CN1508253A - 奈瑟球菌蛋白质的杂交表达 - Google Patents

奈瑟球菌蛋白质的杂交表达 Download PDF

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CN1508253A
CN1508253A CNA2003101036036A CN200310103603A CN1508253A CN 1508253 A CN1508253 A CN 1508253A CN A2003101036036 A CNA2003101036036 A CN A2003101036036A CN 200310103603 A CN200310103603 A CN 200310103603A CN 1508253 A CN1508253 A CN 1508253A
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M·B·阿科
M·科曼杜西
C·加莱蒂
V·马斯格阿尼
M·M·朱利亚尼
M·皮扎
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Abstract

以单个杂交蛋白形式表达两种或多种奈瑟球菌的蛋白质(如A和B)，其可以式NH2－A－B－COOH简单地表示。

Description

奈瑟球菌蛋白质的杂交表达
本案是申请号为01808738.8，名为奈瑟球菌蛋白质的杂交表达的分案申请。本文将所引用的全部文献都纳入作为参考。
技术领域
本发明涉及蛋白质表达的领域。具体说，本发明涉及奈瑟球菌(如淋病奈瑟球菌或较佳地为脑膜炎奈瑟球菌)的蛋白质的异源表达。
背景技术
国际专利申请WO 99/24578、WO 99/36544、WO 99/57280和WO 00/22430公开了脑膜炎奈瑟球菌(Neisseria meningitidis)和淋病奈瑟球菌(Neisseriagonorrhoeae)的蛋白质。这些蛋白质一般是以N-未端GST-融合体或C-未端His-标记融合体在大肠杆菌中表达的(即异源表达)，虽然也公开了其它表达系统(包括在天然的奈瑟球菌中的表达)。
本发明的目的是提供这些蛋白质的异源表达的其它或改进方法。这些方法通常影响表达的水平、纯化的简易程度、表达的细胞内定位和/或表达的蛋白质的免疫学特性。
发明的公开
在本发明中，以单杂交蛋白表达本发明的两种或多种(如3、4、5、6或更多)蛋白质。较佳地，不使用非奈瑟球菌的融合配体(如GST或聚-His)。
这有两个优点。其一，可能不稳定或本身表达很差的蛋白质可以通过加入合适的克服该问题的杂交配体予以帮助。其二，简化工业生产，即制备两种分别有用的蛋白质只需要一次表达和纯化。
因此，本发明提供了同时异源表达本发明的两种或多种蛋白质的方法，其中所述的本发明的两种或多种蛋白质是融合的(即，它们是作为单多肽链翻译的)。
该方法通常包括如下步骤：获得编码本发明第一种蛋白质的第一核酸；获得编码本发明第二种蛋白质的第二核酸；连接第一和第二核酸。将得到的核酸插入表达载体中，或已作为表达载体的一部分。
当仅连接两种蛋白质时，可以式NH2-A-B-COOH简单地表示杂交蛋白。A和B各选自任何奈瑟球菌蛋白质，尤其是那些以SEQ#1-4326表示的。该方法非常适合表达蛋白质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个杂交体是优选的：
     ↓A   B→    ORF46.1     287     741     919     953    961     983
      ORF46.1     ×     ×     ×     ×     ×     ×
          287    ×     ×     ×     ×     ×     ×
          741    ×     ×     ×     ×     ×     ×
          919    ×     ×     ×     ×     ×     ×
          953    ×     ×     ×     ×     ×     ×
          961    ×     ×     ×     ×     ×     ×
          983    ×     ×     ×     ×     ×     ×
因此，优选的以杂交体表达的蛋白质是ORF46.1，287，741，919，953，961和983。它们可以全长形式或多-甘氨酸缺失(ΔG)形式使用(如ΔG-287、ΔGTbp2、ΔG741、ΔG983等)、或以截短形式使用(如Δ1-287、Δ2-287等)，或以结构域缺失的形式使用(如287B、287C、287BC、ORF461-433、ORF46433-608、ORF46、961c等)等。
特别优选的是：(a)含919和287的杂交蛋白；(b)含953和287的杂交蛋白；(c)含287和ORF46.1的杂交蛋白；(d)含ORF1和ORF46.1的杂交蛋白；(e)含919和ORF46.1的杂交蛋白；(f)含ORF46.1和919的杂交蛋白；(g)含ORF46.1、287和919的杂交蛋白；(h)含919和519的杂交蛋白；和(i)含ORF97和225的杂交蛋白。
附图中显示了其它优选例，它们包括ΔG287-919、ΔG287-953、ΔG287-961、ΔG983-ORF46.1、ΔG983-741、ΔG983-961、ΔG983-961C、ΔG741-961、ΔG741-961C、ΔG741-983、ΔG741-ORF46.1、ORF46.1-741、ORF46.1-961、ORF46.1-961C、961-ORF46.1、961-741、961-983、961C-OR961C-983、961CL-ORF46.1、961CL-741和961CL-983。
当使用287时，其优先位于杂交体的C-未端；如果在N-未端使用它，则优先使用287的ΔG形式(如与ORF46.1、919、953或961杂交的杂交体的N-未端)。
当使用287时，其优先为菌株2996或菌株394/98的。
当使用961时，其优先在N-未端。可使用961的结构域形式。
WO 99/66741公开了ORF46、287、919和953的多态形式的排序。本发明可以使用这些多态形式中的任一形式。
较佳地，本发明杂交蛋白中的组成蛋白质(A和B)来源于是同一菌株。
杂交体中的融合蛋白可以是直接相连的，或者是通过接头肽相连的，如通过聚-甘氨酸接头(即，Gn，其中n＝3、4、5、6、7、8、9、10或更多)或通过协助克隆的短肽序列相连。显然不宜将ΔG蛋白质连接于聚-甘氨酸接头的C-未端。
融合蛋白可以缺失天然前导肽或可以包含N-未端融合配体的前导肽序列。
宿主
较佳地，利用异源宿主。异源宿主可以是原核的或真核的。优先为大肠杆菌，但其它合适的宿主包括枯草芽孢杆菌(Bacillus subtilis)、霍乱弧菌(Vibriocholerae)、伤寒沙门氏菌(Salmonella typhi)、鼠伤寒沙门氏菌(salmonellatyphimurium)、脑膜炎奈瑟球菌、淋病奈瑟球菌、乳糖奈瑟球菌(Neisserialactamica)、灰色奈瑟球菌(Neisseria cinerea)、分枝杆菌(如结核分枝杆菌(Mtuberculosis)、酵母等。
载体、宿主等
如上所述的方法，本发明提供了(a)用于这些方法的核酸和载体；(b)含所述载体的宿主细胞；(c)可用这些方法表达的或可表达的蛋白质；(d)包含这些蛋白质的组合物，其可能适合作为疫苗、或例如诊断剂或免疫原性的组合物；(e)用作药物(如疫苗)或诊断剂的组合物；(f)这些组合物在制备以下物质中的用途(1)用于治疗或预防奈瑟球菌引起的感染的药物(2)检测奈瑟球菌或由奈瑟球菌引起的抗体存在与否的诊断剂，和/或(3)能产生抗奈瑟球菌抗体的药物：和(g)治疗患者的方法，其包括对该患者施用治疗有效量的这些组合物。
序列
本发明还提供了具有以下实施例中所列出的任何的蛋白质或核酸。本发明还提供了具有与这些序列是序列相同性的蛋白质和核酸。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
本文参考在WO 99/24578、WO 99/36544和WO 99/57280中公开的2 166个蛋白质序列，并将它们编号为如下的SEQ#：
申请 蛋白质序列  本文的SEQ#
WO 99/24578 偶SEQ ID 2-892  SEQ#1-446
WO 99/36544 偶SEQ ID 2-90  SEQ#447-491
WO 99/57280 偶SEQ ID 2-3020偶SEQ ID 3040-3114SEQ ID 3115-3241  SEQ#492-2001SEQ#2002-2039SEQ#2040-2166
除了这种SEQ#编号外，本文还使用了WO 99/24578、WO 99/36544和WO99/27280中的命名约定(如WO 99/24578和WO 99/36544中用的‘ORF4’、‘ORF40’、‘OFR40-1’等；WO 99/57280中用的‘m919’、‘g919’和‘a919’等)。
在本文中将Tettelin等[Science(2000)287：1809-1815]中的从NMB0001到NMB2160的2160个蛋白质称为SEQ#2167-4326[参见WO 00/66791]。
本文采用的术语“本发明的蛋白质”指包含以下的蛋白质：
(a)SEQ#1-4326中的一个序列；或
(b)与SEQ#1-4326中的一个序列相同的序列；或
(c)SEQ#1-4326中的一个序列的片段。
(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)。其包括突变体和等位基因变体[如，参见WO 00/66741]。相同性最好用Smith-Waterman同源性搜寻算法确定，如在MPSRCH程序(OxfordMolecular)中执行的，采用参数“缺口罚分(gap open penalty)”为12，“缺口延伸罚分(gap extension penalty)”为1进行缺口仿射搜索。通常，将两种蛋白质之间50％或更高的相同性视为功能等效的指示。
(c)中的“片段”应包含SEQ#1-4326中一个序列的至少n个连续的氨基酸，且根据具体的序列n为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)。较佳地，片段包含SEQ#1-4326中一个序列的表位。优选的片段是在WO 00/71574和WO 01/04316中公开的那些。
本发明优选的蛋白质是在脑膜炎奈瑟球菌血清群B中发现的。
根据本发明使用的优选蛋白质是血清群B脑膜炎奈瑟球菌菌株2996或菌株394/98(新西兰菌株)。除非特别指出，本文所述的蛋白质是脑膜炎奈瑟球菌菌株2996的蛋白质。但是，应该理解通常本发明并不受菌株的限制。参考具体的蛋白质(如‘287’、‘919’等)可以包括任何菌株的该蛋白质。
应该理解术语“核酸”包括DNA和RNA，以及它们的类似物，如含有修饰骨架的那些类似物，还包括肽核酸(PNA)等。
附图简述
图1-26显示本发明的杂交蛋白。
本发明的进行模式
实施例1-ORF46的杂交体
脑膜炎奈瑟球菌(血清群B，菌株2996)的完整ORF46蛋白质具有如下序列：
  1  LGISRKISLI  LSILAVCLPM  HAHASDLAND SFIRQVLDRQ HFEPDGKYHL
 51  FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE
101  VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY
151  PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV
201  GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG
251  ISEGSNIAVM HGlGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP
301  NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP
351  KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI TSSTVPPSNG
401  KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAKP
451  VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS QHAQLEREIN
501  KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PVVEYVEING
551  KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV
601  KRPRYRSK*
在前导肽下加下划线。
可以在WO 00/66741中发现其它菌株的ORF46的序列。
ORF46在其C-未端和N-未端与287、919和ORF1融合。该杂交蛋白通常是不溶解的，但产生一些良好的ELISA和杀菌结果(针对同源2996菌株)：
 蛋白质     ELISA     杀菌Ab
 Orf1-Orf46.1-His     850     256
 919-Orf46.1-His     12900     512
 919-287-Orf46-His     n.d.     n.d.
 Orf46.1-287His     150     8192
 Orf46.1-919His     2800     2048
 Orf46.1-287-919His     3200     16384
为了比较，构建了ORF46.1、287(以GST融合体或ΔG287的形式)和919的“三”杂交体，并针对各种菌株(包括同源2996菌株)将其与三种抗原的简单混合物相比。FCA用作佐剂：
 2996  BZ232  MC58  NGH38  F6124  BZ133
 混合物  8192  256  512  1024  ＞2048  ＞2048
 ORF46.1-287-919his  16384  256  4096  8192  8192  8192
 ΔG287-919-ORF46.1his  8192  64  4096  8192  8192  16384
 ΔG287-ORF46.1-919his  4096  128  256  8192  512  1024
同样，这些杂交体显示相当等的或更佳的免疫活性。
针对各种异源菌株，将两种蛋白质(菌株2996)的杂交体与单种蛋白质相比：
 1000  MC58  F6124(MenA)
 ORF46.1-His  ＜4  4096  ＜4
 ORF1-His  8  256  128
 ORF1-ORF46.1-His  1024  512  1024
再次，这些杂交体显示相等的或更佳的免疫活性。
实施例2-ΔG287的杂交体
发现287中(Gly)6序列的缺失对蛋白质的表达有显著影响。将缺失N-未端氨基酸多达GGGGGG的蛋白质称为‘ΔG287’。在菌株MC58中，它的基本序列(前导肽有下划线)为：
    SPDVKS ADTLSKPAAP VVSEKETEAK EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT
GNGGAVTADN PKNEDEVAQN DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP
ANQPDMANAA DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS
NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS DADKISNYKK
DGKNDKFVGL VADSVQMKGI NQYIIFYKPK FTSFARFRRS ARSKRSLPAE MPLIPVNQAD
TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY
NGEVLJFJTE NGRPYPTKGR FAAKVDFGSK SVDGIIDSGD DIHMGTQKFK AAIDGNGFKG
TWTENGSGDV SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*
与‘287-His’或‘287未标记的’相比，有或无His-标记的ΔG287(分别为‘ΔG287-His’和‘ΔG287K’)以很好的水平表达。
在基因变异性数据的基础上，从许多MenB菌株(尤其是从菌株2996、MC58、100和BZ232)的大肠杆菌中表达ΔG287-His的变体。结果也好-它们都具有很高的ELISA滴定度，且血清杀菌滴定度＞8192。由pET-24b表达的ΔG287K在ELISA和血清杀菌实验中有极佳滴定度。
还将聚-Gly序列的缺失应用于Tbp2(NMB0460)、741(NMB1870)和983(NMB1969)。在不编码其前导肽的序列且没有聚-Gly(即，以“ΔG形式”)，在pET载体中克隆并在大肠杆菌中表达时，观察到相同的作用-在携带聚-甘氨酸段缺失的克隆中表达很好，若在表达的蛋白中存在甘氨酸时则表达差或不表达。
将ΔG287直接融合于919、953、961(如下所示的序列)和ORF46.1的符合读框的上游：
ΔG287-919
ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAG
GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCA
GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT
GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC
GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG
CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG
TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT
ACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATAATGTCATCTTTATAAAGACAAGTCCGCTTCA
TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT
CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGG
AATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCA
AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC
CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGAT
TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC
GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG
GAAAAGGGCGGATTCGGCGTGTTTGCCGGGCCAAAAAGGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGC
ATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGA
ACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTC
GCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTTGCGCCCAA
GCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGC
AACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCC
CGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTT
GTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGA
TTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAAC
CAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATG
CACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACAT
CCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAA
GCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTT
GCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGG
CACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATT
ATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGC
GAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCG
TAACTCGAG
  1    MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM
 51    AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS
101    DSAPASNPAP ANGGSNFGRV DLANGVIDG PSQNITLLTHC KGDSCNGDNL
151    LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI
201    IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG
251    HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE
301    VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI
351    DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG
401    KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA
451    VYTVVPHLSL PHWAAQDFAK SLQSFRLGCA NLKNRQGWQD VCAQAFQTPV
501    HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI
551    YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP
601    ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM
651    HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG
701    IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG
751    AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFMG
801    YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*
ΔG287-953
ATGGCTGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCCGGCGCTCCTGTTGTTGCTGAAAAAGAGACCAGAG
GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCCGCCTCCACACAAGGCAGCCAAGATATGGCGGCA
GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT
GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC
GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG
CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG
TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT
ACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCA
TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT
CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGG
AATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCA
AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC
CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGAT
TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC
GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG
GAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTG
GACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTG
ACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGC
GGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTT
TCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCC
GTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGC
ACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATC
CAAATCGAGGCAGCCAAACAATAACTCGAG
 1     MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM
 51    AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS
101    DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL
151.   LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI
201    IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG
251    HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE
301    VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI
351    DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG
401    KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ
451    AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF
501    NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID
551    RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*
ΔG287-961
ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAG
GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCA
GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT
GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC
GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG
CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG
TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACCAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT
ACTATTTGGTTGCCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATTCATTTATAAAGACAAGTCGCTTCA
TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT
CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCCGAAGGG
AATTACCGGTATCTGACTTACGGGGCGCGGAAAAATTGCCGGCGGATCGTATGCCCTCCGTGTGCAGGCGAACCGGCA
AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC
CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGCGGGCATTATCGACAGCGGCGATGAT
TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC
GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG
GAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGAC
GATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGA
GAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGAC
TTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCC
AAAGTAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGGCGGCTTTAGCAGATACT
GATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAG
ACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGAT
ATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCC
GAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGC
ACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAAC
AAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATT
GATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACT
CGCCGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAACAAGCCGCCGCGCTC
TCCGGTCTGTTCCAACCTTACAACGTGGGTTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATGGCA
GTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGT
TCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAACTCGAG
  1    MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM
 51    AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS
101    DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL
151    LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI
201    IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG
251    HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE
301    VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI
351    DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG
401    KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE
451    DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE
501    SEIEKLTTKL ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV
551    KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE
601    TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKAKIATN
651    KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAKKSIADH
701    DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK
751    SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*
 ELISA  杀菌
ΔG287-953-His  3834  65536
ΔG287-961-His  108627  65536
对919和ORF46.1而言，将针对杂交蛋白产生的抗体的杀菌效力(同源菌株)与针对组分抗原(用287-GST)的简单混合物产生的抗体相比：
 与287的混合物  与ΔG287的杂交体
919  32000  128000
ORF46.1  128  16000
还获得了针对异源MenB菌株和针对血清型A和C的杀菌活性的数据：
        919      ORF46.1
 菌株 混合物  杂交体 混合物  杂交体
 NGH38 1024  32000 -  16384
 MC58 512  8192 -  512
 BZ232 512  512 -  -
 Men(F6124) 512  32000 -  8192
 MenC(C11) ＞2048  ＞2048 -  -
 MenC(BZ133) ＞4096  64000 -  8192
因此，在N-未端与ΔG287的杂交蛋白在免疫学上优于与ΔG287-ORF46.1的简单混合物，即使针对异源菌株也特别有效。可以在pET-24b中表达ΔG287-ORF46.1。
用新西兰菌株394/98，而非2996，制备相同的杂交蛋白：
ΔG287NZ-919
ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAG
GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG
GTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAAT
GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT
ATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCCGACGGA
ATGCAGGGTGACGATTCCGTCGGCAGGGCGGGGAAAATGCCGGCAATACGGCTCCCAAGGTACAATCAAGCCGAAAAC
AATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGG
ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT
AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAAAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT
AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG
GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG
CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGC
CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCC
GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC
GAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCCAGAGGCAGGTTTGCCGCAAAAGCGATTTCGGC
AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT
GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG
GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG
CAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAAC
GGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTA
CCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGCATTTCGCCAAAAGCCTCATCCTTCCGCCTCGCCTGGCGCCAAT
TTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAG
TTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAG
CCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCC
GTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATC
GACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGG
TTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATA
CTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGC
AAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGC
TACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTT
TTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGC
ACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACC
GCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTG
CGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAACAGAAAACCACGGGTTACGTCTTGG
CAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAAAAGCTT
  1   MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM
 51   AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM
101   PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG KNAGNTAAQG
151   TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL
201   THCKGDSCSG NNFLDEEVQL KSEREKLSDA DKISNYKKDG KNDGKNDKFV
251   GLVADSVQMK GINQYIIFYK FKPTSFARFR RSARSRRSLP AEMPLIPVNQ
301   ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP
351   SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS
401   GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY
451   RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV
501   GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR
551   QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV
601   LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI
651   DNTGGTHTAD LSRFPITART TAIKGRFEGS RFLPYHTRNQ INGGALDGKA
701   PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM
751   ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG
801   PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT
851   GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP*
ΔG287NZ-953
ATGGCTAGCCCCGATGTCAAGTCGGCGGCGGACACGTGTCAAAACCTGCCGCCCCTGTTTCTGAAAAAGAGACAGAAG
GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG
GTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAAT
GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT
ATGGAAAACCAAGCACCGGATGCCGGGGGAATCGGAGCAGCCGGCAAACCAACCGGATTGGCAAATACGGCGGACGGA
ATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAAC
AATCAAACCGCCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACAATAGCGTGGTGATTTTGGAAGGG
ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT
AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT
AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG
GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG
CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGC
CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCC
GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC
GAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGC
AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT
GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG
GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG
CAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCAT
TTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGT
AAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGAC
ATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCC
GTTGACGCGCAACCTGACCATGCACGGCAAAACGCCCCCCGTCAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGC
CCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTC
GTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAAAAGCTT
1    MASPVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM
 51    AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAAKTDSL TPNHTPASNM
101    PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG
151    TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL
201    THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV
251    GLVADSVQMK GINQYIIFYK PKPTSFARFR RRARSRRSLP AEMPLIPVNQ
301    ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP
351    SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS
401    GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY
451    RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN
501    VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA
551    AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA KKFNCYQSPM
601    AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*
ΔG287NZ-961
ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAG
GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG
GTTTCGGAAGAAAATCAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCCGCAAAAT
GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT
ATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGA
ATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAAC
AATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCCGTGGTGATTTTGGAAGG
ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT
AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT
AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG
GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG
CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCCGATACGCTGATTGTCGATGGGGAAGCGGTCAGC
CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGGAAGGGAATTACCGGTATCTGACTTCGGGGCGGAAAAATTGCCC
GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC
GAAGTGCTGCATTTTCATACGGAAAACGGCCGCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCCGGC
AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT
GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG
GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG
CAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCC
TACAACATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACCATTGATGAAGACGGCACAATTACC
AAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTG
ACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACA
ACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGAGGCAACCACCAACGCCTTGAATAAA
TTGGGGAGAAAATATAACGACATTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTG
GCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGAC
GAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCT
GCAGAAACTGCAGCAGGCAAAGCCGAAGTGCCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCT
GCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTG
TACACCAGAGAAGACTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGC
TTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGC
AAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAAT
GTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTT
GCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGG
TAAAAGCTT
  1    MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM
 51    AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM
101    PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG
151    TNQFENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL
201    THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNKDFV
251    GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ
301    ADTLIVDGEA VSLTGHSGHI FAPEGNYRYL TYGAEKLPGG SYALLVQGEP
351    SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS
401    GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY
451    RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE
501    INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV
551    NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG
601    ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD
651    EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA
701    VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK
751    LDTRLASAEK SIADHDTRLN GLDKTVSDLR DETRQGLAEQ AALSGLFQPY
801    NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY
851    HVGVNYEW*
                                                              实施例3-ΔG983的杂交体
蛋白质983具有如下序列：
 983                                           →ΔG983
   1    MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS
  51    NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN
 101    LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE
 151    LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT
 201    DIRHVKEIGH IDLVSEIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM
 251    MVAAIRNAWV KAGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL
 301    DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN DAQAQPNTYA
 351    LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA
 401    MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN
 451    LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT
 501    SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN
 551    KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ
 601    LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS
 651    AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART
 701    ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT
 751    DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG
 801    RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA
 851    KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL
 901    FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE
 951    GGLRYKLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL
1001    FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE
1051    FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*
因此ΔG983具有如下基本序列：
                                      TSAPD FNAGGTGIGS
NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN
LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE
LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT
DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM
MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL
DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN DAQAQPNTYA
LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA
MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN
LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT
SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN
KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ
LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS
AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSWGDTLSY YVRRGNAART
ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT
DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG
RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA
KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL
FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE
GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL
FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE
 FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*
将ΔG983作为在其C-未端与ORF46.1、741、961或961c的杂交体表达：
ΔG983-ORF46.1
ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA
GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG
GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA
TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC
ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA
AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG
GCCGTTATAGAGACTGAAGCAAAGCCGAGGATATCCGCCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT
ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG
AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC
ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG
TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC
GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC
AACACATATGCCCTATTGCCATTTTATGGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACGCAGT
GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT
ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA
ACATCCTTTTCCGCCACCCATCGTAACCGGCACGGCGGCTTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC
CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG
GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT
ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC
GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG
CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT
CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTCCACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG
CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC
GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT
TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA
GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC
GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC
GCAACACCCGAGACGGTTGAAACTGCGCAGCCGACCGCACAGATATGCCGGGCATCCCGCCCCTACGGCGCAACTTTC
CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT
GCCGACAGTACCGCCGCCCAGGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGC
ACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC
GGCAGTACCCAAACCGTCGGCATTGCCCGCGAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC
AGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGC
GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAA
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG
GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG
GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT
AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGGCGACTACACGGTAACGGGCGGTTTACC
GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT
GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA
CGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATC
CGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAG
CGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAA
GGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACAT
TCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCAT
CCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGAC
ATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACACCCGCAGCACCGGACAACGGCTTGCCGACCGTTTC
CACAATGCCGGTAGTTGCGACGGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCCGATACAGCCCCGAGCTGGAC
AGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATT
GTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCC
ACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGC
GATGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCCATCCCCATC
AAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGC
GCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCT
TACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCG
TCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCG
AATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA
  1    MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD
 51    VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV
101    GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE
151    ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD
201    ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD
251    LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF
301    IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP
351    GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG
401    TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG
451    PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT
501    GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT
551    DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK
601    GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE
651    GDTLSYYVRR GNAARTASAA AHSAPAFLKH AVEQGGSNLE NLMVELDASE
701    SSATPETVET AAAGRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA
751    ATVYADSTAA HADMOGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG
 801    VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF
 851    AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL
 901    GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG
 951    LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR
1001    NMPHTRLVAG LAADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD
1051    GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG
1101    KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA
1151    GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DIYSYDIKGV
1201    AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS
1251    GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS
1301    TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI
1351    PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK
1401    YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF
1451    DGKGFPNFEK HVKYDTLEHH HHHH*
ΔG983-741
ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA
GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG
GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA
TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC
ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA
AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG
GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT
ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG
AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC
ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG
TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC
GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC
AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT
GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGGCTTGAGTATGGCTCCAACCATTCGGAATT
ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCCTTTCACCCGTACAAACCCGATTCAAATTGCCGGA
ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC
CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG
GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT
ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC
GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG
CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT
CTGGCAGATACCGACCATCCGGCGCAAACGAAACCGTACACCATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG
CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC
GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT
TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA
GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC
GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACTGATGGTGCGAACTGGATGCCTCCGAATCATCC
CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT
GCCGACAGTACCGCCGCCCATGCCGTATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCCACAACGGC
ACGGGTCTGCGCGTCATCGGCGCAAACCCAACGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC
GGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC
AGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGC
GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAA
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGCTGTCAACGTTCCGTTTGCCGCAACG
GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT
AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC
GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT
GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA
CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCC
GATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAAC
GAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAG
AACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAG
TTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGG
AAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGC
GGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCC
GCCAAGACAGGGAAACGGCAATCGGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAG
CCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGT
ATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTT
GCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA
   1    MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD
  51    VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV
 101    GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE
 151    ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD
 201    ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD
 251    LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF
 301    IFSTGNDAQA QPNTYALLPF YKKDAQKGII TVAGVDRSGE KFKREMYGEP
 351    GTEPLEYGSN HCGITAMWCL SAFYEASVRF TRTNPIQIAG TSFSAPIVTG
 401    TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG
 451    PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT
 501    GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT
 551    DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK
 601    GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE
 651    GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE
 701    SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA
 751    ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG
 801    VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF
 851    AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL
 901    GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG
 951    LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR
1001    NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE
1051    GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ
1101    GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ
1151    SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT
1201    YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD
1251    GKRHAVISGS VLYNQAEKGS YSLGIFGGDA QEVAGSAEVK TVNGIRHIGL
1301    AAKQLKHHHH HH*
ΔG983-961
ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA
GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG
GTTACAGACAGGGTGCCAAAATCAATGCCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA
TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC
ACAGGGCGATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA
AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG
GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT
ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG
AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC
ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG
TACCGCCAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGGAGGGTATCCGCCTGATGCAACAGAGCGATTAC
GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC
AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT
GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT
ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA
ACATCCTTTTCCGCACCCCTCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC
CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG
GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT
ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC
GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATGGGATATG
CGCGTCGAAACCAAAGGTGCGCTGATTTATAACCGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGCATTGTCTAT
CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG
CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC
GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT
TCTTTCTTCACAAACATCGAAACCGACGGCGGCTGCTGGCTTCCCTCGACAGCGTCGAAAAAAACAGCGGGCAGTGAA
GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGATGCCTTCGGCAGCGGCCACATTCCGCGCCGCC
GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG
GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG
GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT
AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC
GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT
GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGYTCCGGAACCACAGCGGA
CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAA
GCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTAC
GACATTGATGAGACGGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTG
GGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCT
GCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTG
GATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTA
AAATTGATAAAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCA
TTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAA
CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACT
GCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTAACCGACATCAAAGTGATATCGCTACGAACAAAGATAATATT
GCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAAC
GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT
TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTC
CAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGT
ACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCC
TACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA
   1    MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD
  51    VAVTDRDAKI NAPPPNLHTG DFPAPNDAYK NLINLKPAIE AGYTGRGVEV
 101    GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE
 151    ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD
 201    ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD
 251    LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF
 301    IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP
 351    GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG
 401    TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG
 451    PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT
 501    GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT
 551    DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG KGLYMSARGK
 601    GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE
 651    GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE
 701    SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA
 751    ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG
 801    VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF
 851    AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL
 901    GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG
 951    LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR
1001    NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE
1051    GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI
1101    TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE
1151    KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE
1201    KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN
1251    VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI
1301    AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL
1351    NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGRFNVTA AVGGYKSESA
1401    VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*
ΔG983-961c
ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA
GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGAAGCG
GTTACAGACAGGGATGCCAATTTCAATGCCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAACCCAAATGACGCA
TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC
ACAGGCAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAA
AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG
GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT
ATTATTGGCGGGCGTTCCGTCGACGGCAGACCTGCAGGCGGTATTGCGCCCCGATGCGACGTACACATAATGAATACG
AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC
ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG
TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC
GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC
AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCCAGT
GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT
ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA
ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC
CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG
GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT
ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC
GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG
CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT
CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG
CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC
GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT
TCTTTCTTACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA
GGCGACCACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGATGCTTCGGCAGCGGCACATTCCGCGCCCGCC
GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC
GCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTC
CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT
GCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTAGCGGACGGGTTGGACCACAACGGC
ACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC
GGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC
AGCACATGGAGCGAAAACAGTGCAAATCAAAAACCCCACAGCATTAGTCTCTTTGCAGGCATACGGCACGATGGCGGC
GATATCGGCTATCTCAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACCGAA
CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG
GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG
GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT
AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC
GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT
GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA
CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAA
GCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTAC
GACATTGATGAAGACGGCACAATTACCAAAAAAGACACAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTG
GGTCTGAAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTGATGCCAAAGTAAAAGCT
GCAGAATCTGAAAATAGAAAAGTTAACAACCAAGTTAGAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTG
GATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTA
AAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATACCGCCGATTCA
TTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAA
CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACT
GCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATT
GCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAAC
GCTACTACCGAAAAATTGGACACACGCTTGCCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT
TTGGATAAAACAGTGTCAGACCTGCGCAAAGAACCCGGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTC
CAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA
   1    MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD
  51    VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV
 101    GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE
 151    ASFDDEVAIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD
 201    ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD
 251    LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF
 301    IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP
 351    GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG
 401    TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG
 451    PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT
 501    GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT
 551    DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK
 601    GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE
 651    GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE
 701    SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA
 751    ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG
 801    VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF
 851    AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL
 901    GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG
 951    LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR
1001    NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE
1051    GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI
1101    TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE
1151    KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE
1201    KLEAVADTVD KHAEAFDNIA DSLDETNTKA DEAVKTANEA KQTAEETKQN
1251    VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI
1301    AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL
1351    NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH*
实施例4-ΔG741的杂交体
蛋白质741具有如下序列：
  1    VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL
 51    QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ
101    IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI
151    GDIAGEHTSY DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI
201    EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA
251    QEVAGSAEVK TVNGIRHIGL AAKQ*
因此，ΔG741具有如下基本序列：
                            VAAD IGAGLADALT APLDHKDKGL
QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ
IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI
GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI
EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA
QEVAGSAEVK TVNGIRHIGL AAKQ*
将ΔG741直接融合于蛋白质961、961c、983和ORF46.1的符合读框的上游：
ΔG741-961
ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG
TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA
AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG
GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG
ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC
GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTCAATGTCGACCTGGCTCCGCCGATATCAAGCCGGATGTGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC
AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG
AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACA
AACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTC
AAAGCTGGAGAGACCTATCACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAA
GCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAACAAAAAC
GTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTA
GCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAA
GAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCA
TTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA
CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT
GCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATC
GCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTT
GTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGAT
CACGATACTCCGCCTAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAA
GCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCC
GAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACT
TCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA
  1    MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT
 51    YGNGDSLNTG KLKNDKVSRF DFIRQUEVDG QLITLESGEF QVYKQSHSAL
101    TAFQTEQIQD SEHSGKMVAK QFFRIGDIAG EHTSFDKLPE GGRATYRGTA
151    FGSDDAGGKL TYTIDFAAKQ GNGKIEHLGS PELNVDLAAA DIKPDGKRHA
201    VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAKVKTVNGI RHIGLAAKQL
251    EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT
301    ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI
351    EKLTTKLADT DAALADTAAI LDATTNALNK LGENTTTFAE ETKTNIVKID
401    EKLEAVADTV DHHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ
451    NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN
501    IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLSSA EKSIADHDTR
551    LNGLDGTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES
601    AVAIGTGFRF TENFAAGAGV AVGTSSGSSA AYHVGVNYEW LEHHHHHH*
ΔG741-961c
ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG
TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA
AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCCACTTTATCCGCCAAATCGAAGTG
GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGAGTTCCAGTATACAACAAAGCCATTCCGCCTTAACCGCCTTTCAG
ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC
GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC
AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG
AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACA
AACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTC
AAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAA
GCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAAC
GTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTA
GCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAA
GAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCA
TTCAACGATATCGCCGATTCATTGGATGAAACCACACTAAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA
CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT
GCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATC
GCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTT
GTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGAT
CACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAA
GCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA
  1    MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KAAAQGAEKT
 51    YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL
101    TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA
151    FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA
201    VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL
251    EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT
301    ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI
351    EELTTKLADT DAALADTDAA LDATTNALNK LGKNITTFAE ETKTNIVKID
401    EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ
451    NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN
501    IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR
551    LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH*
ΔG741-983
ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG
TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA
AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG
GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG
ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC
GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC
AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG
AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGATCCGGCGGAGGCGGCACTTCTGCG
CCCGACTTCAATGAGGCGGTACCGGTAATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTAC
GCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGG
GATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTG
ATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCC
GTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCG
TATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAG
ACGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGG
CGTTCCGTGGAGCGGCAGACCTGCAGGCGGTATTGCGCCGGATGCGACGCTCACATAATGAATACGAATGATGAAACC
AAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAAC
AGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCG
TTGCTCGACTATTCCGGCGGTGATAAAACAGACAGGGTATCCGCCTGATGCAACGAGAGCGATTACGGCAACCTGTCC
TACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCC
CTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTC
AAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGG
TGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCC
GCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACG
TTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAG
GCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCC
TTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACC
TATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACC
AAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACC
GACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGT
TTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCA
GGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACA
AACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTG
TCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACAC
GCCGTAGAACAGGGCGGCAGCAACTCTGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAG
ACGGTTGAAACTGCGGCAGCCGACCGCACAAATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCA
GCCGTACAGCATGCGAATGCCGCCGACGGTGTACGGATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACC
GCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGC
GTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAA
ACCGTCGGCATTGCCGCCAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGC
GAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTAT
CTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGC
AGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACG
GTCGAAGGGGTCTGCGCTACTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAGGCAGTGCTTTGGGCTGAGCGGC
AACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTG
TTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCA
GCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGCCCTGGGCGCGGATGTCGAATTCGGC
AACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTA
GGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA
   1    MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT
  51    YGNGDSLNTG KLKNDKVSRF DFIRQIRVDG QLITLESGEF QVYKQSHSAL
 101    TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA
 151    FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA
 201    VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGIAAKQL
 251    EGSGGGGTSA PDFNAGGTGI GSNSRATTAK SAAVSYAGIK NEMCKDRSML
 301    CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT
 351    GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG
 401    GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA
 451    GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS
 501    RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR
 551    NKNMLFIFST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR
 601    EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSFS
 651    APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA
 701    GKAMNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSQLQLH
 751    GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI
 801    VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY
 851    MSARGKGAGY LNSTGRRVPF LSAAKIGQDY SFFTNIETDG GLLASLDSVE
 901    KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLMV
 951    ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR
1001    IFNSLAATVY ADSTAAHADM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG
1051    TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT
1101    DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL
1151    MQLGAIGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT
1201    EGTLVGLAGL KLSQPLSDKA VLFATAGVER DLNGRDYYVT GGFTGATAAT
1251    GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV
1301    GYRFLEHHHH HH*
ΔG741-ORF46.1
ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG
TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA
AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCACGCCGTTTCGACTTTACCGCCAAATCGAAGTG
GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG
ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC
GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC
GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA
CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC
AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG
AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGACGGTGGCGGAGGCACTGGATCCTCAGAT
TTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACGGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGC
AGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAGGTACAAAGCCATCAGTTGGGCAACCTGATG
ATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCC
TTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCAT
TGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCG
AGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGA
CAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGATTCAAAACGCGCCACC
CGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATC
ATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATG
CACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGAC
TATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATC
TTTATGGCAGCCATCCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGCGGCATCACGGCACATCCT
ATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCA
TACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATC
ACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCG
TTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA
  1    MVAACIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT
 51    YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLLTLESGEF QVYKQSHSAL
101    TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA
151    FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA
201    VISGSVLYNQ AEKGSYSLGI PGGKAQEVAG SAEVKTVNGI RHIGLAAKQL
251    DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL
301    GKIQSHQLGN LMIQQAAIKG NIGYTVRFSD HGHEVHSPFD NHASHSDSDE
351    AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG
401    VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR
451    SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHGLGLL
501    STENKMARIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA
551    IPIKGIGAVR GKYGLGGITA HPIKRSQNGA IALPKGKSAV SDNFADAAYA
601    KYPSPYHSRN IRSNLEQRYG KENITSATVP PSNGKNVKLA DQRHPKTGVP
651    FDGKGFPNFE KHVKYDTLEH HHHHH*
实施例5-287的杂交体
以全长、具有C-未端His-标记或无其前导肽但具有C-未端His-标记的287的表达水平非常低。用N-未端GST-融合实现了较好的表达。将GST用作N-未端融合配体的替代方法，将287置于蛋白质919的C-未端(‘919-287’)、蛋白质953的C-端(‘953-287’)和蛋白质ORF46.1的C-未端(‘ORF46.1-287’)。在这两种方法中，前导肽都是缺失的，且杂交体是直接的符合读杠的融合。
为了制备953-287杂交体，通过设计正向引物自各序列前导区的下游，除去两种蛋白质的前导肽；在953反向引物中除去终止密码子序列，但在287反向引物中包含终止密码子序列。对953基因而言，用于扩增的5′和3′引物分别包括NdeI和BamHI限制酶切位点，而对287基因的扩增而言，5′和3′引物分别包括BamHI和XhoI限制酶切位点。用这种方法，用NdeI-BamHI(克隆第一个基因)且随后用BamHI-XhoI(克隆第二个基因)，可以实现pET21b+中两个基因的顺序定向克隆。
通过将编码287的成熟部分的序列克隆入pET21b+中的919-His克隆的3′-端的XhoI位点，可以得到919-287杂交体。设计用于扩增287基因的引物，从而在PCR片段的5′-引入SalI限制酶切位点并在3′-引入XhoI位点。因为由SalI和XhoI限制酶产生的粘性末端是相容的，因此可以将由SalI-XhoI消化的287PCR产物插入到由XhoI切割的pET21b-919克隆中。
类似地得到ORF46.1-287杂交体。
将针对杂交蛋白产生的抗体的杀菌效力(同源菌株)与针对组分抗原的简单混合物产生的抗体相比：
 与287的混合物  与287的杂交体
 919  32000  16000
 953  8192  8192
 ORF46.1  128  8192
对919-287和953-287而言，还获得了针对异源MenB菌株和针对血清型A和C的杀菌活性的数据：
 919  953 ORF46.1
 菌株  混合物  杂交体  混合物  杂交体 混合物  杂交体
 MC58  512  1024  512  1024 -  1024
 NGH38  1024  2048  2048  4096 -  4096
 BZ232  512  128  1024  16 -  -
 MenA(F6124)  512  2048  2048  32 -  1024
 MenC(C11)  ＞2048  n.d.  ＞2048  n.d. -  n.d.
 MenC(BZ133)  ＞4096  ＞8192  ＞4096 ＜16 -  2048
还构建了ORF46.1和919的杂交体。在N-未端用919得到了最佳结果(高4倍的滴定度)。
还测试了杂交体919-519His、ORF97-225His和225-ORF97His。它们的ELISA滴定度和杀菌抗体应答结果中等。
两种蛋白质A和B的杂交体可以是NH2-A-B-COOH或NH2-B-A-COOH，用ΔG287还制得了在N-未端与287“反向”杂交的杂交体。使用一系列菌株，包括同源菌株2996。FCA用作佐剂：
         287 & 919          287 & 953        287 & ORF46.1
 菌株  ΔG287-919  919-287   ΔG287-953  953-287   ΔG287-461  46.1-287
 2996  128000  16000   65536  8192   16384  8192
 BZ232  256  128   128  ＜4   ＜4  ＜4
 1000  2048  ＜4   ＜4  ＜4   ＜4  ＜4
 MC58  8192  1024   16384  1024   512  128
 NGH38  32000  2048   ＞2048  4096   16384  4096
 394/98  4096  32   256  128   128  16
 MenA(F6124)  32000  2048   ＞2048  32   8192  1024
 MenC(BZ133)  64000  ＞8192   ＞8192  ＜16   8192  2048
通常在N-未端用287观察到较好的杀菌滴定度。
当融合于蛋白质961[如上所示的NH2-ΔG287-961-COOH-序列]时，得到的蛋白质是不溶解的，纯化必须将其变性和复性。在复性后，发现约50％蛋白质仍不溶解。比较可溶和不溶蛋白质，用可溶蛋白质得到更好的杀菌滴定度(FCA作为佐剂)：
2996  BZ232  MC58  NGH38  F6124  BZ133
可溶的 65536  128  4096  ＞2048  ＞2048  4096
不溶的 8192  ＜4  ＜4  16  n.d.  n.d.
但用明矾佐剂替代，可以改善不溶形式的蛋白质的滴定度：
不溶的 32768  128  4096 ＞2048 ＞2048  2048
还在杂交蛋白中使用961c(见上)。由于961及其结构域变体指导有效的表达，它们非常适合作为杂交蛋白的N-未端部分。
实施例23-其它杂交体
附图中显示本发明的其它杂交蛋白，它们具有如下所示的序列。当与单独蛋白质相比时，这此杂交蛋白是有利的：
ORF46.1-741
ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC
CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC
AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC
CATTCCCCCTTGGACAACCATGCCTCCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTAC
CGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCATATGACGGCCACAGGGCGGCGGCTATCCCGCTCCC
AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC
AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA
CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT
AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT
GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA
CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC
AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGGCGGCTCACG
GCACATCCTACAAGCGGTCGCCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC
GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA
GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA
GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGT
GTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCT
TTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAAC
GGTGACAGCCTCATACGGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGAC
GGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACC
GAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAA
CATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGC
GGAAAACTGACCTACCACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAATCGCCAGAACTC
AATGTCGACCTGGCCGCCGCCGATATCAAGCCGATGGAAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAAC
CAAGCCGACAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAA
ACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA
  1    MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ
 51    LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG
101    FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL
151    NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA
201    FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA
251    RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG
301    AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH
351    SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP
401    NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR
451    KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL
501    ESGEFQVYKQ SHSALTAFQT KQIQDSEHSG KMVAKRQFRI GDIAGEHTSF
551    DCLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV
601    DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK
651    TVNGIRHIGL AAKQLEHHHH HH*
ORF46.1-961
ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC
CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC
AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC
CATTCCCCCTTCGACAACCATGCCTCAAATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC
CGCATCCATTGGGACGGATACGAACACCAACCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC
AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC
AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA
CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT
AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT
GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA
CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC
AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG
GCATCCTTCATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC
GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA
GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA
GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA
GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAAC
GGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGAT
GTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAA
CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCC
GCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT
GCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCC
GAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAA
GCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCC
GAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT
GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGC
AAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATT
GCCGATCACGATACTCGCCTGAACGGTTTGGATAAATCAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCA
GAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTAC
AAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAAACTTTGCCGCCAAAGCAGGCGTGCAGTC
GGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCAC
TGA
  1    MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ
 51    LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG
101    FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL
151    NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA
201    FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA
251    RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG
301    AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH
351    SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP
401    NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI
451    YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK
501    VKAAESEIEK LTTDLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET
551    KTNIVKIDEK LEAVADTVDK EAEAFNDIAD SLDETNTKAD EAVKTANEAK
601    QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA
651    DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK
701    SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGRFNVTAA
751    VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE
801    HHHHHH*
ORF46.1-961c
ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC
CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC
AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC
CATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC
CGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC
AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC
AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA
CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT
AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT
GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA
CTCAAAGACTATGGCCGCAGCAGCCATCCGCGTTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC
AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG
GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATGGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC
GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA
GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA
GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA
GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAAC
GGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGAT
GTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCCGTCATGAAAACAAA
CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCC
GCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT
GCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCC
GAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAA
GCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCC
GAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT
GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGC
AAATTTGTCAGAATTGATGGTCTGAACCCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATT
GCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCA
GAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA
  1    MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ
 51    LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG
101    FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL
151    NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA
201    FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA
251    RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG
301    AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH
351    SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP
401    NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI
451    YDIDEDGTTT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK
501    VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET
551    KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK
601    QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDTKA
651    DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK
701    SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH
751    H*
961-ORF46.1
ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC
AACGGTTTCAAAGCTGGAGACACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATGGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA
TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAAGGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC
TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGAAGGCGTGGCA
GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGAGGAGGATCA
GATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTC
GGCAGCAGGGGGAACTTGCCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTG
ATGGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCAGAAGTCCATTCC
CCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATC
CATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGC
GCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACC
GGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCC
ACCCGATACGCCCCGAGCTGGACAGATCGGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAAC
ATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTC
ATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAA
GACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAAT
ATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACAT
CCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCG
GCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAAC
ATCACCTCCTCCACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTA
CCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCAC
TGA
  1    MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
 51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL
101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA
151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK
201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS
251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
301    VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG
351    FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ
401    VLDRQHFEPD GKYHFLGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK
451    GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE
501    HHPADGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL
551    ADRFHNAGSM LTQGVGDGFK RATRYSPELD RSGNAAEAFN GTADIVKNII
601    GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK
651    DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT
701    AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY
751    GKENITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE
801    HHHHHH*
961-741
ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAATATAACGGACA
TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC
TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCA
GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGGGGTGGTGTC
GCCGCCGACTCGGTGGCGGGGGCTTGCCGATGCACTAACCGCACCGCCGACCATAAAGACAAAGGTTTGCAGTCTTTG
ACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGT
GACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGG
CAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAG
CAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACAT
ACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGA
AAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAAT
GTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAA
GCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACC
GTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA
 1     MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
 51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL
101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA
151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK
201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS
251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
301    VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG
351    FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD
401    ALTAPLDHKD KGLQSLTQDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL
451    KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE
501    HSGRMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY
551    TIDFAAKQGN GKIEELKSPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE
601    KGSYSLGIFG GKAQEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH*
961-983
ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA
TTTGCTGAACAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC
TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCCCCAAAGCAGGCGTGGCA
GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGCGGAGGCGGCACT
TCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTA
TCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACA
GACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAG
AATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGC
GAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTAT
ACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTT
ATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATT
GGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGAT
GAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTC
AATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCQACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGC
CAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAAC
CTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACA
TATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAA
AAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCC
ATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCC
TTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGT
ACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCG
GGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCC
TACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAAC
AACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTC
GAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCA
GATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTAC
ACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAG
GGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTC
TTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGAC
ACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTG
AAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACA
CCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCA
GCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGAC
AGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGT
CTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGT
ACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACA
TGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCGACGATGCGGCGATATC
GGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCG
GAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGAT
TTGACGGTCGAAGGCGGTCTGCGCTACGGCCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGG
AGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCC
GTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCG
ACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAA
TTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTC
GGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA
   1    MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
  51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL
 101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA
 151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK
 201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS
 251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
 301    VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG
 351    FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT
 401    GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA
 451    PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI
 501    SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE
 551    AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMNTNDET
 601    KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR
 651    QALLDYSGGD KTDEGIRLMQ QSDYGNLSYH IRNKNMLFIF STGNDAQAQP
 701    NTYALLPFYE KDAQKGIITV AGVDRSGKKF FREMYGEPGT EPLEYGSNHC
 751    GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM
 801    SNDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD
 851    TKGTSDIAYS FRNDISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL
 901    YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK
 951    GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA GYLNSTGRRV
1001    PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN
1051    AARTASAAAH SAPAGLKHAV EQGGSNLENL MVELDASESS ATPETVETAA
1101    ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA
1151    DMQGRRLKAV SDGLDHNGTG LKVIAQTQQD GGTWEQGGVE GKMRGSTQTV
1201    GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY
12S1    LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD
1301    LTVEGGLRYD LLKQADFAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD
1351    KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG
1401    ADVEFGNGWN GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH*
961c-ORF46.1
ATGGCCACAAACGACGACGATGTTAAAAAAGCGTCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA
TTTGCTGAAGAGACTAAGACAAATATGTAAAAATTGATGAAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGGCCAT
GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCA
AACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGG
GGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAA
CAGGCGGCCTTAAAGGAAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGAC
AACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGAC
GGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGAT
ATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGG
CTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATAC
AGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGC
GCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGC
TTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCC
GCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATG
GCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAG
CGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCC
AAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCC
TCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGAC
GGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA
  1    MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
 51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE AEIEKLTTKL
101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNTV KIDEKLEAVA
151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEALQTAEE TKQNVDAKVK
201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS
251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
301    VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRQ
351    HFEPDGKYHL FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY
401    IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD
451    GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH
501    NAGSMLTQGV GDGGFRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE
551    IVGAGDAVQG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA
601    AIRDWAVQNP NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK
651    RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI
701    TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH
751    H*
961c-741
ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA
TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGAC
ATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGAT
CAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTC
AATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATT
ACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAA
GATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTT
GACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACC
TACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTG
GCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAA
GGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGC
ATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA
  1    MATNDDDVKK AATVATAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
 51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVKAKVKAAE SEIEKLTTKL
101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNTV KIDEKLEAVA
151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK
201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDTKADIATN KDNIAKKANS
251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
301    VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP
351    LDHKDKGLQS LTLDQSVRKN EKLDLAAQGA EKTYGNGDSL NTGKLKNDKV
401    SRFDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ IQDSEHSGKM
451    VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA
501    AKQGNGKIEH LKSPELNVDL AAADIKPDGK RHAVISGSVL YNQAEKGSYS
551    LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH*
961c-983
ATGGCCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAATC
AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC
GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC
AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT
GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA
TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT
GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT
GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA
GCCGAAGCTGCCGCCTGGACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA
GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC
AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC
ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT
GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCC
GACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCC
GGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGAT
GCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATCAAGAAGTTTGATC
AACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTC
GGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTAT
ATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACT
GAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGT
TCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAG
AACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGT
TTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTG
CTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTAC
CACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTA
TTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAA
CGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGC
CTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCA
CCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTG
CTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCC
ATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTC
CGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTAT
ACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAA
GGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGAC
CAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCCTGGACGGCAAAGGTACGCTGTACACACGTTG
GGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGC
TATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAAC
ATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCC
TATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCC
GTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACG
GTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCC
GTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCC
GCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAAGGGCACGGGTCTGCGCGTC
ATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACC
GTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAA
AACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTC
AAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGC
GTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTC
GAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAAC
AGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTT
GCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCA
ACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAAC
GGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGC
TACCGGTTCCTCGAGCACCACCACCACCACCACTGA
  1    MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT
 51    AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL
101    ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA
151    DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK
201    AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS
251    ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT
 301    VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN
 351    SRATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL
 401    HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL
 451    YGRKEHGYNE NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD
 501    IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM
 551    VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD
 601    YSGGDKTDEG IRLMQQSDYG NLSYHIRNKN MLFIFSTGND AQAQPNTYAL
 651    LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNHCGITAM
 701    WCLSAPYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL
 751    RTTLLTTAQD IGAVGVDSKF GWGLLDAGKA MNGPASFPFG DFTADTKGTS
 801    DIAYSFRNDI SGTGGLIKKG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK
 851    SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL
 901    DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA
 951    AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA
1001    SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD
1051    MPGIRPYGAT FRAAAAVQHA NAADGVRIFN SLAATVYADS TAAHADMQGR
1101    RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGKMRG STQTVGIAAK
1151    TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF
1201    SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG
1251    GLRYDLLKQD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS QPLSDKAVLF
1301    ATAGVERDLN GRDYTVTGGF TGATAATGDT GARNMPHTRL VAGLGADVEF
1351    GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH*
961cL-ORF46.1
ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC
GACGACGATGTTAAAAAAGCTGCCACCTGTGGCCATGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA
GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC
GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC
GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA
GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG
ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC
AACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAG
ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC
GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT
ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC
AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC
GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC
GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTT
ATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCC
GAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATT
AAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACGATGCCTCA
CATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACAC
CATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTAC
GACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGT
TTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTG
GAAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGGAA
ATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTT
TCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATC
CGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCC
ATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATG
GGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCC
CCTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCCTCAACCGTGCCG
CCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTT
CCGAATTTTGAGAAGCACGTGAAATATGATACGTAACTCGAG
  1    MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING
 51    FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN
101    KQNVDAKVDA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI
151    TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV
201    KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA
251    KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT
301    RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG
351    GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFGSRGELA ERSGHIGLGK
401    IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDEAG
451    SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIKGVA
501    QNIRLNLTDN RSTGQRLADR FHNAGSMLTQ GVGKGFKRAT RYSPELDRSG
551    NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VAHGLGLLST
601    ENKMARINDL ADMAQLKDYA AAAIRDWAVQ NPNAAQGIEA VSNITMAAIP
651    IKGIGAVRGK YGLGGITAHP IKRSQMGAIA LPKGKSAVSD NFADAAYAKY
701    PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGKNVKLADQ RHPKTGVPFD
751    GKGFPNFEKH VKYDT*
961cL-741
ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC
GACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA
GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC
GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC
GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA
GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG
ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC
AACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAG
ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC
GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT
ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC
AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC
GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC
GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGG
CTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGG
AAAAACGAGAACTGAAGCCTGGCGGCACAAGGTGCGGAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAAA
TTGAAGAACGACAAGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGAACGGGCAGCTCATTACCTTGGAGAGT
GGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCAT
TCCCGGGAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCC
GAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGAT
TTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGAT
ATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCC
CTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATC
GGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA
  1    MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING
 51    FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN
101    KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI
151    TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKAKEAV
201    DTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA
251    KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT
301    RLASAEKSIA DHDTRLNGLD KTVSDLRDET RQGLAEQAAL SGLFQPYNVG
351    GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ
401    GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ
451    SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT
501    YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD
551    GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QKVAGSAEVK TVNGIRHIGL
601    AAKQLEHHHH HH*
961cL-983
ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC
GACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA
GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC
GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC
GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA
GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG
ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC
AACGATATCGCCGATTCATTGGATGAAACCAACACTTAAGGCAGACGAAGCCGTCAAAACGCCAATGAAGCCAAACAG
ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC
GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT
ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC
AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC
GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC
GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCA
GGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAAC
GAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAAT
GCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCT
GCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCC
TTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAA
GCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCG
ACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGC
AGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATG
GTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACA
TCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCC
GGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAAT
AAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTAT
GAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTAT
GGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCC
TATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACC
GGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCT
CAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCC
GCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATT
TCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACC
ATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATT
TATAACGGGGCGGCAATCCGGCGGAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCA
AACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTG
AAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGT
ACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGAC
GGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGT
CGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGC
GGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCG
GCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCG
AATGCCGCCGACGGTGTACGCACTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCCGCCCATGCCGAT
ATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACC
CAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCC
GCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAAT
GCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTC
TCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACG
CTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTG
CGCTACGACCCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTACTGAA
GGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGC
GTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACG
GGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGC
TTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCTGA
CTCGAG
   1    MKHFPSHVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING
  51    FKAGETIYDI DEDGTITKKD ATAADVKADD FKGLGLKKVV TNLTKTVNEN
 101    KQNVDAKVKA AESEIKKLTT KLADTDAALA DTDAALDATT NALNKLGENI
 151    TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV
 201    KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA
 251    KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT
 301    RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG
 351    GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC
 401    AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG
 451    RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG
 501    GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG
 551    GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR
 601    AGTADAFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNLSYHIRN
 651    KNMLFIFSTG NDAQAQPNTY ALLPFYKKDA QKGIITVAGV DRSGEKFKRE
 701    MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFTRTNP IQIAGTSFSA
 751    PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG
 801    KAMNGPASFP FGKFTADTKG TSDIAYSFRN DISGTGGLIK KGGSQLQLHG
 851    NNTYTGKTII EGGSLVLYGN NKSKMRVETK GALIYNGAAS GGSLNSDGIV
 901    YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLYM
 951    SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK
1001    TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE
1051    LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI
1101    FNSLAATVYA DTAAAHADMQ GRRLKAVSDG LDHNGTGLRV IAQTQQDGGT
1151    WEQGGVEGKM RGSTQTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD
1201    SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM
1251    QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE
1301    GTLVGLAGLK LSQPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG
1351    KTGARNMPHT RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG
1401    YRF*
可以理解本发明仅以实施例的方式进行描述，在本发明的范围和精神内还可进行改变。例如，设想可以使用其它菌株的蛋白质[如，参见WO 00/66741，ORF4、ORF40、ORF46、225、235、287、519、726、919和953的多态序列]。
实验详述
克隆策略和寡核苷酸设计
用以脑膜炎奈瑟球菌B MC58的基因组序列为基础设计的寡核苷酸，通过PCR扩增编码感兴趣的抗原的基因。除非特别指出，通常将菌株2996的基因组DNA用作PCR反应的模板，将扩增的片段克隆入表达载体pET21b+(Novagen)，从而以C-未端His标记的产物形式表达该蛋白，或将其克隆入pET-24b+(Novagen)以‘未标记的’形式(如ΔG287K)表达该蛋白。
不用融合配体和用其自身前导肽(如果存在时)表达蛋白质，进行开放读框(ATG到终止密码子)的扩增。
当蛋白质以‘未标记的’的形式表达时，通过从预定的前导序列设计5′-端扩增引物下游除去前导肽。
用于PCR的引物的解链温度取决于整个引物中杂交核苷酸的数量和类型，并用以下公式确定：
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)
对整个低聚物而言，所选寡核苷酸的解链温度通常为65-70℃，仅针对杂交区域，解链温度为50-60℃。
用Perkin Elmer 394 DNA/RNA合成仪合成寡核苷酸，将其从柱上洗脱到2.0ml NH4OH中，在56℃培育5小时以去保护。加入0.3M乙酸钠和2体积的乙醇沉淀寡聚物。将样品离心，将沉淀物重悬浮于水中。
 序列 限制酶切位点
 fu(961)-  Fwd  CGC GGATCC-GGAGGGGGTGGTGTCG BamHI
 741(MC58)-His
 Rev  CCCG CTCGAG-TTGCTTGGCGGCAAGGC XhoI
 fu(961)-983-His  Fwd  CGC GGATCC-GGCGGAGGCGGCACTT BamHI
 Rev  CCCG CTCGAG-GAACCGGTAGCCTACG XhoI
  fu(961)-Orf46.1-His  Fwd  CGC GGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC BamHI
 Rev  CCCG CTCGAG-CGTATCATATTTCACGTGC XhoI
  fu(961c-L)-741(MC58)  Fwd  CGC GGATCC-GGAGGGGGTGGTGTCG BamHI
 Rev  CCCG CTCGAG-TTATTGCTTGGCGGCAAG XhoI
  fu(961c-L)-983  Fwd  CGC GGATCC-GGCGGAGGCGGCACTT BamHI
 Rev  CCCG CTCGAG-TCAGAACCGGTAGCCTAC XhoI
  fu(961c-L)-Orf46.1  Fwd  CGC GGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC BamHI
 Rev  CCCG CTCGAG-TTACGTATCATATTTCACGTGC XgoI
  Fu-(ΔG287-919-His  Fwd  CGC GGATCCGGTGGTGGTGGT-CAAAGCAAGAGCATCCAAACC BamHI
 Rev  CCC AAGCTT-TTCGGGCGGTATTCGGGCTTC HindIII
  fu-(ΔG287)-953-His  Fwd  CGC GGATCCGGTGGTGGTGGT-GCCACCTACAAAGTGGAC BamHI
 Rev  GCCC AAGCTT-TTGTTTGGCTGCCTCGGAT HindIII
  fu-(ΔG287)-961-His  Fwd  CGC GGATCCGGTGGTGGTGGT-ACAAGCGACGACG BamHI
 Rev  GCCC AAGCTT-CCACTCGTAATTGACGCC HindIII
  fu-(ΔG287)-Orf46.1-His  Fwd  CGC GGATCCGGTGGTGGTGCT-TCAGATTTGGCAAACGATTC BamHI
 Rev  CCC AAGCTT-CGTATCATATTTCACGTGC HindIII
  fu-(ΔG287-919)-Orf46.1-His  Fwd  CCC AAGCTTGGTGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC HindIII
 Rev  CCC GCTCGAG-CGTATCATATTTCACGTGC XboI
  fu-(ΔG287-Orf46.1)-919-His  Fwd  CCC AAGCTTGGTGGTGGTGGTGGT-CAAAGCAAGAGCATCCAAACC HindIII
 Rev  CCC GCTCGAG-CGGGCGGTATTCGGGCTT XhoI
  fuΔG287(394.98)-…  Fwd  CGCGGATCC GCTAGC-CCCGATGTTAAATCGGC NheI
 Rev  CGG GGATCC-ATCCTGCTCTTTTTTGCCGG BamHI
  fu Orf1-(Orf64.1)-His  Fwd  CGCGGATCC GCTAGC-GGACACACTTATTTCGGCATC NheI
 Rev  CGCGGATCC-CCAGCGGTAGCCTAATTTGAT
  fu(Orf1)-orf6.1-His  Fwd  CGC GGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC BamHI
 Rev  CCC AAGCTT-CGTATCATATTTCACGTGC HindIII
  fu(919))-Orf46.1-His  Fwd1  GCGGC GTCGACGGTGGCGGAGGCACTGGATCCTCAG SalI
 Fwd2  GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
 Rev  CCC GCTCGAG-CGTATCATATTTCACGTGC XhoI
  Fu(orf46)-287-His  Fwd  CGG GGATCCGGGGGCGGCGGTGGCG BamHI
 Rev  CCC AAGCTTATCCTGCTCTTTTTTGCCGGC HindIII
  Fu(orf46)-919-His  Fwd  CGC GGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACC BamHI
 Rev  CCC AAGCTTCGGGCGGGTATTCGGGCTTC HindIII
  Fu(orf46-919)-287-His Fwd  CCCC AAGCTTGGGGGCGGCCGGTGGCG HindIII
Rev  CCCG CTCGAGATCCTGCTCTTTTTTGCCGGC XhoI
  Fu(orf46-287)-919-His Fwd  CCC AAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACC HindIII
Rev  CCCG CTCGAGCGGGCGGGTATTCGGGCTT XhoI
  (ΔG741)-961c-His Fwd1Fwd2  GGAGGCACTGGATCCGCAGCCACAAACGACGACGAGCGGC CTCGAG-GGTGGCGGAGGCACTGGATCCGCAG XhoI
Rev  CCCG CTCGAG-ACCCAGCTTGTAAGGTTG XhoI
  (ΔG741)-961-His Fwd1Fwd2  GGAGGCACTTGGATCCGCAGCCACAAACGACGACGAGCGGC CTCGAG-GGTGGCGGAGGCACTGGATCCGCAG XhoI
Rev  CCCG CTCGAG-CCACTCGTAATTGACGCC XhoI
  (ΔG741)-983-His Fwd  GCGG CTCGAG-GGATCCGGCGGAGGCGGCACTTCTGCG XhoI
Rev  CCCG CTCGAG-GAACCGGTAGCCTACG XhoI
  (ΔG741)-orf46.1-His Fwd1Fwd2  GGAGGCACTGGATCCTCAGATTTGGCAAACGATTCGCGGC GTCGACGGTGGCGGAGGCACTGGATCCTCAGA SalI
Rev  CCCG CTCGAG-CGTATCATATTTCACGTGC XhoI
  (ΔG983)-741(MC58)-His Fwd  GCGGC CTCGAG-GGATCCGGAGGGGGTGGTGTCGCC XhoI
Rev  CCCG CTCGAG-TTGCTTGGCGGCAAG XhoI
  (ΔG983)-961c-His Fwd1Fwd2  GGAGGCACTGGATCCGCAGCCACAAACGACGACGAGCGGC CTCGAG-GGTGGCGGAGGCACTGGATCCGCAG XhoI
Rev  CCCG CTCGAG-ACCCAGCTTGTAAAGGTG XhoI
  (ΔG983)-961-His Fwd1Fwd2  GGAGGCACTGGATCCCGCAGCCCACAAACGACGACGAGCGGC CTCGAG-GGTGGCGGAGGCACTGGATCCGCAG XhoI
Rev  CCCG CTCGAG-CCACTCGTAATTGACGCC XhoI
  (ΔG983)-Orf46.1-His Fwd1Fwd2  GGAGGCACTGGATCCTCAGATTTGGCAAACGATTCGCGGC GTCGACGGTGGCGGAGGCACTGGATCCTCAGA SalI
Rev  CCCG CTCGAG-CGTATCATATTTCACGTGC XhoI
*将该引物用作反向引物，将所有287的C未端融合于His-标记。
§与287-His反向引物联用的正向引物。
NB-所有PCR反应使用菌株2996，除非特别指出(如菌株MC58)。
在所有以ATG起始不跟随唯一NheI位点的构建物中，ATG密码子是用于克隆的NdeI位点的一部分。在5′端用NheI作为克隆位点制备的构建物(如所有那些在N-未端包含287的)另有两个融合于抗原的编码序列的密码子(GCTAGC)。
染色体DNA模板的制备
在100ml GC培养基中使脑膜炎奈瑟球菌前株2996、MC58、394.98、1000和BZ232(及其它)生长至指数期，离心收获，并重悬浮于5ml缓冲液(20％w/v蔗糖、50mM Tris-HCl、50mM EDTA、pH8)中。在冰上培育10分钟后，加入10ml裂解液(50mM NaCl、1％十二烷基肌氨酸钠(Na-Sarkosyl)、50μg/ml蛋白酶K)裂解细菌，悬浮液在37℃培育2小时。进行2次苯酚提取(平衡至pH8)和一次CHCl3/异戊醇(24∶1)提取。加入0.3M乙酸钠和2体积乙醇沉降DNA，并离心收集。用70％(v/v)乙醇洗涤沉淀物1次，并重溶解于4.0ml TE缓冲液(10mM Tris-HCl、1mM EDTA、pH8.0)。读取OD260测定DNA浓度。
PCR扩增
标准的PCR过程：进行如下在40μM各寡核苷酸引物、400-800μM dNTP溶液、1x PCR缓冲液(包含1.5mM MgCl2)、2.5单位TaqI DNA聚合酶(用Perkin-Elmer AmpliTaQ，Boerhingher Mannheim ExpandTM长模板)存在时，将200ng 2996、MC581000、或BZ232菌株的基因组DNA或10ng重组克隆的质粒DNA制备物用作模板。
将整个混合物在95℃初步培育3分钟后，每份样品进行2-步的扩增：用除去引物(Tm1)的限制酶尾部的杂交温度进行前5轮。然后按全长低聚物(Tm2)计算的杂交温度进行30轮。根据要扩增的Orf的长度，在68℃或72℃进行的延伸时间各不相同。对Orf1而言，自3分钟开始的延伸时间每轮递增15秒。以在72℃ 10分钟延伸步骤完成循环。
将扩增的DNA直接加到1％琼脂糖凝胶上。按制造商的说明，用Qiagen凝胶提取试剂盒纯化相应于正确大小条带的DNA片段。
PCR片段和克隆载体的消化
用适合的限制酶消化相应于扩增片段的纯化DNA，从而克隆入pET-21b+、pET22b+、或pET-24b+。用QIAquick PCR纯化试剂盒(按制造商的说明)纯化消化的片段，用H2O或10mM Tris(pH8.5)洗脱。用适合的限制酶消化质粒载体，加到1.0％琼脂糖凝胶上，用Qiagen AIQquick凝胶提取试剂盒纯化相应于消化的载体的条带。
克隆
将预先消化和纯化的、相应于各基因的片段连接到pET21b+、pET22b+或pET-24b+中。将在连接缓冲液(由制造商提供)中的T4 DNA连接酶用于摩尔比为3∶1的片段/载体。
通过在冰上培育连接酶反应溶液和细菌40分钟，然后在37℃培育3分钟，将重组质粒转化入感受态大肠杆菌DH5或HB101中。
然后添加800μl LB肉汤，并在37℃培育20分钟。在Eppendorf微型离心机中以最高速度离心这些细胞，并重悬浮于约200μl的上清液中，并涂布在LB氨苄青霉素(100mg/ml)琼脂上。
在4.0ml LB肉汤+100μg/ml氨苄青霉素中培育随机选择的集落过夜，进行重组集落的筛选。使细胞沉淀，并按制造商的说明用Qiagen QIAprep SpinMiniprep试剂盒提取质粒DNA。用适合的限制酶消化约1μg的各微型制备物，将消化物加到1-1.5％琼脂糖凝胶(取决于预计的插入大小)与分子量标记(1kbDNA Ladder，GIBCO)平行。根据插入的大小选择阳性克隆。
表达
各基因克隆入表达载体后，将重组质粒转化入适合表达重组蛋白的大肠杆菌菌株中。如上所述，用1μl各构建物转化大肠杆菌BL21-DE3。将单重组集落接种入2ml LB+Amp(100μg/ml)中，在37℃培育过夜，然后在100ml烧瓶中用20ml LB+Amp(100μg/ml)以1∶30稀释，使OD600在0.1-0.2之间。将烧瓶置于旋转式水浴摇床中于30℃或37℃培育，直到OD600显示适合诱导表达的指数生长期(0.4-0.8OD)。加入1.0mM IPTG诱导蛋白质表达。在30℃或37℃培育3小时后，测定OD600并检测表达。用微型离心机离心1.0ml各样品，将沉淀物重悬浮于PBS中，用SDS-PAGE和考马斯蓝染色分析。
His-标记的蛋白质的纯化
从菌株2996和MC58中克隆了287的各种形式。用C-未端His标记的融合体进行构建，其包括成熟形式(aa18-427)、含缺失(Δ1、Δ2、Δ3和Δ4)的构建物和由B或C结构域组成的克隆。对以His-融合体纯化的各克隆而言，划线接种单集落，并且在37℃ LB/Amp(100μg/ml)琼脂板上培育过夜。将从该平板上分离的集落接种到20ml LB/Amp(100μg/ml)的液体培养基中，并在37℃振荡生长过夜。以1∶30将过夜培养物稀释到1.0L LB/Amp(100μg/ml)液体培养基中，让其在最佳温度(30或37℃)生长，直到OD550达到0.6-0.8。添加IPTG(终浓度为1.0mM)诱导重组蛋白的表达，再培育培养物3小时。于4℃，以8000g离心15分钟收获细菌。将细菌沉淀物重悬浮于7.5ml(i)冷缓冲液A(300mMNaCl、50mM磷酸盐缓冲液、10mM咪唑、pH8.0)，用于可溶性蛋白质；或(ii)缓冲液B(10mM Tirs-HCl、100mM磷酸盐缓冲液，pH 8.8和任选地8M尿素)，用于不溶性蛋白质。以可溶形式纯化的蛋白质包括287-His、Δ1、Δ2、Δ3和Δ4287-His、Δ4287MC58-His、287c-His和287cMC58-His。蛋白质287bMC58-His是不溶的并相应地纯化。用Branson Sonifier 450，在冰上以40W、30秒超声处理破坏细胞4次，并于4℃以13000xg离心30分钟。对于不溶蛋白质，将沉淀物重悬浮于2.0ml缓冲液C(6M盐酸胍、100mM磷酸盐缓冲液、10mM Tris-HCl、pH7.5)，并用Dounce匀浆器处理10次。以13000g离心匀浆30分钟并保留上清液。将可溶的和不溶制备物的上清液与150μl Ni2+-树脂(预先用缓冲液A或缓冲液B平衡)混合，并在室温温和振荡培育30分钟。树脂是按制造商说明制备的Chelating Sepharose Fast Flow(Pharmcia)。于4℃，以700g离心分批制备物5分钟，弃去上清液。用10ml缓冲液A或B洗涤树脂2次(分批)10分钟，重悬浮于1.0ml缓冲液A或B中，加到一次性柱上。用(i)缓冲液A(4℃)或(ii)缓冲液B(室温)持续洗涤树脂，直到流出物的OD280达到0.02-0.01。再用(i)冷缓冲液C(300mM NaCl、50mM磷酸盐缓冲液、20mM咪唑、pH8.0)或(ii)缓冲液D(10mM Tris-HCl、100mM磷酸盐缓冲液、pH6.3和任选地8M尿素)进一步洗涤树脂，直到流出物的OD280达到0.02-0.01。加入700μl(i)冷洗脱缓冲液A(300mM NaCl、50mM磷酸盐缓冲液、250mM咪唑、pH8.0)或(ii)洗脱缓冲液B(10mM Tris-HCl、100mM磷酸盐缓冲液、pH4.5和任选地8M尿素)洗脱His-融合蛋白，收集组分直到OD280显示获得了所有重组蛋白。用SDS-PAGE分析20μl量的各洗脱组分。用Bradford试验法计算蛋白质浓度。
变性的His-融合蛋白的复性
需要变性以稳定287bMC8，因此在免疫接种前需进行复性步骤。将甘油加到上述得到的变性组分中，使终浓度为10％v/v。用透析缓冲液I(10％v/v甘油，0.5M精氨酸、50mM磷酸盐缓冲液、5.0mM还原的谷胱甘肽、0.5mM氧化的谷胱甘肽、2.0M尿素，pH8.8)将蛋白质稀释至200μg/ml，用相同的缓冲液在4℃透析12-14小时。于4℃，用缓冲液II(10％v/v甘油，0.5M精氨酸、50mM磷酸盐缓冲液、5.0mM还原的谷胱甘肽、0.5mM氧化的谷胱甘肽、pH8.8)再进行透析12-14小时。用以下公式计算蛋白质的浓度：
蛋白质(mg/ml)＝(1.55×OD280)-(0.76×OD260)
免疫接种
在第0、21和35天，用抗原免疫接种Balb/C小鼠，在第49天分析血清。
血清分析-ELISA
将不包囊的MenB M7和包囊的菌株置于巧克力琼脂板上，在37℃、5％CO2中培育过夜。用无菌Dracon刷从琼脂板上收集细菌菌落，并接种到含有0.25％葡萄糖的Mueller-Hinton肉汤(Difco)中。每30分钟监测一次细菌的生长，随后测定OD620。让细菌生长直到OD达到0.4-0.5。将培养物以4000rpm离心10分钟。弃去上清液，用PBS洗涤细菌2次，重悬浮于含有0.025％甲醛的PBS中，并在37℃培育1小时，然后在4℃搅拌培育过夜。在96孔Greiner板的各孔中添加100μl细菌细胞，并在4℃培育过夜。然后用PBT洗涤缓冲液(0.1％Tween-20的PBS溶液)冲洗这些孔3次。在各孔中加入200μl饱和缓冲液(2.7％聚乙烯吡咯烷酮10的水溶液)，将这些平板在37℃培育2小时。用PBT冲洗这些孔3次。在各孔中加入200μl稀释的血清(稀释缓冲液：1％BSA、0.1％Tween-20、0.1％NaN3的PBS溶液)，将这些平板在37℃培育2小时。用PBT冲洗这些孔3次。在各孔中加入100μl HRP-缀合的兔抗-小鼠(Dako)血清(用稀释缓冲液以1∶2000稀释)，将这些平板置于37℃培育90分钟。用PBT缓冲液洗涤这些孔3次。在各孔中加入100μl HRP的底物缓冲液(25ml柠檬酸缓冲液pH5，10mg邻-苯二胺(phenildiamine)和10μl H2O2)，并将平板在室温中静置20分钟。在各孔中加入100μl 12.5％H2SO4，随后测定OD490。计算ELISA滴定度，即高于预先免疫血清稀释度的、OD490值为0.4的血清稀释度。当OD490值为0.4的血清稀释度高于1∶400时，将ELISA视为阳性。
血清分析-FACS扫描细菌结合分析
将不包囊的MenB M7菌株置于巧克力琼脂板上，在37℃、5％CO2中培育过夜。用无菌Dracon刷从琼脂板上收集菌落，并接种到4支装有0.25％葡萄糖的8ml Mueller-Hinton肉汤(Difco)试管中。每30分钟监测细菌的生长，然后测定OD620。让细菌生长直到OD达到0.35-0.5。将培养物以4000rpm离心10分钟。弃去上清液，用封阻缓冲液(1％BSA的PBS溶液，0.4％NaN3)重悬浮沉淀物，并以4000rpm离心5分钟。将细胞重悬浮于封阻缓冲液，使OD620达到0.05。在96孔Costar板的各孔中添加100μl细菌细胞。在各孔中加入100μl稀释的(1∶100、1∶200、1∶400)血清(以封阻缓冲液配制)，并将这些平板在4℃培育2小时。以4000rpm离心细胞5分钟，吸出上清液，在各孔中加入200μl/孔封阻缓冲液洗少涤细胞。在各孔中加入100μl R-Phicoerytrin缀合的F(ab)2山羊抗-小鼠(以1∶100稀释)，将平板置于4℃培育1小时。以4000rpm离心5分钟以沉淀细胞，添加200μl/孔封阻缓冲洗涤细胞。吸出上清液，细胞重悬浮于200μl/孔PBS、0.25％甲醛。将样品转移到FACScan管中并读数。FACScan(LaserPower 15mW)的条件设定为：FL2开；FSC-H阈值：92；FSC PMT电压：E01；SSCPMT：474；Amp.Gains 6.1；FL-2 PMT：586；补偿值：0。
血清分析-杀菌试验
于37℃，在5％CO2、在巧克力琼脂板(以冷冻原液起始)上培育脑膜炎奈瑟球菌菌株2996过夜。收集菌落，并将其接种到7ml含有0.25％葡萄糖的Mueller-Hinton肉汤中，使OD620达到0.05-0.08。在37℃振荡培育约1.5小时，直到OD620达到0.23-0.24。用50mM磷酸盐缓冲液(pH7.2，含10mM MgCl2、10mMCaCl2和0.5％(w/v)BSA(分析缓冲液))以105CFU/ml工作稀释度稀释细菌。最终反应混合物的总体积为50μl，其中25μl连续2倍稀释的测试血清，12.5μl工作稀释度的细菌，12.5μl幼兔补体(终浓度25％)。
对照包括：用补体血清培育的细菌、用细菌培育并在56℃加热30分钟补充灭活的免疫血清。在加入幼兔补体后，用斜置方法立即将10μl对照置于Mueller-Hinton琼脂板上(0时间)。37℃旋转培育96孔板1小时。将每份样品的7μl涂布在Mueller-Hinton琼脂板上作为斑点，而用斜置方法将10μl对照涂布在Mueller-Hinton琼脂板上(1时间)。37℃培育琼脂板18小时，计算相应于0时间和1时间的菌落数量。
血清分析-Western印迹法
将纯化的蛋白质(500ng/泳道)、外膜小泡(5μg)和MenB菌株2996衍生的全细胞提取物(25μg)加到12％SDS-聚丙烯酰胺凝胶上，并转移到硝基纤维素膜上。用转化缓冲液(0.3％Tris碱，1.14％甘氨酸、20％(v/v)甲醇)在4℃、150mA进行转化2小时。于4℃，在饱和缓冲液(10％脱脂乳、0.1％Triton X100的PBS溶液)中培育过夜使膜饱和。用冲洗缓冲液(3％脱脂乳、0.1％Triton X100的PBS溶液)冲洗膜2次，并在37℃与用洗涤缓冲液以1∶200稀释的小鼠血清一起培育2小时。冲洗膜2次，与1∶2000稀释的辣根过氧化物酶标记的抗-小鼠Ig一起培育90分钟。用0.1％Triton X100的PBS溶液冲洗膜2次，用Opti-4CN底物试剂盒(Bio-Rad)显色。加入水终止反应。
如下制备OMV：于37℃、5％CO2，在5个GC板上让脑膜炎奈瑟球菌2996生长过夜，用接种环收获，并重悬浮于10ml 20mM Tris-HCl pH7.5、2mM EDTA中。在56℃热灭活45分钟，在冰上超声处理破碎细胞5分钟(50％负载循环(dutycycle)、50％输出，Branson超声仪3mm微型针头)。以5000g离心10分钟除去未被破坏的细胞，回收含有完整细胞包膜组分的上清液，于4℃以50000g进一步离心过夜。将含有膜的沉淀物重悬浮于2％二烷基肌氨酸钠、20mM Tris-HClpH7.5、2mM EDTA中，在室温培育20分钟以溶解内膜。以10000g离心上清液10分钟除去聚集体，以50000g进一步离心上清液3小时。用PBS冲洗含有外膜的沉淀物，并重悬浮于相同的缓冲液中。用BSA作为标准，由D.C.Bio-Rad蛋白分析(改进的Lowry方法)测定蛋白质的浓度。
如下制备全细胞提取物：使脑膜炎奈瑟球菌在GC板上培育过夜，用接种环收获，并重悬浮于1ml 20mM Tris-HCl中。在56℃热灭活30分钟。

Claims (28)

1.一种同时异源表达本发明两种或多种蛋白质的方法，其特征在于，所述的方法中(a)所述的本发明的两种或多种蛋白质是融合的。
2.如权利要求23所述的方法，其特征在于，所述的两种或多种蛋白质是：(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.如权利要求24所述的方法，其特征在于，所述的287在蛋白质(a)、(b)或(c)的C-未端。
4.如权利要求1-3任一所述的方法，其特征在于，所述的表达是在大肠杆菌宿主中。
5.由权利要求1-4任一所述的方法表达的蛋白质。
6.式NH2-A-B-COOH的杂交蛋白，其特征在于，所述的A和B是不同的奈瑟球菌蛋白。
7.如权利要求6所述的蛋白质，其特征在于，所述的A和B各选自：orf1、orf4、orf25、orf40、Orf46、orf83、233、287、2921、564、687、741、907、919、953、961和983。
8.权利要求7所述的蛋白质，其特征在于，所述的A和B各选自：ORF46.1、287、741、919、953、961和983。
9.如权利要求8所述的蛋白质，其特征在于，所述的ORF46.1、287、741、919、953、961和983中的至少一种是以其基本全长的形式使用的。
10.如权利要求8或9所述的蛋白质，其特征在于，所述的ORF46.1、287、741、919、953、961和983中的至少一种有缺失。
11.如权利要求10所述的蛋白质，其特征在于，所述的A和/或B具有聚-甘氨酸缺失(‘ΔG’)。
12.如权利要求11所述的蛋白质，其特征在于，所述的A和/或B是ΔG-287、ΔGTbp2、ΔG741或ΔG983。
13.如权利要求10所述的蛋白质，其特征在于，所述的A和/或B是截短的蛋白质。
14.如权利要求13所述的蛋白质，其特征在于，所述的A和/或B是Δ1-287、Δ2-287、Δ3-287或Δ4-287。
15.如权利要求10所述的蛋白质，其特征在于，所述的A和/或B的结构域有缺失。
16.如权利要求15所述的蛋白质，其特征在于，所述的A和/或B是287B、287C、287BC、ORF461-433、ORF46433-608、ORF46或961c。
17.如权利要求6所述的蛋白质，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.如权利要求17所述的蛋白质，其特征在于，所述的蛋白质是ΔG287-919、ΔG287-953、ΔG287-961、ΔG983-ORF46.1、ΔG983-741、ΔG983-961、ΔG983-961C、ΔG741-961、ΔG741-961C、ΔG741-983、ΔG741-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.如权利要求8所述的蛋白质，其特征在于，所述的A或B是287。
20.如权利要求19所述的蛋白质，其特征在于，所述的B是287。
21.如权利要求19所述的蛋白质，其特征在于，所述的A是ΔG-287。
22.如权利要求21所述的蛋白质，其特征在于，所述的B是ORF46、919、953或961。
23.如权利要求19-22任一所述的蛋白质，其特征在于，所述的287来自菌株2996或394/98。
24.如权利要求8所述的蛋白质，其特征在于，所述的A是961。
25.如权利要求6-24任一所述的蛋白质，其特征在于，所述的A和B来自相同的菌株。
26.如权利要求6-24任一所述的蛋白质，其特征在于，所述的A和B直接相连。
27.如权利要求6-24任一所述的蛋白质，其特征在于，所述的A和B通过接头肽相连。
28.如权利要求27所述的蛋白质，其特征在于，所述的接头肽是聚-甘氨酸接头，但条件是B不是ΔG蛋白。
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