CN1154742C - 重组mva病毒及其应用 - Google Patents
重组mva病毒及其应用 Download PDFInfo
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- CN1154742C CN1154742C CNB961952547A CN96195254A CN1154742C CN 1154742 C CN1154742 C CN 1154742C CN B961952547 A CNB961952547 A CN B961952547A CN 96195254 A CN96195254 A CN 96195254A CN 1154742 C CN1154742 C CN 1154742C
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Abstract
包含并能表达插入改良安卡拉痘苗病毒(MVA)基因组自然发生缺失位点的外源基因的重组MVA病毒,以及应用这类重组MVA病毒生产多肽,例如抗原或治疗剂,和重组病毒以用作疫苗或产生病毒载体用于基因治疗。
Description
本发明涉及从改良安卡拉痘苗病毒(MVA)衍生的重组痘苗病毒及其包含且能够表达插入到MVA染色体上自然发生缺失位点上的外源基因;涉及使用这类重组MVA病毒产生多肽,例如抗原或治疗剂,或产生用于基因治疗的病毒载体,以及使用这类编码抗原的重组MVA病毒作为疫苗。
本发明的目的之一是提供一个重组MVA病毒,它可作为有效且特别安全的表达载体。
本发明的另一个目的是提供一种简单、有效、安全的方法产生多肽,例如抗原或治疗剂,产生重组病毒用作疫苗以及产生病毒载体用于基因治疗。
本发明的再一个目的是提供一个基于表达T7RNA聚合酶的重组MVA病毒的表达系统,以及基于此表达系统产生多肽,例如抗原或治疗药剂,或产生病毒载体用于基因治疗或用作疫苗。
痘苗病毒是痘病毒科正痘病毒属的一员,曾用作活疫苗免疫人天花病。世界范围用痘苗病毒成功的接种最终导致天花病因-天花病毒的绝迹[天花的全球绝迹。全球天花绝迹鉴定委员会的最后报告。公共卫生历史,第4号(History ofPublic Health,No.4),日内瓦:世界卫生组织,1980]。因而世界卫生组织(WHO)宣告,除了有高度危险痘病毒感染的人群(例如实验室人员)外,已全世界性地停止接种。
最近,痘苗病毒也已用于设计病毒载体,以进行重组基因表达和可能用作重组活疫苗[Mackett,M.,Smith,G.L.和Moss,B.[1982美国国家科学院院报(P.N.A.S.USA)79,7415-7419;Smith,G.L.,Mackett,M.和Moss,B.[1984]生物技术和遗传工程评论2(Biotechnology and Genetic Engineering Reviews2),383-407]。这必然伴有在DNA重组技术帮助下把编码外源抗原的DNA序列(基因)引入痘苗病毒基因组。如果基因整合到病毒DNA中对病毒生活周期非必需的位点,可能新产生的重组痘苗病毒是感染性的,即能够感染外源细胞并从而表达整合的DNA序列[欧洲专利申请83,286号和110,385号(EP PatentApplications No.83,286 and No.110,385)]。用这种方法制备的重组痘苗病毒一方面可用作活疫苗预防传染性疾病,另一方面可用于在真核细胞中制备异源蛋白质。
表达噬菌体T7 RNA聚合酶基因的重组痘苗病毒可以建立广泛应用的表达系统以用于在哺乳动物细胞中合成重组蛋白(Moss,B.,Elroy-Stein,O.,Mizukami,T.,Alexander,W.A.,和Fuerst T.R.[1990]自然(Nature)348,91-92.)。在所有方案中,重组基因表达依赖于真核细胞细胞质中T7 RNA聚合酶的合成。最普遍的一个方案是用于瞬时表达(Fuerst,T.R.,Niles,E.G.,Studier,F.W.和Moss,B.[1986]美国国家科学院院报(Proc.Natl.Acad.)Sci.USA 83,8122-8126和美国专利申请(US patent application)7.648.971)。首先,一个感兴趣的外源基因插入质粒并在T7 RNA聚合聚启动子控制之下。随后,质粒引入到细胞的原生质体中,该细胞通过标准转染方法由产生T7 RNA聚合酶的重组痘苗病毒预先感染。
此转染方案非常简单因为不需构建新的重组病毒并且特别有效,有超过80%的细胞表达感兴趣的基因[Elroy-Stein,O.和Moss,B.[1990]美国国家科学院院报(Proc.Natl.Acad.Sci.USA)87,6743-6747]。痘苗病毒/T7 RNA聚合酶杂合系统比其它瞬时表达系统的优势很可能在于它不依赖于质粒到细胞核的转运。在过去,该系统在病毒学和细胞生物学中对检测目的是极其有用的(Bunocore,L.和Rose,J.K.[1990]自然(Nature)345,625-628,Pattnaik,A.K.和Wertz,G.W.[1991]美国国家科学院院报(Proc Natl.Acad.Sci.USA)88,1379-1383,Karschin,A.,Aiyar,J.,Gouin,A.,Davidson,N.和Lester,H.A.[1991]欧洲生化学会联合会快报(FEBS Lett.)278,229-233,Ho,B.Y.,Karschin,A.,Raymond,J.,Branchek,T.,Lester,H.A.和Davidson,N.[1992]欧洲生化学会联合会快报(FEBS Lett.)301,303-306,Buchholz,C.J.,Retzler C.,Homann,H.E.,和Neubert,W.J.[1994]病毒学(Virology)204,770-776)。然而,痘苗病毒/T7 RNA聚合聚杂合系统的重要前景应用,例如产生重组蛋白或重组病毒颗粒用于人体的新的治疗或预防方法,可能由于重组痘苗载体的大量复制而受到阻碍。
痘苗病毒对人是感染性的并且在根除天花运动中的接种时偶而会观察到严重的并发症。关于并发症发病率的最好综述由一项美国国家调查提供,该调查检测了大约一千二百万接种疫苗的人,接种疫苗基于纽约城市卫生委员会痘苗病毒菌株(Lane,J.,Ruben,F.,Neff,J.和Millar,J.[1969]新英格兰医学杂志(New Engl.J.Med.)281,1201-1208)。因而安全性考虑和规范影响了使用痘苗病毒作为载体发展重组活疫苗的最令人鼓舞的可能性。此外,文献中描述的大多数重组痘苗病毒基于西方保藏的痘苗病毒菌株(Western Reservestrain of vaccinia virus)。另一方面,众所周知此菌株有较高的神经毒性因而不适用于人类和动物(Morita等,疫苗(Vaccine)5,65-70[1987])。对于载体应用,通过使用高度减毒的痘苗病毒菌株可以减小健康危险。特别发展了许多这类痘苗病毒以避免天花接种中的不希望的副作用。因而,通过痘苗病毒安卡拉菌株(CVA)在鸡胚成纤维细胞中长期的连续传代产生了改良安卡拉痘苗病毒(MVA)(综述参看Mayr,A.,Hochstein-Mintzel,V和Stickl,H.[1975]感染(Infection)3,6-14;瑞士专利号(Swiss Patent No.)568,392)。MVA病毒按照布达佩斯条约(Budapest Treaty)于1987年11月15日保藏于CNCM(巴斯德研究所,微生物菌种国家收藏馆(Institut Pasteur,Collection Nationale de Culturesde Microorganisms,)25,rue du Docteur Roux,75724 Paris Cedex 15),保藏号1-721。MVA的特点是强减毒性,也就是说通过减少的毒性或感染性同时保持良好的免疫原性。检测了MVA病毒以确定它相对于野生型CVA菌株基因组的变化。检测到六个主要的共31,000碱基对的基因组DNA缺失(缺失I,II,III,IV,V和VI)(Meyer,H.,Sutter,G.和Mayr A.[1991]普通病毒学杂志(J.Gen.Virol.)72,1031-1038)。产生的MVA病毒其宿主细胞严格限于禽源细胞。另外,MVA具有高度减毒的特征。在多种动物模型中的检验证明MVA甚至在免疫抑制的动物中都是无毒性的。更重要的是,MVA菌株的优秀特性已在广泛的临床试验中得到证实(Mayr等,Zbl.Bakt.Hyg.l,Abt.Org.B167,375-390[1987],Stickl等,Dtsch.med.Wschr.99,2386-2392[1974])。在这些超过120,000人的研究中,包括高危病人,没发现有与MVA疫苗相关的副作用。
已发现MVA在人细胞中的复制在感染后期受到阻碍,防止了成熟感染性病毒粒子的组装。尽管如此,MVA甚至在非允许细胞中也能高水平表达病毒的和重组的基因,并建议作为有效的和特别安全的基因表达载体(Sutter,G.和Moss,B.[1992]美国国家科学院院报(Proc.Natl.Acad.Sci.USA)89,10847-10851)。最近,在MVA基础上构建了新的痘病毒载体系统,外源DNA序列插入MVA基因组的缺失III或TK基因(Sutter,G.和Moss,B.[1995]发育生物学标准(Dev.Biol.Stand.)Basel,Karger 84,195-200和美国专利(USpatent)5,185,146)。
为了进一步开发MVA的应用,发现了一个通过DNA重组把外源基因引入痘苗病毒MVA菌株的新的可能方法。由于发明不是要改变MVA病毒的基因组,因此必需使用符合此需要的方法。依据本发明,一个外源DNA序列准确地在MVA基因组天然发生缺失位点重组到病毒DNA。
本发明从而特别地包括单独的下述内容或它们的组合:
一个重组MVA病毒包含并能够表达至少一种插入MVA基因组自然发生缺失位点的外源基因;
一个如上所述的重组MVA病毒包含并能够表达至少一种插入MVA基因组缺失II位点的外源基因;
一个如上所述的重组MVA病毒,其中的外源基因编码一个标记,一个治疗基因或一个抗原决定簇;
一个如上所述的重组MVA病毒,其中的外源基因编码一个来自于病原性病毒、细菌、或其它微生物,或来自于寄生虫,或肿瘤细胞的抗原决定簇;
一个如上所述的重组MVA病毒,其中的外源基因编码一个来自于恶性疟虫(Plasmodium Falciparum),分枝杆菌(Mycobacteria),疱疹病毒,流感病毒(influenza virus),肝炎或人免疫缺陷病毒的抗原决定簇;
一个如上所述的重组MVA病毒,其中的抗原决定簇是人免疫缺陷病毒(HIV)的nef或人酪氨酸酶;
一个如上所述的重组MVA病毒,其为MVA-LAI nef或MVA-hTYR;
一个如上所述的重组MVA病毒,其中的外源基因编码T7 RNA聚合酶;
一个如上所述的重组MVA病毒,其为MVA-T7 pol;
一个如上所述的重组MVA病毒,其中的外源基因处于痘苗病毒早期/晚期启动子P7.5的转录控制之下;
如上所述的重组MVA病毒,基本上不含能够在人细胞中复制的病毒;
一个如上所述的重组MVA病毒用于处在T7 RNA聚合酶启动子转录控制之下的DNA序列的转录;
由任一上述重组MVA病毒感染的真核细胞;
由一种上述重组MVA病毒感染的细胞,其中的外源基因编码T7 RNA聚合酶;
由一种上述重组MVA病毒感染的细胞,其中的外源基因编码T7 RNA聚合酶,此外还包含载有一种或几种处于T7 RNA聚合酶启动子转录控制之下的外源基因的一种或几种表达载体;
如上所述细胞的应用以产生由上述外源基因编码的多肽,包括:
a)在适宜条件下培养上述的细胞,以及
b)分离由上述的外源基因编码的多肽。
由一种上述重组MVA病毒感染的细胞,其中的外源基因编码T7 RNA聚合酶,此外还包含表达载体,该表达载体载有处于T7 RNA聚合酶启动子控制之下的病毒基因和/或编码一病毒载体基因组的一个病毒载体构建体;
如上所述细胞的应用以产生病毒颗粒,包括:
a)在适宜条件下培养上述的细胞,以及
b)分离病毒颗粒;
由一种如上所述MVA病毒感染的细胞,其中的外源基因编码T7 RNA聚合酶,此外还含有:
a)载有一种逆转录病毒载体构建体的表达载体,它能够感染和指导由上
述逆转录病毒载体构建体携带的一种或几种外源基因在靶细胞中的
表达,以及
b)一种或几种表达载体,其载有的基因处于T7 RNA聚合酶启动子转录
控制之下并编码上述逆转录病毒载体构建体的基因组包装所必需的
多肽;
如上所述细胞的应用以产生逆转录病毒粒子,包括:
a)在适宜条件下培养上述的细胞,以及
b)分离逆转录病毒颗粒;
含有一种上述重组MVA病毒的疫苗,其中的外源基因在生理可接受载体中编码一抗原决定簇;
一种如上所述重组MVA病毒的应用,其中的外源基因编码一种疫苗制剂中的抗原决定簇;
一种如上所述疫苗的应用以免疫活的动物体,包括人;
一种如上所述包括MVA-LAInef的疫苗的应用以防止或治疗人免疫缺陷病毒(HIV)感染或爱滋病(AIDS);
一种如上所述包括MVA-hTYR的疫苗的应用以防止或治疗黑素瘤;
一种疫苗,其第一种组分包括一种如上所述的重组MVA病毒,其中包括的外源基因在生理可接受载体中编码T7 RNA聚合酶;第二种组分包括一个载有在生理可接受载体中处于T7 RNA聚合酶启动子转录控制之下的抗原决定簇的DNA序列,两种组分可同时包含或分别包含;
如上所述疫苗的应用以免疫活的动物体,包括人,包含用疫苗的第一种和第二种组分对上述活的动物体,包括人,进行同时接种或在同一接种部位延时接种;以及
术语“基因”表示编码一种蛋白或肽的任何DNA序列。
术语“外源基因”表示插入一DNA序列的基因,该序列中通常未发现该基因。
外源基因可以是例如标记基因、治疗基因、编码抗原决定簇的基因或病毒基因。这类基因在本技术领域众所周知。
改良安卡拉痘苗病毒(MVA),一个宿主范围局限的和高度减毒的痘苗病毒茵株,在测试的人和大多数其它哺乳动物细胞系中不能增殖。但由于在非允许细胞中病毒基因表达未受损伤,依据本发明的重组MVA病毒可能用作特别安全和有效的表达载体。
编码抗原决定簇的重组MVA病毒
在一个实施方案中,本发明涉及重组痘苗病毒,其含有编码外源抗原,优选地属于致病病原体的基因,以及以生理可接受形式包含此类病毒的疫苗。本发明也涉及制备此类重组MVA痘苗病毒或疫苗的方法;涉及应用这些疫苗预防那些致病病原体引起的感染。
在本发明的一个优选实施方案中,插入MVA病毒的外源基因是一个编码HIVnef的基因。
我们已构建了重组MVA病毒,允许HIV-Inef基因在痘苗病毒早/晚期启动子P7.5的控制下进行表达。灵长类慢病毒的调节性Nef蛋白合成于病毒复制周期的早期并已表明对高滴度病毒复制和体内疾病诱导起重要作用。这提示HIV Nef可能在爱滋病病理中有关键作用。Nef促进病毒感染性增加和HIV致病性的分子机制需要进一步阐明。但是,Nef是免疫原性的并且Nef特异抗原可用作疫苗防治HIV感染和爱滋病。
在本文中,表达HIVnef基因的重组MVA病毒可用于人体免疫,一方面作为预防疫苗防止人HIV病毒,另一方面用于HIV感染或爱滋病人的免疫治疗。另外,表达HIVnef基因的MVA病毒也可用于重组HIV Nef蛋白的产生。
本发明的另一个优选实施方案中,插入MVA病毒的外源基因是一编码人酪氨酸酶的基因。
我们已构建了重组MVA病毒,允许人酪氨酸酶基因在痘苗病毒早/晚期启动子P7.5的控制下进行表达。近来。人酪氨酸酶被鉴定为黑素瘤特异性肿瘤抗原,它可造成抗肿瘤的溶细胞T-淋巴细胞的产生(Brichard,V等[1993]实验医学杂志(J.Exp.Med.)178,489-495)。由于正常细胞中看来只有黑素细胞表达酪氨酸酶基因,因而酪氨酸酶成为黑素瘤免疫治疗的有效目标抗原。所以,表达人酪氨酸酶基因的重组MVA病毒可用于黑素瘤病人以诱导激发肿瘤排斥或防止转移的免疫反应。表达人酪氨酸酶基因的重组MVA病毒可直接用作抗黑素瘤疫苗,或用病毒制备抗黑素瘤疫苗。在一个例子中,表达人酪氨酸酶基因的重组MVA病毒可用于产生重组酪氨酸酶蛋白,它作为抗原用于疫苗制剂。在另一个例子中,用表达人酪氨酸酶基因的重组MVA病毒作为表达载体,来自肿瘤病人的细胞体外修饰以表达酪氨酸酶并继而转移回病人来诱导抗肿瘤免疫反应。基于表达人酪氨酸酶基因的重组MVA病毒的制备疫苗可用于肠胃外或肿瘤部位。可以防止肿瘤转移或肿瘤的诸如大小、形状、稠度、血管形成或其它表型变化。以表达人酪氨酸酶基因的重组MVA为基础制备的疫苗可用于手术切除肿瘤前、期间或切除后。
为了制备疫苗,依据本发明的MVA痘苗病毒要转换成生理可接受形式。这可基于制备MVA疫苗以免疫天花的经验进行(如Stickl,H.等[1974]Dtsch.Med.Wschr.99,2386-2392所述)。典型地,大约106-108重组MVA颗粒在含2%胨和1%人白蛋白的100毫升磷酸盐缓冲液(PBS)中冻干并置于一个安瓿管中,优选地是一玻璃安瓿管。冻干品可含有补充剂(例如甘露糖醇、葡聚糖、蔗糖、甘氨酸、乳糖或聚乙烯吡咯烷酮)或其它助剂(例如抗氧化剂,稳定剂等,以适用于肠胃外给药。玻璃安瓿管继而密封并可优选地在-20℃以下贮存几个月。
用于接种或治疗的冻干品可溶于0.1到0.5毫升水溶液,优选地是生理盐水中,并经肠胃外给药例如肌肉内接种或局部给药例如接种到肿瘤或在肿瘤部位局部给药。根据本发明的疫苗或治疗优选地采用肌肉内注射(Mayr,A.等[1978]Zbl,Bakt.Hyg.,I.Abt.Orig.B167,375-390)。给药方式,剂量和给药次数可由本领域熟练技术人员对所知方式进行优化。合适的话,在长时间内接种多次以获得对外源抗原合适的免疫反应是适宜的。
使用重组MVA病毒产生异源多肽
依据本发明的重组MVA痘苗病毒也可用于在真核细胞中制备异源多肽。这导致产生被重组痘苗病毒感染的细胞。编码异源多肽的基因在细胞中表达,并分离所表达的异源多肽。产生此类异源多肽的方法通常为本领域熟练技术人员熟知(EP-A-206和EP-A-205,939)。由于MVA病毒的特殊属性,在重组MVA病毒帮助下产生的多肽非常适于用作人和动物药物。
编码T7 RNA聚合酶的重组MVA病毒及其
应用于表达在T7 RNA聚合酶启动子转录
控制下的DNA序列
在本发明的进一个实施方案中,我们构建了重组MVA病毒,允许在痘苗病毒早期/晚期启动子P7.5的控制下表达噬茵体T7 RNA聚合酶。MVA-T7 pol重组病毒作为表达系统的用途已经过诱导处于T7 RNA聚合酶启动子控制之下的重组基因表达的瞬间转染分析得到证实。使用大肠杆菌(E.coli)氯霉素乙酰转移酶(CAT)基因作为报告基因,我们发现MVA-T7聚合酶诱导的CAT基因表达就象衍生于痘苗病毒复制型WR菌株的痘苗病毒/T7pol重组体一样有效。
依据本发明的MVA/T7聚合酶杂合系统从而可用作简单、有效和安全的哺乳动物表达系统,以在缺乏大量痘苗病毒复制的情况下产生多肽。
此表达系统也可用于在T7 RNA聚合酶启动子转录控制下产生重组病毒粒子以用于免疫接种或基因治疗,上述病毒粒子通过被表达T7 RNA聚合酶的重组MVA或包含所有或部分基因的DNA构建体以及为产生诸如MVA粒子或逆转录病毒粒子等的病毒粒子所必需的基因组或重组基因组感染的细胞系的转化来产生。
逆转录病毒载体系统包括两种组分:
1)逆转录病毒载体本身是一个修饰的逆转录病毒(载体质粒),其中编码病毒蛋白的基因被将要传递给靶细胞的治疗基因和标记基因代替。由于编码病毒蛋白的基因替代损坏了病毒,必须在系统中存在第二种组分提供给修饰逆转录病毒丢失的病毒蛋白以拯救该病毒。
第二种组分是:
2)产生大量病毒蛋白但又缺乏产生可复制型病毒能力的细胞系。此细胞系称为包装细胞系并含有被一个或多个携带能够包装修饰的逆转录病毒载体的基因(编码gag,pol和env多肽的基因)的质粒所转染的一细胞系。
为了产生包装的载体,载体质粒转染入包装细胞系。在这种情况下,含有插入的治疗和标记基因的改良逆转录病毒基因组从载体质粒转录并包装入改良逆转录病毒粒子(重组病毒粒子)。然后用此重组病毒感染靶细胞,其载体基因组和任何携带的标记或治疗基因整合到靶细胞DNA。受此重组病毒粒子感染的细胞不能产生新的载体病毒,因为这些细胞中没有病毒蛋白质。但携带治疗和标记基因的载体DNA整合到了细胞DNA并能在感染细胞中表达。
依据本发明表达T7 RNA聚合酶的重组MVA病毒可用于产生包装逆转录病毒载体所需的蛋白。为此目标,把一个逆转录病毒(例如鼠白血病病毒(MLV))的gag,pol和env基因置于一个或多个表达载体(例如质粒)中T7 RNA聚合酶启动子转录控制之下。表达载体随后引入用重组MVA病毒感染的表达T7 RNA聚合酶的细胞中,同时转入载有逆转录病毒载体构建体的一个表达载体,通常处于T7 RNA聚合酶启动子的转录控制之下。
WO 94/29437,WO 89/11539和WO 96/07748描述了不同类型的逆转录病毒载体构建体,它们可用上述包装系统进行包装。
表达T7 RNA聚合酶的重组MVA病毒的再一个应用是产生重组蛋白,非感染性病毒粒子,或感染性突变病毒粒子,以用于生产疫苗或治疗剂(Buchholz等,病毒学(Virology),204,770-776(1994)和EP-B1-356695)。为此目的,把病毒基因(例如HIV-1的gag-pol和env基因)置于一个表达载体(例如质粒或其它重组MVA病毒)中T7启动子的转录控制之下。此构建体随后引入用重组MVA病毒感染的表达T7 RNA聚合酶的细胞中。重组病毒基因高效转录,重组蛋白大量制造并可纯化。此外,表达的重组病毒蛋白(例如HIV-1的env,gag)可能组装成假病毒粒子从细胞出芽并可从组织培养物中分离。在另一个实施方案中,由MVA-T7pol系统表达的病毒蛋白(来源于例如人免疫缺陷病毒(HIV),猴免疫缺陷病毒(SIV),麻疹病毒(Measles virus))通过克服病毒增殖中吸附和感染,脱壳,核酸复制,病毒基因表达,装配,出芽或其它步骤中的缺陷来拯救引入的突变病毒,从而可产生并纯化所述的突变病毒。
MVA-T7pol也可与携带感兴趣抗原基因(例如,HIV的nef,tat,gag,pol或env或其它基因)的DNA序列同时使用进行免疫。首先,给定抗原(例如人免疫缺陷病毒(HIV),丙型肝炎病毒(HCV),人乳头瘤病毒(HPV),单纯疱疹病毒(HSV),麻疹病毒,流感病毒或其它)的编码序列克隆到处于T7 RNA聚合酶启动子控制之下优选地是质粒的载体中并使用常规实验室方法扩增和纯化产生的DNA构建体。其次,载体DNA同时或在适当时间延迟后与MVA-T7pol一起免疫接种。在接种部位,感兴趣的重组基因在含有载体DNA和MVA-T7pol的细胞中瞬时表达并且相应抗原存在于宿主免疫系统刺激了抗原特异性免疫反应。这种使用非复制痘苗载体MVA-T7pol的方案代表了一种提供给定抗原有效瞬时表达而又避免了组成型基因表达潜在危险的核酸免疫接种的有前途的新方法。
重组MVA痘苗病毒可按下文陈述进行制备。
一个包含侧翼为MVA基因组中邻近自然发生缺失例如缺失II的MVADNA序列的编码一个外源多肽DNA序列的DNA构建体,引入到用MVA感染的细胞中,以发生同源重组。一旦把DNA构建体引入真核细胞并且外源DNA与病毒DNA重组,就可能通过对所需重组痘苗病毒用已知的方式,优选地在标记帮助下,分离到所需重组痘苗病毒(对比Nakano等,美国国家科学院院报(Proc.Natl.Acad.Sci.USA)79,1593-1596[1982],Franke等,分子细胞生物学(Mol.Cell.Biol.)1918-1924[1985],Chakrabarti等,分子细胞生物学(Mol.Cell.Biol.)3403-3409[1985],Fathi等,病毒学(Virology)97-105[1986])。要插入的DNA-构建体可以是线状的或环状的。环状DNA是优选的,特别是质粒。DNA-构建体包含位于MVA基因组自然发生缺失例如缺失II左方和右方侧翼的序列(Altenburger,W,Suter,C.P.和AltenburgerJ.(1989)病毒学集刊(Arch.Virol.)105,15-27)。外源DNA序列插入位于自然发生缺失侧翼的序列之间。外源DNA序列可以是编码治疗多肽,例如组织血纤维溶酶原激活剂(t-PA)或干扰素,或来自致病病原体的抗原决定簇的基因。致病病原体可以是导致疾病的病毒、细菌和寄生虫,也可能是在有机体中无限制增殖从而可能导致病理生长的肿瘤细胞。这些致病病原体的例子由Davis,B.D.等做过描述(微生物学(Microbiology)第三版,HarperIntemational Edition)。优选的致病病原体抗原来自人免疫缺陷病毒(例如HIV-1和HIV-2),导致肺结核的分枝杆菌,寄生虫恶性疟虫,以及黑素瘤细胞。
为了表达一个DNA序列或基因,DNA中必须存在基因转录必需的调节序列。这些调节序列(称为启动子)为本技术领域技术人员所熟知并包含例如EP-A-198,328中所述的痘苗病毒11kDa基因和那些7.5kDa基因(EP-A-110,385)。DNA构建体可通过转染,例如通过磷酸钙沉淀方法(Graham等,病毒学(Virol.)52,456-467[1973];Wigler等,细胞(Cell)777-785[1978])通过电穿孔方法(Neumann等,欧洲分子生物学组织杂志(EMBO J.I,)841-845[1982]),通过微注射方法(Graessmann等,酶学方法(Meth.Enzymology)101,482-492(1983)),通过脂质体方法(Straubinger等,酶学方法(Methods inEnzymology 101,512-527(1983),通过原生质球方法(Schaffner,美国国家科学院院报(Proc.Natl.Acad.Sci.USA)77,2163-2167(1980))或其它本技术领域熟知的方法引入到MVA感染的细胞。通过磷酸钙沉淀进行转染是优选的方法。
下面的详细实施例是为了有助于对本发明的更好理解。但不是说本发明仅局限于实施例内容。
附图说明图1:图示MVA基因组图谱和用于通过同源重组插入外源DNA的质粒图
谱:MVA基因组中的HindIII限制性位点标在上面。显示了重叠MVA
基因组中缺失II接界的900 bp HindIII-HindIII N片段。邻近缺失II
的MVADNA序列通过PCR(聚合酶链式反应)扩增并用于构建插入质
粒pUCIILZ。图2:pUCIILZP7.5:用于插入缺失II的MVA载体质粒,包含P11-Lac Z
表达盒和痘苗病毒早期/晚期启动子P7.5以表达可克隆到质粒SmaI位
点的感兴趣基因。图3:pUCIILZdel P7.5:用于在MVA基因组缺失II位点插入外源基因的
MVA载体质粒,含有自缺失P11-Lac Z表达盒和痘苗病毒早期/晚
期启动子P7.5以表达可克隆到质粒SmaI/NotI克隆位点的感兴趣基
因。图4:重组病毒MVA-T7 pol的构建:图示MVA基因组(HindII限制性内
切核酸酶位点)图谱以及使T7 RNA聚合酶基因插入MVA基因组
HingIIIN片段中缺失II位点的载体质粒pUCIILZT7pol的图谱。图5:MVA-T7pol病毒DNA的Sourthern印迹检测图6:用[35S]甲硫氨酸对蛋白进行代谢标记。SDS聚丙烯酰胺(PAGE)凝胶
电泳检测。泳道1:MVAT7pol,泳道2:MVA,泳道3:CV-1细
胞。图7:氯霉素乙酰基转移酶(CAT)检测:用含有处于T7 RNA聚合酶启动子控
制下的CAT基因的质粒转染的以及用MVA-T/pol或WR-T7pol
感染的CV-1细胞。裂解物用于检测CAT活性。C是指氯霉素,1
-AcC和3-AcC是指单和三乙酰化形式的氯霉素。Cat活性用60
分钟内形成的乙酰化产物百分数表示。图8:MVA-LAInef的构建:图示MVA基因组(HindIII限制性内切核酸酶
位点)图谱以及使HIV-1LAI的nef基因插入MVA基因组HindIIIN
片段中缺失II位点上的载体质粒pUCIILZdel P7.5-LAInef的图谱。图9:MVA-hTYR的构建:图示MVA基因组(HindIII限制性内切核酸酶
位点)图谱以及使人酪氨酸酶基因插入MVA基因组HindIIIN片段中缺
失II位点上的载体质粒pUCIILZdel P7.5-TYR的图谱。
实施例
1.病毒的生长和纯化
1.1 MVA病毒的生长
MVA病毒是一种高度减毒的痘苗病毒,它由安卡拉痘苗病毒(CVA)通过在原代鸡胚成纤维细胞(CEF)培养物中长期连续传代衍生而来。关于产物的历史、特性及MVA菌株的应用的概括性综述参考Mayr等在感染(Infection)3,6-14[1975]中发表的总结。由于在CEF中的减毒,MVA病毒在这种禽宿主细胞中复制到很高的滴度。然而在哺乳动物细胞中MVA是严格生长局限的,并且检测不到病毒引起的典型噬斑形成。所以,MVA病毒要生长于CEF细胞。为了制备CEF细胞,从孵化11天的鸡蛋中分离胚,去除末端,并把胚绞碎后在含有0.25%胰蛋白酶的溶液中37℃解离20分钟。过滤产生的细胞悬液并用SorvaURC-3B离心机在室温下2000rpm离心5分钟沉淀细胞,重悬于10体积培养基A(Eagle基础培养基(MEM Eale),例如可获取于LifeTechnologies GmbH,Eggenstein,Germany),并再用Sorvall RC-3B离心机在室温下2000rpm离心5分钟进行沉淀。细胞沉淀重悬于含10%胎牛血清(FCS),青霉素(100单位/毫升),链霉素(100毫克/毫升)和2mM谷氨酰氨的培养基A中以获得含500000细胞/毫升的细胞悬液。用这种方法获得的CEF细胞铺于细胞培养皿中。把它们放到培养基A中,按照所需细胞浓度在二氧化碳培养箱中37℃生长1-2天,并直接或再进行一次细胞传代后用于感染。制备原代培养物的详细描述可在R.I.Freshney,“动物细胞培养”(“Culture ofanimal cell”),Alan R.Liss Verlag,New York[1983]一书中第11章99页及其下列等等中找到。
MVA病毒按下面方法用于感染。CEF细胞培养在175厘米2细胞培养瓶。在90%-100%铺满时,去除培养基并且细胞在培养基A中与MVA病毒悬液(每个细胞0.01感染单位(IU),0.02毫升/厘米2)共培养1小时。然后添加更多的培养基A(0.2毫升/厘米2)并且把培养瓶在37℃培养2-3天(直到大约90%的细胞表现细胞病变效应)。通过刮细胞单层到培养基并在Sorvall RC-3B离心机中4℃3000rpm离心5分钟沉淀细胞物质制备粗病毒贮存物。粗病毒制剂在进一步处理(例如病毒纯化)前贮存于-20℃。
1.2病毒的纯化
为获得尽可能纯的并且不含对宿主细胞特异组分的病毒所采用的纯化步骤类似于Joklik所述(病毒学(Virology)18,9-18[1962])。融解贮存于-20℃的粗病毒保存物并在PBS中悬浮一次(10-20倍沉淀体积),悬浮液如上述方式离心。新沉淀悬浮于10倍体积Tris缓冲液1(10mM Tris-HCl pH9.0),悬浮液用超声波简短处理(Labsonic L,B.Braun Biotech Intemational,Melsungen Germany;2×10秒,60瓦,室温)以进一步离解细胞碎片并从细胞物质中释放病毒粒子。细胞核和较大细胞碎片继而通过悬浮液的简短离心去除(获取于杜邦公司(Du Pont Co.)的Sorvall GSA转头,D-6353 BadNauheim,FRG;3000rpm 10℃离心3分钟)。沉淀再一次悬浮于Tris缓冲液1,用超声波处理并离心,如上所述。收集的含游离病毒粒子的上清液联合并铺层于10mM Tris-HCl,pH9.0的10毫升36%蔗糖垫层上,在Beckman SW27/SW 28离心机转头中4℃13,500rpm离心80分钟。去掉上清液,含有病毒粒子的沉淀溶解于10毫升pH9.0的1mM Tris-HCl,用超声波简短处理(室温下2×10秒,仪器如上所述)均匀,加样到20%-40%蔗糖梯度(蔗糖溶于1mM Tris-HCl,pH9.0)以进一步纯化。梯度液在Beckmann SW41离心机转头中4℃13,000rpm离心50分钟。离心后,含有病毒粒子的分离带在抽吸去带上面体积后用移液管收集。获得的蔗糖溶液用三倍体积PBS稀释然后病毒粒子再通过离心沉淀(Beckmann SW 27/28,60分钟,13,500rpm,4℃)。现在,沉淀绝大部分由纯病毒粒子构成,它重悬于PBS并平衡至病毒浓度平均相当于1-5×109IU/ml。纯化的病毒贮存液贮存于-80℃并直接或用PBS稀释后用于随后的实验。
1.3 MVA病毒的克隆
为了产生均匀贮存病毒制剂,获取于Anton Mayr教授的MVA病毒通过在96孔组织培养板上CEF培养物中连续三次传代过程中的有限稀释进行克隆化。筛选到MVA克隆F6并在CEF中扩增以获得病毒的工作贮存物,作为产生本发明申请中描述的重组MVA病毒以及用于产生以前描述的重组MVA病毒的起始材料(Sutter,G.andMoss,B.[1992]美国国家科学院院报(Proc.Natl.Acad.Sci.USA)89,10847-10851;Sutter,G.,Wyatt,L.,Foley,P.,Bennink,J.和Moss,B.[1994]疫苗(Vaccine)12,1032-1040;Hirsch,V,Fuerst,T.,Sutter,G.,Carroll,M.,Yang,L.,Goldstein,S.,Piatak,M.,Elkins,W.,Alvord,G.,Montefiori,D.,Moss,B.and Lifson,J.[1996]病毒学杂志(J.Virol.)70,3741-3752)。
2.重组MVA病毒的构建及其特征
2.1.载体质粒构建
为了实现重组MVA病毒的产生,构建了新的载体质粒。插入到MVA基因组的外源基因准确定位于MVA基因组自然发生缺失II位点。位于MVA基因组HindIIIN片段中2500bp缺失位点侧翼的MVA DNA序列(Altenburger,W.,Suter,C.P.和Altenburger,J.[1989],病毒学集刊(J.Arch.Virol.)105,15-27)用PCR法扩增并克隆到质粒pUCl8的多克隆位点。用于左方600bp DNA侧翼的引物是5′-CAGCAG
GGTACCCTCATCGTACAGGACGTTCTC-3′和5′-CAGCAG
CCCGGGTATTCGATGATTATTTTTAACAAAATAACA-3′(限制性酶KpnI和SmaI位点下面划线)。用于右方550bp DNA侧翼的引物是5′-CAGCAG
CTGCAGGAATCATCCATTCCACTGAATAGC-3′和5′AGCAGGCATGCCGACGAACAAGGAACTGTAGCAGA-3′(限制性酶PstI和SphI位点下面划线)。在插入到pUCl8的MVADNA这两段侧翼之间,利用BamHI位点插入处于痘苗病毒晚期启动子P11控制之下的大肠杆菌(Escherichiacoli)Lac Z基因(通过pIIILZ限制性消化制备,Sutter,G.和Moss,B.[1992]美国国家科学院院报(PNAS USA)89,10847-10851),从而产生MVA插入载体pUCIILZ[图1]。接下来一段含有痘苗病毒早期一晚期启动子P7.5以及用于克隆的SmaI位点的289bp片段(通过EcoRI和XbaI限制性消化质粒载体pSCII制备[Chakra barti等,1985,分子和细胞生物学(Molecular and CellularBiology)5,3403-3409])插入到pUCIILZ的SmaI位点产生了MVA载体pUCIILZ P7.5[图2]。为了构建一个载体质粒以通过报告酶β-半乳糖苷酶的瞬时合成实现重组MVA病毒的分离,一个来自大肠杆菌(E.coli)Lac Z开放阅读框3′末端的330bp DNA片段用PCR扩增(引物是5′-CAGCAGGTCGACCCCGACCGCCTTACTGCCGCC-3′和5′-GGGGGGCTGCAGATGGTAGCGACCGGCGCTCAG-3′)并克隆到pUCIILZ P7.5的SalI和PstI位点,获得MVA载体pUCIILZdel P7.5[图3]。利用SmaI位点,此载体质粒可用于把处于痘苗病毒启动子P7.5转录控制之下的编码外源基因的DNA序列插入到MVA染色体。在通过筛选β-半乳糖苷酶活性分离到所需重组病毒后,重组病毒的进一步增殖导致通过同源重组产生再改造P11-LacZ表达盒的自缺失。
2.2.重组病毒MVAT7pol的构建和特征
一个含有处于痘苗病毒早期/晚期启动子P7.5控制之下的噬茵体T7RNA聚合酶完整基因的3.1Kbp DNA片段用EcoR1从质粒pTFF3上切下(Fuerst,T.R.,Niles,E.G.,Studier,F.W.和Moss,B.,1986,美国国家科学院院报(P.N.A.S.USA)83,8122-8126),与KlenowDNA聚和酶一起温育修饰产生平头末端,并克隆到pUCIILZ的单独SmaI限制性位点。产生质粒转移载体pUCIILZ T7pol[图4]。选择痘苗病毒早期/晚期启动子P7.5作为T7 RNA聚合酶基因表达的转录调节子。与较强的痘苗病毒晚期启动子(例如P11)相反,此启动子系统使重组基因在靶细胞感染后立即表达。指导外源基因插入MVA基因组缺失II位点的质粒pUCIILZ T7pol用于产生重组病毒MVAT7pol。
用MVA在每细胞感染复数为0.05TCID50情况下感染的CEF细胞,按照前面所述使用质粒pUCIILZ T7pol DNA进行转染(Sutter,G.,Wyatt,L.,Foley,P.,Bennink,J.和Moss,B.(1994)疫苗(Vaccine)12,1032-1040)。表达T7RNA聚合酶并共表达β-D-半乳糖苷酶的重组MVA病毒(MVA P7.5-T7pol)通过在CEF细胞中用5-溴-4-氯-3-吲哚β-D-半乳糖苷(300μg/ml)进行连续五轮噬斑纯化选择出来。随后,通过感染CEF单层扩增重组病毒,并用PCR检测DNA以证实病毒贮存物的遗传均一性。MVA-T7pol病毒DNA的Southem印迹检测证明了重组基因在MVA基因组中缺失II位点的稳定整合[图5]。
为检测重组MVAT7pol的T7 RNA聚合酶表达,测定了来自病毒感染的组织培养物中的[35S]甲硫氨酸标记多肽。生长于12孔板上的单层猴肾细胞系CV-1用病毒以每细胞感染复数20TCID50进行感染。感染3到5小时后,去除培养基,并用1毫升无甲硫氨酸培养基冲洗培养物一次。每孔加入补充有50微居里(μCi)[35S]甲硫氨酸的0.2毫升无甲硫氨酸培养基并在37℃温育30分钟。感染细胞的胞质抽提物通过每孔在0.2毫升0.5%Nonidet P-40裂解缓冲液中37℃温育10分钟制备,样品用SDS-聚丙烯酰胺凝胶电泳检测。CV-1细胞与MVAT7pol的代谢标记显示了两种额外多肽的合成(i)一个大约116,000道尔顿的蛋白代表共表达的大肠杆菌β-半乳糖苷酶以筛选重组病毒以及(ii)一个98,000道尔顿的蛋白,同噬菌体T7 RNA聚合酶预期大小一致[图6]。由MVA T7pol产生的大量β-半乳糖苷酶是令人注目的。体内实验的结果表明,当插入到MVA基因组缺失II位点时有P11-Lac Z基因构建体的强表达,揭示MVA载体病毒中的重组基因当插入到MVA基因组的该位置时可能会更有效地表达。
MVA-T7pol重组病毒作为表达系统的用途与WR-T7pol重组病毒vTF 7-3(Fuerst等1986)的比较通过一个质粒载体DNA的共转染进行了测定,该质粒载体衍生于pTMl(Moss,B.,Elroy-Stein,O.,Mizukami,T.,Alexander,W.A.,和Fuerst T.R.(1990)自然(Nature)348,91-92)并含有(克隆到pTM1多克隆位点的NcoI和BamHI位点)处于T7 RNA聚合酶启动子(PT7)控制之下的大肠杆菌氯霉素乙酰转移酶(CAT)基因。转染和感染的CV-1细胞悬浮于0.2毫升0.25M Tris-HCl(pH7.5)。三轮冻融循环后,通过离心澄清裂解液,测定了上清液中蛋白含量,并且含0.5,0.25,0.1微克总蛋白的样品按Mackett,M.,Smith,G.L.和Moss,B.(1984)病毒学杂志(J.Virol.)49,857-864所述检测了酶活性。放射自显影后,用Fuji成像检测系统定量标记斑点。结果表明通过使用高减毒痘苗载体MVA,可能利用痘苗病毒-T7 RNA聚合酶系统时就象使用完全的可复制型痘苗病毒重组体一样有效[图7]。
2.3.重组病毒MVA-LAInef的构建及其特征
一个含有HIV-1 LAI完整nef基因的648bp DNA片段通过PCR从质粒DNA制备(pTG 1166由M.-P.Kieny,Transgene S.A.,Strasbourg惠赠;PCR引物为5′-CAGCAGGGATCCATGGGTGGCAAGTGGTCAAAAAGTAGT-3′和5′-CAGCAGGGATCCATGTCAGCAGTTCTTGAAGTACTCCGG-3′),用限制性内切核酸酶BamHI消化,与KlenowDNA聚合酶一起温育修饰以产生平头末端,并克隆到pUCIIL ZdelP7.5的SmaI位点以产生载体pUCIIL Zdel P7.5-LAInef[图8]。此质粒可用于改造MVA重组病毒以在痘苗病毒早期/晚期启动子P7.5控制下表达HIV-1 LAI的nef基因。
用MVA在每细胞感染复数0.05 TCID50感染的CEF细胞再按前述方法用质粒pUCIIL Zdel P7.5-LAInef的DNA进行转染(Sutter,G.,Wyatt,L.,Foley,P.,Bennink,J.和Moss,B.[1994]疫苗(Vaccine)12,1032-1040)。含有nef基因和瞬时共表达大肠杆菌Lac Z标记基因的重组MVA通过在CEF细胞中用5-溴-4-氯-3-吲哚β-D半乳糖苷(300μg/ml)染色的连续几轮噬斑纯化选择出来。此后含有nef基因和缺失Lac Z标记基因的重组MVA病毒在5-溴-4-氯-3-吲哚β-D-半乳糖苷(300μg/ml)存在下通过三轮附加的连续噬斑纯化筛选以寻找CEF细胞中的非染色病毒病灶而得以分离。随后,重组病毒感染CEF单层进行扩增,并且用PCR检测MVA-LAInef病毒DNA以证实病毒贮存物的遗传均一性。病毒DNA的Southem印迹检测证实了MVA-LAInef的遗传稳定性并精确表明nef基因和大肠杆菌Lac Z标记基因缺失整合到了病毒基因组缺失II位点。
重组Nef蛋白的有效表达通过对来自MVA-LAInef感染的CEF细胞的蛋白裂解液使用引导抗HIV-1 Nef的鼠单克隆抗体(由K.Krohm惠赠并按照Ovod,V.,Lagerstedt,A.,Ranki,A.,Gombert,F.,Spohn,R.,Tahtinen,M.,Jung,G.,和Krohn,K.[1992]爱滋病(AIDS)6,25-34所述使用)进行Western印迹检测得以证实。
2.4.重组病毒MVA-hTYR的构建及特征
一个含有编码人酪氨酸酶完整基因的1.9kb DNA片段[酪氨酸酶C-DNA克隆123,B2分离自病人SK 29(AV)的黑素细胞系SK 29-MEL,GenBank Acc.no.UO 1873;Brichard,V,Van Pel,A.,Wolfel,T.,Wolfel,C.,DePlaen,E.,Lethe,B.,Coulie,P.和Boon,B.(1993)实验医学杂志(J.Exp.Med.)178,489-495]通过EcoR1消化从质粒pc DNAI/Amp-Tyr[Wolfel,T.,Van Pel,A.,Brichard,V.,Schneider,J.,Seliger,B.,Meyer zum Buschenfelde,K.和Boon,T.(1994)欧洲免疫学杂志(Eur.J.Immunol)24,759-764]制备,与Klenow DNA聚合酶一起温育修饰产生平头末端,并克隆到pUCIIL Zdel P7.5的SmaI位点以产生载体pUCIIL Zdel P7.5-TYR[图9]。此质粒可用于改造MVA重组病毒以在痘苗病毒早期/晚期启动子P7.5控制下表达人酪氨酸酶基因。
MVA以每细胞感染复数0.05 TCI D50感染的CEF细胞用前述的质粒pUCIIL Zdel P7.5-TYR的DNA转染(Sutter,G,Wyatt,L.,Foley,P.,Bennink,J.和Moss,B.(1994)疫苗(Vaccine)12,1032-1040)。稳定表达人酪氨酸酶基因并瞬时共表达大肠杆菌Lac Z基因的重组MVA病毒通过在CEF细胞中用5-溴-4-氯-3-吲哚-D-半乳糖苷(300μg/ml)染色的连续几轮噬斑纯化选择出来。此后,表达编码人酪氨酸酶基因并有缺失的Lac Z标记基因的重组MVA病毒在5-溴-4-氯-3-吲哚β-D-半乳糖苷(300μg/ml)存在下通过三轮附加的连续噬斑纯化以筛选CEF细胞中的非染色病毒病灶而得以分离。随后,重组病毒感染CEF单层进行扩增,并且用PCR检测MVA-hTYR病毒DNA以证实病毒贮存物的遗传均一性。病毒DNA的Southern印迹检测证实了MVA-hTYR的遗传稳定性并精确表明重组酪氨酸酶基因和大肠杆菌Lac Z标记基因缺失整合到了病毒基因组中缺失II位点
重组人酪氨酸酶的有效表达通过对来自MVA-hTYR感染的CEF细胞的蛋白裂解液使用兔多克隆抗体(由V.Hearing惠赠并按照Jimenez,M.,Kameyama,K.,Maloy,L.,Tomita,Y.和Hearing,V.[1988]美国国家科学院院报(P.N.A.S.USA)85,3830-3834所述使用)或引导抗酪氨酸酶的鼠单克隆抗体(由L.Old惠赠并按照Chen,Y.,Stockert,E.,E.,Tsang,S.,Coplan,K.和Old,L.[1995]美国国家科学院院报(P.N.A.S.USA)92,8125-8129所述使用)进行Westem印迹检测得以证实。
序列表
(1)一般资料
(i)申请人
(A)名称:GSF-Forschungszentrumfuer Umwelt und Gesundheit
GmbH
(B)街道:Ingolstaedter Landstr.1,Neuherberg
(C)城市:Oberschleissheim
(E)国家:德国
(F)邮编(ZIP):85764
(ii)发明名称:重组MVA病毒及其应用
(iii)系列数目:8
(iv)计算机可读形式:
(A)媒介类型:软盘
(B)计算机:IBM兼容PC
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,#1.30版本(EPO)
(Vi)在先申请资料:
(A)申请号:DK 0782/95
(B)申请日:1995,7,4
(2)序列资料1:
(i)序列特征:
(A)长度:33碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列1:
CAGCAGGGTA CCCTCATCGT ACAGGACGTT CTC 33
(2)序列资料2:
(i)序列特征:
(A)长度:42碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列2:
CAGCAGCCCG GGTATTCGAT GATTATTTTT AACAAAATAA CA 42
(2)序列资料3:
(i)序列特征:
(A)长度:36碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列3:
CAGCAGCTGC AGGAATCATC CATTCCACTG AATAGC 36
(2)序列资料4:
(i)序列特征:
(A)长度:36碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列4:
CAGCAGGCAT GCCGACGAAC AAGGAACTGT AGCAGA 36
(2)序列资料5:
(i)序列特征:
(A)长度:33碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列5:
CAGCAGGTCG ACCCCGACCG CCTTACTGCC GCC 33
(2)序列资料6:
(i)序列特征:
(A)长度:33碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列6:
GGGGGGCTGC AGATGGTAGC GACCGGCGCT CAG 33
(2)序列资料7:
(i)序列特征:
(A)长度:39碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列7:
CAGCAGGGAT CCATGGGTGG CAAGTGGTCA AAAAGTAGT 39
(2)序列资料8:
(i)序列特征:
(A)长度:39碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:其它核酸
(A)描述:/desc=“DNA-引物”
(xi)序列描述:序列8:
CAGCAGGGAT CCATGTCAGC AGTTCTTGAA GTACTCCGG 39
Claims (71)
1.一种重组MVA病毒,含有并能够表达至少一种外源基因,所述外源基因已插入MVA基因组中除了天然缺失位点III以外的天然缺失位点。
2.权利要求1的重组MVA病毒,含有并能够表达至少一种插入到MVA基因组中缺失位点II的外源基因。
3.权利要求1的重组MVA病毒,其中的外源基因编码一种标记,一种治疗剂或一种抗原或抗原决定簇。
4.权利要求3的重组MVA病毒,其中的外源基因编码来源于致病性病毒,细菌,或其它微生物,或来源于寄生虫,或肿瘤细胞的抗原或抗原决定簇。
5.权利要求4的重组MVA病毒,其中的外源基因编码来源于恶性疟原虫,分枝杆菌,疱疹病毒,流感病毒,肝炎病毒,或人类免疫缺陷病毒的抗原或抗原决定簇。
6.权利要求3的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
7.权利要求4的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
8.权利要求5的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
9.权利要求6的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
10.权利要求7的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
11.权利要求8的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
12.权利要求2的重组MVA病毒,其中的外源基因编码一种标记,一种治疗剂或一种抗原或抗原决定簇。
13.权利要求12的重组MVA病毒,其中的外源基因编码来源于致病性病毒,细菌,或其它微生物,或来源于寄生虫,或肿瘤细胞的抗原或抗原决定簇。
14.权利要求13的重组MVA病毒,其中的外源基因编码来源于恶性疟原虫,分枝杆菌,疱疹病毒,流感病毒,肝炎病毒,或人类免疫缺陷病毒的抗原或抗原决定簇。
15.权利要求12的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
16.权利要求13的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
17.权利要求14的重组MVA病毒,其中的抗原或抗原决定簇是HIV nef或人的酪氨酸酶。
18.权利要求15的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
19.权利要求16的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
20.权利要求17的重组MVA病毒,其是MVA-LAInef或MVA-hTYR。
21.权利要求1的重组MVA病毒,其中的外源基因编码T7 RNA聚合酶。
22.权利要求21的重组MVA病毒,其是MVA-T7pol。
23.权利要求2的重组MVA病毒,其中的外源基因编码T7 RNA聚合酶。
24.权利要求23的重组MVA病毒,其是MVA-T7pol。
25.权利要求1的重组MVA病毒,其中的外源基因处于痘苗病毒早期/晚期启动子P7.5的转录控制之下。
26.权利要求25的重组MVA病毒,基本上不含有能在人的细胞中进行复制的病毒。
27.权利要求1的重组MVA病毒,基本上不含有能在人的细胞中进行复制的病毒。
28.权利要求2-24任一项的重组MVA病毒,其中的外源基因处于痘苗病毒早期/晚期启动子P7.5的转录控制之下。
29.权利要求28的重组MVA病毒,基本上不含有能在人的细胞中进行复制的病毒。
30.权利要求2-24任一项的重组MVA病毒,基本上不含有能在人的细胞中进行复制的病毒。
31.一种真核细胞,已被权利要求1的重组MVA病毒感染。
32.权利要求31的细胞,它另外含有一种或多种表达载体,所述载体携带一或多种处于T7 RNA聚合酶启动子的转录控制之下的外源基因。
33.一种真核细胞,已被权利要求2-24之一的重组MVA病毒感染。
34.权利要求33的细胞,它另外含有一种或多种表达载体,所述载体携带一或多种处于T7 RNA聚合酶启动子的转录控制之下的外源基因。
35.一种制备多肽的方法,包括:
a)在适宜条件下培养权利要求31或33的细胞,以及
b)分离由所述外源基因编码的多肽。
36.权利要求31或33的细胞,它另外含有一种表达载体,该载体携带病毒基因和/或编码病毒载体基因组的病毒载体构建体,它们处于T7 RNA聚合酶启动子的转录控制之下。
37.生产病毒粒子的方法,包括:
a)在适宜条件下培养权利要求36所述细胞,以及
b)分离所述病毒粒子。
38.权利要求31或33的细胞,它还包含
a)一种表达载体,载有逆转录病毒载体构建体,能够感染并指导靶细胞中由上述逆转录病毒载体构建体所携带的一或多种外源基因的表达,以及
b)一或多种表达载体,它们携带在T7 RNA聚合酶启动子的转录控制之下编码多肽的基因,所述多肽是装配该逆转录病毒载体构建体的基因组所必需的。
39.生产逆转录病毒粒子的方法,包括
a)在适宜条件下培养权利要求38所述细胞,以及
b)分离所述逆转录病毒粒子。
40.一种制备多肽的方法,包括:
a)在适宜条件下培养权利要求32或34的细胞,以及
b)分离由所述外源基因编码的多肽。
41.权利要求32或34的细胞,它另外含有一种表达载体,该载体携带病毒基因和/或编码病毒载体基因组的病毒载体构建体,它们处于T7 RNA聚合酶启动子的转录控制之下。
42.生产病毒粒子的方法,包括:
a)在适宜条件下培养权利要求41所述细胞,以及
b)分离所述病毒粒子。
43.权利要求32或34的细胞,它还包含
a)一种表达载体,载有逆转录病毒载体构建体,能够感染并指导靶细胞中由上述逆转录病毒载体构建体所携带的一或多种外源基因的表达,以及
b)一或多种表达载体,它们携带在T7 RNA聚合酶启动子的转录控制之下编码多肽的基因,所述多肽是装配该逆转录病毒载体构建体的基因组所必需的。
44.生产逆转录病毒粒子的方法,包括
a)在适宜条件下培养权利要求43所述细胞,以及
b)分离所述逆转录病毒粒子。
45.一种疫苗,在生理可接受的载体中含有权利要求1,所述的重组MVA病毒。
46.一种疫苗,在生理可接受的载体中含有权利要求2-24之一所述的重组MVA病毒。
47.权利要求45或46的疫苗,为人或动物的疫苗。
48.权利要求45或46的疫苗,所述疫苗含有MVA-LAInef。
49.权利要求45或46的疫苗,所述疫苗含有MVA-hTYR。
50.权利要求45或46的疫苗,所述疫苗含有重组MVA,所述重组MVA包含编码人的酪氨酸酶抗原或抗原决定簇的基因。
51.权利要求45或46的疫苗,所述疫苗含有重组MVA,所述重组MVA包含编码HIV nef抗原或抗原决定簇的基因。
52.权利要求47的疫苗,所述疫苗含有MVA-LAInef。
53.权利要求47的疫苗,所述疫苗含有MVA-hTYR。
54.权利要求47的疫苗,所述疫苗含有重组MVA,所述重组MVA包含编码人的酪氨酸酶抗原或抗原决定簇的基因。
55.权利要求47的疫苗,所述疫苗含有重组MVA,所述重组MVA包含编码HIV nef抗原或抗原决定簇的基因。
56.权利要求1的重组MVA病毒在疫苗制备中的用途。
57.权利要求2-24之一的重组MVA病毒在疫苗制备中的用途。
58.权利要求56或57的用途,其中所述疫苗为人或动物的疫苗。
59.权利要求56或57的用途,其中所述疫苗含有MVA-LAInef。
60.权利要求56或57的用途,其中所述疫苗含有MVA-hTYR。
61.权利要求56或57的用途,其中所述疫苗含有重组MVA,所述重组MVA包含编码人的酪氨酸酶抗原或抗原决定簇的基因。
62.权利要求56或57的用途,其中所述疫苗含有重组MVA,所述重组MVA包含编码HIV nef抗原或抗原决定簇的基因。
63.权利要求58的用途,其中所述疫苗含有MVA-LAInef。
64.权利要求58的用途,其中所述疫苗含有MVA-hTYR。
65.权利要求58的用途,其中所述疫苗含有重组MVA,所述重组MVA包含编码人的酪氨酸酶抗原或抗原决定簇的基因。
66.权利要求58的用途,其中所述疫苗含有重组MVA,所述重组MVA包含编码HIV nef抗原或抗原决定簇的基因。
67.一种疫苗,含有第一种组分和第二种组分,所述第一种组分是处在生理可接受的载体中的权利要求21-24之一所述重组MVA病毒,所述第二种组分是处在生理可接受的载体中的DNA序列,该序列携带处于T7RNA聚合酶启动子的转录控制之下的抗原或抗原决定簇,所述两种组分在疫苗中是处在一起或被分开的状态。
68.权利要求67的疫苗,所述疫苗用于免疫接种包括人在内的活体动物,所述免疫包括用所述疫苗的第一种和第二种组分同时或先后在同一部位对上述包括人在内的活体动物进行接种。
69.权利要求21-24之一的重组MVA病毒的用途,用于制备权利要求67的疫苗。
70.权利要求21-24之一的重组MVA病毒的用途,用于制备权利要求68的疫苗。
71.权利要求21-24之一的重组MVA病毒的用途,用于在T7 RNA聚合酶启动子的转录控制之下转录DNA序列。
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CNB961952547A Expired - Lifetime CN1154742C (zh) | 1995-07-04 | 1996-07-03 | 重组mva病毒及其应用 |
CN2004100367144A Expired - Lifetime CN1554764B (zh) | 1995-07-04 | 1996-07-03 | 重组改良安卡拉痘苗病毒及其应用 |
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