CN1279718A - 包含HIV－1Tat和/或Nef蛋白的融合蛋白 - Google Patents
包含HIV－1Tat和/或Nef蛋白的融合蛋白 Download PDFInfo
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- CN1279718A CN1279718A CN98811432A CN98811432A CN1279718A CN 1279718 A CN1279718 A CN 1279718A CN 98811432 A CN98811432 A CN 98811432A CN 98811432 A CN98811432 A CN 98811432A CN 1279718 A CN1279718 A CN 1279718A
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Abstract
本发明提供(a)与或者(i)融合配偶体或者(ii)HIV Nef蛋白或其衍生物连接的HIV Tat蛋白或其衍生物;或(b)与或者(i)融合配偶体或者(ii)HIV Tat蛋白或其衍生物连接的HIV Nef蛋白或其衍生物;或(c )与HIV Tat蛋白或其衍生物和融合配偶体连接的HIV Nef蛋白或其衍生物。本发明还提供编码这种蛋白的核酸和用前述核酸转化的宿主细胞,诸如巴斯德毕赤酵母细胞。
Description
本发明涉及新的HIV蛋白构成物、它们在药物中的应用、包含它们的药用组合物以及生产它们的方法。
具体而言，本发明涉及包含HIV-1 Tat和／或Nef蛋白的融合蛋白。
HIV-1是获得性免疫缺陷综合征(AIDS)的主要病因，AIDS被认为是世界主要健康问题之一。尽管全世界为生产疫苗已经进行了广泛的研究，但迄今为止，这种努力仍未成功。
已经描述了HIV-1的非包膜蛋白，它包括例如内部结构蛋白和其它非结构蛋白，所述内部结构蛋白诸如gag和pol基因产物，所述非结构蛋白诸如Rev、Nef、Vif和Tat(Greene等，New England J．Med，324，5，308以及下列等等(1991)和Bryant等(Pizzo编辑)，Pediatr．Infect．Dis．J．，11，5，390以及下列等等(1992)。
HIV Nef和Tat蛋白是早期蛋白，即是说，它们在感染的早期和在没有结构蛋白的情况下表达。
按照本发明提供一种蛋白，它包括：
(a)与或者(ⅰ)融合配偶体或者(ⅱ)HIV Tat蛋白或其衍生物连接的HIV Nef蛋白或其衍生物；或
(b)与或者(ⅰ)融合配偶体或者(ⅱ)HIV Nef蛋白或其衍生物连接的HIV Tat蛋白或其衍生物；或
(c)与或者HIV Tat蛋白或其衍生物和融合配偶体连接的HIVNef蛋白或其衍生物。
所述“融合配偶体”是指任何非Tat或Nef的蛋白序列。所述融合配偶体最好是来自B型流感嗜血杆茵的D蛋白或其脂质化(1ipidate)衍生物脂蛋白D。具体的说，最好是使用N-末端三分之一，即大约前100-130个氨基酸。这在本文中用Lipo D 1／3表示。在本发明一个优选的实施方案中，Nef蛋白或其衍生物可以连接于Tat蛋白或其衍生物。这种Nef-Tat融合体也可以可选地连接融合配偶体，诸如D蛋白。
融合配偶体一般连接于Nef或Tat蛋白的N-末端。
本发明包括的衍生物包括具有C末端组氨酸尾(最好含有5-10个组氨酸残基)的分子。一般来说，本文用His(n)表示包含n个残基的组氨酸尾。组氨酸(或“His”)尾的存在有助于纯化。更具体的说，本发明提供具有下列结构的蛋白：
Lipo D 1／3 - Nef - His(6)
Lipo D 1／3 - Nef-Tat - His(6)
Prot D 1／3 - Nef - His(6)
Prot D 1／3 - Nef-Tat - His(6)
- Nef-Tat - His(6)
图1提供了用于这些构成物的融合配偶体的氨基酸序列(Seq．ID．No．7)和DNA序列(Seq．ID．No．6)。
在一个优选的实施方案中，表达具有包含5至10个(最好为6个)组氨酸残基的组氨酸尾的蛋白。其优势在于有助于纯化。已经报导了在酵母(酿酒酵母)中分别表达Nef(Macreadia I．G．等，1993，Yeast9(6)565-573)和Tat(Braddock M等，1989，Cell 58(2)269-79)。只有Nef蛋白是豆蔻基化(myristilate)的。本发明提供首次在毕赤酵母属(Pichia)表达系统中分别表达Nef和Tat(Nef-His和Tat-His构成物)，和成功表达融合构成物Nef-Tat-His。在图2中显示了代表Nef-His(Seq．ID．No．8和9)、Tat-His(Seq．ID．No．10和11)和Nef-Tat-His融合蛋白(Seq．ID．No．12和13)的DNA序列和氨基酸序列。
本发明包括的衍生物也包括突变的蛋白。术语“突变的”在本文中用来指一个分子，该分子已使用广为人知的用于定点诱变的技术或任何其它常规方法，经受了一个或多个氨基酸的缺失、添加或置换。
在图2中(Seq．ID．No．22和23)描述了突变的Tat以及Nef-Tat突变体-His(Seq．ID．No．24和25)。
本发明也提供编码本发明蛋白的DNA。这类序列可以插入到合适的表达载体中，并可以在合适的宿主中表达。
可以使用标准的DNA合成技术，诸如通过由D．M．Roberts等，在Biochemistry 1985，24，5090-5098中所述的酶连接、化学合成、体外酶聚合，或者使用例如热稳定聚合酶的PCR技术，或者这些技术的组合，合成编码本发明蛋白的DNA序列。
可以使用DNA聚合酶(诸如DNA聚合酶Ⅰ(克列诺片段))，在包含三磷酸核苷dATP、dCTP、dGTP和dTTP的合适缓冲液(温度需要在10℃-37℃，体积一般为50 μl或更少)中，体外进行DNA的酶聚合。可以使用DNA连接酶(诸如T4 DNA连接酶)，在合适的缓冲液(诸如0．05M Tris(pH7．4)、0．01M MgCl2、0．01M二流苏糖醇、1mM亚精胺、1mM ATP和0．1mg／ml牛血清白蛋白，温度为4℃至室温，体积一般为50 ml或更少)中，进行DNA片段的酶连接。可以通过使用固相技术，诸如在“Chemical and Enzymatic Synthesis of GeneFragments-A Laboratory Manual”(H．G．Gassen和A．lang编辑)，VerlagChemie，Weinheim(1982)中，或在其它的科学刊物，例如M．J．Gait，H．W．D．Matthes，M．Singh，B．S．Sproat，和R．C．Titmas，Nucleic AcidsResearch，1982，10，6243；B．S．Sproar和W．Bannwarth，TetrahedronLetters，1983，24，5771；M．D．Matteucci和M．H．Caruthers，TetrahedronLetters，1980，21，719；M．D．Matteucci和M．H．Caruthers，Journal of theAmerican Chemical Society，1981，103，3185；S．P．Adams等，Journal ofthe American Chemical Society，1983，105，661；N．D．Sinha，J．Biernat，J．McMannus，和H．Koester，Nucleic Acids Research，1984，12，4539；和H．W．D．Matthes等，EMBO Journal，1984，3，801中描述的那些技术，通过常规磷酸三酯、亚磷酸酯或亚磷酰胺化学法进行DNA聚合体或片段的化学合成。
本发明也提供制备本发明蛋白的方法，所述方法包括以下步骤：
ⅰ) 能够表达在宿主细胞中制备DNA聚合体的复制型或整合型表达载体，所述DNA聚合体包括编码所述蛋白或其衍生物的核苷酸序列；
ⅱ)用所述载体转化宿主细胞；
ⅲ)在允许所述DNA聚合体表达以产生所述蛋白的条件下培养所述转化的宿主细胞；和
ⅳ)回收所述蛋白。
本发明所述方法可以通过诸如在Maniatis等，Molecular Cloning-A Laboratory Manual；Cold Spring Harbor，1982-1989中描述的常规重组技术进行。
术语“转化”在本文中用来指将外源DNA引入到宿主细胞中。这可以例如通过用合适的质粒或病毒载体，使用如在GeneticEngineering；S．M．Kingsman和A．J．Kingsman编辑；Blackwell ScientificPublications；Oxford，England，1988中描述的常规技术转化、转染或感染来实现。所述术语“转化的”和“转化体”在下文将适用于产生的包含和表达目的外源基因的宿主细胞。
所述表达载体是新的，并且也构成本发明的一部分。
可以按照本发明，通过切割与宿主细胞相适合的载体以提供一个具有完整复制子的线性DNA区段，并将所述线性区段与一个或多个与所述线性区段一起编码所需产物的DNA分子(诸如编码本发明的蛋白或其衍生物的DNA聚合体)在连接条件下结合，制备复制型表达载体。
因此，可以在构建载体的过程中根据需要预制或形成所述DNA聚合体。
载体的选择将部分由宿主细胞决定，所述宿主细胞可以是原核细胞或真核细胞，但最好是大肠杆茵或酵母细胞。合适的载体包括质粒、噬菌体、粘粒和重组病毒。
可以用合适的用于DNA限制、聚合和连接的酶，通过在例如上文中提及的Maniatis等描述的步骤，常规进行复制型表达载体的制备。
按照本发明，通过在转化条件下，用本发明的复制型表达载体转化宿主细胞，制备重组的宿主细胞。合适的转化条件是常规的，并在例如上文提及的Maniatis等或“DNA Cloning”第Ⅱ卷，D．M．Glover编辑，IRL Press Ltd，1985中进行了描述。
根据宿主细胞确定转化条件的选择。因此，可以用CaCl2溶液(Cohen等，Proc．Nat．Acad．Sci．，1973，69，2110)，或用包含RbCl、MnCl2、乙酸钾和甘油的混合物的溶液处理诸如大肠杆菌的细菌宿主，然后对其用3-[N-吗啉代]-丙磺酸、RbCl和甘油处理。可以利用钙与载体DNA共沉淀到细胞上转化在培养物中的哺乳动物细胞。本发明也扩展到用本发明的复制型表达载体转化宿主细胞。
如在例如上文中提及的Maniatis等和“DNA Cloning”中的描述，在允许DNA聚合体表达的条件下，常规进行转化的宿主细胞的培养。因此，最好向所述细胞提供营养，并在低于50℃的温度下培养。
根据宿主细胞通过常规方法回收产物。因此，当所述宿主细胞是细菌(诸如大肠杆菌)或酵母(诸如毕赤酵母属(Pichia))时，可以将其物理、化学或酶促溶解，并从产生的裂解液中分离出蛋白产物。当所述宿主细胞是哺乳动物细胞时，一般从营养培养基中或从无细胞提取物中分离产物。常规的蛋白分离技术包括选择性沉淀、吸附色谱分析和包括单克隆抗体亲和柱在内的亲和层析。
对于具有组氨酸尾的本发明的蛋白，通过使用金属离子亲和柱，可以轻易实现纯化。在一个优选的实施方案中，所述蛋白通过阳离子交换层析和／或凝胶过滤层析进一步纯化。然后将所述蛋白通过0．22μm膜除菌。
本发明所述蛋白然后可以制备成疫苗，或酶促清除组氨酸残基。
提供的本发明的蛋白，通过SDS PAGE目测，优选至少80％纯度，更优选90％纯度。最好是，所述蛋白通过SDS PAGE似乎是一条单带。
本发明也提供含有在药学上可接受的赋形剂中的本发明蛋白的药用组合物。
在New Trends and Developments in Vaccines，Voller等(编辑)，University Park Press，Baltimore，Maryland，1978中一般描述了疫苗制剂。Fullerton，美国专利第4，235，877号描述了于脂质体中的囊封。
在本发明的疫苗制剂中，本发明所述蛋白最好是佐剂化(adjuvanted)的。合适的佐剂包括铝盐，诸如氢氧化铝凝胶(alum)或磷酸铝，但也可以是钙盐、铁盐或锌盐，或者可以是酰化酪氨酸或酰化糖的不溶性悬浮液，阳离子或阴离子衍生化的多糖或聚磷腈。
在本发明的制剂中，最好是佐剂组合物优先诱导TH1应答。合适的佐剂系统包括例如单磷酰脂A或其衍生物(最好是3-脱-O-酰化单磷酰脂A(3D-MPL))与铝盐一起的组合物。
增强系统包括单磷酰脂A和皂苷衍生物的组合，特别是在WO94／00153中公开的QS21和3D-MPL的组合，或在WO 96／33739中公开的反应原性较小的组合物，其中QS21用胆固醇猝灭。
在WO 95／17210中描述了特别有效的佐剂制剂，它包括QS21、3D-MPL和维生素E的水包油乳状液，并且是优选的制剂。
因此在本发明一个实施方案中，提供含有按照本发明的蛋白的疫苗，所述蛋白用单磷酰脂A或其衍生物(特别是3D-MPL)佐剂化。
所述疫苗优选另外包括皂苷，更优选是QS21。
所述制剂优选另外包括水包油乳状液和维生素E。本发明也提供生产疫苗制剂的方法，所述方法包括将本发明的蛋白和药学上可接受的赋形剂(诸如3D-MPL)混合在一起。
本发明所述疫苗可以另外包括更多的HIV蛋白，诸如包膜糖蛋白gp160或其衍生物gp120。
在另一方面，本发明涉及在巴斯德毕赤酵母（Pichia pastoris)中表达的HIV Nef或HIV Tat蛋白或它们的衍生物。
参照以下实施例进一步描述本发明：实施例：一般描述
在大肠杆茵和甲基营养酵母巴斯德毕赤酵母中，生产由人免疫缺陷病毒(HIV-1)编码的两种调节蛋白-Nef和Tat蛋白。
选择来自Bru／Lai分离物(Cell 40：9-17，1985)的nef基因用于这些构成物，因为该基因属于那些与共有Nef最密切相关的基因。
用于Bru／Lai nef基因的原材料是在哺乳动物表达载体pcDNA3(pcDNA3／nef)上克隆的1170bp DNA片段。
tat基因源自BH10分子克隆。该基因被公认为是称作pCV1的HTLV Ⅲ cDNA克隆，并在Science，229，1985第69-73页中进行了描述。1．在大肠杆菌中表达HIV-1 nef和tat序列
在质粒载体：pRIT14586和pRIT14589中置入编码Nef蛋白的序列以及nef和tat序列的融合体(参见图1)。
作为融合蛋白生产Nef和Nef-Tat融合体，使用D蛋白的一部分作为融合配偶体。D蛋白是暴露在革兰氏阴性茵流感嗜血杆茵(Haemophilus influenzae)表面的免疫球蛋白D结合蛋白。
pRIT14586包括在λPL启动子控制下衍生自流感嗜血杆茵的编码D蛋白的前127个氨基酸的DNA序列(Infect．Immun．60：1336-1342，1992)，紧接着一个多克隆位点区加上编码一个甘氨酸、6个组氨酸残基和一个终止密码子的DNA序列(图1A)。
设计该载体以表达加工的脂质化His尾融合蛋白(LipoD融合蛋白)。将该融合蛋白具合成为有18个氨基酸残基的长信号序列的前体，并且在加工后，在前体分子的位置19的半胱氨酸成为氨基末端残基，然后通过共价结合的脂肪酸将其修饰(图1B)。
pRIT14589和pRIT14586几乎一样，除了在半胱氨酸19密码子后紧接着开始protD衍生的序列。
该载体的表达产生His尾的非脂质化融合蛋白(Prot D融合蛋白)。
制成四种构成物：LipoD-nef-His、LipoD-nef-tat-His、ProtD-nef-His和ProtD-nef-tat-His。
使用表达载体pRIT14586制成前两种构成物，使用pRIT14589制成后两种构成物。1．1 构建产生LIPOD-Nef-HIS融合蛋白的重组菌株ECLD-N1。1．1．1构建LipoD-nef-His表达质粒pRIT14595
通过PCR，用引物01和02由pcDNA3／Nef质粒扩增nef基因(Bru／Lai分离物)。
NcoⅠ引物01(Seq ID NO 1)：5’ATCGTCCATG．GGT．GGC．AAG．TGG．T3’SpeⅠ引物02(Seq ID NO 2)：5’CGGCTACTAGTGCAGTTCTTGAA3’
扩增的nefDNA区在核苷酸8357起始，在核苷酸8971终止(Cell，40：9-17，1985)。
在所述PCR片段的5’末端引入一个NcoⅠ限制位点(它携带nef基因的ATG密码子)，而在3’末端引入一个SpeⅠ位点。
获得的PCR片段和表达质粒pRIT14586都用NcoⅠ和SpeⅠ限制，在琼脂糖凝胶上纯化，连接和在合适的大肠杆菌宿主细胞-菌株AR58中转化。该菌株是衍生自N99的隐蔽λ溶原体，N99是galE∷Tn10，Δ-8(chlD-pgl)，Δ-H1(cro-chlA)，N+和cI857。
在通过自动测序证实所述nef扩增区后(参见下文的1．1．2单元)，命名产生的重组质粒为pRIT14595。1．1．2选择具有pRIT14595的大肠杆菌茵株AR58转化体
当在AR58大肠杆茵宿主茵株中转化时，重组质粒指导热诱导产生异源蛋白。
通过考马斯蓝染色的SDS-PAGE分析几个重组lipoD-Nef-His转化体的热诱导蛋白产生。所有分析的转化体均显示产生热诱导异源蛋白。估计重组Lipo D-Nef-Tat-His融合蛋白的多度为总蛋白的10％。
选择一种转化体，并给予其实验室编号ECLD-N1。
从菌株ECLD-N1中再分离重组质粒，通过自动测序确认nef-His编码区的序列。该质粒的正式命名为pRIT14595。
由茵株ECLD-N1产生的完全加工和酰化的重组Lipo D-nef-His融合蛋白由以下物质组成：
°脂肪酸
°D蛋白的109个氨基酸(在氨基酸19开始，并延伸至氨基酸127)。
°蛋氨酸，利用pRIT14586的NcoⅠ克隆位点产生(图1)。
°Nef蛋白的205个氨基酸(在氨基酸2开始，并延伸至氨基酸206)。
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸(在pRIT14586的SpeⅠ位点克隆)。
°一个甘氨酸和六个组氨酸。1．2 构建产生PROT D-Nef-His融合蛋白的重组茵株ECD-N1。
编码Prot D-Nef-His融合蛋白的表达质粒pRIT14600的构建与在实施例1．1．1中描述的质粒构建一致，除了pRIT14589用作PCR扩增的nef片段的受体质粒。
用pRIT14600转化大肠杆茵AR58菌株，并按实施例1．1．2所述分析转化体。选定的转化体的实验室编号为ECD-N1。1．3 构建产生LIPO D-Nef-Tat-His融合蛋白的重组茵株ECLD-
NT6。1．3．1构建lipo D-Nef-Tat-His表达质粒pRIT14596
通过PCR，用引物03和04由pCV1质粒的衍生物扩增tat基因(BH10分离物)。在PCR片段的两端都引入SpeⅠ限制位点。
SpeⅠ引物03(Seq ID NO 3)：5’ATCGTACTAGT．GAG．CCA．GTA．GAT．C3’
SpeⅠ引物04(Seq ID NO 4)：5’CGGCTACTAGTTTCCTTCGGGCCT3’
在pCV1克隆中描述了扩增的tat基因的核苷酸序列(Science229：69-73．1985)，包括核苷酸5414至核苷酸7998。
获得的PCR片段和质粒pRIT14595(表达lipoD-Nef-His蛋白)都用SpeⅠ限制酶消化，在琼脂糖凝胶上纯化，连接和在感受态AR58细胞中转化。在通过自动测序确认tat扩增序列后(参见下文1．3．2单元)，命名产生的重组质粒为pRIT14596。1．3．2选择具有pRIT14596的菌株AR58转化体
对转化体进行培养、热诱导，通过考马斯蓝染色凝胶分析它们的蛋白。估计重组蛋白的生产水平为总蛋白的1％。选择一种重组菌株，并给予实验室命名ECLD-NT6。
由ECLD-NT6茵株中再分离lipoD-nef-tat-His重组质粒，测序并正式命名为pRIT14596。
由ECLD-NT6茵株产生的完全加工和酰化的重组Lipo D-Nef-Nef-Tat-His融合蛋白由以下物质组成：
°脂肪酸
°D蛋白的109个氨基酸(在氨基酸19开始，并延伸至氨基酸127)。
°蛋氨酸，利用pRIT14586的NcoⅠ克隆位点产生。
°Nef蛋白的205个氨基酸(在氨基酸2开始，并延伸至氨基酸206)。
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸。
°Tat蛋白的85个氨基酸(在氨基酸2开始，并延伸至氨基酸86)。
°通过克隆步骤引入的一个苏氨酸和一个丝氨酸。
°一个甘氨酸和六个组氨酸。1．4 构建产生PROT D-Nef-Tat-His融合蛋白的重组茵株ECD-
NT1．
编码Prot D-Nef-Tat-His融合蛋白的表达质粒pRIT14601的构建与在实施例1．3．1中描述的质粒构建一致，除了pRIT14600用作PCR扩增的nef片段的受体质粒。
用pRIT14601转化大肠杆菌AR58菌株，并按先前所述分析转化体。选定的转化体的实验室编号为ECD-NT1。2．在巴斯德毕赤酵母中表达HIV-1 nef和tat序列
在甲基营养酵母巴斯德毕赤酵母中，在诱导型醇氧化酶(AOX1)启动子的控制下，表达Nef蛋白、Tat蛋白和融合体Nef-Tat。
为表达这些HIV-1基因，使用修饰的整合型载体PHIL-D2(INVITROGEN)。该载体以这样一种方式修饰：异源蛋白的表达紧接着AOX1基因的天然ATG密码子开始，并将产生具有一个甘氨酸和六个组氨酸残基尾的重组蛋白。通过在PHIL-D2载体的相邻AsuⅡ和EcoRⅠ位点之间克隆寡核苷酸接头，构建此PHIL-D2-MOD载体(参见图3)。除了His尾之外，此接头还携带NcoⅠ、SpeⅠ和XbaⅠ限制位点，在所述限制位点之间插入nef、tat和nef-tat融合体。2．1 构建整合型载体pRIT14597(编码Nef-His蛋白)、pRIT14598(编码Tat-His蛋白)和pRIT14599(编码融合体Nef-Tat-His)。
利用PCR，用引物01和02(参见1．1．1单元pRIT14595的构建)由pcDNA3／Nef质粒扩增nef基因。获得的PCR片段和整合型PHIL-D2-MOD载体都用NcoⅠ和SpeⅠ限制，在琼脂糖凝胶上纯化，连接产生整合型质粒pRIT14597(参见图3)。
利用PCR，用引物05和04(参见1．3．1单元pRIT14596的构建)由pCV1质粒的衍生物扩增tat基因：
NcoⅠ引物05(Seq ID NO 5)：5’ATCGTCCATGGAGCCAGTAGATC 3’
在所述PCR片段的5’末端引入一个NcoⅠ限制位点，而在3’末端用引物04引入一个SpeⅠ位点。获得的PCR片段和PHIL-D2-MOD载体都用NcoⅠ和SpeⅠ限制，在琼脂糖凝胶上纯化，连接产生整合型质粒pRIT14598。
为构建pRIT14599，在PHIL-D2-MOD载体的EcoRⅠ平端(T4聚合酶)和NcoⅠ位点之间连接对应于nef-tat-His编码序列的910bp DNA片段。通过XbaⅠ平端(T4聚合酶)和NcoⅠ消化pRIT14596获得nef-tat-His编码片段。2．2 转化巴斯德毕赤酵母菌株GS115(his4)。
为获得表达Nef-His、Tat-His和融合体Nef-Tat-His的巴斯德毕赤酵母菌株，用携带相应表达盒加上HIS4基因的线性NotⅠ片段转化茵株GS115，以互补宿主基因组中的his4。具有NotI-线性片段的GS115转化体偏向于在AOXI基因座重组。
通过定量的斑点印迹分析选择多拷贝整合克隆，并确定整合、插入(Mut+表现型)或置换(Muts表现型)的类型。
从每次转化中选择显示高水平产生重组蛋白的一种转化体：
产生重组Nef-His蛋白(一种豆蔻基化的215个氨基酸的蛋白)的茵株Y1738(Mut+表现型)，所述重组Nef-His蛋白由以下物质组成：
°豆蔻酸
°蛋氨酸，利用PHIL-D2-MOD载体的NcoⅠ克隆位点产生。
°Nef蛋白的205个氨基酸(在氨基酸2开始，并延伸至氨基酸206)。
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸(在PHIL-D2-MOD载体的SpeⅠ位点克隆)。
°一个甘氨酸和六个组氨酸。
产生Tat-His蛋白(一种95个氨基酸的蛋白)的茵株Y1739(Mut+表现型)，所述Tat-His蛋白由以下物质组成：
°蛋氨酸，利用NcoⅠ克隆位点产生。
°Tat蛋白的85个氨基酸(在氨基酸2开始，并延伸至氨基酸86)。
°通过克隆步骤引入的一个苏氨酸和一个丝氨酸
°一个甘氨酸和六个组氨酸
产生重组Nef-Tat-His融合蛋白(一种豆蔻基化的302个氨基酸的蛋白)的菌株Y1737(Muts表现型)，所述Nef-Tat-His融合蛋白由以下物质组成：
°豆蔻酸
°蛋氨酸，利用NcoⅠ克隆位点产生。
°Nef蛋白的205个氨基酸(在氨基酸2开始，并延伸至氨基酸206)。
°通过克隆步骤产生的一个苏氨酸和一个丝氨酸
°Tat蛋白的85个氨基酸(在氨基酸2开始，并延伸至氨基酸86)。
°通过克隆步骤引入的一个苏氨酸和一个丝氨酸
°一个甘氨酸和六个组氨酸3． 在巴斯德毕赤酵母中表达HIV-1 Tat-突变体
除了已经表达了Nef-Tat突变体融合蛋白之外，也已经表达了突变重组Tat蛋白。所述突变Tat蛋白在保持其免疫原性表位的同时，必须是生物学上无活性的。
选择由D．Clements(Tulane大学)构建的双突变tat基因用于这些构成物。
该tat基因(源自BH10分子克隆)在活性位点区(Lys41→Ala)和在RGD基元(Arg78→Lys和Asp80→Glu)中具有突变(Virology 235：48-64，1997)。
所述突变tat基因被公认为是在CMV表达质粒(pCMVLys41／KGE)中于EcoRⅠ和HindⅢ位点之间亚克隆的cDNA片段。3．1 构建整合型载体pRIT14912(编码Tat突变体-His蛋白)和pRIT14913(编码融合体Nef-Tat突变体-His)。
利用PCR，用引物05和04(参见2．1单元pRIT14598的构建)由pCMVLys41／KGE质粒扩增tat突变体基因。
在所述PCR片段的5’末端引入一个NcoⅠ限制位点，而在3’末端用引物04引入一个SpeⅠ位点。获得的PCR片段和PHIL-D2-MOD载体都用NcoⅠ和SpeⅠ限制，在琼脂糖凝胶上纯化，连接产生整合型质粒pRIT14912。
为构建pRIT14913，利用PCR，用引物03和04(参见1．3．1单元pRIT14596的构建)由pCMVLys41／KGE质粒扩增tat突变体基因。
获得的PCR片段和质粒pRIT14597(表达Nef-His蛋白)都用SpeⅠ限制酶消化，在琼脂糖凝胶上纯化，连接产生整合型质粒pRIT14913。3．2 巴斯德毕赤酵母菌株GS115的转化。
通过使用先前在2．2单元中描述的整合和重组茵株选择策略，获得表达Tat突变体-His蛋白和融合体Nef-Tat突变体-His的巴斯德毕赤酵母菌株。
选择两种产生Tat突变体-His蛋白(一种95个氨基酸的蛋白)的重组茵株：Y1775(Mut+表现型)和Y1776(Muts表现型)。
选择一种产生Nef-Tat突变体-His融合蛋白(一种302个氨基酸的蛋白)的重组菌株：Y1774(Mut+表现型)。4．纯化Nef-Tat-His融合蛋白(巴斯德毕赤酵母)
由146克重组巴斯德毕赤酵母细胞(湿重)或2L Dyno-mill匀浆OD55进行纯化流程。在室温下进行层析步骤。在步骤之间，Nef-Tat阳性组分整夜保存在冷室(+4℃)中；对于更长的时间，将样品于-20℃冷冻。 
*比率：0．5M精氨酸的蛋白浓度为1600μg／ml。纯度
在图4中通过Daiichi银染和在图5中通过考马斯蓝G250显示了由SDS-PAGE估计的纯度水平。
Superdex200步骤后： ＞95％
透析和除菌过滤步骤后： ＞95％回收
由146克重组巴斯德毕赤酵母细胞(=2L Dyno-mill匀浆OD 55)纯化得到51mg Nef-Tat-his蛋白。5．疫苗制备
按照本发明制备的疫苗包括编码抗原(如在实施例1或2中作为实例的抗原)的DNA重组体的表达产物和作为佐剂的制剂(包含在油／水乳状液中的3脱氧酰化单磷酰脂A(3D-MPL)和QS21的混合物)。
3D-MPL：是革兰氏阴性茵明尼苏达沙门氏茵(Salmonellaminnesota)的化学解毒形式的脂多糖(LPS)。
在Smith Kline Beecham Biologicals进行的实验已经显示，与各种载体组合的3D-MPL强有力地增加了体液免疫和TH1型的细胞免疫。
QS21：是一种由Quillaja Saponaria Molina树的树皮的粗提物纯化的皂苷，它具有很强的佐剂活性：它激活对几种抗原的抗原特异性淋巴组织增生和CTL。
在Smith Kline Beecham Biologicals进行的实验已经证明，3D-MPL和QS21的组合在诱导体液免疫应答和TH1型细胞免疫应答中有明显的协同作用。
油／水乳状液包含2种油(维生素E和鲨烯)和含吐温80作为乳化剂的PBS。所述乳状液包含5％鲨烯、5％维生素E、0．4％吐温80，平均颗粒大小为180 nm(参见WO 95／17210)。
在Smith Kline Beecham Biologicals进行的实验已经证明，将此O／W乳状液添加到3D-MPL／QS21中，进一步增加了它们的免疫刺激物性质。制备油／水乳状液(2倍浓缩物)
将吐温80溶于磷酸缓冲盐水(PBS)中，得到在PBS中的2％溶液。为提供100ml两倍浓缩的乳状液，涡旋混合5克DLα-维生素E和5ml鲨烯，以完全混合。加入90ml的PBS／吐温溶液并充分混合。然后将得到的乳状液通过注射器，最后将其用M110S显流化设备显流化。得到的小油滴大小约为180nm。制备水包油制剂。
在以5分钟的间隔连续加入SB62、3D-MPL(5μg)、QS21(5μg)和50μg／ml硫柳汞(作为防腐剂)之前，在10倍浓缩的PBS pH 6．8和水中稀释按照实施例1或2制备的抗原(5μg)。乳状液体积等于总体积的50％(对于100μl剂量为50μl)。
所有的温育都在室温搅拌下进行。6．在啮齿动物中Tat和Nef-Tat的免疫原性
对用Tat和Nef-Tat免疫后诱导的免疫应答进行特征鉴定。为得到关于同种型分布和细胞介导免疫(CMI)的信息，在小鼠中进行两个免疫实验。在第一个实验中，分别用氧化或还原形式的Tat或Nef-Tat相隔两周对小鼠爪垫免疫两次。在水包油乳状液中制备抗原，所述乳状液包含鲨烯、吐温80TM(聚氧乙烯山梨聚糖一油酸酯)、QS21、3D-MPL和α-维生素E，而对照组接受单独的佐剂。在最后一次免疫后两周，得到血清，并进行Tat特异性的ELISA(用还原的Tat包被)，测定抗体效价和同种型(图6a)。在接受氧化的Tat的小鼠中抗体效价最高。一般来说，氧化分子诱导的抗体效价比还原形式高，单独的Tat诱导的抗体效价比Nef-Tat高。在第二个实验中证实了后者。更有意思的是，Tat特异性的抗体的同种型分布根据用于免疫的抗原而不同。单独的Tat诱导平衡的IgGl和IgG2a分布，而Nef-Tat诱导强得多的TH2偏向(图6b)。这在第二个实验中再次得到证实。
在第二个小鼠实验中，动物只接受还原形式的分子或单独的佐剂。除了血清学分析(参见上文)之外，还评估了淋巴节细胞的淋巴组织增生反应。用Tat或Nef-Tat在体外再次刺激这些细胞后，在培养4天后检测3H-胸苷掺入。显示的刺激指数的结果表明，在两组接受抗原的小鼠中都诱导很强的应答(图7)。
总之，小鼠实验表明，Tat以及Nef-Tat是高度免疫原性的候选疫苗抗原。通过具有至少50％IgGl的高抗体应答，特征鉴定了针对这两种分子的免疫应答。而且，观察到很强的CMI应答(通过淋巴组织增生检测)。7．Tat和Nef-Tat蛋白的功能性质
研究氧化或还原形式的Tat和Nef-Tat分子结合人T细胞系的能力。此外，测定对这些细胞系生长的影响。用不同浓度的Tat和Nef-Tat蛋白、Ⅱ型单纯疱疹病毒的无关gD或用单独的缓冲液对照整夜包被ELISA板。除去包被溶液后，向孔中加入HUT-78细胞。孵育两小时后，洗涤各孔，显微测定细胞与孔底部的结合。作为定量测定，用甲苯胺蓝染色细胞，通过SDS溶解细胞，并用ELISA读板仪检测上清液中的甲苯胺蓝浓度。结果表明，所有四种细胞(氧化或还原形式的Tat和Nef-Tat)都介导细胞与ELISA板的结合(图8)。所述无关蛋白(未列出数据)和缓冲液不固定所述细胞。这表明，含重组表达的Tat的蛋白特异性结合于人T细胞系。
在第二个实验中，让HUT-78细胞接触所述蛋白16小时。在孵育阶段结束时，用[3H]-胸苷标记细胞，并检测掺入率来测定细胞生长。根据放射性掺入减少(图9)判断，在此检测中包括的所有四种蛋白都抑制细胞生长。缓冲液对照没有介导此作用。这些结果证明，含重组Tat的蛋白能够抑制人T细胞系的生长。
总之，Tat和Nef-Tat蛋白的功能性特征鉴定显示，这些蛋白能够结合于人T细胞系。而且，所述蛋白能够抑制这类细胞系的生长。
序列表(1)一般资料(ⅰ)申请人：SmithKline Beecham Biologicals S．A．(ⅱ)发明名称：疫苗(ⅲ)序列数：27(ⅳ)通信地址：
(A)收信人：SmithKline Beecham
(B)街道：Two New Horizons Court
(C)城市：Brentford
(D)州：
(E)国家：Middx，英国
(F)邮政编码：TW8 9EP(ⅴ)计算机可读形式
(A)媒体类型：软盘
(B)计算机：IBM兼容机
(C)操作系统：DOS
(D)软件：FastSEQ for Windows，版本2．0(ⅵ)当前申请数据
(A)申请号：
(B)提交日期：1997年9月26日
(C)分类：(ⅶ)在先申请数据
(A)申请号：
(B)提交日期：(ⅷ)代理律师／代理人资料
(A)姓名：Bor，Fiona R
(B)注册号：
(C)参考／档案号：(ⅸ)电讯资料
(A)电话：0181 975 2817
(B)传真：0181 975 6141
(C)电报：(2)SEQ ID NO：1的信息：(ⅰ) 序列特征：
(A)长度：28个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性
(ⅹⅰ) 序列描述：SEQ ID NO：l：ATCGTCCATG．GGT．GGC．A AG．TGG．T 28(2)SEQ ID NO：2的信息：(ⅰ) 序列特征：
(A)长度：23个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：2：CGGCTACTAG TGCAGTTCTT GAA 23
(2)SEQ ID NO：3的信息：
(ⅰ)序列特征：
(A)长度：29个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性
(ⅹⅰ)序列描述：SEQ ID NO：3：ATCGTACTAG T．GAG．CCA．GTA．GAT．C 29(2)SEQ ID NO：4的信息：
(ⅰ) 序列特征：
(A)长度：24个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性
(ⅹⅰ) 序列描述：SEQ ID NO：4：CGGCTACTAG TTTCCTTCGG GCCT 24(2)SEQ ID NO：5的信息：
(ⅰ) 序列特征：
(A)长度：23个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性
(ⅹⅰ) 序列描述：SEQ ID NO：5：ATCGTCCATG GAGCCAGTAG ATC 23(2)SEQ ID NO：6的信息：(ⅰ) 序列特征：
(A)长度：441个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：6：ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCTTTTGCA 180CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360ATGACAGAAA ACTTTGAAAC CATGGCCACG TGTGATCAGA GCTCAACTAG TGGCCACCAT 420CACCATCACC ATTAATCTAG A 441(2)SEQ ID NO：7的信息：(ⅰ) 序列特征：
(A)长度：144个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：7：Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Ala Gly Val Leu1 5 10 15Ala Gly Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys
20 25 30Ser Asp Lys Ile Ile Ile Ala His Arq Gly Ala Ser Gly Tyr Leu Pro
35 40 45Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
50 55 60Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val65 70 75 80Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe
85 90 95Pro His Arg His Arg Lys Asp Gly Arq Tyr Tyr Val Ile Asp Phe Thr
100 105 110Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
115 120 125Ala Thr Cys Asp Gln Ser Ser Thr Ser Gly His His His His His His
130 135 140(2)SEQ ID NO：8的信息：(ⅰ) 序列特征：
(A)长度：648个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：8：ATGGGTGGCA AGTGGTCAAA AAGTAGTGTG GTTGGATGGC CTACTGTAAG GGAAAGAATG 60AGACGAGCTG AGCCAGCAGC AGATGGGGTG GGAGCAGCAT CTCGAGACCT GGAAAAACAT 120GGAGCAATCA CAAGTAGCAA TACAGCAGCT ACCAATGCTG CTTGTGCCTG GCTAGAAGCA 180CAAGAGGAGG AGGAGGTGGG TTTTCCAGTC ACACCTCAGG TACCTTTAAG ACCAATGACT 240TACAAGGCAG CTGTAGATCT TAGCCACTTT TTAAAAGAAA AGGGGGGACT GGAAGGCTA 300ATTCACTCCC AACGAAGACA AGATATCCTT GATCTGTGGA TCTACCACAC ACAAGGCTAC 360TTCCCTGATT GGCAGAACTA CACACCAGGG CCAGGGGTCA GATATCCACT GACCTTTGGA 420TGGTGCTACA AGCTAGTACC AGTTGAGCCA GATAAGGTAG AAGAGGCCAA TAAAGGAGAG 480AACACCAGCT TGTTACACCC TGTGAGCCTG CATGGAATGG ATGACCCTGA GAGAGAAGTG 540TTAGAGTGGA GGTTTGACAG CCGCCTAGCA TTTCATCACG TGGCCCGAGA GCTGCATCCG 600GAGTACTTCA AGAACTGCAC TAGTGGCCAC CATCACCATC ACCATTAA 648(2)SEQ ID NO：9的信息：(ⅰ) 序列特征：
(A)长度：216个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：9：Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val1 5 10 15Arq Glu Arq Met Arg Arq Ala Glu Pro Ala Ala Asp Gly Val Gly Ala
20 25 30Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr
35 40 45Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arq Pro Met Thr65 70 75 80Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile His Ser Gln Arq Arq Gln Asp Ile Leu Asp Leu
100 105 110Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
115 120 125Pro Gly Pro Gly Val Arq Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys
130 135 140Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu145 150 155 160Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro
165 170 175Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His
180 185 190His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser
95 200 205Gly His His His His His His
210 215(2)SEQ ID NO：10的信息：(ⅰ) 序列特征：
(A)长度：288个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：1O：ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 60GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 120AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 180GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAACCCA CCTCCCAATC CCGAGGGGAC 240CCGACAGGCC CGAAGGAAAC TAGTGGCCAC CATCACCATC ACCATTAA 288(2)SEQ ID NO：11的信息：(ⅰ) 序列特征：
(A)长度：96个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：11：Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly
35 40 45Arg Lys Lys Arq Arg Gln Arg Arq Arg Pro Pro Gln Gly Ser Gln Thr
50 55 60His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arq Gly Asp65 70 75 80Pro Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
85 90 95(2)SEQ ID NO：12的信息：(ⅰ) 序列特征：
(A)长度：909个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：12：ATGGGTGGCA AGTGGTCAAA AAGTAGTGTG GTTGGATGGC CTACTGTAAG GGAAAGAATG 60AGACGAGCTG AGCCAGCAGC AGATGGGGTG GGAGCAGCAT CTCGAGACCT GGAAAAACAT 120GGAGCAATCA CAAGTAGCAA TACAGCAGCT ACCAATGCTG CTTGTGCCTG GCTAGAAGCA 180CAAGAGGAGG AGGAGGTGGG TTTTCCAGTC ACACCTCAGG TACCTTTAAG ACCAATGACT 240TACAAGGCAG CTGTAGATCT TAGCCACTTT TTAAAAGAAA AGGGGGGACT GGAAGGGCTA 300ATTCACTCCC AACGAAGACA AGATATCCTT GATCTGTGGA TCTACCACAC ACAAGGCTAC 360TTCCCTGATT GGCAGAACTA CACACCAGGG CCAGGGGTCA GATATCCACT GACCTTTGGA 420TGGTGCTACA AGCTAGTACC AGTTGAGCCA GATAAGGTAG AAGAGGCCAA TAAAGGAGAG 480AACACCAGCT TGTTACACCC TGTGACCCTG CATGGAATGG ATGACCCTGA GAGAGAAGTG 540TTAGAGTGGA GGTTTGACAG CCGCCTAGCA TTTCATCACG TGGCCCGAGA GCTGCATCCG 600GAGTACTTCA AGAACTGCAC TAGTGAGCCA GTAGATCCTA GACTAGAGCC CTGGAAGCAT 660CCAGGAAGTC AGCCTAAAAC TGCTTGTACC AATTGCTATT GTAAAAAGTG TTGCTTTCAT 720TGCCAAGTTT GTTTCATAAC AAAAGCCTTA GGCATCTCCT ATGGCAGGAA GAAGCGGAGA 780CAGCGACGAA GACCTCCTCA AGGCAGTCAG ACTCATCAAG TTTCTCTATC AAAGCAACCC 840ACCTCCCAAT CCCGAGGGGA CCCGACAGGC CCGAAGGAAA CTAGTGGCCA CCATCACCAT 900CACCATTAA 909(2)SEQ ID NO：13的信息：(ⅰ)序列特征：
(A)长度：303个氨基酸
(B)类型：氨基酸
(c)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：13：Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val1 5 10 15Arg Glu Arg Met Arg Arg Ala Glu PrO Ala Ala Asp Gly Val Gly Ala
20 25 30Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr
35 40 45Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr65 70 75 80Tyr Lys Ala Ala Val Asp Leu Sar His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile His Ser Gln Arg Arg Gln ASp Ile Leu Asp Leu
100 105 110Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
115 120 125Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys
130 135 140Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu145 150 155 160Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro
165 170 175Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Set Arg Leu Ala Phe His
180 185 190His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser
195 200 205Glu Pro Val Asp Pro Arq Leu Glu Pro Trp Lys His Pro Gly Ser Gln
210 215 220Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His225 230 235 240Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg
245 250 255Lys Lys Arg Arg Gln Arg Arq Arq Pro Pro Gln Gly Ser Gln Thr His
260 265 270Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro
275 280 285Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
290 295 300(2)SEQ ID NO：14的信息：(ⅰ) 序列特征：
(A)长度：1029个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：14：ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCTTTTGCA 180CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360ATGACAGAAA ACTTTGAAAC CATGGGTGGC AAGTGGTCAA AAAGTAGTGT GGTTGGATGG 420CCTACTGTAA GGGAAAGAAT GAGACGAGCT GAGCCAGCAG CAGATGGGGT GGGAGCAGCA 480TCTCGAGACC TGGAAAAACA TGGAGCAATC ACAAGTAGCA ATACAGCAGC TACCAATGCT 540GCTTGTGCCT GGCTAGAAGC ACAAGAGGAG GAGGAGGTGG GTTTTCCAGT CACACCTCAG 600GTACCTTTAA GACCAATGAC TTACAAGGCA GCTGTAGATC TTAGCCACTT TTTAAAAGAA 660AAGGGGGGAC TGGAAGGGCT AATTCACTCC CAACGAAGAC AAGATATCCT TGATCTGTGG 720ATCTACCACA CACAAGGCTA CTTCCCTGAT TGGCAGAACT ACACACCAGG GCCAGGGGTC 780AGATATCCAC TGACCTTTGG ATGGTGCTAC AAGCTAGTAC CAGTTGAGCC AGATAAGGTA 840GAAGAGGCCA ATAAAGGAGA GAACACCAGC TTGTTACACC CTGTGAGCCT GCATGGAATG 900GATGACCCTG AGAGAGAAGT GTTAGAGTGG AGGTTGACA GCCGCCTAGC ATTTCATCAC 960GTGGCCCGAG AGCTGCATCC GGAGTACTTC AAGAACTGCA CTAGTGGCCA CCATCACCAT 1020CACCATTAA 1029(2)SEQ ID NO：15的信息：(ⅰ) 序列特征：
(A)长度：325个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：15：Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp1 5 10 15Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arq Leu Val Val Ile His
50 55 60Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His65 70 75 80Arq His Arq Lys Asp Gly Arq Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met Gly Gly
l00 105 110Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg Glu Arg
115 120 125Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg
130 135 140Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr145 150 155 160Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
165 l70 175Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala
180 185 190Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
195 200 205Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr
210 215 220His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro225 230 235 240Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro
245 250 255Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn Thr Ser
260 265 270Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg Glu
275 280 285Val Leu Glu TRP Arg Phe Asp Ser Arg Leu Ala Phe His His Val Ala
290 295 300Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Gly His His305 310 315 320His His His His(2)SEQ ID NO：16的信息：(ⅰ) 序列特征：
(A)长度：1290个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：16：ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCGTTTGCA 180CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360ATGACAGAAA ACTTTGAAAC CATGGGTGGC AAGTGGTCAA AAAGTAGTGT GGTTGGATGG 420CCTACTGTAA GGGAAAGAAT GAGACGAGCT GAGCCAGCAG CAGATGGGGT GGGAGCAGCA 480TCTCGAGACC TGGAAAAACA TGGAGCAATC ACAAGTAGCA ATACAGCAGC TACCAATGCT 540GCTTGTGCCT GGCTAGAAGC ACAAGAGGAG GAGGAGGTGG GTTTTCCAGT CACACCTCAG 600GTACCTTTAA GACCAATGAC TTACAAGGCA GCTGTAGATC TTAGCCACTT TTTAAAAGAA 660AAGGGGGGAC TGGAAGGGCT AATTCACTCC CAACGAAGAC AAGATATCCT TGATCTGTGG 720ATCTACCACA CACAAGGCTA CTTCCCTGAT TGGCAGAACT ACACACCAGG GCCAGGGGTC 780AGATATCCAC TGACCTTTGG ATGGTGCTAC AAGCTAGTAC CAGTTGAGCC AGATAAGGTA 840GAAGAGGCCA ATAAAGGAGA GAACACCAGC TTGTTACACC CTGTGAGCCT GCATGGAATG 900GATGACCCTG AGAGAGAATG GTTAGAGTGG AGGTTTGACA GCCGCCTAGC ATTTCATCAC 960GTGGCCCGAG AGCTGCATCC GGAGTACTTC AAGAACTGCA CTAGTGAGCC AGTAGATCCT 1020AGACTAGAGC CCTGGAAGCA TCCAGGAAGT CAGCCTAAAA CTGCTTGTAC CAATTGCTAT 1080TGTAAAAAGT GTTGCTTTCA TTGCCAAGTT TGTTTCATAA CAAAAGCCTT AGGCATCTCC 1140TATGGCAGGA AGAAGCGGAG ACAGCGACGA AGACCTCCTC AAGGCAGTCA AAGGCAGTCA 1200GTTTCTCTAT CAAAGCAACC CACCTCCCAA TCCCGAGGGG ACCCGACAGG CCCGAAGGAA 1260ACTAGTGGCC ACCATCACCA TCACCATTAA 1290(2)SEQ ID N0：17的信息：(ⅰ) 序列特征：
(A)长度：412个氨基酸
(B)类型：氨基酸
(c)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID No：17：Cys Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys Ser Asp1 5 10 15Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro Glu His
20 25 30Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp Tyr Leu
35 40 45Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val Ile His
50 55 60Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe Pro His65 70 75 80Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr Leu Lys
85 90 95Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met Gly Gly
100 105 110Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg Glu Arg
115 120 125Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg
130 135 140Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr145 150 155 160Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
165 170 175Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala
180 185 190Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
195 200 205Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr
210 215 220Hie Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro225 230 235 240Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro
245 250 255Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn Thr Ser
260 265 270Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg Glu
275 280 285Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His Val Ala
290 295 300Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Glu Pro Val305 310 315 320Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys Thr
325 330 335Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val
340 345 350Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg
355 360 365Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser
370 375 380Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro385 390 395 400Lys Glu Thr Ser Gly His His His HiS His His
405 410(2)SEQ ID NO：18的信息：(ⅰ) 序列特征：
(A)长度：981个碱基对
(B)类型：核酸
(c)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：18：ATGGATCCAA GCAGCCATTC ATCAAATATG GCGAATACCC AAATGAAATC AGACAAAATC 60ATTATTGCTC ACCGTGGTGC TAGCGGTTAT TTACCAGAGC ATACGTTAGA ATCTAAAGCA 120CTTGCGTTTG CACAACAGGC TGATTATTTA GAGCAAGATT TAGCAATGAC TAAGGATGGT 180CGTTTAGTGG TTATTCACGA TCACTTTTTA GATGGCTTGA CTGATGTTGC GAAAAAATTC 240CCACATCGTC ATCGTAAAGA TGGCCGTTAC TATGTCATCG ACTTTACCTT AAAAGAAATT 300CAAAGTTTAG AAATGACAGA AAACTTTGAA ACCATGGGTG GCAAGTGGTC AAAAAGTAGT 360GTGGTTGGAT GGCCTACTGT AAGGGAAAGA ATGAGACGAG CTGAGCCAGC AGCAGATGGG 420GTGGGAGCAG CATCTCGAGA CCTGGAAAAA CATGGAGCAA TCACAAGTAG CAATACAGCA 450GCTACCAATG CTGCTTGTGC CTGGCTAGAA GCACAAGAGG AGGAGGAGGT GGGTTTTCCA 540GTCACACCTC AGGTACCTTT AAGACCAATG ACTTACAAGG CAGCTGTAGA TCTTAGCCAC 600TTTTTAAAAG AAAAGGGGGG ACTGGAAGGG CTAATTCACT CCCAACGAAG ACAAGATATC 660CTTGATCTGT GGATCTACCA CACACAAGGC TACTTCCCTG ATTGGCAGAA CTACACACCA 720GGGCCAGGGG TCAGATATCC ACTGACCTTT GGATGGTGCT ACAAGCTAGT ACCAGTTGAG 780CCAGATAAGG TAGAAGAGGC CAATAAAGGA GAGAACACCA GCTTGTTACA CCCTGTGAGC 840CTGCATGGAA TGGATGACCC TGAGAGAGAA GTGTTAGAGT GGAGGTTTGA CAGCCGCCTA 900GCATTTCATC ACGTGGCCCG AGAGCTGCAT CCGGAGTACT TCAAGAACTG CACTAGTGGC 960CACCATCACC ATCACCATTA A 991(2)SEQ ID N0：19的信息：(ⅰ) 序列特征：
(A)长度：327个氨基酸
(B)类型：氨基酸
(c)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID No：19：Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys1 5 10 15Ser Asp Lys Ile Ile Ile Ala His Arq Gly Ala Ser Gly Tyr Leu Pro
20 25 30Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arq Leu Val Val
50 55 60Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe65 70 75 80Pro Hls Arq His Arg Lys Asp Gly Arq Tyr Tyr Val Ile Asp Phe Thr
85 90 95Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arq
115 120 125Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala
130 135 140Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala145 150 155 160Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu
165 170 175Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arq Pro Met Thr Tyr
180 185 190Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
195 200 205Glu Gly Leu Ile His Ser Gln Arq Arq Gln Asp Ile Leu Asp Leu Trp
210 215 220Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro225 230 235 240Gly Pro Gly Val Arq Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu
245 250 255Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn
260 265 270Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu
275 280 285Arq Glu Val Leu Glu Trp Arq Phe Asp Ser Arq Leu Ala Phe His His
290 295 300Val Ala Arq Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Gly305 310 315 320His His His His His His
325(2)SEQ ID NO：20的信息：(ⅰ) 序列特征：
(A)长度：1242个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：20：ATGGATCCAA GCAGCCATTC ATCAAATATG GCGAATACCC AAATGAAATC AGACAAAATC 60ATTATTGCTC ACCGTGGTGC TAGCGGTTAT TTACCAGAGC ATACGTTAGA ATCTAAAGCA 120CTTGCGTTTG CACAACAGGC TGATTATTTA GAGCAAGATT TAGCAATGAC TAAGGATGGT 180CGTTTAGTGG TTATTCACGA TCACTTTTTA GATGGCTTGA CTGATGTTGC GAAAAAATTC 240CCACATCGTC ATCGTAAAGA TGGCCGTTAC TATGTCATCG ACTTTACCTT AAAAGAAATT 300CAAAGTTTAG AAATGACAGA AAACTTTGAA ACCATGGGTG GCAAGTGGTC AAAAAGTAGT 360GTGGTTGGAT GGCCTACTGT AAGGGAAAGA ATGAGACGAG CTGAGCCAGC AGCAGATGGG 420GTGGGAGCAG CATCTCGAGA CCTGGAAAAA CATGGAGCAA TCACAAGTAG CAATACAGCA 480GCTACCAATG CTGCTTGTGC CTGGCTAGAA GCACAAGAGG AGGAGGAGGT GGGTTTTCCA 540GTCACACCTC AGGTACCTTT AAGACCAATG ACTTACAAGG CAGCTGTAGA TCTTAGCCAC 600TTTTTAAAAG AAAAGGGGGG ACTGGAAGGG CTAATTCACT CCCAACGAAG ACAAGATATC 60CTTGATCTGT GGATCTACCA CACACAAGGC TACTTCCCTG ATTGGCAGAA CTACACACCA 720GGGCCAGGGG TCAGATATCC ACTGACCTTT GGATGGTGCT ACAAGCTAGT ACCAGTTGAG 780CCAGATAAGG TAGAAGAGGC CAATAAAGGA GAGAACACCA GCTTGTTACA CCCTGTGAGC 840CTGCATGGAA TGGATGACCC TGAGAGAGAA GTGTTAGAGT GGAGGTTTGA CAGCCGCCTA 900GCATTTCATC ACGTGGCCCG AGAGCTGCAT CCGGAGTACT TCAAGAACTG CACTAGTGAG 960CCAGTAGATC CTAGACTAGA GCCCTGGAAG CATCCAGGAA GTCAGCCTAA AACTGCTTGT 1020ACCAATTGCT ATTGTAAAAA GTGTTGCTTT CATTGCCAAG TTTGTTTCAT AACAAAAGCC 1080TTAGGCATCT CCTATGGCAG GAAGAAGCGG AGACAGCGAC GAAGACCTCC TCAAGGCAGT 1140CAGACTCATC AAGTTTCTCT ATCAAAGCAA CCCACCTCCC AATCCCGAGG GGACCCGACA 1200GGCCCGAAGG AAACTAGTGG CCACCATCAC CATCACCATT AA 1242(2)SEQ ID No：21的信息：(ⅰ) 序列特征：
(A)长度：414个氨基酸
(B)类型：氨基酸
(c)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID N0：21：Met Asp Pro Ser Ser His Ser Ser Asn Met Ala Asn Thr Gln Met Lys1 5 10 15Ser Asp Lys Ile Ile Ile Ala His Arg Gly Ala Ser Gly Tyr Leu Pro
20 25 30Glu His Thr Leu Glu Ser Lys Ala Leu Ala Phe Ala Gln Gln Ala Asp
35 40 45Tyr Leu Glu Gln Asp Leu Ala Met Thr Lys Asp Gly Arg Leu Val Val
50 55 60Ile His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe65 70 75 80Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val Ile Asp Phe Thr
85 90 95Leu Lys Glu Ile Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met
100 105 110Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val Arg
115 120 125Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Ala
130 135 140Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala145 150 155 160Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu
165 170 175Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
180 185 190Lys Ala Ala VaI Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
195 200 205Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp
210 215 220Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro225 230 235 240Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu
245 250 255Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn
260 265 270Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu
275 280 285Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His
290 295 300Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser Glu305 310 315 320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro
325 330 335Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys
340 345 350Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys
355 360 365Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln
370 375 380Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr385 390 395 400Gly Pro Lys Glu Thr Ser Gly His His His His His His
405 410(2)SEQ ID NO：22的信息：(ⅰ) 序列特征：
(A)长度：288个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：22：ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 60GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 120GCTGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 180GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAACCCA CCTCCCAATC CAAAGGGGAG 240CCGACAGGCC CGAAGGAAAC TAGTGGCCAC CATCACCATC ACCATTAA 288(3)SEQ ID NO：23的信息：(ⅰ) 序列特征：
(A)长度：96个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：23：Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30His Cys Gln Val Cys Phe Ile Thr Ala Ala Leu Gly Ile Ser Tyr Gly
35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr
50 55 60His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Lys Gly Glu65 70 75 80Pro Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
85 90 95(2)SEQ ID NO：24的信息：(ⅰ) 序列特征：
(A)长度：909个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：24：ATGGGTGGCA AGTGGTCAAA AAGTAGTGTG GTTGGATGGC CTACTGTAAG GGAAAGAATG 60AGACGAGCTG AGCCAGCAGC AGATGGGGTG GGAGCAGCAT CTCGAGACCT GGAAAAACAT 120GGAGCAATCA CAAGTAGCAA TACAGCAGCT ACCAATGCTG CTTGTGCCTG GCTAGAAGCA 180CAAGAGGAGG AGGAGGTGGG TTTTCCAGTC ACACCTCAGG TACCTTTAAG ACCAATGACT 240TACAAGGCAG CTGTAGATCT TAGCCACTTT TTAAAAGAAA AGGGGGGACT GGAAGGGCTA 300ATTCACTCCC AACGAAGACA AGATATCCTT GATCTGTGGA TCTACCACAC ACAAGGCTAC 360TTCCCTGATT GGCAGAACTA CACACCAGGG CCAGGGGTCA GATATCCACT GACCTTTGGA 420TGGTGCTACA AGCTAGTACC AGTTGAGCCA GATAAGGTAG AAGAGGCCAA TAAAGGAGAG 480AACACCAGCT TGTTACACCC TGTGAGCCTG CATGGAATGG ATGACCCTGA GAGAGAAGTG 540TTAGAGTGGA GGTTTGACAG CCGCCTAGCA TTTCATCACG TGGCCCGAGA GCTGCATCCG 600GAGTACTTCA AGAACTGCAC TAGTGAGCCA GTAGATCCTA GACTAGAGCC CTGGAAGCAT 660CCAGGAAGTC AGCCTAAAAC TGCTTGTACC AATTGCTATT GTAAAAAGTG TTGCTTTCAT 720TGCCAAGTTT GTTTCATAAC AGCTGCCTTA GGCATCTCCT ATGGCAGGAA GAAGCGGAGA 780CAGCGACGAA GACCTCCTCA AGGCAGTCAG ACTCATCAAG TTTCTCTATC AAAGCAACCC 840ACCTCCCAAT CCAAAGGGGA GCCGACAGGC CCGAAGGAAA CTAGTGGCCA CCATCACCAT 900CACCATTAA 909(2)SEQ ID NO：25的信息：(ⅰ) 序列特征：
(A)长度：303个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：25：Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val1 5 10 15Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala
20 25 30Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr
35 40 45Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr65 70 75 80Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu
100 105 110Trp Ile Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr
115 120 125Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys
130 135 140Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu145 150 155 160Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro
165 170 175Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arq Leu Ala Phe His
180 185 190His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser
195 200 205Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln
210 215 220Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His225 230 235 240Cys Gln Val Cys Phe Ile Thr Ala Ala Leu Gly Ile Ser Tyr Gly Arg
245 250 255Lys Lys Arg Arg Gln Arg Arg Arq Pro Pro Gln Gly Ser Gln Thr His
260 265 270Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Lys Gly Glu Pro
275 280 285Thr Gly Pro Lys Glu Thr Ser Gly His His His His His His
290 295 300(2)SEQ ID NO：26的信息：(ⅰ) 序列特征：
(A)长度：57个碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：26：TTCGAAACCA TGGCCGCGGA CTAGTGGCCA CCATCACCAT CACCATTAAC GGAATTC 57(2)SEQ ID NO：27的信息：(ⅰ) 序列特征：
(A)长度：17个氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性(ⅹⅰ) 序列描述：SEQ ID NO：27：Thr Ser Gly is His His His His Hia1 5
Claims (24)
1．一种蛋白，包含
(a)与或者(ⅰ)融合配偶体或者(ⅱ)HIV Nef蛋白或其衍生物连接的HIV Tat蛋白或其衍生物；或
(b)与或者(ⅰ)融合配偶体或者(ⅱ)HIV Tat蛋白或其衍生物连接的HIV Nef蛋白或其衍生物；或
(c)与HIV Tat蛋白或其衍生物和融合配偶体连接的HIVNef蛋白或其衍生物。
2．权利要求1中要求保护的蛋白，它是Tat-Nef融合蛋白或其衍生物。
3．权利要求1中要求保护的蛋白，它是Nef-Tat融合蛋白或其衍生物。
4．按照权利要求1的蛋白，其中所述Tat蛋白的衍生物是突变的Tat蛋白。
5．按照权利要求1的蛋白，其中所述Nef蛋白的衍生物是突变的Nef蛋白。
6．在权利要求1-5的任一项中要求保护的蛋白，其中所述融合配偶体是脂蛋白或其衍生物。
7．在权利要求6中要求保护的蛋白，其中所述脂蛋白是B型流感嗜血杆菌D蛋白或其衍生物。
8．在权利要求7中要求保护的蛋白，其中所述融合配偶体包括来自B型流感嗜血杆菌D蛋白的N末端的100-130个氨基酸。
9．在权利要求1至8的任一项中要求保护的蛋白，其中所述Tat蛋白是完整的Tat蛋白。
10．在权利要求1至8的任一项中要求保护的蛋白，其中所述Nef蛋白是完整的Nef蛋白。
11．在权利要求1至10的任一项中要求保护的蛋白，其中所述Tat蛋白与HIV Nef蛋白和融合配偶体融合。
12．在权利要求1至11的任一项中要求保护的蛋白，其中所述蛋白具有组氨酸尾。
13．编码权利要求1至12的蛋白的核酸。
14．用权利要求13的核酸转化的宿主。
15．在权利要求14中要求保护的宿主，其中所述宿主或者是巴斯德毕赤酵母或者是大肠杆菌。
16．包含与药学上可接受的赋形剂混合的权利要求1至12的任何一个的蛋白的疫苗。
17．权利要求16的疫苗，所述疫苗还包含佐剂。
18．权利要求17的疫苗，其中所述佐剂是TH1诱导佐剂。
19．在权利要求17或18中要求保护的疫苗，其中佐剂包含单磷酰脂A或其衍生物，诸如3脱氧-酰化单磷酰脂A。
20．在权利要求16至19的任一项中要求保护的疫苗，所述疫苗另外包含皂苷佐剂。
21．生产权利要求1至12的蛋白的方法，所述方法包括以下步骤：用编码所述蛋白的核酸转化宿主；表达所述蛋白和回收所述蛋白。
22．在权利要求21中要求保护的方法，其中所述宿主是大肠杆菌或巴斯德毕赤酵母。
23．生产权利要求16至20的疫苗的方法，所述方法包括将权利要求1至12的蛋白与药学上可接受的稀释剂混合。
24．在巴斯德毕赤酵母中制备(ⅰ)HIV Nef蛋白或其衍生物或(ⅱ)HIV Tat蛋白或其衍生物的方法，所述方法包括以下步骤：用编码所述HIV Nef蛋白或其衍生物或HIV Tat蛋白或其衍生物的DNA转化巴斯德毕赤酵母；表达所述蛋白并回收所述蛋白。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9720585.0A GB9720585D0 (en) | 1997-09-26 | 1997-09-26 | Vaccine |
| GB9720585.0 | 1997-09-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1279718A true CN1279718A (zh) | 2001-01-10 |
| CN1188519C CN1188519C (zh) | 2005-02-09 |
Family
ID=10819735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988114321A Expired - Fee Related CN1188519C (zh) | 1997-09-26 | 1998-09-17 | 包含HIV－1Tat和/或Nef蛋白的融合蛋白 |
Country Status (28)
| Country | Link |
|---|---|
| EP (1) | EP1015596B2 (zh) |
| JP (2) | JP2001518300A (zh) |
| KR (1) | KR100554487B1 (zh) |
| CN (1) | CN1188519C (zh) |
| AR (2) | AR017147A1 (zh) |
| AT (1) | ATE338128T1 (zh) |
| AU (1) | AU746564B2 (zh) |
| BR (1) | BR9812547A (zh) |
| CA (1) | CA2305013C (zh) |
| CO (1) | CO4810339A1 (zh) |
| CZ (1) | CZ302878B6 (zh) |
| DE (1) | DE69835756T3 (zh) |
| DK (1) | DK1015596T4 (zh) |
| ES (1) | ES2272012T5 (zh) |
| GB (1) | GB9720585D0 (zh) |
| HK (1) | HK1030431A1 (zh) |
| HU (1) | HUP0004896A3 (zh) |
| IL (1) | IL135102A0 (zh) |
| NO (2) | NO328824B1 (zh) |
| NZ (1) | NZ503482A (zh) |
| PL (1) | PL195243B1 (zh) |
| PT (1) | PT1015596E (zh) |
| SA (1) | SA98190877B1 (zh) |
| SI (1) | SI1015596T2 (zh) |
| TR (1) | TR200000864T2 (zh) |
| TW (1) | TW499436B (zh) |
| WO (1) | WO1999016884A1 (zh) |
| ZA (1) | ZA988789B (zh) |
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| CN107708671A (zh) * | 2015-04-16 | 2018-02-16 | 纽约州立大学研究基金会 | 包含钴卟啉‑磷脂缀合物和聚组氨酸标签的纳米结构 |
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| GB9820525D0 (en) * | 1998-09-21 | 1998-11-11 | Allergy Therapeutics Ltd | Formulation |
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| GB0000891D0 (en) | 2000-01-14 | 2000-03-08 | Allergy Therapeutics Ltd | Formulation |
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| GB9003010D0 (en) * | 1990-02-09 | 1990-04-04 | Glaxo Group Ltd | Gene expression in yeast cells |
| AU681572B2 (en) * | 1991-10-21 | 1997-09-04 | Med Immune, Inc. | Bacterial expression vectors containing DNA encoding secretion signals of lipoproteins |
| DK0656950T3 (da) * | 1992-08-21 | 1999-07-19 | Biogen Inc | TAT-afledte transportpolypeptider |
| ATE302854T1 (de) * | 1993-01-26 | 2005-09-15 | Univ Pennsylvania | Zusammensetzungen und verfahren zur verabreichung von genetischem material |
| JPH11501310A (ja) * | 1995-03-08 | 1999-02-02 | ネオバック | レトロウイルスの調節タンパク質から誘導された無毒の免疫原、抗体、製造方法およびそれらを含んでなる医薬組成物 |
| GB9720585D0 (en) * | 1997-09-26 | 1997-11-26 | Smithkline Beecham Biolog | Vaccine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318091C (zh) * | 2001-07-26 | 2007-05-30 | 高等健康研究院 | 生物活性HIV－1Tat、其片段或衍生物用于预防或治疗性免疫接种和/或治疗其它疾病中靶向和/或激活抗原呈递细胞、和/或运送货物分子的用途 |
| CN1325512C (zh) * | 2002-05-16 | 2007-07-11 | 巴法里安诺迪克有限公司 | Hiv调节/辅助蛋白的融合蛋白 |
| CN107708671A (zh) * | 2015-04-16 | 2018-02-16 | 纽约州立大学研究基金会 | 包含钴卟啉‑磷脂缀合物和聚组氨酸标签的纳米结构 |
| US11207421B2 (en) | 2015-04-16 | 2021-12-28 | The Research Foundation For The State University Of New York | Nanostructures comprising cobalt porphyrin-phospholipid conjugates and polyhistidine-tags |
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