgt
atttcgcaagtgaacaaaaaagaagctaaatttcaacaaatagcatacattaaccct aat
ggctatttaaatggcagggggacaattgcagttaatggtgaagtggtcagtggtacg act aaagacttaatgcaacctacagtgcgtgtatatcaatataaaggacaaggtgttcctcct gaaagtattactatagaccctaatatgtgggaagaaatcagcataaacgatactatggta
agaaaatatgatggtggctatagcttgaatctggataccagcaagaatcaaaaatat gcc
atctattatgaaggggcatatgatgcgcaagctgacacactgttgtatagaacatat ata
cagtcattaaacagttactatccgttcagttaccaaaaaatgaacggtgtgaagttt tac
gaaaacagtgcgagtggaagcggtgagttgaaaccgaaaccacctgaacaaccaaaa cca
gaacctgaaattcaagctgatgtagtagatattattgaagatagccatgtgattgat ata
ggatggaatacagcagttggagaagaaagtggagcaaaccaaggccctcaagaagaa atc
acggaaaa cacgacatcgaagtcattgaggaaaacaacttggtggaaatgacagaagat
acagcagttggagaagaaagtggagcaaaccaaggccctcaagaagaaatcacggaa aat
cacgacatcgaagtcattgaagaaaacaacttagtggaaatgacagaagatacagcg ttg
gaagaagaaagtggagcaaatcaaggtcctcaagaagagatcacagaaaaccacgat atc
gaagtcattgaagaaaacaacttggtggaaatgacagaagatacagcgttggaagaa gaa
agtggagcaaatcaaggtcctcaagaagagatcacagaaaaccacgacatcgaagtc att
gaagaaaataacttagtagaaatgacagaagatacagcagttggagaagaaagtgga gca
aatcaaggtcctcaagaagagatcacagaaaaccacgatatcgaagtcattgaggaa aac
aacttagtggaaatgacagaagatacagcagttggagaagaaagtggagcaaaccaa ggt
cctcaagaagaaatcacggaaaatcacgacatcgaagtcattgaagaaaacaacttg gtg
gaaatgacagaagatacagcgttggaagaagaaagtggagcaaatcaaggtcctcaa gaa
gagatcacagaaaaccacaacatcgaagtcattgaagaaaacaacttggtggaaatg aca
gaagatacagcagttggagaagaaagtggagcaaacccaggacctcaagaagaagta aca
gagaatcaacctcagcaagaagaaatcatggaaaaccaagaagtcgaaaagaaaggc gat
agtaacttggtagaaagtacaaaaactccaaaggccgaagaatcagttagcgttcag cca
actttagaagacaaaaacacaaagaaccacgttaacacagtagtagtgaatacgaag gta
tctgaagttaaagaaaaggatccccaccatacaaaagcactaccagatacggggaca acc
tctcgaagtcattccatgatgattcctctccttc tgttgctgggtcagtagtgttgtta
cgtcgaaagaaaaagcatagtaaggtgaattaa
The protein sequence translated from SEQ ID NO 5 is designated SEQ ID NO: 6 and is shown below:
SEQ ID NO: 6
VYKNEEEKHSIR LSIGAASVIVGGLMYGVLGNDEAQANEDVTETTGRNSVTTQASEQHLQVEAVPQEGNNVN VSSVKVPTNTATQAQEDVASVSDVKAHADDALQVQESSHTDGVSSEFKQETAYANPQTAE TVKPNSEAVHQSE YED QKPVSSSRKEDETMLQQQQVEAKNVVSAEEVSKEENTQVMQSPQDVEQHVGG DISNEVVVDRSDIKGF NSETTIRPHQGQGGRLNYQLKFPSNVKPGDQFTIKLSDNINTHGVSVERTAPRIMA NTEGATDVIAEGLVLE DGKTIVYTFKDYVNGKQNLTAELSVSYFVSPEKVLTTGTQTFTT IGNHSTQSNIDVYYDNSHYVDGRISQVN KKEAKFQQIAYINPNGYLNGRGTIAVNGEVVSGTTKDLMQPTVRVYQYKGQGVPPESITI DPN EEISINDT MVRKYDGGYSLNLDTSKNQKYAIYYEGAYDAQADTLLYRTYIQSLNSYYPFSYQKMNGVK FYENSASGSGELK PKPPEQPKPEPEIQADVVDI IEDSHVIDIG NTAVGEESGANQGPQEEITENHDIEVIEENNLVEMTEDTAVG EESGANQGPQEEITENHDIEVIEENNLVEMTEDTALEEESGANQGPQEEITENHDIEVIE ENNLVEMTEDTAL EEESGANQGPQEEITENHDIEVIEENNLVEMTEDTAVGEESGANQGPQEEITENHDIEVI EENNLVEMTEDTA VGEESGANQGPQEEITENHDIEVIEENNLVEMTEDTALEEESGANQGPQEEITENHNIEV IEENNLVEMTEDT AVGEESGANPGPQEEVTENQPQQEEIMENQEVEKKGDSNLVESTKTP AEESVSVQPTLEDKNTKNHVNTVVV NTKVSEVKEKDPHHTKALPDTGTTSRSHSMMIPLLLVAGSVVLLRRKKKHSKVN
SEQ ID NO: 7
atgaataaatcaagaactaaacattttaattttttatcaaaacgtcagaatcggtat gct
attcgccacttttcagctggtactgtgtcagtgcttgtaggagcagctttcttgcta ggt
gtccatacgagtgatgcatctgctgcagaacaagatcaaacatctgaagcaaagcaa aac
ctctttgatgcttccgctatttttggcgctttaacagagacgaacgaaaaggtagca caa
gtgacgccaacagaaaaaaatctttcatcagttgaagaaatgagagataaaggcgca act
ggaaatggaccatcaataacatcactacaaactgtagaacaaaataatgcagtacaa cct
acagcaacacctattaatgacacagaaaattcaaccgaagcccctatgaaagaacaa tcg
aatgatgcacaaacgactgacgaaagtaacaatgccactcagaaaaataatactgaa ccc
caagcaaacaatgaaatatcagcgcgtaatgcaaaaacaacagcatatttaacaagt gaa
acctttacaacagcaacgtctacaactgatatgcctacacagaaacaagaatatcca tct
ttagaaaatccaacaaatcaatcgcaaacgaacagagcacaaccaccaacaatggaa gca
cccaaactggcagaaggattagacaatctattaaaaaaatcaactttcgaaagtatg tac
gtgacaaaaagaaatcaatttgacaaagagacggcttctaaaacaaaagcatggccg agt
gatgttgttccagaaaatcaagtagagatacttgctgatgcaattcaaaatggctat atc
aaatctgtaaatgatgtgaccaataaagcacatacgttatctggacgtgcatggatg tta
gaccacggaacaccaacgacaatagctaatggtttaacacctgttccagagggcact aaa
gtttatttgcggtggatagatcaagatggtgccacttcgccaatgtatacagcaaaa acg
acaagtagattaagcgccgcggatggtaatcaagtgggtccaggtgc tatgctttcgat
ttacgcacaggttggatagatgctaaaggaaaacaccacgtatatagagcagtaaag ggt
caatattataaaatatggatcaatgattttagaactaaagacggtaatatcgctaca atg
ttacgtgttgcaggaggatatgttccgggaacgtacgtggattctgtgacatacaac aat
atgggccaatttccattaattggtacaaatatgc acgtacaggtatctttatgacaacg
ataccttcagaaaaatatttaatatcaaaacattacgtgaaagatacaaaaggtgct gca
gcaaatccagccgtcacgataattgaaaataactttgtgagcggcaaagtttggata gaa
acaggtgctggagattatgtgaactcagcgacaggtccaaaccacaatgcgaaagat gtc
gttgcctctggatacaaagtggtcatgtcatcattaacagatcaaggtgctaaagcc tac
gatgcgcaagtcaatcgcttgccgaagaaagatcgagcagaagcagcacgtcaatta t a
ataaaacatccagaatatatcgcagcaactgtagaagggataacgaatgagtggggg aga
tatacattgcgtttccctaaaggcacattcaacaaagaccatctttacggttacgta ttg
gattttgatggtgaaattgtaaaaacttattcaggttttacttcaccagagttccgg aga
ccgaattataatttgaccgttacaccgcaaacagctccctattatagacccgttcga cgt
gcatgggtcaatgttaattttgcggttattgaagcaccacaatctcaaatcgaaata aaa
gaatttgatgcaacctctaaccctgcgcatcgtgggcaaacagcaactattgatatc ata
ggtatgcctaaaacttcattacttacacgtgtacaatggaaagattcatcgggcagt att
gttgaggatagtggtcctgtttttacggaagaagaggctgaacatatagcggaattt gta
ataccgtctagcgcaaaatcaggcgaagtgtatactgtacaactcgtggtaggtaat cat
atcgtagcttcggactctcttattgtacatgtcaatgaagaagcggcgacatatcat ccg atatacccatcgacaacagtagaatcaggtcaaagagtaacgattccagcacctaagaat
atggatggcaaacctttactagatggcacaacttttgaaaaaggtcatcacgtacca act
tgggctttagtgaatggtgatggctcgattacagtaaaacctggagaaaaagtagca gag
ggtgagtatgatattccagtgattgtgacatatccagatggttctaaaaacacaatc ttt
gcacctgtgaccgttgaagaaaaacaaccaatggcatcgcaatatgagccaataaca act
ggagtatcgaaaccatttggaaacccagtaatgccaactgatgtaacagattcaatt caa
gtaccgaactatccattggaagggcaacaaccgacagtaacagtggatgatgaaagt caa
ttaccagatggaacaacagaaggttacaaggatatagatgtaacagtgacataccca gac ggaacaaaggatcgtgtcaaagttccagtcgtaacggaacaacaattagatagtgataaa
tatgatccggtcgcaacaggtatcttgaaaccgtttggtactccaacaacagaggaa gac gttataaaattagtggagataccgaaatatccaacagacttaacacaaccaaaagtaaca gtgacggttccaaatactttaccggatgggcaaacgccaggtaaagtagacgttgatgtg acagtaacgtatccagatggttccacagatcacatttcagttccagtttggacaaacaag catctggataaagacaaatataacccaataacgactggggtatcgaaaccatttggaatc ccagtaacgccaactgatgtaacagattcaattcaagtaccgaactatccattggaaggg caacaactgacagtaacagtggatgatgaaacacaattaccagatggaacaacagaaggt cacaaggatatagatgtaacagtgacatacccagacggaacaaaggatcatatcaaagt t ccagtcgtaacggaaaaacaatcagataatgaaaaatatgagccaacaactaacggaatc acgaaaaagtacggtatccctacgacagaggatgaagtgatagatatagttcgaattcca tattttccagtagatggcgtgcaacctattgtaacggtaaatgatcctagactattgcca aatggtcaaaaagaaggtcaaatcaatgttccagtcacagtgacgtatccggatggcaca aaagatctcatgacagttccggttattacaggtaagcaagcagaaaatgaaaaatacgat ccaatcacattaggagtaactaaagattatggtgatcctacaactgcaaacgatgtgaca aagtcaatccaaataccaacatatccagcaggtggcgaacaaccaatcgcaacagcggat gatgaaagtcaattaccggatggcacagtagaaggtaaagtggatattccagtcacagtg acgtatccggatggtactcaggatcatatcactgtcccagtatttaccaatcaacaacga gataatcaaaaagccagtaaagctgtgacgaaaatacatggtatatcggtaacaggcact gatatgacagatactaagaaaaatcataactatccagcaggtggtgaacaacctaaagtt actgtgaaagatgacgatcaattatcagagggtaaagtcgattcaacagtgggtgcggat aatgtgacaactacagatgatttatcaagcgtaactgcggtatctcatggtcatcaaaca agtgtacaaacaacaaaagagaaccaatcagtgcatgatgaagaggtgacgatcccaaca gttgcacatgtgtctacaataatgacaggtgtggtaaagggtgagcaagaagcgacggat atcgtggctagaccacatgttgaaacaactcaactcccatcaatttcagctcaagcaaca gttaaaaaactaccagaaacgggtgaaaacaatgaacaatcaggtgttttattaggtgga tttattgcgttcatgggtagcttacttttattcggcagacgtcgcaaaccaaagaaagat taa
The protein sequence translated from SEQ ID NO 7 is designated SEQ ID NO: 8 and is shown below: SEQ ID NO:
MNKSRTKHFN FLSKRQNRYA IRHFSAGTVS VLVGAAFLLG VHTSDASAAE QDQTSEAKQN LFDASAIFGA LTETNEKVAQ VTPTEKNLSS VEEMRDKGAT GNGPSITSLQ TVEQNNAVQP TATPINDTEN STEAPM EQS NDAQTTDESN NATQKNNTEP QANNEISARN AKTTAYLTSE TFTTATSTTD MPTQKQEYPS LENPTNQSQT NRAQPPTMEA PKLAEGLDNL LKKSTFESMY VTKRNQFDKE TASKTKAWPS DVVPENQVEI LADAIQNGYI KSVNDVTNKA HTLSGRAWML DHGTPTTIAN GLTPVPEGTK VYLRWIDQDG ATSPMYTAKT TSRLSAADGN QVGPGAYAFD LRTGWIDAKG KHHVYRAVKG QYYKIWINDF RTKDGNIATM LRVAGGYVPG TYVDSVTYNN MGQFPLIGTN MQRTGIFMTT IPSEKYLISK HYVKDTKGAA ANPAVTIIEN NFVSGKV IE TGAGDYVNSA TGPNHNA DV VASGYKVVMS SLTDQGAKAY DAQVNRLP K DRAEAARQLL IKHPEYIAAT VEGITNEWGR YTLRFPKGTF NKDHLYGYVL DFDGEIV TY SGFTSPEFRR PNYNLTVTPQ TAPYYRPVRR AWVNVNFA I EAPQSQIEI EFDATSNPAH RGQTATIDII GMP TSLLTR VQWKDSSGSI VEDSGPVFTE EEAEHIAEFV IPSSAKSGEV YTVQLVVGNH IVASDSLIVH VNEEAATYHP IYPSTTVESG QRVTI PAP N MDG PLLDGT TFEKGHHVPT WALVNGDGSI TVKPGEKVAE GEYDIPVIVT YPDGSKNTIF APVTVEEKQP MASQYEPITT GVSKPFGNPV MPTDVTDSIQ VPNYPLEGQQ PTVTVDDESQ LPDGTTEGYK DIDVTVTYPD GTKDRVKVPV VTEQQLDSDK YDPVATGILK PFGTPTTEED VIKLVEIPKY PTDLTQPKVT VTVPNTLPDG QTPG VDVDV TVTYPDGSTD HISVPVWTNK HLDKDKYNPI TTGVSKPFGI PVTPTDVTDS IQVPNYPLEG QQLTVTVDDE TQLPDGTTEG HKDIDVTVTY PDGTKDHIKV PVVTEKQSDN EKYEPTTNGI TKKYGIPTTE DEVIDIVRIP YFPVDGVQPI VTVNDPRLLP NGQKEGQINV PVTVTYPDGT KDLMTVPVIT GKQAENEKYD PITLGVTKDY GDPTTANDVT KSIQIPTYPA GGEQPIATAD DESQLPDGTV EGKVDIPVTV TYPDGTQDHI TVPVFTNQQR DNQKASKAVT KIHGISVTGT DMTDTKKNHN YPAGGEQPKV TVKDDDQLSE GKVDSTVGAD NVTTTDDLSS VTAVSHGHQT SVQTTKENQS VHDEEVTIPT VAHVSTIMTG VVKGEQEATD IVARPHVETT QLPSISAQAT VKKLPETGEN NEQSGVLLGG FIAFMGSLLL FGRRRKPKKD
SEQ ID NO: 9
atgtttaatcaacaaaaacaacactatggtatccggaaatatgcaatcgggacttca tca gtattattaggcatgacattatttatcacacatgacgcaactgcatctgcagctgaaaac aatacaactgcaaagacagagacaaatcaagcagcaacaatttcttctcgcacttcgcca accgacgtcgctcaacctaatgcagacacgaatgctacaacggcgactaaagagacaaca ccacaatcagattcaacagcattaccgcaagcagcagcgcaacctcaaacgggccaaaca gcatcgaaagacacagtagatacaaataaaacgcaaacagcagattccacaaccgctcct cctgtgacagacgcgccaaaagctaatgacgacacaacacagccagaagctgcgactgta gccaaaaaagaagatgctcagacaccatcgactgcagaccctacaccacaagcgcaacaa ccgcctcagtcaaaagcacctcaagaaacgcaacaacaatcaacagttgaagatacaacg ccacaacaaaacgcatcaactgaagcacaccctaaaaatgtagataccgcttcaacaaaa caacaacaaacaacgccatcaaccgcaccgacaccttacacacaacaagcagacgaagca atgacagatgtcacaacaaccagtgtcgacagcaacgtacagccgttagcccctgcagaa gatcaacctaaaaatacgaacacagctgacaaagcaaccgttgcgacaecaccacgtgac aatgctaagactgctgatccgaacaaaaagatgacacgtgcagcaacgacacaacaagat gatgccgtcgatacattgaagtcaaaagaaatgacagcaacgatcgataaaagttttcca gccgttaaatattacacgttgaaaaatggtaaaaaagtcgatgcacaactgacggatgca cgtcaaatcatcgtcaatggtgaagtcattacaccaacagtcaaatacaacaaaattgat gatcatacggctgaatatgacttaacagcacaaaatgattcacgttcgattgatgccaat tttaaatttcgtttatcagttgaaggtaagaccgttgatttacaaatgacagattacacg aacaacaacacagatccacaaaacgtcattcgcaactttagctttgtaagtcaatcgctc gtatctgtaaacaatcaacagaaaaatgccaaactgcaaacatcgaaactgtctacaaat acaatgaaaagcggcgataaatcatatcatatcgatgaaaatttcaaaaacgacttcaac gactttatgatgtacggtttcgtgtcaaatgatgattacagtgcaggattgtggagtaac gcacaaattggcgtcggcattggtgaacaagacttcttacgtgtctacgcacagtctata caaacagatatcggggtcgctgtcggtttaggctcaatgccatggtttatccaaaaagac gctgcacatccagatgcgaaaaatcaaggactactcccacatgtcaaagttgcaattgcg gaagatgaaaatcaagatggtgaaattaactggcaagacggtgcaattgcttatcgtagc attatgaacaatccatatggtgccgaagaagtacctgaccttgttgggtaccgtatcgcg atgaactttggttctcaagcgcaaaacccatttttaaagacgttagatggtgtgaaaaaa ttctatctcaatacagatggtttagggcaatccattttattaaaaggttataacagtgaa ggccacgactctggtcatttagattacgcgaatattggtcaacgtataggtggcgtgaaa gactttaaaacgttacttcaaaaaggggcagattatggcgcacgtttcggtcttcatgtg aatgcatctgaaacatatccagagtctcaagcattcaatcctgccctcttacgtaaagat gcgaatggaaactatatgtatggctggaactggctcgatcaaggctttaacatcgatgca gattacgatttaatacacgggcgtaaagaacgcttcgaagcactcaaacaaattgtcggt gatgacctcgactttatttatgtcgatgtatgggggaatggacaatccggcgacaataca gcttggccatcacatcaattagccaaagaaatcaacgacttaggatggcgcgtcggtgtc gaatggggtcacggtatggaatatgactccacgttccaacattgggcagccgacttaacg tatggatcgtaccaaaataaagggattaactcagaggtagcacgcttcttacgcaaccat caaaaagattcatgggtcggtaactatccaaaatactcaggtgcagctgacttcccattg ctcggcggttatgacatgaaagattttgaaggttggcaaggtcgtaacgattactctgct tacattaaaaatattttcaatgttgatgtaccaacaaagtttttacaacattataaagtg atgcgtattgtcgatggtgagcctgttaaaatgactgccaatggtcaaacgattgactgg acaccagaaatgcaagtcgatttacaaaatgaagccggtgatcaagtcactgttaaacgt aaatctaacgactatgaaaacgacactgacaactaccgctcacgtacaatcgaattgaat ggtcgcacagtactcgatggcgattcataccttttaccatggaattgggatgcgaacggc caaccattaactggcgataacgaaaaattatatcactggaataaaaaaggcggttcaacg acttggacactgcctgaatcatgggatacagaccaagtcgtgctatacgaattatctgaa acgggtcgtaagtcaccacgtacagtggcagtgaaagaccatcaagtgacactcgataat attaaagcagacacaccgtatgtcgtttataaagtcgcacaaccagacaacacagatgtg aactggagcgaagacatgcacgtgaaagatgccggcttcaactcacaacaactgacacct
tggacaatcgaaggcaatcgagataaagtgagcatcgaaaagtcgacaacatcaaat gaa
atgctaaaaatcgatagtccaacaaaaacaacgcaattaacgcaacaattgacaggt tta
gtgccaggacaacgttacgctgtctatgttggcatcgataaccgcagtgatgcagcg gcg
catattgcagtgacacataacggtaaaacgctcgcaagtaacgaaacaggtcaatcg atc
gcgaaaaactatgtgaaagcagatgcacatagtaacaatgctgcgacgtttaaaaat ggc
ggtagttacttccaaaacatgtacgtgtacttcgttgcgccagaagatggtaaagca gac
ttgacgattcaacgcgacccaggtgaaggggccacttatttcgatgatattcgtgtg tta
gaaaataacgcgaatctccttcaaaacggcacattcaaccaagacttcgaaaatgta cca
caagggttattcccgttcgtcgtgtcagaagttgaaggcgttgaagataatcgcgtt cac
ttatctgaaaagcacgcaccgtatacacaacgcggatggaataataaacgtgtcgat gat
gtcattgatggcaaatggtcacttaaagtaaacggtcaaacaggtaaagataaaatg gtc
atccaaacgattccgcaaaacttctacttcgaaccaggaaaaacgtatgaagtgtca ttt
gattatgaagcaggttctgatgatacgtatgcatttgcgacaggtagtggggacatt tct
aaaaatcgtaactttgaaaagacaccattgaaaaatacagtcgatggtggcaaagcg aaa
cgggtgacatttaaagtgacgggtgatgaaaatggtcaaacttggatcggtatttac tca
acgaaaacacccaatgatccacgaggcgtgaaaaatggcaatcaaatcaacttcgaa ggg
acgaaagatttcattctagacaacctttctatccgtgaaattgacgcaccgaagcct gat
gccacacaagaaagcggtgatagcgcaccaatgaatgaaacagatgagcgtaacgtc aat
tcaaacggtacattagccgatcatagtgagacaactgatgtcaatgtcagtgcaacg gca
gatgatacagcagtcaaaggcgaaatgacgacaaacagaacagatgcaccaactgtt aca
ctgcctgaagcaacgatagtagatgaaggcacgtcaaatcctgtcactacaacacca acg
aatacaacacaagctatgacaaataaggctgatgagatgccacaaacgatgaacaat gtt
cctttaactagcatcgctaccgatatgatgcagtctcatgcggtggattccatggca gca
acactagcagctacaaatcaagtggcggcacctgtgcgtcaaacagcaggacctatg caa
catggtatggacagtgcttcaacgcaacacgcacccatacaagttgacaatgtcaca gca
ccaccattaccagatgaacagtttgccgaattacctaaaactggggatacgactcca aat
acacgtggacctttaatggcgatgatagttggcgcagtcttaacagcattcggattc aga
cgccaacgtaaagaaaaatag
The protein sequence translated from SEQ ID NO 9 is designated SEQ ID NO: 10 and is shown below:
SEQ ID NO: 10
MFNQQKQHYGIRKYAIGTSSVLLGMTLFITHDATASAAENNTTAKTETNQAATISSRTSP TDVAQPNADTNAT TATKETTPQSDSTALPQAAAQPQTGQTASKDTVDTNKTQTADSTTAPPVTDAPKANDDTT QPEAATVAKKEDA QTPSTADPTPQAQQPPQSKAPQETQQQSTVEDTTPQQNASTEAHPKNVDTASTKQQQTTP STAPTPYTQQADE AMTDVTTTSVDSNVQPLAPAEDQPKNTNTADKATVATPPRDNAKTADPNKKMTRAATTQQ DDAVDTLKSKEMT ATIDKSFPAVKYYTLKNGKKVDAQLTDARQI IVNGEVITPTVKYNKIDDHTAEYDLTAQNDSRSIDANFKFRL SVEGKTVDLQMTDYTNNNTDPQNVIRNFSFVSQSLVSVNNQQKNAKLQTSKLSTNTMKSG DKSYHIDENFKND FNDFMMYGFVSNDDYSAGLWSNAQIGVGIGEQDFLRVYAQSIQTDIGVAVGLGSMP FIQ DAAHPDAKNQGL LPHVKVAIAEDENQDGEIN QDGAIAYRSIMNNPYGAEEVPDLVGYRIAMNFGSQAQNPFLKTLDGVKKFYLN TDGLGQSILLKGYNSEGHDSGHLDYANIGQRIGGVKDFKTLLQKGADYGARFGLHVNASE TYPESQAFNPALL RKDANGNYMYGWNWLDQGFNIDADYDLIHGRKERFEALKQIVGDDLDFIYVDVWGNGQSG DNTAWPSHQLAKE INDLGWRVGVEWGHGMEYDSTFQHWAADLTYGSYQNKGINSEVARFLRNHQKDS VGNYP YSGAADFPLLGG YDMKDFEGWQGRNDYSAYIKNIFNVDVPTKFLQHYKVMRIVDGEPVKMTANGQTID TPEMQVDLQNEAGDQV TVKRKSNDYENDTDNYRSRTIELNGRTVLDGDSYLLPWN DANGQPLTGDNEKLYH NKKGGSTTWTLPES D TDQVVLYELSETGRKSPRTVAV DHQVTLDNIKADTPYVVYKVAQPDNTDVN SEDMHVKDAGFNSQQLTPWT IEGNRDKVSIEKSTTSNEMLKIDSPT TTQLTQQLTGLVPGQRYAVYVGIDNRSDAAAHIAVTHNGKTLASNE TGQSIAKNYVKADAHSNNAATFKNGGSYFQNMYVYFVAPEDGKADLTIQRDPGEGATYFD DIRVLENNANLLQ NGTFNQDFENVPQGLFPFVVSEVEGVEDNRVHLSEKHAPYTQRGWNNKRVDDVIDGKWSL KVNGQTGKDKMVI QTIPQNFYFEPG TYEVSFDYEAGSDDTYAFATGSGDISKNRNFEKTPLKNTVDGGKAKRVTFKVTGDENGQT IGIYSTKTPNDPRGV NGNQINFEGTKDFILDNLSIREIDAPKPDATQESGDSAPMNETDERNVNSNGTLAD HSETTDVNVSATADDTAVKGEMTTNRTDAPTVTLPEATIVDEGTSNPVTTTPTNTTQAMT NKADE PQTMNNV PLTSIATDMMQSHAVDSMAATLAATNQVAAPVRQTAGPMQHGMDSASTQHAPIQVDNVTA PPLPDEQFAELPK TGDTTPNTRGPLMAMIVGAVLTAFGFRRQRKEK
SEQ ID NO: 1 1
atgacaagaaaatttagggaatttaagaaaagtttaagtgaagaaaaagcaagagtg aaa
ctttacaagtcaggtaaaaactgggttaaagctggaattaaagaatttcagttatta aaa
gcattaggcttatcttttttaagccatgacattgtaaaggatgaaaatggagaagta acg
acacaatttggggaacagttgaagaaaaatgcattaagaacaactgcttt tgcgggtgga
atgttcacagttaatatgttgcatgaccaacaagcatttgcggcgtcggatgcacct ata
acttctgaactggcaaccaaaagtcaaactattggcgatcaaacatcaa tgttattgaa
aagtctacatcgtcagatcaatcaacgaacccaataacagaaagtgaaagtaaacac gat
tctgaaagtatctcattatctgagcatcaaacatcagagtcaacaagtctttcaacg tca
acttccaaatcaatatcaactt cagtagaggaatcagaatcaacatcaaaagattctcat
actaaaactcaagatagtcaatcagatagtcatcagtcaacaagtcaagaggtaaat ggc
tcttccaaccacgagcaatcaacaccacacactgcacaaagtcttacgagcctatct att
gagagccaaacgtcgactt caaatacatcattgaaggaaactaaagaaggggaattgtca
aaaaacctttcgaagttatctcaaaatcaaaacatcaaacttcatgaagaacatacg atg
cgttcagcagatttgagctcaggttatacaggatttagagcggcttactatgtacca aga
tcaagaacaacaccaacgacaaaagtctacacagggcaaggaagcttcagaggtaga ggt
agaattaaatataatattttctacaaagttgtcgttacaagtaatggcaaagaaatg aag
atccgctatacattgagtcaagatgatccaaacacgtctaatgttgaaaaacctagg tgg
gcaggacagaaacgatttggtattcataatacttgggatgaaggtcctggtcgcggg caa
ttaaagttagggtcggcattcggcaaaccaacagttatacaaggagaaactagaccg aat
tatggtagctgggttggcacacctataacgaaatatgtttcaggcgatcgtacaaat ggt
ttttactggcaagctgctgtacttgcaccgagacatggagagaagggagaaggaatc aca
gcagaaattacagttcctattgttaacccttctggaagatttaattgggaattccat cct gtcggtcaacaggacggagttggtggcaaaactgactactttgaaaatgtatggattcga gactatgacccatattacaaatatattcaaactaaggaaggcagagcctcagtttcgcac tctatttctcaggtgaaagcaagtgaatcgagatcgacatcgctcatacaatcggagtct attagaagatcacagtccatatctgagagtgaatctattgtagccgcaagtcattcggca agtgtagcaaaatcgcaatccatctcgagaagtcaatctgtggcgaaatcacaatcgatc tcaagaagtcagtcgatcgcacacagccgatcagcaagtgtggcaaaatcgcaatccatc tcaagaagtcagtcgatcgcacacagccgatcagcaagtgtggcaaaatcacaatcgatt tcaagaagtcagtcgatcgcacacagccgatcagcaagtgtggcgaaatctcaatcgatt tcaagaagtcagtcaattgcgcagagccaatcagcaagtgtggcaaaatcacagtcgatt tcaagaagtcagtcaattgcgcagagccaatcagcaagtgtggcgaaatcgcaatcgatt tcaagaagtcagtcgattgcacatagccgatcagcaagtgtagcggaatcacagtcgatt tcaagaagtcagtcgattgcgaatagccaatctgtagcagcgagtgaatcagagagtcta tcaatatcattgtctaaaaagcagtcaatatcgatgagtaattctgaaagtgcagcaaaa tcacactcgctttcggtgaaaaggtctaactggattaaaaagtcaaaagcggcttcagta agaaagtcacattcactttcggtaagaaaatctaattcggcgaaaaggtcacatgctatt tcggtaagaaagtcaaagtcattatcagttaaaaagtcaatttcgcagagccaatcagca agtgtggcgaaatcgcaatcgatttcaagaagtcaatcagtagcagcgagtgagtcggca tcgctaag aagtcgaagagcacatcgctcagtaactcagtgagtgcagagaaatcgacg tcattaagtcgttcagcaagtgtagcaaaatcgcaatcgatttcaagaagccaatcagta gtagcgagcgaatcggcatcgttaagtaagtcgaagagcacatcgctcagtaactcagtg agtgcagagaaatcgacgtcattaagtcgatcagcaagtgtagcaaaatcgcaatcgatt tcaagaagccaatcggtggcagcgagcgaatcggcatcgttaagtaagtcgaagagcaca tcgctcagtaactcagtgagtgcagagaaatcgacgtcattaagtcgatcagcaagtgta gcaaaatcgcaatcgatttcaagaagccaatcggtggcagcgagcgaatcggcatcgtta agtaagtcgaagagcacatcgctcagtaactcagtgagtgcagagaaatcgacgtcatta agtcgatcagcaagtgtggcaaaatcgcaatcgatttcaagaagccaatcagtagtagcg agcgaatcggcatcgttaagtaagtcgaagagcacatcgctcagtaactcagtgagtgca gagaaatcgacgtcattaagtcgatcagcaagtgtagcaaaatcgcaatcgatttcaaga agccaatcggtggcagcgagcgaatcggcatcgttaagtaagtcgaagagcacatcgctc agtaactcagtgagtgcagagaaatcgacgtcattaagtcgatcagcaagtgtggcaaaa tcgcaatcgatttcaagaagccagtcagtagcagcaagtgagtcggcatcattaagtaag tcgaagagcacatctttaagcaactcagtgagtgtagagaaatcgacgtcattaagtcga tcagcaagtgtggcgaaatcgcaatcgatttcaagaagtcaatcagtagcagcgagtgag tcggcatcgctaagtaagtcgaagagcacatcgctcagtaactcagtgagtgcagagaaa tcgacgtcattaagtcgttcagcaagtgtagcaaaatcgcaatcgatttcaagaagccag tcagtagcagcaagtgagtcggcatcattgagtaaatcaacaagtacgtcaacaagtgac tcagatagcgcgtcaacatcaacatctgtatcagatagcgattcagcttcattgagtaag tcgactagtacatcaacaagcgattcagacagcgcgtcagcatcattgagcaagtcaaca agtacatcaacgagcgactcagatagcgcatcgacatcaacatcagtatcagatagcgac tccgcatcgttgagtaaatcgacaagcacgtcaacaagtgattcagacagcacgtctact tcattgagtaagtcgacaagtacatcgacaagtgattcagatagtgcg caaaatcaacg tcagtatcagacagtacgtccgcatcattgagtaaatcgacaagcacgtcaacaagtgat tcagatagtgcatcaaaatcaacgtcggtatcagatagcacgtcagcatcattaagaaag tcggcaagtacgtcaacgagtgactcagacagcacgtctacttcattgagtaagtcgaca agtacatcgacaagtgattcagatagtgcatcaaaatcaacatcagtatcagatagcgat tcagcttcattgagtaagtcgactagtacatcaacaagcgattcagatagtgcgtcaaaa tcaacgtcggtatcagatagcgactccgcatcgttgagtaagtcgacaagtacgtcaaca agcgattcagacagtgcatcaaaatcaacgtcggtatcagacagtacgtcaacatcatta agtaagtcgacaagtacatcaacaagcgattcagatagtgcgtcaacatcgacatcagta tcggacagtacgtctgcatcattgagtaagtcgacaagcacatcgacaagtgattcagat agcgcatcaacatcagtgtcagatagcgattcagcatcactaagcaagtcaacaagtaca tcgacaagcgattcagacagcgtatcaacatcaacatcagtatcagatagtgattccgcg tcattaagtaagtcgacaagtacgtcaacaagcgattcagatagtgcgtcaaaatcaaca tcagtatcagatagcacgtcaacatcattgagtaaatcaacaagtacatcgacaagtgac tcagatagtgcgtcaacatcggtatcagacagtacgtccgcatcattgagtaaatcgaca agcacgtcaacaagtgattcagatagtgcatcaaaatcaacatcagtatcagatagcgat tcagcatcattaagcaagtcgacaagtacatcgacaagtgattcagatagtgcgtcaaca tcaacgtcagtgtcagatagcgattcagcttcattaagcaaatcaacaagtacgtcaaca agtgactcagatagcgcatcaacatcattaagcaagtcaacaagtacatcgacaagcgat tcagacagtacgtctacatcattaagtaagtcaacaagtacatcaacaagtgattcggat agtgcgtcaaaatcaacatcagtatcagatagcgactcagcttcattaagcaagtcgaca agtacgtcaacaagtgactcagacagtgcgtcaaaatcaacatctgtgtcagatagcgac tccgcatcgttgagtaagtcgacaagtacgtcaacgagcgattcggatagtgcgtcaaaa tcaacatcagtatcagatagtgaatccgcgtcattaagcaagtcgacaagcacatcgaca agtgactcagatagtgcgtcaacatcgacatcggtatcagacagcacatcagtttcatta agcaagtcgacaagcacgtcaacaagcgattcagacagtacgtctacttcattaagcaag tcgacaagcacgtcaacaagtgactcagatagtgactcagcttcgttgagtaaatcgaca agcacgtcaacgagcgattcagatagcgtgtcaacatcaacatctgtgtcagatagcgat tcagcttcattaagcaaatcgacaagtacatcaacaagcgattcagatagtgcgtcaaca tcaacgtcggtatcagatagcggctccgcatcgttgagtaagtcgacaagtacgtcaacg agcgattcagacagtgcatcaaaatcaacgtcggtatcagatagtgattcagcatcacta agcaaatcgacaagcacgtcaacaagtgactcagacagtgcgtcaacatcgacatcggta tcagatagcacatccgcgtcgttaagcaagtcgacaagtacgtcaacaagtgattcagac agcgcatcgacatcaacatcagtatcagatagcgactccgcatcgttgagtaaatcgaca agcacgtcaacaagtgattcggacagtgcgtcaaaatcaacatcagtgtcagatagcgat tcagcttcattgagtaagtcgacaagcacgtcaacaagcgaatcagacagcgcgtcaaaa tcaacgtcagtgtcagatagcgattccgcatcattaagtaaatcgacaagcacgtcaaca agtgactcagatagtgcatcgacatcaacgtcagtatcagatagtgattccgcgtcatta agcaagtcgacaagtacgtcaacaagtgactcagacagtgcgtcaaaatcaacatcagta tcagatagcgattccgcatcattgagtaagtcgacaagcacgtcaacaagcgaatcagac agtgcgtcaacatcgacattagtatcggatagtacgtcggtttcattgagccaatcaaca agtgtggataaagatagtacagcgaagggatcgacagaattagtaaatgttgcatcactt tcaatcagtgcgagtcaatcaagtagtttatctgcttcaacatccacatcgattgaaaag tctgagtctacatcaacaagtggctcaaattcaactaatgcgtcgttaagtagctcatct tcacttagtacatcagcaagtacttctgtaagcgaagtgacatctgtcacacattctgaa aatgatttaagtgcatctaacgatagagatacatccggatcagtaagtcaatttgcttct gaaaatacatcattaagtgattctgcatcaattagtggcgaagtttctagtagtacgtcc gcgtcaacttcgaaatcatcatcactttcagcaagcgcgttacatgataagcatgtatca gaaagcacttctgcatcattaagtagtggagattcaagtcgtgcttcggcatcagtgtca acgtcattatcagaatcagatagtgcgttaatagactctgaatcaattagcgtttccgag cacacatcaacattacaatcaggtagtcattcactatcacaacaacaatcagcagaatta tcacaatcagagcaaacatcacaatcacaacgcatttcaacaagtgcgtcagtatcggct atgaaatcagaaagtgctgctaaggtatctgaatcgctatctacgtctcaatcaaaagta gatagtcaatcacaatcggtatctgaatcagcgagcaactcacgagtgtcaagagattca aaatcaacaagcgcttcaatgcatcgatcattgtcagagtcagtatctcaaagtatgtca cttattgatcagtcagaaagtgattcaacatctatatcgatttcgacgtcaatcagtgat gaagactctatgctgtattctatgagtgattccgcatcgatcagtactaaggcatcaagt agtatgtctacttcgacaagcgaagagcatgccaacagtcattctcagtctgaatcgaca gcatcggttgaagtatctcaagaaatgagtgcatcggcttcaacaagcaaatctgagtct caatcagagtcagtatcagtaagtaacgaagaatcaaatatctcatctatgcaagagtct tttgtagagagtgcaaaagcatcgcgtagtgcatctatgagcgttgcaaaatctgaagcc tctgaatcacagctattaagtgagtctaatgcttcggtaagccaatcagcaagcacaagt agtaaagcatcagcaagtacgtcagaatctatttcaacgtcactcagcgtatctgaagca actcatggaaaaccgagaaatcattcagaaagtgcatcagcaagtcaattattagaagaa aatgagtcattaagcgattcagcatcaacaagtgttgaagattcagaaagtgcatcagca tctctgtcggtgtatcaatcacaatcagcaagtgcattgaaatcaacacatgcatcagaa aaagcttcagtgaatacaagtgcaaacgcatcgaagcgtgcatcagcatcgacatctatc tctaactcgaaatctaaagtcattgcgagtgaatcgaagtcaacaagcatatcaacatat gaatcgttgtcaatatcgactagtaaagaacaatcaacgcgtgtatcagtgagtgagtcg acatcaacgtctaaagtgaagtcagaaagcgactcggcatcaacgtcgacatctgaatca atctcaattagcgcaaatcgttcaggttacacatcgtctaaacgttcggtacaaatgagt gaagcacaatcaacgagcgattcattatcagtaatgcaatctgaaggttcagtaagtgta tcgcaatctttaagtatatcagataagacatcacagtccttatcggaatcaatatcgcat tcagaaagtgactctgatagtaactcagtgtctattagtcaagagacatctgaacaacat tcggtgtcagacagtgactcgatgtcaatttcggaaagcgaatctattgcatatagtcaa tcagcgagtgaatcagaatcaacaagtatcgcaaaatctgatagtatttcgaactcatta tctgtttcattaagtgaatcagaaagtgaagcaagcacatcagcttcagtgagtacatct gaaagtacgtctgtaaagggttctctatcaacaagtatcttgaacagtcaatcagcatct actcatcaatcaacagaagcttctcaaagtacatcaacttcaaaagttgaggaagcatca ttgagtgactctgcttctgtatcagattcacaatcactttcaatgagtcatgagaaatca caaagtgcatcgacttcaaaatctacgagtctgtcaaaaactatttctgagtcagagtct gtgagtgcatcaacatcaacaagtgaagctgtaagtacagaagcaagcgaatttgtatca gcagtagactcattgagtcaagtaacttctaacggaagcacaacgaaagaagatgcgagt acatttgtatccacagtagattcattgaaagacaaagcatcaaataatggtacaccatca gagtttgcgtcagcagtgaaatcaacacacgcatcagtgagtgtgtcagcatcagaaagt acgtcagcatcaacatcaacaagtgaagctgtaagtacagaagcaagcgaatttgtatca gcagtgaattcgttgagtgaagcgacttctaacggaagcacaacgaaagaagatgcgagt acatttgtatccacagtagattcattgaaagacaaagcatcaaataatggtacaccatca gagtttgcgtcagcagtgaaatcaacacacgcatcagtgagtgtgtcagcatcagaaagt acatcagcatcaacatcaacaagtgaagctgtaagtacagaagcaagcgaatttgtatca gcagtagactcattgagtcaagtaacttctaacggaagcacaacgaaagaagatgcaagc acatttgtatccacagtagattcattgaaagataaagcatcaaacaatggtacaccatca gaatttgaatcagttgtgaaatcagtacacggatcaatgagtgcatcagcaagtgcgtca acatcagcatctacatcagcatctacatctacaagtgaagctgcaagtgcagaagcaagc gaattagaatcagtaaggaaatcattatccaatggagcatcaaacggtagcacagcaaga gaaggtgcaagcacatttgtatcaacggtagattcattgaaagataaagcatcaaacaat ggtacagcatcagaatttgaatcagttgtgaagtcagtacacggatcaacaagtgcatca gcaagtgcgtcaacgtcagcatcaacatcagcaagtgaatcagcaagtacagaagcaagt gaatttgtatcagcagtggcatcattaagcagttcagcatggaacggaagcactacagga gaaggtgcaagcacatttgtatcaacagttgattcatcgaaagatt cagcgtcagacaaa gettcaecatcagaatcagaatcagttgtgaagtcagtacacggatcaacgagtacatca gcaagtgtgtcagcgtcggcaagtacatcagcatcgacatcaacaagtgaagctgtaagc acagaagcaagtgagtttgtatcagcagtgaactcattaagcagtgaagcatcgaacggc agcacaacaagagaaggtgcaagcacatt tgtatcaacagtagat tcattgaaagacaaa gcatcaaacaatggtacagcatcagaatttgaatcagttgtgaagtcagtacacggatca atgagtacatcagcaagtgtgtcagcatcagaaagtacgtcggcatcgacatcgacaagt gaagctgtaagtacagaagcaagcgagtcagcatcgataagtgtatcaatgtcagtgagc gcatcaacaagtgcttcaatgagcgtatcagtgtcaaacagtgtgtcagtgagtgactct atttcagtaagtgcatcaacaagtgaacctaactcggtaagcacttctatgagtagttct ctttcaacatcggcatcaacgccatcagaaattacttcaagttcgtcatcaagcgattca gcgacagttcaaaaagtagtttctaaagatgaacagcacgctacaaataaagttgaaaaa ttacctgacacaggtcaatcaacgacacaaactggtttattgggtggagtaggtgcttta cttacaggccttggtttactcaaaaaatcaagaaaacaaaaagatgaagaaacatcatca catgaataa
The protein sequence translated from SEQ ID NO 1 1 is designated SEQ ID NO and is shown below:
SEQ ID NO: 12
MTRKFREFKK SLSEEKARVK LYKSGKN VK AGIKEFQLL ALGLSFLSHD IVKDENGEVT TQFGEQLKKN ALRTTAFAGG MFTVNMLHDQ QAFAASDAPI TSELATKSQT IGDQTSIVIE KSTSSDQSTN PITESESKHD SESISLSEHQ TSESTSLSTS TSKSISTSVE ESESTSKDSH TKTQDSQSDS HQSTSQEVNG SSNHEQSTPH TAQSLTSLSI ESQTSTSNTS LKETKEGELS KNLSKLSQNQ NIKLHEEHTM RSADLSSGYT GFRAAYYVPR SRTTPTTKVY TGQGSFRGRG RIKYNIFYKV VVTSNGKEMK IRYTLSQDDP NTSNVEKPRW AGQKRFGIHN TWDEGPGRGQ LKLGSAFGKP TVIQGETRPN YGS VGTPIT KYVSGDRTNG FYWQAAVLAP RHGEKGEGIT AEITVPIVNP SGRFNWEFHP VGQQDGVGGK TDYFENV IR DYDPYYKYIQ TKEGRASVSH SISQVKASES RSTSLIQSES IRRSQSISES ESIVAASHSA SVAKSQSISR SQSVAKSQSI SRSQSIAHSR SASVA SQSI SRSQSIAHSR SASVAKSQSI SRSQSIAHSR SASVAKSQSI SRSQSIAQSQ SASVAKSQSI SRSQSIAQSQ SASVAKSQSI SRSQSIAHSR SASVAESQSI SRSQSIANSQ SVAASESESL SISLSKKQSI SMSNSESAAK SHSLSVKRSN WIKKSKAASV RKSHSLSVRK SNSAKRSHAI SVRKSKSLSV KKSISQSQSA SVAKSQSISR SQSVAASESA SLSKSKSTSL SNSVSAEKST SLSRSASVAK SQSISRSQSV VASESASLSK SKSTSLSNSV SAEKSTSLSR SASVAKSQSI SRSQSVAASE SASLSKSKST SLSNSVSAEK STSLSRSASV AKSQSISRSQ SVAASESASL SKSKSTSLSN SVSAEKSTSL SRSASVAKSQ S SRSQSVVA SESASLSKSK STSLSNSVSA EKSTSLSRSA SVAKSQSISR SQSVAASESA SLSKSKSTSL SNSVSAEKST SLSRSASVAK SQSISRSQSV AASESASLSK SKSTSLSNSV SVEKSTSLSR SASVAKSQSI SRSQSVAASE SASLSKSKST SLSNSVSAEK STSLSRSASV AKSQSISRSQ SVAASESASL SKSTSTSTSD SDSASTSTSV SDSDSASLSK STSTSTSDSD SASASLSKST STSTSDSDSA STSTSVSDSD SASLSKSTST STSDSDSTST SLSKSTSTST SDSDSASKST SVSDSTSASL SKSTSTSTSD SDSASKSTSV SDSTSASLRK SASTSTSDSD STSTSLSKST STSTSDSDSA SKSTSVSDSD SASLSKSTST STSDSDSASK STSVSDSDSA SLSKSTSTST SDSDSASKST SVSDSTSTSL SKSTSTSTSD SDSASTSTSV SDSTSASLSK STSTSTSDSD SASTSVSDSD SASLSKSTST STSDSDSVST STSVSDSDSA SLSKSTSTST SDSDSASKST SVSDSTSTSL SKSTSTSTSD SDSASTSVSD STSASLSKST STSTSDSDSA SKSTSVSDSD SASLSKSTST STSDSDSAST STSVSDSDSA SLSKSTSTST SDSDSASTSL SKSTSTSTSD SDSTSTSLSK STSTSTSDSD SASKSTSVSD SDSASLSKST STSTSDSDSA SKSTSVSDSD SASLSKSTST STSDSDSASK STSVSDSESA SLSKSTSTST SDSDSASTST SVSDSTSVSL SKSTSTSTSD SDSTSTSLSK STSTSTSDSD SDSASLSKST STSTSDSDSV STSTSVSDSD SASLSKSTST STSDSDSAST STSVSDSGSA SLSKSTSTST SDSDSASKST SVSDSDSASL SKSTSTSTSD SDSASTSTSV SDSTSASLSK STSTSTSDSD SASTSTSVSD SDSASLSKST STSTSDSDSA SKSTSVSDSD SASLSKSTST STSESDSASK STSVSDSDSA SLSKSTSTST SDSDSASTST SVSDSDSASL SKSTSTSTSD SDSASKSTSV SDSDSASLSK STSTSTSESD SASTSTLVSD STSVSLSQST SVDKDSTAKG STELVNVASL SISASQSSSL SASTSTSIEK SESTSTSGSN STNASLSSSS SLSTSASTSV SEVTSVTHSE NDLSASNDRD TSGSVSQFAS ENTSLSDSAS ISGEVSSSTS ASTSKSSSLS ASALHDKHVS ESTSASLSSG DSSRASASVS TSLSESDSAL IDSESISVSE HTSTLQSGSH SLSQQQSAEL SQSEQTSQSQ RISTSASVSA MKSESAAKVS ESLSTSQSKV DSQSQSVSES ASNSRVSRDS KSTSASMHRS LSESVSQSMS LIDQSESDST SISISTSISD EDSMLYSMSD SASISTKASS SMSTSTSEEH ANSHSQSEST ASVEVSQEMS ASASTSKSES QSESVSVSNE ESNISSMQES FVESAKASRS ASMSVAKSEA SESQLLSESN ASVSQSASTS SKASASTSES ISTSLSVSEA THGKPRNHSE SASASQLLEE NESLSDSAST SVEDSESASA SLSVYQSQSA SALKSTHASE KASVNTSANA SKRASASTSI SNSKSKVIAS ESKSTSISTY ESLSISTSKE QSTRVSVSES TSTSKVKSES DSASTSTSES ISISANRSGY TSSKRSVQMS EAQSTSDSLS VMQSEGSVSV SQSLSISDKT SQSLSESISH SESDSDSNSV SISQETSEQH SVSDSDSMSI SESESIAYSQ SASESESTSI AKSDSISNSL SVSLSESESE ASTSASVSTS ESTSVKGSLS TSILNSQSAS THQSTEASQS TSTSKVEEAS LSDSASVSDS QSLSMSHEKS QSASTSKSTS LSKTISESES VSASTSTSEA VSTEASEFVS AVDSLSQVTS NGSTTKEDAS TFVSTVDSLK DKASNNGTPS EFASAVKSTH ASVSVSASES TSASTSTSEA VSTEASEFVS AVNSLSEATS NGSTTKEDAS TFVSTVDSLK DKASNNGTPS EFASAVKSTH ASVSVSASES TSASTSTSEA VSTEASEFVS AVDSLSQVTS NGSTTKEDAS TFVSTVDSLK DKASNNGTPS EFESVVKSVH GSMSASASAS TSASTSASTS TSEAASAEAS ELESVRKSLS NGASNGSTAR EGASTFVSTV DSLKDKASNN GTASEFESVV KSVHGSTSAS ASASTSASTS ASESASTEAS EFVSAVASLS SSAWNGSTTG EGASTFVSTV DSSKDSASDK ASPSESESVV KSVHGSTSTS ASVSASASTS AS STSEAVS TEASEFVSAV NSLSSEASNG STTREGASTF VSTVDSLKDK ASNNGTASEF ESVVKSVHGS MSTSASVSAS ESTSASTSTS EAVSTEASES ASISVSMSVS ASTSASMSVS VSNSVSVSDS ISVSASTSEP NSVSTSMSSS LSTSASTPSE ITSSSSSSDS ATVQKVVSKD EQHATNKVEK LPDTGQSTTQ TGLLGGVGAL LTGLGLLKKS RKQKDEETSS HE
SEQ ID NO: 13
atgaaaaagtctagaaaaaagcgtatcgattttttacctaaccgtcaaaatcgatat gcg atacgtcgtttttcagtaggcactgcgtcaattctcgttggagcaacattaatttttgga attcattcaaatgatgcatcggcagcagtagaagacgcaacatctcaagaagcaggaaca actaacgaaaattcaaatagtacagaagaagcaacaacaaacgaaagtacaactgttgaa gcaccaacaagtgaagaagcaacaacggaagagcaatcagtagaggcgccaacaagtgaa gaagtaacaacggaagagcaatcagtagaggcaccaacaagtgaagaagtaacaacggaa gagcaatcagtagaagcgccaacaagtgaagaagtaacaacggaagagcaatcagtagaa gcgccaacaagtgaagaagtaacaacggaagagcaatcagtagaggcaccaacaagtgaa gaagtaacaacggaagagcaatcagtagaggcaccaactagtgaagaagtaactacggaa gagcaatcagtagaagcaccaacaagtgaagaagcaacaacggaagagcaatcagtagaa gcaccaacaagtgaagaagcaactacaaaaactcctgtaaaagaagaaacatcctcaaca caagaaaattcacccacgactacactagaagaacaattttcaaatgaattcaatcagtta acatctacagaagataaaacaaactacacacgtgaatatttaactcaaaacacaaatctt tcggcagaacaagtggaagcaacagttgaacgcttgaatttaagtcaagaaaatgtaaca gcccaagatatctatttcgcattacttaaagatttagctgatcaacaagatgccttatta ccacgtgtaacacttttggccgctagagattctgagctcacaaacgaagcgtctatcgct ttaactgaaaatagtccaatgttccgcgcagcattagcgaatagtccttctggcaatgat gtggtgtcagaagaagataatattattgtggctgatgcactcgcaaatggatacatcaat tcacaaacagatgcaacaaatgcggcaaatacattgtctggtcgtgcatgggttgtggat acagggacaccagcgacaatgtcaaacggcttaacagctgttccagaaggcacaaaagtc tacatgcaatggattgatacagatggcgcggtttcaccagtgtatcaagcaagcacaaca aataaattgagttcaagtggtggtagccaagtaggtccaggtgcatatgcatttgattta cgtgaagcatggatagactcaaatggcaaagcgcacagatatgaagcgtcaagtggccaa tattatcgtttatggattgatgactacaaaacagtagatgggaatacggcaaccatgtta cgccaagcaggtggtttcttccctggttcatatgttaattcggtgacaggtaacaatatt ggtcaattcccacttatcggaacgaacatgcaacgtacaggtatctttatgggtgtgata ccaacgaacgattacatgactacagatacaagcaattggattcaagataatgaaggacct atttcaaacccagcagtaacgagcacaagtgaatttgtcagtggtaaagtatggtctgag acaggttcaggtgactatgcgaactctgcgacaggtccaaactttaactcaggtgatatt gcacgtgaaggttatcaagttgtcatgtcttcattaacaagtgctggtgcccaagcgtat aaagcacaagtcgaatcgttgccaacagaccaacaagcggcagcagcacaccaattattc aaagaccacccagaatttatttctgcgacagtgacgggtaaaactgatgcaaacggtgcg tatacattacgtttcccttcaggctcattgagtaaagattatctttatggttatgtgatg gataataagggcaacttggttaagggctattcatcattcacgtcacctttattccgttcg cctaacagtaacttatctttcgcgccacaaacagcgccatatcatagaccagccaaaaat gcttgggtgaatgtgaactttgcgcttgtagaaacaattgaaacaagtatagacatcacg aactttgatgtgacagccaacccagcgcaacgtggtgatacggctatcattgatgtgact tctacagcattgtcaccattacctacgcatgttgagtggagagattcaaaagggaatgtc gttcaaaaaagtggagatgtcactacggtagaagaagctgaaacggcaggcacatttact attcctgatgatgcgaaaacaggtgaaatctatacagtttatattgtttcaggaggcaat gaagttgcagcagactcactgattgtccaagtgcaagaaaatgcggcaacctatgaacct gtatatccaacaacaacagttgaacaagaccaaactgtaacaattcctacacctacaaat gaagatggtttagcattaccagacggaacaaagttcgaaggtggcaacaatgtacctgaa tgggcaactgtgaatgaagatggttctatttcaatttcaccaaatcaagatgtggaaaaa ggtaactataatgtgcctgttgtcgtcacatatccagatggttcaaaagaaacagtattt gcaccagttttagttcaagaagctgttccaactgcagaacaatacgatccaacaattgaa acaattaataaggaatatggtactactgcaacagaagatgaaattaaaggcgcaatcaca attccggattacccaacagatggagatcaaccaacaatcacgattgacgacccaactcaa attccaaatggaacagaagaaggcacagtgaatgtaggtgtcactgtcacttatccagat ggttcaacagacaaattaacagtaccagtcgttacaggtaagcaagcggataacgataag tacacaccagaaacaacaccaattacgaaagacttcggtacaggtgtaacagaagacgaa gtgaaaggtgcagtcactgttccggattacccaacagatggagaccaaccaacaattacg attgacgacccaagtcagttgcctgatggttcaaaagaaggaacaacggatgtcgacgta acagtggaatatccagacggcacaacagatcacatcacagttccagtgactgttggaaag caagcggataatgataagtacacaccagaaacaacaccaattacgaaagacttcggtaca ggtgtaacagaagacgaagtgaaaggtgcagtcactgttccggattacccaacagacggt gaccaaccaacaattacaattgatgatccaaatcaattaccggacggttcacaagaaggt acgactgatgtaaatgtaacagtggaatatccagatggcacaacagatcacatcacagtt ccagtgactgttggaaagcaagcggataatgataagtacacaccagaaacaacaccaatt acgaaagacttcggtacaggtgtaacagaagacgaagtgaaaggtgcagtcactgttccg gattacccaacagatggagatcaaccaacggttacaattgatgatccaaatcaattaccg gacggttcacaagaaggtacgactgatgtaaatgtaacagtggaatatccagacggcaca acagatcacatcacagttccagtgactgttggaaagcaagcggataatgataagtacaca ccagaaacaacaccaattacgaaagacttcggtacaggtgtaacagaagacgaagtgaaa ggtgcagt cactgttccggattacccaacagacggtgaccaaccaacggttacaattgat gatccaaatcaattaccggacggttcacaagaaggtacgactgatgtaaatgtaacagtg gaatatccagatggcacaacagatcacatcacagttccagtgactgttggaaagcaagcg gataacgataagtacacaccagaaacaacaccaattacgaaagacttcggtacaggtgta acagaagacgaagtgaaaggtgcagtcactgttccggattacccaacagatggagatcaa ccaacggttacaattgacgatccgagtcagttaccagatggctcacaagaaggcacaaca gatgtgaatgtaacagtggaatatccagatggcacaacagaccacatcacagttccagtg actgttggtaagcaagcagataacgataaqtacacgccagaaacaacaccaattacgaaa gacttcggtacaggtgtaacagaagacgaagtgaaaggtgcagtcactgttccggattac ccaacagatggagaccaaccaacaattacaattgacgatccgagtcagttaccagacggt tcacaagaaggtacgactgatgtaaatgtaacagtggaatatccagatggcacaacagat cacatcacagttccagtgactgttggtaagcaagcagataacgataagtacacaccagaa acaacaccaattacgaaagacttcggtacaggtgtaacagaagacgaagtgaaaggtgca gtcactgttccggattacccaacagatggagaccaaccaacaattacaattgacgatccg agtcagttaccagacggttcacaagaaggtacgactgatgtaaatgtaacagtggaatat ccagatggcacaacaga cacatcacagttccagtgactgttggaaagcaagcagataac gataagtacacaccagaaacaacaccaattacgaaagacttcggtacaggtgtaacagaa ggcgaagtgaaagattcaatcacaattcccggttacccaacagatggagaccaaccaaca attacaattgacgacccaagtcagttaccagatggttcacaagaaggtacgactgatgtc gatgtaacagtggaatatccagacggcacaacagatcacattacagttccagtgactgtt ggaaagcaagcagataacgataagtacacaccagaaacagaaggtgtcaacaaagatcat ggtacgtcagtaacagaagatgaagtgaaaggtgcagtcactgttccgggatacccaaca gatggagatcaaccaacggttacaattgatgatccaagtcaattgccggacggttcacaa gaaggtacgactgatgtaaatgtaacagtggaatatccagacggcacaacagaccacatt acagtcccagtaactgttggtaaacaacctactaaagataacggggctacagataatgat ggcgacatgaatcaaggcacagatgaaggaaatagtgctactgatcatggcgacaatgta aaacaagattcaaacggaaactatacgccggttgaacaacgtgacaatcatgcgacttca cctgcaacagatatggatccaatgccaagcaatagccaaacaacttttgatggcataaat gcaaaaggttcaacttcagagaaagcaaaccataaacaacagtctgagcaattaccagac acaggtgaaagcaatacacaaaatggtgcacttttaggcggattatttgcagcacttgga ggcttattcttaatcggcagacgtcgtaaagaaaaagaaggcaaataa The protein sequence translated from SEQ ID NO 13 is designated SEQ ID NO: 14 and is shown below:
SEQ ID NO: 14
MKKSRKKRID FLPNRQNRYA IRRFSVGTAS ILVGATLIFG IHSNDASAAV EDATSQEAGT TNENSNSTEE ATTNESTTVE APTSEEATTE EQSVEAPTSE EVTTEEQSVE APTSEEVTTE EQSVEAPTSE EVTTEEQSVE APTSEEVTTE EQSVEAPTSE EVTTEEQSVE APTSEEVTTE EQSVEAPTSE EATTEEQSVE APTSEEATTK TPVKEETSST QENSPTTTLE EQFSNEFNQL TSTEDKTNYT REYLTQNTNL SAEQVEATVE RLNLSQENVT AQDIYFALLK DLADQQDALL PRVTLLAARD SELTNEASIA LTENSPMFRA ALANSPSGND VVSEEDNIIV ADALANGYIN SQTDATNAAN TLSGRAWVVD TGTPATMSNG LTAVPEGTKV YMQWIDTDGA VSPVYQASTT NKLSSSGGSQ VGPGAYAFDL REAWIDSNGK AHRYEASSGQ YYRLWIDDYK TVDGNTATML RQAGGFFPGS YVNSVTGNNI GQFPLIGTNM QRTGIFMGVI PTNDYMTTDT SNWIQDNEGP ISNPAVTSTS EFVSGKVWSE TGSGDYANSA TGPNFNSGDI AREGYQVVMS SLTSAGAQAY KAQVESLPTD QQAAAAHQLF KDHPEFISAT VTGKTDANGA YTLRFPSGSL SKDYLYGYVM DNKGNLVKGY SSFTSPLFRS PNSNLSFAPQ TAPYHRPAKN AWVNVNFALV ETIETSIDIT NFDVTA PAQ RGDTAIIDVT STALSPLPTH VEWRDS GNV VQKSGDVTTV EEAETAGTFT I PDDAKTGEI YTVYIVSGGN EVAADSLIVQ VQENAATYEP VYPTTTVEQD QTVTIPTPTN EDGLALPDGT KFEGGNNVPE WATVNEDGSI SISPNQDVEK GNYNVPVVVT YPDGSKETVF APVLVQEAVP TAEQYDPTIE TINKEYGTTA TEDEIKGAIT IPDYPTDGDQ PTITIDDPTQ IPNGTEEGTV NVGVTVTYPD GSTDKLTVPV VTGKQADNDK YTPETTPITK DFGTGVTEDE VKGAVTVPDY PTDGDQPTIT IDDPSQLPDG SKEGTTDVDV TVEYPDGTTD HITVPVTVGK QADNDKYTPE TTPITKDFGT GVTEDEVKGA VTVPDYPTDG DQPTITIDDP NQLPDGSQEG TTDVNVTVEY PDGTTDHITV PVTVGKQADN DKYTPETTPI TKDFGTGVTE DEVKGAVTVP DYPTDGDQPT VTIDDPNQLP DGSQEGTTDV NVTVEYPDGT TDHITVPVTV GKQADNDKYT PETTPIT DF GTGVTEDEVK GAVTVPDYPT DGDQPTVTID DPNQLPDGSQ EGTTDVNVTV EYPDGTTDHI TVPVTVGKQA DNDKYTPETT PITKDFGTGV TEDEVKGAVT VPDYPTDGDQ PTVTIDDPSQ LPDGSQEGTT DVNVTVEYPD GTTDHITVPV TVGKQADNDK YTPETTPITK DFGTGVTEDE VKGAVTVPDY PTDGDQPTIT IDDPSQLPDG SQEGTTDVNV TVEYPDGTTD HITVPVTVGK QADNDKYTPE TTPITKDFGT GVTEDEVKGA VTVPDYPTDG DQPTITIDDP SQLPDGSQEG TTDVNVTVEY PDGTTDHITV PVTVGKQADN DKYTPETTPI TKDFGTGVTE GEVKDSITIP GYPTDGDQPT ITIDDPSQLP DGSQEGTTDV DVTVEYPDGT TDHITVPVTV GKQADNDKYT PETEGVNKDH GTSVTEDEVK GAVTVPGYPT DGDQPTVTID DPSQLPDGSQ EGTTDVNVTV EYPDGTTDHI TVPVTVGKQP TKDNGATDND GDMNQGTDEG NSATDHGDNV KQDSNGNYTP VEQRDNHATS PATDMDPMPS NSQTTFDGIN AKGSTSEKAN HKQQSEQLPD TGESNTQNGA LLGGLFAALG GLFLIGRRRK EKEGK
SEQ ID NO: 15
atgacagaacgaaaatccccttcatctcaaaacatgcgtcatcgtttagtcaaagct ggt actgtccttttattggttggtagtggactgcaaatgccttcaacattgtcacacgaaatg acagcgatagctcagacagatgcgactgatgatttgaaaacattacgtgaaaatgcagat aaaaaagtgaaagcgttacaatatttaaatacggattataaaaatgaatttcttgcgtta attcgtgaatatgatacgtcgtcaaaaaatattgaagtggttgttgacgaagcagaagca gccaatcgtctagctcatgacgctcaatcggacgatgaaatacaacctgaattagatgcc attgatgaaaaaattagcgcgttaaaggcaaaggttgatgaaggtcaacgagaatcaact gaagcgcgtcaagatgtaacgtcaacagagacaaagagtgctgaatcagaaggaagagag ccatccactgaaggcgagagcaaagtaaaggagtcatcttcagcacaaacgattgtagca cctcatcatggtcaacaagatgtgagcgcactgaaagaccatattaagaacgatgtcgat acacttaaacaagactatgcaacgcaagacaagcaagtgacaccactccagggcattgac agtgcaatcacacgcattgaccatttcgtttcagaaagcgtggatcacaagtctgacaat tattttgaagaaaaacgtcaacatttacaaaactttgaacaagacattaaaaaacgtacg gacatttctgggactgagaaggcgactttgcttgatgatgcgaaaacggtagccaaccaa ctgaacgcgcaaaatgatacgattttaactgaacttcaacagcatgacgataaacgtgca gcagttgaatcgatattaggtgagatttttaatgcacaagaagcggctgaacgtgcgaaa cagatagatgttaaaggtaaaacagatcaacaattggcaaacgaaattcatcaacaagcg gacggacttatcaaaacgtcgagtgatgatttattgttaggaatgttggaaaataattca aatacacaaggtctagtggaaagcattttacgaacacgctttgacaaacaagaagcgcac aaaattgccggcgaaatcatgcaaggcaagccttcaaatacagcgatactcgaccgcttg aaagaccattttaaagcgaatggtaaggcgagtggagatgatattttaaatgcgttaatt aataatacggatgcagatgctgaagtgattgaatcaattctagggggccgtcttaatgca gaaaatgcaaaattgattgccgatcgtgtacagcaagataaaaagaagacacatcaaaac ttaaaggcgattgaagacgaacttagtgcgcaagcgaatcgattgttaacgttacggaag caattgcaacaaatccgtcataatacgcaaacagatatgaatgacttgtttgcaccactg cgtcgtattgcaaatattctcggtggtggtttaaatcgtgacgacattcactcttcaggt cgtacgaatgacaaattgcagcaactgttaaatcgtgatcattcgttgttaggtcgtggt ggtgatttattcaaacatgattttgcgccaaagccgaatatcgatccatatcaagcgatt aatagtcaaacggcatcagaaggttttttagatggtttatttgatcaaaatggcgatttc aatttaccgaatacaggtgaaatagtgaagcggacttggctaccgttgggtattttagtc gttgcaatcggtgtactgatcttaacggtgagatttcataaaaaaacacgcaaacaataa
The protein sequence translated from SEQ ID NO 15 is designated SEQ ID NO: 16 and is shown below: SEQ ID NO: 16
MTERKSPSSQ NMRHRLVKAG TVLLLVGSGL QMPSTLSHEM TAIAQTDATD DLKTLRENAD KVKALQYLN TDYKNEFLAL IREYDTSSKN IEVVVDEAEA ANRLAHDAQS DDEIQPELDA IDEKISALKA KVDEGQREST EARQDVTSTE TKSAESEGRE PSTEGESKVK ESSSAQTIVA PHHGQQDVSA LKDHIKNDVD TLKQDYATQD KQVTPLQGID SAITRIDHFV SESVDHKSDN YFEEKRQHLQ NFEQDIKKRT DISGTEKATL LDDAKTVANQ LNAQNDTILT ELQQHDDKRA AVESILGEIF NAQEAAERAK QIDVKG TDQ QLANEIHQQA DGLIKTSSDD LLLGMLENNS NTQGLVESIL RTRFDKQEAH KIAGEIMQGK PSNTAILDRL KDHFKANGKA SGDDILNALI NNTDADAEVI ESILGGRLNA ENAKLIADRV QQDKKKTHQN LKAIEDELSA QANRLLTLRK QLQQIRHNTQ TDMNDLFAPL RRIANILGGG LNRDDIHSSG RTNDKLQQLL NRDHSLLGRG GDLFKHDFAP KPNIDPYQAI NSQTASEGFL DGLFDQNGDF NLPNTGEIVK RT LPLGILV VAIGVLILTV RFHKKTRKQ
SEQ ID NO: 17
atgttaaaaaaattaattgttacaggtttgattgctacagcggcgacacaagtttat gcg catgacacgcaagcggcggaaaagggtgctacagatgctccgaatgtgatggt taaggat gaggcgaaaaaagaagtgacaccgataatccataaaccgacttgcatttacccgcatcta gaaggcgaagatgatgctgcgtatttaaaacgtatggcaacgaatccaccagaaggcgca gtgccgtacggtgtattgaataaagatggatcgattacagaaccgaatacaaatccacat tttgatgttttaaaaattgaagatccaaatgcgatgaaagatttggttgatacaccggca gatgatcaagatacggtaccgagtgatttacaaattgaaccaccagcattaataggacca gctactaaacatacggatggtacgggagacgcaaaatctaatgatgaccacaaagtaaca aaatcttcgggagcgtcagcccaagatatgaagaaaaaagacgtgacaacacaaactgca caaccaaaagcagataaaaagatggcgactgcaaaagtagcaccagcgaaacaacaagat aaagcagccaaaatgttaccagcagcaggggaaccacaagtgaatgcaatcagtcaaaca gcacttgcactttcaatgatcgcattaggtgtcatcgcgttctttacacgacgacgcaaa acaaattaa
The protein sequence translated from SEQ ID NO 17 is designated SEQ ID NO: 18 and is shown below:
SEQ ID NO: 18
MLKKLIVTGL IATAATQVYA HDTQAAEKGA TDAPNVMVKD EAKKEVTPI I HKPTCIYPHL EGEDDAAYLK RMATNPPEGA VPYGVLNKDG SITEPNTNPH FDVLKIEDPN AMKDLVDTPA DDQDTVPSDL QIEPPALIGP ATKHTDGTGD AKSNDDH VT SSGASAQDM KKKDVTTQTA QPKADKKMAT AKVAPAKQQD KAAKMLPAAG EPQVNAISQT ALALSMIALG VIAFFTRRRK TN SEQ ID NO: 19
atggtagaatataaaaaagaacatagcgtaaagcgactattaaaattaggaatcggt tca acgagtattttatgtgttgtatcacctcttttattaacacatgacgttgttcaagcagca gatatcaataacaggatgccagctttgaatacattgaagaccacttcttcatatgatcaa agggcacacatggatgaattacgaaacgccattacttcagatagtgacactactcaaaca ccatcattcaatgagataactgtgtcttcaactaatgaaacggatgcagcgtcaacggaa aatgtgaacccgagtgatgaggtcccggcaaaggatgaaagtgaatcaacgacaccgagt acagaacaagacacatctatagaagaaacgggtactgaagaagtgccatctcatgaagac aatcatcacaacaccccaagtcaagaagagcaaccgtctccgcctgatcaaccaggaaca aacaaagatgaagagagtggagaaaaaccgaataaagaaaatcatcggaagccgaatcaa ccgaacaaagaccaaccttcaaaagatgagaataaaaaacctgacaaaggaaacaaacca gcaccaccgtctaaaatgccaaatcgcccggatcaaaaggaagatggttcaaacaacacc ccaccacctgccactgataacggtggaaacagtaatgacggtacaacaacgggtcccaat ggtggaggtggcagtgaagcaagtccaccaccgaatgagcaaccgtcaaatggcaatgca agcgatacccatcaaaacggttcagtttcaagcaccaatcattcgaatcagtatggtaca tcggcttatgatgaatacgcaggtttattgaataataattataaatataatccattgttt aaagaagaggttgcgcgtttaagtcaatttggaagtcaagatcaacatgatattgcaagt ttgagtcgtaaagaacaattttctcaaaatgcatttttagatgacttgcaacaaagtaca gattattttagatatcaatattttaacccgctttccacagagcaatactatcatcgttta gataaacaagtattagcactcgttacgggggaatttggttcgatgccagatttcaagaaa agtggtgataagtcattggttaataagcatcagcaagataaagtgaagaaaattgaacag caaggagaaaatattaatacgcatcatatgaaaaatacgaaagaagatacaggaaaatca ttaagttacaagccgatgatatatattggcattgtcatggtcggttttgtcggcctgatc agtatgattttatggaaacgactgcatcatttttggaaataa
The protein sequence translated from SEQ ID NO 19 is designated SEQ ID NO: 20 and is shown below:
SEQ ID NO: 20
MVEYKKEHSV KRLLKLGIGS TSILCVVSPL LLTHDVVQAA DINNR PALN TLKTTSSYDQ RAHMDELRNA ITSDSDTTQT PSFNEITVSS TNETDAASTE NVNPSDEVPA KDESESTTPS TEQDTSIEET GTEEVPSHED NHHNTPSQEE QPSPPDQPGT NKDEESGE P NKENHRKPNQ PNKDQPSKDE NKKPDKGNKP APPSKMPNRP DQKEDGSNNT PPPATDNGGN SNDGTTTGPN GGGGSEASPP PNEQPSNGNA SDTHQNGSVS STNHSNQYGT SAYDEYAGLL NNNYKYNPLF KEEVARLSQF GSQDQHDIAS LSRKEQFSQN AFLDDLQQST DYFRYQYFNP LSTEQYYHRL DKQVLALVTG EFGSMPDFKK SGDKSLVNKH QQDKVKKIEQ QGENINTHHM KNTKEDTGKS LSYKPMIYIG IVMVGFVGLI SMILWKRLHH FWK SEQ ID NO: 21
gtgattacaaataaaaatatatatagtattcgaaagcataaacttggcgtggcatca ttc ttattggggacattatttgttgtagggcatgcaaataatgctgaagcttcagaagtgagc gcaacaacacaagaacataatgtcgagactgagcaaacaaaaactgagggcgaactaaca actgaggtagcacaacaagcagtcagcgaatcagcacctatagctgaaaacatgcagaaa acaacatcagtggcaagtgaaaatgcgaaagaggttacagcttctgatagcacacaagaa gtcacaaaaactgaagcaaaagatacagcaacaatgaaagattcagaaattgcacaacct gtatcagaagtgaataaacctgttactcaaacagctgcacccgtagcagaaccatcaaca gcaaacaaacaaacttcaccacgacaagtacaagaacttactgcaccaatggacacaaaa gtaattaatgtagaaaacggaacagatgtgacaagtaaagtgaaagttgaaaaatcgtca attacagggcatcagaataaagataaaacatatcatcaatcgaacactgtaaatccacat aaagctgaacgtgtgacattaaattatgattggtcatttgaaaatggaattaaagctggt gattattttgacttccaattaagcgataatgtcgatacaaatggaatatcaacaataaaa aaagtcccacacattatggatagtcaaaatagcgaacaaattattgcttacggggaaatt aatgaaaacaaccgtgtccgttaccgatttatggactatgtaaatcaaaaagaaaattta aaaggtaaattgtcattaaacttatttattaaaccagataaagttcaagatgaaggaaaa atcactgtcacttcacaattgggcaaggaaatgacaagtcaggaatttgacattaaatat attgatggtgtaaaaagcccttcaggtatcacattaaacggtcgtcttgatgaattatca aaagcagatcaatcatttacgcattattctatatttaaacctaagcataataacttaact aatgtaactttaagaggcacagtttcaaataacgcacagcaaaatgaaaaaaatggtcaa gttaatgtttacgaatatattggtcaaggagaattgccacaaagtgcttatgccaatgta aatgatacgaagcagttcaatgacattactaagagtatgaaatcaatcaaaaataacagt aatggctatgaaattacttttgacatgaacaaagacaatcatccttatatcatagtatat caaggtcactttaacaataatgcaaaagactttgatttctcaacaaatgcgacaggttat caaaatttaaatcaatcggaatatagttattattggccttacaattattcattcaattta acatgggataatggtgttgctttctactctaataatgcaagtggggaagggaacgacaaa cctgtaccgccgacttatggatatagtccgacagtaaatacaattcaagatactcatgcg gattatcctgtaatgactttccaacaacctggaactctagaggagacagaagacagtatg ccaatcactacacttaccgaatctggtgaggatcgtggtgaaaatacttctccaattatc gagacaacagaagattcacagcctgttgagtttgaagaagagacaaatcatggcattcaa gacgtgacacttcatgcagatgctgttgattttgaggaagaaacaaaccatggtgaacaa gacacggtacaccactctgatgtcgttgaatacgacgaagatacgacaactggcatgtta acaggtgccatttctgaccatacaacagaagaaggcacgatggagtacacaactgatggc ttattgattgagtttgatgatgaaatgaatcctaatgtgagcggtcagtacgatgacatc acaacggatacgatagaggaatcatctcatattgacacattcactgaacttgaatctgaa tttggtcaacatgacggtatagtgacatttgaagaagatactatcgttgagaagccgaaa acagaaaagggtaaccgagtaccacttgtaattgatttatcaacaccaaaacataaccat cagttcaatattcaacctaccgatccaaatattgatacctctgctacgtatcgaattggc aattttgtatggcgcgatgaagatcacaatggcgtacaaaatgatggtgaacatggtctt gaaggtgttcttgtcacacttaaaacagctgatggtgtcgttttaaatacaacgacaagt gatgccaatggacactaccagttcactaatgttcaaaaaggaaaatatattgttgaattc actacacctgaaggttatgaagcaacaagcaaacatactacagcgaatactgaaaaagac tctgatgggttaatcgcaaatatcgatgttactcaagatgatatgtcaatcgatgctggt ttcttcccgttagaaaactggaatcctcagccagagccgaaaaaccctgatgatagagag aaaccggcacctgagcaacctgatgtacctcagccagaaccgaaaaaccctgatgataga gagaaaccggcacctgagcaacctgatgtacctcagccagaaccgaaaaatcctgatgat agagagaaaccggcacctgagcaacctgatgtacctcaaccagagccgaaaaatcctgat gataaagagaaaccggcacctgagcaacctgatgtacctcaaccagagccgaaaaatcct gatgataaagagaaaccggcacctgagcaacctgatgcacctcaaccaaagccgatgctc ccaggtgaaaaggtgaaacccaaaccaactcatcccggtgaagctatgcaaacaacacct caggacaaatcaacatctcaaacagatgaagcacttcctaaaacaggtgaateatcatca caatcatctgctttaatcttcggtggtttactcagtctattaggacttggtttattacgt cgatcatctaaacaaaaccgttcttcaatgaaataa
The protein sequence translated from SEQ ID NO 21 is designated SEQ ID NO: 22 and is shown below:
SEQ ID NO: 22
VITNKNIYSI RKHKLGVASF LLGTLFVVGH ANNAEASEVS ATTQEHNVET EQTKTEGELT TEVAQQAVSE SAPIAENMQK TTSVASENAK EVTASDSTQE VTKTEAKDTA TMKDSEIAQP VSEVNKPVTQ TAAPVAEPST ANKQTSPRQV QELTAPMDTK VINVENGTDV TSKVKVEKSS ITGHQNKDKT YHQSNTVNPH KAERVTLNYD SFENGIKAG DYFDFQLSDN VDTNGISTIK KVPHIMDSQN SEQI IAYGEI NENNRVRYRF MDYVNQKENL KGKLSLNLFI KPDKVQDEGK ITVTSQLGKE MTSQEFDIKY IDGVKSPSGI TLNGRLDELS KADQSFTHYS IFKPKHNNLT NVTLRGTVSN NAQQNEKNGQ VNVYEYIGQG ELPQSAYANV NDTKQFNDIT KSMKSIKNNS NGYEITFD N KDNHPYI IVY QGHFNNNAKD FDFSTNATGY QNLNQSEYSY YWPYNYSFNL T DNGVAFYS NNASGEGNDK PVPPTYGYSP TVNTIQDTHA DYPVMTFQQP GTLEETEDSM PITTLTESGE DRGENTSPII ETTEDSQPVE FEEETNHGIQ DVTLHADAVD FEEETNHGEQ DTVHHSDVVE YDEDTTTGML TGAISDHTTE EGTMEYTTDG LLIEFDDEMN PNVSGQYDDI TTDTIEESSH IDTFTELESE FGQHDGIVTF EEDTIVEKPK TEKGNRVPLV IDLSTPKHNH QFNIQPTDPN IDTSATYRIG NFVWRDEDHN GVQNDGEHGL EGVLVTL TA DGVVLNTTTS DANGHYQFTN VQKGKYIVEF TTPEGYEATS KHTTANTEKD SDGLIANIDV TQDDMSIDAG FFPLENWNPQ PEPKNPDDRE KPAPEQPDVP QPEPKNPDDR EKPAPEQPDV PQPEPKNPDD REKPAPEQPD VPQPEPKNPD DKEKPAPEQP DVPQPEPKNP DDKEKPAPEQ PDAPQPKPML PGEKVKPKPT HPGEAMQTTP QDKSTSQTDE ALPKTGESSS QSSALI FGGL LSLLGLGLLR RSSKQNRSSM K
SEQ ID NO: 23 atggcatttgatggtatgtttacaagaaaaatggtagaagatttacaatttctcgtttct gggcgtattcataaaatcaatcaaccggaaaacgatacaatcatcatggttataagacag caacgccaaaatcatcaattgttgttgtcgattcacccgaattttgcacggattcacctc actacaaaaaaatatgataatccatttgaaccgccgatgtttgcgcgcgtctttcgtaaa catttagaaggtggacgtatccttgccattcgccaaatcggaaatgaccgtcgcatcgaa atggacgtggaaagtaaagatgaaattggtgacacgattcatcgtacagtgattttagaa attatgggcaaacatagtaatctcattctcgttaatgaagaacgtaaaattttagaaggt tttaaacaccttacaccaaatacgaatcaatttagaaccgtgatgccaggttttcaatat gaagtgccgccaacacaacataaacagaacccttatgcatatactggtgcgcaagtgctc caacatattgatttcaatgcgggcaaaattgatcgccaactgcttcaaacgtttgaaggt ttttcaccgttaatcacaaaagaaatcacatcaagacgccattttatgaccacacaaact ttacctgaagcttttgacgaagtgatggccgaaacgaaagcgacaccccaaccggtattt cataaaaataacgaaacaggtaaagaagacttttattttatgaagttacatcagttttac gatgattgcgtcacatatgattcactccatgaactgctcgaccgtttttatgatgcacgc ggtgaacgtgaacgcgtcaaacaacgtgcaaacgatttagtcaaactcgtccaacaatta cttcaaaaatatcaaaataaattaagtaagctcgtcgatgaacaagcggggactgaagaa aaagaaaatcaacaattgtacggcgagttaatcacagcgaatatttatcaactcaaacct ggagatcgccagttagaaacagtaaattattatacaggagaaaacgtgactattccgtta aatccacaaaagtcacctgctgaaaatgcgcaatactattacaagcaatacaaccgaatg aaaacacgtgagcgcgaattgacccatcaaattactttaacggaagaaaatatcgcttat tttgaaaatatcgagcaacagttgtcacaca tcaagttcatgaaattgacgatattcgt gaagaactagcagaacaaggctttatcaaacaaaagaaacagcagaaaaagaaaaagcaa caaaaaatccagttacaatcctacgtttcgactgatggcgatacgattttagtcggtaaa aataataagcaaaatgattatttaacgaataaacgtgcgcaaaaatcgcatttatggttc catacaaaagatatcccaggaagccatgtcgtgattttaaatgatgcgccaagtgacaaa acgattgaagaagcggcgatgattgcagcgtacttttcaaaggcggggcaatcgggacaa attccagtggattatacaacaattcgcaatgtgcataagccgagtggcagtaaacctgga tttgtaacgtacgataaccagaagacgctttacgcaacgccggattatgacatgattcgt cgattgaaagctgaagaagcgtaa
The protein sequence translated from SEQ ID NO 23 is designated SEQ ID NO: 24 and is shown below:
SEQ ID NO: 24
MAFDGMFTRK MVEDLQFLVS GRIHKINQPE NDTIIMVIRQ QRQNHQLLLS IHPNFARIHL TTKKYDNPFE PPMFARVFRK HLEGGRILAI RQIGNDRRIE MDVESKDEIG DTIHRTVILE IMGKHSNLIL VNEERKILEG FKHLTPNTNQ FRTVMPGFQY EVPPTQHKQN PYAYTGAQVL QHIDFNAGKI DRQLLQTFEG FSPLITKEIT SRRHFMTTQT LPEAFDEVMA ETKATPQPVF HKNNETGKED FYFMKLHQFY DDCVTYDSLH ELLDRFYDAR GERERVKQRA NDLVKLVQQL LQKYQN LSK LVDEQAGTEE KENQQLYGEL ITANIYQLKP GDRQLETVNY YTGENVTIPL NPQKSPAENA QYYYKQYNRM KTRERELTHQ ITLTEENIAY FENIEQQLSH IQVHEIDDIR EELAEQGFIK QKKQQKK KQ QKIQLQSYVS TDGDTILVGK NNKQNDYLTN KRAQKSHLWF HTKDI PGSHV VILNDAPSDK TIEEAAMIAA YFSKAGQSGQ IPVDYTTIRN VHKPSGSKPG FVTYDNQKTL YATPDYDMIR RLKAEEA
SEQ ID NO: 25
atggtcaaaaaatttggttataaaacacctacaatcgttgcacttactttggctgga act gcattttctgcacaccaagccaatgccgctgaacaagttgcacctgaaaaaacacctacg aatgtacttgatgatcaatacgcattaaaacaagctgatgatgcgaaacaaacgacacaa ggaacaacacttgcaggttcaaaagaatacaaggatccttcacaaattgatacgactcaa gtcgatacagcagcacaaactgaaacgcccgtagaaggagggcaacaagacgcacaacaa cctactacaactgatgaagcgacatcaacagatcatactgtatcaaaaggtacaaacgaa agtgcatcacctgcaacagcttctatagatgaaggaacattaaacgcacaagtcaattca gatgaaacggctactaaccgtacacaagacgtcactgaaaatgtgacaaaatatccttat cattcaagtgaaatcgatacacatgaagacgcaactgtgtcaccagatacatatcatgca ctggacacgcatgcgcaacaaccttcagcaatggatgtaagcgattcaacatcagcacaa actgaagcgacgcaagtaaatacgtcaacaaatgtaaatgacaaagaggccgtttcgaca acagaagatgcacctactacacaacttcaagcagctgtacaatctgaagccaacaaagaa gcgaaggcaactactgaaacagctcaaaataaaacacctcaagttgaaaagaaagcaaca gcaactcaaaatacagcacagttagcaacggggcatcaggatattactgacaaagtctca aaacgcgtagcagtgacaaatgaaacgaaagcggatgccacaacagcgaaaacacaagca cctacttcagtgacacatcaagctgatacacaagcaaaaacgataacagacaagaaggca acaacttacagtgcacaaaccgcaactgaccaagacataaatgcgaatccggacggtcca acacctccacgcgttggcggtaaagggggtccccctgcttcactttcactccaatcgact ggtcaaacagcattccgttcagctgtcgctagtaaaccgagtgcatatcaacctaaagtg aaatcgtctattaatgactatattcgtaagcaaaactacaaagtgcctgtatatgaagaa gattattcaagttacttccctaaatacggttatcgtaatggtgtcggtaaacctgagggc atcatcgtgcatgatacagcaaatgacaactctacaattgatggcgaaatcagttacatg aaaagaaattatcaaaatgctttcgtacatggctttattaatggtcaacgtattgttgaa acgcaacctacagattatttagcatggggtgcaggtgcgattgcgaatgaacgctttatt catatcgaactcgttcatgttcacagtaaagaagatttcgcacgtcaaatgaacaatatg gcagattatgcggcgacgaacttacaatattatggcctttctccagatagtgcggaatat gatggtcgtgggacagtttggacacatgatgctgtttctagatttttaggtggtacagac cataccgatccgcacggctatttaaaacaacatggttattcctttgatgcgttgtatgat ttaatcaatgaaaaatatcaagtgaaaatgggttatgcctcacctgctaactcgtcttca aaaccatcaacaaatactggcttaacagttaaaaacacaacaggtttcggccgtattaac acaacaaatagcggtttatatacgaccgtttatgatcaaaaaggtaaagcgacgaatcaa acgaatcaaacgttaaaagttacaaaagaagcgacgttaaatggcaacaaattctattta atgagtgatgcaaaatctaatcaaacactcggttgggtcaaatcaaacgacgcaacatat caagctgcccaagctgagaaaaaagtaacgaaaacgtatactgtcaaaccaggaacaaca gtatatcaagtgccttggggtgcctcatctcaaacagtaggcaaagctccaggtacgtca aaccaatcattcaaatcaacgaaagaacaaactgttgcgaaaacgaaatggctttatggg acagttggcaaagtgacaggctggattaatgcaagtagtgttgtagcaaatgatcaaaaa ccatcgacgaataccgcactaaaagtaacaactgacactggtctcggtcgcattaaagac aaaaatagtggtttatacgcaacggtatatgataaaactggtaaaagcacttcagccact aaccaaacattaaaagtaacgaaaaaagcaagtgtcaatggccaatcattctatttagta tcagattatgctaaaggtacaaatgttggttgggtgaaacagtcagatgtcgaatatcaa acaagtaaagccccttctaaagtgaatcaaaattatacgattaaatcgggtgcgaaattg tatcaagtgccttggggtacaagtaaacaagttgccggtacagtgacaggtgctgcgaca caaacatttaaggcaacacaatctcaaactgtaggtaaagcaacatacttgtatgggaca gttggcaaattatctggttggattaattcaacagcattagcagctcaaaaaacaacaacg aatgttactaaaacaatttctcaaatcggtcaactgaacacgaaaaatagcggtgtcaaa gcttctatttatgacaaaacagcaaaagatgcatccaaatgggcaggtcaaacttataaa attactaaaacagcttctgccaataacgaagactatgtattactgcaaaatagtacagga ggcacgccactcggttggttcaatgttaaagacgtcacaacacgcaacttaggtgctgaa acagctgttaaagggcggtacactgttaatagtaaaacatctggactctacgctatgcct tggggtacaacgaagcaacgtgtcgatacattaaaaaatgccacaagtcgtttatttaca gcttcaaaatcagttaaagtcggtaatgatacattcttattcggtacagtgaatcaaaaa ttgggctggattaatcaaaaagacttaacagctgtagcagcaaaagttgcaaacatgaaa actgcatcgaatagcgcagtcaaaggtgccgcaatcacaactttgaaaaaagtagaagat tatgtgattacgaataaaaatggttattattacactaaagttggagattcaaaaacagct ggtgctttaaaaggtttttatcaacaaatttttaaagtcgaaaaaacatctttactgaac ggcattacttggtactatggcgcattccaaaacgggacgaaaggatggattaaagcagct gacatacgttcatcattcattcaacatactgcggtcagtagcacattgaaagcagcactc gataaacaaatggcgctgacttacccgcctcaagttcaacgtgtagccggtaaatgggtc aatgcgaatcgtgcagaaactgaaaaagcaatgaataccgcagcaattgaaaaagatccg actctcatttaccaatttttaaaacttgataaataccaaggtcttggcgtagaagaactt aataaattgttaagaggcaaaggcattttagaaggtcaaggtgccgcatttaaagaagcc gcacaaaaacacaatattaatgaggtttacttaatgtctcacgcatttttagaaacaggt aacgggacttctcaattagccaatggcggtcacgtagataaaaataataaagtcgtaaca aacggtaaaccgaagtattacaacatgttcggtatcggggcaattgatacagacgcttta cgcaatggctttaaaactgctgaaaaatatggttggaatacggtcagcaaagcgattatc ggtggcgcaaaattcatccgtgatcagtacatcggttcaggacaaaacacattgtatcgt atgcgttggaatccagaacaccctgccacacatcagtatgcgactgatattaattgggca aatgtaaacgcacaacgcatgaaatatttctatgatcaaattggtgaaacaggtaaatat ttcgacgtcgatgtatataagaagtag
The protein sequence translated from SEQ ID NO 25 is designated SEQ ID NO: 26 and is shown below: SEQ ID NO: 26
MVKKFGYKTP TIVALTLAGT AFSAHQANAA EQVAPE TPT NVLDDQYALK QADDAKQTTQ GTTLAGSKEY KDPSQIDTTQ VDTAAQTETP VEGGQQDAQQ PTTTDEATST DHTVS GTNE SASPATASID EGTLNAQVNS DETATNRTQD VTENVTKYPY HSSEIDTHED ATVSPDTYHA LDTHAQQPSA DVSDSTSAQ TEATQVNTST NVNDKEAVST TEDAPTTQLQ AAVQSEANKE AKATTETAQN KTPQVEKKAT ATQNTAQLAT GHQDITDKVS KRVAVTNETK ADATTAKTQA PTSVTHQADT QAKTITDKKA TTYSAQTATD QDINANPDGP TPPRVGG GG PPASLSLQST GQTAFRSAVA SKPSAYQPKV KSSINDYIRK QNYKVPVYEE DYSSYFPKYG YRNGVGKPEG I IVHDTANDN STIDGEISYM KRNYQNAFVH GFINGQRIVE TQPTDYLA G AGAIANERFI HIELVHVHSK EDFARQMNNM ADYAATNLQY YGLSPDSAEY DGRGTV THD AVSRFLGGTD HTDPHGYLKQ HGYSFDALYD LINEKYQVKM GYASPANSSS KPSTNTGLTV KNTTGFGRIN TTNSGLYTTV YDQKGKATNQ TNQTLKVTKE ATLNGNKFYL MSDAKSNQTL G VKSNDATY QAAQAEKKVT KTYTVKPGTT VYQVPWGASS QTVGKAPGTS NQSFKSTKEQ TVAKTKWLYG TVGKVTGWIN ASSVVANDQK PSTNTALKVT TDTGLGRIKD NSGLYATVY DKTGKSTSAT NQTLKVTKKA SVNGQSFYLV SDYAKGTNVG WVKQSDVEYQ TSKAPSKVNQ NYTIKSGAKL YQVPWGTSKQ VAGTVTGAAT QTFKATQSQT VGKATYLYGT VGKLSG INS TALAAQKTTT NVTKTISQIG QLNTKNSGVK ASIYDKTAKD ASKWAGQTYK ITKTASANNE DYVLLQNSTG GTPLGWFNVK DVTTRNLGAE TAVKGRYTVN SKTSGLYAMP WGTTKQRVDT LKNATSRLFT ASKSVKVGND TFLFGTVNQK LG INQKDLT AVAA VANMK TASNSAVKGA AITTLKKVED YVITNKNGYY YTKVGDSKTA GALKGFYQQI FKVEKTSLLN GIT YYGAFQ NGTKGWIKAA DIRSSFIQHT AVSSTL AAL DKQMALTYPP QVQRVAGK V NANRAETEKA MNTAAIEKDP TLIYQFLKLD KYQGLGVEEL NKLLRGKGIL EGQGAAFKEA AQ HNINEVY LMSHAFLETG NGTSQLANGG HVDKNNKVVT NGKPKYYNMF GIGAIDTDAL RNGFKTAEKY GWNTVSKAII GGAKFIRDQY IGSGQNTLYR RWNPEHPAT HQYATDI A NVNAQRMKYF YDQIGETGKY FDVDVYKK
SEQ ID NO: 27
gtgtcgacagaaaaacaagatgatacacaagcaaaagcgaatgcactttctacagat gat tcaacacctacaacagaacaatcaaaaagtgataccgaaccaacgcaaaatcaagaagtg aatgaaaaagaagcaacacaagttgagcaaactccagataatgcatcatcagaatttaaa gacagtgcagcacaagatgaaacaacatcgaaagacgctgacattgctcaaacaaaagaa gcaaaaaatgaagcattgcaaagtgactcatcagcaaacctatcaaatcaagaagcagaa aaagaaaacacaactaacagtgaatctcaagtaaatgaacaacctaaagcagatacaact tctgattcacaagtttcaaatacacctcaacaagatcctacatcgacagtaccttcaeca gaaacatcagaagacaatcgaccttcaacagaattaaaaaatagtgaaacaactgcttct caaacaactttaaacgaacaacctactgaatcaacatccaatcaaactgaaacgacaaaa gcaccaacaaatacaacagtcgcaaacaaaaaagcacctgcacaattaaaagacattaaa ggtacaactcaacttcgcgcagtcagtgcaagtcaacctactgctgttgcagctggtggg acaaacgtaaatgacaaagtaacagcatcaaatatgaaaataactgaatcttatatcgag ccaaacaactcaggaaacttttatttaaaaagtaactttaacgtaaacgggactgttaaa gaaggtgactactttactgtaaaaatgcctgacactgtcaatacttttggtgacacgcgc cattcacctgactttagagaaaaaattacaaatcaaaaaggtgaagttgtggctttaggt gaatatgatgttgccaaccatactatgacatacacgttcactaatgtcgttaataattta gaaaatgtgtccggttcgtttaacttgactcaatttatggatcgtaaagtggcaacagat tctcaaacatatccattaaaatacgacattgcaggcgaatctttagatacacaaattaaa gtgaattacggtcaatattacagtgaaggtgattctaacttaaaatcaatgatcacttca gaagatcctaaaactggggaatatgatcaatacatttatgtcaacccattacaaaaaacg gcaaacggtacagttgtaagagttcaagggttccaagttgatccaactaagagtaatggg caagtgaaaccagatacaacgcagatcaagattttaaaagttgctgatggtcaaccactt aatagtagtttcggtgtgaatgacagtgaatatgaagatgtcacaaaacaatttaatatt gtttatcgtgataataatttggcagatatttactttggaaacttaaatgggcaacgctat atcgttaaagtgacgagcaaagaaaatttggattctaaagaggatttaaacttgcgtgct attatggccactcaaaaccgatatggtcaatataactatattacttgggataacgatatt gtgaaaagctcttctggtggtacagccgacggaaatgaagcatcatatcaattaggcgac aaagtttggaatgatgtgaataaaaatggtatccaagatcaaggtgaaactggtattgct gatgtaaaggttactttaaaagatcttgatggcaacattt ggatacaacttatacaaac acgaatggtaaatatatctttgataatttaaaaaatggtaattatcaagtgggttttgaa acaccggaaggctatgctgcaagtccatccaaccaaggtaatgacgcccttgactctgat ggtcctacaaatgtacaagctgtcattagtgatgggaacaacttaactatcgaccaaggt ttttaccaaactgaaacaccaacacacaacgtcggcgacaaagtttgggaagacttaaat aaagatggcatccaagaccaaaatgaaccaggtatcgctaacgttaaggtcactttaaaa gacgcggatggtaacgttgtggatacacgtacgactgatgataaagggaattacttattc gaaaaagttaaagaaggcgaatatacaattgaatttgaaacgcctgaaggttatacaccg acacaaacaggccaaggcagagtcagcactgactctaatgggacatcttcccttatttta gtcgaaggtaacgatgacttaacaatcgatagcggtttctacaaagaacctgttacacac aaagttggcgacaaagtttgggatgacttaaataaagacggtatccaagatgacaatgaa ccaggcatctctgacgttaaagtcactttaaaagatgcggatggtaacgtcgtagataca cgtacaactgatgctaacggtaactatttatttgaaaacgtgaaagaaggcgactatacg attgaatttgaaacgcctgaaggttacacaccgactgttacaggtcaaggtacagctgat aatgactctaacggtacatctacaaaagttacagttaaagatggcgatgacttaacaatt gacagtggtttcactcaagttacacctgagccaccgacacataatgttggcgacaaagtt tgggatgacttaaataaagacggtatccaagatgacaatgaaccaggcatctctgacgtt aaagtcactttaaaagatgcggatggtaacgtcgtagatacacgtacaactgatgctaac ggtaactatttatttgaaaacgtgaaagaaggcgactatacgattgaatttgaaacgcct gaaggttacacaccgactgttacaggtcaaggtacagctgataatgactctaacggtaca tctacaaaagttacagttaaagatggcgatgacttaacaattgacagtggtttcactcaa gttacacctgagccaccgactgaacctgaaaaccctagtccagagcaaccttctgaaccg ggtcaacctgaaaatcctagtccagagcaaccttctgaaccaggtcaacctgaaaatcct agt ccagagcaaccttctgaaccaggtcaacctgaaaatcctagtccagaacaaccttct gaaccgggtcaacctgaaaatcctagt ccagaacagccttctgagccaggacaacctaaa aatcctagtccagaacagccaaataatccaagtgtgccaggtgttcaaaatcctgaaaaa ccaagcttaactccagtcacacaaccggttcattcaaacggcaataaagcaaaaccatct caacaacaaaaagctttacctgaaacaggtgaaactgaatcacatcaaggtacattattc ggtggtattttagctgctttaggcgcattactctttgcacgtaaaaaacgccacgataaa aaacaatcacactaa
The protein sequence translated from SEQ ID NO 27 is designated SEQ ID NO: 28 and is shown below:
SEQ ID NO: 28
VSTEKQDDTQ AKANALSTDD STPTTEQSKS DTEPTQNQEV NEKEATQVEQ
TPDNASSEFK DSAAQDETTS KDADIAQTKE AKNEALQSDS SANLSNQEAE
KENTTNSESQ VNEQPKADTT SDSQVSNTPQ QDPTSTVPSP ETSEDNRPST
ELKNSETTAS QTTLNEQPTE STSNQTETTK APTNTTVANK KAPAQLKDIK
GTTQLRAVSA SQPTAVAAGG TNVNDKVTAS NMKITESYIE PNNSGNFYLK
SNFNVNGTVK EGDYFTVKMP DTVNTFGDTR HSPDFREKIT NQKGEVVALG
EYDVANHTMT YTFTNVVNNL ENVSGSFNLT QFMDRKVATD SQTYPLKYDI
AGESLDTQIK VNYGQYYSEG DSNLKSMITS EDPKTGEYDQ YIYVNPLQKT
ANGTVVRVQG FQVDPTKSNG QVKPDTTQIK ILKVADGQPL NSSFGVNDSE
YEDVTKQFNI VYRDNNLADI YFGNLNGQRY IVKVTSKENL DSKEDLNLRA
IMATQNRYGQ YNYITWDNDI VKSSSGGTAD GNEASYQLGD KVWNDVNKNG
IQDQGETGIA DVKVTLKDLD GNILDTTYTN TNGKYIFDNL KNGNYQVGFE
TPEGYAASPS NQGNDALDSD GPTNVQAVIS DGNNL IDQG FYQTETPTHN
VGDKVWEDLN KDGIQDQNEP GIANVKVTLK DADGNVVDTR TTDDKGNYLF
EKVKEGEYTI EFETPEGYTP TQTGQGRVST DSNGTSSLIL VEGNDDLTID
SGFYKEPVTH KVGDKVWDDL NKDGIQDDNE PGISDVKVTL KDADGNVVDT
RTTDANGNYL FENVKEGDYT IEFETPEGYT PTVTGQGTAD NDSNGTSTKV
TVKDGDDLTI DSGFTQVTPE PPTHNVGDKV WDDLNKDGIQ DDNEPGISDV
KVTLKDADGN VVDTRTTDAN GNYLFENVKE GDYTIEFETP EGYTPTVTGQ
GTADNDSNGT STKVTVKDGD DLTIDSGFTQ VTPEPPTEPE NPSPEQPSEP
GQPENPSPEQ PSEPGQPENP SPEQPSEPGQ PENPSPEQPS EPGQPENPSP
EQPSEPGQPK NPSPEQPNNP SVPGVQNPEK PSLTPVTQPV HSNGNKAKPS
QQQKALPETG ETESHQGTLF GGILAALGAL LFARKKRHDK KQSH
SEQ ID NO: 29
atgaagaaaacaatttcagtacttggtctagggctattagcaacattttttgtaagt aac gaatcatatgccgcagaaacgattcaaaacaatacgtcatcaagtgaaacgaatcaaaat tcagatcagacgccgttagatcattatattcgaaaagcagatggcacactggttgaaccg aacgtgtacccacataaagattatgtagagaatgaaggacctttaccagagtttaaattt caagttgactctaagaaagattcatctgatccaaatcaagcaccgttagatcattatatt cgaaaagcggatggcacgttggttgaaccgaatgtatatccacacaaagattatgtcgaa aatgaagggcctttaccagagtttaaatttatgtatgctgacaaacaaaatcatcatgac caacagagtaaaaacaacaaggataagcagcgtgcaaattacagtgacaaaaagcataat gatcagccgggtcatccaaaagcagtcacgccagctgtacaacatgataaagcagtcact tcaaacgctactgtaaaagcattgccaaacacaggtgaatctgataaaacaacacaatta ccaatcgtattatcattgttatctgtggggattttagttttattaaaattgagaaaataa
The protein sequence translated from SEQ ID NO 29 is designated SEQ ID NO: 30 and is shown below:
SEQ ID NO: 30
MKKTISVLGL GLLATFFVSN ESYAAETIQN NTSSSETNQN SDQTPLDHYI
RKADGTLVEP NVYPHKDYVE NEGPLPEFKF QVDSKKDSSD PNQAPLDHYI
RKADGTLVEP NVYPHKDYVE NEGPLPEFKF MYADKQNHHD QQSKNNKDKQ
RANYSDKKHN DQPGHPKAVT PAVQHDKAVT SNATVKALPN TGESDKTTQL PIVLSLLSVG ILVLLKLRK
SEQ ID NO: 31
atgaaaagtaaatatgattttttacctaatagacttaataaattttctatacgaaaa ttt actgttggtagtgtatcagtgctaataggagccactttattattcgggtttgtagaagga gaagcatcagcatcagtaaaagaaggtcaacaaagtataaattctagtgagaaagaaagc gccgatcctacagtagttgatttaattagtaagaaagaaacaaatttagatggactagat gtatcaagagaagaaacgaccaaagtaccaataaatgaaaacaaaagaggtgaggaacaa agtatttctgataaagctataacagaaaaagctgatacaccagtaagcaatttatcaagt aaggaagttgaggagcaaggtgtttctgataaagctataacagaaaaagctgatacacca gtaaccaatttatcaagtaaggaagctaaggagcaaggtgcttctgatagagttataaca gaaaaagctgatacaccagtaagcaatttatcaagtaaggaagctaaggagcaaggtgct tctgatagagttataacagaaaaagctgatacaccagtaagcaatttatcaagtaaggaa gttgaggagcaaggtgtttctgataaagctatagagaaaatagctgatgcatcagctact gatttgtcaagtaaggaagaagtagaacaagatatatctacacaaggtaaagtaaaatca aaggaagcagtacaagtagaaagtagtcagttacaaaatttaaatagtgaaataaatgct gaacctaatgaaattaaggcaatagatagaagttcaatattacctttaaatttaaatgat gaagaaaataacaaaaaagttaataaagggactcgggttccagaagctacattaagaaat gcctctaataaccaactcaatacacgaatgagatcagtgagtttatttagagttgctaga ctaacagaaatcaatagaaatgttaatgataaagtaaaggtttcggatatcgacatcgca atagccccaccgcatactaaccctaaaactggaaaagaagaattttgggcgacatcttct tcagttttaaagttaaaggcaagctatgaattggataatagcatttctaaaggggatcaa tttactattcaatttggtcaaaatattcgtccaggtggattaaatttaccaagaccttat aattttttatatgataaggataaaaaattagttgcaactggccgttacaataaagaatca aatacaatcacatatacatttacggattatgtagataaacatcaaaacattaaaggtagt tttgagatgaatgcattttctagaaaggaaaatgctactactgacaaaacagcatatcca atggatgt actattgcgaatcaaaaatatagtgaaaatattattgtagactatggtaat aaaaagaatgctgctattatttcaagtacagaatatattgatttagatggtagtagaaaa atgacaacatatattaatcaaaatggtagtaaaaattccatctatcgtgctgatatgcaa attgatttgaacggttataaatttgatccatccaaaaacaattttaaaatttatgaagtg gaaaatagcagtgactttgtggatagcttttcaccagatgtgagcaagttaagggatgtt acgagtcaatttaatattcaatatacaaataataatacaatggcaaaagtggattttggt actaacctttggaggggtaaaaaatatattattcagcaagtggcgaatatagacgacagt aaattagtgaaaaatgcttcaatcaattatacattgaataaaatggattttaataataaa agaacggtagaaacacataacaatacttattctacagtgaaagataaatcaacagcacta ggtgacgtacaggaaagtcaatctattagtgagagccaatcagttagtgaaagcgagtca ctaagtgagagccaatcaatcagtgaaagcgaatcattaagtgagagccaatcaatcagt gaaagcgaatcattaagtgaaagtcaatcaatctcagagagcgaatcactaagtgaaagt cagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatctcagagagtgaatca ttaagtgaaagtcagtcaatttcagagagtgaatcactaagtgaaagtcagtcaatttca gaaagcgaatcattaagcgagagtcagtcaatttcagaaagcgaatcattaagcgagagt cagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatcagtgaaagcgaatca ctaagcgagagccaatcaatctcagagagtgaatcattaagcgagagtcaatcaatctca gagagcgaatcattaagtgagagtcaatcaatcagtgaaagcgagtcactaagtgagagt caatcaatttcagagagcgaatcattaagtgaaagccaatcaatctcagagagtgaatca ctaagtgagagccaatcaatctcagagagtgaatcattaagtgagagccaatcaatctca gagagcgagtcactaagcgagagccaatcaatttcagagagtgaatcactaagtgaaagt caatcaatttcagagagcgaatcactaagtgagagccaatcaatctcagagagcgaatca ctaagtgaaagtcaatcaatttcagagagtgaatcactaagcgagagccaatcaatctca gagagtgaatcattaagtgaaagtcagtcaatttcagagagtgaatcactaagtgaaagt cagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatcagtgaaagcgaatca ctaagcgagagtcaatcaatctcagagagcgaatcattaagtgaaagtcaatcaatttca gaaagcgagtcattaagcgagagtcagtcaatctcagagagcgaatcactaagcgagagt caatcaatctcagagagtgaatcattaagtgagagccaatcagttagtgaaagcgaatca ctaagtgaaagtcagtcaatttcagaaagcgaatcattaagtgagagtcaatcaatttca gaaagcgaatcattaagtgaaagccaatcaatcagtgaaagcgaatcactaagcgagagc caatcaatcagtgaaagcgaatcattaagtgagagtcaatcaatctcagaaagcgaatca ttaagtgagagtcaatcaatcagtgaaagcgaatcactaagcgagagccaatcaatctca gagagcgaatcactaagcgagagccaatcaatctcagagagcgagtcactaagcgagagc caatcaatcagtgaaagcgaatcattaagtgagagtcaatcaatcagtgaaagcgagtca ctaagtgagagccaatcaatctcagagagtgaatcattgagtgagagccaatcaatctca gagagcgagtcactaagtgagagtcaatcaatttcagagagcgaatcattaagtgaaagc caatcaatctcagagagtgaatcattgagtgagagccaatcagttagtgaaagcgagtca ctaagtgagagtcaatcaatcagtgaaagcgagtcactaagtgagagtcaatcaatttca gagagcgaatcattaagcgagagtcagtcaatctcagagagtgaatcactaagtgagagc caatcaatctcagagagtgaatcattaagtgagagccaatcaatctcagagagtgaatca ctaagtgagagtcaatcaatcagtgaaagcgaatcactaagcgagagccaatcaatttca gagagtgaatcattaagtgagagccaatcagttagtgaaagcgaatcactaagcgagagc caatcaatctcagagagcgaatcattgagtgagagccaatcaatctcagagagtgaatca ttgagtgagagtcaatcaatcagtgaaagcgaatcactaagcgaaagtcaatcaatttca gagagtgaatcattgagtgagagccaatcaatttcagagagtgaatcactaagtgaaagt cagtcaatttcagaaagcgaatcactaagcgagagccaatcaatctcagagagcgaatca ctaagtgaaagtcagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatctca gagagtgaatcattaagtgaaagtcagtcaatttcagagagtgaatcactaagtgaaagt cagtcaatttcagaaagcgaatcattaagcgagagtcagtcaatttcagaaagcgaatca ttaagtgaaagccaatcaatcagtgaaagcgaatcactaagcgagagccaatcaatctca gagagcgaatcactaagcgagagccaatcaatctcagagagcgaatcactaagtgaaagt caatcaatttcagagagtgaatcattgagtgagagtcaatcaatttcagagagtgaatca ctaagtgaaagtcaatcaatttcagagagtgaatcactaagcgagagccaatcaatctca gagagtgaatcattaagtgaaagtcagtcaatttcagagagggaatcactaagtgaaagt cagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatcagtgaaagcgaatca ctaagtgaaagtcaatcaatctcagagagtgaatcactaagtgagagccaatcaatctca gagagtgaatcattgagtgagagccaatcaatctcagagagcgaatcactaagtgaaagt caatcaatttcagaaagcgagtcattaagcgagagtcagtcaatctcagagagtgaatca ctaagtgagagccaatcaatctcagagagtgaatcactaagtgagagtcaatcaatcagt gaaagcgaatcactaagcgagagccaatcaatttcagagagtgaatcattaagtgagagc caatcagttagtgaaagcgaatcactaagcgagagccaatcaatctcagagagcgagtca ctaagcgagagtcaatcaatctcagagagtgaatcactaagtgaaagtcagtcaatttca gaaagcgagtcactaagcgagagtcaatcaatctcagagagtgaatcattgagtgagagc caatcaatctcagagagcgaatcattgagtgagagccaatcaatctcagagagtgaatca ttgagtgagagccaatcaatttcagagagcgaatcactaagcgagagccaatcaatcagt gaaagcgaatcattaagtgagagtcagtcaattagcgaaagcgaatcactaagtgagagt caatcaatctcagagagtgaatcactaagtgaaagtcagtcaatcagcgaaagcgaatct aaatctttacctaataccggtactggagaaaagatttctaattatccaggtattttagga ggattattaagcatattaggtataagtttgcttaaaagaaaagacagagagaaaaaatta ggacaaaaatctaataagtag
The protein sequence translated from SEQ ID NO 31 is designated SEQ ID NO: 32 and is shown below:
SEQ ID NO: 32 MKSKYDFLPN RLNKFSIRKF TVGSVSVLIG ATLLFGFVEG EASASVKEGQ QSINSSEKES ADPTVVDLIS KKETNLDGLD VSREETTKVP INENKRGEEQ SISDKAITEK ADTPVSNLSS KEVEEQGVSD KAITEKADTP VTNLSSKEAK EQGASDRVIT EKADTPVSNL SSKEAKEQGA SDRVITEKAD TPVSNLSSKE VEEQGVSDKA IEKIADASAT DLSSKEEVEQ DISTQGKVKS KEAVQVESSQ LQNLNSEINA EPNEIKAIDR SSILPLNLND EENNKKVNKG TRVPEATLRN ASNNQLNTRM RSVSLFRVAR LTEINRNVND KVKVSDIDIA IAPPHTNPKT GKEEFWATSS SVLKLKASYE LDNSISKGDQ FTIQFGQNIR PGGLNLPRPY NFLYDKDK L VATGRYNKES NTITYTFTDY VDKHQNIKGS FEMNAFSRKE NATTDKTAYP MDVTIANQKY SENIIVDYGN KKNAAIISST EYIDLDGSRK MTTYINQNGS KNSIYRADMQ IDLNGYKFDP SKNNFKIYEV ENSSDFVDSF SPDVSKLRDV TSQFNIQYTN NNTMAKVDFG TNLWRGKKYI IQQVANIDDS KLVKNASINY TLNKMDFNNK RTVETHNNTY STVKDKSTAL GDVQESQSIS ESQSVSESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSVSESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSVSESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS VSESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISERESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSVSESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES LSESQSISES ESLSESQSIS ESESLSESQS ISESESLSES QSISESESLS ESQSISESES KSLPNTGTGE KISNYPGILG GLLSILGISL LKRKDREKKL GQKSNK
SEQ ID NO: 33
atgttaagaacaaattataaactaagaaagcttaaagtaggtttagtatcgacaggt gtg gcgttgacttttgtgatggcaagtgggaatgcagaggcgtcggagaacgagcagactgaa gtaaaaggggaggcgcaagttgcttctgtgaatgaaaaagagagtgaagcagaattacct gtagcgcaacaagaagcatctattcaactagacaaagtacaaccaggcgatgcacagctt tcaggctatacacagccaaacaaagcgatttctgtaaagatcgacaataaagatattgtg tctgtagatgatggctatgaagaggtattatcggatgatacaggtaaatttgtatatgat ttgaaagggcgtcaaattgtttacaatcaaaaagttgatgttgaagcgatgacgccattt aattttgaagattttgatgaatcagcacttgagagcgaagaggcattggaggcgttaggt caattggaagacgaagaaacagcgacagcttctgtgacgacgcctagatatgaaggtgcg tatacagttcctgaagaacgcttgacacccattcaaggccaacagcaagtattcatcgaa cctattttagaaggggcaagtaaaatcaaaggacatacatctgtacaaggtaaagtcgcg ttagcaatcaatcaagaacatgtgcacctaggtgatacgttagaagaacaagcagcactc actgatcaagagtggcaaggtcgttatgacgggatttggcgccatattgatgatcaaggg tttttcgagtttgacttgaaccgtctttacaataaatcttacccattgaagtctggcgat ttagtgactttatcttttaaatctaatgacgaagtaggcccattattcaatgtgaacgtt gagcctttcgaacgtgtggcacaagctaaaacaaagtatgagcagaatgacagtccagta gtcaacaaattggatgatactaaaagtgacttggaggttcaacctatctatggagacctt acacaagcagcagtacatggcgagtcgaaagtgttgataccggggacgtcaaaagttgaa ggacgtacgaattatgcacatgcatggatagagatggcatctaatttaggggaatatcgt agtttccctaaattacaagctgatgcgacaggtgcgtttatatttgatttaaaagcggca gacatacaattgttaaacggagaacgtttgacattcagagccgttgacccacatacaaaa caacagttagctgaaactacatcagaagtacgcccagtagatatgcaagatgaagagtca gaggttgtgcagacttcaagcactgagaaatcagcacttgcggatgaaattcttcgttct atgacaattgacaaatcatttaatcctgaagttaccgagataccgggtcatgtatatcct aagaaaacagaggataaaggtgctgaaaatacagaacaagcctcagagaattctgagaag ccatctcagactacagaatctcaaaatgatgccgtacaagatgtagagaaatcctctgtt aatgaggaggttacgccaccttcaacagaatctgctcaagttgaaaaggggcaaaataca gaaggggctttgcttccaaaaaatgtagaacaacatgtagagagtataccataccaaaaa cgtaaagcgttgataggactgacaaaacatcaaggatcagggcacatgccgccattttct ttaagctttaataataaagaagatgacgtatccacaaaggttaacgaagcaaacgagcat gaacgtaagcagggtacagtttatccagagcaaatagaacaattacctcaaacaggttta actgaaaaatcgccattctgggcattgttatttgttgtatcaggcacaggtttattatta ttcaaacgttctagacgacaacgccaatcttaa
The protein sequence translated from SEQ ID NO 33 is designated SEQ ID NO: 34 and is shown below:
SEQ ID NO: 34
MLRTNYKLRKLKVGLVSTGVALTFVMASGNAEASENEQTEVKGEAQVASV NEKESEAELPVAQQEASIQLDKVQPGDAQLSGYTQPNKAISVKIDNKDIV SVDDGYEEVLSDDTGKFVYDLKGRQIVYNQKVDVEAMTPFNFEDFDESAL ESEEALEALGQLEDEETATASVTTPRYEGAYTVPEERLTPIQGQQQVFIE
PILEGASKIKGHTSVQGKVALAINQEHVHLGDTLEEQAALTDQEWQGRYD GIWRHIDDQGFFEFDLNRLYN SYPLKSGDLVTLSFKSNDEVGPLFNVNV EPFERVAQAKTKYEQNDSPVVNKLDDTKSDLEVQPIYGDLTQAAVHGES VLIPGTS VEGRTNYAHAWIEMASNLGEYRSFP LQADATGAFIFDLKAA DIQLLNGERLTFRAVDPHTKQQLAETTSEVRPVDMQDEESEVVQTSSTEK SALADEILRSMTIDKSFNPEVTEIPGHVYPKKTED GAENTEQASENSEK PSQTTESQNDAVQDVEKSSVNEEVTPPSTESAQVEKGQNTEGALLPKNVE QHVESI PYQKRKALIGLTKHQGSGHMPPFSLSFNNKEDDVSTKVNEANEH ERKQGTVYPEQIEQLPQTGLTEKSPFWALLFVVSGTGLLLFKRSRRQRQS
SEQ ID NO: 35
atgaaaactaaatacacagcaaaattattaattggggcagcaacaatatctttagca aca tttatttcacaagggaacgcacatgcgagcgaacaaactacaggactcgcaccggcacaa cctgtcaactt gattcaatcaatgtaacgccagaccaaaaaacattctatcaagtctta catatggaaggcatttcagaagaccaacgtgaacaatatttgaaacaattgcacgaagac ccaagtagcgcacaaaatgttttttcagaatcaattaaagatgccatccacccggaacgt cgtgttgcgcaacaaaatgcgttttacagcgtattacacaacgatgacttatccgaagag caacgtgatgcatacattggtagaattaaagaagatccagatcaaagccaagaagtattt gttgagtctttaaatgtggcacctaaagcagaatcacatgaagatcgcctcattgaatta caaaacaaaaatttaatggaagcgaatgaagcacttaaagcgttacaacaagaagacagc attcagaatagacgtgcggctcaacgtgctgtcaacaaattgacgccggatagcgcgaac gcattccaaaaagaattagatcaaatcaatgccccacgcgacgctaaaattaaagctgac gctgaagcaaaaaaacaagcacctgaagtaagcgcaccacaaattgaagatgcacctact actgaagttgcaccatctccaaaacaagatatgccaaaagtagataaaaaagaagaagat aaagtagaaagtgatactgaggtcaaagaagtacctaaagctgatacagagaaaaaccct caatctaaagacacttctaaaactgaacaagctaaagaaacacctaaagtagagcaatca cctaaaacagaaaaggctgaagaagcacctaaagcagaaacacctcaaaatggaaataaa gcacaaactgaagaagctaaaccagaagtaaaagacaatgtgaaaaacactccatctgca cctgtgt acctgaaacaggaaaagcaacaacttcaacacttgaaagctactggaattct ttcaaagacagtgtgaataaaggttatacttacattaaacaaagcttagaaagtggttat caatatttaaaaggtcaatacgactatatcactaaaaaatacaatgatgcgaaatactat acaaaaatgtattcaaatcataagtctacaattgatcagtctgtattagctatattaggt aaaactggatctagcgcatatatcaagccattaaatatcgaagaaaattcaaacgtattt tacaaagcttatgcaaaaacaagaaactttgctacagaaagcattaacacaggaaaagta ttatacacattatatcaaaaccctactgtagttaaatctgctttcactgcaattgaaaca gcaaatacagtaaaaaatgcaataagcaatcttttctctctcttcaaataa The protein sequence translated from SEQ ID NO 35 is designated SEQ ID NO: 36 and is shown below:
SEQ ID NO: 36
MKTKYTAKLLIGAATISLATFISQGNAHASEQTTGLAPAQPVNFDSINVT PDQKTFYQVLHMEGISEDQREQYLKQLHEDPSSAQNVFSESIKDAIHPER RVAQQNAFYSVLHNDDLSEEQRDAYIGRIKEDPDQSQEVFVESLNVAPKA ESHEDRLIELQNKNLMEANEAL ALQQEDSIQNRRAAQRAVNKLTPDSAN AFQKELDQINAPRDAKIKADAEAKKQAPEVSAPQIEDAPTTEVAPSPKQD MPKVDKKEEDKVESDTEVKEVPKADTEKNPQS DTSKTEQAKETPKVEQS PKTEKAEEAPKAETPQNGNKAQTEEAKPEVKDNVKNTPSAPVLPETGKAT TSTLESYWNSFKDSVNKGYTYIKQSLESGYQYLKGQYDYITKKYNDAKYY TKMYSNHKSTIDQSVLAILGKTGSSAYIKPLNIEENSNVFYKAYAKTRNF ATESINTGKVLYTLYQNPTVVKSAFTAIETANTVKNAISNLFSLFK
Since each of the abovementioned proteins/nucleic acid sequences is derived from Staphylococcus pseudintermedius, the inventors have designated these (and the corresponding protein sequences) Staphylococcus pseudintermedius surface genes/nucleic acids/proteins (Sps). For simplicity, the bulk of this specification will use the term "Sps" or "Sps genes" or "Sps nucleic acids" which are intended to encompass all of the nucleic acid sequences described above (i.e. SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33 and 35.
Furthermore, in addition to encompassing the entire or complete gene/nucleic sequences listed above, it is to be understood that the designation "Sps" also encompasses fragments, portions, mutants, derivatives and/or homologoues/orthologues of any of these genes.
In addition, the term "Sps" or "Sps proteins" encompasses the proteinaceous products of the Sps genes/nucleic acids or fragments, portions, analogues, variants or derivatives thereof (for example short peptide fragments). In particular, the term "Sps proteins" encompasses the sequences given as SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 above.
Typically, the gene/nucleic acid fragments, portions, mutants, variants, derivatives and/or homologues/orthologues of the invention are functional or active - that is, they retain the function and/or activity of the wild type or native Sps genes/nucleic acids. Advantageously, fragments, portions, mutants, variants, derivatives and/or homologues/orthologues of any of the Sps genes/nucleic acids provided by this invention, encode proteins (or peptides, peptide fragments) retaining the ability to bind to or associate with extracellular matrix proteins such as, for example, fibrinogen, fibronectin and/or collagen. In other embodiments, the proteins and/or peptides encoded by the nucleic acid sequences described herein are immunogenic or antigenic. Furthermore, fragments, portions, variants or derivatives of any of the proteins encoded by the nucleic acid sequences described herein may also retain the immunogenicity and/or antigenicity of a corresponding wild type Sps protein (for example the proteins listed above). Where the invention relates to immunogenic compositions and/or vaccines, the use of proteins and/or peptides which are immunogenic (or antigenic) is important.
The term "mutants" may encompass naturally occurring mutants or those artificially created by the introduction of one or more nucleic acid additions, deletions, substitutions or inversions.
Homologous or identical genes, nucleic acid or protein sequences may exhibit as little as approximately 20 or 30% sequence homology or identity to certain reference sequnces, however, in other cases, homologous or identical genes/nucleic acids and/or proteins may exhibit at least 40, 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology or identity to the various sequences given above as SEQ ID NOS: 1-36. It should be understood that mutant, variant, derivative and/or orthologuous sequences may exhibit similar levels of homology/identity to each other and/or to the Sps genes/nucleic acids shown as SEQ ID NOS 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33 and/or 35 above.
One of skill in this field will readily understand that genes/nucleic acids homologous/identical to the Sps genes detailed herein may be found in other bacterial species. As such, homologous genes from other species may be included within the scope of this invention. Using the various nucleic acid and amino acid sequences described herein, one of skill in the art could readily identify related sequences in other microbial (particularly bacterial) species. For example, nucleic acid obtained from a particular bacterial species may be probed using the probes derived from the sequences of this invention, to identify homologous or closely related sequences.
It should be understood that Sps nucleic acid sequences of this invention may be single-stranded or double-stranded and a single-stranded nucleic acid molecule may include a polynucleotide fragment having a nucleotide sequence that is complementary to a nucleotide sequence that encodes a Sps protein or fragment thereof. As used herein, the term "complementary" refers to the ability of two single stranded polynucleotide fragments to base pair with each other.
A single-stranded nucleic acid molecule of the invention may further include a polynucleotide fragment having a nucleotide sequence that is substantially complementary to a nucleotide sequence that encodes a Sps protein or fragment thereof according to the invention, or to the complement of the nucleotide sequence that encodes said Sps protein or fragment thereof. Substantially complementary polynucleotide fragments can include at least one base pair mismatch, such that at least one nucleotide present on a first polynucleotide fragment will not base pair to at least one nucleotide present on a second polynucleotide fragment, however the two polynucleotide fragments will still have the capacity to hybridize. The present invention therefore encompasses polynucleotide fragments which are substantially complementary. Two polynucleotide fragments are substantially complementary if they hybridize under hybridization conditions exemplified by 2xSSC (SSC: 150 mM NaCl, 15 mM trisodium citrate, pH 7.6) at 55°C. Substantially complementary polynucleotide fragments for purposes of the present invention may preferably share at least about 60, 65, 70, 75, 80 or 85% nucleotide identity, preferably at least about 90%, 95% or 99% nucleotide identity. Locations and levels of nucleotide sequence identity between two nucleotide sequences can be readily determined using, for example, CLUSTALW multiple sequence alignment software.
In addition, it should be understood that the present invention also relates to the products of the genes/nucleic acids encompassed by this invention and in particular to proteins or peptides homologous/identical to those having sequences provided by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. Furthermore, fragments, portions, analogues, variants, derivatives of any of these or homologous and/or identical or modified proteins are also within the scope of this invention. Typically, fragments, portions, derivatives, variants and/or homologous or modified proteins or peptides of the invention are functional or active - that is they retain the function of a wild type Sps protein. In certain embodiments fragments, portions, derivatives or variants of, and/or modified sequences or sequences with homology or identity to, the amino acid sequences provided by this invention, retain the ability to bind to or associate with extracellular matrix proteins such as, for example, fibrinogen, fibronectin and/or collagen. Additionally or alternatively, fragments, portions, mutants, variants, derivatives and/or homologues/orthologues of the Sps genes provided by this invention, may encode proteins (or peptide fragments) that are antigenically similar or identical to the proteins encoded by the genes described herein. Similarly, fragments, portions, derivatives and/or variants of and/or modified sequences or sequences with homology or identity to, the amino acid sequences provided by this invention are also antigenically similar or identical to the proteins encoded by the genes described herein. It should be understood that the term "antigenically similar or identical" may encompass proteins or peptides eliciting an immune response similar or identical to the immune response elicited by any of the Sps proteins described herein. In certain embodiments fragments, portions, derivatives and/or variants of and/or modified sequences or sequences with homology or identity to, the amino acid sequences described herein, elicit immune responses which protect against Staphylococcus pseudintermedius infection and/or prevent, reduce or neutralise Staphylococcus pseudintermedius cell/tissue adhesion and/or colonisation. One of skill will readily understand that the antigenicity of a polypeptide can be evaluated in vitro by, for example, performing a Western blot on the purified polypeptide (for example, an affinity purified polypeptide) using polyclonal antisera from an animal, such as a rabbit that was vaccinated with at least an antigenic portion of an Sps protein of the present invention.
One of skill in this field will readily understand that for the various nucleic acid sequences and polypeptides described herein, natural variations due to, for example, polymorphisms, may exist between Sps genes and proteins isolated from different microbial species and even different strains of the same species. Gene or protein variants may manifest as proteins and/or genes that exhibit one or more amino acid/nucleic acid substitutions, additions, deletions and/or inversions relative to a reference sequence (for example any of the sequences described above). As such, it is to be understood that all such natural variants, especially those that are functional or display the desired activity, are to be included within the scope of this invention.
In another embodiment, the invention relates to derivatives of any of the Sps sequences described herein. The term "derivatives" may encompass Sps genes or peptide sequences which, relative to those described herein, comprise one or more amino acid substitutions, deletions, additions and/or inversions. Additionally, or alternatively, analogues of the various peptides described herein may be produced by introducing one or more conservative amino acid substitutions into the primary sequence. One of skill in this field will understand that the term "conservative substitution" is intended to embrace the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physcio-chemical properties and/or structure or function of the native (or wild type) protein. Analogues of this type are also encompassed with the scope of this invention. In one embodiment, substitute amino acids may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of preferred conservative substitutions include Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free N¾.
As is well known in the art, the degeneracy of the genetic code permits substitution of one or more bases in a codon without changing the primary amino acid sequence. Consequently, although the sequences described in this application are known to encode the Sps proteins described herein, the degeneracy of the code may be exploited to yield variant nucleic acid sequences which encode the same primary amino acid sequences.
The present invention may further provide modified Sps proteins. For example, a "modified" Sps protein may be chemically and/or enzymatically derivatised at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, phosphorylation and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
One of skill in this field will appreciate that the amino acid and/or nucleic acid sequences described herein may be used to generate recombinant Sps genes/proteins and as such, the present invention further contemplates methods of generating and/or expressing recombinant Sps genes and/or proteins, and products for use in such methods. Accordingly, in addition to providing substantially purified or isolated recombinant Sps sequences, a second aspect of this invention provides DNA constructs comprising a replicable expression vector and nucleic acid encoding one or more of the Sps protein(s) described herein.
Expression vectors for the production of the molecules of the invention include plasmids, phagemids, viruses, bacteriophages, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognised in a suitable host cell to effect expression of the desired genes.
Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such systems typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.
A vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector containing cells.
Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (ads.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston. Mass (1988).
In general, such vectors may contain specific genes, which are capable of providing phenotypic selection in transformed cells. The use of prokaryotic and eukaryotic viral expression vectors to express the nucleic acid sequences coding for the recombinant proteins of the present invention are also contemplated.
The vector is introduced into a host cell by methods known to those of skill in the art. Introduction of the vector into the host cell can be accomplished by any method that introduces the construct into the cell, including, for example, electroporation, heat shock, chemical compounds such, for example, calcium phosphate, stronitium phosphate, microinjection techniques and/or gene guns. See, e.g., Current Protocols in Molecular Biology, Ausuble, F.M., ea., John Wiley & Sons, N.Y. (1989).
Another aspect relates to a host cell transformed with any one of the nucleic acid constructs of the present invention. Suitable host cells include prokaryote cells, lower eukaryotic and higher eukaryotic cells. Prokaryotes include Gram negative and Gram positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes include yeast, S. cerevisiae and Pichia, and species of the genus Diclyostelium.
"Host cell" as used herein refers to cell which can be recombinantly transformed with vectors constructed using recombinant DNA techniques.
A drug resistance or other selectable marker is intended in part to facilitate the selection of the transformants. Additionally, the presence of a selectable marker, such as a drug resistance marker may be of use in keeping contaminating microorganisms from multiplying in the culture medium. Such a pure culture of the transformed host cells would be obtained by culturing the cells under conditions which require the induced phenotype for survival.
PCR techniques may be exploited to selectively obtain Sps gene sequences from samples of Staphylococcal DNA. These amplified sequences may be introduced into any of the vectors described above. In one embodiment, the vector may further comprise a nucleotide sequence of a tag or label to assist in protein purification procedures.
Techniques used to purify recombinant proteins generated in this way are known and, where the recombinant protein is tagged or labelled, these may include the use of, for example, affinity chromatography techniques.
In view of the above, a fourth aspect of this invention provides a process for the production of recombinant Sps protein(s) or peptide(s) of the invention, said process comprising the steps of (a) transforming a host cell with the nucleotide sequence of the invention or transfecting a host cell with a nucleic acid construct of the invention; (b) culturing the cells obtained in (a) under conditions in which expression of the protein takes place; and (c) isolating the expressed recombinant protein or peptide from the cell culture or/and the culture supernatant.
The polypeptide may be partially purified from the host and where the polypeptide is secreted from the host cell, the cells may be separated from the media by centrifugation, the cells being pelleted. Alternatively, the polypeptide may be partially purified from this supernatant, for example using affinity chromatography.
A fifth aspect of this invention provides monoclonal or polyclonal antibodies, whether derived from rodents, mammals, avians, ungulates, or other organisms, that bind to the Sps proteins described herein. Production and isolation of monoclonal and polyclonal antibodies to a selected polypeptide sequence is routine in the art see for example "Basic methods in Antibody production and characterisation" Howard & Bethell, 2000, Taylor & Francis Ltd. Such antibodies may be used in diagnostic procedures, as well as for passive immunisation.
Staphylococcus pseudintermedius is known to cause cutaneous inflammatory diseases in a variety of animals. One such cutaneous inflammatory disease is canine pyoderma which is a major cause or morbidity in dogs. Pydoderma associated with Staphylococcus pseudintermedius infection is common among dogs and is often associated with puritis, alopecia, erythema and swelling. At present, the treatment of this infection is difficult, requiring the use of aggressive, systemically administered antibiotics. The present inventors have discovered that Sps genes (and their protein products) play a role in Staphylococcus pseudintermedius colonisation and pathogenesis. As such, the Sps genes and proteins described herein may find application in the treatment and/or prevention of cutaneous disorders such as canine pyoderma.
Accordingly, a sixth aspect of this invention provides an Sps protein or gene as substantially defined above, for use in raising an immune response in an organism. The proteins and genes described herein may find particular application as a vaccine, but could also be used to obtain an immune serum potentially useful in passive vaccination techniques.
Advantageously, the invention may provide a vaccine for use in preventing or controlling disease in canine species caused or contributed to by Staphylococcus pseudintermedius. In other embodiments, the vaccines provided by this invention may be used to protect against the development of infections caused or contributed to by Staphylococcus pseudintermedius. In other embodiments, the vaccines may be used to protect against instances of canine pyoderma.
In one embodiment, the vaccine may be a polypeptide and/or polynucleotide vaccine. A polynucleotide vaccine may comprise a polynucleotide fragment, preferably a DNA fragment, having a nucleotide sequence encoding an antigenic polypeptide comprising at least an antigenic portion any one or more of the Sps proteins described herein. Vaccines of this type may otherwise be referred to as "DNA vaccines" - such vaccines may be introduced to host cells (such as mammalian, for example, canine cells) where they express antigens which elicit immune responses.
A polypeptide or protein vaccine may comprises one or more of the Sps proteins (or antigentic fragments or portions) described herein. One of skill will appreciate that the one or more Sps protein(s) may be naturally occurring and isolated from Staphylococcus pseudintermedius, or recombinant.
A protein vaccine may be administered by any suitable route. Advantageously, a protein vaccine may be administered orally (by ingestion), topically or by direct injection - preferably intraperitoneal or intramuscular injection. A protein subunit vaccine formulated for oral administration can contain the polypeptide encapsulated in for example, a biodegradable polymer as described hereinafter.
In view of the above, the invention further provides a method of immunising a dog against Staphylococcus pseudintermedius, said method comprising administering to the dog a DNA or protein vaccine of the invention.
Conveniently, the protein vaccines described herein may further include or comprise one or more adjuvant(s). Further, one or more booster vaccinations are preferably administered at time periods subsequent to the initial administration to create a higher level of immune response in the animal.
In yet another aspect, the vaccine of the invention may comprise a fusion protein comprising a carrier polypeptide and one or more Sps protein(s) of the invention. The Sps protein(s) for use in this aspect of the invention can itself be antigenic or non-anti genie; in embodiments wherein the protein is non-antigenic, the carrier polypeptide is antigenic, stimulating the immune system to react to the fusion protein thereby generating an immune response in an organism - such as, for example a canine immune response to Staphylococcus pseudintermedius. A non-antigenic protein thus functions as a hapten. An example of an antigenic carrier polypeptide is keyhole limpet hemocyanim (KLH). Conventional fusion constructs between carriers such as glutathione sulfotransferase (GST) and said Sps protein(s) of the invention are also included as protein vaccines according to the invention, as are fusions of the Sps protein(s) and an affinity tag such as a polyhistidine sequence. A fusion construct may be preferred for use as a protein vaccine when the antigenic Sps analog, fragment, or modification thereof is small.
In a seventh aspect, the present invention provides a method for immunising dogs against Staphylococcus pseudintermedius, said method comprising administering to the dog a vaccine of the invention.
A polynucleotide vaccine may further comprises a promoter, such as the CMV promoter, operably linked to the coding sequence for the Sps polypeptide or antigenic fragment thereof (e.g., U.S. Pat. No. 5,780,44, Davis). The polynucleotide may be cloned within a vector such as a plasmid. There are numerous plasmids known to those of ordinary skill in the art useful for the production of polynucleotide vaccines.
Other possible additions to the polynucleotide vaccine constructs include nucleotide sequences encoding cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12) and co-stimulatory molecules such B7-1 , B7-2, CD40. The cytokines can be used in various combinations to fine- tune the response of the animal's immune system, including both antibody and cytotoxic T lymphocyte responses, to bring out the specific level of response needed to affect the animal's reproductive system. A polynucleotide vaccine of the invention can also encode a fusion product containing the antigenic polypeptide and a molecule, such as CTLA-4, that directs the fusion product to antigen-presenting cells inside the host.
Plasmid DNA can also be delivered using attenuated bacteria as delivery system, a method that is suitable for DNA vaccines that are administered orally. Bacteria are transformed with an independently replicating plasmid, which becomes released into the host cell cytoplasm following the death of the attenuated bacterium in the host cell. An alternative approach to delivering the polynucleotide to an animal involves the use of a viral or bacterial vector. Examples of suitable viral vectors include adenovirus, polio virus, pox viruses such as vaccinia, canary pox, and fowl pox, herpes viruses, including catfish herpes virus, adenovirus-associated vector, retroviruses and bacteriophage. Exemplary bacterial vectors include attenuated forms of Salmonella, Shigella, Edwardsiella ictaluri, and Yersinia ruckeri. Preferably, the polynucleotide is a vector, such as a plasmid, that is capable of autologous expression of the nucleotide sequence encoding said Sps protein or fragment thereof.
In one embodiment, the vaccine may be a DNA vaccine comprising a DNA fragment having a nucleotide sequence that encodes a polypeptide having an amino acid sequence homologous or identic to a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34 or an antigenic analog, fragment, or modified version thereof.
Polynucleotide-based immunisation induces an immune response to an antigen expressed in vivo from a heterologous polynucleotide fragment introduced into a cell. DNA vaccine may be particularly useful as the heterologous nucleic acid expression may continue for a length of time sufficient to induce a relatively strong and sustained immune response without the need for subsequent "booster" vaccinations, as may be required when using protein based vaccines. A polynucleotide vaccine comprising a polynucleotide fragment having a nucleotide sequence encoding said Sps can be administered to dog (or rather to a particular tissue or cells thereof) using biolistic bombardment, ingestion or direct injection, as described for example, in U.S. Pat. No. 5,780,448 (Davis), preferably intraperitoneal or intramuscular injection. A preferred method of administration is biolistic bombardment, as with a "gene gun". A polynucleotide vaccine formulated for oral administration preferably contains DNA encapsulated in a biodegradable polymer. Examples of a suitable biodegradable polymer include chitosan and homo- or co-polyers of polylactic acid and polyglycolic acid. Accordingly, the present invention further provides a method for immunising dogs against Staphylococcus pseudintermedius by administering to the dog a polynucleotide vaccine of the invention, preferably a DNA vaccine.
Other methods of administering nucleic acid vaccines of the type described herein may include, for example, use of the technology described in WO02/076498.
The amount of protein/polynucleotide vaccine to be administered to an animal depends on the type and size of animal, the condition being treated, and the nature of the protein/polynucleotide, and can be readily determined by one of skill in the art. In some applications, one or more booster administrations of the protein/polynucleotide vaccine at time periods subsequent to the initial administration are useful to create a higher level of immune response in the animal.
In one embodiment of the vaccine of the invention and/or Sps proteins described herein (including antigenic fragments, analogs or modified version thereof) may be linked, for example, at its carboxy-terminus, to a further component. The further component may serve to facilitate uptake of the Sps protein, or enhance its immunogenicity/processing. The immune-stimulating compositions of the invention may be optionally mixed with excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the active component(s). The term "pharmaceutically acceptable carrier" refers to a carrier(s) that is "acceptable" in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Suitable excipients are well known to the person skilled in the art. Examples include; water, saline (e.g. 0.85% sodium chloride; see Ph.Eur. monograph 2001 :0062), buffered saline, fish oil with an emulsifier (e.g. a lecithin, Bolec MT), inactivant (e.g. formaldehyde; see Ph.Eur. monograph 1997:0193), mineral oils, such as light mineral oils, alhydrogel, aluminium hydroxide. Where used herein, the term "oil adjuvant" to embraces both mineral oils and synthetic oils. A preferred adjuvant is Montanide ISA 71 1 (SeppicQuai D'Orsay, 75321 Paris, France) which is a manide oleate in an oil suspension. In addition, if desired, the immune-stimulating composition (including vaccine) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the immune-stimulating composition.
A vaccine composition may be administered as a course of a number of discrete doses over a period of time. For example it may be administered over a period of around 2-21 days.
Vaccination may be repeated at daily, twice-weekly, weekly or monthly intervals. For example a boost vaccination may be administered after the initial dose. For example a boost may be administered at around 4-14 weeks after the vaccination. The initial vaccination and any boost may be carried out using the same or different modes of administration. For example, the initial may be by injection and the boost may be by oral administration. An example regime includes a first vaccination by injection, followed by a course of orally administered boost vaccine, or a booster prior to an expected outbreak. However, it will be appreciated that any suitable route of administration(s) and/or regime(s) may be employed.
Additionally, knowledge of the Sps protein nucleotide and amino acid sequences set forth herein opens up new possibilities for detecting, diagnosing and characterising Staphylococcus pseudintermedius in canine populations. For example, an oligonucleotide probe or primer based on a conserved region of one or more of the Sps proteins described herein, may be used to detect the presence of the Sps protein in or on a canine host. Vaccines may contain one or more of the Sps proteins/nucleic acids/genes described herein (i.e. those shown as SEQ ID NOS: 1-38). In one embodiment, the vaccine may comprise a cocktail of Sps proteins/peptides and or nucleic acids. Typically, a cocktail may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 Sps nucleic acid and/or protein/peptide components (for example (2 or more) components having sequences homologous or identical to any of SEQ ID NOS: 1 -36).
Furthermore, the vaccines may contain bacterial antigens used to control other diseases, for example diseases caused by other Staphylococcal species and/or antigens to treat, prevent or control diseases and/or conditions with other aetiologies or caused or contributed to by other pathogens. As such, the vaccine compositions described herein may find application in multivalent vaccines including antigens against other canine diseases.
In addition to vaccines and/or immunogenic compositions comprising one or more of the Sps proteins described herein, the present invention further provides compounds for treating infections caused or contributed to by Staphylococcus pseudintermedius or compounds for the preparation of medicaments for treating the same.
In one embodiment, the compound may be a small organic molecule, antibody, peptide or carbohydrate which antagonises the interaction between the Sps protein and its ligand (an extracellular matrix (ECM) protein). For example, the compound may be a synthetic peptide comprising or based on, the sequence of an ECM protein known to interact with a particular Sps protein, or the sequence of a protein given above which may interfere with binding between the wild type 5. psedintermedius protein and its ligand. Additionally or alternatively, binding agents, such as for example, antibodies with specificity or affinity for one or more Sps protein ligands, may also be used to antagonise the Sps/ligand interaction. Therapeutic approaches of this type may prevent Staphylococcus pseudintermedius colonising or binding/adhering to cells.
In view of the above, the invention may relate to methods of treating infections caused or contributed to by Staphylococcus pseudintermedius, said method comprising administering to an animal a therapeutically effective amount of a compound which antagonises Sps/ligand interactions. In a further aspect, the present invention provides pharmaceutical compositions comprising a compound which antagonises Sps/ligand interactions together with a pharmaceutical excipient, carrier or diluent.
One of skill will appreciate that the vaccines, methods, uses or medicaments comprising any of the Sps genes/nucleic acids and/or proteins and/or antagonistic compounds (for example Sps protein/nucleic acid fragments and/or antibodies) described herein may be combined with one or more other compounds for treating one or more other conditions - in particular one or more other skin conditions. Said other skin condition may be, for example, atopic dermatitis.
In a further aspect, the present invention provides methods of diagnosing infections, diseases and/or conditions caused or contributed to by S. pseudintermedius, said methods comprising the steps of identifying in a sample provided by a subject suspected of suffering from an infection, disease and/or conditions S. pseudintermedius caused or contributed to by S. pseudintermedius, a level of a protein, peptide or nucleic acid (for example a gene) encoded by a sequence provided by SEQ ID NOS: 1-36 or a fragment, portion, mutant, derivative and/or homologoue/orthologue thereof.
It should be understood that all methods of diagnosis or detection described herein, may include an optional step in which the results are compared with the results of a control sample, which does not comprise sequences derived from S. pseudintermedius, in particular sequences corresponding to those provided as SEQ ID NOS: 1 -36 disclosed herein.
The term "sample" may be taken to mean any sample comprising protein and/or nucleic acid. For example, a "sample" may comprise a bodily fluids such as whole blood, plasma, serum, saliva, sweat and/or semen. In other instances "samples" such as tissue biopsies and/or scrapings may be used. In particular, cutaneous (i.e. skin) tissue biopsies and/or scrapings may be used. Advantageously such biopsies may comprise cells obtained from lesions suspected of resulting from or bein associated with a S. pseudintermedius. Specifically, a biopsy, tissue sample or scraping may comprise cells derived from lesions exhibiting pathology characteristic of the S. pseudintermedius disease, pyoderma (particularly caninine pyoderma).
In addition, a sample may comprise a tissue or gland secretion and washing protocols may be used to obtain samples of fluid secreted into or onto various tissues, including, for example, the skin. One of skill in this field will appreciate that the samples described above may yield or comprise quantities of nucleic acid (i.e. D A or RNA) encoding all or part of the various proteins described herein as well as quantities of proteins or peptides (or fragments thereof) encoded thereby. In one embodiment, the sample may comprise quantities of nucleic acid/peptide having or comprising the sequences given as SEQ ID NOS: 1-36.
One of skill in the art will be familiar with the techniques that may be used to identify levels of certain nucleic acid sequences and/or proteins, such as, for example, levels of the sequences given as SEQ ID NOS: 1-36 described herein (or a fragment, portion, mutant, derivative and/or homologoue/orthologue thereof).
For example, PCR based techniques may be used to detect levels of gene expression or gene quantity in a sample. Useful techniques may include, for example, polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR based techniques in combination with real-time PCR (otherwise known as quantitative PCR).
Additionally, or alternatively, a level of gene/protein expression may be identified by way of microarray analysis. Such a method would involve the use of a DNA micro-array which comprises nucleic acid derived from one or more of the nucleic acid sequences described herein (for example SEQ ID NOS: 1, 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35). To identify a level of gene expression, one of skill in the art may extract nucleic acid, preferably mRNA, from a sample and subject it to an amplification protocol such as, for example RT- PCR to generate cDNA. Preferably, primers specific for a certain mRNA sequence - in this case a S. pseudintermedius sequence comprised with any of, for example, SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35.
The amplified cDNA may be subjected to a further amplification step, optionally in the presence of labelled nucleotides (as described above). Thereafter, the optionally labelled amplified cDNA may be contacted with the microarray under conditions which permit binding with the DNA of the microarray. In this way, it may be possible to identify a level of S. pseudintermedius gem expression.
Further information regarding the PCR based techniques described herein may be found in, for example, PCR Primer: A Laboratory Manual, Second Edition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold Spring Harbour Laboratory Press and Molecular Cloning: A Laboratory Manual by Joseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press. In addition, other techniques such as deep sequencing and/or pyrosequencing may be used to detect cSCC sequences in any of the samples described above. Further information on these techniques may be found in "Applications of next-generation sequencing technologies in functional genomics", Olena Morozovaa and Marco A. Marra, Genomics Volume 92, Issue 5, November 2008, Pages 255-264 and "Pyrosequencing sheds light on DNA sequencing", Ronaghi, Genome Research, Vol. 1 1, 2001, pages 3-1 1.
In addition to the molecular detection methods described above, one of skill will also appreciate that immunological detection techniques such as, for example, enzyme-linked immunosorbent assays (ELISAs) may be used to identify levels of S. pseudintermedius proteins in samples. In other embodiments, ELISPOT, dot blot and/or Western blot techniques may also be used. In this way, samples provided by subjects suffering from S. pseudintermedius related diseases and/or infections (for example canine subjects suffereing from canine pyoderma), may be probed for levels of one or more S. pseudintermedius proteins, particularly those encoded by SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36, so as to detect the presence of such proteins in a sample which may indicate a S. pseudintermedius infection.
Immunological detection techniques, may require the use of a substrate to which an antibody and/or antigen may be bound, conjugated or otherwise immobilised.
Suitable substrates may comprise, for example, glass, nitrocellulose, paper, agarose and/or plastic. A substrate which comprises, for example, a plastic material, may take the form of a microtitre plate.
Further information regarding ELISA procedures and protocols relating to the other immunological techniques described herein may be found in Using Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1999) and Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1988).
The present invention also extends to kits comprising reagents and compositions suitable for diagnosing, detecting or evaluating possible S. pseudintermedius infections, diseases and/or conditions. Kits according to this invention may be used to identify and/or detect levels of S. pseudintermedius gene(s)/ 5. pseudintermedius protein(s) in samples. Depending on whether or not the kits are intended to be used to identify levels of S. pseudintermedius genes and/or S. pseudintermedius proteins in samples, the kits may comprise substrates having S. pseudintermedius proteins or agents capable of binding S. pseudintermedius proteins, bound thereto. In addition, the kits may comprise agents capable of binding S. pseudintermedius proteins - particularly where the kit is to be used to identify levels of one or more S. pseudintermedius proteins in samples. In other embodiments, the kit may comprise polyclonal antibodies or monoclonal antibodies which exhibit specificity and/or selectivity for one or more 5. pseudintermedius proteins. Antibodies for inclusion in the kits provided by this invention may be conjugated to detectable moieties. Kits for use in detecting the expression of genes encoding S. pseudintermedius proteins may comprise one or more oligonucleotides/primers for detecting/amplifying/probing samples for S. pseudintermedius protein encoding sequences. The kits may also comprise other reagents to facilitate, for example, sequencing, PCR and/or RFLP analysis. In one embodiment, the kits may comprise one or more oligonucleotides/primers for detecting/amplifying/probing nucleic acid samples (for example nucleic acid derived from canine skin) for levels of sequences corresponding to all or part of those described as SEQ ID NOS: 1-36 herein.
DETAILED DESCRIPTION
The invention will now be described in more detail with reference to the following Figures which show:
Figure 1. Genomic location of the 17 genes encoding putative CWA proteins in S. pseudintermedius strain ED99. Eight genes are situated in the oriC environ, indicated in orange, and nine are located in the core genome, sps = S. pseudintermedius surface protein.
Figure 2. Distribution of the genes encoding putative CWA proteins among 20 S. pseudintermedius strains, representatives of the closely related S. delphini and S. intermedius, and other staphylococcal species associated with animal skin disease. The diversity of strains is represented a phylogenetic tree; grey squares indicate that the gene is present, blank squares that the gene is absent based on Southern blot analysis (for spsA to spsO), or PCR amplification (for spsP and spsQ).
Figure 3. Western blot analysis of cell wall-associated proteins of S. pseudintermedius ED99 and L. lactis expressing SpsD, SpsL, and SpsO with sera from dogs diagnosed with pyoderma. (A) SDS PAGE analysis and (B) Western blot analysis of protein fractions from S. pseudintermedius ED99 in exponential phase of growth (lane 1 ); L. lactis expressing SpsL (lane 2); L. lactis expressing SpsD (lane 3); L. lactis expressing SpsO (lane 4); and L. lactis with pOri23 alone (lane 5).
Figure 4. Adherence of L. lactis expressing specified MSCRAMMs to human Fn. Plates were coated with 1 μg of human Fn or 1 g of BSA per well. Absorbance was measured at 590 nm and results are expressed as mean values of triplicate samples. Error bars indicate standard deviation. L. lactis expressing FnbpA from S. aureus and PBS were included as controls.
Figure 5. Adherence of L. lactis expressing specified MSCRAMMs to Fg from different animal sources. Plates were coated with 1 μg of Fg or 1 μg of BSA per well. Absorbance was measured at 590 nm and results are expressed as mean values of triplicate samples. Error bars indicate standard deviation. L. lactis expressing FnbpA from S. aureus and PBS were included as controls.
Figure 6. Adherence of L. lactis expressing specified MSCRAMMs to CK10. Plates were coated with 1 g of recombinant CK10 or 1 μg of BSA per well. Absorbance was measured at 590 nm and results are expressed as mean values of triplicate samples. Error bars indicate standard deviation. S. aureus strain SHI 000 in exponential and stationary phases of growth and PBS were included as controls.
Figure 7. Adherence of L. lactis expressing different MSCRAMMs to canine corneocytes of five dogs. Bacterial adherence is calculated as percentage area covered with bacterial cells per field of corneocytes (ROI = 500 μιη 2 ). Results are based on the arithmetic mean of duplicate experiments. The bottom of each box represents the first quartile (Ql), the top of the box the third quartile (Q3), the bold lines the median, and the black circles the mean values. The whiskers define the range of the data.
Figure 8: Reactivity of canine convalescent serum from pyoderma cases to Sps D, Sps L and Sps O recombinant A domain. 1 ug aliquots of rSps D and rSps L, and 10 μΐ volumes of purified rSps O were subjected to SDS-PAGE under standard conditions and Coomassie stained (A) or Western blot transferred onto a nitrocellulose membrane. Membranes were probed with a 1 : 1000 dilution of canine serum, followed by a 1 :3000 dilution of HRP- conjugated sheep anti-canine IgG. Reactive bands were visualized on Chemi-luminescent Film (B). 5 μΐ aliquots of recombinant ClfB and the superantigen SEI from S. aureus were included in the terminal lanes of each gel as negative controls.
Figure 9: Inhibition of adherence of L. lactis expressing SpsD (A) and SpsL (B) to fibrinogen (2 g per well) by canine convalescent serum from pyoderma cases. Bacterial cultures, normalised to an OD600 of 1 in PBS were pre-incubated for 2 h with doubling dilutions of serum ranging from 2% to -0.01 % (v/v), prior to inoculation into fibrinogen coated wells. Results (n = 3, ± SD) are expressed as absorbance readings at 590 nm minus background levels of fluorescence. Background fluorescence was measured by inoculating control cultures, incubated for 2 h in the absence of serum, into wells coated with BSA (2 g per well). Incubations of S. aureus Newman (C) were included as a negative control.
Materials and Methods
Genome-wide screen for genes encoding for cell-wall anchored proteins
The S. pseudintermedius strain ED99 draft genome was interrogated for homologous sequences using position specific iterative basic local alignment search tool (PSI-BLAST), available from the National Center for Biotechnology Information (NCBI), USA (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and for the presence of a LPX[TSA][GANS] motif pattern by pattern hit initiated basic local alignment search tool (PHI-BLAST), available from NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Signal sequences were predicted by employing the SignalP server (http://www.cbs.dtu.dk/services/SignalP/), provided by the Center for Biological Sequence Analysis (CBS), Technical University of Denmark.
In silico structural analysis of cell-wall anchored proteins
The predicted CWA proteins were searched for functional domains using EMBL-EBI InterPro Scan (http://www.ebi.ac.ulc/interpro). Structural analysis was carried out with the PHYRE (Protein Homology/analogY Recognition Engine) fold recognition server, available from the Structural Bioinformatics Group, Imperial College London, UK (http://www.sbg.bio.ic.ac.uk/phyre/). Repeat sequences were predicted by generating nucleic acid dot plots, using software available from Colorado State University, USA (http://www.vivo.colostate.edu/molkit/dnadot/), applying tandem repeats finder software from Boston University, USA (http://tandem.bu.edu trf/trf.html), and variable sequence tandem repeats extraction and architecture modelling (XSTREAM), available from the University of California, USA (http://jimcooperlab.mcdb.ucsb.edu xstream/). Sequence alignments and pair- wise sequence comparisons were generated with ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2). Amino acid composition and molecular weight predictions were generated using ProtParam on the ExPASy Proteomics Server (http : //www .expasy.ch/tools/protparam.html). Cloning of selected genes encoding putative MSCRAMMs of S. pseudintermedius ED99 into L. lactis MG1363
Oligonucleotides were designed for PCR amplification of the full-length spsD, spsL and spsO genes and either Pstl, Sail or BamHl specific restriction sites were inserted on both sides of the DNA fragments. 50 μΐ PCR reactions contained 2 μΐ (approximately 100 ng) genomic DNA template, 0.25 μΜ forward primer, 0.25 μΜ reverse primer, l PfuUltra™ II reaction buffer (Stratagene, USA), 0.25 mM dNTP's (Promega, USA) and 1 μΐ PfuUltra™ II Fusion HS DNA polymerase (Stratagene, USA). The thermocycler programme included an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 50°C for 20 s and extension at 72°C for 2 min, followed by a final extension step at 72°C for 3 min. PCR products were visualised on 0.8% (w/v) agarose gels, gel extracted under avoidance of UV light exposure and purified using QIAquick Gel Extraction Kit (Qiagen, UK) according to the manufacturer's instructions. Purified DNA fragments were cloned into the StrataClone™ Blunt PCR cloning vector pSC-B (Stratagene, USA) using the StrataClone Ultra™ Blunt PCR Cloning Kit (Stratagene, USA) according to the manufacturer's instructions. Each cloning reaction consisted of 3 μΐ Strataclone Buffer Blunt (Stratagene, USA), 2 μΐ purified PCR product and 1 μΐ Strataclone™ Blunt Vector Mix (Stratagene, USA). StrataClone™ SoloPack ® competent cells (Stratagene, USA) were transformed according to the manufacturer's instructions and colonies selected using blue- white screening on LB-ampicillin (100 μg/ml)-X-gal plates. White colonies were transferred into 5 ml LB-ampicillin (100 μg/ml) broth and grown overnight at 37°C with shaking at 200 rpm. Plasmid was isolated using QIAprep Spin Miniprep Kit (Qiagen, UK) according to the manufacturer's instructions. Purified plasmids were digested using appropriate restriction endonucleases (New England Biolabs, UK), and diagnostic digests were analysed on 0.8% (w/v) agarose gels. For generating DNA constructs, the E. coli - L. lactis shuttle vector pOri23 (kindly provided by P. Moreillon, University of Lausanne, Switzerland) was used. The pOri23 vector carries the ermAM gene for erythromycin resistance, the high-copy-number oriColEl replicon for autonomous replication in E. coli and the constitutive lactococcal promoter P23 (Que et al, 2000). The multiple cloning site of pOri23 consists of restriction sites for endonucleases Pstl, Sail and Ba Rl (Que et al, 2000). StrataClone™ plasmids containing the DNA inserts of interest and the E. coli - L. laclis shuttle vector pOri23 were each digested in a 100 μΐ total reaction volume containing 10 μΐ plasmid (approximately 2.5 μg), 20 units appropriate restriction endonucleases (New England Biolabs, UK), and suitable buffers (New England Biolabs, UK) according to the manufacturer's instructions. Restriction digestions were performed at 37°C for 16 h. The restriction fragments to be cloned were extracted from 0.8% (w/v) agarose gels without UV exposure as described in the general Material and Methods and purified using QIAquick Gel Extraction Kit (Qiagen, UK) according to the manufacturer's instructions. DNA inserts and restriction-digested pOri23 plasmid were quantified using spectrophotometry (NanoDrop ND-1000, Thermo Scientific, USA) and ligation reactions were carried out with a plasmid to insert ratio of 1 :3 in a 10 μΐ total ligation reaction volume, consisting of 1 μΐ vector (approximately 10 ng), 400 units T4 DNA ligase (New England Biolabs, UK), l x T4 DNA ligase reaction buffer (New England Biolabs, UK), x μΐ DNA insert (depending on DNA concentration), and x μΐ sterile water (depending on the volume of DNA insert). Ligations were incubated at 16°C for 16 h.
One 50 μΐ aliquot of electrocompetent L. lactis cells was thawed on ice and 2 μΐ (~20 ng) pOri23 plasmid carrying the DNA insert of interest was added. Electroporation cuvettes (Sigma-Aldrich, UK) were pre-chilled and L. lactis cells plus plasmid were transferred into the cuvettes. Electroporation was performed at standard settings (25 μΡ, 2.5 kV, 200 Ohm) and 1 ml GM17 was added immediately. Cells were incubated at 30°C in a static incubator for 2 h prior to spreading 250 μΐ of cell suspension per plate onto GM17 plates containing 5 μg/ml erythromycin. Plates were incubated overnight at 30°C.
For screening of L. lactis transformants, plasmid was isolated using the Qiagen MiniPrep Kit (Qiagen, UK) with addition of 100 U/ml mutanolysin (Sigma-Aldrich, UK) and 100 μg ml lysozyme (Sigma-Aldrich, UK) to buffer PI . Diagnostic digests of purified plasmids were carried out with appropriate restriction enzymes and analysed on 0.8% (w/v) agarose gels.
Additionally, colony PCR was performed for pOri23 carrying spsD and spsO using gene-specific oligonucleotides (Table 5.3). L. lactis colonies were resuspended in 10 μΐ 10% (v/v) IGEPAL (Sigma-Aldrich, UK) and incubated for 10 min at 95°C in a thermocycler machine. 40 μΐ master mix containing 0.3 μΜ forward primer, 0.3 μΜ reverse primer, 0.2 mM dNTP's (Promega, USA), 1 x reaction buffer (Promega, USA), 1.5 mM MgCl 2 (Promega, USA) and 0.025 u/μΐ taq polymerase (Promega, USA) was added. The thermocycler programme included an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min, followed by a final extension step at 72°C for 7 min. PCR products were visualised on 0.8% (w/v) agarose gels.
Western blot analysis of L. lactis constructs
Samples were dissolved in lx Laemmli sample buffer (Sigma- Aldrich, UK), boiled for 10 min and resolved by SDS-PAGE in 10% polyacrylamide gels by standard procedures, and Western blot analysis was carried out as described in the general Materials and Methods. Three canine sera samples from pyoderma cases (obtained from patients at the Hospital for Small Animals, The Royal (Dick) School of Veterinary Studies, The University of Edinburgh) were pooled and used as primary antibody in a 1 : 1000 dilution. HRP-conjugated sheep anti-dog antibody was used as a secondary antibody in a 1 :5000 dilution (Bethyl Laboratories Inc., USA).
Canine corneocyte adherence assay
For preliminary experiments to confirm adherence of S 1 . pseudintermedius ED99 and non-adherence of L. lactis, corneocytes were obtained from a seven-year-old male neutered Border collie cross-breed with no history or physical signs of systemic or cutaneous disease. Corneocytes for the L. lactis adherence study were obtained from five dogs of different breeds (one Labrador retriever, two Border collies and two cross-breeds). Three dogs were ovariohysterectomised females and two were entire males. The median age was seven years (range one to twelve years). The dogs showed no abnormalities on general physical examination and had no history or physical signs of skin disease at the time of corneocyte collection. All dogs were privately owned by staff or students of the Royal (Dick) School of Veterinary Studies, The University of Edinburgh. None of the dogs had received topical or systemic drug treatments for at least three weeks prior to the day of corneocyte collection.
Samples were taken from the ventral abdomen and inner thigh. If necessary, sample sites were clipped with Oster clippers (Oster Cryotech, USA) using a number 40 blade. For collection of corneocytes, the method described by Forsythe et al. (2002) was used. Briefly, the area was cleaned of surface debris and commensal bacteria by applying four strips of single sided adhesive tape (Cellux, Henkel Consumer Adhesives, UK), using each strip once. To collect corneocytes, double- sided, clear, adhesive wig tape (Tropical Tape Super Grip, USA) was mounted onto a microscope slide in 1 cm 2 pieces and applied to the same area of skin surface 10 times with gentle force. Slides were investigated by microscopic examination and only slides with at least 75% corneocyte coverage were used in the study.
The corneocyte slides were positioned in moisture chambers (Nunc™, Thermo Fisher Scientific, Denmark) as described by Forsythe et al. (2002). The moisture chambers consisted of 30 cm x 30 cm plastic trays with lids and were prepared by lining the trays with moistened paper towels. S. pseudintermedius ED99 stationary or exponential (OD 00 of 0.5) phase cultures and L. lactis exponential phase cultures (OD 6 oo 0.6 to 0.8) were centrifuged at 4000 rpm for 5 min, washed with PBS and resuspended in PBS to a final OD 600 of 0.5. The moisture chambers were placed in a static incubator and 250 μΐ of bacterial suspension was added to each 1 cm 2 of tape, forming a meniscus on the tape. Slides incubated with 250 μΐ of sterile PBS were included as a control. The slides were incubated at 37°C for 90 min and washed in PBS. Each slide was stained with 0.5% (w/v) crystal violet (Sigma-Aldrich, UK) for 90 s before rinsing off with PBS. The slides were air-dried and a drop of immersion oil (Cargille Laboratories Inc., USA) and a cover slip (Scientific Laboratory Supplies, UK) were added before microscopic quantification. All slides were prepared in duplicate on the same day and incubated at the same time. Prior to incubation with bacterial suspensions or PBS, each slide was labelled with a letter code to allow identification after the microscopic analysis. The identification code on each slide was hidden by a third party for subsequent image acquisition so that the investigator was blinded to the origin of the slide. For quantification of adherent bacteria, computerised image analysis was used as described previously by Forsythe et al. (2002) with minor modifications. For each slide, bright field images of lOOOx oil-immersion fields were acquired with a Sony DXC-390P 3CCD colour video camera (Scion Corporation, USA) connected to a Leica Laborlux S microscope (Leica Microsystems UK Ltd., UK). The RGB video signal from the camera was digitised using Scion Image (Scion Corporation, USA) installed in a G4 Macintosh computer (Apple Computer, USA) fitted with a CG-7 frame grabber (Scion Corporation, USA). For image acquisition, fields equivalent to 14.4 mm 2 were selected randomly by starting in the bottom left corner of each slide and moving through the slide in a defined way using the scale on the microscope stage. A field was discarded if the corneocyte layer was not confluent, the bacteria were poorly stained against the background or the field could not be focused properly. The software used for quantification of bacterial adherence was set to calculate the percentage area that was covered by bacteria per confluent layer of corneocytes in a defined region of interest (ROl) of 500 μηι 2 within each image field acquired. Previous studies by Forsythe et al. (2002) using the same technique and software have demonstrated that 15 replicates of each duplicate slide resulted in acceptable coefficients of variation of approximately 10%. In this study, 25 replicates of each slide were acquired and the overall mean percentage area of adherence was determined by calculating the mean of all replicates.
Results
Identification of genes encoding; 17 putative cell-wall-anchored proteins in the S. pseudinlermedi s ED99 genome
The initial search for putative CWA proteins identified 34 sequences that fulfilled at least one of the search criteria (homology to characterised MSCRA Ms in the database, predicted LPXTG motif or variant near the C terminus, predicted signal peptide at the N terminus). After gap closure and combination of incomplete sequences, a total of 17 ORFs encoding putative CWA proteins with a predicted minimum length of approximately 250 amino acids was determined. The 17 predicted CWA proteins were designated 'Sps' for ' Staphylococcus pseudintermedius surface proteins', followed by a capital letter (SpsA to SpsQ). Their position in the S. pseudintermedius ED99 genome is indicated in Figure 1. Of note, eight genes encoding putative CWA proteins are located near the oriC environ (Figure 1). Homology searches in the database resulted in sequence identities with known staphylococcal proteins ranging from -30% to -80% (Table 1). Signal sequences, necessary for Sec-dependent protein secretion (Foster and Hook, 1998), were predicted for 14 putative Sps proteins, consisting of 29 aa for SpsC and SpsK, 33 aa for SpsN, SpsP, and SpsQ, 36 aa for SpsD, 37 for SpsG, 38 aa for SpsA, SpsB, and SpsL, 39 aa for SpsH, 44 aa for SpsO, and 48 aa for SpsF and SpsM. No signal sequence was predicted for SpsE, Spsl, and SpsJ (Figure 4.3).
The putative CWA proteins SpsD, SpsL, and SpsO have several MSCRAMM features.
Out of the 17 putative CWA proteins of S. pseudintermedius ED99, SpsD, SpsL, and SpsO contained each of the MSCRAMM features screened for, including a signal sequence at the N-terminus, followed by a non-repeated A domain with two IgG-like folds, dividing the A domain into Nl , N2, and N3 subdomains, a tandemly repeated domain at the C-terminus (and at the N-terminus for SpsO), and a C-terminal LPXTG-anchor motif. The main characteristics of SpsD, SpsL, and SpsO are summarised in Table 2. Of interest, a TYTFTDYVD motif or variant, important for the 'dock, lock and latch' ligand-binding mechanism (Ponnuraj et al., 2003), was found in SpsD, SpsL, and SpsO, and putative latching sequences were identified (Table 2). Further, putative Fn-binding motifs with weak homology to FnbpA-10 of FnbpA in S. aureus were detected in the repeat region of SpsL (24% identity in pair- wise alignments for SpsLl -SpsL6, and 21% for SpsL-7). No homology to Fn-binding motifs of FnbpA was detected in the repeat regions of SpsD and SpsO. Of note, the genes encoding for SpsD, SpsL, and SpsO in the S. pseudintermedius ED99 genome are situated in different genomic contexts. While spsD is located in a well-conserved region of the core genome, spsL is part of the oriC environ (Takeuchi et al. , 2005) (Figure 1 ). The spsO gene appears to be species-specific as it is not present in the genomes of other staphylococcal species. The region contains two putative transposases, suggesting that the whole region might be subjected to horizontal gene transfer.
Distribution of the 17 genes encoding putative cell-wall-anchored proteins among the S. intermedius group
In order to investigate the distribution of the 17 genes encoding putative CWA proteins identified in the S. pseudintermedius ED99 genome among other members of the SIG and closely related staphylococcal species, Southern blot analysis and PCR amplification were performed. A total of 20 5 1 . pseudintermedius strains representing the breadth of diversity within the species, representatives of the closely related S. delphini and S. intermedius species, and other staphylococcal species associated with animal hosts (Figure 2) were screened for the presence of the putative CWA encoding genes by Southern blot analysis (spsA to spsO). For the S. aureus spa orthologues spsP and spsQ, PCR amplification was employed, as the genes share 70% nucleotide identity which precluded design of gene-specific probes for Southern blot analysis. For similar reasons, the primers designed for PCR amplification were located upstream of spsP (spsP-F), in the non-repeated region of spsP (spsP-R), in the unique region between spsP and spsQ (spsQ-F), and in a region unique for spsQ (spsQ-R).
Of the 17 genes examined, 13 were found in all S. pseudintermedius strains investigated. The remaining 4 (spsF, spsO, and the S. aureus spa orthologues spsP and spsQ) were present in 1 1 , 6, 7, and 11 of the 20 S. pseudintermedius strains, respectively. Furthermore, 8 of the 17 genes were detected in S. delphini and 6 in S. intermedins, and 9 genes were exclusive to & pseudintermedius. None of the genes encoding putative CWA proteins was detected in the non-SIG staphylococcal species examined. Results are summarised in Figure 2. Of note, it cannot be excluded at this point that DNA sequence variation in PCR primer annealing sites for spsP and spsQ, and weak homology (less than approximate 70%) for spsA to spsO among different strains have influenced the results.
Expression of CWA proteins on the S. pseudintermedius bacterial cell surface.
The in silico identification of 17 putative CWA proteins in S. pseudintermedius ED99 raises questions about the expression of these proteins and their role in colonisation and disease. Surface proteome analysis of early-, mid-, and late exponential phase S. pseudintermedius ED99 was performed in collaboration with the Moredun Research Institute, Penicuik, Scotland, UK, using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS- MS). Six out of the 17 putative CWA proteins predicted in the 5. pseudintermedius ED99 genome were detected on the bacterial surface, including SpsD, SpsK, SpsL, SpsN, SpsO, and SpsQ. The putative CWA proteins SpsL, SpsN, and SpsQ were identified in all three phases of growth; SpsK was lacking in early-, SpsO in mid-, and SpsD in late exponential phase. The 1 1 undetected CWA proteins might not have been expressed under the conditions tested, or the expression level might have been below the detection threshold of the LC-ESI-MS-MS method used.
Cloning and expression of SpsD, SpsL, and SpsO in L. lactis.
In order to examine the role of putative selected MSCRAMMs independently on the bacterial cell surface, the full-length spsD (3096 bp), spsL (2793 bp) and spsO (5538 bp) genes were cloned into L. lactis using the shuttle vector pOri23 (Que et al , 2000). Positive clones were identified by restriction digestion of purified pOri23 plasmids from single colonies of transformed L. lactis cells (data not shown). The pOri23 construct inserts were verified by DNA sequencing for spsL and spsD. For spsO, DNA sequence was generated for approximately 3000 bp of the total length of 5538 bp. A segment of the repeat region corresponding to -2500 bp could not be determined due to the existence of identical tandem repeats which did not allow directed sequencing. As a negative control for subsequent MSCRAMM characterisation studies, L. lactis was transformed with the empty vector pOri23, confirmed by restriction digestion analysis. The predicted molecular weights were 1 15 kDa for SpsD, 103 kDa for SpsL, and 198 kDa for SpsO. L. lactis expressing SpsD and SpsL demonstrated seroreactivity with canine sera from pyoderma cases.
The potential antibody response to SpsD, SpsL, and SpsO in vivo was investigated by Western blot analysis employing canine sera from staphylococcal pyoderma cases. The pyoderma was clinically manifested at the time of blood sampling and the dogs were also diagnosed with AD (Neuber et ah, 2008). Cell wall- associated protein fractions of the L. lactis constructs and of S. pseudintermedi s ED99 were subjected to SDS-PAGE, transferred to nitrocellulose membrane and incubated with pooled canine sera from three pyoderma cases as described in Materials and Methods. An array of immunoreactive bands was detected for S. pseudintermedius ED99, ranging from 24 kDa to 102 kDa in molecular weight (Figure 3). For L. lactis expressing SpsD and L. lactis expressing SpsL, multiple seroreactive bands in the range of 38 kDa to 225 kDa for SpsD, and 38 kDa and 52 kDa for SpsL were detected, which were absent in the protein fractions of L. lactis carrying pOri23 alone (Figure 3). In contrast, L. lactis expressing SpsO did not demonstrate seroreactivity with sera from dogs diagnosed with pyoderma (Figure 3).
Adherence of L. lactis constructs to extracellular matrix proteins.
L. lactis expressing SpsO, SpsD, SpsL, and L. lactis carrying the vector pOri23 alone were tested for their ability to adhere to human Fn, human, canine, feline, and bovine Fg, and to recombinant mouse C 10 in solid phase assays.
The putative MSCRAMMs SpsD and SpsL mediate binding of L. lactis to fibronectin.
L. lactis expressing SpsD and SpsL demonstrated adherence to human Fn, whereas L. laclis expressing SpsO demonstrated increased binding to Fn, but also to BSA, indicative of a non-specific interaction (Figure 4).
The putative MSCRAMMs SpsD and SpsL mediate binding of L. lactis to fibrinogen, and SpsL demonstrates canine host-specificity.
L. lactis expressing SpsD strongly adhered to Fg from several animal sources (Figure 5). In contrast, L. lactis expressing SpsL adhered to canine and feline Fg only, and did not bind to human and bovine Fg (Figure 5), indicating a host-specific interaction. L. lactis expressing SpsO did not bind to Fg from any source compared to L. lactis with the pOri23 vector alone (Figure 5).
The putative MSCRAMM SpsD mediates binding of L. lactis to cvtokeratin 10. L. lactis expressing SpsD demonstrated strong adherence to CK10, whereas L. lactis expressing SpsO and SpsL did not show increased binding compared to L. lactis with the vector pOri23 alone (Figure 6).
The putative MSCRAMMs SpsD and SpsO. but not SpsL, mediate adherence of L. lactis to ex vivo canine corneocytes.
L. lactis expressing SpsD, SpsL, and SpsO were tested for their ability to adhere to ex vivo canine corneocytes in comparison to L. lactis with the empty vector pOri23 and S. pseudintermedius ED99. L. lactis carrying the empty vector pOri23 adhered poorly to canine corneocytes (Figure 7). For S. pseudintermedius ED99, the mean percentage adherence to canine corneocytes was 4.24% which was significantly different to L. lactis carrying pOri23 alone (P = 0.001) (Figure 7). L. lactis expressing SpsD and L. lactis expressing SpsO adhered to ex vivo canine corneocytes (Figure 7). The increase in adherence was approaching significance for SpsD (P = 0.050), and was significant for SpsO when expressed in L. lactis compared to L. lactis carrying pOri23 alone (P = 0.004). Binding of L. lactis expressing SpsL was not significantly different to L. lactis carrying pOri23 alone (P = 0.108), indicating that SpsL does not promote adherence to canine corneocytes (Figure 7).
Purified recombinant Sps D, Sps L and Sps O demonstrate reactivity with canine convalescent serum.
Reactivity of recombinant A domain from Sps D, Sps L and Sps O with canine convalescent serum from pyoderma cases was examined by Western affinity blot analysis (Figure 8). rSpsD, rSpsL and rSpsO all crossreacted with IgG present in the canine serum (Figure 8).
Pre-incubation with canine convalescent serum inhibits SpsL-mediated binding to fibrinogen.
The ability of the reactive antibody present in convalescent serum to inhibit SpsD and SpsL ligand binding was investigated using a modified solid phase adherence assay. Prior to inoculation into fibrinogen coated wells, PBS normalised cultures of L. lactis expressing SpsD and SpsL were incubated for 2 h with doubling dilutions of convalescent serum at 28 °C (Figure 9). Convalescent serum inhibited binding of L. lactis expressing SpsL, but not SpsD to canine fibrinogen, with complete inhibition at a final concentration of 2% v/v (Figure 9).
Discussion In summary, genome-wide analysis of S. pseudintermedius ED99 revealed the presence of 17 genes encoding putative CWA proteins based on typical MSCRAM features. All MSCRAMM characteristics searched for were identified for SpsD, SpsL, and SpsO, including a signal sequence, a non-repeated A domain with two IgG-like folds, tandemly repeated regions, and a C-terminal LPXTG-anchor motif. Interestingly, SpsD, SpsL, and SpsO belong to different groups based on Southern blot analysis, with SpsD being present in all SIG members, SpsL in S. pseudintermedius only, and SpsO in only six of the S. pseudintermedius strains investigated, and not in the other SIG species tested. Based on in silico analysis and in vitro expression data, SpsD, SpsL, and SpsO were selected for functional characterisation.
All CWA proteins and in particular, SpsD, SpsL, and SpsO could be employed in passive and active immunisation studies to test their antigenic properties, either singular or in combination, in a similar fashion as proposed for S. aureus ClfA (Josefsson et al., 2001 ; Hall et al , 2003; Patti, 2004; Nanra et al., 2009). Further, a combinatory vaccine of S. aureus surface proteins IsdA, iron-regulated surface determinant protein B (IsdB), SdrD, and SdrE has proven to be highly protective in a mouse infection model (Stranger-Jones et ai, 2006), demonstrating the promising potential of vaccine preparations containing multiple staphylococcal CWA proteins.
In addition, MSCRAMMs with known ligands could be targets of anti- staphylococcal drug development, e.g. by generating synthetic peptides based on the interacting ECM proteins, which antagonise the MSCRAMM-host protein interaction, but do not interfere profoundly with physiological processes in the host. An excellent example is provided by Ganesh et al. (2008) who demonstrated that synthetic peptides, based on the Fg-binding site for ClfA, hinder the ClfA interaction, but do not block binding of the platelet integrin an b p 3 to Fg. Recently, Stranger-Jones et al screened the genome of the human pathogen S. aureus for all genes predicted to encode CWA proteins, and immunized mice with each protein to determine their capacity to protect against lethal or invasive infection (Stranger-Jones et al, 2006). Four of the proteins were combined into a multiple protein vaccine which induced high levels of protection against S. aureus invasive disease of mice. These data have stimulated renewed optimism in a vaccine for the prevention of human S. aureus infections. A similar approach could be used to design an effective vaccine for the prevention of S. pseudintermedius canine pyoderma. References
Bannoehr J, Ben Zakour NL, Waller AS, Guardabassi L, Thoday KL, van den Broek AH, Fitzgerald JR. (2007). Population genetic structure of the Staphylococcus intermedius group: insights into agr diversification and the emergence of methicillin- resistant strains. J Bacteriol. 189:8685-92
Ben Zakour, N. L., Guinane, C. M. & Fitzgerald, J. R. (2008) Pathogenomics of the staphylococci: insights into niche adaptation and the emergence of new virulent strains. FEMS Microbiol Lett, 289, 1-12.
Clarke, S. R. & Foster, S. J. (2006) Surface adhesins of Staphylococcus aureus. Adv Microb Physiol, 51 , 187-224.
Corrigan, R. M., Miajlovic, H. & Foster, T. J. (2009) Surface proteins that promote adherence of Staphylococcus aureus to human desquamated nasal epithelial cells. BMC Microbiol, 9, 22.
Clarke, S. R., Andre, G., Walsh, E. J., Dufrene, Y. F., Foster, T. J. & Foster, S. J. (2009) Iron-regulated surface determinant protein A mediates adhesion of Staphylococcus aureus to human corneocyte envelope proteins. Infect Immun, 77, 2408-16.
Curtis, C.F.,et al (2006) Masked, controlled study to investigate the efficacy of a Staphylococcus intermedius autogenous bacterin for the control of canine idiopathic recurrent superficial pyoderma. Vet Dermatol 17, 163-8 (2006).
Forsythe, P. J., Hill, P. B., Thoday, . L. & Brown, J. (2002) Use of computerized image analysis to quantify staphylococcal adhesion to canine corneocytes: does breed and body site have any relevance to the pathogenesis of pyoderma? Vet Dermatol, 13, 29-36.
Foster, T. J. & Hook, M. (1998) Surface protein adhesins of Staphylococcus aureus. Trends Microbiol, 6, 484-8.
Ganesh, V. K., Rivera, J. J., Smeds, E., Ko, Y. P., Bowden, M. G., Wann, E. R.,
Gurusiddappa, S., Fitzgerald, J. R. & Hook, M. (2008) A structural model of the
Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics. PLoS Pathog, 4, el 000226
Guardabassi, L., Schwarz, S. & Lloyd, D. H. (2004b) Pet animals as reservoirs of antimicrobial-resistant bacteria. J Antimicrob Chemother, 54, 321-32.
Hall, A. E., Domanski, P. J., Patel, P. R., Vernachio, J. H., Syribeys, P. J., Gorovits,
E. L., Johnson, M. A., Ross, J. M, Hutchins, J. T. & Patti, J. M. (2003) Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A. Infect I m n, 71 , 6864-70.
Hill, P.B. et al. (2006) Survey of the prevalence, diagnosis and treatment of
dermatological conditions in small animals in general practice Vet Rec 158, 533-9 (2006).
Josefsson, E., Hartford, O., O'brien, L., Patti, J. M. & Foster, T. (2001) Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis, 184, 1572-80.
Lindsay, J. A., Moore, C. E., Day, N. P., Peacock, S. J., Witney, A. A., Stabler, R. A., Husain, S. E., Butcher, P. D. & Hinds, J. (2006) Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface- associated and regulatory genes. J Bacteriol, 188, 669-76.
Mazmanian, S.K., et al. ( 1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760-3 ( 1999).
Nanra, J. S., Timofeyeva, Y., Buitrago, S. M., Sellman, B. R., Dilts, D. A., Fink, P., Nunez, L., Hagen, M., Matsuka, Y. V., Mininni, T., Zhu, D., Pavliak, V., Green, B. A., Jansen, K. U. & Anderson, A. S. (2009) Heterogeneous in vivo expression of clumping factor A and capsular polysaccharide by Staphylococcus aureus: implications for vaccine design. Vaccine, 27, 3276-80.
Otto, M. (2008) Targeted immunotherapy for staphylococcal infections : focus on anti- MSCRAMM antibodies. BioDrugs 22, 27-36 (2008)
Patti, J. M. (2004) A humanized monoclonal antibody targeting Staphylococcus aureus. Vaccine, 22 Suppl 1 , S39-43.
Pizza, M. et al. (2008) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1 816-1820
Table 1. Sequence homology of the 17 predicted cell-wall-anchored proteins against known proteins in the public domain.
Putative CWA Best Hit (BLAST) Identity Similarity protein (%) (%)
SpsA LPXTG cell-wall surface anchor family protein of S. aureus COL 3 1.2 56.9
SpsB RodA, a rod shape determining protein of S. epidermidis ATCC 12228 69.7 87.8
SpsC Afunctional autolysin precursor of S. epidermidis ATCC 12228 50.7 65.9
SpsD Fnbp protein homolog of S. aureus Mu50 40.7 59.1
SpsE Fibrinogen binding protein of S. epidermidis ATCC 12228 78.6 90.1
SpsF hypothetical protein, similar to the putative cell-surface adhesin SdrF of S. haemolyticus JCSC I435 52.8 69.3
SpsG hypothetical protein, cell-wall surface anchor family of Streptococcus pneumoniae D39 47.7 63.6
SpsH Sdr-repeat family protein SdrH, S. aureus USA300 36.0 53.1
Spsl serine-aspartate rich, fibrinogen-binding, bone sialoprotein-binding protein S. epidermidis ATCC 12228 37.3 55.5
SpsJ precursor of a serine-rich adhesin for platelets of 5. haemolyticus JCSC1435 52.2 61 .2
SpsK IgG-binding protein of S. aureus COL 50.4 71 .1
SpsL Fnbp protein homolog of S. aureus u50 33.4 5 1.7
SpsM hypothetical protein, similar to the putative cell-surface adhesin SdrF, S. haemolyticus JCSC 1435 44.4 61 .7
SpsN probable exported protein of S. aureus RF 122 38.0 60.0
SpsO serine-aspartate repeat-containing protein C precursor of Staphylococcus warneri L37603 50.0 68
SpsP LPXTG-motif cell wall anchor domain of S. aureus JH9 60.6 74.3
SpsQ IgG-binding protein A precursor of S. aureus MRSA252 57.0 71.7
Table 2. Main characteristics of the predicted CWA proteins SpsD, SpsL, and SpsO of S. pseudintermedius ED99.
Amino MW Signal LPXTG Ig-like fold b TYTFTDYVD- Putative latching Repeat Copy acids (kDa) a peptide motif (position) like motif sequence region number
(position) b (position) b (position) repeats
SpsD 1031 1 15 36 aa LPDTG 167-320 aa RYRFMDYVN NNASGEG 867-959 aa
322-519 aa (267-275 aa) (491-497 aa)
SpsL 930 103 38 aa LPKTG 220-363 aa VYTFKDYVN NSASGSG 543-818 aa
364-531 aa (298-306 aa) (502-508 aa)
SpsO 1846 198 44 aa LPNTG 339-492 aa TYTFTDYVD DKSTALG 661-1800 aa
487-659 aa (424-432 aa) (635-641 aa) 97-216 aa c
aa = amino acids; a MW = predicted approximate molecular weight in kDa (kilo dalton); b within the A domain; c N-terminal repeats