CN101085346A - 减毒瘟病毒 - Google Patents
减毒瘟病毒 Download PDFInfo
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- CN101085346A CN101085346A CNA2007101042355A CN200710104235A CN101085346A CN 101085346 A CN101085346 A CN 101085346A CN A2007101042355 A CNA2007101042355 A CN A2007101042355A CN 200710104235 A CN200710104235 A CN 200710104235A CN 101085346 A CN101085346 A CN 101085346A
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- lys
- leu
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Abstract
本发明涉及减毒瘟病毒(其特征为其糖蛋白ERNS的酶活性已经失活),其制备方法,其使用及其检测方法。
Description
此案是申请日为1999年05月26日、中国申请号为99807052.1、发明名称为“减毒瘟病毒”的发明申请的分案申请。
发明领域
本发明涉及通过使糖蛋白ERNS的RNase活性失活以减毒瘟病毒的方法。本发明亦涉及根据本发明减毒的瘟病毒,制备此种瘟病毒的核酸,含本发明减毒瘟病毒的疫苗及药用组合物。本发明亦涉及区分本发明减毒病毒与致病性病毒的方法。
发明背景
在许多国家中,瘟病毒是经济动物重要疾病的致病原,现今已知的病毒株可分成三种,此三种病毒为黄病毒科中的同一属。
I.牛病毒性腹泻病毒(BVDV)会使牛罹患牛病毒性腹泻(BVD)及粘膜疾病(MD)(Baker,1987;Moennig and Plagemann,1992;Thiel等人,1996)。
II.典型猪瘟病毒(CSFV),曾用名为猪霍乱病毒,会引起典型猪瘟(CSF)或猪霍乱(HC)(Moennig and Plagemann,1992;Thiel等人,1996)。
III.边界病病毒(BDV)主要发现于羊,并引起边界病(BD),已有报道称,羊的子宫内发生BDV感染后出现类似于MD的症状(Moennig amdPlagemann,1992;Thiel等人,1996)。
Becher等人(1995)或其他人提供瘟病毒的另一种分类方法。
瘟病毒为小包膜病毒,含有单股正链RNA基因组,但缺乏5′帽结构及3′多聚(A)序列。病毒基因组编码约4000个氨基酸的一种多聚蛋白,经细胞蛋白酶和病毒蛋白酶所参与的共同-及翻译后加工,产生最终的切割产物。病毒蛋白按NH2-Npro-C-ERNS-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH的顺序排列为多聚蛋白(Rice,1996)。蛋白C及糖蛋白ERNS,E1及E2代表瘟病毒体的结构成分。(Thiel等人,1991)。E2及小部分的ERNS发现是抗体中和作用的标靶(Donis等人,1988;Paton等人,1992;van Rijn等人,1993;Weiland等人,1990,1992)。ERNS缺少膜锚着点(membraneanchor),因此可由受染细胞大量分泌;已有报告指出此蛋白具有RNase活性(Hulst等人1994;Schneider等人,1993;Windisch等人,1996)。这种酶活性对病毒生活周期的影响目前尚不得知。已有报告指出,利用定点诱变法将CSFV疫苗株的RNase破坏后,会产生使细胞病变的病毒,此病毒于细胞培养上的生长特性与野生型病毒株无异(Hulst等人,1998)。酶活性依赖于两段氨基酸存在,所述氨基酸在瘟病毒ERNS及已知不同的植物及真菌来源的RNase之间是保守的,这两种保守序列均含有一个组氨酸残基(Schneider等人,1993)。将CSFV疫苗株的ERNS蛋白上的这些残基置换成赖氨酸后,会破坏RNase活性(Hulst等人,1998)。在CSFV疫苗株上的基因组中引入这些突变不会影响病毒活力或生长特性,但会使病毒表现出轻微致细胞病变的表型(Hulst等人,1998)。
已制备出包含减毒病毒或死病毒,或于异源表达***中表达的病毒蛋白的CSFV及BVDV疫苗并已投入使用。使这些作为活疫苗的病毒减毒的结构基础尚未知。因此,接种疫苗后如发生回复突变(backmutation)或重组会有产生不可预期的回复株的危险,而另一方面,灭活疫苗或异源表达的病毒蛋白(亚单位疫苗)诱发免疫力的效力相当低。
通常,用带特定突变的活疫苗作为减毒的基础可避免目前疫苗制备的缺点,但目前瘟病毒中尚未找道减毒突变作用的潜在靶。
上述减毒突变作用的另一优点是其分子的单一性,可作为减毒瘟病毒的区分标志,并使它们与本领域的瘟病毒区分。
由于疫苗的效价及安全性以及对瘟病毒感染的可测性预防与治疗的重要性,因此急需能高效诱发免疫力并具有特定减毒作用基础以区分致病性瘟病毒的特异性减毒活疫苗。
因此,本发明的技术问题是提供特异地减毒并带有可测性标记的瘟病毒,其用以作为诱发免疫力的高效价减毒活疫苗,此方法可使此减毒活病苗与本领域的致病性瘟病毒相区别。
发明公开
在权利要求中得到定性的说明及实施方案可解决上述技术问题。
本发明令人惊讶地发现通过使糖蛋白ERNS的RNase活性失活可特异性减毒瘟病毒。
减毒瘟病毒现在可提供具有较强免疫原性的活疫苗。
因此,本发明一个方面是提供内含瘟病毒的活疫苗,其中糖蛋白ERNS的RNase活性已失活。
本专利说明书中所用“疫苗”一词指内含至少一种可诱发动物体产生免疫反应的免疫活性成分的药用组合物,其中可含(但非必须)一种或多种额外成分,以增进该活性成分的免疫活性,疫苗中可额外含有药用组合物的其它常用成分。疫苗的免疫活性成分可包括完整的活的有机体,为其原形或为减毒有机体(此疫苗称为修饰的活疫苗(MLV)),或包括利用适当方法灭活的有机体(此疫苗称为死疫苗(KV))。疫苗免疫活性成分的另一种形式包括该有机体中适当的元件(亚单位疫苗),这些元件的制备是破坏有机体或此种有机体的培养物,随后纯化产生所要的结构,或对相应***(如(但不限于)细菌,昆虫,哺乳动物或其他种类)进行适当的操作而诱发合成过程,而后分离纯化,或利用合适的药用组合物(多核苷酸疫苗)直接引入基因物质至需要疫苗的动物体中,以诱发该合成过程。疫苗可包括一种或同时包括多种上述的元件。
促进免疫反应的额外成分通常指佐剂,例如氢氧化铝,矿物油或其他油,或加至疫苗中的辅助分子或此种额外成分诱发身体所产生的辅助分子,例如(但不局限于)干扰素,白细胞介素或生长因子。
“药用组合物”基本上包括能改变给药生物体或在其内部或表面生长的生物体的生理功能如免疫功能的一或多个组分,如,但不限于,抗生素或抗寄生物药物,以及用于其它特定目的的其它添加成分,所述目的如,但不限于,改变形状、无菌性、稳定性、经肠道或非肠道途径(如经口、鼻内、静脉、肌肉、皮下、皮内或其它适当途径)给予该组合物的可行性、给药后的耐受特性、控释特性。
本发明疫苗指上述定义的疫苗,其中一种免疫活性成分是瘟病毒或来源于瘟病毒。
“活疫苗”指一种包括活病毒,特别是活病毒活性成分的疫苗。
本专利说明书中所用“瘟病毒”指所有瘟病毒,其特征是在黄病毒科中与BVDV,CSFV及BDV同属一属,并可表达糖蛋白ERNS。当然,该名词亦包括Becher等人(1995)或其他人所界定的所有能表达糖蛋白ERNS的瘟病毒。本专利说明书中所用“RNase活性”一词指糖蛋白ERNS水解RNA的能力。
需注意的是在发表的文献中,糖蛋白E0常为糖蛋白ERNS的同义词。
“失活该糖蛋白上的RNase活性”指与未经修饰的该糖蛋白ERNS的野生型相比,修饰后的糖蛋白ERNS水解RNA的能力丧失或减小。
利用在此所证实的方法或Hulst等人(1998)的方法,使该糖蛋白上的至少一个氨基酸删除和/或突变可失活该糖蛋白的RNase活性。因此,本发明一个优选实施方案涉及活疫苗,其中删除和/或突变该糖蛋白至少一个氨基酸后,该RNase活性失活。
现已证明,糖蛋白ERNS形成一个约97kD的二硫键连接的同型二聚体,其中每个单体是由对应于CSFV多聚蛋白268至494位氨基酸(Rumenapf等人(1993)所述)的227个氨基酸所组成。CSFV的Alfort株所表达的前495个氨基酸列于图1中,仅供参考。CSFV的Alfort株的基因组序列可购自GenBank/EMBL资料库,录入号J04358;另一种是BVDV的CP7株的氨基酸序列,可购自GenBank/EMBL资料库(录入号U63479)。这两段氨基酸在糖蛋白ERNS及某些植物与真菌RNase活性蛋白上高度保守(Scheider等人,1993)。这两段氨基酸对RNase酶活性特别重要。第一段由该病毒多聚蛋白的295至307位氨基酸组成,第二段由338至357位氨基酸组成(如图1中所示的CSFV Alfort株,根据所发表的CSFV Alfort株推演出的氨基酸序列(Meyers等人1989)编号)。上述对RNase活性特别重要的氨基酸不限于CSFVAlfort株的指定位置,而只是举例说明优选该位置的氨基酸或在其他病毒株(如BVDV,BDV及普通瘟病毒)对应位置的氨基酸,通常因为其高度保守。对除CSFV Alfort株以外的瘟病毒而言,优选氨基酸的位置编号常不同,但精通瘟病毒分子生物领域者很容易就能利用其相对于该糖蛋白高度保守的特点找出优选氨基酸的位置。在一个非限制性实例中,CSVF Alfort的346位与BVDV cp7株的349位相同。
因此,在一个更优选实施方案中,本发明涉及一种本发明疫苗,其中该失活性删除作用及/突变作用位于该糖蛋白(如图1中例示的CSFV Alfort株)295至307位和/或338至357位的氨基酸,或其他病毒株对应位置。
在一个非常优选实施方案中,本发明揭示删除或突变该糖蛋白346位的氨基酸后,可失活该RNase活性,进而产生特别有用的活疫苗。因此,本发明涉及根据本发明的疫苗,其中通过删除或突变该糖蛋白(如图1中例示的CSFV Alfort株)346位的氨基酸,或其他病毒株对应位置的氨基酸可失活RNase活性。
本发明证明删除病毒多聚蛋白346位的组氨酸残基(编号是根据CSFVAlfort/Tubingen的公开序列(Meyers等人,1989),其代表一个推想的ERNSRNase活性位置保守残基)后,瘟病毒仍是具有生命的,并可编码不具RNase活性的ERNS蛋白。本发明亦证明删除BDV瘟病毒对应的ERNS组氨酸(位置349,编号根据BVDV CP7 GenBank/EMBL资料库(录入号U63479))后,会产生有生命的病毒颗粒,但其ERNS糖蛋白已失去RNase活性。与造成一个氨基酸置换成另一个氨基酸的点突变作用不同的是,通常删除突变较稳定不易回复,用带有该删除但能表达ERNS的致病性CSFV Alfort/Tubingen突变株感染猪后,不会产生发烧或其他CSFV感染后的典型临床症状,但用野生株感染猪后,会产生发烧,腹泻,食欲减退,呆滞,B-细胞减少及中枢神经***疾病。在受感染14天后垂死期时,将这些猪处死发现,在其皮肤及内部器官中有严重的出血。以突变病毒株感染猪后,第3,5,7,10,14天检测时,则不会出现病毒血症及B-细胞减少,但利用野生病毒株感染猪之后,则可轻易的自猪血液样品中分离出CSFV。删除突变株明显可在动物体中复制,其证据是产生了中和抗体(见实施例3,表3C)。病毒突变株所引起的免疫反应足以让动物经2×105TCID50的高度致病性CSFV Eystrup株(此与Alfort株来源不同)(Konig,1994)感染后存活。然而,受测动物却无CSFV感染后的典型临床症状,诸如发烧,腹泻,出血,B-细胞减少,或厌食。此资料证明以删除突变株感染猪后诱发了足以使猪产生免于强力再感染的保护作用的免疫反应。
因此,在一个最优选实施方案中,本发明涉及根据本发明的疫苗,其中通过删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他病毒株的对应位置,以失活RNase活性。
在另一个最优选实施方案中,本发明涉及根据本发明的BVDV疫苗,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他BVDV病毒株的对应位置,以失活RNase活性。
本发明另一个方面涉及减毒瘟病毒,其中删除和/或突变糖蛋白ERNS的至少一个氨基酸,以失活该糖蛋白上的RNase活性,但该糖蛋白例如图1中所述的CSFV糖蛋白的297和/或346位氨基酸不是赖氨酸。Hulst等人在1998年已描述了在该糖蛋白297和/或346位为赖氨酸的重组瘟病毒,证明这些特殊的瘟病毒在猪肾细胞中会产生细胞病变效应。截至目前为止,对因ERNS酶活性的失活所产生的令人惊奇且新颖的减毒特性完全不清楚。
在上述涉及疫苗的优选实施方案中,本发明还涉及根据本发明的瘟病毒,其中删除和/或突变该糖蛋白(如图1中例示的CSFV Alfort株)295至307位和/或338至357位的氨基酸,或其他毒株的对应位置,以失活该RNase活性。
在上述涉及疫苗的更优选实施方案中,本发明还涉及根据本发明的瘟病毒,其中删除或突变该糖蛋白(如图1中例示的CSFV Alfort株)346位的氨基酸,或其他毒株的对应位置,以失活该RNase活性。
在上述涉及疫苗的最优选实施方案中,本发明还涉及根据本发明的瘟病毒,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他毒株的对应位置,以失活该RNase活性。
在另一个最优选实施方案中,本发明涉及BVDV瘟病毒,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他BVDV病毒株的对应位置,以失活该RNase活性。
利用核酸-修饰重组技术使糖蛋白ERNS上的突变氨基酸序列表达,可轻易制备根据本发明的减毒瘟病毒及疫苗活性成分。因此,本发明另一个方面涉及编码糖蛋白ERNS的核苷酸,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活糖蛋白ERNS的RNase活性,但图1中所述的CSFV Alfort株糖蛋白297位和/或346位氨基酸不是赖氨酸。
基于上述原理,在一个较优选实施方案中,本发明涉及根据本发明的核酸,其中删除和/或突变该糖蛋白(如图1中例示的CSFV Alfort株)295至307位和/或338至357位的氨基酸,或其他毒株的对应位置,以失活该RNase活性。
基于上述疫苗原理,在一个更优选实施方案中,本发明涉及根据本发明的核酸,其中删除或突变该糖蛋白(如图1中例示的CSFV Alfort株)346位的氨基酸,或其他毒株的对应位置,以失活该RNasde活性。
在一个最优选实施方案中,本发明涉及根据本发明的核酸,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他毒株的对应位置,以失活该RNase活性。
在一个最优选实施方案中,本发明涉及根据本发明的BVDV核酸,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他BVDV毒株的对应位置,以失活该RNase活性。
核苷酸(例如DNA或RNA)亦可用于制备DNA-,RNA-和/或载体-疫苗,在这些疫苗中,核苷酸可直接应用于动物体中,或是间接经由除源病毒以外的载体应用于动物体中,核苷酸疫苗及载体疫苗是目前技艺所熟知的,不再进一步详细说明。
本发明另一个方面涉及本发明核酸用于制备核苷酸-和/或载体-疫苗的用途。
根据本发明的疫苗,减毒瘟病毒,和/或核酸特别可用于制备药用组合物。
因此,本发明另一个方面涉及包含根据本发明的疫苗,和/或根据本发明的瘟病毒,和/或根据本发明的核苷酸序列的药用组合物。此种药用组合物的一个非限制性实施例(只用于说明)可根据下列配制:受染细胞的培养上清液与稳定剂(例如亚精胺和/或BSA(牛血清白蛋白))混合,而后将混合物冷冻干燥,或用其他方法脱水。进行疫苗接种前,将该混合物溶于水溶液(例如生理盐水,PBS(磷酸盐缓冲液))或非水溶液(例如油乳化液,以铝为基础的佐剂)中。
本发明另一个方面涉及一种使瘟病毒减毒的方法。本发明提供一种独特且出人意表的减毒瘟病毒的方法,其特征是失活糖蛋白ERNS的RNase活性。
特异地减毒的瘟病毒尤其可用于制备疫苗,因此本发明另一个方面涉及制备特异地减毒的瘟病毒疫苗的方法,其特征是失活糖蛋白ERNS的RNase活性。
失活糖蛋白ERNS的RNase活性为瘟病毒的可检测性标记工作提供了一个令人惊讶且新颖的方法。本发明另一个方面是提供使瘟病毒带上可测性标记的方法,所述病毒的特征是糖蛋白ERNS的RNase活性失活。本发明瘟病毒缺乏糖蛋白ERNS的RNase活性的特性能使这些瘟病毒带上可测性标记。通过分离并分析糖蛋白ERNS上RNase活性的有无,可明确区分有标记及没有标记的瘟病毒或瘟病毒感染细胞所分泌的ERNS。
许多技术可用于通过删除和/或突变失活糖蛋白ERNS的RNase活性,以产生灭活的瘟病毒,由于此种删除作用和/或突变作用对结构的影响,因此可轻易的检测此种瘟病毒。可如实施例8所示用核酸测序或PCR-技术(聚合酶链式反应)检测出已改变的糖蛋白ERNS上核酸序列的序列差异;利用不识别未变蛋白的特异性单克隆抗体可检测出已改变的蛋白序列。相反的,在证实有瘟病毒的条件下,利用不能结合至识别未经改变的糖蛋白ERNS的特异性单克隆抗体的特性,亦可检测改变了并因此带有结构标记的蛋白。当然,与未标记瘟病毒的感染比较,造成标记病毒上的RNase活性破坏的删除和/或突变会使动物产生不同的免疫应答。
涉及减毒瘟病毒方法中所有方面的优选实施方案中,制备根据本发明特异地减毒的瘟病毒疫苗的方法及使瘟病毒带上可测性标记的方法均涉及糖蛋白ERNS失活的方法,其中是通过删除和/或突变该糖蛋白至少一个氨基酸,以失活该RNase活性。
涉及减毒瘟病毒方法中所有方面的更优选实施方案中,制备根据本发明特异地减毒的瘟病毒疫苗的的方法及使瘟病毒带上可测性标记的方法涉及糖蛋白ERNS失活的方法,其中删除和/或突变位于该糖蛋白(如图1中例示的CSFV Alfort株)295至307位及/338至357位,或其他毒株的对应位置的氨基酸。
涉及减毒瘟病毒方法中所有方面的非常优选实施方案中,制备根据本发明特别减毒的瘟病毒疫苗的方法及使瘟病毒带上可测性标记的方法涉及糖蛋白ERNS失活的方法,其中删除或突变该糖蛋白(如图1中例示的CSFVAlfort株)346位的氨基酸,或其他毒株的对应位置,以失活RNase活性。
涉及减毒瘟病毒方法中所有方面的最优选实施方案中,制备根据本发明特别减毒的瘟病毒疫苗的方法及使瘟病毒带上可测性标记的方法涉及糖蛋白ERNS失活的方法,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸残基,或其他毒株对应位置的组氨酸,以失活RNase活性。
本发明提供疫苗和/或其他药用组合物,其特别有用于预防及治疗动物的瘟病毒感染。因此,本发明另一个方面涉及预防及治疗动物体内瘟病毒感染的方法,其特征在于对需要此种预防或治疗的动物投与一种根据本发明的疫苗或其他根据本发明的药用组合物。
本发明另一个方面是提供一种制备特异性减毒的瘟病毒的方法,所述病毒特征在于失活糖蛋白ERNS的RNase活性。
本发明一个方面是提供一种制备特异性标记瘟病毒的方法,所述病毒特征在于失活糖蛋白ERNS的RNase活性。
涉及制备特异性减毒瘟病毒的方法的优选实施方案中,制备特异性标记的本发明瘟病毒的方法,是涉及糖蛋白ERNS失活的那些方法,其中删除和/或突变该糖蛋白至少一个氨基酸,以失活RNase活性。
涉及制备特异性减毒瘟病毒的方法的优选实施方案中,制备特异性标记的根据本发明的瘟病毒的方法,是涉及糖蛋白ERNS失活的方法,其中所述删除和/或突变位于该糖蛋白(如图1中例示的CSFV Alfort株)295至307和/或338至357位的氨基酸,或其他毒株的对应位置。
涉及制备特异性减毒瘟病毒的方法的优选实施方案中,制备特异性标记的根据本发明的瘟病毒的方法,是涉及糖蛋白ERNS失活的方法,其中删除或突变该糖蛋白(如图1中例示的CSFV Alfort株)346位的氨基酸,或其他毒株的对应位置,以失活RNase活性。
涉及制备特异性减毒瘟病毒的方法的优选实施方案中,制备特异性标记的根据本发明的瘟病毒的方法,是涉及糖蛋白ERNS失活的方法,其中删除该糖蛋白(如图1中例示的CSFV Alfort株)346位的组氨酸,或其他毒株的对应位置,以失活RNasc活性。
本发明的疫苗及其他药用组合物可用以预防及治疗动物体内的瘟病毒感染。
因此,本发明一个方面涉及根据本发明的疫苗预防及治疗动物体内瘟病毒感染的用途,本发明另一个方面涉及根据本发明药用组合物预防及治疗动物体内瘟病毒感染的用途。
根据本发明的瘟病毒和/或核酸是药用组合物或疫苗的有效活性成分,因此,本发明另一个方面涉及本发明瘟病毒和/或本发明核酸在制备疫苗或药用组合物上的用途。
一如前述,糖蛋白ERNS上的RNase活性的灭活提供了一种能标记瘟病毒的令人惊讶的新方法。
因此,本发明的一个方面涉及区分根据本发明可检测性标记的瘟病毒与未标记并可能具有致病性的瘟病毒。此种方法特别可用于追踪动物体中标记的瘟病毒的效力。接种疫苗的动物在从它的体内取样并分析该标记后要证实其为标记阳性。而未标记的动物特别是证实带有瘟病毒的未标记动物可立即区分,隔离或捕杀,以便消除病原传播给其他动物的紧急危险性。
本发明提供一种检测标记瘟病毒的方法,其特征在于使糖蛋白ERNS的RNase活性失活。由于本发明瘟病毒缺乏糖蛋白ERNS的RNase活性,因此可用以使这些瘟病毒带上可测性标记。因此,通过分离及分析此种糖蛋白ERNs的RNase活性的有无,可明确区分标记及未标记的瘟病毒。根据标准方法(例如实施例2中所述或Hu等人(1994)所述)测定含目的瘟病毒或其物质的样品中有无该酶活性。
因此,本发明一个较优选的实施方案涉及一种区分瘟病毒感染动物及接种根据本发明特异性减毒的瘟病毒的动物的方法,其包括的步骤如下:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的目的动物采样:
(2)测定该样品中有无糖蛋白ERNS的RNase活性;
(3)调查糖蛋白ERNS的RNase活性的缺乏与接种疫苗的动物之间的相关性及该活性的存在与该动物的瘟病毒感染之间的相关性。
本发明提供因删除和/或突变而使其糖蛋白ERNs上的RNase活性失活的瘟病毒。由于此种删除和/或突变对结构的影响,使此种瘟病毒可轻易的检测到。利用测序技术或PCR-技术可检测编码已变糖蛋白ERNS的ERNS基因的基因序列差异。因此,在一个优选的实施方案中,本发明提供一种区分瘟病毒感染动物及接种根据本发明特异性减毒的瘟病毒的动物的方法,其包括下列的步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的目的动物采样;
(2)鉴定该样品中瘟病毒的基因组核苷酸序列或蛋白质;
(3)调查接种疫苗的动物与疫苗中呈现的ERNS核苷酸删除和/或突变之间的相关性,及该动物的瘟病毒感染与缺乏该删除和/或突变之间的相关性。
此外,利用不能识别未变蛋白的特异性单克隆抗体或多克隆抗体检测根据本发明的瘟病毒在改变糖蛋白ERNS的蛋白序列后所产生的结构变化。因此,在另一个实施方案中,本发明涉及一种区分瘟病毒感染动物及接种根据本发明减毒瘟病毒的方法,其包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的目的动物采样;
(2)因单克隆抗体或多克隆抗体特异性结合至该样品中的ERNS糖蛋白,而鉴定减毒瘟病毒的修饰的ERNs糖蛋白,该糖蛋白已根据本发明的方法修饰,因此该单克隆抗体或多克隆抗体不会结合至未经修饰的ERNS糖蛋白上。
(3)调查所述单克隆抗体或多克隆抗体的特异性结合与疫苗接种动物的相关性,及抗体未发生结合与该动物的瘟病毒感染之间的相关性,条件是已另行证实该动物和/或该样品中存在瘟病毒物质。
反之,若能另行证实瘟病毒的存在,也可通过只识别糖蛋白ERNS的特异性单克隆或多克隆抗体的无法结合而测定已改变并因此产生结构标记的蛋白质。在一个优选的实施方案中,本发明涉及一种区分瘟病毒感染动物及接种根据本发明的减毒瘟病毒的动物的方法,其包括下列的步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的目的动物采样;
(2)因单克隆抗体或多克隆抗体特异性结合至该样品中的ERNS糖蛋白,而鉴定瘟病毒未经修饰的ERNS糖蛋白,该糖蛋白未以本发明的方法修饰,因此该单克隆抗体或多克隆抗体不会结合至修饰的ERNS糖蛋白上;
(3)调查所述单克隆抗体或多克隆抗体的特异性结合与该动物体中瘟病毒感染的相关性,及抗体未发生结合与动物的疫苗接种之间的相关性,条件是已另行证实该动物和/或该样品中存在瘟病毒物质。
当然,与没有标记的瘟病毒感染的反应结果比较,本发明标记病毒的结构修饰及RNase活性的缺乏会在动物体中产生不同的免疫反应。本发明瘟病毒会引发不同且显著的免疫反应(包括细胞免疫反应及体液免疫反应),其与未修饰者及可能为病原者所引发的免疫反应不同。例如,根据本发明的糖蛋白ERNS所产生的多克隆抗体在其结合特异性上与未修饰糖蛋白所产生的多克隆抗体的结合特异性不同。此结合特异性的差异提供一种区分接种了本发明瘟病毒的动物与其他瘟病毒感染动物的标记。筛选血清中能结合至野生型表位或该表位的标志物删除突变的特异性多克隆抗体,可用以区分受染的动物及接种疫苗的动物,已有对这类检查的报告,例如假性狂犬病-感染的猪及接种疫苗的猪(Kit等人,1991)。
本发明优选的一个实施方案涉及一种用以区分瘟病毒感染动物及接种本发明减毒瘟病毒的动物的方法,其步骤包括如下:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物采样;
(2)鉴定该多克隆抗体对未经修饰的糖蛋白ERNS或根据本发明修饰的糖蛋白ERNS的任何特异性结合;
(3)调查该多克隆抗体结合至未经修饰的糖蛋白ERNS与瘟病毒感染之间的相关性,及该多克隆抗体结合至根据本发明修饰的糖蛋白ERNS与疫苗接种动物之间的相关性。
参考文献
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实施例
实施例1 产生RNase阴性的瘟病毒突变株
自全长cDNA克隆pA/CSFV(Meyers等人,1996a)或pA/BVDV(Meyers等人,1996b)(其感染性cRNA可通过体外转录获得)开始制备亚克隆。以CSFV而言,将pA/CSFV的XhoI/SspI切割片段克隆至经Xhol及Smal切割的pBluescript SK+上。以BVDV而言,取自pA/BVDV的XhoI/BglII片段克隆至以相同酶切割的质粒pCITE-2C上。利用大肠杆菌CJ 236细胞(BioRad)及VCMS单链噬菌体(Stratagene),根据Kunkel等人(1987)的方法,从这些构建体制备单股的质粒DNA,利用“噬菌粒体外诱变试剂盒”(BioRad)将此单股DNA转换成双股DNA。生成目的瘟病毒突变体所需的某些合成寡核苷酸如下例示:
C-297-L:AGGAGCTTACTTGGGATCTG
C-346-L:GGAACAAACTTGGATGGTGT
C-297-K:ACAGGAGCTTAAAAGGGATCTGGC
C-346-K:ATGGAACAAAAAGGGATGGTGTAA
C-346-d:GAATGGAACAAAGGATGGTGTAAC
B-346-d:CATGAATGGAACAAAGGTTGGTGCAACTGG
双股质粒DNA用以转染大肠杆菌XL1-Blue(Stratagene)。利用氨苄青霉素筛选带有质粒的细菌菌落。制备质粒DNA,并进一步利用T7聚合酶测序试剂盒(Pharmacia)分析核苷酸序列。内含所要的突变且无第二个位置改变的质粒用以构建全长的cDNA克隆。以CSFV的例子而言,取自突变质粒的XhoI/NdeI片段与取自质粒578(pCITE 2A,含有pA/CSFV的XhoI/BglII片段)的NdeI/BglII片段同时引入经XhoI及BglII切割的pA/CSFV。为制得BVDV CP7突变株,将内含删除的XhoI/BglII片段及分离自pA/BVDV/Ins-的BglII/NcoI片段同时引入经XhoI及NcoI切割的pA/BVDV。构建体pA/BVDV/Ins-转染至适当细胞后,转录形成的cRNA会产生非致细胞病变的BVDV(Mcyers等人,1996b)。扩增不同全长的克隆,并分离质粒。利用DNA测序证明所要突变作用的存在。用SrfI(对CSFV全长克隆)及SmaI(对BVDV全长克隆)线性化后,如前述转录cRNA(Meyers等人,1996ab),利用凝胶过滤法及酚/氯仿抽提作用纯化RNA,并用以转染猪肾(PK15)细胞或牛肾(MDBK克隆B2)细胞(分别为CSFV或BVDV构建体)。利用病毒特异性抗血清,以免疫荧光法分析转染作用。如果收获的是所需突变体(免疫荧光为阳性反应),在如转染实验所用的相同细胞上进行传代,以扩增病毒。CSFV突变株的进一步分析包括一步生长曲线的测定,及利用病毒特异性cDNA探针进行的Northern blot分析,和用逆转录聚合酶链式反应(RT-PCR)鉴定病毒RNA的特性,然后测序PCR片段,以确定病毒基因组中存有所要的突变。所有的例子均要证明存有所要的突变。所有收获的病毒生长相当良好,所产生的RNA量与带有展示野生型序列的质粒的病毒所产生者相似。
用相应的cRNA进行转染,3天后,分离细胞。部分细胞接种在3.5cm的培养皿中,一天后用丙酮/甲醇固定,并以BVDV-特异性单克隆抗体混合物进行免疫荧光分析,以测定BVDV突变株的生存力(Weiland等人,1989)。所有的细胞均为阳性反应,而用不具感染性的RNA转染的细胞则无讯号产生。部分用相应cRNA转染的细胞经一轮冻融后产生抽提液。用此细胞抽提液感染新鲜的细胞,于感染3天后,以BVDV特异性免疫荧光法证明其为BVDV阳性反应。
表1摘录了ERNS保守序列中代表假定的RNase活性位点处所引入的不同变化,其由所示的病毒突变株编码。
表1
名称 | 在Rnase基元中的序列 | RNase活性 | 突变株的生存力 |
pA/CSFVC-297-LC-346-LC-297-L/346-LC-297-KC-346-KC-297-dC-346-dC-296/7/8-dC-345/6/7-dC-345/6-dC-346/7-dC-342-dC-342/6-dC-301-dC-295-S/GC-300-W/GC-302-E/AC-305-C/GC-300-W/G-302-E/AC-340-R/GC-343-W/GC-345-K/AC-297-K/346-KC-297-K/346-LpA/BVDVB-346-d | ...S L H G I W P E K I C... ...R H E W N K H G W C N W.....S L L G I W P E K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E W N K L G W C N W.....S L L G I W P E K I C... ...R H E W N K L G W C N W.....S L K G I W P E K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E W N K K G W C N W.....S L _ G I W P E K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E W N K _ G W C N W.....S _ _ _ I W P E K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E W N _ _ _ W C N W.....S L H G I W P E K I C... ...R H E W N _ _ G W C N W.....S L H G I W P E K I C... ...R H E W N K _ _ W C N W.....S L H G I W P E K I C... ...R H _ W N K H G W C N W.....S L H G I W P E K I C... ...R H _ W N K _ G W C N W.....S L H G I W _ E K I C... ...R H E W N K H G W C N W.....G L H G I W P E K I C... ...R H E W N K H G W C N W.....S L H G I G P E K I C... ...R H E W N K H G W C N W.....S L H G I W P A K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I G... ...R H E W N K H G W C N W...S L H G I G P A K I C.. ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...G H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E G N K H G W C N W.....S L H G I W P E K I C... ...R H E W N A H G W C N W.....S L K G I W P E K I C... ...R H E W N K K G W C N W.....S L K G I W P E K I C... ...R H E W N K K G W C N W.....S L H G I W P E K I C... ...R H E W N K H G W C N W.....S L H G I W P E K I C... ...R H E W N K _ G W C N W.. | +-----------------------+- | ++++++-+-------++------++++ |
表1说明:RNase活性测试在瞬时试验中进行。用痘苗病毒vTF7-3(Fuerst等人,1986)感染BHK21细胞,而后用相应cDNA构建体(5微克质粒DNA,转染用Superfect如供应商所建议的进行)转染细胞。置于37℃二氧化碳培养箱中10小时后,裂解转染的细胞,进行加工以测定RNase活性。生存力如下述测定。
实施例2 ERNS上的不同突变对RNase活性的影响
用突变病毒感染适当的细胞以测试不同突变对ERNS的RNase活性的效应。以CSFV而言,用感染复数(m.o.i.)为0.01进行感染。以野生株病毒进行感染作为阳性对照,而未感染的细胞则为阴性对照。感染48小时后,用磷酸盐缓冲液清洗细胞2次,并用0.4毫升的裂解缓冲液(20mM Tris/HCl;100mM NaCl,1mM EDTA,2毫克/毫升牛血清白蛋白;1%Triton X100;0.1%去氧胆酸;0.1%十二烷基磺酸钠)裂解细胞。将细胞裂解液装入1.5毫升的反应试管中,进行超声震荡[Branso超声波仪B12,120瓦特,20秒水浴(cuphorn water bath)],离心去除杂质(5分钟,14,000rpm,Eppendorf离心机,4℃),并将上清液再进行超速离心(Beckmann桌上型超速离心机,60分钟,4℃,45,000rpm,TLA 45转子)。测定总体积为200微升的内含5或50微升第二次离心步骤所得之上清液及80微克Poly(rU)(Pharmacio)的RNase分析缓冲液(40mMFris-醋酸(pH6.5),0.5mM EDTA,5mM二硫苏糖醇(DTT)中的RNase活性。将反应混合物置于37℃下培养1小时,加入200微升的1.2M高氯酸,20mM LaSO4。置于冰上培养15分钟后,以14,000rpm速度,4℃条件,将混合物在Eppendort离心机上离心15分钟。在上清液中加入3倍体积的水,取出少量的混合物,利用Ultrospec 3000分光光度计(Pharmacia)测定混合物在260nm的吸收值。所有的例子中,引入至ERNS基因的突变均完全失去RNase活性(表1)。
以BVDV突变株而言,在RNA转染作用后,回收的病毒不经转代,测定所得物质的RNase活性。用适当RNA转染的细胞在转染后72小时后分离并分别转种于两个培养皿中。24小时后,从其中一个培养皿中制备细胞抽提物,并用上述的方法分析RNase活性。为了证明感染作用,利用BVDV特异性单克隆抗体(Weiland等人,1989)以免疫荧光法分析第二个培养皿中的细胞,结果发现100%为阳性反应。以pA/BVDV/Ins-及pA/B-346-d(该质粒相当于pA/BVDV/Ins-,但删除了相当于CSFV Alfort株基因组中密码子346的密码子)转录所得的RNA完成转染作用。未转染的MDBK细胞为阴性对照。
表2A 不同病毒RNase活性的测定
Alfort | C-WT | C-297-L | C-346-L | C-346-d | C-346-d/Rs | 对照 | |
OD260 | 2.4 | 2.3 | 1.1 | 1.1 | 1.1 | 2.3 | 1.1 |
Alfort | C-WT | C-297-L | C-346-L | C-297-K | C-346-K | C-297-L/346-L | |
OD260 | 2.09 | 2.16 | 0.715 | 0.77 | 0.79 | 0.766 | 0.77 |
C-297-K/346-L | C-297-K/346-K | C-346-d | 对照 | ||||
OD260 | 0.725 | 0.835 | 0.8 | 0.84 |
表2A的说明:
PK 15细胞用所示的病毒以0.01的m.o.i.(感染复数)感染,置于37℃的二氧化碳培养箱中培养48小时,而后裂解,并进行RNase测试。以260nm的光密度测定不同细胞抽提液所得的酸可溶性RNA的含量。结果发现RNase活性的差异不是因为样品中ERNS蛋白含量的不同,因为对ERNS进行放射活性标记,免疫沉淀,并以磷光计分析放射活性所得的ERNS定量值与此相似。此外,分析时,将ERNS浓度降至正常含量的十分之一时,并不会明显改变所得的OD值,此结果表示在所选的分析条件下实验中ERNS为饱和。
CSFV Alfort株;其他所有的病毒收获自以下质粒体外转录的RNA:例如C-WT得自pA/CSFV;C-297-L得自pA/C-297-L等;C-346-d/Rs病毒得自pA/C-346-d/Rs(通过将相应cDNA片段替换为衍生自pA/CSFV的等价片段,使pA/C-346-d产生回复突变,从而产生该质粒)等;对照:未感染的PK15细胞的抽提液。
表2B
B-WT | B-346-d | 对照 | |
OD260 | 2.5 | 1.1 | 1.1 |
表2B的说明
以体外转录的RNA感染MDBK细胞,转染后72小时分离细胞,并于24小时后分析RNase活性。利用正文中所述的免疫荧光分析法证明细胞的感染。
B-WT:回收自pA/BVDV/Ins-的病毒;B-346-d:回收自pA/B-346-d的病毒;对照:未感染的MDBK细胞的抽提物。
实施例3 RNase失活后CSFV的致病性
为了测试是否破坏RNase活性后会影响瘟病毒在其自然宿主中的致病性,以突变株病毒V(pA/C-346-d)(表中的C-346-d)进行动物实验。回收自CSFV全长克隆且不具突变的病毒(V(pA/CSFV))作为阳性对照(表中的C-WT)。每个突变株使用三只小猪(品系:德国当地品种:体重约25公斤)。每只动物的感染剂量为1×105TCID50;三分之二的接种物以鼻腔内方式投与(每个鼻孔三分之一),三分之一的接种物以肌肉内方式投与。两组动物分别豢养于相隔离的地方,在感染之前及感染后第3,5,7,10,12及14天时收集动物血液。此外,每天记录体温(图2)。以野生型病毒感染的动物呈现出主要的典型猪瘟病征,如发烧,共济失调,食欲减退,腹泻,中枢神经疾病,皮肤出血(表3a)。第3天时自血液中回收病毒(动物#68),在第5,7,10,14天时自血液中回收病毒(动物#68,#78,#121)(表3b),感染14天后的垂死期杀死动物,此时,未能检测到病毒中和抗体。相反地,用突变株感染的动物未有临床病征产生(表3a)。而在整个实验期间的体温维持正常(图2),且这些动物未曾停止进食。任何时间自血液中均无法回收出病毒,然而,这些动物却明显受到感染,且病毒很可能进行了复制,因为所有的动物均产生中和抗体(表3c)。
表3a:感染后临床病征:
动物实验1 | |||||||||
动物编号: | 感染病毒株 | 临床病征 | |||||||
发烧 | 腹泻 | CNS疾病 | 食欲减退 | 皮肤出血 | 神情呆滞 | 安死术日已垂死 | 尸检时器官有出血 | ||
#68 | C-WT | + | + | + | + | + | + | + | + |
#78 | C-WT | + | + | + | + | + | + | + | + |
#121 | C-WT | + | + | + | + | + | + | + | + |
#70 | C-346-d | - | - | - | - | - | - | - | n.a. |
#72 | C-346-d | - | - | - | - | - | - | - | n.a. |
#74 | C-346-d | - | - | - | - | - | - | - | n.a. |
表3a说明:
本实验包括六只小猪(德国当地品种,体重约25公斤),分成两组,每组单独豢养。三只小猪用CSFV-WT(1×105TCID50)感染,而另三只则以C-346-d(1×105TCID50)感染。记录肛温及临床病征,摘录于表中;n.a:未进行尸检。
表3b:测试感染后血细胞病毒血症
动物实验1 | ||||||
动物编号 | 感染病毒株 | 感染后产生病毒血症日 | ||||
3 | 5 | 7 | 10 | 14 | ||
#68 | C-WT | + | + | + | + | + |
#78 | C-WT | - | + | + | + | + |
#121 | C-WT | - | + | + | + | + |
#70 | C-346-d | - | - | - | - | - |
#72 | C-346-d | - | - | - | - | - |
#74 | C-346-d | - | - | - | - | - |
表3b的说明:
将血与PK 15细胞共同培养后检测血细胞病毒血症。在37℃下培养72小时后,用PBS清洗细胞,以冰冷的丙酮/甲醇固定,并用特异于糖蛋白E2的单克隆抗体(mAb A18,Weiland等人,1990)经免疫荧光法分析感染。
表3c:CSFV特异性血清中和效价的生成
感染后天数 | -3 | 0 | 17 | 25 | 69 | 76 | 79 | 87 |
70#猪 | - | - | 1∶18 | 1∶162 | 1∶162 | 1∶162 | 1∶486 | 1∶1458 |
72#猪 | - | - | 1∶18 | 1∶54 | 1∶486 | 1∶1458 | 1∶1458 | 1∶1374 |
74#猪 | - | - | 1∶6 | 1∶54 | 1∶162 | 1∶162 | 1∶486 | 1∶1458 |
表3c说明:
动物实验期间,在不同的时间点测试病毒突变株C-346-d感染猪后的抗体效价:
50微升的稀释血清与50微升内含30 TCID50病毒(CSFV Alfort/Tubingen)的培养基混合。在37℃下培养90分钟后,加入100微升细胞(1.5×104细胞),将混合物接种于96孔板中。72小时后,细胞以冰冷的丙酮/甲醇固定,并用特异于糖蛋白E2的单克隆抗体(mAb A18,Weiland等人,1990)以免疫荧光法分析感染。动物在感染后69天时,以2×105TCID50的CSFV Eystrup株感染。表中所示为对进入体内的病毒可以产生完全中和作用的最高血清稀释度。
实施例4 RNase阴性病毒感染所诱发的保护性免疫
为了分析以病毒突变株感染后是否可以产生保护性免疫反应,在以CSFV突变株感染约9周后,利用高度致病性异源CSFV病毒株(Eystrup,取自Behring)进行功击实验。以2×105TCID50病毒进行感染。在数个前述实验中(Konig,1994)发现此含量足以诱发致死性疾病。然而,事先用CSFVRNase突变株感染的动物,在进行攻击感染后,未呈现出疾病的病征。动物并未检测到发烧(图3)及病毒血症,但其中和抗体量增加,此表示用于攻击的病毒确实造成增殖性感染并发生了复制。
实施例5 减毒原理的确认
为了证明所发现的病毒突变株的减毒作用确实是因为多聚蛋白346位的组氨酸删除所致,而非另一个未确定位置的突变所造成,通过将全长克隆pA/C-346-d的1.6kb XhoI/NdeI片段置换为展示野生株序列的pA/CSFV的相对片段从而恢复野生型序列。利用核苷酸测序法分析切割自pA/C-346-d片段的突变。结果除了编码多聚蛋白346位的组氨酸的三联密码删除外,并未发现与野生株序列有其他的差异。用回复突变株的cDNA构建体可获得病毒V(pA/C-346-d/Rs),它的生长会恢复成野生株一般,且呈现等同的RNase活性(表2A)。
第二个动物实验中,回复病毒可用于感染猪。删除突变株可作为对照,同样,共有两组(每组三只动物)进行实验。因为动物比第一次实验所用的动物年幼(德国本地品种,体重约20公斤),此时所用的病毒含量为5×104TCID50。同样,以突变病毒株感染的动物并未出现临床症状(表5,图4)。只有其中一只动物发烧一天。虽然如此,这些动物均产生了中和抗体,且可对抗致死性CSFV感染。用含量为2×105TCID50的Eystrup株病毒进行感染,感染后,这些动物并未出现临床症状,且体温维持正常状态(图5)。与用删除突变株感染的猪相比,接种回复的野生病毒株的猪会产生致死性典型猪瘟。其中一只动物在感染后11天不得不杀死,另两只则于3天后杀死。所有的动物均出现典型猪瘟的典型病征,即发烧、腹泻,食欲减退,及在不同的器官(包括肾脏)中出现出血等病理现象。
表5a:试验感染后的临床症状
动物实验2 | |||||||||
动物编号: | 感染病毒株 | 临床病征 | |||||||
发烧 | 腹泻 | CNS疾病 | 食欲减退 | 皮肤出血 | 神情呆滞 | 安死术日已垂死 | 尸检时器官有出血 | ||
#43 | C-346-d | +* | - | - | - | - | - | - | n.a. |
#47 | C-346-d | - | - | - | - | - | - | - | n.a. |
#87 | C-346-d | - | - | - | - | - | - | - | n.a. |
#27 | C-346-d/RS | + | + | + | + | - | + | + | + |
#28 | C-346-d/RS | + | + | + | + | - | + | + | + |
#30 | C-346-d/RS | + | + | + | + | - | + | + | - |
*只发烧一天 |
表5a:
此实验包括六只小猪(德国当地品种;体重约20公斤),分成两组,每组单独豢养。三只动物以突变株C-346-d(5×104 TCID50)感染,而另三只以C-346-d/RS(5×104 TCID50)感染。C-346-d/RS是由突变株C-346-d中ERNS基因恢复成野生型序列而产生。记录肛温及临床症状并摘录之;n.a.:未进行尸检。
表5b用回收自感染动物病毒血症期的病毒所进行的诊断性RNase测试
Alfort | 动物#3C-297-K | 动物#5C-297-K | 动物#27C-346-d/RS | 动物#28C-346-d/RS | 动物#30C-346-d/RS | 对照 | |
OD260 | 1.84 | 0.60 | 0.56 | 1.84 | 1.93 | 1.94 | 0.49 |
从感染后5天的编号3及5的动物及动物实验#2(实施例5所述)中感染后7天的编号27,28及30的动物血中所回收的病毒于组织培养中繁殖,定量并以前述的方法测定RNase活性。未感染的PK15细胞及以野生株CSFV(Alfort)感染的细胞(对照)作为对照。编号3及5的动物以突变株C-297-K感染,而编号27,28及30的动物以突变株C-346-d/RS感染,如表中所示。
实施例6 ERNS中双突变作用的效应
为了测试ERNS中双突变作用在相关病毒于其自然宿主中复制及在致病性上的效应,用突变的V(pA/C-297-L/346-L)进行动物实验。从无突变的CSFV全长克隆(V(pA/CSFV)所回收的病毒作为阳性对照。每个突变株以三只小猪进行实验(品系:德国当地品种;体重约25公斤)。每只动物的感染剂量为1×105TCID50;三分之二的接种量以鼻腔内方式投与(每个鼻孔三分之一),而三分之一的接种量以肌肉内方式投与。在感染前(第0天)及感染后第5,8,12及20天自动物采血样。此外,每天记录体温(图6)。用双突变株感染的动物并未产生任何临床病状,且动物未曾停止进食。除了动物45/1在第8天时有发烧外,其他的动物(动物45/2及45/3)在整个实验期间并未出现发烧症状,这可能是因为注射右后脚时造成的细菌感染所致。当此动物在第10天以抗生素治疗后,一天内体温恢复成正常(图6)。在第5天时,所有动物的血液均可回收病毒,然而在稍后的时间点并未检测到病毒血症(表6a)。所有的动物均产生中和抗体(表6b),约在感染后4.5个月后测定动物45/1的中和抗体效价仍高达1∶4374。因此,以双突变株进行感染后会延长免疫记忆。
表6a病毒血症测试
感染后天数 | 5 | 8 | 12 |
45/1猪 | + | - | - |
45/2猪 | + | - | - |
45/3猪 | + | - | - |
表6b中和抗体效价
动物 | 第0天 | 感染后20天 |
45/1 | - | 1∶128 |
45/2 | - | 1∶256 |
45/3 | - | 1∶256 |
实施例7 BVDV病毒“B-346-d”的免疫力及减毒原理
本实验通过比较回收自pA/B-346-d的B-346-d病毒与回收自pA/BVDB/Ins-的B-WT病毒,研究VBDV病毒的减毒原理及免疫力。当然,病毒B-346-d的突变位置位于BVDV的349位,但命名为B-346是指对应于图1中所示的CSFV Alfort的346位。
挑选三组3-6个月大而BVDV血清为阴性反应的动物进行实验。第1组及第2组各包括5只动物,而第3组则包括3只动物。第1组及第2组的动物投与2×106TCID50的B-346-d(第1组)或B-WT(第2组),每次投与量为5毫升,以肌肉内方式(臀肌),鼻内内方式及皮下方式(肩胛骨处)感染动物。感染后14天后,以参数监测两组的病毒血症,例如血细胞病毒血症及鼻拭子的病毒分泌量等。此外,监测临床参数,如肛温,血细胞数目及常态健康参数。
第1组动物以B-346-d感染后77天,进行攻击性感染,以探讨其对具有不同抗原并具有毒性的BVDV-分离株(#13)感染的保护性免疫能力。第3组的动物作为攻击性实验的对照,并利用根据第1组的感染步骤以毒性BVDV-分离株感染。
B-346-d病毒属于按Pellerin,C.等人,1994所述分类法中的抗原组(I型),而BVDV病毒(#13)属于另一抗原组(II型)。动物投与2×106TCID50的BVDV分离株(#13)进行感染,每次投与5毫升。动物经由肌肉内方式(臀肌),鼻腔内方式及皮下途径(肩胛骨处)感染。感染14天后,以参数监测两组的病毒血症,例如血细胞病毒血症及鼻拭子的病毒分泌量等。此外,监测临床参数,如肛温,白细胞数目及常态健康参数。
用B-346-d感染后的动物未呈现BVDV感染的典型临床症状,诸如肛温上升(表7a),或任何呼吸道的临床症状(未列)。
血细胞病毒血症(表7b)及鼻拭子病毒分泌量(表7c)的减低,明确显示B-346-d与B-WT比较后确实有减毒作用的存在。
毒性BVDV分离株#13确实诱发第3组动物产生带有典型BVDV感染症状的严重病毒血症,如肛温上升数天(表7d),严重的白细胞减少症(表7e),广泛的血细胞病毒血症(表7f),以及病毒分泌于鼻拭液中(表7g)。而以B-346-d感染进行接种的第1组动物,在以毒性BVDV分离株#13攻击后,几乎未有BVDV感染的临床典型症状出现。感染后肛温未有明显的上升(表7d),所观察到的白细胞减少症就过程及时间而言都非常不明显(表7e)。血液中未分离出BVDV(表7f),只有一只动物的鼻腔分泌物中检测到病毒(表7g)。
因此,用B-346-d感染会诱发强烈的免疫力,当以异源BVDV分离株攻击性感染后,发现可明显降低临床症状,病毒分泌量及血细胞病毒血症。
表7a:第1组(B-346-d)及第2组(B-WT)的平均肛温
研究天数 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
第1组 | 38.8 | 39.1 | 39.0 | 38.7 | 38.8 | 38.7 | 38.7 | 38.5 | 38.7 | 38.5 | 38.5 | 38.5 | 38.4 | 38.9 | 38.7 |
第2组 | 38.8 | 39.0 | 38.9 | 38.6 | 38.6 | 38.7 | 38.6 | 38.4 | 39.1 | 38.4 | 38.7 | 38.6 | 38.7 | 38.6 | 38.6 |
第1组的动物在第0天时,以6×106 TCID50的B-346-d感染,而第2组的动物则以6×106 TCID50的B-WT感染。
表7b:第1组及第2组的血细胞病毒血症
组别 | 动物 | 鼻腔分泌首日记录 | 鼻腔分泌末日记录 | 鼻腔分泌时间(天数) | 平均分泌时间(天数) |
1 | 1 | 6 | 6 | 1 | 1.4 |
2 | 4 | 6 | 2 | ||
3 | 5 | 5 | 1 | ||
4 | - | - | 0 | ||
5 | 6 | 9 | 3 | ||
2 | 6 | 4 | 8 | 5 | 4.4 |
7 | 4 | 7 | 4 | ||
8 | 4 | 7 | 4 | ||
9 | 4 | 7 | 4 | ||
10 | 4 | 8 | 5 |
分别用B-346-d及B-WT感染后,每日采集EDTA血液样品直至10天。在3个牛睾丸细胞(Cte)培养(培养基中含有肝素(1单位/毫升)以防凝血)中各加入0.2毫升的血液。隔夜培养后,用新鲜不含肝素的培养基置换原有的接种物培养基。培养4至6天后,利用对BVDV有特异性的多克隆血清,以免疫荧光法检测BVDV感染细胞。冷冻阴性组织,而后将其解冻。取其0.2毫升在Cte细胞上传一代,以确认没有BVDV。
表7c:鼻分泌物中的病毒量
组别 | 动物 | 鼻腔分泌的首日记录 | 鼻腔分泌最末日记录 | 分泌物中检测到病毒的天数 | 每组检测到病毒的平均天数 |
1 | 1 | 4 | 8 | 4 | 2.6 |
2 | 6 | 6 | 1 | ||
3 | 4 | 4 | 1 | ||
4 | 5 | 7 | 3 | ||
5 | 3 | 6 | 4 | ||
2 | 6 | 6 | 8 | 3 | 3.6 |
7 | 5 | 7 | 3 | ||
8 | 5 | 8 | 4 | ||
9 | 5 | 6 | 2 | ||
10 | 3 | 9 | 6 |
将鼻腔分泌物离心(1000g),以去除大块的残渣及污染物。移出上清液,取0.2毫升接种至三种细胞培养的每一种中。隔夜培养后,用2毫升的新鲜培养基置换原有的接种物/培养基,培养4至6天后,利用对BVDV具有特异性的多克隆血清,以免疫荧光法检测BVDV感染细胞。
表7d:第1组及第3组的平均肛温
研究天数 | -2 | -1 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 12 | 14 |
第1组 | 38.4 | 38.6 | 38.5 | 38.5 | 38.6 | 38.4 | 38.4 | 38.4 | 38.3 | 38.4 | 38.4 | 38.4 | 38.4 | 38.4 | 38.5 |
第3组 | 38.8 | 39.1 | 38.8 | 39.1 | 39.4 | 39.7 | 40.2 | 40.2 | 40.4 | 41.3 | 40.2 | 40.1 | 40.2 | 40.8 | 40.4 |
攻击性感染后,肛温记录至16天。第1组及第3组的动物以6×106TCID50的毒性BVDV分离株#13进行感染。
表7e:平均的白血球细胞计数
研究天数 | -2 | -1 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 12 | 14 |
第1组 | 11.9 | 11.9 | 11.3 | 10.8 | 9.2 | 8.2 | 8.9 | 9.9 | 11.2 | 11.6 | 11.6 | 10.6 | 10.8 | 10.8 | 9.4 |
第3组 | 11.7 | 15.8 | 13.8 | 11.1 | 7.7 | 9.8 | 7.4 | 6.8 | 7.5 | 8.7 | 7.0 | 8.1 | 6.2 | 6.4 | 6 2 |
两组动物在攻击性感染前2天至攻击性感染后14天,每日采集EDTA血液样品。利用Sysmex Micro-Cell-计数器F800测定EDTA血液样品中白细胞数目。
表7f:自血液样品中分离的BVDV
组别 | 动物 | 血液中测出病毒的首日 | 血液中测出病毒的末日 | 血液中病毒存在时间(天数) | 平均天数 |
1 | 1 | - | - | 0 | 0 |
2 | - | - | 0 | ||
3 | - | - | 0 | ||
4 | - | - | 0 | ||
5 | - | - | 0 | ||
3 | 11 | 3 | 10 | 8 | 9.7 |
12 | 3 | 14 | 12 | ||
13 | 3 | 9 | 9 |
感染后10天内,每日采集EDA血液样品。在3个牛睾丸细胞(Cte)培养(培养基中含有肝素(1单位/毫升)以预防凝血)中各加入0.2毫升的血液。隔夜培养后,用新鲜不含肝素的培养基置换原有的接种物/培养基。培养4至6天后,利用对BVDV有特异性的多克隆血清,以免疫荧光法检测BVDV感染细胞。冷冻阴性组织,而后将其解冻。取其0.2毫升在Cte细胞上传一代,以确认没有BVDV。
表7g:鼻分泌物中的病毒量
组别 | 动物 | 鼻腔分泌首日记录 | 鼻腔分泌末日记录 | 鼻腔分泌天数 | 每组平均天数 |
1 | 1 | 3 | 4 | 2 | 0.8 |
2 | - | - | 0 | ||
3 | - | - | 0 | ||
4 | - | - | 0 | ||
5 | 4 | 5 | 2 | ||
3 | 11 | 3 | 14 | 12 | 10 |
12 | 3 | 14 | 12 |
13 | 3 | 8 | 6 |
将鼻腔分泌物离心(1000g),以去除大块的残渣及污染物。移出上清液,取0.2毫升接种至三种细胞组织培养的每一种中。隔夜培养后,用2毫升的新鲜培养基置换原有的接种物/培养基。培养4至6天后,利用对BVDV具有特异性的多克隆血清,以免疫荧光法检测BVDV感染细胞。
实施例8 利用RT-PCR区分C-346-d及没有删除346位组氨酸密码子的CSFV
编码CSFV糖蛋白ERNS上保守性RNase基元的RNA序列为高度保守的序列,所有已知的CSFV序列中,对应于所发表的CSFV Alfort株(Meyers等人,1987)序列中1387至1416位残基的区域并无核苷酸的变化。因此,源自此基因组保守区域的寡核苷酸引物可用于RT-PCR中,以检测所有的CSFV分离株(参考图7)。因此,利用适当设计的引物,以RT-PCR分析可检测到编码组氨酸346的三联密码(核苷酸1399-1401)的缺失。合成含有保守区域的不同的寡核苷酸,可包括该组氨酸密码或不含该组氨酸密码。这些寡核苷酸在RT-PCR反应中作为上游引物,而寡核苷酸ERNS-终止密码作为下游引物。分别自以C-346-d,C-WT,C-346-L或C-346-K感染的组织培养细胞中纯化RNA,此RNA作为模板。逆转录2微克加热变性的RNA(2分钟92℃,5分钟冰浴,溶于11.5微升的水中,内含30pMol逆转录引物)在加入8微升的RT混合物(125mM Tris/HCl pH8.3,182.5mM KCl,7.5mMMgCl2,25mM二硫苏糖醇,dATP,dTTP,dCTP,dGTP各1.25mM),15单位的RNAguard(Pharmacia,Freiburg,德国)及50单位的Superscript(LifeFechnologies/BRL。Eggenstein,德国)后,于37℃进行45分钟。完成逆转录作用后,将试管置于冰上,加入30微升PCR混合物(8.3mM Tris/HCl,pH8.3;33.3mM KCl;2.2mM MgCl2;dATP,dTTP,dCTP,dGTP各0.42mM;0.17%Triton×100;0.03%牛血清白蛋白;5单位Taq聚合酶(Appligene,Heidelberg,德国)及16.7%DMSO)。当使用引物OI H+3时,用于扩增的反应混合物不含DMSO。扩增作用共进行36个循环(94℃30秒;57℃30秒;74℃45秒)。取1微升的扩增反应物上样于1%琼脂糖凝胶上,利用电泳法将扩增的产物分开,并以溴化乙啶染色。结果如图7所示,OI H-3/OI ERNS终止密码这一引物对,能特异性扩增删除了346位的密码子的RNA产生的条带,而其他两对引物组合物可以增殖含有密码346的产物,但以删除了此密码的RNA为模板时,则无条带呈现。
RT-PCR引物:
上游:
OI H-3:TGGAACAAAGGATGGTGT
OI H+2:TGGAACAAACATGGATGG
OI H+3:GAATGGAACAAACATGGA
下游:
OI ERNS终止密码:GGAATTCTCAGGCATAGGCACCAAACCAGG
附图简述
图1:CSFV Alfort株所表达的前495个氨基酸
所列的序列为CSFV Alfort株表达的前495个氨基酸(Mayers等人,1989)。该病毒株糖蛋白ERNS的一个单体对应于Rumenapf等人(1993)所述的268至494位氨基酸。下画线的295至307及338至357位残基的区域与植物及真菌RNase具有同源性(Schneider等人,1993)。
图2:试验性感染后动物的肛温曲线图
从感染前2天至感染后第18天每天记录肛温。肛温曲线详细记录每组中每只动物经病毒V(pA/CSFV)(源自质粒pA/CSFV)感染后(实线)及病毒V(pA/C-346-d)(源自质粒pA/C-346-d)感染后(虚线)的肛温变化。
图3:攻击性感染后动物的肛温曲线
病毒攻击性感染后1-2 1天的每日肛温记录。在69天前动物经突变的病毒C-346-d[V(pA/C-346-d)]感染,现在用CSFV Eystrup株以致死剂量攻击性感染动物(如正文中所详述)。肛温曲线详细记录每组中每只动物以2×105TCID50的CSFV Eystrup株攻击性感染后的肛温变化。
图4:试验感染后动物的肛温曲线图
感染后0-18天所记录的每天肛温图。肛温曲线详细记录两组中每只动物经病毒C-346-d[V(pA/C-346-d)]感染后(虚线)或经回复病毒C-346-d/RS[V(pA/C-346-d/RS)感染后(实线)的肛温变化。
图5:动物实验#2攻击性感染后的肛温
病毒攻击性感染后1-10天的每日肛温记录。动物37天前经突变的C-346-d感染,现在以致死剂量(2×105TCID50)的CSFV Eystrup株病毒攻击性感染动物。
图6:以实施例6的双突变病毒株感染动物后的肛温
记录了以突变株V(pA/C-297-L/346-L)进行病毒攻击性感染之前和之后的每日肛温。
图7:利用实施例8的RT-PCR技术区分C-346-d及未删除346位的组氨酸密码的CSFV
a)OIH-3/OI ERNS终止密码这一引物对可特异性扩增已删除346位密码的RNA(C-346-d)所产生的条带,详述如实施例8。相反地,不具有该删除的RNA不会与该引物对反应(C-WT,C-346-L,C-346-K)。
b)及c)其他两个引物组合(OI H+2及OI H+3)扩增未删除346位密码的RNA(OI H+2及OI H+3)产生的条带。当以删除了346-的突变株(C-346-d)的RNA为模板时,则没有条带产生。
序列表
<110>Boehringer Ingelheim Vetmedica GmbH
<120>减毒瘟病毒
<130>减毒瘟病毒-PCT/EP
<140>PCT/EP99/03642
<141>1999-05-27
<150>EP98110356.7
<151>1998-06-05
<160>27
<170>PatentIn Ver.2.1
<210>1
<211>494
<212>PRT
<213>BVDV Erns在346位缺失
<400>1
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Pro Ala Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asn Pro Leu
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His Lys Arg Gly Glu Arg Glu Val Pro Thr Asn Leu Ala Ser Leu Pro
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Asn Lys Pro Gln Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala
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Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro
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Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr Asp Thr Glu
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Leu Lys Ala Ile His Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr
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Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys Gly Trp Cys Asn
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<211>495
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<400>3
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<210>4
<211>494
<212>PRT
<213>CSFV Erns在297位缺失的突变株
<400>4
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Gly Ile Trp Pro Glu Lys Ile Cys
290 295 300
Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu Ile
305 310 315 320
Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys Arg
325 330 335
Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr Asn
340 345 350
Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr
355 360 365
Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys
370 375 380
Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr
385 390 395 400
Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val
405 410 415
Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr
420 425 430
Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu
435 440 445
Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala
450 455 460
Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys
465 470 475 480
Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>5
<211>495
<212>PRT
<213>CSFV Erns的297位赖氨酸突变株
<400>5
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Va1 Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Iys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Lys Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Va1 Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>6
<211>495
<212>PRT
<213>CSFV Erns的297位壳氨酸突变株
<400>6
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Leu Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>7
<211>494
<212>PRT
<213>CSFV Erns的301位缺失的突变株
<400>7
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Glu Lys Ile Cys
290 295 300
Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu Ile
305 310 315 320
Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys Arg
325 330 335
Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr Asn
340 345 350
Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr
355 360 365
Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys
370 375 380
Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr
385 390 395 400
Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val
405 410 415
Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr
420 425 430
Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu
435 440 445
Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala
450 455 460
Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys
465 470 475 480
Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>8
<211>495
<212>PRT
<213>CSFV Erns的302位丙氨酸突变株
<400>8
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Ala Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu GLu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>9
<211>495
<212>PRT
<213>CSFV Erns的305位甘氨酸突变株
<400>9
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Gly Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>10
<211>495
<212>PRT
<213>CSFV Erns的340位甘氨酸的突变株
<400>10
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Gly His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>11
<211>493
<212>PRT
<213>CSFV Erns的342-346位缺失的突变株
<400>11
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Trp Asn Lys Gly Trp Cys Asn Trp Tyr Asn Ile
340 345 350
Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr Glu
355 360 365
Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Asn
370 375 380
Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr Leu
385 390 395 400
Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val Ile
405 410 415
Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly
420 425 430
Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Leu
435 440 445
Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg
450 455 460
Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys Leu
465 470 475 480
Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>12
<211>494
<212>PRT
<213>CSFV Erns 342位缺失的突变株
<400>12
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Trp Asn Lys His Gly Trp Cys Asn Trp Tyr Asn
340 345 350
Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr
355 360 365
Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys
370 375 380
Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr
385 390 395 400
Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val
405 410 415
Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr
420 425 430
Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu
435 440 445
Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala
450 455 460
Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys
465 470 475 480
Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>13
<211>495
<212>PRT
<213>CSFV Erns 343位甘氨酸的突变株
<400>13
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala ser Glu Arg Thr AsnTyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Gly Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>14
<211>492
<212>PRT
<213>CSFV Erns 345-347位缺失的突变株
<400> 14
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Trp Cys Asn Trp Tyr Asn Ile Asp
340 345 350
Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr Glu Gly
355 360 365
Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Asn Thr
370 375 380
Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr Leu Thr
385 390 395 400
Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val Ile Glu
405 410 415
Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly Asp
420 425 430
His Glu Cys Gly ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Leu Asp
435 440 445
Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg Val
450 455 460
Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys Leu Glu
465 470 475 480
Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>15
<211>493
<212>PRT
<213>CSFV Erns 345-346位缺失的突变株
<400>15
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Ihr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Gly Trp Cys Asn Trp Tyr Asn Ile
340 345 350
Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr Glu
355 360 365
Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Asn
370 375 380
Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr Leu
385 390 395 400
Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val Ile
405 410 415
Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly
420 425 430
Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Leu
435 440 445
Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg
450 455 460
Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys Leu
465 470 475 480
Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>16
<211>495
<212>PRT
<213>CSFV Erns 345位丙氨酸突变株
<400>16
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Ala His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>17
<211>493
<212>PRT
<213>CSFV Erns 346-347缺失的突变株
<400>17
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Trp Cys Asn Trp Tyr Asn Ile
340 345 350
Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr Glu
355 360 365
Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Asn
370 375 380
Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr Leu
385 390 395 400
Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val Ile
405 410 415
Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly
420 425 430
Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Leu
435 440 445
Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg
450 455 460
Val Thr Ser Trp Leu Gly Arg Gln Lau Ser Thr Ala Gly Lys Lys Leu
465 470 475 480
Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>18
<211>494
<212>PRT
<213>CSFV Erns 346位缺失的突变株
<400>18
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Gly Trp Cys Asn Trp Tyr Asn
340 345 350
Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu Thr
355 360 365
Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys
370 375 380
Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr Thr
385 390 395 400
Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr Val
405 410 415
Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr
420 425 430
Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu
435 440 445
Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala
450 455 460
Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys Lys
465 470 475 480
Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490
<210>19
<211>495
<212>PRT
<213>CSFV Erns 346位赖氨酸突变株
<400>19
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Lys Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>20
<211>495
<212>PRT
<213>CSFV Erns 346亮氨酸突变株
<400>20
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Leu Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>21
<211>495
<212>PRT
<213>CSFV Erns 297位赖氨酸及346位赖氨酸的突变株
<400>21
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val A5p Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Lys Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Lys Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>22
<211>495
<212>PRT
<213>CsFV Erns 297位赖氨酸及346位亮氨酸的突变株
<400>22
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Lys Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Leu Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>23
<211>495
<212>PRT
<213>CSFV Erns 297位亮氨酸及346位亮氨酸的突变株
<400>23
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu Leu Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys Leu Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Va1 Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>24
<211>495
<212>PRT
<213>BVDV的Ern蛋白
<400>24
Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys
1 5 10 15
Pro Ala Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asn Pro Leu
20 25 30
Phe Gly Glu Arg Gly Val Ile His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Lys Arg Gly Glu Arg Glu Val Pro Thr Asn Leu Ala Ser Leu Pro
50 55 60
Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Lys Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Leu Lys Pro Gly Pro Leu Phe Tyr Gln Asp Tyr Lys Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Glu Glu Ala Ser Met Cys
100 105 110
Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Ser Arg Leu
115 120 125
Tyr His Ile Tyr Val Cys Ile Asp Gly Cys Ile Ile Val Lys Ser Ala
130 135 140
Thr Lys Asp Arg Gln Lys Val Leu Lys Trp Val His Asn Lys Leu Asn
145 150 155 160
Cys Pro Leu Trp Val Ser Ser Cys Ser Asp Thr Lys Asp Glu Gly Val
165 170 175
Val Arg Lys Lys Gln Gln Lys Pro Asp Arg Leu Glu Lys Gly Arg Met
180 185 190
Lys Ile Thr Pro Lys Glu Ser Glu Lys Asp Ser Lys Thr Lys Pro Pro
195 200 205
Asp Ala Thr Ile Val Val Asp Gly Val Lys Tyr Gln Val Lys Lys Lys
210 215 220
Gly Lys Val Lys Ser Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys
225 230 235 240
Asn Lys Pro Gln Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala
245 250 255
Trp Ala Ile Ile Ala Leu Val Phe Phe Gln Val Thr Met Gly Glu Asn
260 265 270
Ile Thr Gln Trp Asn Leu Gln Asp Asn Gly Thr Glu Gly Ile Gln Arg
275 280 285
Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro
290 295 300
Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr Asp Thr Glu
305 310 315 320
Leu Lys Ala Ile His Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr
325 330 335
Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys
340 345 350
Asn Trp Tyr Asn Ile Glu Pro Trp Ile Leu Leu Met Asn Lys Thr Gln
355 360 365
Ala Asn Leu Thr Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys
370 375 380
Arg Tyr Asp Arg Asp Ser Asp Leu Asn Val Val Thr Gln Ala Arg Asp
385 390 395 400
Ser Pro Thr Pro Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser phe
405 410 415
Ala Gly Ile Leu Val Gln Gly Pro Cys Asn Phe Glu Ile Ala Val Ser
420 425 430
Asp Val Leu Phe Lys Glu His Asp Cys Thr Ser Val Ile Gln Asp Thr
435 440 445
Ala His Tyr Leu Val Asp Gly Met Thr Asn Ser Leu Glu Ser Ala Arg
450 455 460
Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Arg Gln Leu Gly Ile
465 470 475 480
Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Thr Trp Phe Gly Ala
485 490 495
<210>25
<211>495
<212>PRT
<213>CSFV的Em蛋白
<400>25
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly AlaTyr Ala Leu
485 490 495
<210>26
<211>495
<212>PRT
<213>CSFV Erns 300位甘氨酸的突变株
<400>26
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Gly Pro Glu Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
<210>27
<211>495
<212>PRT
<213>CSFV Erns 300位甘氨酸及302位丙氨酸的突变株
<400>27
Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Ser Lys Gln Lys
1 5 10 15
Pro Val Gly Val Glu Glu Pro Val Tyr Asp Thr Ala Gly Arg Pro Leu
20 25 30
Phe Gly Asn Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro
35 40 45
His Asp Arg Gly Arg Gly Asp Ile Arg Thr Thr Leu Arg Asp Leu Pro
50 55 60
Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly
65 70 75 80
Ile Tyr Ile Lys Pro Gly Pro Val Tyr Tyr Gln Asp Tyr Thr Gly Pro
85 90 95
Val Tyr His Arg Ala Pro Leu Glu Phe Phe Asp Glu Ala Gln Phe Cys
100 105 110
Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125
Tyr His Ile Tyr Val Cys Val Asp Gly Cys Ile Leu Leu Lys Leu Ala
130 135 140
Lys Arg Gly Thr Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asn
145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser
165 170 175
Lys Asp Lys Lys Pro Asp Arg Met Asn Lys Gly Lys Leu Lys Ile Ala
180 185 190
Pro Arg Glu His Glu Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr
195 200 205
Ile Val Val Glu Gly Val Lys Tyr Gln Ile Lys Lys Lys Gly Lys Val
210 215 220
Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro
225 230 235 240
Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Val
245 250 255
Ile Thr Ile Leu Leu Tyr Gln Pro Val Ala Ala Glu Asn Ile Thr Gln
260 265 270
Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln Arg Ala Met Tyr
275 280 285
Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Gly Pro Ala Lys Ile
290 295 300
Cys Lys Gly Val Pro Thr His Leu Ala Thr Asp Thr Glu Leu Lys Glu
305 310 315 320
Ile Arg Gly Met Met Asp Ala Ser Glu Arg Thr Asn Tyr Thr Cys Cys
325 330 335
Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr
340 345 350
Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Thr Asn Leu
355 360 365
Thr Glu Gly Pro Pro Asp Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp
370 375 380
Lys Asn Thr Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr
385 390 395 400
Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr
405 410 415
Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu
420 425 430
Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr
435 440 445
Leu Leu Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala
450 455 460
Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Ser Thr Ala Gly Lys
465 470 475 480
Lys Leu Glu Arg Arg Ser Iys Thr Trp Phe Gly Ala Tyr Ala Leu
485 490 495
Claims (52)
1.一种包括瘟病毒的活疫苗,其中糖蛋白ERNS的RNase活性已失活。
2.权利要求1的疫苗,其中通过删除和/或突变该糖蛋白的至少一个氨基酸,以失活该RNase活性。
3.权利要求2的疫苗,其中的删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
4.权利要求1至3中任一项的疫苗,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
5.权利要求1至4中任一项的疫苗,其中删除该糖蛋白如图1的CSFVAlfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
6.权利要求l至5中任一项的包含BVDV瘟病毒的疫苗,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
7.一种瘟病毒,其中当糖蛋白ERNS上如图1的CSFV Alfort株所例示的297和/或346位不是赖氨酸时,删除和/或突变该糖蛋白的至少一个氨基酸,或其他病毒株的相应氨基酸,以失活该糖蛋白的RNase活性。
8.权利要求7的瘟病毒,其中删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
9.权利要求7或8的瘟病毒,其中删除或突变该糖蛋白如图1的CSFVAlfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
10.权利要求7至9中任一项的瘟病毒,其中删除该糖蛋自如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
11.权利要求7至10中任一项的BVDV瘟病毒,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
12.一种编码糖蛋白ERNS的核酸,其中当该糖蛋白上如图1的CSFVAlfort株所例示的297和/或346位不是赖氨酸时,删除和/或突变该糖蛋白的至少一个氨基酸,或其他病毒株的相应氨基酸,以失活该糖蛋白的RNase活性。
13.权利要求12的核酸,其中删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
14.权利要求12或13的核酸,其中删除或突变该糖蛋白如图1的CSFVAlfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
15.权利要求12至14中任一项的核酸,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
16.权利要求12至15中任一项的BVDV核酸,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
17.权利要求12至16中任一项的核酸用于制备核苷酸-和/或载体-疫苗的应用。
18.一种药用组合物,其包括权利要求1至6中任一项的疫苗,和/或权利要求7至11中任一项的瘟病毒,和/或权利要求12至16中任一项的核苷酸序列。
19.一种使瘟病毒减毒的方法,其特征在于使糖蛋白ERNS的RNase活性失活。
20.权利要求19的方法,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活该RNase活性。
21.权利要求19或20的方法,其中该删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
22.权利要求19至21中任一项的方法,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
23.权利要求19至22中任一项的方法,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
24.一种制备特异地减毒的疫苗的方法,其特征在于使糖蛋白ERNS的RNase活性失活。
25.权利要求24的方法,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活RNase活性。
26.权利要求24或25的方法,其中的删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
27.权利要求24至26中任一项的方法,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
28.权利要求24至27中任一项的方法,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
29.一种使瘟病毒带上可检测性标记的方法,其特征在于失活糖蛋白ERNS上的RNase活性。
30.权利要求29的方法,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活该RNase活性。
31.权利要求29或30的方法,其中的删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
32.权利要求29至31中任一项的方法,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
33.权利要求29至32中任一项的方法,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
34.一种预防和治疗动物体内瘟病毒感染的方法,其特征在于将权利要求1至6中任一项的疫苗或权利要求18的药用组合物投予需要此类预防或治疗的动物。
35.一种制备特异地减毒之瘟病毒的方法,其特征在于失活糖蛋白ERNS的RNase活性。
36.权利要求35的方法,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活该RNase活性。
37.权利要求35或36的方法,其中的删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
38.权利要求35至37中任一项的方法,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
39.权利要求36至38中任一项的方法,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
40.一种制备特异性标记的瘟病毒的方法,其特征在于失活糖蛋白ERNS的RNase活性。
41.权利要求40的方法,其中删除和/或突变该糖蛋白的至少一个氨基酸,以失活RNase活性。
42.权利要求40或41的方法,其中的删除和/或突变位于该糖蛋白如图1的CSFV Alfort株所例示的295至307位和/或338至357位的氨基酸,或其他病毒株上的相应氨基酸。
43.权利要求40至42中任一项的方法,其中删除或突变该糖蛋白如图1的CSFV Alfort株所例示的346位的氨基酸,或其他病毒株上的相应氨基酸,以失活该RNase活性。
44.权利要求40至43中任一项的方法,其中删除该糖蛋白如图1的CSFV Alfort株所例示的346位的组氨酸残基,或其他病毒株上的相应氨基酸,以失活该RNase活性。
45.权利要求1至6中任一项的疫苗用于预防和治疗动物体内瘟病毒感染的应用。
46.权利要求18的药用组合物用于预防和治疗动物体内瘟病毒感染的应用。
47.权利要求7至11中任一项的瘟病毒和/或权利要求12至16中任一项的核苷酸序列用于制备疫苗或药用组合物的应用。
48.一种区分瘟病毒感染的动物和接种了特异地减毒的瘟病毒的动物的方法,其中所述特异地减毒的瘟病毒是根据权利要求19至23中任一项的方法减毒,包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物采样;
(2)鉴定该样品中瘟病毒的核苷酸序列;
(3)调查疫苗中ERNS核苷酸序列的删除和/或突变的出现与接种疫苗的动物之间的相关性,及所述删除和/或突变的缺乏与该动物的瘟病毒感染之间的相关性。
49.权利要求48的方法,其包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物采样;
(2)利用单克隆抗体或多克隆抗体对该样品中出现的ERNS糖蛋白的特异性结合,鉴定减毒瘟病毒上修饰的ERNS糖蛋白,所述糖蛋白已根据权利要求19至23中任一项的方法修饰,因此所述单克隆抗体或多克隆抗体不与未修饰的ERNS糖蛋白结合;
(3)调查所述单克隆抗体或多克隆抗体的特异性结合与疫苗接种动物的相关性,及抗体无法结合与该动物的瘟病毒感染之间的相关性,条件是已另行证实该动物和/或该样品中存在瘟病毒物质。
50.权利要求49的方法,其包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物采样;
(2)利用单克隆抗体或多克隆抗体对该样品中出现的ERNS糖蛋白的特异性结合,鉴定瘟病毒上未修饰的ERNS糖蛋白,该糖蛋白未以权利要求19至23中任一项的方法修饰,因此所述单克隆抗体或多克隆抗体不与修饰的ERNS糖蛋白结合;
(3)调查所述单克隆抗体或多克隆抗体的特异性结合与该动物体内瘟病毒感染之间的相关性,及抗体无法结合与接种疫苗的动物之间的相关性,条件是已另行证实该动物和/或该样品中存在瘟病毒物质。
51.权利要求48的方法,其包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物采样;
(2)测定该样品中有无糖蛋白ERNS的RNase活性;
(3)调查糖蛋白ERNS的RNase活性的缺乏与接种疫苗的动物之间的相关性及该活性的存在与该动物的瘟病毒感染之间的相关性。
52.权利要求48的方法,其包括下列步骤:
(1)自疑有瘟病毒感染的目的动物或接种疫苗的动物获取多克隆抗体的样品;
(2)鉴定所述多克隆抗体对未修饰的糖蛋白ERNS或根据本发明修饰的糖蛋白ERNS的任何特异性结合;
(3)调查所述多克隆抗体对未修饰的糖蛋白ERNS的结合与瘟病毒感染之间的相关性,及所述多克隆抗体对根据本发明修饰的糖蛋白ERNS的结合与疫苗接种动物之间的相关性。
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CN103882051A (zh) * | 2014-03-20 | 2014-06-25 | 北京市农林科学院 | 一种检测猪瘟病毒抗体的elisa方法及检测试剂盒 |
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US7179473B2 (en) | 1998-06-05 | 2007-02-20 | Boehringer Ingelheim Vetmedica Gmbh | Attenuated pestiviruses |
EP1104676A1 (en) * | 1999-11-30 | 2001-06-06 | Boehringer Ingelheim Vetmedica Gmbh | Safe attenuated bovine viral diarrhea viruses for use in pregnant cows |
DE60118456T2 (de) | 2000-04-21 | 2006-08-24 | Akzo Nobel N.V. | Pestvirus Mutanten und diese enthaltende Impfstoffe |
US7135561B2 (en) | 2001-09-06 | 2006-11-14 | Boehringer Ingelheim Vetmedica Gmbh | Infectious bovine viral diarrhea virus clone |
US20090068223A1 (en) | 2005-11-15 | 2009-03-12 | Boehringer Ingelheim Vetmedica, Inc. | Combination vaccine comprising an attenuated bovine viral diarrhea virus |
AR069087A1 (es) | 2007-10-29 | 2009-12-30 | Boehringer Ingelheim Vetmed | Cepa bacteriana de m. bovis atenuada avirulenta obtenida por pases, vacuna de micoplasma boris y metodos de uso de la misma |
UY31930A (es) | 2008-06-25 | 2010-01-29 | Boheringer Ingelheim Pharma Kg | Pestivirus atenuados recombinantes, en particular a csfv, bvdv o bdv atenuado recombinante |
NZ591950A (en) | 2008-10-31 | 2012-12-21 | Boehringer Ingelheim Vetmed | Use of various antigens including antigens from mycoplasma bovis in multivalent vaccine composition |
US8846054B2 (en) | 2009-01-09 | 2014-09-30 | Boehringer Ingelheim Vetmedica, Inc. | Method of treating pregnant cows and/or heifers |
UY32570A (es) | 2009-04-24 | 2010-11-30 | Boehringer Ingelheim Vetmed | Vacuna viva modificada de mycoplasma bovis mejorada |
CN101915837B (zh) * | 2010-07-28 | 2013-07-03 | 中国兽医药品监察所 | 一种猪瘟兔化弱毒活疫苗效力检验方法 |
CN103338785B (zh) | 2010-09-21 | 2015-01-14 | 英特维特国际股份有限公司 | Bvdv疫苗 |
WO2020211801A1 (en) * | 2019-04-18 | 2020-10-22 | Boehringer Ingelheim Vetmedica (China) Co., Ltd. | Recombinant classical swine fever virus |
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CN103882051A (zh) * | 2014-03-20 | 2014-06-25 | 北京市农林科学院 | 一种检测猪瘟病毒抗体的elisa方法及检测试剂盒 |
CN103882051B (zh) * | 2014-03-20 | 2016-04-06 | 北京市农林科学院 | 一种检测猪瘟病毒抗体的elisa方法及检测试剂盒 |
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