# Patent

## Title
Staphylococcal antigens

## Abstract

The present invention provides novel sequences encoding Staphylococcus pseudintermedius proteins/nucleic acids potentially useful in the treatment and/or prevention of canine disorders. In particular, the various protein and/or nucleic acid sequences described herein may find application as vaccines for use in treating and/or preventing a variety of canine diseases and/or conditions caused or contributed to by Staphylococcus pseudintermedius.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of PCT/GB2010/001916, filed Oct. 15, 2010, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/252,026, filed Oct. 15, 2009, both of which are hereby incorporated by reference in their entirety.


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 desirable 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: 1-38 below.







SEQ ID NO: 1







atggaaaacaaaaacttttttagtattcgtaaactatctattggtgtagg


 


ttcttgcttaatcgcgagttctttacttgtaaacacgccaagttttgctg


 


aagaaacagataatgcgaacattaatgacgcacaacaaaacgccttttat


 


gaaattttacatttgccaaacttaactgaagagcaacaaaatggattcat


 


ccaaagtcttaaagatgatccaagtgtgagcaacgacattttagtagaag


 


ctaagaaattaaatgacactcaagctaaacctgattacagtgaagcacaa


 


caaaatgcattttatgaaattttacatttgtcaaacttaactgaagagca


 


acaaaatggattcatccaaagtcttaaagatgatccaagtgtgagcaacg


 


acattttagtagaagctaagaagttaaatgacactcaagctaaacctgat


 


tacagtgaagcacaacaaaatgcattttatgaaattttacatttgtcaaa


 


cttaactgaagagcaacaaaatgggttcatccaaagccttaaagatgatc


 


caagtgtaagtaaagaaattttagcagaagctaagaagttaaatgatagt


 


caagcacctaaagttgataaagctaaaaaaactgacaaagctgaagcgaa


 


agcagatgataaagctaaaggtgaagaagccaaaaaatctgaagacaaaa


 


aagatagcaaagcagataaggcaaaatcgaaaaacgctacacatgttgtt


 


aaacctggtgaaactttagataatattgctaaagatcatcatacaactgt


 


tgataaaattgctaaagataacaaaataaaagataaaaatgtgattaaac


 


taggtcaaaaacttgttgttgataaacaaaaagcaactcaaggaaaacaa


 


gaagctgtagcgaaaaatgaagtgaaggctttacctaatactggtgaaaa


 


tgatgatatcgcattattcagcacaacagttgcgggtggcgtaagtatcg


 


ctttaggttcattattattaggaagaaacagaaaaactagctaa






The protein sequence translated from SEQ ID NO 1 is designated SEQ ID NO: 2 and is shown below:








SEQ ID NO: 2







MENKNFFSIRKLSIGVGSCLIASSLLVNTPSFAEETDNANINDAQQNAFY


 


EILHLPNLTEEQQNGFIQSLKDDPSVSNDILVEAKKLNDTQAKPDYSEAQ


 


QNAFYEILHLSNLTEEQQNGFIQSLKDDPSVSNDILVEAKKLNDTQAKPD


 


YSEAQQNAFYEILHLSNLTEEQQNGFIQSLKDDPSVSKEILAEAKKLNDS


 


QAPKVDKAKKTDKAEAKADDKAKGEEAKKSEDKKDSKADKAKSKNATHVV


 


KPGETLDNIAKDHHTTVDKIAKDNKIKDKNVIKLGQKLVVDKQKATQGKQ


 


EAVAKNEVKALPNTGENDDIALFSTTVAGGVSIALGSLLLGRNRKTS


 







SEQ ID NO: 3







atggaaaacaaaaactttttcagcattcgtaaattatcaattggggtggg


 


ttcatgtttaatcgcgagctctttacttgtgaatacaccaagtttcgcag


 


aagaaggagataataacgcagaagcgcaacaaaacgctttctctgaggta


 


gtaaaattacctaaccttagcgaagaacaacgtaatggtttcattcaaag


 


ccttaaagatgatccaagtacaagtcaagatgtgcttaatgaagctaaaa


 


aattaaatgatagtcaagagggatctcaacctgctcctgattacagtgat


 


gaacaacaaaatgcattttatgaaattttacaccttccaaacttaactga


 


agaacaacgcaatggctatattcaaagtcttaaagatgacccaagtgtaa


 


gcgctaatattcttgttgaagctaaaaatatgaatgttaaccaaacacct


 


acacaacctgcgccaagtttcgatgaagcgcaacaaaatgcattctatga


 


gattgtaaacttaccaaatcttactgaagagcaacgtaacggtttcatcc


 


aaagccttaaagacgatccaagtgtaagtaaagatatccttgttgaagct


 


aaaaagttaaatgacagccaagcaaaacctgattacagtgaagcgcaaca


 


aaatgcattttatgaaattttacaccttccaaacttaactgaagaacaac


 


gtaacggtttcatccaaagccttaaagacgatccgagtgtaagtagtgat


 


attcttgctgaagctaagaaattaaatgacagccaagcgcctaaagaaga


 


caacaacgtaaaagacaataattcaggtgaaaacaaagctgaagacaaag


 


gcaacaaagaaaacaaagctgaagataaaggcagcaaagaagacaaagct


 


gaagataaaggcagcaaagaagacaaagctgaagataaaggcagcaaaga


 


agacaaagctgaagataaaggcagcaaagaagacaaagctgaagataaag


 


gcagcatagaagataaagctaaagacaaagacaacaaagaaggcaaagct


 


gcagacaaaggtatggacaaagcgaaagatgcaatgcatgtcgttcaacc


 


tggtgaaacagtagaaaaaattgctaaagctaataacacaactgtagaac


 


aaatcgctaaagataatcatttagaagataaaaacatgattttaccaggt


 


caaaaacttgttgttgacaaccaaaaagcaatgaaagacagccaagaagc


 


taaagcaaaccacgaaatgaaagctttacctgaaacaggtgaagaaaacg


 


atatggcattattcggtacatcacttacaggtggtcttagcttagcatta


 


ggtttatacatcttaggacgtggcagaaaaacaaactaa






The protein sequence translated from SEQ ID NO 3 is designated SEQ ID NO: 4 and is shown below:








SEQ ID NO: 4







MENKNFFSIRKLSIGVGSCLIASSLLVNTPSFAEEGDNNAEAQQNAFSEV


 


VKLPNLSEEQRNGFIQSLKDDPSTSQDVLNEAKKLNDSQEGSQPAPDYSD


 


EQQNAFYEILHLPNLTEEQRNGYIQSLKDDPSVSANILVEAKNMNVNQTP


 


TQPAPSFDEAQQNAFYEIVNLPNLTEEQRNGFIQSLKDDPSVSKDILVEA


 


KKLNDSQAKPDYSEAQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSSD


 


ILAEAKKLNDSQAPKEDNNVKDNNSGENKAEDKGNKENKAEDKGSKEDKA


 


EDKGSKEDKAEDKGSKEDKAEDKGSKEDKAEDKGSIEDKAKDKDNKEGKA


 


ADKGMDKAKDAMHVVQPGETVEKIAKANNTTVEQIAKDNHLEDKNMILPG


 


QKLVVDNQKAMKDSQEAKANHEMKALPETGEENDMALFGTSLTGGLSLAL


 


GLYILGRGRKTN


 







SEQ ID NO: 5







gtgtacaaaaatgaagaagaaaagcattcaataagaaagttatctatagg


 


agccgcatctgtcattgttgggggactcatgtatggtgttttgggaaatg


 


atgaagctcaagcgaatgaagatgtcactgaaacaactgggagaaattca


 


gtgacaacgcaagcttctgagcaacatttgcaagtggaagcagtacctca


 


agaaggcaataatgtaaatgtatcctctgtaaaagtacctacgaatacgg


 


caacgcaagcacaagaagatgttgcaagtgtatccgatgttaaagcacat


 


gctgatgatgcattacaagtacaagaaagtagtcatactgatggtgtttc


 


ttcagaattcaagcaggagacagcttatgcgaatcctcaaacagctgaga


 


cagttaaacctaatagtgaagcagtgcatcagtctgaatacgaggataag


 


caaaaacccgtatcatctagccgcaaagaagatgagactatgcttcagca


 


gcaacaagttgaagccaaaaatgttgtgagtgcggaggaagtgtctaaag


 


aagaaaatactcaagtgatgcaatcccctcaagacgttgaacaacatgta


 


ggtggtaaagatatctctaatgaggttgtagtggataggagtgatatcaa


 


aggatttaacagcgaaactactattcgacctcatcagggacaaggtggta


 


ggttgaattatcaattaaagtttcctagcaatgtaaagccaggcgatcag


 


tttactataaaattatctgacaatatcaatacacatggtgtttctgttga


 


aagaaccgcaccgagaatcatggctaaaaatactgaaggtgcgacggatg


 


taattgctgaaggtctagtgttggaagatggtaaaaccatcgtatataca


 


tttaaagactatgtaaatggcaagcaaaatttgactgctgagttatcagt


 


gagctatttcgtaagtccggaaaaagtcttgactactgggacacaaacat


 


tcacgacgatgatcggtaatcattcaacgcaatccaatattgacgtttat


 


tatgataatagtcattatgtagatggacgtatttcgcaagtgaacaaaaa


 


agaagctaaatttcaacaaatagcatacattaaccctaatggctatttaa


 


atggcagggggacaattgcagttaatggtgaagtggtcagtggtacgact


 


aaagacttaatgcaacctacagtgcgtgtatatcaatataaaggacaagg


 


tgttcctcctgaaagtattactatagaccctaatatgtgggaagaaatca


 


gcataaacgatactatggtaagaaaatatgatggtggctatagcttgaat


 


ctggataccagcaagaatcaaaaatatgccatctattatgaaggggcata


 


tgatgcgcaagctgacacactgttgtatagaacatatatacagtcattaa


 


acagttactatccgttcagttaccaaaaaatgaacggtgtgaagttttac


 


gaaaacagtgcgagtggaagcggtgagttgaaaccgaaaccacctgaaca


 


accaaaaccagaacctgaaattcaagctgatgtagtagatattattgaag


 


atagccatgtgattgatataggatggaatacagcagttggagaagaaagt


 


ggagcaaaccaaggccctcaagaagaaatcacggaaaatcacgacatcga


 


agtcattgaggaaaacaacttggtggaaatgacagaagatacagcagttg


 


gagaagaaagtggagcaaaccaaggccctcaagaagaaatcacggaaaat


 


cacgacatcgaagtcattgaagaaaacaacttagtggaaatgacagaaga


 


tacagcgttggaagaagaaagtggagcaaatcaaggtcctcaagaagaga


 


tcacagaaaaccacgatatcgaagtcattgaagaaaacaacttggtggaa


 


atgacagaagatacagcgttggaagaagaaagtggagcaaatcaaggtcc


 


tcaagaagagatcacagaaaaccacgacatcgaagtcattgaagaaaata


 


acttagtagaaatgacagaagatacagcagttggagaagaaagtggagca


 


aatcaaggtcctcaagaagagatcacagaaaaccacgatatcgaagtcat


 


tgaggaaaacaacttagtggaaatgacagaagatacagcagttggagaag


 


aaagtggagcaaaccaaggtcctcaagaagaaatcacggaaaatcacgac


 


atcgaagtcattgaagaaaacaacttggtggaaatgacagaagatacagc


 


gttggaagaagaaagtggagcaaatcaaggtcctcaagaagagatcacag


 


aaaaccacaacatcgaagtcattgaagaaaacaacttggtggaaatgaca


 


gaagatacagcagttggagaagaaagtggagcaaacccaggacctcaaga


 


agaagtaacagagaatcaacctcagcaagaagaaatcatggaaaaccaag


 


aagtcgaaaagaaaggcgatagtaacttggtagaaagtacaaaaactcca


 


aaggccgaagaatcagttagcgttcagccaactttagaagacaaaaacac


 


aaagaaccacgttaacacagtagtagtgaatacgaaggtatctgaagtta


 


aagaaaaggatccccaccatacaaaagcactaccagatacggggacaacc


 


tctcgaagtcattccatgatgattcctctccttcttgttgctgggtcagt


 


agtgttgttacgtcgaaagaaaaagcatagtaaggtgaattaa






The protein sequence translated from SEQ ID NO 5 is designated SEQ ID NO: 6 and is shown below:








SEQ ID NO: 6







VYKNEEEKHSIRKLSIGAASVIVGGLMYGVLGNDEAQANEDVTETTGRNS


 


VTTQASEQHLQVEAVPQEGNNVNVSSVKVPTNTATQAQEDVASVSDVKAH


 


ADDALQVQESSHTDGVSSEFKQETAYANPQTAETVKPNSEAVHQSEYEDK


 


QKPVSSSRKEDETMLQQQQVEAKNVVSAEEVSKEENTQVMQSPQDVEQHV


 


GGKDISNEVVVDRSDIKGFNSETTIRPHQGQGGRLNYQLKFPSNVKPGDQ


 


FTIKLSDNINTHGVSVERTAPRIMAKNTEGATDVIAEGLVLEDGKTIVYT


 


FKDYVNGKQNLTAELSVSYFVSPEKVLTTGTQTFTTMIGNHSTQSNIDVY


 


YDNSHYVDGRISQVNKKEAKFQQIAYINPNGYLNGRGTIAVNGEVVSGTT


 


KDLMQPTVRVYQYKGQGVPPESITIDPNMWEEISINDTMVRKYDGGYSLN


 


LDTSKNQKYAIYYEGAYDAQADTLLYRTYIQSLNSYYPFSYQKMNGVKFY


 


ENSASGSGELKPKPPEQPKPEPEIQADVVDIIEDSHVIDIGWNTAVGEES


 


GANQGPQEEITENHDIEVIEENNLVEMTEDTAVGEESGANQGPQEEITEN


 


HDIEVIEENNLVEMTEDTALEEESGANQGPQEEITENHDIEVIEENNLVE


 


MTEDTALEEESGANQGPQEEITENHDIEVIEENNLVEMTEDTAVGEESGA


 


NQGPQEEITENHDIEVIEENNLVEMTEDTAVGEESGANQGPQEEITENHD


 


IEVIEENNLVEMTEDTALEEESGANQGPQEEITENHNIEVIEENNLVEMT


 


EDTAVGEESGANPGPQEEVTENQPQQEEIMENQEVEKKGDSNLVESTKTP


 


KAEESVSVQPTLEDKNTKNHVNTVVVNTKVSEVKEKDPHHTKALPDTGTT


 


SRSHSMMIPLLLVAGSVVLLRRKKKHSKVN


 







SEQ ID NO: 7







atgaataaatcaagaactaaacattttaattttttatcaaaacgtcagaa


 


tcggtatgctattcgccacttttcagctggtactgtgtcagtgcttgtag


 


gagcagctttcttgctaggtgtccatacgagtgatgcatctgctgcagaa


 


caagatcaaacatctgaagcaaagcaaaacctctttgatgcttccgctat


 


ttttggcgctttaacagagacgaacgaaaaggtagcacaagtgacgccaa


 


cagaaaaaaatctttcatcagttgaagaaatgagagataaaggcgcaact


 


ggaaatggaccatcaataacatcactacaaactgtagaacaaaataatgc


 


agtacaacctacagcaacacctattaatgacacagaaaattcaaccgaag


 


cccctatgaaagaacaatcgaatgatgcacaaacgactgacgaaagtaac


 


aatgccactcagaaaaataatactgaaccccaagcaaacaatgaaatatc


 


agcgcgtaatgcaaaaacaacagcatatttaacaagtgaaacctttacaa


 


cagcaacgtctacaactgatatgcctacacagaaacaagaatatccatct


 


ttagaaaatccaacaaatcaatcgcaaacgaacagagcacaaccaccaac


 


aatggaagcacccaaactggcagaaggattagacaatctattaaaaaaat


 


caactttcgaaagtatgtacgtgacaaaaagaaatcaatttgacaaagag


 


acggcttctaaaacaaaagcatggccgagtgatgttgttccagaaaatca


 


agtagagatacttgctgatgcaattcaaaatggctatatcaaatctgtaa


 


atgatgtgaccaataaagcacatacgttatctggacgtgcatggatgtta


 


gaccacggaacaccaacgacaatagctaatggtttaacacctgttccaga


 


gggcactaaagtttatttgcggtggatagatcaagatggtgccacttcgc


 


caatgtatacagcaaaaacgacaagtagattaagcgctgcggatggtaat


 


caagtgggtccaggtgcttatgctttcgatttacgcacaggttggataga


 


tgctaaaggaaaacaccacgtatatagagcagtaaagggtcaatattata


 


aaatatggatcaatgattttagaactaaagacggtaatatcgctacaatg


 


ttacgtgttgcaggaggatatgttccgggaacgtacgtggattctgtgac


 


atacaacaatatgggccaatttccattaattggtacaaatatgcaacgta


 


caggtatctttatgacaacgataccttcagaaaaatatttaatatcaaaa


 


cattacgtgaaagatacaaaaggtgctgcagcaaatccagccgtcacgat


 


aattgaaaataactttgtgagcggcaaagtttggatagaaacaggtgctg


 


gagattatgtgaactcagcgacaggtccaaaccacaatgcgaaagatgtc


 


gttgcctctggatacaaagtggtcatgtcatcattaacagatcaaggtgc


 


taaagcctacgatgcgcaagtcaatcgcttgccgaagaaagatcgagcag


 


aagcagcacgtcaattattaataaaacatccagaatatatcgcagcaact


 


gtagaagggataacgaatgagtgggggagatatacattgcgtttccctaa


 


aggcacattcaacaaagaccatctttacggttacgtattggattttgatg


 


gtgaaattgtaaaaacttattcaggttttacttcaccagagttccggaga


 


ccgaattataatttgaccgttacaccgcaaacagctccctattatagacc


 


cgttcgacgtgcatgggtcaatgttaattttgcggttattgaagcaccac


 


aatctcaaatcgaaataaaagaatttgatgcaacctctaaccctgcgcat


 


cgtgggcaaacagcaactattgatatcataggtatgcctaaaacttcatt


 


acttacacgtgtacaatggaaagattcatcgggcagtattgttgaggata


 


gtggtcctgtttttacggaagaagaggctgaacatatagcggaatttgta


 


ataccgtctagcgcaaaatcaggcgaagtgtatactgtacaactcgtggt


 


aggtaatcatatcgtagcttcggactctcttattgtacatgtcaatgaag


 


aagcggcgacatatcatccgatatacccatcgacaacagtagaatcaggt


 


caaagagtaacgattccagcacctaagaatatggatggcaaacctttact


 


agatggcacaacttttgaaaaaggtcatcacgtaccaacttgggctttag


 


tgaatggtgatggctcgattacagtaaaacctggagaaaaagtagcagag


 


ggtgagtatgatattccagtgattgtgacatatccagatggttctaaaaa


 


cacaatctttgcacctgtgaccgttgaagaaaaacaaccaatggcatcgc


 


aatatgagccaataacaactggagtatcgaaaccatttggaaacccagta


 


atgccaactgatgtaacagattcaattcaagtaccgaactatccattgga


 


agggcaacaaccgacagtaacagtggatgatgaaagtcaattaccagatg


 


gaacaacagaaggttacaaggatatagatgtaacagtgacatacccagac


 


ggaacaaaggatcgtgtcaaagttccagtcgtaacggaacaacaattaga


 


tagtgataaatatgatccggtcgcaacaggtatcttgaaaccgtttggta


 


ctccaacaacagaggaagacgttataaaattagtggagataccgaaatat


 


ccaacagacttaacacaaccaaaagtaacagtgacggttccaaatacttt


 


accggatgggcaaacgccaggtaaagtagacgttgatgtgacagtaacgt


 


atccagatggttccacagatcacatttcagttccagtttggacaaacaag


 


catctggataaagacaaatataacccaataacgactggggtatcgaaacc


 


atttggaatcccagtaacgccaactgatgtaacagattcaattcaagtac


 


cgaactatccattggaagggcaacaactgacagtaacagtggatgatgaa


 


acacaattaccagatggaacaacagaaggtcacaaggatatagatgtaac


 


agtgacatacccagacggaacaaaggatcatatcaaagttccagtcgtaa


 


cggaaaaacaatcagataatgaaaaatatgagccaacaactaacggaatc


 


acgaaaaagtacggtatccctacgacagaggatgaagtgatagatatagt


 


tcgaattccatattttccagtagatggcgtgcaacctattgtaacggtaa


 


atgatcctagactattgccaaatggtcaaaaagaaggtcaaatcaatgtt


 


ccagtcacagtgacgtatccggatggcacaaaagatctcatgacagttcc


 


ggttattacaggtaagcaagcagaaaatgaaaaatacgatccaatcacat


 


taggagtaactaaagattatggtgatcctacaactgcaaacgatgtgaca


 


aagtcaatccaaataccaacatatccagcaggtggcgaacaaccaatcgc


 


aacagcggatgatgaaagtcaattaccggatggcacagtagaaggtaaag


 


tggatattccagtcacagtgacgtatccggatggtactcaggatcatatc


 


actgtcccagtatttaccaatcaacaacgagataatcaaaaagccagtaa


 


agctgtgacgaaaatacatggtatatcggtaacaggcactgatatgacag


 


atactaagaaaaatcataactatccagcaggtggtgaacaacctaaagtt


 


actgtgaaagatgacgatcaattatcagagggtaaagtcgattcaacagt


 


gggtgcggataatgtgacaactacagatgatttatcaagcgtaactgcgg


 


tatctcatggtcatcaaacaagtgtacaaacaacaaaagagaaccaatca


 


gtgcatgatgaagaggtgacgatcccaacagttgcacatgtgtctacaat


 


aatgacaggtgtggtaaagggtgagcaagaagcgacggatatcgtggcta


 


gaccacatgttgaaacaactcaactcccatcaatttcagctcaagcaaca


 


gttaaaaaactaccagaaacgggtgaaaacaatgaacaatcaggtgtttt


 


attaggtggatttattgcgttcatgggtagcttacttttattcggcagac


 


gtcgcaaaccaaagaaagattaa






The protein sequence translated from SEQ ID NO 7 is designated SEQ ID NO: 8 and is shown below:








SEQ ID NO: 8







MNKSRTKHFN FLSKRQNRYA IRHFSAGTVS VLVGAAFLLG


 


VHTSDASAAE QDQTSEAKQN LFDASAIFGA LTETNEKVAQ


 


VTPTEKNLSS VEEMRDKGAT GNGPSITSLQ TVEQNNAVQP


 


TATPINDTEN STEAPMKEQS 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 NFVSGKVWIE


 


TGAGDYVNSA TGPNHNAKDV VASGYKVVMS SLTDQGAKAY


 


DAQVNRLPKK DRAEAARQLL IKHPEYIAAT VEGITNEWGR


 


YTLRFPKGTF NKDHLYGYVL DFDGEIVKTY SGFTSPEFRR


 


PNYNLTVTPQ TAPYYRPVRR AWVNVNFAVI EAPQSQIEIK


 


EFDATSNPAH RGQTATIDII GMPKTSLLTR VQWKDSSGSI


 


VEDSGPVFTE EEAEHIAEFV IPSSAKSGEV YTVQLVVGNH


 


IVASDSLIVH VNEEAATYHP IYPSTTVESG QRVTIPAPKN


 


MDGKPLLDGT TFEKGHHVPT WALVNGDGSI TVKPGEKVAE


 


GEYDIPVIVT YPDGSKNTIF APVTVEEKQP MASQYEPITT


 


GVSKPFGNPV MPTDVTDSIQ VPNYPLEGQQ PTVTVDDESQ


 


LPDGTTEGYK DIDVTVTYPD GTKDRVKVPV VTEQQLDSDK


 


YDPVATGILK PFGTPTTEED VIKLVEIPKY PTDLTQPKVT


 


VTVPNTLPDG QTPGKVDVDV 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







atgtttaatcaacaaaaacaacactatggtatccggaaatatgcaatcgg


 


gacttcatcagtattattaggcatgacattatttatcacacatgacgcaa


 


ctgcatctgcagctgaaaacaatacaactgcaaagacagagacaaatcaa


 


gcagcaacaatttcttctcgcacttcgccaaccgacgtcgctcaacctaa


 


tgcagacacgaatgctacaacggcgactaaagagacaacaccacaatcag


 


attcaacagcattaccgcaagcagcagcgcaacctcaaacgggccaaaca


 


gcatcgaaagacacagtagatacaaataaaacgcaaacagcagattccac


 


aaccgctcctcctgtgacagacgcgccaaaagctaatgacgacacaacac


 


agccagaagctgcgactgtagccaaaaaagaagatgctcagacaccatcg


 


actgcagaccctacaccacaagcgcaacaaccgcctcagtcaaaagcacc


 


tcaagaaacgcaacaacaatcaacagttgaagatacaacgccacaacaaa


 


acgcatcaactgaagcacaccctaaaaatgtagataccgcttcaacaaaa


 


caacaacaaacaacgccatcaaccgcaccgacaccttacacacaacaagc


 


agacgaagcaatgacagatgtcacaacaaccagtgtcgacagcaacgtac


 


agccgttagcccctgcagaagatcaacctaaaaatacgaacacagctgac


 


aaagcaaccgttgcgacaccaccacgtgacaatgctaagactgctgatcc


 


gaacaaaaagatgacacgtgcagcaacgacacaacaagatgatgccgtcg


 


atacattgaagtcaaaagaaatgacagcaacgatcgataaaagttttcca


 


gccgttaaatattacacgttgaaaaatggtaaaaaagtcgatgcacaact


 


gacggatgcacgtcaaatcatcgtcaatggtgaagtcattacaccaacag


 


tcaaatacaacaaaattgatgatcatacggctgaatatgacttaacagca


 


caaaatgattcacgttcgattgatgccaattttaaatttcgtttatcagt


 


tgaaggtaagaccgttgatttacaaatgacagattacacgaacaacaaca


 


cagatccacaaaacgtcattcgcaactttagctttgtaagtcaatcgctc


 


gtatctgtaaacaatcaacagaaaaatgccaaactgcaaacatcgaaact


 


gtctacaaatacaatgaaaagcggcgataaatcatatcatatcgatgaaa


 


atttcaaaaacgacttcaacgactttatgatgtacggtttcgtgtcaaat


 


gatgattacagtgcaggattgtggagtaacgcacaaattggcgtcggcat


 


tggtgaacaagacttcttacgtgtctacgcacagtctatacaaacagata


 


tcggggtcgctgtcggtttaggctcaatgccatggtttatccaaaaagac


 


gctgcacatccagatgcgaaaaatcaaggactactcccacatgtcaaagt


 


tgcaattgcggaagatgaaaatcaagatggtgaaattaactggcaagacg


 


gtgcaattgcttatcgtagcattatgaacaatccatatggtgccgaagaa


 


gtacctgaccttgttgggtaccgtatcgcgatgaactttggttctcaagc


 


gcaaaacccatttttaaagacgttagatggtgtgaaaaaattctatctca


 


atacagatggtttagggcaatccattttattaaaaggttataacagtgaa


 


ggccacgactctggtcatttagattacgcgaatattggtcaacgtatagg


 


tggcgtgaaagactttaaaacgttacttcaaaaaggggcagattatggcg


 


cacgtttcggtcttcatgtgaatgcatctgaaacatatccagagtctcaa


 


gcattcaatcctgccctcttacgtaaagatgcgaatggaaactatatgta


 


tggctggaactggctcgatcaaggctttaacatcgatgcagattacgatt


 


taatacacgggcgtaaagaacgcttcgaagcactcaaacaaattgtcggt


 


gatgacctcgactttatttatgtcgatgtatgggggaatggacaatccgg


 


cgacaatacagcttggccatcacatcaattagccaaagaaatcaacgact


 


taggatggcgcgtcggtgtcgaatggggtcacggtatggaatatgactcc


 


acgttccaacattgggcagccgacttaacgtatggatcgtaccaaaataa


 


agggattaactcagaggtagcacgcttcttacgcaaccatcaaaaagatt


 


catgggtcggtaactatccaaaatactcaggtgcagctgacttcccattg


 


ctcggcggttatgacatgaaagattttgaaggttggcaaggtcgtaacga


 


ttactctgcttacattaaaaatattttcaatgttgatgtaccaacaaagt


 


ttttacaacattataaagtgatgcgtattgtcgatggtgagcctgttaaa


 


atgactgccaatggtcaaacgattgactggacaccagaaatgcaagtcga


 


tttacaaaatgaagccggtgatcaagtcactgttaaacgtaaatctaacg


 


actatgaaaacgacactgacaactaccgctcacgtacaatcgaattgaat


 


ggtcgcacagtactcgatggcgattcataccttttaccatggaattggga


 


tgcgaacggccaaccattaactggcgataacgaaaaattatatcactgga


 


ataaaaaaggcggttcaacgacttggacactgcctgaatcatgggataca


 


gaccaagtcgtgctatacgaattatctgaaacgggtcgtaagtcaccacg


 


tacagtggcagtgaaagaccatcaagtgacactcgataatattaaagcag


 


acacaccgtatgtcgtttataaagtcgcacaaccagacaacacagatgtg


 


aactggagcgaagacatgcacgtgaaagatgccggcttcaactcacaaca


 


actgacaccttggacaatcgaaggcaatcgagataaagtgagcatcgaaa


 


agtcgacaacatcaaatgaaatgctaaaaatcgatagtccaacaaaaaca


 


acgcaattaacgcaacaattgacaggtttagtgccaggacaacgttacgc


 


tgtctatgttggcatcgataaccgcagtgatgcagcggcgcatattgcag


 


tgacacataacggtaaaacgctcgcaagtaacgaaacaggtcaatcgatc


 


gcgaaaaactatgtgaaagcagatgcacatagtaacaatgctgcgacgtt


 


taaaaatggcggtagttacttccaaaacatgtacgtgtacttcgttgcgc


 


cagaagatggtaaagcagacttgacgattcaacgcgacccaggtgaaggg


 


gccacttatttcgatgatattcgtgtgttagaaaataacgcgaatctcct


 


tcaaaacggcacattcaaccaagacttcgaaaatgtaccacaagggttat


 


tcccgttcgtcgtgtcagaagttgaaggcgttgaagataatcgcgttcac


 


ttatctgaaaagcacgcaccgtatacacaacgcggatggaataataaacg


 


tgtcgatgatgtcattgatggcaaatggtcacttaaagtaaacggtcaaa


 


caggtaaagataaaatggtcatccaaacgattccgcaaaacttctacttc


 


gaaccaggaaaaacgtatgaagtgtcatttgattatgaagcaggttctga


 


tgatacgtatgcatttgcgacaggtagtggggacatttctaaaaatcgta


 


actttgaaaagacaccattgaaaaatacagtcgatggtggcaaagcgaaa


 


cgggtgacatttaaagtgacgggtgatgaaaatggtcaaacttggatcgg


 


tatttactcaacgaaaacacccaatgatccacgaggcgtgaaaaatggca


 


atcaaatcaacttcgaagggacgaaagatttcattctagacaacctttct


 


atccgtgaaattgacgcaccgaagcctgatgccacacaagaaagcggtga


 


tagcgcaccaatgaatgaaacagatgagcgtaacgtcaattcaaacggta


 


cattagccgatcatagtgagacaactgatgtcaatgtcagtgcaacggca


 


gatgatacagcagtcaaaggcgaaatgacgacaaacagaacagatgcacc


 


aactgttacactgcctgaagcaacgatagtagatgaaggcacgtcaaatc


 


ctgtcactacaacaccaacgaatacaacacaagctatgacaaataaggct


 


gatgagatgccacaaacgatgaacaatgttcctttaactagcatcgctac


 


cgatatgatgcagtctcatgcggtggattccatggcagcaacactagcag


 


ctacaaatcaagtggcggcacctgtgcgtcaaacagcaggacctatgcaa


 


catggtatggacagtgcttcaacgcaacacgcacccatacaagttgacaa


 


tgtcacagcaccaccattaccagatgaacagtttgccgaattacctaaaa


 


ctggggatacgactccaaatacacgtggacctttaatggcgatgatagtt


 


ggcgcagtcttaacagcattcggattcagacgccaacgtaaagaaaaata


 


g






The protein sequence translated from SEQ ID NO 9 is designated SEQ ID NO: 10 and is shown below:








SEQ ID NO: 10







MFNQQKQHYGIRKYAIGTSSVLLGMTLFITHDATASAAENNTTAKTETNQ


 


AATISSRTSPTDVAQPNADTNATTATKETTPQSDSTALPQAAAQPQTGQT


 


ASKDTVDTNKTQTADSTTAPPVTDAPKANDDTTQPEAATVAKKEDAQTPS


 


TADPTPQAQQPPQSKAPQETQQQSTVEDTTPQQNASTEAHPKNVDTASTK


 


QQQTTPSTAPTPYTQQADEAMTDVTTTSVDSNVQPLAPAEDQPKNTNTAD


 


KATVATPPRDNAKTADPNKKMTRAATTQQDDAVDTLKSKEMTATIDKSFP


 


AVKYYTLKNGKKVDAQLTDARQIIVNGEVITPTVKYNKIDDHTAEYDLTA


 


QNDSRSIDANFKFRLSVEGKTVDLQMTDYTNNNTDPQNVIRNFSFVSQSL


 


VSVNNQQKNAKLQTSKLSTNTMKSGDKSYHIDENFKNDFNDFMMYGFVSN


 


DDYSAGLWSNAQIGVGIGEQDFLRVYAQSIQTDIGVAVGLGSMPWFIQKD


 


AAHPDAKNQGLLPHVKVAIAEDENQDGEINWQDGAIAYRSIMNNPYGAEE


 


VPDLVGYRIAMNFGSQAQNPFLKTLDGVKKFYLNTDGLGQSILLKGYNSE


 


GHDSGHLDYANIGQRIGGVKDFKTLLQKGADYGARFGLHVNASETYPESQ


 


AFNPALLRKDANGNYMYGWNWLDQGFNIDADYDLIHGRKERFEALKQIVG


 


DDLDFIYVDVWGNGQSGDNTAWPSHQLAKEINDLGWRVGVEWGHGMEYDS


 


TFQHWAADLTYGSYQNKGINSEVARFLRNHQKDSWVGNYPKYSGAADFPL


 


LGGYDMKDFEGWQGRNDYSAYIKNIFNVDVPTKFLQHYKVMRIVDGEPVK


 


MTANGQTIDWTPEMQVDLQNEAGDQVTVKRKSNDYENDTDNYRSRTIELN


 


GRTVLDGDSYLLPWNWDANGQPLTGDNEKLYHWNKKGGSTTWTLPESWDT


 


DQVVLYELSETGRKSPRTVAVKDHQVTLDNIKADTPYVVYKVAQPDNTDV


 


NWSEDMHVKDAGFNSQQLTPWTIEGNRDKVSIEKSTTSNEMLKIDSPTKT


 


TQLTQQLTGLVPGQRYAVYVGIDNRSDAAAHIAVTHNGKTLASNETGQSI


 


AKNYVKADAHSNNAATFKNGGSYFQNMYVYFVAPEDGKADLTIQRDPGEG


 


ATYFDDIRVLENNANLLQNGTFNQDFENVPQGLFPFVVSEVEGVEDNRVH


 


LSEKHAPYTQRGWNNKRVDDVIDGKWSLKVNGQTGKDKMVIQTIPQNFYF


 


EPGKTYEVSFDYEAGSDDTYAFATGSGDISKNRNFEKTPLKNTVDGGKAK


 


RVTFKVTGDENGQTWIGIYSTKTPNDPRGVKNGNQINFEGTKDFILDNLS


 


IREIDAPKPDATQESGDSAPMNETDERNVNSNGTLADHSETTDVNVSATA


 


DDTAVKGEMTTNRTDAPTVTLPEATIVDEGTSNPVTTTPTNTTQAMTNKA


 


DEMPQTMNNVPLTSIATDMMQSHAVDSMAATLAATNQVAAPVRQTAGPMQ


 


HGMDSASTQHAPIQVDNVTAPPLPDEQFAELPKTGDTTPNTRGPLMAMIV


 


GAVLTAFGFRRQRKEK


 







SEQ ID NO: 11







atgacaagaaaatttagggaatttaagaaaagtttaagtgaagaaaaagc


 


aagagtgaaactttacaagtcaggtaaaaactgggttaaagctggaatta


 


aagaatttcagttattaaaagcattaggcttatcttttttaagccatgac


 


attgtaaaggatgaaaatggagaagtaacgacacaatttggggaacagtt


 


gaagaaaaatgcattaagaacaactgcttttgcgggtggaatgttcacag


 


ttaatatgttgcatgaccaacaagcatttgcggcgtcggatgcacctata


 


acttctgaactggcaaccaaaagtcaaactattggcgatcaaacatcaat


 


tgttattgaaaagtctacatcgtcagatcaatcaacgaacccaataacag


 


aaagtgaaagtaaacacgattctgaaagtatctcattatctgagcatcaa


 


acatcagagtcaacaagtctttcaacgtcaacttccaaatcaatatcaac


 


ttcagtagaggaatcagaatcaacatcaaaagattctcatactaaaactc


 


aagatagtcaatcagatagtcatcagtcaacaagtcaagaggtaaatggc


 


tcttccaaccacgagcaatcaacaccacacactgcacaaagtcttacgag


 


cctatctattgagagccaaacgtcgacttcaaatacatcattgaaggaaa


 


ctaaagaaggggaattgtcaaaaaacctttcgaagttatctcaaaatcaa


 


aacatcaaacttcatgaagaacatacgatgcgttcagcagatttgagctc


 


aggttatacaggatttagagcggcttactatgtaccaagatcaagaacaa


 


caccaacgacaaaagtctacacagggcaaggaagcttcagaggtagaggt


 


agaattaaatataatattttctacaaagttgtcgttacaagtaatggcaa


 


agaaatgaagatccgctatacattgagtcaagatgatccaaacacgtcta


 


atgttgaaaaacctaggtgggcaggacagaaacgatttggtattcataat


 


acttgggatgaaggtcctggtcgcgggcaattaaagttagggtcggcatt


 


cggcaaaccaacagttatacaaggagaaactagaccgaattatggtagct


 


gggttggcacacctataacgaaatatgtttcaggcgatcgtacaaatggt


 


ttttactggcaagctgctgtacttgcaccgagacatggagagaagggaga


 


aggaatcacagcagaaattacagttcctattgttaacccttctggaagat


 


ttaattgggaattccatcctgtcggtcaacaggacggagttggtggcaaa


 


actgactactttgaaaatgtatggattcgagactatgacccatattacaa


 


atatattcaaactaaggaaggcagagcctcagtttcgcactctatttctc


 


aggtgaaagcaagtgaatcgagatcgacatcgctcatacaatcggagtct


 


attagaagatcacagtccatatctgagagtgaatctattgtagccgcaag


 


tcattcggcaagtgtagcaaaatcgcaatccatctcgagaagtcaatctg


 


tggcgaaatcacaatcgatctcaagaagtcagtcgatcgcacacagccga


 


tcagcaagtgtggcaaaatcgcaatccatctcaagaagtcagtcgatcgc


 


acacagccgatcagcaagtgtggcaaaatcacaatcgatttcaagaagtc


 


agtcgatcgcacacagccgatcagcaagtgtggcgaaatctcaatcgatt


 


tcaagaagtcagtcaattgcgcagagccaatcagcaagtgtggcaaaatc


 


acagtcgatttcaagaagtcagtcaattgcgcagagccaatcagcaagtg


 


tggcgaaatcgcaatcgatttcaagaagtcagtcgattgcacatagccga


 


tcagcaagtgtagcggaatcacagtcgatttcaagaagtcagtcgattgc


 


gaatagccaatctgtagcagcgagtgaatcagagagtctatcaatatcat


 


tgtctaaaaagcagtcaatatcgatgagtaattctgaaagtgcagcaaaa


 


tcacactcgctttcggtgaaaaggtctaactggattaaaaagtcaaaagc


 


ggcttcagtaagaaagtcacattcactttcggtaagaaaatctaattcgg


 


cgaaaaggtcacatgctatttcggtaagaaagtcaaagtcattatcagtt


 


aaaaagtcaatttcgcagagccaatcagcaagtgtggcgaaatcgcaatc


 


gatttcaagaagtcaatcagtagcagcgagtgagtcggcatcgctaagta


 


agtcgaagagcacatcgctcagtaactcagtgagtgcagagaaatcgacg


 


tcattaagtcgttcagcaagtgtagcaaaatcgcaatcgatttcaagaag


 


ccaatcagtagtagcgagcgaatcggcatcgttaagtaagtcgaagagca


 


catcgctcagtaactcagtgagtgcagagaaatcgacgtcattaagtcga


 


tcagcaagtgtagcaaaatcgcaatcgatttcaagaagccaatcggtggc


 


agcgagcgaatcggcatcgttaagtaagtcgaagagcacatcgctcagta


 


actcagtgagtgcagagaaatcgacgtcattaagtcgatcagcaagtgta


 


gcaaaatcgcaatcgatttcaagaagccaatcggtggcagcgagcgaatc


 


ggcatcgttaagtaagtcgaagagcacatcgctcagtaactcagtgagtg


 


cagagaaatcgacgtcattaagtcgatcagcaagtgtggcaaaatcgcaa


 


tcgatttcaagaagccaatcagtagtagcgagcgaatcggcatcgttaag


 


taagtcgaagagcacatcgctcagtaactcagtgagtgcagagaaatcga


 


cgtcattaagtcgatcagcaagtgtagcaaaatcgcaatcgatttcaaga


 


agccaatcggtggcagcgagcgaatcggcatcgttaagtaagtcgaagag


 


cacatcgctcagtaactcagtgagtgcagagaaatcgacgtcattaagtc


 


gatcagcaagtgtggcaaaatcgcaatcgatttcaagaagccagtcagta


 


gcagcaagtgagtcggcatcattaagtaagtcgaagagcacatctttaag


 


caactcagtgagtgtagagaaatcgacgtcattaagtcgatcagcaagtg


 


tggcgaaatcgcaatcgatttcaagaagtcaatcagtagcagcgagtgag


 


tcggcatcgctaagtaagtcgaagagcacatcgctcagtaactcagtgag


 


tgcagagaaatcgacgtcattaagtcgttcagcaagtgtagcaaaatcgc


 


aatcgatttcaagaagccagtcagtagcagcaagtgagtcggcatcattg


 


agtaaatcaacaagtacgtcaacaagtgactcagatagcgcgtcaacatc


 


aacatctgtatcagatagcgattcagcttcattgagtaagtcgactagta


 


catcaacaagcgattcagacagcgcgtcagcatcattgagcaagtcaaca


 


agtacatcaacgagcgactcagatagcgcatcgacatcaacatcagtatc


 


agatagcgactccgcatcgttgagtaaatcgacaagcacgtcaacaagtg


 


attcagacagcacgtctacttcattgagtaagtcgacaagtacatcgaca


 


agtgattcagatagtgcgtcaaaatcaacgtcagtatcagacagtacgtc


 


cgcatcattgagtaaatcgacaagcacgtcaacaagtgattcagatagtg


 


catcaaaatcaacgtcggtatcagatagcacgtcagcatcattaagaaag


 


tcggcaagtacgtcaacgagtgactcagacagcacgtctacttcattgag


 


taagtcgacaagtacatcgacaagtgattcagatagtgcatcaaaatcaa


 


catcagtatcagatagcgattcagcttcattgagtaagtcgactagtaca


 


tcaacaagcgattcagatagtgcgtcaaaatcaacgtcggtatcagatag


 


cgactccgcatcgttgagtaagtcgacaagtacgtcaacaagcgattcag


 


acagtgcatcaaaatcaacgtcggtatcagacagtacgtcaacatcatta


 


agtaagtcgacaagtacatcaacaagcgattcagatagtgcgtcaacatc


 


gacatcagtatcggacagtacgtctgcatcattgagtaagtcgacaagca


 


catcgacaagtgattcagatagcgcatcaacatcagtgtcagatagcgat


 


tcagcatcactaagcaagtcaacaagtacatcgacaagcgattcagacag


 


cgtatcaacatcaacatcagtatcagatagtgattccgcgtcattaagta


 


agtcgacaagtacgtcaacaagcgattcagatagtgcgtcaaaatcaaca


 


tcagtatcagatagcacgtcaacatcattgagtaaatcaacaagtacatc


 


gacaagtgactcagatagtgcgtcaacatcggtatcagacagtacgtccg


 


catcattgagtaaatcgacaagcacgtcaacaagtgattcagatagtgca


 


tcaaaatcaacatcagtatcagatagcgattcagcatcattaagcaagtc


 


gacaagtacatcgacaagtgattcagatagtgcgtcaacatcaacgtcag


 


tgtcagatagcgattcagcttcattaagcaaatcaacaagtacgtcaaca


 


agtgactcagatagcgcatcaacatcattaagcaagtcaacaagtacatc


 


gacaagcgattcagacagtacgtctacatcattaagtaagtcaacaagta


 


catcaacaagtgattcggatagtgcgtcaaaatcaacatcagtatcagat


 


agcgactcagcttcattaagcaagtcgacaagtacgtcaacaagtgactc


 


agacagtgcgtcaaaatcaacatctgtgtcagatagcgactccgcatcgt


 


tgagtaagtcgacaagtacgtcaacgagcgattcggatagtgcgtcaaaa


 


tcaacatcagtatcagatagtgaatccgcgtcattaagcaagtcgacaag


 


cacatcgacaagtgactcagatagtgcgtcaacatcgacatcggtatcag


 


acagcacatcagtttcattaagcaagtcgacaagcacgtcaacaagcgat


 


tcagacagtacgtctacttcattaagcaagtcgacaagcacgtcaacaag


 


tgactcagatagtgactcagcttcgttgagtaaatcgacaagcacgtcaa


 


cgagcgattcagatagcgtgtcaacatcaacatctgtgtcagatagcgat


 


tcagcttcattaagcaaatcgacaagtacatcaacaagcgattcagatag


 


tgcgtcaacatcaacgtcggtatcagatagcggctccgcatcgttgagta


 


agtcgacaagtacgtcaacgagcgattcagacagtgcatcaaaatcaacg


 


tcggtatcagatagtgattcagcatcactaagcaaatcgacaagcacgtc


 


aacaagtgactcagacagtgcgtcaacatcgacatcggtatcagatagca


 


catccgcgtcgttaagcaagtcgacaagtacgtcaacaagtgattcagac


 


agcgcatcgacatcaacatcagtatcagatagcgactccgcatcgttgag


 


taaatcgacaagcacgtcaacaagtgattcggacagtgcgtcaaaatcaa


 


catcagtgtcagatagcgattcagcttcattgagtaagtcgacaagcacg


 


tcaacaagcgaatcagacagcgcgtcaaaatcaacgtcagtgtcagatag


 


cgattccgcatcattaagtaaatcgacaagcacgtcaacaagtgactcag


 


atagtgcatcgacatcaacgtcagtatcagatagtgattccgcgtcatta


 


agcaagtcgacaagtacgtcaacaagtgactcagacagtgcgtcaaaatc


 


aacatcagtatcagatagcgattccgcatcattgagtaagtcgacaagca


 


cgtcaacaagcgaatcagacagtgcgtcaacatcgacattagtatcggat


 


agtacgtcggtttcattgagccaatcaacaagtgtggataaagatagtac


 


agcgaagggatcgacagaattagtaaatgttgcatcactttcaatcagtg


 


cgagtcaatcaagtagtttatctgcttcaacatccacatcgattgaaaag


 


tctgagtctacatcaacaagtggctcaaattcaactaatgcgtcgttaag


 


tagctcatcttcacttagtacatcagcaagtacttctgtaagcgaagtga


 


catctgtcacacattctgaaaatgatttaagtgcatctaacgatagagat


 


acatccggatcagtaagtcaatttgcttctgaaaatacatcattaagtga


 


ttctgcatcaattagtggcgaagtttctagtagtacgtccgcgtcaactt


 


cgaaatcatcatcactttcagcaagcgcgttacatgataagcatgtatca


 


gaaagcacttctgcatcattaagtagtggagattcaagtcgtgcttcggc


 


atcagtgtcaacgtcattatcagaatcagatagtgcgttaatagactctg


 


aatcaattagcgtttccgagcacacatcaacattacaatcaggtagtcat


 


tcactatcacaacaacaatcagcagaattatcacaatcagagcaaacatc


 


acaatcacaacgcatttcaacaagtgcgtcagtatcggctatgaaatcag


 


aaagtgctgctaaggtatctgaatcgctatctacgtctcaatcaaaagta


 


gatagtcaatcacaatcggtatctgaatcagcgagcaactcacgagtgtc


 


aagagattcaaaatcaacaagcgcttcaatgcatcgatcattgtcagagt


 


cagtatctcaaagtatgtcacttattgatcagtcagaaagtgattcaaca


 


tctatatcgatttcgacgtcaatcagtgatgaagactctatgctgtattc


 


tatgagtgattccgcatcgatcagtactaaggcatcaagtagtatgtcta


 


cttcgacaagcgaagagcatgccaacagtcattctcagtctgaatcgaca


 


gcatcggttgaagtatctcaagaaatgagtgcatcggcttcaacaagcaa


 


atctgagtctcaatcagagtcagtatcagtaagtaacgaagaatcaaata


 


tctcatctatgcaagagtcttttgtagagagtgcaaaagcatcgcgtagt


 


gcatctatgagcgttgcaaaatctgaagcctctgaatcacagctattaag


 


tgagtctaatgcttcggtaagccaatcagcaagcacaagtagtaaagcat


 


cagcaagtacgtcagaatctatttcaacgtcactcagcgtatctgaagca


 


actcatggaaaaccgagaaatcattcagaaagtgcatcagcaagtcaatt


 


attagaagaaaatgagtcattaagcgattcagcatcaacaagtgttgaag


 


attcagaaagtgcatcagcatctctgtcggtgtatcaatcacaatcagca


 


agtgcattgaaatcaacacatgcatcagaaaaagcttcagtgaatacaag


 


tgcaaacgcatcgaagcgtgcatcagcatcgacatctatctctaactcga


 


aatctaaagtcattgcgagtgaatcgaagtcaacaagcatatcaacatat


 


gaatcgttgtcaatatcgactagtaaagaacaatcaacgcgtgtatcagt


 


gagtgagtcgacatcaacgtctaaagtgaagtcagaaagcgactcggcat


 


caacgtcgacatctgaatcaatctcaattagcgcaaatcgttcaggttac


 


acatcgtctaaacgttcggtacaaatgagtgaagcacaatcaacgagcga


 


ttcattatcagtaatgcaatctgaaggttcagtaagtgtatcgcaatctt


 


taagtatatcagataagacatcacagtccttatcggaatcaatatcgcat


 


tcagaaagtgactctgatagtaactcagtgtctattagtcaagagacatc


 


tgaacaacattcggtgtcagacagtgactcgatgtcaatttcggaaagcg


 


aatctattgcatatagtcaatcagcgagtgaatcagaatcaacaagtatc


 


gcaaaatctgatagtatttcgaactcattatctgtttcattaagtgaatc


 


agaaagtgaagcaagcacatcagcttcagtgagtacatctgaaagtacgt


 


ctgtaaagggttctctatcaacaagtatcttgaacagtcaatcagcatct


 


actcatcaatcaacagaagcttctcaaagtacatcaacttcaaaagttga


 


ggaagcatcattgagtgactctgcttctgtatcagattcacaatcacttt


 


caatgagtcatgagaaatcacaaagtgcatcgacttcaaaatctacgagt


 


ctgtcaaaaactatttctgagtcagagtctgtgagtgcatcaacatcaac


 


aagtgaagctgtaagtacagaagcaagcgaatttgtatcagcagtagact


 


cattgagtcaagtaacttctaacggaagcacaacgaaagaagatgcgagt


 


acatttgtatccacagtagattcattgaaagacaaagcatcaaataatgg


 


tacaccatcagagtttgcgtcagcagtgaaatcaacacacgcatcagtga


 


gtgtgtcagcatcagaaagtacgtcagcatcaacatcaacaagtgaagct


 


gtaagtacagaagcaagcgaatttgtatcagcagtgaattcgttgagtga


 


agcgacttctaacggaagcacaacgaaagaagatgcgagtacatttgtat


 


ccacagtagattcattgaaagacaaagcatcaaataatggtacaccatca


 


gagtttgcgtcagcagtgaaatcaacacacgcatcagtgagtgtgtcagc


 


atcagaaagtacatcagcatcaacatcaacaagtgaagctgtaagtacag


 


aagcaagcgaatttgtatcagcagtagactcattgagtcaagtaacttct


 


aacggaagcacaacgaaagaagatgcaagcacatttgtatccacagtaga


 


ttcattgaaagataaagcatcaaacaatggtacaccatcagaatttgaat


 


cagttgtgaaatcagtacacggatcaatgagtgcatcagcaagtgcgtca


 


acatcagcatctacatcagcatctacatctacaagtgaagctgcaagtgc


 


agaagcaagcgaattagaatcagtaaggaaatcattatccaatggagcat


 


caaacggtagcacagcaagagaaggtgcaagcacatttgtatcaacggta


 


gattcattgaaagataaagcatcaaacaatggtacagcatcagaatttga


 


atcagttgtgaagtcagtacacggatcaacaagtgcatcagcaagtgcgt


 


caacgtcagcatcaacatcagcaagtgaatcagcaagtacagaagcaagt


 


gaatttgtatcagcagtggcatcattaagcagttcagcatggaacggaag


 


cactacaggagaaggtgcaagcacatttgtatcaacagttgattcatcga


 


aagattcagcgtcagacaaagcttcaccatcagaatcagaatcagttgtg


 


aagtcagtacacggatcaacgagtacatcagcaagtgtgtcagcgtcggc


 


aagtacatcagcatcgacatcaacaagtgaagctgtaagcacagaagcaa


 


gtgagtttgtatcagcagtgaactcattaagcagtgaagcatcgaacggc


 


agcacaacaagagaaggtgcaagcacatttgtatcaacagtagattcatt


 


gaaagacaaagcatcaaacaatggtacagcatcagaatttgaatcagttg


 


tgaagtcagtacacggatcaatgagtacatcagcaagtgtgtcagcatca


 


gaaagtacgtcggcatcgacatcgacaagtgaagctgtaagtacagaagc


 


aagcgagtcagcatcgataagtgtatcaatgtcagtgagcgcatcaacaa


 


gtgcttcaatgagcgtatcagtgtcaaacagtgtgtcagtgagtgactct


 


atttcagtaagtgcatcaacaagtgaacctaactcggtaagcacttctat


 


gagtagttctctttcaacatcggcatcaacgccatcagaaattacttcaa


 


gttcgtcatcaagcgattcagcgacagttcaaaaagtagtttctaaagat


 


gaacagcacgctacaaataaagttgaaaaattacctgacacaggtcaatc


 


aacgacacaaactggtttattgggtggagtaggtgctttacttacaggcc


 


ttggtttactcaaaaaatcaagaaaacaaaaagatgaagaaacatcatca


 


catgaataa






The protein sequence translated from SEQ ID NO 11 is designated SEQ ID NO: 12 and is shown below:








SEQ ID NO: 12







MTRKFREFKK SLSEEKARVK LYKSGKNWVK AGIKEFQLLK


 


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 YGSWVGTPIT KYVSGDRTNG


 


FYWQAAVLAP RHGEKGEGIT AEITVPIVNP SGRFNWEFHP


 


VGQQDGVGGK TDYFENVWIR DYDPYYKYIQ TKEGRASVSH


 


SISQVKASES RSTSLIQSES IRRSQSISES ESIVAASHSA


 


SVAKSQSISR SQSVAKSQSI SRSQSIAHSR SASVAKSQSI


 


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 SISRSQSVVA


 


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 ASTSTSEAVS


 


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







atgaaaaagtctagaaaaaagcgtatcgattttttacctaaccgtcaaaa


 


tcgatatgcgatacgtcgtttttcagtaggcactgcgtcaattctcgttg


 


gagcaacattaatttttggaattcattcaaatgatgcatcggcagcagta


 


gaagacgcaacatctcaagaagcaggaacaactaacgaaaattcaaatag


 


tacagaagaagcaacaacaaacgaaagtacaactgttgaagcaccaacaa


 


gtgaagaagcaacaacggaagagcaatcagtagaggcgccaacaagtgaa


 


gaagtaacaacggaagagcaatcagtagaggcaccaacaagtgaagaagt


 


aacaacggaagagcaatcagtagaagcgccaacaagtgaagaagtaacaa


 


cggaagagcaatcagtagaagcgccaacaagtgaagaagtaacaacggaa


 


gagcaatcagtagaggcaccaacaagtgaagaagtaacaacggaagagca


 


atcagtagaggcaccaactagtgaagaagtaactacggaagagcaatcag


 


tagaagcaccaacaagtgaagaagcaacaacggaagagcaatcagtagaa


 


gcaccaacaagtgaagaagcaactacaaaaactcctgtaaaagaagaaac


 


atcctcaacacaagaaaattcacccacgactacactagaagaacaatttt


 


caaatgaattcaatcagttaacatctacagaagataaaacaaactacaca


 


cgtgaatatttaactcaaaacacaaatctttcggcagaacaagtggaagc


 


aacagttgaacgcttgaatttaagtcaagaaaatgtaacagcccaagata


 


tctatttcgcattacttaaagatttagctgatcaacaagatgccttatta


 


ccacgtgtaacacttttggccgctagagattctgagctcacaaacgaagc


 


gtctatcgctttaactgaaaatagtccaatgttccgcgcagcattagcga


 


atagtccttctggcaatgatgtggtgtcagaagaagataatattattgtg


 


gctgatgcactcgcaaatggatacatcaattcacaaacagatgcaacaaa


 


tgcggcaaatacattgtctggtcgtgcatgggttgtggatacagggacac


 


cagcgacaatgtcaaacggcttaacagctgttccagaaggcacaaaagtc


 


tacatgcaatggattgatacagatggcgcggtttcaccagtgtatcaagc


 


aagcacaacaaataaattgagttcaagtggtggtagccaagtaggtccag


 


gtgcatatgcatttgatttacgtgaagcatggatagactcaaatggcaaa


 


gcgcacagatatgaagcgtcaagtggccaatattatcgtttatggattga


 


tgactacaaaacagtagatgggaatacggcaaccatgttacgccaagcag


 


gtggtttcttccctggttcatatgttaattcggtgacaggtaacaatatt


 


ggtcaattcccacttatcggaacgaacatgcaacgtacaggtatctttat


 


gggtgtgataccaacgaacgattacatgactacagatacaagcaattgga


 


ttcaagataatgaaggacctatttcaaacccagcagtaacgagcacaagt


 


gaatttgtcagtggtaaagtatggtctgagacaggttcaggtgactatgc


 


gaactctgcgacaggtccaaactttaactcaggtgatattgcacgtgaag


 


gttatcaagttgtcatgtcttcattaacaagtgctggtgcccaagcgtat


 


aaagcacaagtcgaatcgttgccaacagaccaacaagcggcagcagcaca


 


ccaattattcaaagaccacccagaatttatttctgcgacagtgacgggta


 


aaactgatgcaaacggtgcgtatacattacgtttcccttcaggctcattg


 


agtaaagattatctttatggttatgtgatggataataagggcaacttggt


 


taagggctattcatcattcacgtcacctttattccgttcgcctaacagta


 


acttatctttcgcgccacaaacagcgccatatcatagaccagccaaaaat


 


gcttgggtgaatgtgaactttgcgcttgtagaaacaattgaaacaagtat


 


agacatcacgaactttgatgtgacagccaacccagcgcaacgtggtgata


 


cggctatcattgatgtgacttctacagcattgtcaccattacctacgcat


 


gttgagtggagagattcaaaagggaatgtcgttcaaaaaagtggagatgt


 


cactacggtagaagaagctgaaacggcaggcacatttactattcctgatg


 


atgcgaaaacaggtgaaatctatacagtttatattgtttcaggaggcaat


 


gaagttgcagcagactcactgattgtccaagtgcaagaaaatgcggcaac


 


ctatgaacctgtatatccaacaacaacagttgaacaagaccaaactgtaa


 


caattcctacacctacaaatgaagatggtttagcattaccagacggaaca


 


aagttcgaaggtggcaacaatgtacctgaatgggcaactgtgaatgaaga


 


tggttctatttcaatttcaccaaatcaagatgtggaaaaaggtaactata


 


atgtgcctgttgtcgtcacatatccagatggttcaaaagaaacagtattt


 


gcaccagttttagttcaagaagctgttccaactgcagaacaatacgatcc


 


aacaattgaaacaattaataaggaatatggtactactgcaacagaagatg


 


aaattaaaggcgcaatcacaattccggattacccaacagatggagatcaa


 


ccaacaatcacgattgacgacccaactcaaattccaaatggaacagaaga


 


aggcacagtgaatgtaggtgtcactgtcacttatccagatggttcaacag


 


acaaattaacagtaccagtcgttacaggtaagcaagcggataacgataag


 


tacacaccagaaacaacaccaattacgaaagacttcggtacaggtgtaac


 


agaagacgaagtgaaaggtgcagtcactgttccggattacccaacagatg


 


gagaccaaccaacaattacgattgacgacccaagtcagttgcctgatggt


 


tcaaaagaaggaacaacggatgtcgacgtaacagtggaatatccagacgg


 


cacaacagatcacatcacagttccagtgactgttggaaagcaagcggata


 


atgataagtacacaccagaaacaacaccaattacgaaagacttcggtaca


 


ggtgtaacagaagacgaagtgaaaggtgcagtcactgttccggattaccc


 


aacagacggtgaccaaccaacaattacaattgatgatccaaatcaattac


 


cggacggttcacaagaaggtacgactgatgtaaatgtaacagtggaatat


 


ccagatggcacaacagatcacatcacagttccagtgactgttggaaagca


 


agcggataatgataagtacacaccagaaacaacaccaattacgaaagact


 


tcggtacaggtgtaacagaagacgaagtgaaaggtgcagtcactgttccg


 


gattacccaacagatggagatcaaccaacggttacaattgatgatccaaa


 


tcaattaccggacggttcacaagaaggtacgactgatgtaaatgtaacag


 


tggaatatccagacggcacaacagatcacatcacagttccagtgactgtt


 


ggaaagcaagcggataatgataagtacacaccagaaacaacaccaattac


 


gaaagacttcggtacaggtgtaacagaagacgaagtgaaaggtgcagtca


 


ctgttccggattacccaacagacggtgaccaaccaacggttacaattgat


 


gatccaaatcaattaccggacggttcacaagaaggtacgactgatgtaaa


 


tgtaacagtggaatatccagatggcacaacagatcacatcacagttccag


 


tgactgttggaaagcaagcggataacgataagtacacaccagaaacaaca


 


ccaattacgaaagacttcggtacaggtgtaacagaagacgaagtgaaagg


 


tgcagtcactgttccggattacccaacagatggagatcaaccaacggtta


 


caattgacgatccgagtcagttaccagatggctcacaagaaggcacaaca


 


gatgtgaatgtaacagtggaatatccagatggcacaacagaccacatcac


 


agttccagtgactgttggtaagcaagcagataacgataagtacacgccag


 


aaacaacaccaattacgaaagacttcggtacaggtgtaacagaagacgaa


 


gtgaaaggtgcagtcactgttccggattacccaacagatggagaccaacc


 


aacaattacaattgacgatccgagtcagttaccagacggttcacaagaag


 


gtacgactgatgtaaatgtaacagtggaatatccagatggcacaacagat


 


cacatcacagttccagtgactgttggtaagcaagcagataacgataagta


 


cacaccagaaacaacaccaattacgaaagacttcggtacaggtgtaacag


 


aagacgaagtgaaaggtgcagtcactgttccggattacccaacagatgga


 


gaccaaccaacaattacaattgacgatccgagtcagttaccagacggttc


 


acaagaaggtacgactgatgtaaatgtaacagtggaatatccagatggca


 


caacagatcacatcacagttccagtgactgttggaaagcaagcagataac


 


gataagtacacaccagaaacaacaccaattacgaaagacttcggtacagg


 


tgtaacagaaggcgaagtgaaagattcaatcacaattcccggttacccaa


 


cagatggagaccaaccaacaattacaattgacgacccaagtcagttacca


 


gatggttcacaagaaggtacgactgatgtcgatgtaacagtggaatatcc


 


agacggcacaacagatcacattacagttccagtgactgttggaaagcaag


 


cagataacgataagtacacaccagaaacagaaggtgtcaacaaagatcat


 


ggtacgtcagtaacagaagatgaagtgaaaggtgcagtcactgttccggg


 


atacccaacagatggagatcaaccaacggttacaattgatgatccaagtc


 


aattgccggacggttcacaagaaggtacgactgatgtaaatgtaacagtg


 


gaatatccagacggcacaacagaccacattacagtcccagtaactgttgg


 


taaacaacctactaaagataacggggctacagataatgatggcgacatga


 


atcaaggcacagatgaaggaaatagtgctactgatcatggcgacaatgta


 


aaacaagattcaaacggaaactatacgccggttgaacaacgtgacaatca


 


tgcgacttcacctgcaacagatatggatccaatgccaagcaatagccaaa


 


caacttttgatggcataaatgcaaaaggttcaacttcagagaaagcaaac


 


cataaacaacagtctgagcaattaccagacacaggtgaaagcaatacaca


 


aaatggtgcacttttaggcggattatttgcagcacttggaggcttattct


 


taatcggcagacgtcgtaaagaaaaagaaggcaaataa






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


 


NFDVTANPAQ RGDTAIIDVT STALSPLPTH VEWRDSKGNV


 


VQKSGDVTTV EEAETAGTFT IPDDAKTGEI 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 PETTPITKDF 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







atgacagaacgaaaatccccttcatctcaaaacatgcgtcatcgtttagt


 


caaagctggtactgtccttttattggttggtagtggactgcaaatgcctt


 


caacattgtcacacgaaatgacagcgatagctcagacagatgcgactgat


 


gatttgaaaacattacgtgaaaatgcagataaaaaagtgaaagcgttaca


 


atatttaaatacggattataaaaatgaatttcttgcgttaattcgtgaat


 


atgatacgtcgtcaaaaaatattgaagtggttgttgacgaagcagaagca


 


gccaatcgtctagctcatgacgctcaatcggacgatgaaatacaacctga


 


attagatgccattgatgaaaaaattagcgcgttaaaggcaaaggttgatg


 


aaggtcaacgagaatcaactgaagcgcgtcaagatgtaacgtcaacagag


 


acaaagagtgctgaatcagaaggaagagagccatccactgaaggcgagag


 


caaagtaaaggagtcatcttcagcacaaacgattgtagcacctcatcatg


 


gtcaacaagatgtgagcgcactgaaagaccatattaagaacgatgtcgat


 


acacttaaacaagactatgcaacgcaagacaagcaagtgacaccactcca


 


gggcattgacagtgcaatcacacgcattgaccatttcgtttcagaaagcg


 


tggatcacaagtctgacaattattttgaagaaaaacgtcaacatttacaa


 


aactttgaacaagacattaaaaaacgtacggacatttctgggactgagaa


 


ggcgactttgcttgatgatgcgaaaacggtagccaaccaactgaacgcgc


 


aaaatgatacgattttaactgaacttcaacagcatgacgataaacgtgca


 


gcagttgaatcgatattaggtgagatttttaatgcacaagaagcggctga


 


acgtgcgaaacagatagatgttaaaggtaaaacagatcaacaattggcaa


 


acgaaattcatcaacaagcggacggacttatcaaaacgtcgagtgatgat


 


ttattgttaggaatgttggaaaataattcaaatacacaaggtctagtgga


 


aagcattttacgaacacgctttgacaaacaagaagcgcacaaaattgccg


 


gcgaaatcatgcaaggcaagccttcaaatacagcgatactcgaccgcttg


 


aaagaccattttaaagcgaatggtaaggcgagtggagatgatattttaaa


 


tgcgttaattaataatacggatgcagatgctgaagtgattgaatcaattc


 


tagggggccgtcttaatgcagaaaatgcaaaattgattgccgatcgtgta


 


cagcaagataaaaagaagacacatcaaaacttaaaggcgattgaagacga


 


acttagtgcgcaagcgaatcgattgttaacgttacggaagcaattgcaac


 


aaatccgtcataatacgcaaacagatatgaatgacttgtttgcaccactg


 


cgtcgtattgcaaatattctcggtggtggtttaaatcgtgacgacattca


 


ctcttcaggtcgtacgaatgacaaattgcagcaactgttaaatcgtgatc


 


attcgttgttaggtcgtggtggtgatttattcaaacatgattttgcgcca


 


aagccgaatatcgatccatatcaagcgattaatagtcaaacggcatcaga


 


aggttttttagatggtttatttgatcaaaatggcgatttcaatttaccga


 


atacaggtgaaatagtgaagcggacttggctaccgttgggtattttagtc


 


gttgcaatcggtgtactgatcttaacggtgagatttcataaaaaaacacg


 


caaacaataa






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 KKVKALQYLN 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


 


QIDVKGKTDQ 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 RTWLPLGILV


 


VAIGVLILTV RFHKKTRKQ


 







SEQ ID NO: 17







atgttaaaaaaattaattgttacaggtttgattgctacagcggcgacaca


 


agtttatgcg catgacacgcaagcggcggaaaagggtgctacagatgct


 


ccgaatgtgatggttaaggatgaggcgaaaaaagaagtgacaccgataat


 


ccataaaccgacttgcatttacccgcatctagaaggcgaagatgatgctg


 


cgtatttaaaacgtatggcaacgaatccaccagaaggcgcagtgccgtac


 


ggtgtattgaataaagatggatcgattacagaaccgaatacaaatccaca


 


ttttgatgttttaaaaattgaagatccaaatgcgatgaaagatttggttg


 


atacaccggcagatgatcaagatacggtaccgagtgatttacaaattgaa


 


ccaccagcattaataggaccagctactaaacatacggatggtacgggaga


 


cgcaaaatctaatgatgaccacaaagtaacaaaatcttcgggagcgtcag


 


cccaagatatgaagaaaaaagacgtgacaacacaaactgcacaaccaaaa


 


gcagataaaaagatggcgactgcaaaagtagcaccagcgaaacaacaaga


 


taaagcagccaaaatgttaccagcagcaggggaaccacaagtgaatgcaa


 


tcagtcaaacagcacttgcactttcaatgatcgcattaggtgtcatcgcg


 


ttctttacacgacgacgcaaaacaaattaa






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


 


EAKKEVTPII HKPTCIYPHL EGEDDAAYLK RMATNPPEGA


 


VPYGVLNKDG SITEPNTNPH FDVLKIEDPN AMKDLVDTPA


 


DDQDTVPSDL QIEPPALIGP ATKHTDGTGD AKSNDDHKVT


 


KSSGASAQDM KKKDVTTQTA QPKADKKMAT AKVAPAKQQD


 


KAAKMLPAAG EPQVNAISQT ALALSMIALG VIAFFTRRRK


 


TN


 







SEQ ID NO: 19







atggtagaatataaaaaagaacatagcgtaaagcgactattaaaattagg


 


aatcggttcaacgagtattttatgtgttgtatcacctcttttattaacac


 


atgacgttgttcaagcagcagatatcaataacaggatgccagctttgaat


 


acattgaagaccacttcttcatatgatcaaagggcacacatggatgaatt


 


acgaaacgccattacttcagatagtgacactactcaaacaccatcattca


 


atgagataactgtgtcttcaactaatgaaacggatgcagcgtcaacggaa


 


aatgtgaacccgagtgatgaggtcccggcaaaggatgaaagtgaatcaac


 


gacaccgagtacagaacaagacacatctatagaagaaacgggtactgaag


 


aagtgccatctcatgaagacaatcatcacaacaccccaagtcaagaagag


 


caaccgtctccgcctgatcaaccaggaacaaacaaagatgaagagagtgg


 


agaaaaaccgaataaagaaaatcatcggaagccgaatcaaccgaacaaag


 


accaaccttcaaaagatgagaataaaaaacctgacaaaggaaacaaacca


 


gcaccaccgtctaaaatgccaaatcgcccggatcaaaaggaagatggttc


 


aaacaacaccccaccacctgccactgataacggtggaaacagtaatgacg


 


gtacaacaacgggtcccaatggtggaggtggcagtgaagcaagtccacca


 


ccgaatgagcaaccgtcaaatggcaatgcaagcgatacccatcaaaacgg


 


ttcagtttcaagcaccaatcattcgaatcagtatggtacatcggcttatg


 


atgaatacgcaggtttattgaataataattataaatataatccattgttt


 


aaagaagaggttgcgcgtttaagtcaatttggaagtcaagatcaacatga


 


tattgcaagtttgagtcgtaaagaacaattttctcaaaatgcatttttag


 


atgacttgcaacaaagtacagattattttagatatcaatattttaacccg


 


ctttccacagagcaatactatcatcgtttagataaacaagtattagcact


 


cgttacgggggaatttggttcgatgccagatttcaagaaaagtggtgata


 


agtcattggttaataagcatcagcaagataaagtgaagaaaattgaacag


 


caaggagaaaatattaatacgcatcatatgaaaaatacgaaagaagatac


 


aggaaaatcattaagttacaagccgatgatatatattggcattgtcatgg


 


tcggttttgtcggcctgatcagtatgattttatggaaacgactgcatcat


 


ttttggaaataa






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


 


DINNRMPALN TLKTTSSYDQ RAHMDELRNA ITSDSDTTQT


 


PSFNEITVSS TNETDAASTE NVNPSDEVPA KDESESTTPS


 


TEQDTSIEET GTEEVPSHED NHHNTPSQEE QPSPPDQPGT


 


NKDEESGEKP 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


gtgattacaaataaaaatatatatagtattcgaaagcataaacttggcgt


 


ggcatcattcttattggggacattatttgttgtagggcatgcaaataatg


 


ctgaagcttcagaagtgagcgcaacaacacaagaacataatgtcgagact


 


gagcaaacaaaaactgagggcgaactaacaactgaggtagcacaacaagc


 


agtcagcgaatcagcacctatagctgaaaacatgcagaaaacaacatcag


 


tggcaagtgaaaatgcgaaagaggttacagcttctgatagcacacaagaa


 


gtcacaaaaactgaagcaaaagatacagcaacaatgaaagattcagaaat


 


tgcacaacctgtatcagaagtgaataaacctgttactcaaacagctgcac


 


ccgtagcagaaccatcaacagcaaacaaacaaacttcaccacgacaagta


 


caagaacttactgcaccaatggacacaaaagtaattaatgtagaaaacgg


 


aacagatgtgacaagtaaagtgaaagttgaaaaatcgtcaattacagggc


 


atcagaataaagataaaacatatcatcaatcgaacactgtaaatccacat


 


aaagctgaacgtgtgacattaaattatgattggtcatttgaaaatggaat


 


taaagctggtgattattttgacttccaattaagcgataatgtcgatacaa


 


atggaatatcaacaataaaaaaagtcccacacattatggatagtcaaaat


 


agcgaacaaattattgcttacggggaaattaatgaaaacaaccgtgtccg


 


ttaccgatttatggactatgtaaatcaaaaagaaaatttaaaaggtaaat


 


tgtcattaaacttatttattaaaccagataaagttcaagatgaaggaaaa


 


atcactgtcacttcacaattgggcaaggaaatgacaagtcaggaatttga


 


cattaaatatattgatggtgtaaaaagcccttcaggtatcacattaaacg


 


gtcgtcttgatgaattatcaaaagcagatcaatcatttacgcattattct


 


atatttaaacctaagcataataacttaactaatgtaactttaagaggcac


 


agtttcaaataacgcacagcaaaatgaaaaaaatggtcaagttaatgttt


 


acgaatatattggtcaaggagaattgccacaaagtgcttatgccaatgta


 


aatgatacgaagcagttcaatgacattactaagagtatgaaatcaatcaa


 


aaataacagtaatggctatgaaattacttttgacatgaacaaagacaatc


 


atccttatatcatagtatatcaaggtcactttaacaataatgcaaaagac


 


tttgatttctcaacaaatgcgacaggttatcaaaatttaaatcaatcgga


 


atatagttattattggccttacaattattcattcaatttaacatgggata


 


atggtgttgctttctactctaataatgcaagtggggaagggaacgacaaa


 


cctgtaccgccgacttatggatatagtccgacagtaaatacaattcaaga


 


tactcatgcggattatcctgtaatgactttccaacaacctggaactctag


 


aggagacagaagacagtatgccaatcactacacttaccgaatctggtgag


 


gatcgtggtgaaaatacttctccaattatcgagacaacagaagattcaca


 


gcctgttgagtttgaagaagagacaaatcatggcattcaagacgtgacac


 


ttcatgcagatgctgttgattttgaggaagaaacaaaccatggtgaacaa


 


gacacggtacaccactctgatgtcgttgaatacgacgaagatacgacaac


 


tggcatgttaacaggtgccatttctgaccatacaacagaagaaggcacga


 


tggagtacacaactgatggcttattgattgagtttgatgatgaaatgaat


 


cctaatgtgagcggtcagtacgatgacatcacaacggatacgatagagga


 


atcatctcatattgacacattcactgaacttgaatctgaatttggtcaac


 


atgacggtatagtgacatttgaagaagatactatcgttgagaagccgaaa


 


acagaaaagggtaaccgagtaccacttgtaattgatttatcaacaccaaa


 


acataaccatcagttcaatattcaacctaccgatccaaatattgatacct


 


ctgctacgtatcgaattggcaattttgtatggcgcgatgaagatcacaat


 


ggcgtacaaaatgatggtgaacatggtcttgaaggtgttcttgtcacact


 


taaaacagctgatggtgtcgttttaaatacaacgacaagtgatgccaatg


 


gacactaccagttcactaatgttcaaaaaggaaaatatattgttgaattc


 


actacacctgaaggttatgaagcaacaagcaaacatactacagcgaatac


 


tgaaaaagactctgatgggttaatcgcaaatatcgatgttactcaagatg


 


atatgtcaatcgatgctggtttcttcccgttagaaaactggaatcctcag


 


ccagagccgaaaaaccctgatgatagagagaaaccggcacctgagcaacc


 


tgatgtacctcagccagaaccgaaaaaccctgatgatagagagaaaccgg


 


cacctgagcaacctgatgtacctcagccagaaccgaaaaatcctgatgat


 


agagagaaaccggcacctgagcaacctgatgtacctcaaccagagccgaa


 


aaatcctgatgataaagagaaaccggcacctgagcaacctgatgtacctc


 


aaccagagccgaaaaatcctgatgataaagagaaaccggcacctgagcaa


 


cctgatgcacctcaaccaaagccgatgctcccaggtgaaaaggtgaaacc


 


caaaccaactcatcccggtgaagctatgcaaacaacacctcaggacaaat


 


caacatctcaaacagatgaagcacttcctaaaacaggtgaatcatcatca


 


caatcatctgctttaatcttcggtggtttactcagtctattaggacttgg


 


tttattacgtcgatcatctaaacaaaaccgttcttcaatgaaataa






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 WSFENGIKAG DYFDFQLSDN VDTNGISTIK


 


KVPHIMDSQN SEQIIAYGEI NENNRVRYRF MDYVNQKENL


 


KGKLSLNLFI KPDKVQDEGK ITVTSQLGKE MTSQEFDIKY


 


IDGVKSPSGI TLNGRLDELS KADQSFTHYS IFKPKHNNLT


 


NVTLRGTVSN NAQQNEKNGQ VNVYEYIGQG ELPQSAYANV


 


NDTKQFNDIT KSMKSIKNNS NGYEITFDMN KDNHPYIIVY


 


QGHFNNNAKD FDFSTNATGY QNLNQSEYSY YWPYNYSFNL


 


TWDNGVAFYS 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


 


EGVLVTLKTA DGVVLNTTTS DANGHYQFTN VQKGKYIVEF


 


TTPEGYEATS KHTTANTEKD SDGLIANIDV TQDDMSIDAG


 


FFPLENWNPQ PEPKNPDDRE KPAPEQPDVP QPEPKNPDDR


 


EKPAPEQPDV PQPEPKNPDD REKPAPEQPD VPQPEPKNPD


 


DKEKPAPEQP DVPQPEPKNP DDKEKPAPEQ PDAPQPKPML


 


PGEKVKPKPT HPGEAMQTTP QDKSTSQTDE ALPKTGESSS


 


QSSALIFGGL LSLLGLGLLR RSSKQNRSSM K


 


SEQ ID NO: 23


atggcatttgatggtatgtttacaagaaaaatggtagaagatttacaatt


 


tctcgtttctgggcgtattcataaaatcaatcaaccggaaaacgatacaa


 


tcatcatggttataagacagcaacgccaaaatcatcaattgttgttgtcg


 


attcacccgaattttgcacggattcacctcactacaaaaaaatatgataa


 


tccatttgaaccgccgatgtttgcgcgcgtctttcgtaaacatttagaag


 


gtggacgtatccttgccattcgccaaatcggaaatgaccgtcgcatcgaa


 


atggacgtggaaagtaaagatgaaattggtgacacgattcatcgtacagt


 


gattttagaaattatgggcaaacatagtaatctcattctcgttaatgaag


 


aacgtaaaattttagaaggttttaaacaccttacaccaaatacgaatcaa


 


tttagaaccgtgatgccaggttttcaatatgaagtgccgccaacacaaca


 


taaacagaacccttatgcatatactggtgcgcaagtgctccaacatattg


 


atttcaatgcgggcaaaattgatcgccaactgcttcaaacgtttgaaggt


 


ttttcaccgttaatcacaaaagaaatcacatcaagacgccattttatgac


 


cacacaaactttacctgaagcttttgacgaagtgatggccgaaacgaaag


 


cgacaccccaaccggtatttcataaaaataacgaaacaggtaaagaagac


 


ttttattttatgaagttacatcagttttacgatgattgcgtcacatatga


 


ttcactccatgaactgctcgaccgtttttatgatgcacgcggtgaacgtg


 


aacgcgtcaaacaacgtgcaaacgatttagtcaaactcgtccaacaatta


 


cttcaaaaatatcaaaataaattaagtaagctcgtcgatgaacaagcggg


 


gactgaagaaaaagaaaatcaacaattgtacggcgagttaatcacagcga


 


atatttatcaactcaaacctggagatcgccagttagaaacagtaaattat


 


tatacaggagaaaacgtgactattccgttaaatccacaaaagtcacctgc


 


tgaaaatgcgcaatactattacaagcaatacaaccgaatgaaaacacgtg


 


agcgcgaattgacccatcaaattactttaacggaagaaaatatcgcttat


 


tttgaaaatatcgagcaacagttgtcacacattcaagttcatgaaattga


 


cgatattcgtgaagaactagcagaacaaggctttatcaaacaaaagaaac


 


agcagaaaaagaaaaagcaacaaaaaatccagttacaatcctacgtttcg


 


actgatggcgatacgattttagtcggtaaaaataataagcaaaatgatta


 


tttaacgaataaacgtgcgcaaaaatcgcatttatggttccatacaaaag


 


atatcccaggaagccatgtcgtgattttaaatgatgcgccaagtgacaaa


 


acgattgaagaagcggcgatgattgcagcgtacttttcaaaggcggggca


 


atcgggacaaattccagtggattatacaacaattcgcaatgtgcataagc


 


cgagtggcagtaaacctggatttgtaacgtacgataaccagaagacgctt


 


tacgcaacgccggattatgacatgattcgtcgattgaaagctgaagaagc


 


gtaa






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 LQKYQNKLSK LVDEQAGTEE


 


KENQQLYGEL ITANIYQLKP GDRQLETVNY YTGENVTIPL


 


NPQKSPAENA QYYYKQYNRM KTRERELTHQ ITLTEENIAY


 


FENIEQQLSH IQVHEIDDIR EELAEQGFIK QKKQQKKKKQ


 


QKIQLQSYVS TDGDTILVGK NNKQNDYLTN KRAQKSHLWF


 


HTKDIPGSHV VILNDAPSDK TIEEAAMIAA YFSKAGQSGQ


 


IPVDYTTIRN VHKPSGSKPG FVTYDNQKTL YATPDYDMIR


 


RLKAEEA


 


SEQ ID NO: 25


atggtcaaaaaatttggttataaaacacctacaatcgttgcacttacttt


 


ggctggaactgcattttctgcacaccaagccaatgccgctgaacaagttg


 


cacctgaaaaaacacctacgaatgtacttgatgatcaatacgcattaaaa


 


caagctgatgatgcgaaacaaacgacacaaggaacaacacttgcaggttc


 


aaaagaatacaaggatccttcacaaattgatacgactcaagtcgatacag


 


cagcacaaactgaaacgcccgtagaaggagggcaacaagacgcacaacaa


 


cctactacaactgatgaagcgacatcaacagatcatactgtatcaaaagg


 


tacaaacgaaagtgcatcacctgcaacagcttctatagatgaaggaacat


 


taaacgcacaagtcaattcagatgaaacggctactaaccgtacacaagac


 


gtcactgaaaatgtgacaaaatatccttatcattcaagtgaaatcgatac


 


acatgaagacgcaactgtgtcaccagatacatatcatgcactggacacgc


 


atgcgcaacaaccttcagcaatggatgtaagcgattcaacatcagcacaa


 


actgaagcgacgcaagtaaatacgtcaacaaatgtaaatgacaaagaggc


 


cgtttcgacaacagaagatgcacctactacacaacttcaagcagctgtac


 


aatctgaagccaacaaagaagcgaaggcaactactgaaacagctcaaaat


 


aaaacacctcaagttgaaaagaaagcaacagcaactcaaaatacagcaca


 


gttagcaacggggcatcaggatattactgacaaagtctcaaaacgcgtag


 


cagtgacaaatgaaacgaaagcggatgccacaacagcgaaaacacaagca


 


cctacttcagtgacacatcaagctgatacacaagcaaaaacgataacaga


 


caagaaggcaacaacttacagtgcacaaaccgcaactgaccaagacataa


 


atgcgaatccggacggtccaacacctccacgcgttggcggtaaagggggt


 


ccccctgcttcactttcactccaatcgactggtcaaacagcattccgttc


 


agctgtcgctagtaaaccgagtgcatatcaacctaaagtgaaatcgtcta


 


ttaatgactatattcgtaagcaaaactacaaagtgcctgtatatgaagaa


 


gattattcaagttacttccctaaatacggttatcgtaatggtgtcggtaa


 


acctgagggcatcatcgtgcatgatacagcaaatgacaactctacaattg


 


atggcgaaatcagttacatgaaaagaaattatcaaaatgctttcgtacat


 


ggctttattaatggtcaacgtattgttgaaacgcaacctacagattattt


 


agcatggggtgcaggtgcgattgcgaatgaacgctttattcatatcgaac


 


tcgttcatgttcacagtaaagaagatttcgcacgtcaaatgaacaatatg


 


gcagattatgcggcgacgaacttacaatattatggcctttctccagatag


 


tgcggaatatgatggtcgtgggacagtttggacacatgatgctgtttcta


 


gatttttaggtggtacagaccataccgatccgcacggctatttaaaacaa


 


catggttattcctttgatgcgttgtatgatttaatcaatgaaaaatatca


 


agtgaaaatgggttatgcctcacctgctaactcgtcttcaaaaccatcaa


 


caaatactggcttaacagttaaaaacacaacaggtttcggccgtattaac


 


acaacaaatagcggtttatatacgaccgtttatgatcaaaaaggtaaagc


 


gacgaatcaaacgaatcaaacgttaaaagttacaaaagaagcgacgttaa


 


atggcaacaaattctatttaatgagtgatgcaaaatctaatcaaacactc


 


ggttgggtcaaatcaaacgacgcaacatatcaagctgcccaagctgagaa


 


aaaagtaacgaaaacgtatactgtcaaaccaggaacaacagtatatcaag


 


tgccttggggtgcctcatctcaaacagtaggcaaagctccaggtacgtca


 


aaccaatcattcaaatcaacgaaagaacaaactgttgcgaaaacgaaatg


 


gctttatgggacagttggcaaagtgacaggctggattaatgcaagtagtg


 


ttgtagcaaatgatcaaaaaccatcgacgaataccgcactaaaagtaaca


 


actgacactggtctcggtcgcattaaagacaaaaatagtggtttatacgc


 


aacggtatatgataaaactggtaaaagcacttcagccactaaccaaacat


 


taaaagtaacgaaaaaagcaagtgtcaatggccaatcattctatttagta


 


tcagattatgctaaaggtacaaatgttggttgggtgaaacagtcagatgt


 


cgaatatcaaacaagtaaagccccttctaaagtgaatcaaaattatacga


 


ttaaatcgggtgcgaaattgtatcaagtgccttggggtacaagtaaacaa


 


gttgccggtacagtgacaggtgctgcgacacaaacatttaaggcaacaca


 


atctcaaactgtaggtaaagcaacatacttgtatgggacagttggcaaat


 


tatctggttggattaattcaacagcattagcagctcaaaaaacaacaacg


 


aatgttactaaaacaatttctcaaatcggtcaactgaacacgaaaaatag


 


cggtgtcaaagcttctatttatgacaaaacagcaaaagatgcatccaaat


 


gggcaggtcaaacttataaaattactaaaacagcttctgccaataacgaa


 


gactatgtattactgcaaaatagtacaggaggcacgccactcggttggtt


 


caatgttaaagacgtcacaacacgcaacttaggtgctgaaacagctgtta


 


aagggcggtacactgttaatagtaaaacatctggactctacgctatgcct


 


tggggtacaacgaagcaacgtgtcgatacattaaaaaatgccacaagtcg


 


tttatttacagcttcaaaatcagttaaagtcggtaatgatacattcttat


 


tcggtacagtgaatcaaaaattgggctggattaatcaaaaagacttaaca


 


gctgtagcagcaaaagttgcaaacatgaaaactgcatcgaatagcgcagt


 


caaaggtgccgcaatcacaactttgaaaaaagtagaagattatgtgatta


 


cgaataaaaatggttattattacactaaagttggagattcaaaaacagct


 


ggtgctttaaaaggtttttatcaacaaatttttaaagtcgaaaaaacatc


 


tttactgaacggcattacttggtactatggcgcattccaaaacgggacga


 


aaggatggattaaagcagctgacatacgttcatcattcattcaacatact


 


gcggtcagtagcacattgaaagcagcactcgataaacaaatggcgctgac


 


ttacccgcctcaagttcaacgtgtagccggtaaatgggtcaatgcgaatc


 


gtgcagaaactgaaaaagcaatgaataccgcagcaattgaaaaagatccg


 


actctcatttaccaatttttaaaacttgataaataccaaggtcttggcgt


 


agaagaacttaataaattgttaagaggcaaaggcattttagaaggtcaag


 


gtgccgcatttaaagaagccgcacaaaaacacaatattaatgaggtttac


 


ttaatgtctcacgcatttttagaaacaggtaacgggacttctcaattagc


 


caatggcggtcacgtagataaaaataataaagtcgtaacaaacggtaaac


 


cgaagtattacaacatgttcggtatcggggcaattgatacagacgcttta


 


cgcaatggctttaaaactgctgaaaaatatggttggaatacggtcagcaa


 


agcgattatcggtggcgcaaaattcatccgtgatcagtacatcggttcag


 


gacaaaacacattgtatcgtatgcgttggaatccagaacaccctgccaca


 


catcagtatgcgactgatattaattgggcaaatgtaaacgcacaacgcat


 


gaaatatttctatgatcaaattggtgaaacaggtaaatatttcgacgtcg


 


atgtatataagaagtag






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 EQVAPEKTPT


 


NVLDDQYALK QADDAKQTTQ GTTLAGSKEY KDPSQIDTTQ


 


VDTAAQTETP VEGGQQDAQQ PTTTDEATST DHTVSKGTNE


 


SASPATASID EGTLNAQVNS DETATNRTQD VTENVTKYPY


 


HSSEIDTHED ATVSPDTYHA LDTHAQQPSA MDVSDSTSAQ


 


TEATQVNTST NVNDKEAVST TEDAPTTQLQ AAVQSEANKE


 


AKATTETAQN KTPQVEKKAT ATQNTAQLAT GHQDITDKVS


 


KRVAVTNETK ADATTAKTQA PTSVTHQADT QAKTITDKKA


 


TTYSAQTATD QDINANPDGP TPPRVGGKGG PPASLSLQST


 


GQTAFRSAVA SKPSAYQPKV KSSINDYIRK QNYKVPVYEE


 


DYSSYFPKYG YRNGVGKPEG IIVHDTANDN STIDGEISYM


 


KRNYQNAFVH GFINGQRIVE TQPTDYLAWG AGAIANERFI


 


HIELVHVHSK EDFARQMNNM ADYAATNLQY YGLSPDSAEY


 


DGRGTVWTHD AVSRFLGGTD HTDPHGYLKQ HGYSFDALYD


 


LINEKYQVKM GYASPANSSS KPSTNTGLTV KNTTGFGRIN


 


TTNSGLYTTV YDQKGKATNQ TNQTLKVTKE ATLNGNKFYL


 


MSDAKSNQTL GWVKSNDATY QAAQAEKKVT KTYTVKPGTT


 


VYQVPWGASS QTVGKAPGTS NQSFKSTKEQ TVAKTKWLYG


 


TVGKVTGWIN ASSVVANDQK PSTNTALKVT TDTGLGRIKD


 


KNSGLYATVY DKTGKSTSAT NQTLKVTKKA SVNGQSFYLV


 


SDYAKGTNVG WVKQSDVEYQ TSKAPSKVNQ NYTIKSGAKL


 


YQVPWGTSKQ VAGTVTGAAT QTFKATQSQT VGKATYLYGT


 


VGKLSGWINS TALAAQKTTT NVTKTISQIG QLNTKNSGVK


 


ASIYDKTAKD ASKWAGQTYK ITKTASANNE DYVLLQNSTG


 


GTPLGWFNVK DVTTRNLGAE TAVKGRYTVN SKTSGLYAMP


 


WGTTKQRVDT LKNATSRLFT ASKSVKVGND TFLFGTVNQK


 


LGWINQKDLT AVAAKVANMK TASNSAVKGA AITTLKKVED


 


YVITNKNGYY YTKVGDSKTA GALKGFYQQI FKVEKTSLLN


 


GITWYYGAFQ NGTKGWIKAA DIRSSFIQHT AVSSTLKAAL


 


DKQMALTYPP QVQRVAGKWV NANRAETEKA MNTAAIEKDP


 


TLIYQFLKLD KYQGLGVEEL NKLLRGKGIL EGQGAAFKEA


 


AQKHNINEVY LMSHAFLETG NGTSQLANGG HVDKNNKVVT


 


NGKPKYYNMF GIGAIDTDAL RNGFKTAEKY GWNTVSKAII


 


GGAKFIRDQY IGSGQNTLYR MRWNPEHPAT HQYATDINWA


 


NVNAQRMKYF YDQIGETGKY FDVDVYKK


 


SEQ ID NO: 27


gtgtcgacagaaaaacaagatgatacacaagcaaaagcgaatgcactttc


 


tacagatgattcaacacctacaacagaacaatcaaaaagtgataccgaac


 


caacgcaaaatcaagaagtgaatgaaaaagaagcaacacaagttgagcaa


 


actccagataatgcatcatcagaatttaaagacagtgcagcacaagatga


 


aacaacatcgaaagacgctgacattgctcaaacaaaagaagcaaaaaatg


 


aagcattgcaaagtgactcatcagcaaacctatcaaatcaagaagcagaa


 


aaagaaaacacaactaacagtgaatctcaagtaaatgaacaacctaaagc


 


agatacaacttctgattcacaagtttcaaatacacctcaacaagatccta


 


catcgacagtaccttcaccagaaacatcagaagacaatcgaccttcaaca


 


gaattaaaaaatagtgaaacaactgcttctcaaacaactttaaacgaaca


 


acctactgaatcaacatccaatcaaactgaaacgacaaaagcaccaacaa


 


atacaacagtcgcaaacaaaaaagcacctgcacaattaaaagacattaaa


 


ggtacaactcaacttcgcgcagtcagtgcaagtcaacctactgctgttgc


 


agctggtgggacaaacgtaaatgacaaagtaacagcatcaaatatgaaaa


 


taactgaatcttatatcgagccaaacaactcaggaaacttttatttaaaa


 


agtaactttaacgtaaacgggactgttaaagaaggtgactactttactgt


 


aaaaatgcctgacactgtcaatacttttggtgacacgcgccattcacctg


 


actttagagaaaaaattacaaatcaaaaaggtgaagttgtggctttaggt


 


gaatatgatgttgccaaccatactatgacatacacgttcactaatgtcgt


 


taataatttagaaaatgtgtccggttcgtttaacttgactcaatttatgg


 


atcgtaaagtggcaacagattctcaaacatatccattaaaatacgacatt


 


gcaggcgaatctttagatacacaaattaaagtgaattacggtcaatatta


 


cagtgaaggtgattctaacttaaaatcaatgatcacttcagaagatccta


 


aaactggggaatatgatcaatacatttatgtcaacccattacaaaaaacg


 


gcaaacggtacagttgtaagagttcaagggttccaagttgatccaactaa


 


gagtaatgggcaagtgaaaccagatacaacgcagatcaagattttaaaag


 


ttgctgatggtcaaccacttaatagtagtttcggtgtgaatgacagtgaa


 


tatgaagatgtcacaaaacaatttaatattgtttatcgtgataataattt


 


ggcagatatttactttggaaacttaaatgggcaacgctatatcgttaaag


 


tgacgagcaaagaaaatttggattctaaagaggatttaaacttgcgtgct


 


attatggccactcaaaaccgatatggtcaatataactatattacttggga


 


taacgatattgtgaaaagctcttctggtggtacagccgacggaaatgaag


 


catcatatcaattaggcgacaaagtttggaatgatgtgaataaaaatggt


 


atccaagatcaaggtgaaactggtattgctgatgtaaaggttactttaaa


 


agatcttgatggcaacattttggatacaacttatacaaacacgaatggta


 


aatatatctttgataatttaaaaaatggtaattatcaagtgggttttgaa


 


acaccggaaggctatgctgcaagtccatccaaccaaggtaatgacgccct


 


tgactctgatggtcctacaaatgtacaagctgtcattagtgatgggaaca


 


acttaactatcgaccaaggtttttaccaaactgaaacaccaacacacaac


 


gtcggcgacaaagtttgggaagacttaaataaagatggcatccaagacca


 


aaatgaaccaggtatcgctaacgttaaggtcactttaaaagacgcggatg


 


gtaacgttgtggatacacgtacgactgatgataaagggaattacttattc


 


gaaaaagttaaagaaggcgaatatacaattgaatttgaaacgcctgaagg


 


ttatacaccgacacaaacaggccaaggcagagtcagcactgactctaatg


 


ggacatcttcccttattttagtcgaaggtaacgatgacttaacaatcgat


 


agcggtttctacaaagaacctgttacacacaaagttggcgacaaagtttg


 


ggatgacttaaataaagacggtatccaagatgacaatgaaccaggcatct


 


ctgacgttaaagtcactttaaaagatgcggatggtaacgtcgtagataca


 


cgtacaactgatgctaacggtaactatttatttgaaaacgtgaaagaagg


 


cgactatacgattgaatttgaaacgcctgaaggttacacaccgactgtta


 


caggtcaaggtacagctgataatgactctaacggtacatctacaaaagtt


 


acagttaaagatggcgatgacttaacaattgacagtggtttcactcaagt


 


tacacctgagccaccgacacataatgttggcgacaaagtttgggatgact


 


taaataaagacggtatccaagatgacaatgaaccaggcatctctgacgtt


 


aaagtcactttaaaagatgcggatggtaacgtcgtagatacacgtacaac


 


tgatgctaacggtaactatttatttgaaaacgtgaaagaaggcgactata


 


cgattgaatttgaaacgcctgaaggttacacaccgactgttacaggtcaa


 


ggtacagctgataatgactctaacggtacatctacaaaagttacagttaa


 


agatggcgatgacttaacaattgacagtggtttcactcaagttacacctg


 


agccaccgactgaacctgaaaaccctagtccagagcaaccttctgaaccg


 


ggtcaacctgaaaatcctagtccagagcaaccttctgaaccaggtcaacc


 


tgaaaatcctagtccagagcaaccttctgaaccaggtcaacctgaaaatc


 


ctagtccagaacaaccttctgaaccgggtcaacctgaaaatcctagtcca


 


gaacagccttctgagccaggacaacctaaaaatcctagtccagaacagcc


 


aaataatccaagtgtgccaggtgttcaaaatcctgaaaaaccaagcttaa


 


ctccagtcacacaaccggttcattcaaacggcaataaagcaaaaccatct


 


caacaacaaaaagctttacctgaaacaggtgaaactgaatcacatcaagg


 


tacattattcggtggtattttagctgctttaggcgcattactctttgcac


 


gtaaaaaacgccacgataaaaaacaatcacactaa






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 DGNNLTIDQG


 


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


atgaagaaaacaatttcagtacttggtctagggctattagcaacattttt


 


tgtaagtaacgaatcatatgccgcagaaacgattcaaaacaatacgtcat


 


caagtgaaacgaatcaaaattcagatcagacgccgttagatcattatatt


 


cgaaaagcagatggcacactggttgaaccgaacgtgtacccacataaaga


 


ttatgtagagaatgaaggacctttaccagagtttaaatttcaagttgact


 


ctaagaaagattcatctgatccaaatcaagcaccgttagatcattatatt


 


cgaaaagcggatggcacgttggttgaaccgaatgtatatccacacaaaga


 


ttatgtcgaaaatgaagggcctttaccagagtttaaatttatgtatgctg


 


acaaacaaaatcatcatgaccaacagagtaaaaacaacaaggataagcag


 


cgtgcaaattacagtgacaaaaagcataatgatcagccgggtcatccaaa


 


agcagtcacgccagctgtacaacatgataaagcagtcacttcaaacgcta


 


ctgtaaaagcattgccaaacacaggtgaatctgataaaacaacacaatta


 


ccaatcgtattatcattgttatctgtggggattttagttttattaaaatt


 


gagaaaataa






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


atgaaaagtaaatatgattttttacctaatagacttaataaattttctat


 


acgaaaatttactgttggtagtgtatcagtgctaataggagccactttat


 


tattcgggtttgtagaaggagaagcatcagcatcagtaaaagaaggtcaa


 


caaagtataaattctagtgagaaagaaagcgccgatcctacagtagttga


 


tttaattagtaagaaagaaacaaatttagatggactagatgtatcaagag


 


aagaaacgaccaaagtaccaataaatgaaaacaaaagaggtgaggaacaa


 


agtatttctgataaagctataacagaaaaagctgatacaccagtaagcaa


 


tttatcaagtaaggaagttgaggagcaaggtgtttctgataaagctataa


 


cagaaaaagctgatacaccagtaaccaatttatcaagtaaggaagctaag


 


gagcaaggtgcttctgatagagttataacagaaaaagctgatacaccagt


 


aagcaatttatcaagtaaggaagctaaggagcaaggtgcttctgatagag


 


ttataacagaaaaagctgatacaccagtaagcaatttatcaagtaaggaa


 


gttgaggagcaaggtgtttctgataaagctatagagaaaatagctgatgc


 


atcagctactgatttgtcaagtaaggaagaagtagaacaagatatatcta


 


cacaaggtaaagtaaaatcaaaggaagcagtacaagtagaaagtagtcag


 


ttacaaaatttaaatagtgaaataaatgctgaacctaatgaaattaaggc


 


aatagatagaagttcaatattacctttaaatttaaatgatgaagaaaata


 


acaaaaaagttaataaagggactcgggttccagaagctacattaagaaat


 


gcctctaataaccaactcaatacacgaatgagatcagtgagtttatttag


 


agttgctagactaacagaaatcaatagaaatgttaatgataaagtaaagg


 


tttcggatatcgacatcgcaatagccccaccgcatactaaccctaaaact


 


ggaaaagaagaattttgggcgacatcttcttcagttttaaagttaaaggc


 


aagctatgaattggataatagcatttctaaaggggatcaatttactattc


 


aatttggtcaaaatattcgtccaggtggattaaatttaccaagaccttat


 


aattttttatatgataaggataaaaaattagttgcaactggccgttacaa


 


taaagaatcaaatacaatcacatatacatttacggattatgtagataaac


 


atcaaaacattaaaggtagttttgagatgaatgcattttctagaaaggaa


 


aatgctactactgacaaaacagcatatccaatggatgttactattgcgaa


 


tcaaaaatatagtgaaaatattattgtagactatggtaataaaaagaatg


 


ctgctattatttcaagtacagaatatattgatttagatggtagtagaaaa


 


atgacaacatatattaatcaaaatggtagtaaaaattccatctatcgtgc


 


tgatatgcaaattgatttgaacggttataaatttgatccatccaaaaaca


 


attttaaaatttatgaagtggaaaatagcagtgactttgtggatagcttt


 


tcaccagatgtgagcaagttaagggatgttacgagtcaatttaatattca


 


atatacaaataataatacaatggcaaaagtggattttggtactaaccttt


 


ggaggggtaaaaaatatattattcagcaagtggcgaatatagacgacagt


 


aaattagtgaaaaatgcttcaatcaattatacattgaataaaatggattt


 


taataataaaagaacggtagaaacacataacaatacttattctacagtga


 


aagataaatcaacagcactaggtgacgtacaggaaagtcaatctattagt


 


gagagccaatcagttagtgaaagcgagtcactaagtgagagccaatcaat


 


cagtgaaagcgaatcattaagtgagagccaatcaatcagtgaaagcgaat


 


cattaagtgaaagtcaatcaatctcagagagcgaatcactaagtgaaagt


 


cagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatctcaga


 


gagtgaatcattaagtgaaagtcagtcaatttcagagagtgaatcactaa


 


gtgaaagtcagtcaatttcagaaagcgaatcattaagcgagagtcagtca


 


atttcagaaagcgaatcattaagcgagagtcagtcaatttcagaaagcga


 


atcattaagtgaaagccaatcaatcagtgaaagcgaatcactaagcgaga


 


gccaatcaatctcagagagtgaatcattaagcgagagtcaatcaatctca


 


gagagcgaatcattaagtgagagtcaatcaatcagtgaaagcgagtcact


 


aagtgagagtcaatcaatttcagagagcgaatcattaagtgaaagccaat


 


caatctcagagagtgaatcactaagtgagagccaatcaatctcagagagt


 


gaatcattaagtgagagccaatcaatctcagagagcgagtcactaagcga


 


gagccaatcaatttcagagagtgaatcactaagtgaaagtcaatcaattt


 


cagagagcgaatcactaagtgagagccaatcaatctcagagagcgaatca


 


ctaagtgaaagtcaatcaatttcagagagtgaatcactaagcgagagcca


 


atcaatctcagagagtgaatcattaagtgaaagtcagtcaatttcagaga


 


gtgaatcactaagtgaaagtcagtcaatttcagaaagcgaatcattaagt


 


gaaagccaatcaatcagtgaaagcgaatcactaagcgagagtcaatcaat


 


ctcagagagcgaatcattaagtgaaagtcaatcaatttcagaaagcgagt


 


cattaagcgagagtcagtcaatctcagagagcgaatcactaagcgagagt


 


caatcaatctcagagagtgaatcattaagtgagagccaatcagttagtga


 


aagcgaatcactaagtgaaagtcagtcaatttcagaaagcgaatcattaa


 


gtgagagtcaatcaatttcagaaagcgaatcattaagtgaaagccaatca


 


atcagtgaaagcgaatcactaagcgagagccaatcaatcagtgaaagcga


 


atcattaagtgagagtcaatcaatctcagaaagcgaatcattaagtgaga


 


gtcaatcaatcagtgaaagcgaatcactaagcgagagccaatcaatctca


 


gagagcgaatcactaagcgagagccaatcaatctcagagagcgagtcact


 


aagcgagagccaatcaatcagtgaaagcgaatcattaagtgagagtcaat


 


caatcagtgaaagcgagtcactaagtgagagccaatcaatctcagagagt


 


gaatcattgagtgagagccaatcaatctcagagagcgagtcactaagtga


 


gagtcaatcaatttcagagagcgaatcattaagtgaaagccaatcaatct


 


cagagagtgaatcattgagtgagagccaatcagttagtgaaagcgagtca


 


ctaagtgagagtcaatcaatcagtgaaagcgagtcactaagtgagagtca


 


atcaatttcagagagcgaatcattaagcgagagtcagtcaatctcagaga


 


gtgaatcactaagtgagagccaatcaatctcagagagtgaatcattaagt


 


gagagccaatcaatctcagagagtgaatcactaagtgagagtcaatcaat


 


cagtgaaagcgaatcactaagcgagagccaatcaatttcagagagtgaat


 


cattaagtgagagccaatcagttagtgaaagcgaatcactaagcgagagc


 


caatcaatctcagagagcgaatcattgagtgagagccaatcaatctcaga


 


gagtgaatcattgagtgagagtcaatcaatcagtgaaagcgaatcactaa


 


gcgaaagtcaatcaatttcagagagtgaatcattgagtgagagccaatca


 


atttcagagagtgaatcactaagtgaaagtcagtcaatttcagaaagcga


 


atcactaagcgagagccaatcaatctcagagagcgaatcactaagtgaaa


 


gtcagtcaatttcagaaagcgaatcattaagtgaaagccaatcaatctca


 


gagagtgaatcattaagtgaaagtcagtcaatttcagagagtgaatcact


 


aagtgaaagtcagtcaatttcagaaagcgaatcattaagcgagagtcagt


 


caatttcagaaagcgaatcattaagtgaaagccaatcaatcagtgaaagc


 


gaatcactaagcgagagccaatcaatctcagagagcgaatcactaagcga


 


gagccaatcaatctcagagagcgaatcactaagtgaaagtcaatcaattt


 


cagagagtgaatcattgagtgagagtcaatcaatttcagagagtgaatca


 


ctaagtgaaagtcaatcaatttcagagagtgaatcactaagcgagagcca


 


atcaatctcagagagtgaatcattaagtgaaagtcagtcaatttcagaga


 


gggaatcactaagtgaaagtcagtcaatttcagaaagcgaatcattaagt


 


gaaagccaatcaatcagtgaaagcgaatcactaagtgaaagtcaatcaat


 


ctcagagagtgaatcactaagtgagagccaatcaatctcagagagtgaat


 


cattgagtgagagccaatcaatctcagagagcgaatcactaagtgaaagt


 


caatcaatttcagaaagcgagtcattaagcgagagtcagtcaatctcaga


 


gagtgaatcactaagtgagagccaatcaatctcagagagtgaatcactaa


 


gtgagagtcaatcaatcagtgaaagcgaatcactaagcgagagccaatca


 


atttcagagagtgaatcattaagtgagagccaatcagttagtgaaagcga


 


atcactaagcgagagccaatcaatctcagagagcgagtcactaagcgaga


 


gtcaatcaatctcagagagtgaatcactaagtgaaagtcagtcaatttca


 


gaaagcgagtcactaagcgagagtcaatcaatctcagagagtgaatcatt


 


gagtgagagccaatcaatctcagagagcgaatcattgagtgagagccaat


 


caatctcagagagtgaatcattgagtgagagccaatcaatttcagagagc


 


gaatcactaagcgagagccaatcaatcagtgaaagcgaatcattaagtga


 


gagtcagtcaattagcgaaagcgaatcactaagtgagagtcaatcaatct


 


cagagagtgaatcactaagtgaaagtcagtcaatcagcgaaagcgaatct


 


aaatctttacctaataccggtactggagaaaagatttctaattatccagg


 


tattttaggaggattattaagcatattaggtataagtttgcttaaaagaa


 


aagacagagagaaaaaattaggacaaaaatctaataagtag






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


 


NFLYDKDKKL 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


atgttaagaacaaattataaactaagaaagcttaaagtaggtttagtatc


 


gacaggtgtggcgttgacttttgtgatggcaagtgggaatgcagaggcgt


 


cggagaacgagcagactgaagtaaaaggggaggcgcaagttgcttctgtg


 


aatgaaaaagagagtgaagcagaattacctgtagcgcaacaagaagcatc


 


tattcaactagacaaagtacaaccaggcgatgcacagctttcaggctata


 


cacagccaaacaaagcgatttctgtaaagatcgacaataaagatattgtg


 


tctgtagatgatggctatgaagaggtattatcggatgatacaggtaaatt


 


tgtatatgatttgaaagggcgtcaaattgtttacaatcaaaaagttgatg


 


ttgaagcgatgacgccatttaattttgaagattttgatgaatcagcactt


 


gagagcgaagaggcattggaggcgttaggtcaattggaagacgaagaaac


 


agcgacagcttctgtgacgacgcctagatatgaaggtgcgtatacagttc


 


ctgaagaacgcttgacacccattcaaggccaacagcaagtattcatcgaa


 


cctattttagaaggggcaagtaaaatcaaaggacatacatctgtacaagg


 


taaagtcgcgttagcaatcaatcaagaacatgtgcacctaggtgatacgt


 


tagaagaacaagcagcactcactgatcaagagtggcaaggtcgttatgac


 


gggatttggcgccatattgatgatcaagggtttttcgagtttgacttgaa


 


ccgtctttacaataaatcttacccattgaagtctggcgatttagtgactt


 


tatcttttaaatctaatgacgaagtaggcccattattcaatgtgaacgtt


 


gagcctttcgaacgtgtggcacaagctaaaacaaagtatgagcagaatga


 


cagtccagtagtcaacaaattggatgatactaaaagtgacttggaggttc


 


aacctatctatggagaccttacacaagcagcagtacatggcgagtcgaaa


 


gtgttgataccggggacgtcaaaagttgaaggacgtacgaattatgcaca


 


tgcatggatagagatggcatctaatttaggggaatatcgtagtttcccta


 


aattacaagctgatgcgacaggtgcgtttatatttgatttaaaagcggca


 


gacatacaattgttaaacggagaacgtttgacattcagagccgttgaccc


 


acatacaaaacaacagttagctgaaactacatcagaagtacgcccagtag


 


atatgcaagatgaagagtcagaggttgtgcagacttcaagcactgagaaa


 


tcagcacttgcggatgaaattcttcgttctatgacaattgacaaatcatt


 


taatcctgaagttaccgagataccgggtcatgtatatcctaagaaaacag


 


aggataaaggtgctgaaaatacagaacaagcctcagagaattctgagaag


 


ccatctcagactacagaatctcaaaatgatgccgtacaagatgtagagaa


 


atcctctgttaatgaggaggttacgccaccttcaacagaatctgctcaag


 


ttgaaaaggggcaaaatacagaaggggctttgcttccaaaaaatgtagaa


 


caacatgtagagagtataccataccaaaaacgtaaagcgttgataggact


 


gacaaaacatcaaggatcagggcacatgccgccattttctttaagcttta


 


ataataaagaagatgacgtatccacaaaggttaacgaagcaaacgagcat


 


gaacgtaagcagggtacagtttatccagagcaaatagaacaattacctca


 


aacaggtttaactgaaaaatcgccattctgggcattgttatttgttgtat


 


caggcacaggtttattattattcaaacgttctagacgacaacgccaatct


 


taa






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


 


GIWRHIDDQGFFEFDLNRLYNKSYPLKSGDLVTLSFKSNDEVGPLFNVNV


 


EPFERVAQAKTKYEQNDSPVVNKLDDTKSDLEVQPIYGDLTQAAVHGESK


 


VLIPGTSKVEGRTNYAHAWIEMASNLGEYRSFPKLQADATGAFIFDLKAA


 


DIQLLNGERLTFRAVDPHTKQQLAETTSEVRPVDMQDEESEVVQTSSTEK


 


SALADEILRSMTIDKSFNPEVTEIPGHVYPKKTEDKGAENTEQASENSEK


 


PSQTTESQNDAVQDVEKSSVNEEVTPPSTESAQVEKGQNTEGALLPKNVE


 


QHVESIPYQKRKALIGLTKHQGSGHMPPFSLSFNNKEDDVSTKVNEANEH


 


ERKQGTVYPEQIEQLPQTGLTEKSPFWALLFVVSGTGLLLFKRSRRQRQS


 


SEQ ID NO: 35


atgaaaactaaatacacagcaaaattattaattggggcagcaacaatatc


 


tttagcaacatttatttcacaagggaacgcacatgcgagcgaacaaacta


 


caggactcgcaccggcacaacctgtcaactttgattcaatcaatgtaacg


 


ccagaccaaaaaacattctatcaagtcttacatatggaaggcatttcaga


 


agaccaacgtgaacaatatttgaaacaattgcacgaagacccaagtagcg


 


cacaaaatgttttttcagaatcaattaaagatgccatccacccggaacgt


 


cgtgttgcgcaacaaaatgcgttttacagcgtattacacaacgatgactt


 


atccgaagagcaacgtgatgcatacattggtagaattaaagaagatccag


 


atcaaagccaagaagtatttgttgagtctttaaatgtggcacctaaagca


 


gaatcacatgaagatcgcctcattgaattacaaaacaaaaatttaatgga


 


agcgaatgaagcacttaaagcgttacaacaagaagacagcattcagaata


 


gacgtgcggctcaacgtgctgtcaacaaattgacgccggatagcgcgaac


 


gcattccaaaaagaattagatcaaatcaatgccccacgcgacgctaaaat


 


taaagctgacgctgaagcaaaaaaacaagcacctgaagtaagcgcaccac


 


aaattgaagatgcacctactactgaagttgcaccatctccaaaacaagat


 


atgccaaaagtagataaaaaagaagaagataaagtagaaagtgatactga


 


ggtcaaagaagtacctaaagctgatacagagaaaaaccctcaatctaaag


 


acacttctaaaactgaacaagctaaagaaacacctaaagtagagcaatca


 


cctaaaacagaaaaggctgaagaagcacctaaagcagaaacacctcaaaa


 


tggaaataaagcacaaactgaagaagctaaaccagaagtaaaagacaatg


 


tgaaaaacactccatctgcacctgtgttacctgaaacaggaaaagcaaca


 


acttcaacacttgaaagctactggaattctttcaaagacagtgtgaataa


 


aggttatacttacattaaacaaagcttagaaagtggttatcaatatttaa


 


aaggtcaatacgactatatcactaaaaaatacaatgatgcgaaatactat


 


acaaaaatgtattcaaatcataagtctacaattgatcagtctgtattagc


 


tatattaggtaaaactggatctagcgcatatatcaagccattaaatatcg


 


aagaaaattcaaacgtattttacaaagcttatgcaaaaacaagaaacttt


 


gctacagaaagcattaacacaggaaaagtattatacacattatatcaaaa


 


ccctactgtagttaaatctgctttcactgcaattgaaacagcaaatacag


 


taaaaaatgcaataagcaatcttttctctctcttcaaataa






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


 


ESHEDRLIELQNKNLMEANEALKALQQEDSIQNRRAAQRAVNKLTPDSAN


 


AFQKELDQINAPRDAKIKADAEAKKQAPEVSAPQIEDAPTTEVAPSPKQD


 


MPKVDKKEEDKVESDTEVKEVPKADTEKNPQSKDTSKTEQAKETPKVEQS


 


PKTEKAEEAPKAETPQNGNKAQTEEAKPEVKDNVKNTPSAPVLPETGKAT


 


TSTLESYWNSFKDSVNKGYTYIKQSLESGYQYLKGQYDYITKKYNDAKYY


 


TKMYSNHKSTIDQSVLAILGKTGSSAYIKPLNIEENSNVFYKAYAKTRNF


 


ATESINTGKVLYTLYQNPTVVKSAFTAIETANTVKNAISNLFSLFK






An active domain from the protein of SEQ ID NO: 6 is designated SEQ ID NO: 37








SEQ ID NO: 37


NEDVTETTGRNSVTTQASEQHLQVEAVPQEGNNVNVSSVKVPTNTATQAQ


 


EDVASVSDVKAHADDALQVQESSHTDGVSSEFKQETAYANPQTAETVKPN


 


SEAVHQSEYEDKQKPVSSSRKEDETMLQQQQVEAKNVVSAEEVSKEENTQ


 


VMQSPQDVEQHVGGKDISNEVVVDRSDIKGFNSETTIRPHQGQGGRLNYQ


 


LKFPSNVKPGDQFTIKLSDNINTHGVSVERTAPRIMAKNTEGATDVIAEG


 


LVLEDGKTIVYTFKDYVNGKQNLTAELSVSYFVSPEKVLTTGTQTFTTMI


 


GNHSTQSNIDVYYDNSHYVDGRISQVNKKEAKFQQIAYINPNGYLNGRGT


 


IAVNGEVVSGTTKDLMQPTVRVYQYKGQGVPPESITIDPNMWEEISINDT


 


MVRKYDGGYSLNLDTSKNQKYAIYYEGAYDAQADTLLYRTYIDSLNSYYP


 


FSYQKMNGVKFYENSASGSGELKPKPPEQPKPEPEIQADVVDIIEDSHVI


 


DIGW






An spsl gene fragment corresponding to A domain is designated SEQ ID NO: 38, which encodes the protein of SEQ ID NO: 37








SEQ ID NO: 38


AATGAAGATGTCACTGAAACAACTGGGAGAAATTCAGTGACAACGCAAGC


 


TTCTGAGCAACATTTGCAAGTGGAAGCAGTACCTCAAGAAGGCAATAATG


 


TAAATGTATCCTCTGTAAAAGTACCTACGAATACGGCAACGCAAGCACAA


 


GAAGATGTTGCAAGTGTATCCGATGTTAAAGCACATGCTGATGATGCATT


 


ACAAGTACAAGAAAGTAGTCATACTGATGGTGTTTCTTCAGAATTCAAGC


 


AGGAGACAGCTTATGCGAATCCTCAAACAGCTGAGACAGTTAAACCTAAT


 


AGTGAAGCAGTGCATCAGTCTGAATACGAGGATAAGCAAAAACCCGTATC


 


ATCTAGCCGCAAAGAAGATGAGACTATGCTTCAGCAGCAACAAGTTGAAG


 


CCAAAAATGTTGTGAGTGCGGAGGAAGTGTCTAAAGAAGAAAATACTCAA


 


GTGATGCAATCCCCTCAAGACGTTGAACAACATGTAGGTGGTAAAGATAT


 


CTCTAATGAGGTTGTAGTGGATAGGAGTGATATCAAAGGATTTAACAGCG


 


AAACTACTATTCGACCTCATCAGGGACAAGGTGGTAGGTTGAATTATCAA


 


TTAAAGTTTCCTAGCAATGTAAAGCCAGGCGATCAGTTTACTATAAAATT


 


ATCTGACAATATCAATACACATGGTGTTTCTGTTGAAAGAACCGCACCGA


 


GAATCATGGCTAAAAATACTGAAGGTGCGACGGATGTAATTGCTGAAGGT


 


CTAGTGTTGGAAGATGGTAAAACCATCGTATATACATTTAAAGACTATGT


 


AAATGGCAAGCAAAATTTGACTGCTGAGTTATCAGTGAGCTATTTCGTAA


 


GTCCGGAAAAAGTCTTGACTACTGGGACACAAACATTCACGACGATGATC


 


GGTAATCATTCAACGCAATCCAATATTGACGTTTATTATGATAATAGTCA


 


TTATGTAGATGGACGTATTTCGCAAGTGAACAAAAAAGAAGCTAAATTTC


 


AACAAATAGCATACATTAACCCTAATGGCTATTTAAATGGCAGGGGGACA


 


ATTGCAGTTAATGGTGAAGTGGTCAGTGGTACGACTAAAGACTTAATGCA


 


ACCTACAGTGCGTGTATATCAATATAAAGGACAAGGTGTTCCTCCTGAAA


 


GTATTACTATAGACCCTAATATGTGGGAAGAAATCAGCATAAACGATACT


 


ATGGTAAGAAAATATGATGGTGGCTATAGCTTGAATCTGGATACCAGCAA


 


GAATCAAAAATATGCCATCTATTATGAAGGGGCATATGATGCGCAAGCTG


 


ACACACTGTTGTATAGAACATATATACAGTCATTAAACAGTTACTATCCG


 


TTCAGTTACCAAAAAATGAACGGTGTGAAGTTTTACGAAAACAGTGCGAG


 


TGGAAGCGGTGAGTTGAAACCGAAACCACCTGAACAACCAAAACCAGAAC


 


CTGAAATTCAAGCTGATGTAGTAGATATTATTGAAGATAGCCATGTGATT


 


GATATAGGATGG






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, 11, 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, 36 and 37 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 sequences, 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 or 37-38. 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, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and/or 38 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 2×SSC(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, 36 and 37. 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 pseudinrermedius 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 Gln for Asn to maintain a free NH2.
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-antigenic; 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, 34, 36 or 37 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 carder(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 711 (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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 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: 1-38).
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 S. 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: 1-38 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: 1-38 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 being 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. DNA 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: 1-38.
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: 1-38 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 microarray 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, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 38). 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, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 38.
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 gene 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. 11, 2001, pages 3-11.
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, 36, or 37 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)/S. 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 S. 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: 1-38 herein.



DETAILED DESCRIPTION
The invention will now be described in more detail with reference to the following Figures which show:
FIG. 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.
FIG. 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).
FIG. 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).
FIG. 4. Adherence of L. lactis expressing specified MSCRAMMs to human Fn. Plates were coated with 1 μg of human Fn 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.
FIG. 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.
FIG. 6. Adherence of L. lactis expressing specified MSCRAMMs to CK10. Plates were coated with 1 μg 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 SH1000 in exponential and stationary phases of growth and PBS were included as controls.
FIG. 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 μm2). Results are based on the arithmetic mean of duplicate experiments. The bottom of each box represents the first quartile (Q1), 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.
FIG. 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 μl 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 μl aliquots of recombinant CIfB and the superantigen SEI from S. aureus were included in the terminal lanes of each gel as negative controls.
FIG. 9: Inhibition of adherence of L. lactis expressing SpsD (A) and SpsL (B) to fibrinogen (2 μg 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 μg per well). Incubations of S. aureus Newman (C) were included as a negative control.



EXAMPLES
Example 1
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, 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. Signal sequences were predicted by employing the SignalP server (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. 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. Repeat sequences were predicted by generating nucleic acid dot plots, using software available from Colorado State University, USA, applying tandem repeats finder software from Boston University, USA, and variable sequence tandem repeats extraction and architecture modelling (XSTREAM), available from the University of California, USA. Sequence alignments and pair-wise sequence comparisons were generated with ClustalW2. Amino acid composition and molecular weight predictions were generated using ProtParam on the ExPASy Proteomics Server.
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 PstI, SalI or BamHI specific restriction sites were inserted on both sides of the DNA fragments. 50 μl PCR reactions contained 2 μl (approximately 100 ng) genomic DNA template, 0.25 μM forward primer, 0.25 μM reverse primer, 1× PFUULTRATM II reaction buffer (Stratagene, USA), 0.25 mM dNTP's (Promega, USA) and 1 μl PFUULTRATM 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 STRATACLONETM Blunt PCR cloning vector pSC-B (Stratagene, USA) using the STRATACLONE ULTRATM Blunt PCR Cloning Kit (Stratagene, USA) according to the manufacturer's instructions. Each cloning reaction consisted of 3 μl Strataclone Buffer Blunt (Stratagene, USA), 2 μl purified PCR product and 1 μl STRATACLONETM Blunt Vector Mix (Stratagene, USA). STRATACLONETM 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 oriColE1 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 PstI, SalI and BamHI (Que et al., 2000).
STRATACLONETM plasmids containing the DNA inserts of interest and the E. coli-L. lactis shuttle vector pOri23 were each digested in a 100 μl total reaction volume containing 10 μl 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 μl total ligation reaction volume, consisting of 1 μl vector (approximately 10 ng), 400 units T4 DNA ligase (New England Biolabs, UK), 1×T4 DNA ligase reaction buffer (New England Biolabs, UK), x μl DNA insert (depending on DNA concentration), and x μl sterile water (depending on the volume of DNA insert). Ligations were incubated at 16° C. for 16 h.
One 50 μl aliquot of electrocompetent L. lactis cells was thawed on ice and 2 μl ( ̃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 μF, 23 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 μl 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 P1. 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 μl 10% (v/v) IGEPAL (Sigma-Aldrich, UK) and incubated for 10 min at 95° C. in a thermocycler machine. 40 μl master mix containing 0.3 μM forward primer, 0.3 μM reverse primer, 0.2 mM dNTP's (Promega, USA), 1× reaction buffer (Promega, USA), 1.5 mM MgCl2 (Promega, USA) and 0.025 u/μl 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 1× 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. 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 cm2 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 (NUNCTM Thermo Fisher Scientific, Denmark) as described by Forsythe et al. (2002). The moisture chambers consisted of 30 cm×30 cm plastic trays with lids and were prepared by lining the trays with moistened paper towels. S. pseudintermedius ED99 stationary or exponential (OD600 of 0.5) phase cultures and L. lactis exponential phase cultures (OD600 0.6 to 0.8) were centrifuged at 4000 rpm for 5 min, washed with PBS and resuspended in PBS to a final OD600 of 0.5. The moisture chambers were placed in a static incubator and 250 μl of bacterial suspension was added to each 1 cm2 of tape, forming a meniscus on the tape. Slides incubated with 250 μl 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 1000× 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 mm2 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 (ROI) of 500 μm2 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. pseudintermedius ED99 Genome
The initial search for putative CWA proteins identified 34 sequences that fulfilled at least one of the search criteria (homology to characterised MSCRAMMs in the database, predicted (SEQ ID NO: 39) 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 FIG. 1. Of note, eight genes encoding putative CWA proteins are located near the oriC environ (FIG. 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, Sps1, and SpsJ (FIG. 4.3).
The Putative CWA Proteins SpsD, SpsL, and SpsO have Several MSCRAMM Features.
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 N1, N2, and N3 subdomains, a tandemly repeated domain at the C-terminus (and at the N-terminus for SpsO), and a C-terminal (SEQ ID NO: 39) LPXTG-anchor motif. The main characteristics of SpsD, SpsL, and SpsO are summarised in Table 2. Of interest, a (SEQ ID NO: 40) 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 SpsL1-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) (FIG. 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 S. 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 (FIG. 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 11, 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. intermedius, and 9 genes were exclusive to S. pseudintermedius. None of the genes encoding putative CWA proteins was detected in the non-SIG staphylococcal species examined. Results are summarised in FIG. 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 S. 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 11 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 115 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 al., 2008). Cell wall-associated protein fractions of the L. lactis constructs and of S. pseudintermedius 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 (FIG. 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 (FIG. 3). In contrast, L. lactis expressing SpsO did not demonstrate seroreactivity with sera from dogs diagnosed with pyoderma (FIG. 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 CK10 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. lactis expressing SpsO demonstrated increased binding to Fn, but also to BSA, indicative of a non-specific interaction (FIG. 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 (FIG. 5). In contrast, L. lactis expressing SpsL adhered to canine and feline Fg only, and did not bind to human and bovine Fg (FIG. 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 (FIG. 5).
The Putative MSCRAMM SpsD Mediates Binding of L. lactis to Cytokeratin 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 (FIG. 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 (FIG. 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) (FIG. 7). L. lactis expressing SpsD and L. lactis expressing SpsO adhered to ex vivo canine corneocytes (FIG. 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 (FIG. 7).
Purified Recombinant Sps D, Sys 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 (FIG. 8). rSpsD, rSpsL and rSpsO all crossreacted with IgG present in the canine serum (FIG. 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. (FIG. 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 (FIG. 9).
Discussion
In summary, genome-wide analysis of S. pseudintermedius ED99 revealed the presence of 17 genes encoding putative CWA proteins based on typical MSCRAMM 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 al., 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 α11bβ3 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.
Example 2
Staphylococcus Pseudintermedius Surface Protein Vaccination Experiment
S. pseudintermedius surface antigens were divided into 2 pools of 3 and 4 antigens, respectively. Vaccine pool 1 contained antigens SpsC, IsaA, and SpsN and vaccine pool 2 contained SpsD A domain, N2,N3 subdomains, SpsL A domain (SEQ ID NO: 37), and SpsA.
Groups of 8 or 9 BalbC mice were vaccinated subcutaneously with pool 1 or pool 2 or PBS, each with complete Freund's adjuvant, followed by additional vaccinations at day 8 and day 23 with incomplete Freund's adjuvant. On day 32, mice were challenged through a subcutaneous route with 107 cfu S. pseudintermedius ED99. Mice were then examined for abscess formation, and weight loss.
Mice vaccinated with pool 2 (comprising the protein having amino acid sequence provided in SEQ ID NO: 37) had significantly reduced lesion size ( ̃50% reduction), and significantly reduced weight loss ( ̃50%) compared to PBS control mock vaccinated animals.
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TABLE 1







Sequence homology of the 17 predicted cell-wall-anchored proteins against known proteins in the public domain.










Putative CWA

Identity
Similarity


protein
Best Hit (BLAST)
(%)
(%)













SpsA
LPXTG cell-wall surface anchor family protein of S. aureus COL
31.2
56.9


SpsB
RodA, a rod shape determining protein of S. epidermidis ATCC 12228
69.7
87.8


SpsC
bifunctional 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 JCSC1435
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


SpsI
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 S. haemolyticus JCSC143S
52.2
61.2


SpsK
IgG-binding protein of S. aureus COL
50.4
71.1


SpsL
Fnbp protein homolog of S. aureus Mu50
33.4
51.7


SpsM
hypothetical protein, similar to the putative cell-surface adhesin SdrF, S. haemolyticus JCSC1435
44.4
61.7


SpsN
probable exported protein of S. aureus RF122
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.























Putative









Ig-like
TYTFTDYVD-
latching
Repeat
Copy



Amino
MW
Signal
LPXTG
foldb
like motif
sequence
region
number



acids
(kDa)a
peptide
motif
(position)
(position)b
(position)b
(position)
repeats



















SpsD
1031
115
36 aa
LPDTG
167-320 aa
RYRFMDYVN
NNASGEG
 867-959 aa
 5







322-519 aa
(267-275 aa)
(491-497 aa)




 


SpsL
930
103
38 aa
LPKTG
220-363 aa
VYTFKDYVN
NSASGSG
 543-818 aa
 7







364-531 aa
(298-306 aa)
(502-508 aa)




 


SpsO
1846
198
44 aa
LPNTG
339-492 aa
TYTFTDYVD
DKSTALG
661-1800 aa
96







487-659 aa
(424-432 aa)
(635-641 aa)
  97-216 aac
 4c





aa = amino acids;


aMW = predicted approximate molecular weight in kDa (kilo dalton);


bwithin the A domain;


cN-terminal repeats

## Claims

1. A vaccine for S. pseudintermedius infections comprising a protein and one or more adjuvant(s); and wherein the protein comprises SEQ ID NO: 37.

2. The vaccine according to claim 1, wherein the vaccine further comprises one or more Sps protein(s) selected from the group consisting of:
(i) a protein comprising SEQ ID NO: 2;
(ii) a protein comprising SEQ ID NO: 4;
(iii) a protein comprising SEQ ID NO: 6;
(iv) a protein comprising SEQ ID NO: 8;
(v) a protein comprising SEQ ID NO: 10;
(vi) a protein comprising SEQ ID NO: 12;
(vii) a protein comprising SEQ ID NO: 14;
(viii) a protein comprising SEQ ID NO: 16;
(ix) a protein comprising SEQ ID NO: 18;
(x) a protein comprising SEQ ID NO: 20;
(xi) a protein comprising SEQ ID NO: 22;
(xii) a protein comprising SEQ ID NO: 24;
(xiii) a protein comprising SEQ ID NO: 26;
(xiv) a protein comprising SEQ ID NO: 28;
(xv) a protein comprising SEQ ID NO: 30;
(xvi) a protein comprising SEQ ID NO: 32;
(xvii) a protein comprising SEQ ID NO: 34; and
(xviii) a protein comprising SEQ ID NO: 36.

3. The vaccine according to claim 1, wherein the protein is encapsulated in a biodegradable polymer.