CN1976717A

CN1976717A - 安全的突变病毒疫苗

Info

Publication number: CN1976717A
Application number: CNA2004800283626A
Authority: CN
Inventors: 万小坤; 杰伊·G·卡尔弗特; 迈克尔·K·奥哈拉; 曹雪梅
Original assignee: Pfizer Products Inc
Current assignee: Pfizer Products Inc
Priority date: 2003-07-29
Filing date: 2004-07-26
Publication date: 2007-06-06
Also published as: PT1651263E; BRPI0413107A; JP4662931B2; KR100810820B1; PL1651263T3; IL173338A0; ATE489107T1; NZ545612A; US7361357B2; CA2533877A1; ES2355276T3; ZA200600585B; NO20060931L; WO2005021034A3; RS20060045A; US20050053621A1; SI1651263T1; TW200514568A; ES2355276T9; EP1651263A2

Abstract

本发明提供了一种安全疫苗以及制备此种疫苗的方法。本发明的疫苗中包含至少两种活的同一科的突变病毒或是编码所述病毒的核酸分子，其中两种中的每一种病毒或其编码核酸分子都包含能够赋予所需表型的突变，而且病毒中的突变都存在于同一基因组位点上从而使突变病毒无法相互重组来抵消突变。

Description

安全的突变病毒疫苗

技术领域：

本发明主要涉及适用于给药动物以抗病毒感染的疫苗。更准确地说，本发明涉及安全疫苗以及制备此种疫苗的方法。本发明的疫苗中包含至少两种活的同一科突变病毒或是编码所述病毒的核酸分子，其中每一种病毒或编码核酸分子中包含能够赋予所需表型的突变，而且病毒中的突变存在于同一基因组位点上从而使突变病毒无法相互重组来抵消(eliminate)突变。

背景技术：

黄病毒科(Flaviviridae)由疫病毒属(Pestivirus)、黄病毒属(Flavivirus)和丙型肝炎病毒属(Hepacivirus)属组成。疫病毒属(Pestivirus)的代表种有牛病毒性腹泻病毒-1(BVDV-1)、BVDV-2、经典猪瘟病毒(classical swinefever virus)以及羊先天性髓质形成不全症病毒(Border disease virus)。这一病毒科中成员的病毒粒子包封着约9.5到12.3kb的正链RNA基因组。该基因组RNA包含邻接的长的开放读码框(ORFs)，其可以被翻译为多蛋白质再经过细胞和病毒蛋白酶的加工而产生成熟的病毒蛋白。对于疫病毒属(Pestivirus)的成员，开放读码框(ORF)编码大约3900个氨基酸的多蛋白质，其可以被协同翻译和后翻译加工成为下列成熟的病毒蛋白质(从5’端到3’端)：N^pro、C、E^ms、E1、E2、NS2-3、NS4A、NS4B、NS5A以及NS5B。

在一些疫病毒属(Pestivirus)成员中，基于它们对组织培养细胞的作用存在两种生物型，即为致细胞病变的(细胞病变的或cp)和非致细胞病变的(非细胞病变的或ncp)。基因组分析显示在致细胞病变的(cp)疫病毒(pestiviruses)基因组中存在细胞序列的插入，有时伴有病毒序列的复制、基因组重排和/或病毒序列的缺失，但在非致细胞病变的(ncp)疫病毒(pestiviruses)相应的RNA中并不存在。这暗示着致细胞病变的(cp)疫病毒(pestiviruses)是由非致细胞病变的(ncp)疫病毒(pestiviruses)通过RNA重组发展而来的。

牛病毒性腹泻病毒(BVDV)是一种广泛分布的牛病原体。BVDV-1在具免疫活性动物体内通常只引起轻度的腹泻，然而BVDV-2则能够引起血小板减少症、大出血以及急性的致命疾病。牛病毒性腹泻病毒(BVDV)能够穿过怀孕牛的胎盘从而引起持续感染(PI)小牛的出生(Malmquist，J.Am.Vet.Mea.Assoc.152：763-768(1968)；Ross，et al.，J.Am.Vet.Med.Assoc.188：618-619(1986))。病毒血症的(Viremic)小牛对病毒具有免疫耐受而且它们此后的生活中会持续存在病毒血症。它们提供了一个粘膜疾病爆发的根源(Liess，et al.，Dtsch.Tieraerztl.Wschr.81：481-487(1974))而且极易被微生物感染而引起例如肺炎或肠道病变这样的疾病(Barber，et al.，Vet.Rec.117：459-464(1985))。任一基因型的病毒都可以以为两生物型中的一种存在，致细胞病变的(cp)或者非致细胞病变的(ncp)。致细胞病变的(cp)显型与NS3的表达有关，虽然细胞感染致细胞病变的(cp)或非致细胞病变的(ncp)牛病毒性腹泻病毒(BVDV)都会表达NS2-3，然而NS3却只能在致细胞病变的(cp)牛病毒性腹泻病毒(BVDV)感染以后被检测到。NS3与NS2-3的C末端部分是线性对应的。NS3的表达呈现了一种在致细胞病变的(cp)牛病毒性腹泻病毒(BVDV)中观察到的基因组改造的结果。

目前可得到的病毒疫苗包括灭活或减毒的活病毒疫苗、活载体疫苗、亚单位疫苗以及DNA或RNA疫苗。见Roth等“New Technology For ImprovedVaccine Safety And Efficacy”，Veterinary Clinics North America：Food AnimalPractice 17(3)：585-597(2001)。病毒的减毒可以通过紫外照射、化学处理或者体外的高连续传代(high serial passage)来达成。这些方法导致的突变其数量、位置和种类在没有进行基因组序列分析的情况下是未知的。减毒还可以通过进行确定的基因改造来完成，例如，对已知能够带来毒性的病毒序列进行特异性缺失，或者在病毒基因组中进行序列的插入。关于应用减毒活病毒疫苗的一种顾虑是减毒的突变病毒具有在体内重组的潜力以抵消减毒突变从而恢复毒性。例如，在有毒性(野生型)野外菌株存在的情况下，病毒基因组中存在缺失的减毒病毒会具有与毒性菌株重组从而恢复缺失序列的潜力。见例如，Roth等，同上。还观察到在细胞培养基中具有细胞插入的致细胞病变疫病毒(cytopathic pestiviruses)通过细胞序列的缺失可以产生非致细胞病变疫病毒(noncytopathic pestiviruses)，可能是通过RNA重组完成的。见例如，Baroth等，“Insertion of cellular NEDD8 codingsequences in a pestivirus”，Virology.278(2)：456-66，(2000)，以及Becher等，“RNA recombination between persisting pestivirus and a vaccine strain：generation of cytopathogenic virus and induction of lethal disease”，Journal ofVirology 75(14)：6256-64(2001)。如果希望在疫苗组合物中包括两种来自于同一种、属或科的减毒突变病毒，那么将存在一种顾虑是两种病毒可能在接种疫苗的动物体内重组从而抵消减毒突变。见例如，Glazenburg等，“Genetic recombination of pseudorabies virus：evidence that homologousrecombination between insert sequences is less frequent than betweenautologous sequences”，Archives of Virology，140(4)：671-85(1995)。

于是，仍在存在着进一步开发能够保护动物抵御病毒感染的安全有效的疫苗的需要。

发明内容：

本发明提供了一种包含至少两种活的同一科突变病毒或是编码所述病毒的核酸分子的安全疫苗，其中每一种病毒或其编码核酸中包含一种能够赋予所需表型的突变，而且病毒中的突变都存在于同一基因组位点上从而使突变病毒无法相互重组来抵消突变。

本发明还提供了一种通过筛选或构建两种或更多活的同一科、属或种的突变病毒制备一种安全病毒疫苗的方法，其中每一种病毒中都包含能够赋予所需表型的突变，而且病毒中的突变都存在于同一基因组位点上从而使突变病毒无法进行同源重组来抵消突变。

本发明进一步还提供了一种通过给动物施用本发明的疫苗组合物来保护动物抵御病毒感染的方法。

附图说明：

图1、BVDV-1的NADL株和BVDV-2的53637株的NS2-3区域的细胞插入和侧翼病毒序列比对。

具体实施方式：

与本发明一致被特别公认的是，在同一病毒基因组位点上包含突变的同一科突变病毒是不能相互进行重组来抵消突变的。

因此，在一种具体实施方式中，本发明提供了一种安全的疫苗组合物，其包含至少两种即两种或更多，活的同一科突变病毒或是编码所述病毒的核酸分子，其中病毒中的突变都存在于同一基因组位点上从而使突变病毒无法彼此间进行重组来抵消突变。

在另一种具体实施方式中，本发明提供了一种制备如上文中描述的一种安全病毒疫苗的方法。尤其是通过筛选或构建两种或更多活的同一科、属或种的突变病毒来制备安全疫苗，其中每一种病毒中都包含能够赋予所需表型(例如减毒作用、细胞趋向性或生物型的改造、物种趋向性的改造、或者外源基因盒的表达)的突变，而且病毒中的突变都存在于同一基因组位点上从而使突变病毒无法进行彼此间进行的同源重组来抵消突变。

术语“疫苗”或“疫苗组合物”是指包含活的突变病毒的组合物，在接种动物后诱导对致病型病毒的完全或部分免疫，或者缓和由致病型病毒引起的疾病症状。疫苗组合物抵御病毒的保护效果一般是由在受试者中诱导免疫应答来达到的，可以是细胞介导或是体液免疫应答，也可以是两者的组合。一般而言，消除或减少病毒感染的发生、症状的改善、或者从感染者体内加速消灭病毒都是疫苗组合物保护效果的体现。

“动物”是指包括鸟类，例如鸡、火鸡、驯养的水禽，和任何一种哺乳动物，例如牛、羊、猪、山羊、狗、猫以及马。

在此所用的术语“病毒”、“病毒隔离群”或者“病毒株”是指包含病毒基因组DNA或RNA、相关蛋白以及其他化学成分(例如脂类)的病毒粒子或病毒体。

“编码病毒的核酸分子”或“病毒的核酸分子”是指病毒的基因组核酸分子，可以是RNA的形式，也可以是DNA的形式。

“突变”是指包括一个或多个核苷酸的缺失、插入或取代，或者它们的组合。根据本发明，突变优选赋予所需表型，例如减毒作用、细胞趋向性或生物型的改造、物种趋向性的改造、或者外源基因盒的表达。更优选的突变是能够赋予削弱的毒性的突变。

“减毒作用”是指在感染病毒的动物体内病毒失去了它的一些或所有的增殖和/或引起疾病的能力。例如，当减毒病毒存在于减毒病毒的野生致病型能够复制的动物体内时，它是完全不能复制或限于一轮或少数轮复制或者在细胞或组织趋向性上受到限制的病毒。

减毒病毒可能在涉及病毒致病性的一个或多个基因中具有突变。此类突变在此也称为“减毒突变”。一种减毒病毒的制备可以是通过对野生型致病病毒在体外进行紫外照射、化学处理或者野生型致病病毒的高连续传代(high serial passage)来产生。二者择一地，一种减毒病毒还可以通过对野生型致病病毒已知能够带来毒性的病毒序列进行特异性缺失、在病毒基因组中进行序列的插入或者在病毒基因组中进行一个或多个点突变来制备。减毒病毒可以是从动物体内获得的病毒隔离群，该隔离群是由野生致病型病毒经过除了人工手段之外的事件而派生出来的，举例来说，发生宿主动物体内如重组这一类的事件。

本发明免疫组合物中存在的两种或更多的活的突变病毒包含着处于同一基因组位点上的突变。“同一基因组位点”是指当病毒的基因组核苷酸序列被对齐时，病毒基因组中的突变彼此重叠从而没有机会在病毒基因组之间和之中进行同源重组来抵消突变。换句话说，当病毒的基因组核苷酸序列被对齐时，被对齐的序列中至少有一处邻接部分中对齐的病毒基因组中的序列是突变序列。本领域技术人员可以利用不少计算机程序来对比和对齐核酸序列。对齐这些序列以达到最佳的对比目的(举例来说，可以在核酸序列中引入缺口以带来和另一个核酸序列的最佳对齐)。例如，Altschul等，1990，J.Mol.Biol.215：403410中描述的NBLAST和XBLAST程序，Altschul等，1997，Nucleic Acids Res.25：33893402中描述的Gapped BLAST程序，以及Altschul等，1997，同上中描述的PSI-Blast程序。当使用BLAST、Gapped BLAST以及PSI-Blast程序时，可以利用相应程序(举例来说，XBLAST和NBLAST)的缺省参数(见http://www.ncbi.nlm.nih.gov)。

一般而言，本发明的概念，即包括在同一疫苗组合物中的两种或更多的具有处于同一基因组位点突变的活的突变病毒，可应用于任一科的突变病毒，其中病毒基因组具有充分的序列同一性以容许同源重组。已经表明，短至15个核苷酸的核苷酸同一性就能够导致有效的同源重组(Nagy andBujarski，J.Virol.69：131-140，1995)。

本发明尤其应用于黄病毒科(Flaviviridae)的病毒。黄病毒科(Flaviviridae)由疫病毒属(Pestivirus)、黄病毒属(Flavivirus)和丙型肝炎病毒属(Hepacivirus)属组成。黄病毒科(Flaviviridae)中成员的病毒粒子包封着约9.5到12.3kb的正链RNA基因组。该基因组RNA包含邻接的长的开放读码框，其可以被翻译为多蛋白质再经过细胞和病毒蛋白酶的加工而产生成熟的病毒蛋白。

优选的，本发明疫苗组合物中的突变病毒是来自于同一属中相同种或不同种的。

在一个优选的具体实施方式中，本发明疫苗组合物包含两种或更多的来自于疫病毒属(Pestivirus)的活的突变病毒。疫病毒属(Pestivirus)由牛病毒性腹泻病毒类型-1(BVDV-1)、牛病毒性腹泻病毒类型-2(BVDV-2)、经典猪瘟病毒(classical swine fever virus)以及羊先天性髓质形成不全症病毒(Border disease virus)种所代表。开放读码框(ORF)编码大约3900个氨基酸的多蛋白质，其可以被协同翻译和后翻译加工成为下列成熟的病毒蛋白质(从5’端到3’端)：N^pro、C、E^rns、E1、E2、NS2-3、NS4A、NS4B、NS5A以及NS5B。

通常情况下，BVDV具有RNA形式的基因组。RNA可以逆转录成DNA以用于克隆。因此，这里所给出的关于核酸和牛病毒性腹泻病毒序列的参考都围绕着病毒RNA序列以及由病毒RNA序列衍生出来的DNA序列。为便利起见，在下文序列表中描述的BVDV的基因组序列只涉及DNA序列。其各自相应的RNA序列对本领域技术人员是显而易见的。

在一个更优选的具体实施方式中，本发明的疫苗组合物包含致细胞病变的BVDV-1和致细胞病变的BVDV-2，其中与致细胞病变生物型相关的两病毒的突变均存在于同一基因组位点上从而使两突变病毒无法重组来抵消突变。

BVDV-1和BVDV-2体现了牛病毒性腹泻病毒(BVDV)的两种非常相关的基因型。在整个基因组中，两种病毒的核苷酸序列共有约70％的同一性，而且在NS2-3区域内有更高一些的同一性比率。可以认为在BVDV-1和BVDV-2病毒基因组之间，至少在NS2-3区域内，同一性比率足以容许发生同源重组。

BVDV-1在动物体内通常只引起轻度的腹泻，然而BVDV-2则是具有高毒性的病毒其能够引起血小板减少症、大出血以及急性的致命疾病(Corapiet al.，J.Virol.63：3934-3943；Bolin et al.，Am.J.Vet.Res.53：2157-2163；Pellerin et al.，Virology 203：260-268，1994；Ridpath et al.，Virology 205：66-74，1994；Carman et al.，J.Vet.Diagn.Invest.10：27-35，1998)。通过一系列单克隆抗体(MAbs)以及利用动物体内得到的病毒特异性抗血清的交叉中和决定了病毒的两种类型具有截然不同的抗原性。(Corapi et al.，Am.J.Vet.Res.51：1388-1394，1990)。任一基因型的病毒都可以两生物型中的一种存在：致细胞病变的(cytopathogenic)(细胞病变的(cytopathic)或cp)或非致细胞病变的(noncytopathic)(非细胞病变(noncytopathic)的或ncp)。致细胞病变病毒对培养细胞产生细胞病变效应(如细胞溶菌[胞溶]作用)，而非致细胞病变病毒不会。

较为理想的是制备提供针对BVDV-1和BVDV-2两者的保护的疫苗。然而，由于两病毒之间高度的序列同一性，存在使包含在同一疫苗组合物中的活的致细胞病变BVDV-1和活的致细胞病变BVDV-2能够在接种疫苗的动物体内互相重组而产生非致细胞病变病毒的可能性。BVDV-1与BVDV-2之间的重组是有文献来证明的。见，例如，Ridpath et al.，Virology212：259-262(1995)。在免疫能力产生以前，用ncP病毒感染怀孕牛体内胎畜能够导致胎畜在怀孕期间保留病毒血症而且在后来小牛的出生也会遗留下持续的病毒血症。这种小牛会由于致细胞病变BVDV的双重感染引起的粘膜病而死亡。因此，本发明提供的疫苗组合物中包含具有处于同一基因组位点的突变的活的致细胞病变BVDV-1和活的致细胞病变BVDV-2，能够特别理想地保护动物抵御BVDV-1和BVDV-2两种病毒。

在一个具体实施方式中，可以将从动物体内获得的牛病毒性腹泻病毒(BVDV)致细胞病变隔离群应用于本发明的疫苗组合物。对本领域技术人员来说，BVDV-1和BVDV-2的致细胞病变隔离群都已经被披露而且能够获得，举例来说，BVDV-1NADL(ATCC#VR1422或VR-534)、BVDV-253637株(保藏于ATCC编号PTA-4859)、以及二型野外隔离群，此类菌种描述于Ridpath and Neill，J.Virol 74：8771-8774，(2000)。迄今为止所报道的致细胞病变隔离群典型地包含异源序列的插入物，例如，一种编码泛激素的序列(Genbank入藏号M96687或De Moerlooze et al.，J.Gen.Virol.74：1433-1438，(1993))、编码牛NEDD8的序列(Baroth et al.，supra)、或者编码Bos taurus DnaJ1的序列(在下述的实施例中有所描述)及其他。

在另一具体实施方式中，致细胞病变牛病毒性腹泻病毒(cp BVDV)是通过在牛病毒性腹泻病毒基因组中进行确定的改造来产生的，例如，通过缺失特异的病毒序列、在特异病毒基因组位点中插入序列、或者进行一个或多个取代、或者上述的组合。

通过在一个特异的基因组位点中嵌入异源(即对病毒而言外来的)序列来产生致细胞病变牛病毒性腹泻病毒(cp BVDV)时，插入序列的性质对于本发明来说通常并不关键。另外，只要插入导致了一种减毒的表现型，插入就并不限于任何特定的位点。由于致细胞病变隔离群中的异源序列经常发现于NS2-3区域中，所以NS2-3区域，尤其是围绕推定的NS2-3分裂位点部分(其相应于BVDV-1NADL株#1679到#1680位的氨基酸残基)(编号方式基于公开的基因组序列Genbank登记号No.M31182，SEQ ID NO：4)，是优选的插入位置。

致细胞病变的BVDV-1可以通过进行确定的基因改造来产生，所述基因改造模拟从动物体内获得的致细胞病变BVDV-2隔离群中识别出的突变，于是这些病毒在同一基因组位点中就具有与致细胞病变生物型相关的突变。类似地，致细胞病变的BVDV-2也可以通过进行确定的基因改造的方法来产生所述改造模拟从动物体内获得的致细胞病变BVDV-1隔离群中识别出的突变。

在优选的具体实施方式中，本发明的疫苗组合物包含NADL(致细胞病变BVDV-1隔离群)和BVDV-2 53637(致细胞病变BVDV-2隔离群)，其中两致细胞病变隔离群均在同一基因组位点上含有引起致细胞病变生物型的突变。在SEQ ID NO：4中阐明了BVDV-1NADL株的基因组序列，而BVDV-2 53637株保藏于ATCC，编号为PTA-4859。两种隔离群在NS2-3区域都含有插入。减毒的致细胞病变BVDV-1在核苷酸位置#4993(NADL序列编号方式)的胸腺嘧啶的3’端含有Bos Taurus DnaJ1编码序列的插入，所述胸腺嘧啶是编码在氨基酸1536位置的甘氨酸残基的密码子第三个核苷酸。减毒的致细胞病变BVDV-2在同一基因组位点含有一个Bos taurusDnaJ1编码序列的插入。

根据本发明，在所述疫苗组合物中使用的致细胞病变牛病毒性腹泻病毒(cp BVDV)隔离群已经得到减毒而且由此成为非致细胞病变的。对本领域技术人员来说减毒的方法是已知的而且下文中也会叙述。

在另一具体实施方式中，本发明的疫苗组合物包含减毒的BVDV-1和减毒的BVDV-2，其中两病毒中的减毒突变都存在于同一基因组位点上从而使两突变病毒无法重组而抵消减毒突变。

减毒的BVDV可以通过在体外进行紫外照射、化学处理或者致病型病毒的高连续传代(high serial passage)来制备。可以用序列分析来确定这些方法产生的突变的性质和基因组位置。突变可以是缺失、插入或者取代一个或多个核苷酸的形式，或是上述的组合。二者择一地，减毒的BVDV还可以通过在BVDV基因组中进行确定的改造来产生，例如通过缺失特异性病毒序列、在特异性病毒基因组位点中插入序列，或制备一个或多个取代或者其组合。

如上所述，用于本发明疫苗组合物的活的突变病毒可以来自同一科、属或种，其中病毒基因组具有充分的序列同一性以容许同源重组。适用于本发明疫苗组合物的病毒组合的其他例子还包括但不限于，不同型脊髓灰质炎病毒的组合、传染性支气管炎病毒多重活突变株的组合、新城鸡瘟病毒多重活突变株的组合、犬腺病毒-1和犬腺病毒-2的组合、马疱疹病毒-1和马疱疹病毒-4的组合、流感病毒多重活突变株的组合、猫杯状病毒多重活减毒株的组合、轮状病毒多重血清型的组合、鼻病毒多重血清型的组合、口蹄疫病毒多重血清型的组合、猪生殖和呼吸综合症病毒欧洲和北美基因型的组合、传染性滑囊症病毒标准和变异株的组合。

按照本发明，病毒粒子虽然是用于疫苗的优选形式，但编码同一科、属或种突变病毒的核酸分子也同样可以直接用于疫苗。DNA或RNA分子可以以“裸”的形式存在或者也可以结合能够帮助细胞吸收的试剂(例如，脂质体或阳离子脂类)。现有技术中已经详细描述了应用核酸(DNA或mRNA)的疫苗和接种工艺，例如，美国专利No.5,703,055、美国专利No.5,580,859、美国专利No.5,589,466、国际专利公开WO 98/35562、以及Ramsayet al.，1997，Immunol.Cell Biol.75：360-363、Davis，1997，Cur.Opinion Biotech.8：635-640、Manickan et al.，1997，Critical Rev.Immunol.17：139-154、Robinson，1997，Vaccine 15(8)：785-787、Robinson et al.，1996，AIDS Res.Hum.Retr.12(5)：455-457、Lai and Bennett，1998，Critical Rev.Immunol.18：449-484、以及Vogel and Sarver，1995，Clin.Microbiol.Rev.8(3)：406-410，所有这些在此合并为参考文献。

除了两种或更多来自同一科、属或种的活突变病毒以外，疫苗组合物还可以包括其他的抗原成分。适用于本发明的其他抗原成分还包括但不限于，产生于下列病原菌的抗原，例如，肺炎支原体(Mycoplasmahyopneumonia)、睡眠嗜血杆菌(Haemophilus somnus)、副猪嗜血杆菌(Haemophilus parasuis)、支气管败血波氏杆菌(Bordetella bronchiseptica)、炭疽芽胞杆菌(Bacillus anthracis)、猪胸膜肺炎放线杆菌(Actinobacilluspleuropneumonie)、多杀性巴氏杆菌(Pasteurella multocida)、Mannhemiahaemolytica、牛支原体(Mycoplasma bovis)、Mycoplasma galanacieum、鸡毒支原体(Mycoplasma gallisepticum)、牛分枝杆菌(Mycobacterium bovis)、副结核分支杆菌(Mycobacterium paratuberculosis)、梭状芽孢杆菌属的种(Clostridial spp.)、乳房链球菌(Streptococcus uberis)、猪链球菌(Streptococcus suis)、金黄色葡萄球菌、Erysipelothrix rhusopathiae、弯曲杆菌属的种(Campylobacter spp.)、坏死梭杆菌(Fusobacterium necrophorum)、埃希氏大肠杆菌、细胞内劳森菌(Lawsonia intracellularis)、单核细胞增生性李斯特菌(Listeria monocytogenes)、立氏立克次体(Rickettsia rickettsii)、疏螺旋体属的种(Borrelia spp.)、艾利希氏体属的种(Ehrlichia spp.)、衣原体属的种(Chlamydia spp.)、布氏杆菌属的种(Brucella spp.)、弧菌属的种(Vibriospp.)、Salmonella enterica serovars、钩端螺旋体属的种(Leptospira spp.)；致病的真菌类如念珠菌属，原生动物如小隐孢子虫(Cryptosporidium parvum)、Neospora canium、刚地弓形虫(Toxoplasma gondii)、爱美尔球虫(Eimeriaspp)、梨浆虫属(Babesia spp)、贾第虫属(Giardia spp)；寄生虫如奥斯特线虫属(Ostertagia)、古柏线虫属(Cooperia)、血矛线虫(Haemonchus)、吸虫(Fasciola)；它们以一种非活性的整个或部分细胞制剂的形式、或者以一种由遗传工程技术或化学合成法获得的抗原分子的形式存在。其他的抗原还包括了致病病毒例如马立克氏病病毒(Marek’s disease virus)、传染性法氏囊病病毒(Infectious bursal disease virus)、新城疫病毒(Newcastle’sdisease virus)、鸡贫血病病毒(chicken anemia virus)、禽痘病毒(fowlpox virus)、禽白血病病毒(avian leukosis virus)、传染性喉气管炎病毒(infectiouslaryngotracheitis virus)、网状内皮病毒(reticuloendothelial virus)、犬小病毒(canine parvovirus)、犬瘟病毒(canine distemper virus)、犬疱疹病毒(canineherpesvirus)、犬冠状病毒(canine coronavirus)、犬副流感病毒-5(canineparainfluenza-5)、猫泛白细胞减少症病毒(feline panleukopenia virus)、猫疱疹病毒(feline herpes virus)、猫卡性病毒(feline calicivirus)、猫免疫缺乏病毒、猫传染性腹膜炎病毒(feline infectious peritonitis virus)、马疱疹病毒、马动脉炎病毒、马传染性贫血病毒、东部马脑炎病毒、西部马脑炎病毒、委内瑞拉马脑炎病毒、西尼罗河病毒、传染性胃肠炎病毒、牛冠状病毒、牛疱疹病毒-1，3，6、牛副流行性感冒病毒、牛呼吸道合胞体病毒、牛白血病病毒、牛疫病毒(rinderpest virus)、口蹄疫病毒、狂犬病病毒、非洲猪瘟病毒(African swine fever virus)、猪细小病毒(Porcine parvovirus)、猪呼吸道冠状病毒(PRRS virus)、猪圆环病毒(Porcine circovirus)、流感病毒、猪水疱病病毒、Techen出血热病毒(Techen frver virus)、伪狂犬病病毒(Pseudorabiesvirus)，它们以一种改良的活的(减毒的)病毒制剂形式、一种非活性的全部或部分病毒制剂形式，或者以一种由遗传工程技术或化学合成法获得的抗原分子的形式存在。当使用其他减毒活病毒时，这些其他病毒优选来自与两种主要的减毒病毒不同的科，如上所述。

在一个优选的实施例中，本发明提供了疫苗组合物，其含有源自BVDV-1NADL株的减毒致细胞病变BVDV-1，和源自BVDV-253637株的减毒致细胞病变BVDV-2，其中两致细胞病变分离株在同一基因组位点均含有与致细胞病变生物型相关的突变，而且还含有至少一个(即：一个或多个)下列处于非活性或改良活性形式的抗原成分：牛疱疹病毒-1、牛呼吸道合胞病毒、副流感病毒-3、胚胎弯曲柑桔、犬钩端螺旋体(Leptospiracanicola)、流感伤寒钩端螺旋体(Leptospira grippotyphosa)、哈德焦钩端螺旋体(Leptospira hardjo)、出血性黄疸钩端螺旋体(Leptospiraicterohaemorrhagiae)、波摩那钩端螺旋体(Leptospira pomona)、或者Mannhemia haemolytica。

此外，本发明的疫苗组合物还可以包括一个或多个兽医学可接受的载体。此处所用的“一种兽医学可接受的载体”包括任何和所有溶剂、分散介质、包覆层、佐剂、稳定剂、稀释剂、防腐剂、抗细菌以及抗真菌药、等渗剂、吸附延迟剂、以及类似物质。稀释剂可以包括水、生理盐水、葡萄糖、乙醇、甘油、以及类似物质。等渗剂可以包括氯化钠、葡萄糖、甘露醇、山梨糖醇、以及乳糖，及其他。稳定剂包括白蛋白，及其他。该疫苗组合物还可以进一步的包括一种或多种其他免疫调节剂例如：白细胞介素、干扰素、或其他细胞活素。

适用于该疫苗组合物的佐剂包括但不限于：RIBI佐剂系统(Ribi Inc.)、明矾、氢氧化铝凝胶、水包油乳剂、油包水乳剂例如完全以及不完全弗氏佐剂、Block共聚物(CytRx，Atlanta GA)，SAF-M(Chiron，Emeryville CA)，AMPHIGEN^佐剂、皂甙、Quil A、胆固醇、QS-21(Cambridge Biotech Inc.，Cambridge MA)、或其他皂甙片段、单磷酰类脂A、Avridine脂类胺(lipid-amine)佐剂、来自于大肠杆菌的热不稳定性肠毒素(重组或别的方式)、霍乱毒素、或胞壁酰二肽，以及许多其他佐剂。

通常情况下，存在于疫苗中的活突变病毒的数量在每剂量大约1×10⁶到大约1×10⁸病毒粒子，配以一种兽医学可接受的载体，体积大约在0.5到5毫升之间。在疫苗组合物中能够有效提供保护效应的病毒的精确的量可以由熟练的兽医来决定。当病毒的DNA或RNA分子用于该疫苗时，核酸的量一般应该在大约0.1μg/ml到大约5.0mg/ml之间。

本发明的疫苗组合物可以根据给药途径制成多种形式。例如，该疫苗组合物可以制成适用于注射用途的无菌水溶液或分散系的形式，或者利用制冷干燥技术制成冻干形式。冻干组合物一般保存在4℃左右，而且可以在配以或不配以佐剂的情况下在稳定溶液例如生理盐水或和HEPES中还原。

本发明的疫苗组合物可以给动物施用以治疗或预防由疫苗组合物中病毒的致病类型所引起的疾病。因此，接种动物以抵抗由病毒所引起疾病的方法也提供于本发明中。

在实施该方法的过程中，本发明疫苗组合物给动物施用优选是通过肠道外途径给药，虽然其他的给药途径也可以同样适用，例如通过口服、鼻腔内、肌肉内、淋巴结内、真皮下、腹膜内、皮下、直肠或阴道给药、或通过联合的途径。为了提供最适宜的疫苗接种，可能需要追加剂量方案，而且还可以调整剂量方案。

本发明通过如下的实施例进行进一步的阐述，但并不意味着限制于此。

实施例：

实施例1

定位BVDV2 53637株中的细胞嵌入位置

确定来自于BVDV2-53637的NS2-3区域的序列的一部分，用于识别和定位在此区域中的任何细胞插入的位置。使用前向引物53637U1(5’-CGTCCACAGATGGTTTGGT-3’；SEQ ID NO：1)和逆向引物53637L(5’-GGCTATGTATTGGACGTAACCC-3’；SEQ ID NO：2)从病毒RNA扩增670碱基的RT-PCR产物。纯化该RT-PCR产物和进行序列分析(SEQ ID NO：3)。当与BVDV1-NADL(Genbank入藏号M31182，SEQ ID NO：4)进行比对时，观察到了显著的相似性(图1)。两种病毒都含有由Bos taurus DnaJ1基因派生出来的框内插入。就NADL来说，插入物的长度为90个氨基酸(270个核苷酸)，定位在NADL多蛋白质中1536位甘氨酸和1627位脯氨酸之间。这些坐标相应于非致细胞病变BVDV1株例如SD-1(Genbank入藏号AAA42860，SEQ ID NO：6)中的1536位甘氨酸和1537位脯氨酸，说明NADL中的基因组改造是简单的嵌入，并不伴有侧翼病毒序列的缺失或复制。与BVDV1-NADL类似，BVDV2-53637中有Bos taurus DnaJ1基因的一部分的插入。细胞插入物会更长(131个氨基酸，393个核苷酸)，相对于BVDV1-NADL中的插入在两个方向上均有延长。在两病毒中NS2-3区域内细胞插入的位置是相同的。不同于BVDV1-NADL，BVDV2-53637的插入伴有侧翼病毒序列的5个氨基酸(15核苷酸)的缺失。在插入物的5’末端侧翼有三个氨基酸残基的缺失，同时在插入物的3’末端侧翼有两个氨基酸残基的缺失。由于该细胞插入在两疫苗病毒中的同一基因组位点上，所以它们无法经过同源重组消除插入来产生非致细胞病变的嵌合病毒。

实施例2

在共传代的BVDV1-NADL/BVDV2-53637培养基中试图

检测非细胞病变BVDV病毒

为了确定两疫苗病毒是否能够通过重组来产生可观测水平的非致细胞病变BVDV，在易感细胞上共同培养病毒，而且使用敏感的半嵌套RT-PCR试验从过量的仍含有细胞插入的较长期致细胞病变产物中来检测潜在的非致细胞病变病毒。为了增加体外类型间重组的可能性，每个病毒都在6孔板中以感染复数2-4同时接种于满板BK-6细胞(每次实验12次重复)。2-3天共同培养以后对细胞进行两次冷冻和解冻，然后通过低速离心清除细胞碎片。将得到的上清液作为下一次传代的接种物。在几次研究中总共进行了七次连续的传代。在传代过程中，BVDV1-NADL远比BVDV2-53637生长得快，但是七次传代以后利用嵌套RT-PCR仍然可以检测出II型病毒。使用敏感的半嵌套RT-PCR试验来检测出任何非致细胞病变病毒的存在。

在第一轮RT-PCR中，将前向引物53637U1(SEQ ID NO：1)或者NADL4744(5’-CGTGGCTTCTTGGTACGGG-3’，SEQ ID NO：7)协同逆向引物53637L(SEQ ID NO：2)或者NADL5305(5’-AGCGGTATATTGTACAAAG CCA-3’，SEQ IDNO：8)一起使用。前向和逆向引物的所有四个组合都是为了检测BVDV1、BVDV2、以及类型间重组的。所需的RT-PCR产物大小对致细胞病变BVDV1-NADL来说是562bp，而对致细胞病变BVDV2-53637来说是670bp。如果存在可观测水平的非致细胞病变病毒，预期应生成第一轮产物292bp(BVDV1-NADL)或者277bp(BVDV2-53637)。类型间重组体的大小应当与其中一个亲本相似，或者介于中间的长度，这取决于重组位点的位置。第一轮RT-PCR后没有检测出非致细胞病变BVDV。

在有过量的致细胞病变BVDV存在情况下为了增加检测非致细胞病变BVDV的敏感度，在嵌套PCR之前还包括了限制性内切酶消化的步骤用以破坏派生于致细胞病变病毒的较大的NS2-3模板。在观察到MspI和DraI切割Bos taurus DnaJ1插入物而并不切割侧翼病毒序列的基础上，选择了MspI和DraI的组合。在第二轮(半嵌套)PCR中，将前向引物53637U2(5’-TGCACGATCTGTGAAGGGAAAGAA-3’，SEQ ID NO：9)或者NADL4844(5’-TGCACTGTATGTGAGGGCCGAGAG-3’，SEQ ID NO：10)协同相同的两个逆向引物53637L或者NADL5305一起使用。使用适当的引物组合来试图检测类型间重组体和BVDV1和BVDV2。所需的RT-PCR产物大小对致细胞病变BVDV1-NADL来说是462bp，而对致细胞病变BVDV2-53637来说是570bp(由于致细胞病变BVDV RT-PCR产物的不完全消化以低水平存在)。如果存在可观测水平的非致细胞病变病毒，预期应产生192bp(BVDV1-NADL)或者177bp(BVDV2-53637)的第二轮产物。类型间重组体的大小应当与其中一个亲本相似，或者介于中间的长度，这取决于重组位点的位置。第二轮RT-PCR后没有检测出非致细胞病变BVDV。在一些个别的反应中，看到了各种大小的异常条带。所有介于100到300bp之间的条带都被认为是潜在的非致细胞病变产物并提交了DNA序列分析。每种情况下异常条带都是PCR过程中错误引发的结果。在所有研究中并没有非致细胞病变病毒存在的证据。

序列表

<110>辉瑞产品公司(Pfizer Products Inc)

Welch，Siao-Kun Wan

Calvert，Jay Gregory

O’Hara，Michael K.

Cao，Xuemei K.

<120>安全的突变病毒疫苗

<130>PC25951A

<140>60/490,834

<141>2003-07-29

<160>10

<170>PatentIn version 3.2

<210>1

<211>19

<212>DNA

<213>人工序列

<220>

<223>合成寡核苷酸前向引物53637U1

<400>1

cgtccacaga tggtttggt 19

<210>2

<211>22

<212>DNA

<213>人工序列

<220>

<223>合成逆向引物53637L

<400>2

ggctatgtat tggacgtaac cc 22

<210>3

<211>670

<212>DNA

<213>牛病毒性腹泻病毒253637株

<400>3

cgtccacaga tggtttggtg aggaggaaat atatggggca cccaaggtga tcaccatcat 60

aaaagctagt accctaagta aaaacaggca ctgcataatc tgcacgatct gtgaagggaa 120

agaatggaac ggagccaact gcccaaagtg tggaagacaa ggaaagccca taacatgtgg 180

aatgacactc gcagactttg aggagaaaca ttacaaaaag atatttataa gagaaggacg 240

ccaagaagca atgaatacga tgatgtgcag ccgatgccag ggaaagcata ggaggtttga 300

aacggaccgg gaacctaaga gtgccagata ctgtgctgag tgtaataggc tgcatcctgc 360

tgaggaaggt gacttttggg cagagtcaag catgttgggc ctcaaaatca cctactttgc 420

gctgatggat ggaaaggtgt atgatatcac agagtgggct ggatgccagc gtgtgggaat 480

ctccccagat acccacagag tcccttgtca catctcattt ggttcacgga tgccaggcac 540

cagtgggcgg cagagagcta ctccagatgc ccctcctgct gaccttcagg atttcttgag 600

ccggatcttt caagtacccc caggccagat gtccagggaa gagtataagg gttacgtcca 660

atacatagcc 670

<210>4

<211>12573

<212>DNA

<213>牛病毒性腹泻病毒NADL 1株

<400>4

gtatacgaga attagaaaag gcactcgtat acgtattggg caattaaaaa taataattag 60

gcctagggaa caaatccctc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120

gactagcata atgagggggg tagcaacagt ggtgagttcg ttggatggct taagccctga 180

gtacagggta gtcgtcagtg gttcgacgcc ttggaataaa ggtctcgaga tgccacgtgg 240

acgagggcat gcccaaagca catcttaacc tgagcggggg tcgcccaggt aaaagcagtt 300

ttaaccgact gttacgaata cagcctgata gggtgctgca gaggcccact gtattgctac 360

taaaaatctc tgctgtacat ggcacatgga gttgatcaca aatgaacttt tatacaaaac 420

atacaaacaa aaacccgtcg gggtggagga acctgtttat gatcaggcag gtgatccctt 480

atttggtgaa aggggagcag tccaccctca atcgacgcta aagctcccac acaagagagg 540

ggaacgcgat gttccaacca acttggcatc cttaccaaaa agaggtgact gcaggtcggg 600

taatagcaga ggacctgtga gcgggatcta cctgaagcca gggccactat tttaccagga 660

ctataaaggt cccgtctatc acagggcccc gctggagctc tttgaggagg gatccatgtg 720

tgaaacgact aaacggatag ggagagtaac tggaagtgac ggaaagctgt accacattta 780

tgtgtgtata gatggatgta taataataaa aagtgccacg agaagttacc aaagggtgtt 840

caggtgggtc cataataggc ttgactgccc tctatgggtc acaacttgct cagacacgaa 900

agaagaggga gcaacaaaaa agaaaacaca gaaacccgac agactagaaa gggggaaaat 960

gaaaatagtg cccaaagaat ctgaaaaaga cagcaaaact aaacctccgg atgctacaat 1020

agtggtggaa ggagtcaaat accaggtgag gaagaaggga aaaaccaaga gtaaaaacac 1080

tcaggacggc ttgtaccata acaaaaacaa acctcaggaa tcacgcaaga aactggaaaa 1140

agcattgttg gcgtgggcaa taatagctat agttttgttt caagttacaa tgggagaaaa 1200

cataacacag tggaacctac aagataatgg gacggaaggg atacaacggg caatgttcca 1260

aaggggtgtg aatagaagtt tacatggaat ctggccagag aaaatctgta ctggcgtccc 1320

ttcccatcta gccaccgata tagaactaaa aacaattcat ggtatgatgg atgcaagtga 1380

gaagaccaac tacacgtgtt gcagacttca acgccatgag tggaacaagc atggttggtg 1440

caactggtac aatattgaac cctggattct agtcatgaat agaacccaag ccaatctcac 1500

tgagggacaa ccaccaaggg agtgcgcagt cacttgtagg tatgataggg ctagtgactt 1560

aaacgtggta acacaagcta gagatagccc cacaccctta acaggttgca agaaaggaaa 1620

gaacttctcc tttgcaggca tattgatgcg gggcccctgc aactttgaaa tagctgcaag 1680

tgatgtatta ttcaaagaac atgaacgcat tagtatgttc caggatacca ctctttacct 1740

tgttgacggg ttgaccaact ccttagaagg tgccagacaa ggaaccgcta aactgacaac 1800

ctggttaggc aagcagctcg ggatactagg aaaaaagttg gaaaacaaga gtaagacgtg 1860

gtttggagca tacgctgctt ccccttactg tgatgtcgat cgcaaaattg gctacatatg 1920

gtatacaaaa aattgcaccc ctgcctgctt acccaagaac acaaaaattg tcggccctgg 1980

gaaatttggc accaatgcag aggacggcaa gatattacat gagatggggg gtcacttgtc 2040

ggaggtacta ctactttctt tagtggtgct gtccgacttc gcaccggaaa cagctagtgt 2100

aatgtaccta atcctacatt tttccatccc acaaagtcac gttgatgtaa tggattgtga 2160

taagacccag ttgaacctca cagtggagct gacaacagct gaagtaatac cagggtcggt 2220

ctggaatcta ggcaaatatg tatgtataag accaaattgg tggccttatg agacaactgt 2280

agtgttggca tttgaagagg tgagccaggt ggtgaagtta gtgttgaggg cactcagaga 2340

tttaacacgc atttggaacg ctgcaacaac tactgctttt ttagtatgcc ttgttaagat 2400

agtcaggggc cagatggtac agggcattct gtggctacta ttgataacag gggtacaagg 2460

gcacttggat tgcaaacctg aattctcgta tgccatagca aaggacgaaa gaattggtca 2520

actgggggct gaaggcctta ccaccacttg gaaggaatac tcacctggaa tgaagctgga 2580

agacacaatg gtcattgctt ggtgcgaaga tgggaagtta atgtacctcc aaagatgcac 2640

gagagaaacc agatatctcg caatcttgca tacaagagcc ttgccgacca gtgtggtatt 2700

caaaaaactc tttgatgggc gaaagcaaga ggatgtagtc gaaatgaacg acaactttga 2760

atttggactc tgcccatgtg atgccaaacc catagtaaga gggaagttca atacaacgct 2820

gctgaacgga ccggccttcc agatggtatg ccccatagga tggacaggga ctgtaagctg 2880

tacgtcattc aatatggaca ccttagccac aactgtggta cggacatata gaaggtctaa 2940

accattccct cataggcaag gctgtatcac ccaaaagaat ctgggggagg atctccataa 3000

ctgcatcctt ggaggaaatt ggacttgtgt gcctggagac caactactat acaaaggggg 3060

ctctattgaa tcttgcaagt ggtgtggcta tcaatttaaa gagagtgagg gactaccaca 3120

ctaccccatt ggcaagtgta aattggagaa cgagactggt tacaggctag tagacagtac 3180

ctcttgcaat agagaaggtg tggccatagt accacaaggg acattaaagt gcaagatagg 3240

aaaaacaact gtacaggtca tagctatgga taccaaactc ggacctatgc cttgcagacc 3300

atatgaaatc atatcaagtg aggggcctgt agaaaagaca gcgtgtactt tcaactacac 3360

taagacatta aaaaataagt attttgagcc cagagacagc tactttcagc aatacatgct 3420

aaaaggagag tatcaatact ggtttgacct ggaggtgact gaccatcacc gggattactt 3480

cgctgagtcc atattagtgg tggtagtagc cctcttgggt ggcagatatg tactttggtt 3540

actggttaca tacatggtct tatcagaaca gaaggcctta gggattcagt atggatcagg 3600

ggaagtggtg atgatgggca acttgctaac ccataacaat attgaagtgg tgacatactt 3660

cttgctgctg tacctactgc tgagggagga gagcgtaaag aagtgggtct tactcttata 3720

ccacatctta gtggtacacc caatcaaatc tgtaattgtg atcctactga tgattgggga 3780

tgtggtaaag gccgattcag ggggccaaga gtacttgggg aaaatagacc tctgttttac 3840

aacagtagta ctaatcgtca taggtttaat catagctagg cgtgacccaa ctatagtgcc 3900

actggtaaca ataatggcag cactgagggt cactgaactg acccaccagc ctggagttga 3960

catcgctgtg gcggtcatga ctataaccct actgatggtt agctatgtga cagattattt 4020

tagatataaa aaatggttac agtgcattct cagcctggta tctgcggtgt tcttgataag 4080

aagcctaata tacctaggta gaatcgagat gccagaggta actatcccaa actggagacc 4140

actaacttta atactattat atttgatctc aacaacaatt gtaacgaggt ggaaggttga 4200

cgtggctggc ctattgttgc aatgtgtgcc tatcttattg ctggtcacaa ccttgtgggc 4260

cgacttctta accctaatac tgatcctgcc tacctatgaa ttggttaaat tatactatct 4320

gaaaactgtt aggactgata cagaaagaag ttggctaggg gggatagact atacaagagt 4380

tgactccatc tacgacgttg atgagagtgg agagggcgta tatctttttc catcaaggca 4440

gaaagcacag gggaattttt ctatactctt gccccttatc aaagcaacac tgataagttg 4500

cgtcagcagt aaatggcagc taatatacat gagttactta actttggact ttatgtacta 4560

catgcacagg aaagttatag aagagatctc aggaggtacc aacataatat ccaggttagt 4620

ggcagcactc atagagctga actggtccat ggaagaagag gagagcaaag gcttaaagaa 4680

gttttatcta ttgtctggaa ggttgagaaa cctaataata aaacataagg taaggaatga 4740

gaccgtggct tcttggtacg gggaggagga agtctacggt atgccaaaga tcatgactat 4800

aatcaaggcc agtacactga gtaagagcag gcactgcata atatgcactg tatgtgaggg 4860

ccgagagtgg aaaggtggca cctgcccaaa atgtggacgc catgggaagc cgataacgtg 4920

tgggatgtcg ctagcagatt ttgaagaaag acactataaa agaatcttta taagggaagg 4980

caactttgag ggtatgtgca gccgatgcca gggaaagcat aggaggtttg aaatggaccg 5040

ggaacctaag agtgccagat actgtgctga gtgtaatagg ctgcatcctg ctgaggaagg 5100

tgacttttgg gcagagtcga gcatgttggg cctcaaaatc acctactttg cgctgatgga 5160

tggaaaggtg tatgatatca cagagtgggc tggatgccag cgtgtgggaa tctccccaga 5220

tacccacaga gtcccttgtc acatctcatt tggttcacgg atgcctttca ggcaggaata 5280

caatggcttt gtacaatata ccgctagggg gcaactattt ctgagaaact tgcccgtact 5340

ggcaactaaa gtaaaaatgc tcatggtagg caaccttgga gaagaaattg gtaatctgga 5400

acatcttggg tggatcctaa gggggcctgc cgtgtgtaag aagatcacag agcacgaaaa 5460

atgccacatt aatatactgg ataaactaac cgcatttttc gggatcatgc caagggggac 5520

tacacccaga gccccggtga ggttccctac gagcttacta aaagtgagga ggggtctgga 5580

gactgcctgg gcttacacac accaaggcgg gataagttca gtcgaccatg taaccgccgg 5640

aaaagatcta ctggtctgtg acagcatggg acgaactaga gtggtttgcc aaagcaacaa 5700

caggttgacc gatgagacag agtatggcgt caagactgac tcagggtgcc cagacggtgc 5760

cagatgttat gtgttaaatc cagaggccgt taacatatca ggatccaaag gggcagtcgt 5820

tcacctccaa aagacaggtg gagaattcac gtgtgtcacc gcatcaggca caccggcttt 5880

cttcgaccta aaaaacttga aaggatggtc aggcttgcct atatttgaag cctccagcgg 5940

gagggtggtt ggcagagtca aagtagggaa gaatgaagag tctaaaccta caaaaataat 6000

gagtggaatc cagaccgtct caaaaaacag agcagacctg accgagatgg tcaagaagat 6060

aaccagcatg aacaggggag acttcaagca gattactttg gcaacagggg caggcaaaac 6120

cacagaactc ccaaaagcag ttatagagga gataggaaga cacaagagag tattagttct 6180

tataccatta agggcagcgg cagagtcagt ctaccagtat atgagattga aacacccaag 6240

catctctttt aacctaagga taggggacat gaaagagggg gacatggcaa ccgggataac 6300

ctatgcatca tacgggtact tctgccaaat gcctcaacca aagctcagag ctgctatggt 6360

agaatactca tacatattct tagatgaata ccattgtgcc actcctgaac aactggcaat 6420

tatcgggaag atccacagat tttcagagag tataagggtt gtcgccatga ctgccacgcc 6480

agcagggtcg gtgaccacaa caggtcaaaa gcacccaata gaggaattca tagcccccga 6540

ggtaatgaaa ggggaggatc ttggtagtca gttccttgat atagcagggt taaaaatacc 6600

agtggatgag atgaaaggca atatgttggt ttttgtacca acgagaaaca tggcagtaga 6660

ggtagcaaag aagctaaaag ctaagggcta taactctgga tactattaca gtggagagga 6720

tccagccaat ctgagagttg tgacatcaca atccccctat gtaatcgtgg ctacaaatgc 6780

tattgaatca ggagtgacac taccagattt ggacacggtt atagacacgg ggttgaaatg 6840

tgaaaagagg gtgagggtat catcaaagat acccttcatc gtaacaggcc ttaagaggat 6900

ggccgtgact gtgggtgagc aggcgcagcg taggggcaga gtaggtagag tgaaacccgg 6960

gaggtattat aggagccagg aaacagcaac agggtcaaag gactaccact atgacctctt 7020

gcaggcacaa agatacggga ttgaggatgg aatcaacgtg acgaaatcct ttagggagat 7080

gaattacgat tggagcctat acgaggagga cagcctacta ataacccagc tggaaatact 7140

aaataatcta ctcatctcag aagacttgcc agccgctgtt aagaacataa tggccaggac 7200

tgatcaccca gagccaatcc aacttgcata caacagctat gaagtccagg tcccggtcct 7260

attcccaaaa ataaggaatg gagaagtcac agacacctac gaaaattact cgtttctaaa 7320

tgccagaaag ttaggggagg atgtgcccgt gtatatctac gctactgaag atgaggatct 7380

ggcagttgac ctcttagggc tagactggcc tgatcctggg aaccagcagg tagtggagac 7440

tggtaaagca ctgaagcaag tgaccgggtt gtcctcggct gaaaatgccc tactagtggc 7500

tttatttggg tatgtgggtt accaggctct ctcaaagagg catgtcccaa tgataacaga 7560

catatatacc atcgaggacc agagactaga agacaccacc cacctccagt atgcacccaa 7620

cgccataaaa accgatggga cagagactga actgaaagaa ctggcgtcgg gtgacgtgga 7680

aaaaatcatg ggagccattt cagattatgc agctggggga ctggagtttg ttaaatccca 7740

agcagaaaag ataaaaacag ctcctttgtt taaagaaaac gcagaagccg caaaagggta 7800

tgtccaaaaa ttcattgact cattaattga aaataaagaa gaaataatca gatatggttt 7860

gtggggaaca cacacagcac tatacaaaag catagctgca agactggggc atgaaacagc 7920

gtttgccaca ctagtgttaa agtggctagc ttttggaggg gaatcagtgt cagaccacgt 7980

caagcaggcg gcagttgatt tagtggtcta ttatgtgatg aataagcctt ccttcccagg 8040

tgactccgag acacagcaag aagggaggcg attcgtcgca agcctgttca tctccgcact 8100

ggcaacctac acatacaaaa cttggaatta ccacaatctc tctaaagtgg tggaaccagc 8160

cctggcttac ctcccctatg ctaccagcgc attaaaaatg ttcaccccaa cgcggctgga 8220

gagcgtggtg atactgagca ccacgatata taaaacatac ctctctataa ggaaggggaa 8280

gagtgatgga ttgctgggta cggggataag tgcagccatg gaaatcctgt cacaaaaccc 8340

agtatcggta ggtatatctg tgatgttggg ggtaggggca atcgctgcgc acaacgctat 8400

tgagtccagt gaacagaaaa ggaccctact tatgaaggtg tttgtaaaga acttcttgga 8460

tcaggctgca acagatgagc tggtaaaaga aaacccagaa aaaattataa tggccttatt 8520

tgaagcagtc cagacaattg gtaaccccct gagactaata taccacctgt atggggttta 8580

ctacaaaggt tgggaggcca aggaactatc tgagaggaca gcaggcagaa acttattcac 8640

attgataatg tttgaagcct tcgagttatt agggatggac tcacaaggga aaataaggaa 8700

cctgtccgga aattacattt tggatttgat atacggccta cacaagcaaa tcaacagagg 8760

gctgaagaaa atggtactgg ggtgggcccc tgcacccttt agttgtgact ggacccctag 8820

tgacgagagg atcagattgc caacagacaa ctatttgagg gtagaaacca ggtgcccatg 8880

tggctatgag atgaaagctt tcaaaaatgt aggtggcaaa cttaccaaag tggaggagag 8940

cgggcctttc ctatgtagaa acagacctgg taggggacca gtcaactaca gagtcaccaa 9000

gtattacgat gacaacctca gagagataaa accagtagca aagttggaag gacaggtaga 9060

gcactactac aaaggggtca cagcaaaaat tgactacagt aaaggaaaaa tgctcttggc 9120

cactgacaag tgggaggtgg aacatggtgt cataaccagg ttagctaaga gatatactgg 9180

ggtcgggttc aatggtgcat acttaggtga cgagcccaat caccgtgctc tagtggagag 9240

ggactgtgca actataacca aaaacacagt acagtttcta aaaatgaaga aggggtgtgc 9300

gttcacctat gacctgacca tctccaatct gaccaggctc atcgaactag tacacaggaa 9360

caatcttgaa gagaaggaaa tacccaccgc tacggtcacc acatggctag cttacacctt 9420

cgtgaatgaa gacgtaggga ctataaaacc agtactagga gagagagtaa tccccgaccc 9480

tgtagttgat atcaatttac aaccagaggt gcaagtggac acgtcagagg ttgggatcac 9540

aataattgga agggaaaccc tgatgacaac gggagtgaca cctgtcttgg aaaaagtaga 9600

gcctgacgcc agcgacaacc aaaactcggt gaagatcggg ttggatgagg gtaattaccc 9660

agggcctgga atacagacac atacactaac agaagaaata cacaacaggg atgcgaggcc 9720

cttcatcatg atcctgggct caaggaattc catatcaaat agggcaaaga ctgctagaaa 9780

tataaatctg tacacaggaa atgaccccag ggaaatacga gacttgatgg ctgcagggcg 9840

catgttagta gtagcactga gggatgtcga ccctgagctg tctgaaatgg tcgatttcaa 9900

ggggactttt ttagataggg aggccctgga ggctctaagt ctcgggcaac ctaaaccgaa 9960

gcaggttacc aaggaagctg ttaggaattt gatagaacag aaaaaagatg tggagatccc 10020

taactggttt gcatcagatg acccagtatt tctggaagtg gccttaaaaa atgataagta 10080

ctacttagta ggagatgttg gagagctaaa agatcaagct aaagcacttg gggccacgga 10140

tcagacaaga attataaagg aggtaggctc aaggacgtat gccatgaagc tatctagctg 10200

gttcctcaag gcatcaaaca aacagatgag tttaactcca ctgtttgagg aattgttgct 10260

acggtgccca cctgcaacta agagcaataa ggggcacatg gcatcagctt accaattggc 10320

acagggtaac tgggagcccc tcggttgcgg ggtgcaccta ggtacaatac cagccagaag 10380

ggtgaagata cacccatatg aagcttacct gaagttgaaa gatttcatag aagaagaaga 10440

gaagaaacct agggttaagg atacagtaat aagagagcac aacaaatgga tacttaaaaa 10500

aataaggttt caaggaaacc tcaacaccaa gaaaatgctc aacccaggga aactatctga 10560

acagttggac agggaggggc gcaagaggaa catctacaac caccagattg gtactataat 10620

gtcaagtgca ggcataaggc tggagaaatt gccaatagtg agggcccaaa ccgacaccaa 10680

aacctttcat gaggcaataa gagataagat agacaagagt gaaaaccggc aaaatccaga 10740

attgcacaac aaattgttgg agattttcca cacgatagcc caacccaccc tgaaacacac 10800

ctacggtgag gtgacgtggg agcaacttga ggcgggggta aatagaaagg gggcagcagg 10860

cttcctggag aagaagaaca tcggagaagt attggattca gaaaagcacc tggtagaaca 10920

attggtcagg gatctgaagg ccgggagaaa gataaaatat tatgaaactg caataccaaa 10980

aaatgagaag agagatgtca gtgatgactg gcaggcaggg gacctggtgg ttgagaagag 11040

gccaagagtt atccaatacc ctgaagccaa gacaaggcta gccatcacta aggtcatgta 11100

taactgggtg aaacagcagc ccgttgtgat tccaggatat gaaggaaaga cccccttgtt 11160

caacatcttt gataaagtga gaaaggaatg ggactcgttc aatgagccag tggccgtaag 11220

ttttgacacc aaagcctggg acactcaagt gactagtaag gatctgcaac ttattggaga 11280

aatccagaaa tattactata agaaggagtg gcacaagttc attgacacca tcaccgacca 11340

catgacagaa gtaccagtta taacagcaga tggtgaagta tatataagaa atgggcagag 11400

agggagcggc cagccagaca caagtgctgg caacagcatg ttaaatgtcc tgacaatgat 11460

gtacggcttc tgcgaaagca caggggtacc gtacaagagt ttcaacaggg tggcaaggat 11520

ccacgtctgt ggggatgatg gcttcttaat aactgaaaaa gggttagggc tgaaatttgc 11580

taacaaaggg atgcagattc ttcatgaagc aggcaaacct cagaagataa cggaagggga 11640

aaagatgaaa gttgcctata gatttgagga tatagagttc tgttctcata ccccagtccc 11700

tgttaggtgg tccgacaaca ccagtagtca catggccggg agagacaccg ctgtgatact 11760

atcaaagatg gcaacaagat tggattcaag tggagagagg ggtaccacag catatgaaaa 11820

agcggtagcc ttcagtttct tgctgatgta ttcctggaac ccgcttgtta ggaggatttg 11880

cctgttggtc ctttcgcaac agccagagac agacccatca aaacatgcca cttattatta 11940

caaaggtgat ccaatagggg cctataaaga tgtaataggt cggaatctaa gtgaactgaa 12000

gagaacaggc tttgagaaat tggcaaatct aaacctaagc ctgtccacgt tgggggtctg 12060

gactaagcac acaagcaaaa gaataattca ggactgtgtt gccattggga aagaagaggg 12120

caactggcta gttaagcccg acaggctgat atccagcaaa actggccact tatacatacc 12180

tgataaaggc tttacattac aaggaaagca ttatgagcaa ctgcagctaa gaacagagac 12240

aaacccggtc atgggggttg ggactgagag atacaagtta ggtcccatag tcaatctgct 12300

gctgagaagg ttgaaaattc tgctcatgac ggccgtcggc gtcagcagct gagacaaaat 12360

gtatatattg taaataaatt aatccatgta catagtgtat ataaatatag ttgggaccgt 12420

ccacctcaag aagacgacac gcccaacacg cacagctaaa cagtagtcaa gattatctac 12480

ctcaagataa cactacattt aatgcacaca gcactttagc tgtatgagga tacgcccgac 12540

gtctatagtt ggactaggga agacctctaa cag 12573

<210>5

<211>3988

<212>PRT

<213>牛病毒性腹泻病毒1NADL株

<400>5

Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys

1 5 10 15

Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asp Pro Leu

20 25 30

Phe Gly Glu Arg Gly Ala Val His Pro Gln Ser Thr Leu Lys Leu Pro

35 40 45

His Lys Arg Gly Glu Arg Asp Val Pro Thr Asn Leu Ala Ser Leu Pro

50 55 60

Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Arg 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 Leu Phe Glu Glu Gly Ser Met Cys

100 105 110

Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu

115 120 125

Tyr His Ile Tyr Val Cys Ile Asp Gly Cys Ile Ile Ile Lys Ser Ala

130 135 140

Thr Arg Ser Tyr Gln Arg Val Phe Arg Trp Val His Asn Arg Leu Asp

145 150 155 160

Cys Pro Leu Trp Val Thr Thr Cys Ser Asp Thr Lys Glu Glu Gly Ala

165 170 175

Thr Lys Lys Lys Thr Gln Lys Pro Asp Arg Leu Glu Arg Gly Lys Met

180 185 190

Lys Ile Val Pro Lys Glu Ser Glu Lys Asp Ser Lys Thr Lys Pro Pro

195 200 205

Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Arg Lys Lys

210 215 220

Gly Lys Thr 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 Ile Val Leu 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 Ile Glu

305 310 315 320

Leu Lys Thr 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 Val Met Asn Arg Thr Gln

355 360 365

Ala Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys Ala Val Thr Cys

370 375 380

Arg Tyr Asp Arg Ala 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 Met Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser

420 425 430

Asp Val Leu Phe Lys Glu His Glu Arg Ile Ser Met Phe Gln Asp Thr

435 440 445

Thr Leu Tyr Leu Val Asp Gly Leu Thr Asn Ser Leu Glu Gly Ala Arg

450 455 460

Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile

465 470 475 480

Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Thr Trp Phe Gly Ala Tyr

485 490 495

Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Ile Gly Tyr Ile Trp

500 505 510

Tyr Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile

515 520 525

Val Gly Pro Gly Lys Phe Gly Thr Asn Ala Glu Asp Gly Lys Ile Leu

530 535 540

His Glu Met Gly Gly His Leu Ser Glu Val Leu Leu Leu Ser Leu Val

545 550 555 560

Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Val Met Tyr Leu Ile

565 570 575

Leu His Phe Ser Ile Pro Gln Ser His Val Asp Val Met Asp Cys Asp

580 585 590

Lys Thr Gln Leu Asn Leu Thr Val Glu Leu Thr Thr Ala Glu Val Ile

595 600 605

Pro Gly Ser Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asn

610 615 620

Trp Trp Pro Tyr Glu Thr Thr Val Val Leu Ala Phe Glu Glu Val Ser

625 630 635 640

Gln Val Val Lys Leu Val Leu Arg Ala Leu Arg Asp Leu Thr Arg Ile

645 650 655

Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Val Cys Leu Val Lys Ile

660 665 670

Val Arg Gly Gln Met Val Gln Gly Ile Leu Trp Leu Leu Leu Ile Thr

675 680 685

Gly Val Gln Gly His Leu Asp Cys Lys Pro Glu Phe Ser Tyr Ala Ile

690 695 700

Ala Lys Asp Glu Arg Ile Gly Gln Leu Gly Ala Glu Gly Leu Thr Thr

705 710 715 720

Thr Trp Lys Glu Tyr Ser Pro Gly Met Lys Leu Glu Asp Thr Met Val

725 730 735

Ile Ala Trp Cys Glu Asp Gly Lys Leu Met Tyr Leu Gln Arg Cys Thr

740 745 750

Arg Glu Thr Arg Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr

755 760 765

Ser Val Val Phe Lys Lys Leu Phe Asp Gly Arg Lys Gln Glu Asp Val

770 775 780

Val Glu Met Asn Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala

785 790 795 800

Lys Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro

805 810 815

Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys

820 825 830

Thr Ser Phe Asn Met Asp Thr Leu Ala Thr Thr Val Val Arg Thr Tyr

835 840 845

Arg Arg Ser Lys Pro Phe Pro His Arg Gln Gly Cys Ile Thr Gln Lys

850 855 860

Asn Leu Gly Glu Asp Leu His Asn Cys Ile Leu Gly Gly Asn Trp Thr

865 870 875 880

Cys Val Pro Gly Asp Gln Leu Leu Tyr Lys Gly Gly Ser Ile Glu Ser

885 890 895

Cys Lys Trp Cys Gly Tyr Gln Phe Lys Glu Ser Glu Gly Leu Pro His

900 905 910

Tyr Pro Ile Gly Lys Cys Lys Leu Glu Asn Glu Thr Gly Tyr Arg Leu

915 920 925

Val Asp Ser Thr Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro Gln

930 935 940

Gly Thr Leu Lys Cys Lys Ile Gly Lys Thr Thr Val Gln Val Ile Ala

945 950 955 960

Met Asp Thr Lys Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile

965 970 975

Ser Ser Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr

980 985 990

Lys Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln

995 1000 1005

Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu

1010 1015 1020

Val Thr Asp His His Arg Asp Tyr Phe Ala Glu Ser Ile Leu Val

1025 1030 1035

Val Val Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu Leu

1040 1045 1050

Val Thr Tyr Met Val Leu Ser Glu Gln Lys Ala Leu Gly Ile Gln

1055 1060 1065

Tyr Gly Ser Gly Glu Val Val Met Met Gly Asn Leu Leu Thr His

1070 1075 1080

Asn Asn Ile Glu Val Val Thr Tyr Phe Leu Leu Leu Tyr Leu Leu

1085 1090 1095

Leu Arg Glu Glu Ser Val Lys Lys Trp Val Leu Leu Leu Tyr His

1100 1105 1110

Ile Leu Val Val His Pro Ile Lys Ser Val Ile Val Ile Leu Leu

1115 1120 1125

Met Ile Gly Asp Val Val Lys Ala Asp Ser Gly Gly Gln Glu Tyr

1130 1135 1140

Leu Gly Lys Ile Asp Leu Cys Phe Thr Thr Val Val Leu Ile Val

1145 1150 1155

Ile Gly Leu Ile Ile Ala Arg Arg Asp Pro Thr Ile Val Pro Leu

1160 1165 1170

Val Thr Ile Met Ala Ala Leu Arg Val Thr Glu Leu Thr His Gln

1175 1180 1185

Pro Gly Val Asp Ile Ala Val Ala Val Met Thr Ile Thr Leu Leu

1190 1195 1200

Met Val Ser Tyr Val Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu

1205 1210 1215

Gln Cys Ile Leu Ser Leu Val Ser Ala Val Phe Leu Ile Arg Ser

1220 1225 1230

Leu Ile Tyr Leu Gly Arg Ile Glu Met Pro Glu Val Thr Ile Pro

1235 1240 1245

Asn Trp Arg Pro Leu Thr Leu Ile Leu Leu Tyr Leu Ile Ser Thr

1250 1255 1260

Thr Ile Val Thr Arg Trp Lys Val Asp Val Ala Gly Leu Leu Leu

1265 1270 1275

Gln Cys Val Pro Ile Leu Leu Leu Val Thr Thr Leu Trp Ala Asp

1280 1285 1290

Phe Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Val Lys

1295 1300 1305

Leu Tyr Tyr Leu Lys Thr Val Arg Thr Asp Thr Glu Arg Ser Trp

1310 1315 1320

Leu Gly Gly Ile Asp Tyr Thr Arg Val Asp Ser Ile Tyr Asp Val

1325 1330 1335

Asp Glu Ser Gly Glu Gly Val Tyr Leu Phe Pro Ser Arg Gln Lys

1340 1345 1350

Ala Gln Gly Asn Phe Ser Ile Leu Leu Pro Leu Ile Lys Ala Thr

1355 1360 1365

Leu Ile Ser Cys Val Ser Ser Lys Trp Gln Leu Ile Tyr Met Ser

1370 1375 1380

Tyr Leu Thr Leu Asp Phe Met Tyr Tyr Met His Arg Lys Val Ile

1385 1390 1395

Glu Glu Ile Ser Gly Gly Thr Asn Ile Ile Ser Arg Leu Val Ala

1400 1405 1410

Ala Leu Ile Glu Leu Asn Trp Ser Met Glu Glu Glu Glu Ser Lys

1415 1420 1425

Gly Leu Lys Lys Phe Tyr Leu Leu Ser Gly Arg Leu Arg Asn Leu

1430 1435 1440

Ile Ile Lys His Lys Val Arg Asn Glu Thr Val Ala Ser Trp Tyr

1445 1450 1455

Gly Glu Glu Glu Val Tyr Gly Met Pro Lys Ile Met Thr Ile Ile

1460 1465 1470

Lys Ala Ser Thr Leu Ser Lys Ser Arg His Cys Ile Ile Cys Thr

1475 1480 1485

Val Cys Glu Gly Arg Glu Trp Lys Gly Gly Thr Cys Pro Lys Cys

1490 1495 1500

Gly Arg His Gly Lys Pro Ile Thr Cys Gly Met Ser Leu Ala Asp

1505 1510 1515

Phe Glu Glu Arg His Tyr Lys Arg Ile Phe Ile Arg Glu Gly Asn

1520 1525 1530

Phe Glu Gly Met Cys Ser Arg Cys Gln Gly Lys His Arg Arg Phe

1535 1540 1545

Glu Met Asp Arg Glu Pro Lys Ser Ala Arg Tyr Cys Ala Glu Cys

1550 1555 1560

Asn Arg Leu His Pro Ala Glu Glu Gly Asp Phe Trp Ala Glu Ser

1565 1570 1575

Ser Met Leu Gly Leu Lys Ile Thr Tyr Phe Ala Leu Met Asp Gly

1580 1585 1590

Lys Val Tyr Asp Ile Thr Glu Trp Ala Gly Cys Gln Arg Val Gly

1595 1600 1605

Ile Ser Pro Asp Thr His Arg Val Pro Cys His Ile Ser Phe Gly

1610 1615 1620

Ser Arg Met Pro Phe Arg Gln Glu Tyr Asn Gly Phe Val Gln Tyr

1625 1630 1635

Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Val Leu Ala

1640 1645 1650

Thr Lys Val Lys Met Leu Met ValGly Asn Leu Gly Glu Glu Ile

1655 1660 1665

Gly Asn Leu Glu His Leu Gly Trp Ile Leu Arg Gly Pro Ala Val

1670 1675 1680

Cys Lys Lys Ile Thr Glu His Glu Lys Cys His Ile Asn Ile Leu

1685 1690 1695

Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr

1700 1705 1710

Pro Arg Ala Pro Val Arg Phe Pro Thr Ser Leu Leu Lys Val Arg

1715 1720 1725

Arg Gly Leu Glu Thr Ala Trp Ala Tyr Thr His Gln Gly Gly Ile

1730 1735 1740

Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys

1745 1750 1755

Asp Ser Met Gly Arg Thr Arg Val Val Cys Gln Ser Asn Asn Arg

1760 1765 1770

Leu Thr Asp Glu Thr Glu Tyr Gly Val Lys Thr Asp Ser Gly Cys

1775 1780 1785

Pro Asp Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn

1790 1795 1800

Ile Ser Gly Ser Lys Gly Ala Val Val His Leu Gln Lys Thr Gly

1805 1810 1815

Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe

1820 1825 1830

Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu

1835 1840 1845

Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn

1850 1855 1860

Glu Glu Ser Lys Pro Thr Lys Ile Met Ser Gly Ile Gln Thr Val

1865 1870 1875

Ser Lys Asn Arg Ala Asp Leu Thr Glu Met Val Lys Lys Ile Thr

1880 1885 1890

Ser Met Asn Arg Gly Asp Phe Lys Gln Ile Thr Leu Ala Thr Gly

1895 1900 1905

Ala Gly Lys Thr Thr Glu Leu Pro Lys Ala Val Ile Glu Glu Ile

1910 1915 1920

Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala

1925 1930 1935

Ala Glu Ser Val Tyr Gln Tyr Met Arg Leu Lys His Pro Ser Ile

1940 1945 1950

Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala

1955 1960 1965

Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Met Pro

1970 1975 1980

Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe

1985 1990 1995

Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile

2000 2005 2010

Gly Lys Ile His Arg Phe Ser Glu Ser Ile Arg Val Val Ala Met

2015 2020 2025

Thr Ala Thr Pro Ala Gly Ser Val Thr Thr Thr Gly Gln Lys His

2030 2035 2040

Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp

2045 2050 2055

Leu Gly Ser Gln Phe Leu Asp Ile Ala Gly Leu Lys Ile Pro Val

2060 2065 2070

Asp Glu Met Lys Gly Asn Met Leu Val Phe Val Pro Thr Arg Asn

2075 2080 2085

Met Ala Val Glu Val Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn

2090 2095 2100

Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ala Asn Leu Arg Val

2105 2110 2115

Val Thr Ser Gln Ser Pro Tyr Val Ile Val Ala Thr Asn Ala Ile

2120 2125 2130

Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Ile Asp Thr

2135 2140 2145

Gly Leu Lys Cys Glu Lys Arg Val Arg Val Ser Ser Lys Ile Pro

2150 2155 2160

Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Val Gly Glu

2165 2170 2175

Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg

2180 2185 2190

Tyr Tyr Arg Ser Gln Glu Thr Ala Thr Gly Ser Lys Asp Tyr His

2195 2200 2205

Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile

2210 2215 2220

Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu

2225 2230 2235

Tyr Glu Glu Asp Ser Leu Leu Ile Thr Gln Leu Glu Ile Leu Asn

2240 2245 2250

Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile

2255 2260 2265

Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn

2270 2275 2280

Ser Tyr Glu Val Gln Val Pro Val Leu Phe Pro Lys Ile Arg Asn

2285 2290 2295

Gly Glu Val Thr Asp Thr Tyr Glu Asn Tyr Ser Phe Leu Asn Ala

2300 2305 2310

Arg Lys Leu Gly Glu Asp Val Pro Val Tyr Ile Tyr Ala Thr Glu

2315 2320 2325

Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Leu Asp Trp Pro Asp

2330 2335 2340

Pro Gly Asn Gln Gln Val Val Glu Thr Gly Lys Ala Leu Lys Gln

2345 2350 2355

Val Thr Gly Leu Ser Ser Ala Glu Asn Ala Leu Leu Val Ala Leu

2360 2365 2370

Phe Gly Tyr Val Gly Tyr Gln Ala Leu Ser Lys Arg His Val Pro

2375 2380 2385

Met Ile Thr Asp Ile Tyr Thr Ile Glu Asp Gln Arg Leu Glu Asp

2390 2395 2400

Thr Thr His Leu Gln Tyr Ala Pro Asn Ala Ile Lys Thr Asp Gly

2405 2410 2415

Thr Glu Thr Glu Leu Lys Glu Leu Ala Ser Gly Asp Val Glu Lys

2420 2425 2430

Ile Met Gly Ala Ile Ser Asp Tyr Ala Ala Gly Gly Leu Glu Phe

2435 2440 2445

Val Lys Ser Gln Ala Glu Lys Ile Lys Thr Ala Pro Leu Phe Lys

2450 2455 2460

Glu Asn Ala Glu Ala Ala Lys Gly Tyr Val Gln Lys Phe Ile Asp

2465 2470 2475

Ser Leu Ile Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp

2480 2485 2490

Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Ala Ala Arg Leu Gly

2495 2500 2505

His Glu Thr Ala Phe Ala Thr Leu Val Leu Lys Trp Leu Ala Phe

2510 2515 2520

Gly Gly Glu Ser Val Ser Asp His Val Lys Gln Ala Ala Val Asp

2525 2530 2535

Leu Val Val Tyr Tyr Val Met Asn Lys Pro Ser Phe Pro Gly Asp

2540 2545 2550

Ser Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe

2555 2560 2565

Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr His

2570 2575 2580

Asn Leu Ser Lys Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr

2585 2590 2595

Ala Thr Ser Ala Leu Lys Met Phe Thr Pro Thr Arg Leu Glu Ser

2600 2605 2610

Val Val Ile Leu Ser Thr Thr Ile Tyr Lys Thr Tyr Leu Ser Ile

2615 2620 2625

Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala

2630 2635 2640

Ala Met Glu Ile Leu Ser Gln Asn Pro Val Ser Val Gly Ile Ser

2645 2650 2655

Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu

2660 2665 2670

Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys

2675 2680 2685

Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn

2690 2695 2700

Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile

2705 2710 2715

Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr

2720 2725 2730

Lys Gly Trp Glu Ala Lys Glu Leu Ser Glu Arg Thr Ala Gly Arg

2735 2740 2745

Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly

2750 2755 2760

Met Asp Ser Gln Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile

2765 2770 2775

Leu Asp Leu Ile Tyr Gly Leu His Lys Gln Ile Asn Arg Gly Leu

2780 2785 2790

Lys Lys Met Val Leu Gly Trp Ala Pro Ala Pro Phe Ser Cys Asp

2795 2800 2805

Trp Thr Pro Ser Asp Glu Arg Ile Arg Leu Pro Thr Asp Asn Tyr

2810 2815 2820

Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala

2825 2830 2835

Phe Lys Asn Val Gly Gly Lys Leu Thr Lys Val Glu Glu Ser Gly

2840 2845 2850

Pro Phe Leu Cys Arg Asn Arg Pro Gly Arg Gly Pro Val Asn Tyr

2855 2860 2865

Arg Val Thr Lys Tyr Tyr Asp Asp Asn Leu Arg Glu Ile Lys Pro

2870 2875 2880

Val Ala Lys Leu Glu Gly Gln Val Glu His Tyr Tyr Lys Gly Val

2885 2890 2895

Thr Ala Lys Ile Asp Tyr Ser Lys Gly Lys Met Leu Leu Ala Thr

2900 2905 2910

Asp Lys Trp Glu Val Glu His Gly Val Ile Thr Arg Leu Ala Lys

2915 2920 2925

Arg Tyr Thr Gly Val Gly Phe Asn Gly Ala Tyr Leu Gly Asp Glu

2930 2935 2940

Pro Asn His Arg Ala Leu Val Glu Arg Asp Cys Ala Thr Ile Thr

2945 2950 2955

Lys Asn Thr Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe

2960 2965 2970

Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr Arg Leu Ile Glu Leu

2975 2980 2985

Val His Arg Asn Asn Leu Glu Glu Lys Glu Ile Pro Thr Ala Thr

2990 2995 3000

Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Val Gly

3005 3010 3015

Thr Ile Lys Pro Val Leu Gly Glu Arg Val Ile Pro Asp Pro Val

3020 3025 3030

Val Asp Ile Asn Leu Gln Pro Glu Val Gln Val Asp Thr Ser Glu

3035 3040 3045

Val Gly Ile Thr Ile Ile Gly Arg Glu Thr Leu Met Thr Thr Gly

3050 3055 3060

Val Thr Pro Val Leu Glu Lys Val Glu Pro Asp Ala Ser Asp Asn

3065 3070 3075

Gln Asn Ser Val Lys Ile Gly Leu Asp Glu Gly Asn Tyr Pro Gly

3080 3085 3090

Pro Gly Ile Gln Thr His Thr Leu Thr Glu Glu Ile His Asn Arg

3095 3100 3105

Asp Ala Arg Pro Phe Ile Met Ile Leu Gly Ser Arg Asn Ser Ile

3110 3115 3120

Ser Asn Arg Ala Lys Thr Ala Arg Asn Ile Asn Leu Tyr Thr Gly

3125 3130 3135

Asn Asp Pro Arg Glu Ile Arg Asp Leu Met Ala Ala Gly Arg Met

3140 3145 3150

Leu Val Val Ala Leu Arg Asp Val Asp Pro Glu Leu Ser Glu Met

3155 3160 3165

Val Asp Phe Lys Gly Thr Phe Leu Asp Arg Glu Ala Leu Glu Ala

3170 3175 3180

Leu Ser Leu Gly Gln Pro Lys Pro Lys Gln Val Thr Lys Glu Ala

3185 3190 3195

Val Arg Asn Leu Ile Glu Gln Lys Lys Asp Val Glu Ile Pro Asn

3200 3205 3210

Trp Phe Ala Ser Asp Asp Pro Val Phe Leu Glu Val Ala Leu Lys

3215 3220 3225

Asn Asp Lys Tyr Tyr Leu Val Gly Asp Val Gly Glu Leu Lys Asp

3230 3235 3240

Gln Ala Lys Ala Leu Gly Ala Thr Asp Gln Thr Arg Ile Ile Lys

3245 3250 3255

Glu Val Gly Ser Arg Thr Tyr Ala Met Lys Leu Ser Ser Trp Phe

3260 3265 3270

Leu Lys Ala Ser Asn Lys Gln Met Ser Leu Thr Pro Leu Phe Glu

3275 3280 3285

Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys Ser Asn Lys Gly

3290 3295 3300

His Met Ala Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro

3305 3310 3315

Leu Gly Cys Gly Val His Leu Gly Thr Ile Pro Ala Arg Arg Val

3320 3325 3330

Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Asp Phe Ile

3335 3340 3345

Glu Glu Glu Glu Lys Lys Pro Arg Val Lys Asp Thr Val Ile Arg

3350 3355 3360

Glu His Asn Lys Trp Ile Leu Lys Lys Ile Arg Phe Gln Gly Asn

3365 3370 3375

Leu Asn Thr Lys Lys Met LeuAsn Pro Gly Lys Leu Ser Glu Gln

3380 3385 3390

Leu Asp Arg Glu Gly Arg Lys Arg Asn Ile Tyr Asn His Gln Ile

3395 3400 3405

Gly Thr Ile Met Ser Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro

3410 3415 3420

Ile Val Arg Ala Gln Thr Asp Thr Lys Thr Phe His Glu Ala Ile

3425 3430 3435

Arg Asp Lys Ile Asp Lys Ser Glu Asn Arg Gln Asn Pro Glu Leu

3440 3445 3450

His Asn Lys Leu Leu Glu Ile Phe His Thr Ile Ala Gln Pro Thr

3455 3460 3465

Leu Lys His Thr Tyr Gly Glu Val Thr Trp Glu Gln Leu Glu Ala

3470 3475 3480

Gly Val Asn Arg Lys Gly Ala Ala Gly Phe Leu Glu Lys Lys Asn

3485 3490 3495

Ile Gly Glu Val Leu Asp Ser Glu Lys His Leu Val Glu Gln Leu

3500 3505 3510

Val Arg Asp Leu Lys Ala Gly Arg Lys Ile Lys Tyr Tyr Glu Thr

3515 3520 3525

Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Gln

3530 3535 3540

Ala Gly Asp Leu Val Val Glu Lys Arg Pro Arg Val Ile Gln Tyr

3545 3550 3555

Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Asn

3560 3565 3570

Trp Val Lys Gln Gln Pro Val Val Ile Pro Gly Tyr Glu Gly Lys

3575 3580 3585

Thr Pro Leu Phe Asn Ile Phe Asp Lys Val Arg Lys Glu Trp Asp

3590 3595 3600

Ser Phe Asn Glu Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp

3605 3610 3615

Asp Thr Gln Val Thr Ser Lys Asp Leu Gln Leu Ile Gly Glu Ile

3620 3625 3630

Gln Lys Tyr Tyr Tyr Lys Lys Glu Trp His Lys Phe Ile Asp Thr

3635 3640 3645

Ile Thr Asp His Met Thr Glu Val Pro Val Ile Thr Ala Asp Gly

3650 3655 3660

Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp

3665 3670 3675

Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Met Tyr

3680 3685 3690

Gly Phe Cys Glu Ser Thr Gly Val ProTyr Lys Ser Phe Asn Arg

3695 3700 3705

Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr

3710 3715 3720

Glu Lys Gly Leu Gly Leu Lys Phe Ala Asn Lys Gly Met Gln Ile

3725 3730 3735

Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Glu Lys

3740 3745 3750

Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His

3755 3760 3765

Thr Pro Val Pro Val Arg Trp Ser Asp Asn Thr Ser Ser His Met

3770 3775 3780

Ala Gly Arg Asp Thr Ala Val Ile Leu Ser Lys Met Ala Thr Arg

3785 3790 3795

Leu Asp Ser Ser Gly Glu Arg Gly Thr Thr Ala Tyr Glu Lys Ala

3800 3805 3810

Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Val

3815 3820 3825

Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Gln Pro Glu Thr Asp

3830 3835 3840

Pro Ser Lys His Ala Thr Tyr Tyr Tyr Lys Gly Asp Pro Ile Gly

3845 3850 3855

Ala Tyr Lys Asp Val Ile Gly Arg Asn Leu Ser Glu Leu Lys Arg

3860 3865 3870

Thr Gly Phe Glu Lys Leu Ala Asn Leu Asn Leu Ser Leu Ser Thr

3875 3880 3885

Leu Gly Val Trp Thr Lys His Thr Ser Lys Arg Ile Ile Gln Asp

3890 3895 3900

Cys Val Ala Ile Gly Lys Glu Glu Gly Asn Trp Leu Val Lys Pro

3905 3910 3915

Asp Arg Leu Ile Ser Ser Lys Thr Gly His Leu Tyr Ile Pro Asp

3920 3925 3930

Lys Gly Phe Thr Leu Gln Gly Lys His Tyr Glu Gln Leu Gln Leu

3935 3940 3945

Arg Thr Glu Thr Asn Pro Val Met Gly Val Gly Thr Glu Arg Tyr

3950 3955 3960

Lys Leu Gly Pro Ile Val Asn Leu Leu Leu Arg Arg Leu Lys Ile

3965 3970 3975

Leu Leu Met Thr Ala Val Gly Val Ser Ser

3980 3985

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Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys

1 5 10 15

Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asn Pro Leu

20 25 30

Phe Gly Glu Arg Gly Ala Ile His Pro Gln Ser Thr Leu Lys Leu Pro

35 40 45

His Lys Arg Gly Glu Arg Asn Val Pro Thr Ser 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 Leu Phe Glu Glu Gly Ser Met Cys

100 105 110

Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu

115 120 125

Tyr His lle Tyr Ile Cys lle Asp Gly Cys Ile Thr Val Lys Ser Ala

130 135 140

Thr Arg Ser His Gln Arg Val Leu Arg Trp Val His Asn Arg Leu Asp

145 150 155 160

Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Thr Lys Glu Glu Gly Ala

165 170 175

Thr Lys Lys Lys Gln Gln Lys Pro Asp Arg Leu Glu Lys Gly Arg Met

180 185 190

Lys Ile Val 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 Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala

245 250 255

Trp Ala Ile Leu Ala Val Val Leu Ile Glu 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 Val Glu

305 310 315 320

Leu Lys Thr 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 Ile Met Asn Arg Thr Gln

355 360 365

Ala Asn Leu Thr Glu Gly Gln Pro Pro 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 Val Leu Thr Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser

420 425 430

Asp Val Leu Phe Lys Glu His Glu Cys Thr Gly Val Phe Gln Asp Thr

435 440 445

Ala His Tyr Leu Val Asp Gly Val Thr Asn Ser Leu Glu Ser Ala Arg

450 455 460

Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile

465 470 475 480

Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Thr Trp Phe Gly Ala Tyr

485 490 495

Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Ile Gly Tyr Ile Trp

500 505 510

Phe Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile

515 520 525

Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu

530 535 540

His Glu Met Gly Gly His Leu Ser Glu Val Leu Leu Leu Ser Leu Val

545 550 555 560

Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Ala Met Tyr Leu Ile

565 570 575

Leu His Phe Ser Ile Pro Gln Ser His Val Asp Ile Thr Asp Cys Asp

580 585 590

Lys Thr Gln Leu Asn Leu Thr Ile Glu Leu Thr Thr Ala Asp Val Ile

595 600 605

Pro Gly Ser Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp

610 615 620

Trp Trp Pro Tyr Glu Thr Ala Ala Val Leu Ala Phe Glu Glu Val Gly

625 630 635 640

Gln Val Val Lys Ile Val Leu Arg Ala Leu Arg Asp Leu Thr Arg Ile

645 650 655

Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Val Cys Leu Ile Lys Met

660 665 670

Val Arg Gly Gln Val Val Gln Gly Ile Leu Trp Leu Leu Leu Ile Thr

675 680 685

Gly Val Gln Gly His Leu Asp Cys Lys Pro Glu Tyr Ser Tyr Ala Ile

690 695 700

Ala Lys Asn Asp Arg Val Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr

705 710 715 720

Val Trp Lys Asp Tyr Ser His Glu Met Lys Leu Glu Asp Thr Met Val

725 730 735

Ile Ala Trp Cys Lys Gly Gly Lys Phe Thr Tyr Leu Ser Arg Cys Thr

740 745 750

Arg Glu Thr Arg Tyr Leu Ala Ile Leu His Ser Arg Ala Leu Pro Thr

755 760 765

Ser Val Val Phe Lys Lys Leu Phe Glu Gly Gln Lys Gln Glu Asp Thr

770 775 780

Val Glu Met Asp Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala

785 790 795 800

Lys Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro

805 810 815

Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys

820 825 830

Met Leu Ala Asn Arg Asp Thr Leu Asp Thr Ala Val Val Arg Thr Tyr

835 840 845

Arg Arg Ser Val Pro Phe Pro Tyr Arg Gln Gly Cys Ile Thr Gln Lys

850 855 860

Thr Leu Gly Glu Asp Leu Tyr Asp Cys Ala Leu Gly Gly Asn Trp Thr

865 870 875 880

Cys Val Thr Gly Asp Gln Ser Arg Tyr Thr Gly Gly Leu Ile Glu Ser

885 890 895

Cys Lys Trp Cys Gly Tyr Lys Phe Gln Lys Ser Glu Gly Leu Pro His

900 905 910

Tyr Pro Ile Gly Lys Cys Arg Leu Asn Asn Glu Thr Gly Tyr Arg Leu

915 920 925

Val Asp Asp Thr Ser Cys Asp Arg Glu Gly Val Ala Ile Val Pro His

930 935 940

Gly Leu Val Lys Cys Lys Ile Gly Asp Thr Thr Val Gln Val Ile Ala

945 950 955 960

Thr Asp Thr Lys Leu Gly Pro Met Pro Cys Lys Pro His Glu Ile Ile

965 970 975

Ser Ser Glu Gly Pro Ile Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr

980 985 990

Arg Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln

995 1000 1005

Gln Tyr Met Leu Lys Gly Asp Tyr Gln Tyr Trp Phe Asp Leu Glu

1010 1015 1020

Val Thr Asp His His Arg Asp Tyr Phe Ala Glu Ser Ile Leu Val

1025 1030 1035

Val Val Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu Leu

1040 1045 1050

Val Thr Tyr Met Val Leu Ser Glu Gln Lys Ala Ser Gly Ala Gln

1055 1060 1065

Tyr Gly Ala Gly Glu Val Val Met Met Gly Asn Leu Leu Thr His

1070 1075 1080

Asp Asn Val Glu Val Val Thr Tyr Phe Phe Leu Leu Tyr Leu Leu

1085 1090 1095

Leu Arg Glu Glu Ser Val Lys Lys Trp Val Leu Leu Leu Tyr His

1100 1105 1110

Ile Leu Val Ala His Pro Leu Lys Ser Val Ile Val Ile Leu Leu

1115 1120 1125

Met Ile Gly Asp Val Val Lys Ala Asp Pro Gly Gly Gln Gly Tyr

1130 1135 1140

Leu Gly Gln Ile Asp Val Cys Phe Thr Met Val Val Ile Ile Ile

1145 1150 1155

Ile Gly Leu Ile Ile Ala Arg Arg Asp Pro Thr Ile Val Pro Leu

1160 1165 1170

Ile Thr Ile Val Ala Ser Leu Arg Val Thr Gly Leu Thr Tyr Ser

1175 1180 1185

Pro Gly Val Asp Ala Ala Met Ala Val Ile Thr Ile Thr Leu Leu

1190 1195 1200

Met Val Ser Tyr Val Thr Asp Tyr Phe Arg Tyr Lys Arg Trp Leu

1205 1210 1215

Gln Cys Ile Leu Ser Leu Val Ser Gly Val Phe Leu Ile Arg Cys

1220 1225 1230

Leu Ile His Leu Gly Arg Ile Glu Thr Pro Glu Val Thr Ile Pro

1235 1240 1245

Asn Trp Arg Pro Leu Thr Leu Ile Leu Phe Tyr Leu Ile Ser Thr

1250 1255 1260

Thr Va1 Val Thr Met Trp Lys Ile Asp Leu Ala Gly Leu Leu Leu

1265 1270 1275

Gln Gly Val Pro Ile Leu Leu Leu Ile Thr Thr Leu Trp Ala Asp

1280 1285 1290

Phe Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Val Lys

1295 1300 1305

Leu Tyr Tyr Leu Lys Thr Ile Lys Thr Asp Ile Glu Lys Ser Trp

1310 1315 1320

Leu Gly Gly Leu Asp Tyr Lys Arg Val Asp Ser Ile Tyr Asp Val

1325 1330 1335

Asp Glu Ser Gly Glu Gly Val Tyr Leu Phe Pro Ser Arg Gln Lys

1340 1345 1350

Ala Gln Lys Asn Phe Ser Met Leu Leu Pro Leu Val Arg Ala Thr

1355 1360 1365

Leu Ile Ser Cys Val Ser Ser Lys Trp Gln Leu Ile Tyr Met Ala

1370 1375 1380

Tyr Leu Ser Val Asp Phe Met Tyr Tyr Met His Arg Lys Val Ile

1385 1390 1395

Glu Glu Ile Ser Gly Gly Thr Asn Met Ile Ser Arg Ile Val Ala

1400 1405 1410

Ala Leu Ile Glu Leu Asn Trp Ser Met Glu Glu Glu Glu Ser Lys

1415 1420 1425

Gly Leu Lys Lys Phe Tyr Leu Leu Ser Gly Arg Leu Arg Asn Leu

1430 1435 1440

Ile Ile Lys His Lys Val Arg Asn Glu Thr Val Ala Gly Trp Tyr

1445 1450 1455

Gly Glu Glu Glu Val Tyr Gly Met Pro Lys Ile Met Thr Ile Ile

1460 1465 1470

Lys Ala Ser Thr Leu Asn Lys Asn Lys His Cys Ile Ile Cys Thr

1475 1480 1485

Val Cys Glu Gly Arg Lys Trp Lys Gly Gly Thr Cys Pro Lys Cys

1490 1495 1500

Gly Arg His Gly Lys ProIle Thr Cys Gly Met Ser Leu Ala Asp

1505 1510 1515

Phe Glu Glu Arg His Tyr Lys Arg Ile Phe Ile Arg Glu Gly Asn

1520 1525 1530

Phe Glu Gly Pro Phe Arg Gln Glu Tyr Asn Gly Phe Ile Gln Tyr

1535 1540 1545

Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala

1550 1555 1560

Thr Lys Val Lys Met Leu Met Val Gly Asn Leu Gly Glu Glu Val

1565 l570 1575

Gly Asp Leu Glu His Leu Gly Trp Ile Leu Arg Gly Pro Ala Val

1580 1585 1590

Cys Lys Lys Ile Thr Glu His Glu Arg Cys His Ile Asn Ile Leu

1595 1600 1605

Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr

1610 1615 1620

Pro Arg Ala Pro Val Arg Phe Pro Thr Ser Leu Leu Lys Val Arg

1625 1630 1635

Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile

1640 1645 1650

Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys

1655 1660 1665

Asp Ser Met Gly Arg Thr Arg Val Val Cys Gln Ser Asn Asn Lys

1670 1675 1680

Leu Thr Asp Glu Thr Glu Tyr Gly Val Lys Thr Asp Ser Gly Cys

1685 1690 1695

Pro Asp Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn

1700 1705 1710

Ile Ser Gly Ser Lys Gly Ala Val Val His Leu Gln Lys Thr Gly

1715 1720 1725

Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe

1730 1735 1740

Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu

1745 1750 1755

Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn

1760 1765 1770

Glu Glu Ser Lys Pro Thr Lys Ile Met Ser Gly Ile Gln Thr Val

1775 1780 1785

Ser Lys Asn Thr Ala Asp Leu Thr Glu Met Val Lys Lys Ile Thr

1790 1795 1800

Ser Met Asn Arg Gly Asp Phe Lys Gln Ile Thr Leu Ala Thr Gly

1805 1810 1815

Ala Gly Lys Thr Thr Glu Leu Pro Lys Ala Val Ile Glu Glu Ile

1820 1825 1830

Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala

1835 1840 1845

Ala Glu Ser Val Tyr Gln Tyr Met Arg Leu Lys His Pro Ser Ile

1850 1855 1860

Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala

1865 1870 1875

Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Met Pro

1880 1885 1890

Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe

1895 1900 1905

Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile

1910 1915 1920

Gly Lys Ile His Arg Phe Ser Glu Ser Ile Arg Val Val Ala Met

1925 1930 1935

Thr Ala Thr Pro Ala Gly Ser Val Thr Thr Thr Gly Gln Lys His

1940 1945 1950

Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Glu Gly Glu Asp

1955 1960 1965

Leu Gly Ser Gln Phe Leu Asp Ile Ala Gly Leu Lys Ile Pro Val

1970 1975 1980

Asp Glu Met Lys Gly Asn Met Leu Val Phe Val Pro Thr Arg Asn

1985 1990 1995

Met Ala Val Glu Val Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn

2000 2005 2010

Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ala Asn Leu Arg Val

2015 2020 2025

Val Thr Ser Gln Ser Pro Tyr Val Ile Val Ala Thr Asn Ala Ile

2030 2035 2040

Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr

2045 2050 2055

Gly Leu Lys Cys Glu Lys Arg Val Arg Val Ser Ser Lys Ile Pro

2060 2065 2070

Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Val Gly Glu

2075 2080 2085

Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg

2090 2095 2100

Tyr Tyr Arg Ser Gln Glu Thr Ala Thr Gly Ser Lys Asp Tyr His

2105 2110 2115

Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile

2120 2125 2130

Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu

2135 2140 2145

Tyr Glu Glu Asp Ser Leu Leu Ile Thr Gln Leu Glu Ile Leu Asn

2150 2155 2160

Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile

2165 2170 2175

Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn

2180 2185 2190

Ser Tyr Glu Val Gln Val Pro Val Leu Phe Pro Lys Ile Arg Asn

2195 2200 2205

Gly Glu Val Thr Asp Thr Tyr Glu Asn Tyr Ser Phe Leu Asn Ala

2210 2215 2220

Arg Lys Leu Gly Glu Asp Val Pro Val Tyr Ile Tyr Ala Thr Glu

2225 2230 2235

Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Leu Asp Trp Pro Asp

2240 2245 2250

Pro Gly Asn Gln Gln Val Val Glu Thr Gly Lys Ala Leu Lys Gln

2255 2260 2265

Val Ala Gly Leu Ser Ser Ala Glu Asn Ala Leu Leu Val Ala Leu

2270 2275 2280

Phe Gly Tyr Val Gly Tyr Gln Ala Leu Ser Lys Arg His Val Pro

2285 2290 2295

Met Ile Thr Asp Ile Tyr Thr Ile Glu Asp Gln Arg Leu Glu Asp

2300 2305 2310

Thr Thr His Leu Gln Tyr Ala Pro Asn Ala Ile Lys Thr Glu Gly

2315 2320 2325

Thr Glu Thr Glu Leu Lys Glu Leu Ala Ser Gly Asp Val Glu Lys

2330 2335 2340

Ile Met Gly Ala Ile Ser Asp Tyr Ala Ala Gly Gly Leu Asp Phe

2345 2350 2355

Val Lys Ser Gln Ala Glu Lys Ile Lys Thr Ala Pro Leu Phe Lys

2360 2365 2370

Glu Asn Val Glu Ala Ala Arg Gly Tyr Val Gln Lys Leu Ile Asp

2375 2380 2385

Ser Leu Ile Glu Asp Lys Asp Val Ile Ile Arg Tyr Gly Leu Trp

2390 2395 2400

Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Ala Ala Arg Leu Gly

2405 2410 2415

His Glu Thr Ala Phe Ala Thr Leu Val Leu Lys Trp Leu Ala Phe

2420 2425 2430

Gly Gly Glu Thr Val Ser Asp His Ile Arg Gln Ala Ala Val Asp

2435 2440 2445

Leu Val Val Tyr Tyr Val Met Asn Lys Pro Ser Phe Pro Gly Asp

2450 2455 2460

Thr Glu Thr Gln Gln Glu Gly Arg Arg Phe Va1 Ala Ser Leu Phe

2465 2470 2475

Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn

2480 2485 2490

Asn Leu Ser Lys Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr

2495 2500 2505

Ala Thr Ser Ala Leu Lys Met Phe Thr Pro Thr Arg Leu Glu Ser

2510 2515 2520

Val Val Ile Leu Ser Thr Thr Ile Tyr Lys Thr Tyr Leu Ser Ile

2525 2530 2535

Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala

2540 2545 2550

Ala Met Glu Ile Leu Ser Gln Asn Pro Val Ser Val Gly Ile Ser

2555 2560 2565

Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu

2570 2575 2580

Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys

2585 2590 2595

Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn

2600 2605 2610

Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile

2615 2620 2625

Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr

2630 2635 2640

Lys Gly Trp Glu Ala Lys Glu Leu Ser Glu Arg Thr Ala Gly Arg

2645 2650 2655

Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly

2660 2665 2670

Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile

2675 2680 2685

Leu Asp Leu Ile His Gly Leu His Lys Gln Ile Asn Arg Gly Leu

2690 2695 2700

Lys Lys Ile Val Leu Gly Trp Ala Pro Ala Pro Phe Ser Cys Asp

2705 2710 2715

Trp Thr Pro Ser Asp Glu Arg Ile Arg Leu Pro Thr Asp Ser Tyr

2720 2725 2730

Leu Arg Val Glu Thr Lys Cys Pro Cys Gly Tyr Glu Met Lys Ala

2735 2740 2745

Leu Lys Asn Val Ser Gly Lys Leu Thr Lys Val Glu Glu Ser Gly

2750 2755 2760

Pro Phe Leu Cys Arg Asn Arg Pro Gly Arg Gly Pro Val Asn Tyr

2765 2770 2775

Arg Val Thr Lys Tyr Tyr Asp Asp Asn Leu Arg Glu Ile Arg Pro

2780 2785 2790

Val Ala Lys Leu Glu Gly Gln Val Glu His Tyr Tyr Lys Gly Val

2795 2800 2805

Thr Ala Arg Ile Asp Tyr Ser Lys Gly Lys Thr Leu Leu Ala Thr

2810 2815 2820

Asp Lys Trp Glu Val Glu His Gly Thr Leu Thr Arg Leu Thr Lys

2825 2830 2835

Arg Tyr Thr Gly Val Gly Phe Arg Gly Ala Tyr Leu Gly Asp Glu

2840 2845 2850

Pro Asn His Arg Asp Leu Val Glu Arg Asp Cys Ala Thr Ile Thr

2855 2860 2865

Lys Asn Thr Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe

2870 2875 2880

Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr Arg Leu Ile Glu Leu

2885 2890 2895

Val His Arg Asn Asn Leu Glu Glu Lys Glu Ile Pro Thr Ala Thr

2900 2905 2910

Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Val Gly

2915 2920 2925

Thr Ile Lys Pro Val Leu Gly Glu Arg Val Ile Pro Asp Pro Val

2930 2935 2940

Val Asp Ile Asn Leu Gln Pro Glu Val Gln Val Asp Thr Ser Glu

2945 2950 2955

Val Gly Ile Thr Ile Ile Gly Lys Glu Ala Val Met Thr Thr Gly

2960 2965 2970

Val Thr Pro Val Met Glu Lys Val Glu Pro Asp Thr Asp Asn Asn

2975 2980 2985

Gln Ser Ser Val Lys Ile Gly Leu Asp Glu Gly Asn Tyr Pro Gly

2990 2995 3000

Pro Gly Val Gln Thr His Thr Leu Val Glu Glu Ile His Asn Lys

3005 3010 3015

Asp Ala Arg Pro Phe Ile Met Val Leu Gly Ser Lys Ser Ser Met

3020 3025 3030

Ser Asn Arg Ala Lys Thr Ala Arg Asn Ile Asn Leu Tyr Thr Gly

3035 3040 3045

Asn Asp Pro Arg Glu Ile Arg Asp Leu Met Ala Glu Gly Arg Ile

3050 3055 3060

Leu Val Val Ala Leu Arg Asp Ile Asp Pro Asp Leu Ser Glu Leu

3065 3070 3075

Val Asp Phe Lys Gly Thr Phe Leu Asp Arg Glu Ala Leu Glu Ala

3080 3085 3090

Leu Ser Leu Gly Gln Pro Lys Pro Lys Gln Val Thr Lys Ala Ala

3095 3100 3105

Ile Arg Asp Leu Leu Lys Glu Glu Arg Gln Val Glu Ile Pro Asp

3110 3115 3120

Trp Phe Thr Ser Asp Asp Pro Val Phe Leu Asp Ile Ala Met Lys

3125 3130 3135

Lys Asp Lys Tyr His Leu Ile Gly Asp Val Val Glu Val Lys Asp

3140 3145 3150

Gln Ala Lys Ala Leu Gly Ala Thr Asp Gln Thr Arg Ile Val Lys

3155 3160 3165

Glu Val Gly Ser Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Phe

3170 3175 3180

Leu Gln Ala Ser Ser Lys Gln Met Ser Leu Thr Pro Leu Phe Glu

3185 3190 3195

Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys Ser Asn Lys Gly

3200 3205 3210

His Met Ala Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro

3215 3220 3225

Leu Gly Cys Gly Val His Leu Gly Thr Val Pro Ala Arg Arg Val

3230 3235 3240

Lys Met His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Asp Leu Val

3245 3250 3255

Glu Glu Glu Glu Lys Lys Pro Arg Ile Arg Asp Thr Val Ile Arg

3260 3265 3270

Glu His Asn Lys Trp Ile Leu Lys Lys Ile Lys Phe Gln Gly Asn

3275 3280 3285

Leu Asn Thr Lys Lys Met Leu Asn Pro Gly Lys Leu Ser Glu Gln

3290 3295 3300

Leu Asp Arg Glu Gly His Lys Arg Asn Ile Tyr Asn Asn Gln Ile

3305 3310 3315

Ser Thr Val Met Ser Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro

3320 3325 3330

Ile Val Arg Ala Gln Thr Asp Thr Lys Ser Phe His Glu Ala Ile

3335 3340 3345

Arg Asp Lys Ile Asp Lys Asn Glu Asn Arg Gln Asn Pro Glu Leu

3350 3355 3360

His Asn Lys Leu Leu Glu Ile Phe His Thr Ile Ala Asp Pro Ser

3365 3370 3375

Leu Lys His Thr Tyr Gly Glu Val Thr Trp Glu Gln Leu Glu Ala

3380 3385 3390

Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe Leu Glu Lys Lys Asn

3395 3400 3405

Ile Gly Glu Val Leu Asp Ser Glu Lys His Leu Val Glu Gln Leu

3410 3415 3420

Val Arg Asp Leu Lys Ala Gly Arg Lys Ile Arg Tyr Tyr Glu Thr

3425 3430 3435

Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Gln

3440 3445 3450

Ala Gly Asp Leu Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr

3455 3460 3465

Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Asn

3470 3475 3480

Trp Val Lys Gln Gln Pro Val Val Ile Pro Gly Tyr Glu Gly Lys

3485 3490 3495

Thr Pro Leu Phe Asn Ile Phe Asn Lys Val Arg Lys Glu Trp Asp

3500 3505 3510

Leu Phe Asn Glu Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp

3515 3520 3525

Asp Thr Gln Val Thr Ser Arg Asp Leu His Leu Ile Gly Glu Ile

3530 3535 3540

Gln Lys Tyr Tyr Tyr Arg Lys Glu Trp His Lys Phe Ile Asp Thr

3545 3550 3555

Ile Thr Asp His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly

3560 3565 3570

Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp

3575 3580 3585

Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr

3590 3595 3600

Ala Phe Cys Glu Ser Thr Gly Val Pro Tyr Lys Ser Phe Asn Arg

3605 3610 3615

Val Ala Lys Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr

3620 3625 3630

Glu Lys Gly Leu Gly Leu Lys Phe Ser Asn Lys Gly Met GlnIle

3635 3640 3645

Leu His Glu Ala Gly Lys Pro Gln Lys Leu Thr Glu Gly Glu Lys

3650 3655 3660

Met Lys Val Ala Tyr Lys Phe Glu Asp Ile Glu Phe Cys Ser His

3665 3670 3675

Thr Pro Val Pro Val Arg Trp Ser Asp Asn Thr Ser Ser Tyr Met

3680 3685 3690

Ala Gly Arg Asp Thr Ala Val Ile Leu Ser Lys Met Ala Thr Arg

3695 3700 3705

Leu Asp Ser Ser Gly Glu Arg Gly Thr Thr Ala Tyr Glu Lys Ala

3710 3715 3720

Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Val

3725 3730 3735

Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Arg Pro Glu Thr Ala

3740 3745 3750

Pro Ser Thr Gln Thr Thr Tyr Tyr Tyr Lys Gly Asp Pro Ile Gly

3755 3760 3765

Ala Tyr Lys Asp Val Ile Gly Arg Asn Leu Ser Glu Leu Lys Arg

3770 3775 3780

Thr Gly Phe Glu Lys Leu Ala Asn Leu Asn Leu Ser Leu Ser Thr

3785 3790 3795

Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Ile Ile Gln Asp

3800 3805 3810

Cys Val Ala Ile Gly Lys Glu Glu Gly Asn Trp Leu Val Asn Ala

3815 3820 3825

Asp Arg Leu Ile Ser Ser Lys Thr Gly His Leu Tyr Ile Pro Asp

3830 3835 3840

Lys Gly Phe Thr Leu Gln Gly Lys His Tyr Glu Gln Leu Gln Leu

3845 3850 3855

Gly Ala Glu Thr Asn Pro Val Met Gly Val Gly Thr Glu Arg Tyr

3860 3865 3870

Lys Leu Gly Pro Ile Val Asn Leu Leu Leu Arg Arg Leu Lys Val

3875 3880 3885

Leu Leu Met Ala Ala Val Gly Ala Ser Ser

3890 3895

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<223>合成寡核苷酸引物53637U2

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Claims

1.一种疫苗组合物，所述组合物包含同一科的至少两种活的突变病毒，其中每一种病毒在病毒基因组中都包含突变，而且病毒中的突变存在于同一基因组位点上从而使突变病毒无法相互重组来抵消突变。

2.如权利要求1所述的疫苗组合物，其中所述的科是黄病毒科。

3.如权利要求1所述的疫苗组合物，其中所述的两种活的突变病毒都来自于疫病毒属。

4.如权利要求1所述的疫苗组合物，其中所述的两种突变的活病毒由突变BVDV-1和突变BVDV-2组成。

5.如权利要求4所述的疫苗组合物，其中所述的两种突变的活病毒由致细胞病变(cp)BVDV-1和致细胞病变BVDV-2组成，而且它们都是被减毒的。

6.如权利要求5所述的疫苗组合物，其中致细胞病变BVDV-1和致细胞病变BVDV-2在NS2-3区域都含有导致致细胞病变生物型的突变。

7.如权利要求6所述的疫苗组合物，其中致细胞病变BVDV-1是BVDV-1 NADL，而致细胞病变BVDV-2是BVDV-2 53637。

8.如权利要求4所述的疫苗组合物，进一步含有下列的至少一种：牛疱疹病毒-1、牛呼吸道合胞体病毒、副流感病毒-3、胚胎弯曲杆菌、犬钩端螺旋体、流感伤寒钩端螺旋体、哈德焦钩端螺旋体、黄疸出血钩端螺旋体、波摩那钩端螺旋体或者Mannhemia haemolytica。

9.如权利要求1所述的疫苗组合物，进一步含有兽医学上可接受的载体。

10.一种制备安全病毒疫苗的方法，包括选择或构建同一科的两种活的突变病毒，其中每一种病毒都包含突变，而且病毒中的突变都存在于同一基因组位点上从而使突变病毒无法进行同源重组来抵消突变。

11.如权利要求10所述的方法，其中所述的突变选自于缺失、插入、取代或者其组合。

12.如权利要求10所述的方法，其中所述突变赋予下列表型，所述表型选自减毒作用、细胞趋向性或生物型的改造、物种趋向性的改造、外源基因盒的表达或者其组合。

13.如权利要求10所述的方法，其中所述的科是黄病毒科。

14.如权利要求10所述的方法，其中两种活的突变病毒都来自于疫病毒属。

15.如权利要求10所述的方法，其中所述的两种突变的活病毒由突变BVDV-1和突变BVDV-2组成。