專利名稱：奈瑟球菌蛋白質(zhì)的雜交表達的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及蛋白質(zhì)表達的領(lǐng)域。具體說，本發(fā)明涉及奈瑟球菌(如淋病奈瑟球菌或較佳地為腦膜炎奈瑟球菌)的蛋白質(zhì)的異源表達。
背景技術(shù)：
國際專利申請WO 99/24578、WO99/36544、WO99/57280和WO 00/22430公開了腦膜炎奈瑟球菌(Neisseria meningitidis)和淋病奈瑟球菌(Neisseriagonorrhoeae)的蛋白質(zhì)。這些蛋白質(zhì)一般是以N-未端GST-融合體或C-未端His-標記融合體在大腸桿菌中表達的(即異源表達)，雖然也公開了其它表達系統(tǒng)(包括在天然的奈瑟球菌中的表達)。
本發(fā)明的目的是提供這些蛋白質(zhì)的異源表達的其它或改進方法。這些方法通常影響表達的水平、純化的簡易程度、表達的細胞內(nèi)定位和/或表達的蛋白質(zhì)的免疫學(xué)特性。
發(fā)明的公開在本發(fā)明中，以單雜交蛋白表達本發(fā)明的兩種或多種(如3、4、5、6或更多)蛋白質(zhì)。較佳地，不使用非奈瑟球菌的融合配體(如GST或聚-His)。
這有兩個優(yōu)點。其一，可能不穩(wěn)定或本身表達很差的蛋白質(zhì)可以通過加入合適的克服該問題的雜交配體予以幫助。其二，簡化工業(yè)生產(chǎn)，即制備兩種分別有用的蛋白質(zhì)只需要一次表達和純化。
因此，本發(fā)明提供了同時異源表達本發(fā)明的兩種或多種蛋白質(zhì)的方法，其中所述的本發(fā)明的兩種或多種蛋白質(zhì)是融合的(即，它們是作為單多肽鏈翻譯的)。
該方法通常包括如下步驟獲得編碼本發(fā)明第一種蛋白質(zhì)的第一核酸；獲得編碼本發(fā)明第二種蛋白質(zhì)的第二核酸；連接第一和第二核酸。將得到的核酸插入表達載體中，或已作為表達載體的一部分。
當(dāng)僅連接兩種蛋白質(zhì)時，可以式NH2-A-B-COOH簡單地表示雜交蛋白。A和B各選自任何奈瑟球菌蛋白質(zhì)，尤其是那些以SEQ#1-4326表示的。該方法非常適合表達蛋白質(zhì)orf1、orf4、orf25、orf40、Orf46/46.1、orf83、233、287、292L、564、687、741、907、919、953、961和983。
由式NH2-A-B-COOH在下表中以‘X′表示的42個雜交體是優(yōu)選的

因此，優(yōu)選的以雜交體表達的蛋白質(zhì)是ORF46.1,287,741,919,953,961和983。它們可以全長形式或多-甘氨酸缺失(△G)形式使用(如△G-287、△GTbp2、△G741、△G983等)、或以截短形式使用(如△1-287、△2-287等)，或以結(jié)構(gòu)域缺失的形式使用(如287B、287C、287BC、ORF461-433、ORF46433-608、ORF46、961c等)等。
特別優(yōu)選的是(a)含919和287的雜交蛋白；(b)含953和287的雜交蛋白；(c)含287和ORF46.1的雜交蛋白；(d)含ORF1和ORF46.1的雜交蛋白；(e)含919和ORF46.1的雜交蛋白；(f)含ORF46.1和919的雜交蛋白；(g)含ORF46.1、287和919的雜交蛋白；(h)含919和519的雜交蛋白；和(i)含ORF97和225的雜交蛋白。
附圖中顯示了其它優(yōu)選例，它們包括△G287-919、△G287-953、△G287-961、△G983-ORF46.1、△G983-741、△G983-961、△G983-961C、△G741-961、△G741-961C、△G741-983、△G741-ORF46.1、ORF46.1-741、ORF46.1-961、ORF46.1-961C、961-ORF46.1、961-741、961-983、961C-ORF46.1、961C-741、961C-983、961CL-ORF46.1、961CL-741和961CL-983。
當(dāng)使用287時，其優(yōu)先位于雜交體的C-未端；如果在N-未端使用它，則優(yōu)先使用287的△G形式(如與ORF46.1、919、953或961雜交的雜交體的N-未端)。
當(dāng)使用287時，其優(yōu)先為菌株2996或菌株394/98的。
當(dāng)使用961時，其優(yōu)先在N-未端?？墒褂?61的結(jié)構(gòu)域形式。
WO 99/66741公開了ORF46、287、919和953的多態(tài)形式的排序。本發(fā)明可以使用這些多態(tài)形式中的任一形式。
較佳地，本發(fā)明雜交蛋白中的組成蛋白質(zhì)(A和B)來源于是同一菌株。
雜交體中的融合蛋白可以是直接相連的，或者是通過接頭肽相連的，如通過聚-甘氨酸接頭(即，Gn，其中n＝3、4、5、6、7、8、9、10或更多)或通過協(xié)助克隆的短肽序列相連。顯然不宜將△G蛋白質(zhì)連接于聚-甘氨酸接頭的C-未端。
融合蛋白可以缺失天然前導(dǎo)肽或可以包含N-未端融合配體的前導(dǎo)肽序列。
宿主較佳地，利用異源宿主。異源宿主可以是原核的或真核的。優(yōu)先為大腸桿菌，但其它合適的宿主包括枯草芽孢桿菌(Bacillus sudtilis)、霍亂弧菌(Vibriocholerae)、傷寒沙門氏菌(Salmonella typhi)、鼠傷寒沙門氏菌(salmonellatyphimurium)、腦膜炎奈瑟球菌、淋病奈瑟球菌、乳糖奈瑟球菌(Neisserialactamica)、灰色奈瑟球菌(Neisseria cinerea)、分枝桿菌(如結(jié)核分枝桿菌(M.tuberculosis)、酵母等。
載體、宿主等如上所述的方法，本發(fā)明提供了(a)用于這些方法的核酸和載體；(b)含所述載體的宿主細胞；(c)可用這些方法表達的或可表達的蛋白質(zhì)；(d)包含這些蛋白質(zhì)的組合物，其可能適合作為疫苗、或例如診斷劑或免疫原性的組合物；(e)用作藥物(如疫苗)或診斷劑的組合物；(f)這些組合物在制備以下物質(zhì)中的用途(1)用于治療或預(yù)防奈瑟球菌引起的感染的藥物(2)檢測奈瑟球菌或由奈瑟球菌引起的抗體存在與否的診斷劑，和/或(3)能產(chǎn)生抗奈瑟球菌抗體的藥物；和(g)治療患者的方法，其包括對該患者施用治療有效量的這些組合物。
序列本發(fā)明還提供了具有以下實施例中所列出的任何的蛋白質(zhì)或核酸。本發(fā)明還提供了具有與這些序列是序列相同性的蛋白質(zhì)和核酸。如上所述，“序列相同性”的程度最好大于50％(如60％、70％、80％、90％、95％、99％或更大)。
本文的命名本文參考在WO 99/24578、WO 99/36544和WO 99/57280中公開的2166個蛋白質(zhì)序列，并將它們編號為如下的SEQ#

除了這種SEQ#編號外，本文還使用了WO 99/24578、WO 99/36544和WO99/27280中的命名約定(如WO99/24578和WO 99/36544中用的‘ORF4′、‘ORF40′、‘OFR40-1′等；WO 99/57280中用的‘m919′、‘g919′和‘a(chǎn)919′等)。
在本文中將Tettelin等[Science(2000)2871809-1815]中的從NMB0001到NMB2160的2160個蛋白質(zhì)稱為SEQ#2167-4326[參見WO 00/66791]。
本文采用的術(shù)語“本發(fā)明的蛋白質(zhì)”指包含以下的蛋白質(zhì)(a)SEQ#1-4326中的一個序列；或(b)與SEQ#1-4326中的一個序列相同的序列；或(c)SEQ#1-4326中的一個序列的片段。
(b)中的“序列相同性”的程度最好大于50％(如60％、70％、80％、90％、95％、99％或更大)。其包括突變體和等位基因變體[如，參見WO 00/66741]。相同性最好用Smith-Waterman同源性搜尋算法確定，如在MPSRCH程序(Oxford Molecular)中執(zhí)行的，采用參數(shù)“缺口罰分(gap open penalty)”為12，“缺口延伸罰分(gap extension penalty)”為1進行缺口仿射搜索。通常，將兩種蛋白質(zhì)之間50％或更高的相同性視為功能等效的指示。
(c)中的“片段”應(yīng)包含SEQ#1-4326中一個序列的至少n個連續(xù)的氨基酸，且根據(jù)具體的序列n為7或更高(如8、10、12、14、16、18、20、25、30、35、40、50、60、70、80、90、100或更高)。較佳地，片段包含SEQ#1-4326中一個序列的表位。優(yōu)選的片段是在WO 00/71574和WO 01/04316中公開的那些。
本發(fā)明優(yōu)選的蛋白質(zhì)是在腦膜炎奈瑟球菌血清群B中發(fā)現(xiàn)的。
根據(jù)本發(fā)明使用的優(yōu)選蛋白質(zhì)是血清群B腦膜炎奈瑟球菌菌株2996或菌株394/98(新西蘭菌株)。除非特別指出，本文所述的蛋白質(zhì)是腦膜炎奈瑟球菌菌株2996的蛋白質(zhì)。但是，應(yīng)該理解通常本發(fā)明并不受菌株的限制。參考具體的蛋白質(zhì)(如‘287′、‘919′等)可以包括任何菌株的該蛋白質(zhì)。
應(yīng)該理解術(shù)語“核酸”包括DNA和RNA，以及它們的類似物，如含有修飾骨架的那些類似物，還包括肽核酸(PNA)等。
附圖簡述

圖1-26顯示本發(fā)明的雜交蛋白。
本發(fā)明的進行模式實施例1-ORF46的雜交體腦膜炎奈瑟球菌(血清群B，菌株2996)的完整ORF46蛋白質(zhì)具有如下序列1LGISRKISLILSILAVCLPMHAHASDLAND SFIRQVLDRQ HFEPDGKYHL51 FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE101 VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY151 PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV201 GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG251 ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP301 NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI TSSTVPPSNG401 KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAKP451 VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS QHAQLEREIN501 KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PVVEYVEING551 KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV601 KRRYRSK*在前導(dǎo)肽下加下劃線。
可以在WO 00/66741中發(fā)現(xiàn)其它菌株的ORF46的序列。
ORF46在其C-未端和N-未端與287、919和ORF1融合。該雜交蛋白通常是不溶解的，但產(chǎn)生一些良好的ELISA和殺菌結(jié)果(針對同源2996菌株)

為了比較，構(gòu)建了ORF46.1、287(以GST融合體或△G287的形式)和919的“三”雜交體，并針對各種菌株(包括同源2996菌株)將其與三種抗原的簡單混合物相比。FCA用作佐劑

同樣，這些雜交體顯示相當(dāng)?shù)鹊幕蚋训拿庖呋钚浴?br> 針對各種異源菌株，將兩種蛋白質(zhì)(菌株2996)的雜交體與單種蛋白質(zhì)相比

再次，這些雜交體顯示相等的或更佳的免疫活性。
實施例2-△G287的雜交體發(fā)現(xiàn)287中(Gly)6序列的缺失對蛋白質(zhì)的表達有顯著影響。將缺失N-未端氨基酸多達GGGGGG的蛋白質(zhì)稱為‘△G287′。在菌株MC58中，它的基本序列(前導(dǎo)肽有下劃線)為
SPDVKS ADTLSKPAAP VVSEKETEAK EDAPQAGSQG QGAPSAQGSQ DMAAVSEENTGNGGAVTADN PKNEDEVAQN DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQPANQPDMANAA DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGSNFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS DADKISNYKKDGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS ARSRRSLPAE MPLIPVNOADTLIVDGEAVS LTGHSGNIFA PEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVYNGEVLHFHTE NGRPYPTRGR FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKGTWTENGSGDV SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*與‘287-His’或‘287未標記的’相比，有或無His-標記的△G287(分別為‘△G287-His’和‘△G287K’)以很好的水平表達。
在基因變異性數(shù)據(jù)的基礎(chǔ)上，從許多MenB菌株(尤其是從菌株2996、MC58、100和Bz232)的大腸桿菌中表達△G287-His的變體。結(jié)果也好-它們都具有很高的ELISA滴定度，且血清殺菌滴定度＞8192。由pET-24b表達的△G287K在ELISA和血清殺菌實驗中有極佳滴定度。
還將聚-Gly序列的缺失應(yīng)用于Tbp2(NMB0460)、741(NMB1870)和983(NMB1969)。在不編碼其前導(dǎo)肽的序列且沒有聚-Gly(即，以“△G形式”)，在pET載體中克隆并在大腸桿菌中表達時，觀察到相同的作用-在攜帶聚-甘氨酸段缺失的克隆中表達很好，若在表達的蛋白中存在甘氨酸時則表達差或不表達。
將△G287直接融合于919、953、961(如下所示的序列)和ORP46.1的符合讀框的上游AG287-919ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAQAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCQATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGOCGGAAAAAATGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCQACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACGCCAAGGCTGGCAGGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCCTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAACTCGAG1 MASPDVKSAD TLSKPAAPVV AEKETEVKEL APQAGSQGQG APSTQGSQDM51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL151 LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI201 IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG401 KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA451 VYTVVPHLSL PHWAAQDFAK SLQSFRLGCA NLKNRQGWQD VCAQAFQTPV501 HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI551 YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP601 ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM651 HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG701 IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG751 AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFWG801 YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*AG287-953ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACCAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCCATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACTAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGAAGCGGTCAGCCTTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCAAACTCAAAGCCCAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCCTGGACTACCTCGTTAACCTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAACTCGAG1MASPDVRSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM51AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS101DSAPASNPAP ANGGSNFGRV DLANDVLIDG PSQNITLTHC KGDSCNGDNL151LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI
201IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG251HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG401KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ451AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF501NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID551RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*△G287-961ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAACACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAACGCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAACTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAACTCGAG1MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM51AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS101DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL151LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI201IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG251HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE301VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI351DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG401KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE451DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE501SEIEKLTTKL ADTDAALADT DAALDATTNA LNKIGENITT FAEETKTNIV551KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTEADEAVKT ANEAKQTAEE601TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKARAVAAKV TDIKADIATN651KDNIAKKANS ADVYTREESD SKFVRIKGLN ATTEKLDTTL ASAEKSIADH701DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK751SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*

對919和ORF46.1而言，將針對雜交蛋白產(chǎn)生的抗體的殺菌效力(同源菌株)與針對組分抗原(用287-GST)的簡單混合物產(chǎn)生的抗體相比

還獲得了針對異源MenB菌株和針對血清型A和C的殺菌活性的數(shù)據(jù)

因此，在N-未端與△G287的雜交蛋白在免疫學(xué)上優(yōu)于與△G287-ORF46.1的簡單混合物，即使針對異源菌株也特別有效。可以在pET-24b中表達△G287-ORF46.1。
用新西蘭菌株394/98，而非2996，制備相同的雜交蛋白△G287NZ-919ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGCTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAAAAGCTT1MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM51AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG151TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV251GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ301ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV501GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR551QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV601LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI651DNTGGTHTAD LSRFPITART TAIKGRFEGS RFLPYHTRNQ INGGALDGKA701PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM751ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG801PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT851GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP*△G287NZ-953ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGGCCGCAAAATGCCGCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAAAAGCTTT1MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM
51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201 THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV251 GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ301 ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351 SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401 GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451 RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN501 VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA551 AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA EKFNCYQSPM601 AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*△G287NZ-961ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGATGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAAAAGCTT1MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM51AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM101PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGTTAAQG151TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL201THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV251GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ301ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP351SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS401GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY451RPTDAEJGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE501INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV551NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG601ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD651EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA701VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK751LDTRLASAEK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY801NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY851HVGVNYEW*實施例3-△G983的雜交體蛋白質(zhì)983具有如下序列983AG9831MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS51NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN101LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE151LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT201DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM251MVAAIRNAWV NNSFGTTSRA GTADLFQIAN GTADLFQIAN SEEQYRQALL301DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN DAQAQPNTYA351LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA401MWCLSAPYEA SVRFTKTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN451LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT501SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN551KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ601LDGKGTLYTR LGKLLKVDGT ARGKGAGYLN ARGKGAGYLN STGRRVPFLS651AAKIGQDYSF FTNIETDGGL AGSEGDTLSY AGSEGDTLSY YVRRGNAART701ASAAAHSAPA GLKHAVEQGG DASESSATPE DASESSATPE TVETAAADRT751DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STGRRVPFLS801RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW AQTQQDGGTW GSTQTVGIAA851KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL901FSYGRYKNSI SRSTGADEHA LGALGGVNVP LGALGGVNVP FAATGDLTVE951GGLRYDLLKQ DAFAEKGSAL TLVGLAGLKL TLVGLAGLKL SQPLSDKAVL1001FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVEI051FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*因此△G983具有如下基本序列將△G983作為在其C．未端與ORF46.1、741、961或961c的雜交體表達△G983-ORF46.1ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAAACAACAGCGAAATCAGAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGCCCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGACAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351 GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKEGGSQ LQLHGNNTYT501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG
801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD1051GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG1101KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA1151GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DIYSYDIKGV1201AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS1251GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS1301TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI1351PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK1401YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF1451DGKGFPNFEK HVKYDTLEHH HHHH*△G983-741ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAACGTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAACTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE1051GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ1101GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ1151SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT1201YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD1251GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL1301AAKQLEHHHH HH*△G983-961ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAAGGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACCAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGCCATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACCTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCCATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCCTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACGTGACCAAAACCGTCAATGAAAACAAACAAAAGCTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTGCGTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGATGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKSMLF301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351 GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801 VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSLQYGNHSG RVGVGYRFLE1051 GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI1101 TKKDATAADV EADDFKGLGL KKVVTNLTKT ANENKQNVDA KVKAAESEIE1151 KLTTKLADTD AALADTDAAL DATTNALNKL GANITTFAEE TATNIVKIDE1201 KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN1251 VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI1301 AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL1351 NGLDKTVSDL RKETRQGLAE QAALSGLFQP UMVGRFMVTA AVGGYKSESA1401 VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*△G983-961cATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCCTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCHGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP351 GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG801 VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR1001 NMPHTKLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE1051 GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI1101 TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE1151 KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE
1201KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN1251VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI1301AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL1351NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH*實施例4-△G741的雜交體蛋白質(zhì)741具有如下序列1VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL51 QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ101 IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI151 GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI201 EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA251 QEVAGSAEVK TVNGIRHIGL AAKQ*因此，△G741具有如下基本序列VAAD IGAGLADALT APLDHKDKGLQSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQIEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRIGDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKIEHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKAQEVAGSAEVK TVNGIRHIGL AAKQ*將△G741直接融合于蛋白質(zhì)961、961C、983和ORF46.1的符合讀框的上游△G741-961ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL251 EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT301 ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI351 EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID401 EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ
451 NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN501 IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR551 LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES601 AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYHVGVNYEW LEHHHHHH*△G741-961cATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTCAAGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL251 EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT301 ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI351 EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID401 EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ451 NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN501 IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR551 LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH*△G741-983ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGATCCGGCGGAGGCGGCAGTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACCACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTCAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAELT51YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL101TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA151FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA201VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL251EGSGGGGTSA PDFNAGGTGI GSNSRATTAK SAAVSYAGIK NEMCKDRSML301CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT351GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG401GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA451GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS501RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR551NKNMLFIFST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR601EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSFS651APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA701GKAMNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSQLQLH751GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI801VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY851MSARGKGAGY LNSTGRRVPF LSAAKIGQDY SFFTNIETDG GLLASLDSVE901KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLMV951ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR1001IFNSLAATVY ADSTAAHADM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG1051TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT1101DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL1151MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT1201EGTLVGLAGL KLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT1251GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV1301GYRFLEHHHH HH*△G741-ORF46.1ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAACTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGACCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT51YGNGDSLATT KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL101TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA151FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRLA201VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL251DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL301GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE351AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG401VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR451SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN LAVMHGLGLL501STENKMARIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA551IPIKGIGAVR GKYGLGGITA HPIKRSQMGA IALPKGKSAV SDNFADAAYA601KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGKNVKLA DQRHPKTGVP651FDGKGFPNFE KHVKYDTLEH HHHHH*實施例5-287的雜交體以全長、具有C-未端His-標記或無其前導(dǎo)肽但具有C-未端His-標記的287的表達水平非常低。用N-未端GST-融合實現(xiàn)了較好的表達。將GST用作N-未端融合配體的替代方法，將287置于蛋白質(zhì)919的C-未端(‘919-287′)、蛋白質(zhì)953的C-端(‘953-287′)和蛋白質(zhì)ORF46.1的C-未端(‘ORF46.1-287′)。在這兩種方法中，前導(dǎo)肽都是缺失的，且雜交體是直接的符合讀杠的融合。
為了制備953-287雜交體，通過設(shè)計正向引物自各序列前導(dǎo)區(qū)的下游，除去兩種蛋白質(zhì)的前導(dǎo)肽；在953反向引物中除去終止密碼子序列，但在287反向引物中包含終止密碼子序列。對953基因而言，用于擴增的5′和3′引物分別包括NdeI和BamHI限制酶切位點，而對287基因的擴增而言，5′和3′引物分別包括BamHI和XhoI限制酶切位點。用這種方法，用NdeI-BamHI(克隆第一個基因)且隨后用BamHI-XhoI(克隆第二個基因)，可以實現(xiàn)pET21b+中兩個基因的順序定向克隆。
通過將編碼287的成熟部分的序列克隆入pET21b+中的919-His克隆的3′-端的XhoI位點，可以得到919-287雜交體。設(shè)計用于擴增287基因的引物，從而在PCR片段的5′-引入SalI限制酶切位點并在3引入XhoI位點。因為由SalI和XhoI限制酶產(chǎn)生的粘性末端是相容的，因此可以將由SalI-XhoI消化的287PCR產(chǎn)物插入到由XhoI切割的pET21b-919克隆中。
類似地得到ORF46.1-287雜交體。
將針對雜交蛋白產(chǎn)生的抗體的殺菌效力(同源菌株)與針對組分抗原的簡單混合物產(chǎn)生的抗體相比

對919-287和953-287而言，還獲得了針對異源MenB菌株和針對血清型A和C的殺菌活性的數(shù)據(jù)

還構(gòu)建了ORF46.1和919的雜交體。在N-未端用919得到了最佳結(jié)果(高4倍的滴定度)。
還測試了雜交體919-519His、ORF97-225His和225-ORF97His。它們的ELISA滴定度和殺菌抗體應(yīng)答結(jié)果中等。
兩種蛋白質(zhì)A和B的雜交體可以是NH2-A-B-COOH或NH2-B-A-COOH，用△G287還制得了在N-未端與287“反向”雜交的雜交體。使用一系列菌株，包括同源菌株2996。FCA用作佐劑

通常在N-未端用287觀察到較好的殺菌滴定度。
當(dāng)融合于蛋白質(zhì)961[如上所示的NH2-△G287-961-COOH-序列]時，得到的蛋白質(zhì)是不溶解的，純化必須將其變性和復(fù)性。在復(fù)性后，發(fā)現(xiàn)約50％蛋白質(zhì)仍不溶解。比較可溶和不溶蛋白質(zhì)，用可溶蛋白質(zhì)得到更好的殺菌滴定度(FCA作為佐劑)

還在雜交蛋白中使用961c(見上)。由于961及其結(jié)構(gòu)域變體指導(dǎo)有效的表達，它們非常適合作為雜交蛋白的N-未端部分。
實施例23-其它雜交體附圖中顯示本發(fā)明的其它雜交蛋白，它們具有如下所示的序列。當(dāng)與單獨蛋白質(zhì)相比時，這此雜交蛋白是有利的ORF46.1-741ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTAATTGTCCGCCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAACGCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTCAGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD LKGVAQNIRL151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP401 NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR451 KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL501 ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF551 DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV601 DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK651 TVNGIRHIGL AAKQLEHHHH HH*ORF46.1-961ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA
1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP401 NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI451 YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK501 VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET551 KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EATKTANEAK601 QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA651 DIVTNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LKTRLASAEK701 SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGRFNVTAA751 VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE801 HHHHHH*ORF46.1-961cATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCCACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCACTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP401 NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI451 YDIDEDGTIT KKDATAADVE ADDFFGLGLK KVVTNLTKTV NENKQNVDAK501 VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET551 KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK601 QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA651 DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK701 SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH751 H*961-ORF46.1ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ401 VLDRQHFEPD GKYHLFGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK451 GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE501 HHPADGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL551 ADRFHNAGSM LTQGVGDGFK RATRYSPELD RSGNAAEAFN GTADIVKNII601 GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK651 DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT701 AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY751 GKENITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE801 HHHHHH*961-741ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SELEKLTTKL101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD401 ALTAPLDHKD KGLQSLTLDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL451 KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE501 HSGKMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY551 TIDFAAKQGN GKIEHLKSPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE601 KGSYSLGIFG GKAQEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH*961-983ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGQCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAAGTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGGGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKTEAVA151 DTVDKHAEAF NDIADSLDET NTKADGAVKT ANEAKQTAEE TKQNVDAKVK201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIALLANS251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT401 GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA~51 PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI501 SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE551 AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMNTNDET601 KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR651 QALLDYSGGD KTDEGIRLMQ QSDYGNLSYH IRNKNMLFIF STGNDAQAQP701 NTYALLPFYE KDAQKGIITV AGVDRSGEKF KREMYGEPGT EPLEYGSNHC751 GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM801 SNDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD851 TKGTSDIAYS FRNDISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL901 YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK951 GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA GYLNSTGRRV1001 PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN1051 AARTASAAAH SAPAGLKHAV EQGGSNLENL MVELDASESS ATPETVETAA1101 ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA1151 DMQGRRLKAV SDGLDHNGTG LRVIAQTQQD GGTWEQGGVE GKMRGSTQTV1201 GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY1251 LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD1301 LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTVLGLA GLKLSQPLSD1351 KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG1401 ADVEFGNGWN GLARYSYAGS KQYGAHSGRV GVGYRFLEHH HHHH*961C-ORF46.1ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCCGTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTCAAGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGSKLAND SFIRQVLDRQ351HFEPDGKYHL FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY401IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRLH WDGYEHHPAD451GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH501NAGSMLTQGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE551IVGAGDAVQG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA601AIRDWAVQNP NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK651RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI701TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH751H*961C-741ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDTDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT301VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP351LDHKDKGLQS LTLDQSVRKN EKLKLAAQGA EKTYGNGDSL NTGKLKNDKV401SRFDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ IQDSEHSGKM
451VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTLDFA501AKQGNGKIEH LKSPELNVDL AAADIKPDGK RHAVISGSVL YNQAEKGSYS551LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH*961c-983ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAACACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTGGGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGACGAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAAGGAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAAGAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA1MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT51AADVEADDFK GLGLKKVVTN LTKTVNEVKQ NVDAKVKAAE SEIEKLTTKL101ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA151DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK201AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS251ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
301 VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN351 SRATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL401 HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL451 YGRKEHGYNE NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD501 IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETANEMM551 VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD601 YSGGDKTDEG IRLMQQSDYG NLSYHIRNKN MLFIFSTGND AQAQPNTYAL651 LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNHCGITAM701 WCLSAPYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL751 RTTLLTTAQD IGAVGVDSKF GWGLLDAGKA MNGPASFPFG DFTADTKGTS801 DIAYSFRNDI SGTGGLIKKG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK851 SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL901 DGKGTLYTRL IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA TGRRVPFLSA951 AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA1001 SAAAHSAPAG LKHAVEQGGS NLENLMVELD ADESSATPET VETAAADRTD1051 MPGIRPYGAT FRAAAAVQHA NAADGVRIFN SLAATVYADS TAAHADMQGR1101 RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGKMRG STQTVGIAAK1151 TGKNTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGULKGLF1201 SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG1251 GLRYDLLKQD AFAEKGSALG WSGNSLTELT LVGLAGLKLS QPLSDKAVLF1301 ATAGVERDLN GRDYTVTGGF TGATAATGKT GARBNOGTRK VAGLGADVEFF1351 GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH*961CL-ORF46.1ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAACAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGTAACTCGAG1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101 KQNVDAKVKA AESEIEKLTT DTDAALDATT DTDAALDATT NALNKLGENI151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSDFVRIDG LNATTEKLDT301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG351 GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFGSRGELA ERSGHIGLGK401 IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDEAG451 SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIKGVA501 QNIRLNLTDN RSTGQRLADR FHNAGAMLTQ GVGDGFKRAT RYSPELDRSG
551 NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VMHGLGLLST601 ENKMARINDL ADMAQLKDYA AAAIRDWAVQ NPNAAQGIEA VSNIFMAAIP651 IKGIGAVRGK YGLGGITAHP IKRSQMGAIA LPKGKSAVSD NFADAAYAKY701 PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGKNVKLADQ RHPKTGVPFD751 GKGFPNFEKH VKYDT*961cL-741ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACGCTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG351 GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ401 GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGGFQVYKQ451 SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT501 YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD551 GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL601 AAKQLEHHHH HH*961cL-983ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAACCTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCTGACTCGAG1MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNNGQEING51FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN101KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI151TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV201KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA251KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT301RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG351GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC401AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG451RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG501GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG551GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR601AGTADLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNLSYHIRN651KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRE701MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFTRTNP IQIAGTSFSA751PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFKWGLLDAG801KAMNGPASFP FGDFTADTKG TSDIAYSFRN DISGTGGLIK KGGSQLQLHG851NNTYTGKTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV901YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLYM951SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK1001TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE1051LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI1101FNSLAATVYA DSTAAHADMQ GRRLEAVSDG LDHNGTGLRV IAQTQQDGGT1151WEQGGVEGKM RGSTQTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD1201SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM1251QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE1301GTLVGLAGLK LSQPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG1351KTGARNMPHT RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG1401YRF*
可以理解本發(fā)明僅以實施例的方式進行描述，在本發(fā)明的范圍和精神內(nèi)還可進行改變。例如，設(shè)想可以使用其它菌株的蛋白質(zhì)[如，參見WO 00/66741，ORF4、ORF40、ORF46、225、235、287、519、726、919和953的多態(tài)序列]。
實驗詳述克隆策略和寡核苷酸設(shè)計用以腦膜炎奈瑟球菌BMC58的基因組序列為基礎(chǔ)設(shè)計的寡核苷酸，通過PCR擴增編碼感興趣的抗原的基因。除非特別指出，通常將菌株2996的基因組DNA用作PCR反應(yīng)的模板，將擴增的片段克隆入表達載體pET21b+(Novagen)，從而以C-未端His標記的產(chǎn)物形式表達該蛋白，或?qū)⑵淇寺∪雙ET-24b+(Novagen)以‘未標記的′形式(如△G287K)表達該蛋白。
不用融合配體和用其自身前導(dǎo)肽(如果存在時)表達蛋白質(zhì)，進行開放讀框(ATG到終止密碼子)的擴增。
當(dāng)?shù)鞍踪|(zhì)以‘未標記的′的形式表達時，通過從預(yù)定的前導(dǎo)序列設(shè)計5′-端擴增引物下游除去前導(dǎo)肽。
用于PCR的引物的解鏈溫度取決于整個引物中雜交核苷酸的數(shù)量和類型，并用以下公式確定Tm1＝4(G+C)+2(A+T) (除去尾部的)Tm2＝64.9+0.41(％GC)-600/N (完整的引物)對整個低聚物而言，所選寡核苷酸的解鏈溫度通常為65-70℃，僅針對雜交區(qū)域，解鏈溫度為50-60℃。
用Perkin Elmer 394 DNA/RNA合成儀合成寡核苷酸，將其從柱上洗脫到2.0ml NH4OH中，在56℃培育5小時以去保護。加入0.3M乙酸鈉和2體積的乙醇沉淀寡聚物。將樣品離心，將沉淀物重懸浮于水中。



*將該引物用作反向引物，將所有287的C未端融合于His-標記。
§與287-His反向引物聯(lián)用的正向引物。
NB-所有PCR反應(yīng)使用菌株2996，除非特別指出(如菌株MC58)。
在所有以ATG起始不跟隨唯一NheI位點的構(gòu)建物中，ATG密碼子是用于克隆的NdeI位點的一部分。在5′端用NheI作為克隆位點制備的構(gòu)建物(如所有那些在N-未端包含287的)另有兩個融合于抗原的編碼序列的密碼子(GCTAGC)。
染色體DNA模板的制備在100ml GC培養(yǎng)基中使腦膜炎奈瑟球菌前株2996、MC58、394.98、1000和BZ232(及其它)生長至指數(shù)期，離心收獲，并重懸浮于5ml緩沖液(20％w/V蔗糖、50mM Tris-HCl、50mM EDTA、pH8)中。在冰上培育10分鐘后，加入10ml裂解液(50mM NaCl、1％十二烷基肌氨酸鈉(Na-Sarkosyl)、50μg/ml蛋白酶K)裂解細菌，懸浮液在37℃培育2小時。進行2次苯酚提取(平衡至pH8)和一次CHCl3/異戊醇(24∶1)提取。加入0.3M乙酸鈉和2體積乙醇沉降DNA，并離心收集。用70％(v/v)乙醇洗滌沉淀物1次，并重溶解于4.0ml TE緩沖液(10mM Tris-HCl、1mM EDTA、pH8.O)。讀取OD260測定DNA濃度。
PCR擴增標準的PCR過程進行如下在40μM各寡核苷酸引物、400-800μM dNTP溶液、1x PCR緩沖液(包含1.5mM MgCl2)、2.5單位TaqI DNA聚合酶(用Perkin-Elmer AmpliTaQ，Boerhingher Mannheim ExpandTM長模板)存在時，將200ng 2996、MC581000、或BZ232菌株的基因組DNA或10ng重組克隆的質(zhì)粒DNA制備物用作模板。
將整個混合物在95℃初步培育3分鐘后，每份樣品進行2-步的擴增用除去引物(Tml)的限制酶尾部的雜交溫度進行前5輪。然后按全長低聚物(Tm2)計算的雜交溫度進行30輪。根據(jù)要擴增的Orf的長度，在68℃或72℃進行的延伸時間各不相同。對Orfl而言，自3分鐘開始的延伸時間每輪遞增15秒。以在72℃10分鐘延伸步驟完成循環(huán)。
將擴增的DNA直接加到1％瓊脂糖凝膠上。按制造商的說明，用Qiagen凝膠提取試劑盒純化相應(yīng)于正確大小條帶的DNA片段。
PCR片段和克隆載體的消化用適合的限制酶消化相應(yīng)于擴增片段的純化DNA，從而克隆入pET-2lb+、pET22b+、或pET-24b+。用QIAquick PCR純化試劑盒(按制造商的說明)純化消化的片段，用H2O或10mM Tris(pH8.5)洗脫。用適合的限制酶消化質(zhì)粒載體，加到1.O％瓊脂糖凝膠上，用Qiagen AIQquick凝膠提取試劑盒純化相應(yīng)于消化的載體的條帶。
克隆將預(yù)先消化和純化的、相應(yīng)于各基因的片段連接到pET2lb+、pET22b+或pET-24b+中。將在連接緩沖液(由制造商提供)中的T4 DNA連接酶用于摩爾比為3∶1的片段/載體。
通過在冰上培育連接酶反應(yīng)溶液和細菌40分鐘，然后在37℃培育3分鐘，將重組質(zhì)粒轉(zhuǎn)化入感受態(tài)大腸桿菌DH5或HBl01中。
然后添加800μl LB肉湯，并在37℃培育20分鐘。在Eppendorf微型離心機中以最高速度離心這些細胞，并重懸浮于約200μl的上清液中，并涂布在LB氨芐青霉素(100mg/ml)瓊脂上。
在4.0ml LB肉湯+100μg/ml氨芐青霉素中培育隨機選擇的集落過夜，進行重組集落的篩選。使細胞沉淀，并按制造商的說明用Qiagen QIAprep SpinMiniprep試劑盒提取質(zhì)粒DNA。用適合的限制酶消化約1μg的各微型制備物，將消化物加到1-1.5％瓊脂糖凝膠(取決于預(yù)計的插入大小)與分子量標記(1kbDNA Ladder，GIBCO)平行。根據(jù)插入的大小選擇陽性克隆。
表達各基因克隆入表達載體后，將重組質(zhì)粒轉(zhuǎn)化入適合表達重組蛋白的大腸桿菌菌株中。如上所述，用1μl各構(gòu)建物轉(zhuǎn)化大腸桿菌BL21-DE3。將單重組集落接種入2ml LB+Amp(100μg/ml)中，在37℃培育過夜，然后在100ml燒瓶中用20ml LB+Amp(100μg/ml)以1∶30稀釋，使OD600在O-1-0.2之間。將燒瓶置于旋轉(zhuǎn)式水浴搖床中于30℃或37℃培育，直到OD600顯示適合誘導(dǎo)表達的指數(shù)生長期(0.4-0.80D)。加入1.0mM IPTG誘導(dǎo)蛋白質(zhì)表達。在30℃或37℃培育3小時后，測定OD600并檢測表達。用微型離心機離心1.0ml各樣品，將沉淀物重懸浮于PBS中，用SDS-PAGE和考馬斯藍染色分析。
His-標記的蛋白質(zhì)的純化從菌株2996和MC58中克隆了287的各種形式。用C-未端His標記的融合體進行構(gòu)建，其包括成熟形式(aa18-427)、含缺失(△1、△2、△3和△4)的構(gòu)建物和由B或C結(jié)構(gòu)域組成的克隆。對以His-融合體純化的各克隆而言，劃線接種單集落，并且在37℃LB/Amp(100μg/ml)瓊脂板上培育過夜。將從該平板上分離的集落接種到20ml LB/Amp(100μg/ml)的液體培養(yǎng)基中，并在37℃振蕩生長過夜。以1∶30將過夜培養(yǎng)物稀釋到1.0L LB/Amp(100μg/ml)液體培養(yǎng)基中，讓其在最佳溫度(30或37℃)生長，直到OD550達到0.6-0.8。添加IPTG(終濃度為1.0mM)誘導(dǎo)重組蛋白的表達，再培育培養(yǎng)物3小時。于4℃，以8000g離心15分鐘收獲細菌。將細菌沉淀物重懸浮于7.5ml(i)冷緩沖液A(300mMNaCl、50mM磷酸鹽緩沖液、10mM咪唑、pH8.0)，用于可溶性蛋白質(zhì)；或(ii)緩沖液B(10mM Tirs-HCl、100mM磷酸鹽緩沖液，pH8.8和任選地8M尿素)，用于不溶性蛋白質(zhì)。以可溶形式純化的蛋白質(zhì)包括287-His、△1、△2、△3和△4287-His、△4287MC58-His、287c-His和287cMC58-His。蛋白質(zhì)287bMC58-His是不溶的并相應(yīng)地純化。用Branson Sonifier 450，在冰上以40W、30秒超聲處理破壞細胞4次，并于4℃以13000×g離心30分鐘。對于不溶蛋白質(zhì)，將沉淀物重懸浮于2.0ml緩沖液C(6M鹽酸胍、100mM磷酸鹽緩沖液、10mM Tris-HCl、pH 7.5)，并用Dounce勻漿器處理10次。以13000g離心勻漿30分鐘并保留上清液。將可溶的和不溶制備物的上清液與150μl Ni2+-樹脂(預(yù)先用緩沖液A或緩沖液B平衡)混合，并在室溫溫和振蕩培育30分鐘。樹脂是按制造商說明制備的Chelating Sepharose Fast Flow(Pharmcia)。于4℃，以700g離心分批制備物5分鐘，棄去上清液。用10ml緩沖液A或B洗滌樹脂2次(分批)10分鐘，重懸浮于1.0ml緩沖液A或B中，加到一次性柱上。用(i)緩沖液A(4℃)或(ii)緩沖液B(室溫)持續(xù)洗滌樹脂，直到流出物的OD280達到0.02-0.01。再用(i)冷緩沖液C(300mM NaCl、50mM磷酸鹽緩沖液、20mM咪唑、pH 8.0)或(ii)緩沖液D(10mM Tris-HCl、100mM磷酸鹽緩沖液、pH6.3和任選地8M尿素)進一步洗滌樹脂，直到流出物的OD280達到0.02-0.01。加入700μl(i)冷洗脫緩沖液A(300mM NaCl、50mM磷酸鹽緩沖液、250mM咪唑、pH 8.0)或(ii)洗脫緩沖液B(10mM Tris-HCl、100mM磷酸鹽緩沖液、pH 4.5和任選地8M尿素)洗脫His-融合蛋白，收集組分直到OD280顯示獲得了所有重組蛋白。用SDS-PAGE分析20μl量的各洗脫組分。用Bradford試驗法計算蛋白質(zhì)濃度。
變性的His-融合蛋白的復(fù)性需要變性以穩(wěn)定287bMC8，因此在免疫接種前需進行復(fù)性步驟。將甘油加到上述得到的變性組分中，使終濃度為10％v/v。用透析緩沖液I(10％v/v甘油，0.5M精氨酸、50mM磷酸鹽緩沖液、5.0mM還原的谷胱甘肽、0.5mM氧化的谷胱甘肽、2.0M尿素，pH 8.8)將蛋白質(zhì)稀釋至200μg/ml，用相同的緩沖液在4℃透析12-14小時。于4℃，用緩沖液II(10％v/v甘油，0.5M精氨酸、50mM磷酸鹽緩沖液、5.0mM還原的谷胱甘肽、0.5mM氧化的谷胱甘肽、pH 8.8)再進行透析12-14小時。用以下公式計算蛋白質(zhì)的濃度蛋白質(zhì)(mg/ml)＝(1.55×OD280)-(0.76×OD260)免疫接種在第0、21和35天，用抗原免疫接種Balb/C小鼠，在第49天分析血清。
血清分析-ELISA將不包囊的MenB M7和包囊的菌株置于巧克力瓊脂板上，在37℃、5％CO2中培育過夜。用無菌Dracon刷從瓊脂板上收集細菌菌落，并接種到含有0.25％葡萄糖的Mueller-Hinton肉湯(Difco)中。每30分鐘監(jiān)測一次細菌的生長，隨后測定OD620。讓細菌生長直到OD達到0.4-0.5。將培養(yǎng)物以4000rpm離心10分鐘。棄去上清液，用PBS洗滌細菌2次，重懸浮于含有0.025％甲醛的PBS中，并在37℃培育1小時，然后在4℃攪拌培育過夜。在96孔Greiner板的各孔中添加100μl細菌細胞，并在4℃培育過夜。然后用PBT洗滌緩沖液(0.1％Tween-20的PBS溶液)沖洗這些孔3次。在各孔中加入200μl飽和緩沖液(2.7％聚乙烯吡咯烷酮10的水溶液)，將這些平板在37℃培育2小時。用PBT沖洗這些孔3次。在各孔中加入200μl稀釋的血清(稀釋緩沖液1％BSA、0.1％Tween-20、0.1％NaN3的PBS溶液)，將這些平板在37℃培育2小時。用PBT沖洗這些孔3次。在各孔中加入100μl HRP-綴合的兔抗-小鼠(Dako)血清(用稀釋緩沖液以1∶2000稀釋)，將這些平板置于37℃培育90分鐘。用PBT緩沖液洗滌這些孔3次。在各孔中加入100μl HRP的底物緩沖液(25ml檸檬酸緩沖液pH 5，10mg鄰-苯二胺(phenildiamine)和10μl H2O2)，并將平板在室溫中靜置20分鐘。在各孔中加入100μl 12.5％H2SO4，隨后測定OD490。計算ELISA滴定度，即高于預(yù)先免疫血清稀釋度的、OD490值為0.4的血清稀釋度。當(dāng)OD490值為0.4的血清稀釋度高于1∶400時，將ELISA視為陽性。
血清分析-FACS掃描細菌結(jié)合分析將不包囊的MenB M7菌株置于巧克力瓊脂板上，在37℃、5％CO2中培育過夜。用無菌Dracon刷從瓊脂板上收集菌落，并接種到4支裝有0.25％葡萄糖的8ml Mueller-Hinton肉湯(Difco)試管中。每30分鐘監(jiān)測細菌的生長，然后測定OD620。讓細菌生長直到OD達到0.35-0.5。將培養(yǎng)物以4000rpm離心10分鐘。棄去上清液，用封阻緩沖液(1％BSA的PBS溶液，0.4％NaN3)重懸浮沉淀物，并以4000rpm離心5分鐘。將細胞重懸浮于封阻緩沖液，使OD620達到0.05。在96孔Costar板的各孔中添加100μl細菌細胞。在各孔中加入100μl稀釋的(1∶100、1∶200、1∶400)血清(以封阻緩沖液配制)，并將這些平板在4℃培育2小時。以4000rpm離心細胞5分鐘，吸出上清液，在各孔中加入200μl/孔封阻緩沖液洗少滌細胞。在各孔中加入100μl R-Phicoerytrin綴合的F(ab)2山羊抗-小鼠(以1∶100稀釋)，將平板置于4℃培育1小時。以4000rpm離心5分鐘以沉淀細胞，添加200μl/孔封阻緩沖洗滌細胞。吸出上清液，細胞重懸浮于200μl/孔PBS、0.25％甲醛。將樣品轉(zhuǎn)移到FACScan管中并讀數(shù)。FACScan(Laser Power15mW)的條件設(shè)定為FL2開；FSC-H閾值92；FSC PMT電壓E01；SSCPMT474；Amp.Gains 6.1；FL-2 PMT586；補償值0。
血清分析-殺菌試驗于37℃，在5％CO2、在巧克力瓊脂板(以冷凍原液起始)上培育腦膜炎奈瑟球菌菌株2996過夜。收集菌落，并將其接種到7ml含有0.25％葡萄糖的Mueller-Hinton肉湯中，使OD620達到0.05-0.08。在37℃振蕩培育約1.5小時，直到OD620達到0.23-0.24。用50mM磷酸鹽緩沖液(pH7.2，含10mM MgCl2、10mM CaCl2和0.5％(w/v)BSA(分析緩沖液))以105CFU/ml工作稀釋度稀釋細菌。最終反應(yīng)混合物的總體積為50μl，其中25μl連續(xù)2倍稀釋的測試血清，12.5μl工作稀釋度的細菌，12.5μl幼兔補體(終濃度25％)。
對照包括用補體血清培育的細菌、用細菌培育并在56℃加熱30分鐘補充滅活的免疫血清。在加入幼兔補體后，用斜置方法立即將10μl對照置于Mueller-Hinton瓊脂板上(0時間)。37℃旋轉(zhuǎn)培育96孔板1小時。將每份樣品的7μl涂布在Mueller-Hinton瓊脂板上作為斑點，而用斜置方法將10μl對照涂布在Mueller-Hinton瓊脂板上(1時間)。37℃培育瓊脂板18小時，計算相應(yīng)于0時間和1時間的菌落數(shù)量。
血清分析-Western印跡法將純化的蛋白質(zhì)(500ng/泳道)、外膜小泡(5μg)和MenB菌株2996衍生的全細胞提取物(25μg)加到12％SDS-聚丙烯酰胺凝膠上，并轉(zhuǎn)移到硝基纖維素膜上。用轉(zhuǎn)化緩沖液(03％Tris堿，1.14％甘氨酸、20％(v/v)甲醇)在4℃、150mA進行轉(zhuǎn)化2小時。于4℃，在飽和緩沖液(10％脫脂乳、0.1％Triton X100的PBS溶液)中培育過夜使膜飽和。用沖洗緩沖液(3％脫脂乳、0.1％Triton X100的PBS溶液)沖洗膜2次，并在37℃與用洗滌緩沖液以1∶200稀釋的小鼠血清一起培育2小時。沖洗膜2次，與1∶2000稀釋的辣根過氧化物酶標記的抗-小鼠Ig一起培育90分鐘。用0.1％Triton X100的PBS溶液沖洗膜2次，用Opti-4CN底物試劑盒(Bio-Rad)顯色。加入水終止反應(yīng)。
如下制備OMV于37℃、5％CO2，在5個GC板上讓腦膜炎奈瑟球菌2996生長過夜，用接種環(huán)收獲，并重懸浮于10ml 20mM Tris-HCl pH 7.5、2mM EDTA中。在56℃熱滅活45分鐘，在冰上超聲處理破碎細胞5分鐘(50％負載循環(huán)(dutycycle)、50％輸出，Branson超聲儀3mm微型針頭)。以5000g離心10分鐘除去未被破壞的細胞，回收含有完整細胞包膜組分的上清液，于4℃以50000g進一步離心過夜。將含有膜的沉淀物重懸浮于2％二烷基肌氨酸鈉、20mM Tris-HClpH 7.5、2mM EDTA中，在室溫培育20分鐘以溶解內(nèi)膜。以10000g離心上清液10分鐘除去聚集體，以50000g進一步離心上清液3小時。用PBS沖洗含有外膜的沉淀物，并重懸浮于相同的緩沖液中。用BSA作為標準，由D.C.Bio-Rad蛋白分析(改進的Lowry方法)測定蛋白質(zhì)的濃度。
如下制備全細胞提取物使腦膜炎奈瑟球菌在GC板上培育過夜，用接種環(huán)收獲，并重懸浮于1ml 20mM Tris-HCl中。在56℃熱滅活30分鐘。
權(quán)利要求
1.一種同時異源表達本發(fā)明兩種或多種蛋白質(zhì)的方法，其特征在于，所述的方法中(a)所述的本發(fā)明的兩種或多種蛋白質(zhì)是融合的。
2.如權(quán)利要求23所述的方法，其特征在于，所述的兩種或多種蛋白質(zhì)是(1)919和287；(b)953和287；(c)287和ORF46.1；(d)ORF1和ORF46.1；(e)919和ORF46.1；(f)ORF46.1、287和919；(g)919和519；和(h)ORF97和225。
3.如權(quán)利要求24所述的方法，其特征在于，所述的287在蛋白質(zhì)(a)、(b)或(c)的C-未端。
4.如權(quán)利要求1-3任一所述的方法，其特征在于，所述的表達是在大腸桿菌宿主中。
5.由權(quán)利要求1-4任一所述的方法表達的蛋白質(zhì)。
6.式NH2-A-B-COOH的雜交蛋白，其特征在于，所述的A和B是不同的奈瑟球菌蛋白。
7.如權(quán)利要求6所述的蛋白質(zhì)，其特征在于，所述的A和B各選自orfl、orf4、orf25、orf40、Orf46、orf83、233、287、2921、564、687、741、907、919、953、961和983。
8.權(quán)利要求7所述的蛋白質(zhì)，其特征在于，所述的A和B各選自O(shè)RF46.1、287、741、919、953、961和983。
9.如權(quán)利要求8所述的蛋白質(zhì)，其特征在于，所述的ORF46.1、287、741、919、953、961和983中的至少一種是以其基本全長的形式使用的。
10.如權(quán)利要求8或9所述的蛋白質(zhì)，其特征在于，所述的ORF46.1、287、741、919、953、961和983中的至少一種有缺失。
11.如權(quán)利要求10所述的蛋白質(zhì)，其特征在于，所述的A和/或B具有聚-甘氨酸缺失(‘△G′)。
12.如權(quán)利要求11所述的蛋白質(zhì)，其特征在于，所述的A和/或B是△G-287、△GTbp2、△G741或△G983。
13.如權(quán)利要求10所述的蛋白質(zhì)，其特征在于，所述的A和/或B是截短的蛋白質(zhì)。
14.如權(quán)利要求13所述的蛋白質(zhì)，其特征在于，所述的A和/或B是△1-287、△2-287、△3-287或△4-287。
15.如權(quán)利要求10所述的蛋白質(zhì)，其特征在于，所述的A和/或B的結(jié)構(gòu)域有缺失。
16.如權(quán)利要求15所述的蛋白質(zhì)，其特征在于，所述的A和/或B是287B、287C、287BC、ORF461-433、ORF46433-608、ORF46或961c。
17.如權(quán)利要求6所述的蛋白質(zhì)，其特征在于，所述的A和B是(a)919和287；(b)953和287；(c)287和ORF46.1；(d)ORF1和ORF46.1；(e)919和ORF46.1；(f)ORF46.1和919；(g)919和519；或(h)ORF97和225。
18.如權(quán)利要求17所述的蛋白質(zhì)，其特征在于，所述的蛋白質(zhì)是△G287-919、△G287-953、△G287-961、△G983-ORF46.1、△G983-741、△G983-961、△G983-961C、△G741-961、△G741-961C、△G741-983、△G741-ORF46.1、ORF46.1-741、ORF46.1-961、ORF46.1-961C、961-ORF46.1、961-741、961-983、961C-ORF46.1、961C-741、961C-983、961CL-ORF46.1、961CL-741或961CL-983。
19.如權(quán)利要求8所述的蛋白質(zhì)，其特征在于，所述的A或B是287。
20.如權(quán)利要求19所述的蛋白質(zhì)，其特征在于，所述的B是287。
21.如權(quán)利要求19所述的蛋白質(zhì)，其特征在于，所述的A是△G-287。
22.如權(quán)利要求21所述的蛋白質(zhì)，其特征在于，所述的B是ORF46、919、953或961。
23.如權(quán)利要求19-22任一所述的蛋白質(zhì)，其特征在于，所述的287來自菌株2996或394/98。
24.如權(quán)利要求8所述的蛋白質(zhì)，其特征在于，所述的A是961。
25.如權(quán)利要求6-24任一所述的蛋白質(zhì)，其特征在于，所述的A和B來自相同的菌株。
26.如權(quán)利要求6-24任一所述的蛋白質(zhì)，其特征在于，所述的A和B直接相連。
27.如權(quán)利要求6-24任一所述的蛋白質(zhì)，其特征在于，所述的A和B通過接頭肽相連。
28.如權(quán)利要求27所述的蛋白質(zhì)，其特征在于，所述的接頭肽是聚-甘氨酸接頭，但條件是B不是△G蛋白。
全文摘要
以單個雜交蛋白形式表達兩種或多種奈瑟球菌的蛋白質(zhì)(如A和B)，其可以式NH
文檔編號C07K19/00GK1426471SQ01808738
公開日2003年6月25日 申請日期2001年2月28日 優(yōu)先權(quán)日2000年2月28日
發(fā)明者M·B·阿科, M·科曼杜西, C·加萊蒂, V·馬斯格阿尼, M·M·朱利亞尼, M·皮扎 申請人:啟龍股份公司