專利名稱：安全的突變病毒疫苗的制作方法
技術領域：
本發(fā)明主要涉及適用于給藥動物以抗病毒感染的疫苗。更準確地說，本發(fā)明涉及安全疫苗以及制備此種疫苗的方法。本發(fā)明的疫苗中包含至少兩種活的同一科突變病毒或是編碼所述病毒的核酸分子，其中每一種病毒或編碼核酸分子中包含能夠賦予所需表型的突變，而且病毒中的突變存在于同一基因組位點上從而使突變病毒無法相互重組來抵消(eliminate)突變。
背景技術：
黃病毒科(Flaviviridae)由疫病毒屬(Pestivirus)、黃病毒屬(Flavivirus)和丙型肝炎病毒屬(Hepacivirus)屬組成。疫病毒屬(Pestivirus)的代表種有牛病毒性腹瀉病毒-1(BVDV-1)、BVDV-2、經(jīng)典豬瘟病毒(classical swinefever virus)以及羊先天性髓質形成不全癥病毒(Border disease virus)。這一病毒科中成員的病毒粒子包封著約9.5到12.3kb的正鏈RNA基因組。該基因組RNA包含鄰接的長的開放讀碼框(ORFs)，其可以被翻譯為多蛋白質再經(jīng)過細胞和病毒蛋白酶的加工而產生成熟的病毒蛋白。對于疫病毒屬(Pestivirus)的成員，開放讀碼框(ORF)編碼大約3900個氨基酸的多蛋白質，其可以被協(xié)同翻譯和后翻譯加工成為下列成熟的病毒蛋白質(從5’端到3’端)Npro、C、Ems、E1、E2、NS2-3、NS4A、NS4B、NS5A以及NS5B。
在一些疫病毒屬(Pestivirus)成員中，基于它們對組織培養(yǎng)細胞的作用存在兩種生物型，即為致細胞病變的(細胞病變的或cp)和非致細胞病變的(非細胞病變的或ncp)?；蚪M分析顯示在致細胞病變的(cp)疫病毒(pestiviruses)基因組中存在細胞序列的插入，有時伴有病毒序列的復制、基因組重排和/或病毒序列的缺失，但在非致細胞病變的(ncp)疫病毒(pestiviruses)相應的RNA中并不存在。這暗示著致細胞病變的(cp)疫病毒(pestiviruses)是由非致細胞病變的(ncp)疫病毒(pestiviruses)通過RNA重組發(fā)展而來的。
牛病毒性腹瀉病毒(BVDV)是一種廣泛分布的牛病原體。BVDV-1在具免疫活性動物體內通常只引起輕度的腹瀉，然而BVDV-2則能夠引起血小板減少癥、大出血以及急性的致命疾病。牛病毒性腹瀉病毒(BVDV)能夠穿過懷孕牛的胎盤從而引起持續(xù)感染(PI)小牛的出生(Malmquist，J.Am.Vet.Mea.Assoc.152763-768(1968)；Ross，et al.，J.Am.Vet.Med.Assoc.188618-619(1986))。病毒血癥的(Viremic)小牛對病毒具有免疫耐受而且它們此后的生活中會持續(xù)存在病毒血癥。它們提供了一個粘膜疾病爆發(fā)的根源(Liess，et al.，Dtsch.Tieraerztl.Wschr.81481-487(1974))而且極易被微生物感染而引起例如肺炎或腸道病變這樣的疾病(Barber，et al.，Vet.Rec.117459-464(1985))。任一基因型的病毒都可以以為兩生物型中的一種存在，致細胞病變的(cp)或者非致細胞病變的(ncp)。致細胞病變的(cp)顯型與NS3的表達有關，雖然細胞感染致細胞病變的(cp)或非致細胞病變的(ncp)牛病毒性腹瀉病毒(BVDV)都會表達NS2-3，然而NS3卻只能在致細胞病變的(cp)牛病毒性腹瀉病毒(BVDV)感染以后被檢測到。NS3與NS2-3的C末端部分是線性對應的。NS3的表達呈現(xiàn)了一種在致細胞病變的(cp)牛病毒性腹瀉病毒(BVDV)中觀察到的基因組改造的結果。
目前可得到的病毒疫苗包括滅活或減毒的活病毒疫苗、活載體疫苗、亞單位疫苗以及DNA或RNA疫苗。見Roth等“New Technology For ImprovedVaccine Safety And Efficacy”，Veterinary Clinics North AmericaFood AnimalPractice 17(3)585-597(2001)。病毒的減毒可以通過紫外照射、化學處理或者體外的高連續(xù)傳代(high serial passage)來達成。這些方法導致的突變其數(shù)量、位置和種類在沒有進行基因組序列分析的情況下是未知的。減毒還可以通過進行確定的基因改造來完成，例如，對已知能夠帶來毒性的病毒序列進行特異性缺失，或者在病毒基因組中進行序列的插入。關于應用減毒活病毒疫苗的一種顧慮是減毒的突變病毒具有在體內重組的潛力以抵消減毒突變從而恢復毒性。例如，在有毒性(野生型)野外菌株存在的情況下，病毒基因組中存在缺失的減毒病毒會具有與毒性菌株重組從而恢復缺失序列的潛力。見例如，Roth等，同上。還觀察到在細胞培養(yǎng)基中具有細胞插入的致細胞病變疫病毒(cytopathic pestiviruses)通過細胞序列的缺失可以產生非致細胞病變疫病毒(noncytopathic pestiviruses)，可能是通過RNA重組完成的。見例如，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 straingeneration of cytopathogenic virus and induction of lethal disease”，Journal ofVirology 75(14)6256-64(2001)。如果希望在疫苗組合物中包括兩種來自于同一種、屬或科的減毒突變病毒，那么將存在一種顧慮是兩種病毒可能在接種疫苗的動物體內重組從而抵消減毒突變。見例如，Glazenburg等，“Genetic recombination of pseudorabies virusevidence that homologousrecombination between insert sequences is less frequent than betweenautologous sequences”，Archives of Virology，140(4)671-85(1995)。
于是，仍在存在著進一步開發(fā)能夠保護動物抵御病毒感染的安全有效的疫苗的需要。

發(fā)明內容
本發(fā)明提供了一種包含至少兩種活的同一科突變病毒或是編碼所述病毒的核酸分子的安全疫苗，其中每一種病毒或其編碼核酸中包含一種能夠賦予所需表型的突變，而且病毒中的突變都存在于同一基因組位點上從而使突變病毒無法相互重組來抵消突變。
本發(fā)明還提供了一種通過篩選或構建兩種或更多活的同一科、屬或種的突變病毒制備一種安全病毒疫苗的方法，其中每一種病毒中都包含能夠賦予所需表型的突變，而且病毒中的突變都存在于同一基因組位點上從而使突變病毒無法進行同源重組來抵消突變。
本發(fā)明進一步還提供了一種通過給動物施用本發(fā)明的疫苗組合物來保護動物抵御病毒感染的方法。


圖1、BVDV-1的NADL株和BVDV-2的53637株的NS2-3區(qū)域的細胞插入和側翼病毒序列比對。
具體實施例方式與本發(fā)明一致被特別公認的是，在同一病毒基因組位點上包含突變的同一科突變病毒是不能相互進行重組來抵消突變的。
因此，在一種具體實施方式
中，本發(fā)明提供了一種安全的疫苗組合物，其包含至少兩種即兩種或更多，活的同一科突變病毒或是編碼所述病毒的核酸分子，其中病毒中的突變都存在于同一基因組位點上從而使突變病毒無法彼此間進行重組來抵消突變。
在另一種具體實施方式
中，本發(fā)明提供了一種制備如上文中描述的一種安全病毒疫苗的方法。尤其是通過篩選或構建兩種或更多活的同一科、屬或種的突變病毒來制備安全疫苗，其中每一種病毒中都包含能夠賦予所需表型(例如減毒作用、細胞趨向性或生物型的改造、物種趨向性的改造、或者外源基因盒的表達)的突變，而且病毒中的突變都存在于同一基因組位點上從而使突變病毒無法進行彼此間進行的同源重組來抵消突變。
術語“疫苗”或“疫苗組合物”是指包含活的突變病毒的組合物，在接種動物后誘導對致病型病毒的完全或部分免疫，或者緩和由致病型病毒引起的疾病癥狀。疫苗組合物抵御病毒的保護效果一般是由在受試者中誘導免疫應答來達到的，可以是細胞介導或是體液免疫應答，也可以是兩者的組合。一般而言，消除或減少病毒感染的發(fā)生、癥狀的改善、或者從感染者體內加速消滅病毒都是疫苗組合物保護效果的體現(xiàn)。
“動物”是指包括鳥類，例如雞、火雞、馴養(yǎng)的水禽，和任何一種哺乳動物，例如牛、羊、豬、山羊、狗、貓以及馬。
在此所用的術語“病毒”、“病毒隔離群”或者“病毒株”是指包含病毒基因組DNA或RNA、相關蛋白以及其他化學成分(例如脂類)的病毒粒子或病毒體。
“編碼病毒的核酸分子”或“病毒的核酸分子”是指病毒的基因組核酸分子，可以是RNA的形式，也可以是DNA的形式。
“突變”是指包括一個或多個核苷酸的缺失、插入或取代，或者它們的組合。根據(jù)本發(fā)明，突變優(yōu)選賦予所需表型，例如減毒作用、細胞趨向性或生物型的改造、物種趨向性的改造、或者外源基因盒的表達。更優(yōu)選的突變是能夠賦予削弱的毒性的突變。
“減毒作用”是指在感染病毒的動物體內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，當減毒病毒存在于減毒病毒的野生致病型能夠復制的動物體內時，它是完全不能復制或限于一輪或少數(shù)輪復制或者在細胞或組織趨向性上受到限制的病毒。
減毒病毒可能在涉及病毒致病性的一個或多個基因中具有突變。此類突變在此也稱為“減毒突變”。一種減毒病毒的制備可以是通過對野生型致病病毒在體外進行紫外照射、化學處理或者野生型致病病毒的高連續(xù)傳代(high serial passage)來產生。二者擇一地，一種減毒病毒還可以通過對野生型致病病毒已知能夠帶來毒性的病毒序列進行特異性缺失、在病毒基因組中進行序列的插入或者在病毒基因組中進行一個或多個點突變來制備。減毒病毒可以是從動物體內獲得的病毒隔離群，該隔離群是由野生致病型病毒經(jīng)過除了人工手段之外的事件而派生出來的，舉例來說，發(fā)生宿主動物體內如重組這一類的事件。
本發(fā)明免疫組合物中存在的兩種或更多的活的突變病毒包含著處于同一基因組位點上的突變。“同一基因組位點”是指當病毒的基因組核苷酸序列被對齊時，病毒基因組中的突變彼此重疊從而沒有機會在病毒基因組之間和之中進行同源重組來抵消突變。換句話說，當病毒的基因組核苷酸序列被對齊時，被對齊的序列中至少有一處鄰接部分中對齊的病毒基因組中的序列是突變序列。本領域技術人員可以利用不少計算機程序來對比和對齊核酸序列。對齊這些序列以達到最佳的對比目的(舉例來說，可以在核酸序列中引入缺口以帶來和另一個核酸序列的最佳對齊)。例如，Altschul等，1990，J.Mol.Biol.215403410中描述的NBLAST和XBLAST程序，Altschul等，1997，Nucleic Acids Res.2533893402中描述的Gapped BLAST程序，以及Altschul等，1997，同上中描述的PSI-Blast程序。當使用BLAST、Gapped BLAST以及PSI-Blast程序時，可以利用相應程序(舉例來說，XBLAST和NBLAST)的缺省參數(shù)(見http://www.ncbi.nlm.nih.gov)。
一般而言，本發(fā)明的概念，即包括在同一疫苗組合物中的兩種或更多的具有處于同一基因組位點突變的活的突變病毒，可應用于任一科的突變病毒，其中病毒基因組具有充分的序列同一性以容許同源重組。已經(jīng)表明，短至15個核苷酸的核苷酸同一性就能夠導致有效的同源重組(Nagy andBujarski，J.Virol.69131-140，1995)。
本發(fā)明尤其應用于黃病毒科(Flaviviridae)的病毒。黃病毒科(Flaviviridae)由疫病毒屬(Pestivirus)、黃病毒屬(Flavivirus)和丙型肝炎病毒屬(Hepacivirus)屬組成。黃病毒科(Flaviviridae)中成員的病毒粒子包封著約9.5到12.3kb的正鏈RNA基因組。該基因組RNA包含鄰接的長的開放讀碼框，其可以被翻譯為多蛋白質再經(jīng)過細胞和病毒蛋白酶的加工而產生成熟的病毒蛋白。
優(yōu)選的，本發(fā)明疫苗組合物中的突變病毒是來自于同一屬中相同種或不同種的。
在一個優(yōu)選的具體實施方式
中，本發(fā)明疫苗組合物包含兩種或更多的來自于疫病毒屬(Pestivirus)的活的突變病毒。疫病毒屬(Pestivirus)由牛病毒性腹瀉病毒類型-1(BVDV-1)、牛病毒性腹瀉病毒類型-2(BVDV-2)、經(jīng)典豬瘟病毒(classical swine fever virus)以及羊先天性髓質形成不全癥病毒(Border disease virus)種所代表。開放讀碼框(ORF)編碼大約3900個氨基酸的多蛋白質，其可以被協(xié)同翻譯和后翻譯加工成為下列成熟的病毒蛋白質(從5’端到3’端)Npro、C、Erns、E1、E2、NS2-3、NS4A、NS4B、NS5A以及NS5B。
通常情況下，BVDV具有RNA形式的基因組。RNA可以逆轉錄成DNA以用于克隆。因此，這里所給出的關于核酸和牛病毒性腹瀉病毒序列的參考都圍繞著病毒RNA序列以及由病毒RNA序列衍生出來的DNA序列。為便利起見，在下文序列表中描述的BVDV的基因組序列只涉及DNA序列。其各自相應的RNA序列對本領域技術人員是顯而易見的。
在一個更優(yōu)選的具體實施方式
中，本發(fā)明的疫苗組合物包含致細胞病變的BVDV-1和致細胞病變的BVDV-2，其中與致細胞病變生物型相關的兩病毒的突變均存在于同一基因組位點上從而使兩突變病毒無法重組來抵消突變。
BVDV-1和BVDV-2體現(xiàn)了牛病毒性腹瀉病毒(BVDV)的兩種非常相關的基因型。在整個基因組中，兩種病毒的核苷酸序列共有約70％的同一性，而且在NS2-3區(qū)域內有更高一些的同一性比率。可以認為在BVDV-1和BVDV-2病毒基因組之間，至少在NS2-3區(qū)域內，同一性比率足以容許發(fā)生同源重組。
BVDV-1在動物體內通常只引起輕度的腹瀉，然而BVDV-2則是具有高毒性的病毒其能夠引起血小板減少癥、大出血以及急性的致命疾病(Corapiet al.，J.Virol.633934-3943；Bolin et al.，Am.J.Vet.Res.532157-2163；Pellerin et al.，Virology 203260-268，1994；Ridpath et al.，Virology 20566-74，1994；Carman et al.，J.Vet.Diagn.Invest.1027-35，1998)。通過一系列單克隆抗體(MAbs)以及利用動物體內得到的病毒特異性抗血清的交叉中和決定了病毒的兩種類型具有截然不同的抗原性。(Corapi et al.，Am.J.Vet.Res.511388-1394，1990)。任一基因型的病毒都可以兩生物型中的一種存在致細胞病變的(cytopathogenic)(細胞病變的(cytopathic)或cp)或非致細胞病變的(noncytopathic)(非細胞病變(noncytopathic)的或ncp)。致細胞病變病毒對培養(yǎng)細胞產生細胞病變效應(如細胞溶菌[胞溶]作用)，而非致細胞病變病毒不會。
較為理想的是制備提供針對BVDV-1和BVDV-2兩者的保護的疫苗。然而，由于兩病毒之間高度的序列同一性，存在使包含在同一疫苗組合物中的活的致細胞病變BVDV-1和活的致細胞病變BVDV-2能夠在接種疫苗的動物體內互相重組而產生非致細胞病變病毒的可能性。BVDV-1與BVDV-2之間的重組是有文獻來證明的。見，例如，Ridpath et al.，Virology212259-262(1995)。在免疫能力產生以前，用ncP病毒感染懷孕牛體內胎畜能夠導致胎畜在懷孕期間保留病毒血癥而且在后來小牛的出生也會遺留下持續(xù)的病毒血癥。這種小牛會由于致細胞病變BVDV的雙重感染引起的粘膜病而死亡。因此，本發(fā)明提供的疫苗組合物中包含具有處于同一基因組位點的突變的活的致細胞病變BVDV-1和活的致細胞病變BVDV-2，能夠特別理想地保護動物抵御BVDV-1和BVDV-2兩種病毒。
在一個具體實施方式
中，可以將從動物體內獲得的牛病毒性腹瀉病毒(BVDV)致細胞病變隔離群應用于本發(fā)明的疫苗組合物。對本領域技術人員來說，BVDV-1和BVDV-2的致細胞病變隔離群都已經(jīng)被披露而且能夠獲得，舉例來說，BVDV-1NADL(ATCC#VR1422或VR-534)、BVDV-253637株(保藏于ATCC編號PTA-4859)、以及二型野外隔離群，此類菌種描述于Ridpath and Neill，J.Virol 748771-8774，(2000)。迄今為止所報道的致細胞病變隔離群典型地包含異源序列的插入物，例如，一種編碼泛激素的序列(Genbank入藏號M96687或De Moerlooze et al.，J.Gen.Virol.741433-1438，(1993))、編碼牛NEDD8的序列(Baroth et al.，supra)、或者編碼Bos taurus DnaJ1的序列(在下述的實施例中有所描述)及其他。
在另一具體實施方式
中，致細胞病變牛病毒性腹瀉病毒(cp BVDV)是通過在牛病毒性腹瀉病毒基因組中進行確定的改造來產生的，例如，通過缺失特異的病毒序列、在特異病毒基因組位點中插入序列、或者進行一個或多個取代、或者上述的組合。
通過在一個特異的基因組位點中嵌入異源(即對病毒而言外來的)序列來產生致細胞病變牛病毒性腹瀉病毒(cp BVDV)時，插入序列的性質對于本發(fā)明來說通常并不關鍵。另外，只要插入導致了一種減毒的表現(xiàn)型，插入就并不限于任何特定的位點。由于致細胞病變隔離群中的異源序列經(jīng)常發(fā)現(xiàn)于NS2-3區(qū)域中，所以NS2-3區(qū)域，尤其是圍繞推定的NS2-3分裂位點部分(其相應于BVDV-1NADL株#1679到#1680位的氨基酸殘基)(編號方式基于公開的基因組序列Genbank登記號No.M31182，SEQ ID NO4)，是優(yōu)選的插入位置。
致細胞病變的BVDV-1可以通過進行確定的基因改造來產生，所述基因改造模擬從動物體內獲得的致細胞病變BVDV-2隔離群中識別出的突變，于是這些病毒在同一基因組位點中就具有與致細胞病變生物型相關的突變。類似地，致細胞病變的BVDV-2也可以通過進行確定的基因改造的方法來產生所述改造模擬從動物體內獲得的致細胞病變BVDV-1隔離群中識別出的突變。
在優(yōu)選的具體實施方式
中，本發(fā)明的疫苗組合物包含NADL(致細胞病變BVDV-1隔離群)和BVDV-2 53637(致細胞病變BVDV-2隔離群)，其中兩致細胞病變隔離群均在同一基因組位點上含有引起致細胞病變生物型的突變。在SEQ ID NO4中闡明了BVDV-1NADL株的基因組序列，而BVDV-2 53637株保藏于ATCC，編號為PTA-4859。兩種隔離群在NS2-3區(qū)域都含有插入。減毒的致細胞病變BVDV-1在核苷酸位置#4993(NADL序列編號方式)的胸腺嘧啶的3’端含有Bos Taurus DnaJ1編碼序列的插入，所述胸腺嘧啶是編碼在氨基酸1536位置的甘氨酸殘基的密碼子第三個核苷酸。減毒的致細胞病變BVDV-2在同一基因組位點含有一個Bos taurusDnaJ1編碼序列的插入。
根據(jù)本發(fā)明，在所述疫苗組合物中使用的致細胞病變牛病毒性腹瀉病毒(cp BVDV)隔離群已經(jīng)得到減毒而且由此成為非致細胞病變的。對本領域技術人員來說減毒的方法是已知的而且下文中也會敘述。
在另一具體實施方式
中，本發(fā)明的疫苗組合物包含減毒的BVDV-1和減毒的BVDV-2，其中兩病毒中的減毒突變都存在于同一基因組位點上從而使兩突變病毒無法重組而抵消減毒突變。
減毒的BVDV可以通過在體外進行紫外照射、化學處理或者致病型病毒的高連續(xù)傳代(high serial passage)來制備?？梢杂眯蛄蟹治鰜泶_定這些方法產生的突變的性質和基因組位置。突變可以是缺失、插入或者取代一個或多個核苷酸的形式，或是上述的組合。二者擇一地，減毒的BVDV還可以通過在BVDV基因組中進行確定的改造來產生，例如通過缺失特異性病毒序列、在特異性病毒基因組位點中插入序列，或制備一個或多個取代或者其組合。
如上所述，用于本發(fā)明疫苗組合物的活的突變病毒可以來自同一科、屬或種，其中病毒基因組具有充分的序列同一性以容許同源重組。適用于本發(fā)明疫苗組合物的病毒組合的其他例子還包括但不限于，不同型脊髓灰質炎病毒的組合、傳染性支氣管炎病毒多重活突變株的組合、新城雞瘟病毒多重活突變株的組合、犬腺病毒-1和犬腺病毒-2的組合、馬皰疹病毒-1和馬皰疹病毒-4的組合、流感病毒多重活突變株的組合、貓杯狀病毒多重活減毒株的組合、輪狀病毒多重血清型的組合、鼻病毒多重血清型的組合、口蹄疫病毒多重血清型的組合、豬生殖和呼吸綜合癥病毒歐洲和北美基因型的組合、傳染性滑囊癥病毒標準和變異株的組合。
按照本發(fā)明，病毒粒子雖然是用于疫苗的優(yōu)選形式，但編碼同一科、屬或種突變病毒的核酸分子也同樣可以直接用于疫苗。DNA或RNA分子可以以“裸”的形式存在或者也可以結合能夠幫助細胞吸收的試劑(例如，脂質體或陽離子脂類)?，F(xiàn)有技術中已經(jīng)詳細描述了應用核酸(DNA或mRNA)的疫苗和接種工藝，例如，美國專利No.5,703,055、美國專利No.5,580,859、美國專利No.5,589,466、國際專利公開WO 98/35562、以及Ramsayet al.，1997，Immunol.Cell Biol.75360-363、Davis，1997，Cur.Opinion Biotech.8635-640、Manickan et al.，1997，Critical Rev.Immunol.17139-154、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.18449-484、以及Vogel and Sarver，1995，Clin.Microbiol.Rev.8(3)406-410，所有這些在此合并為參考文獻。
除了兩種或更多來自同一科、屬或種的活突變病毒以外，疫苗組合物還可以包括其他的抗原成分。適用于本發(fā)明的其他抗原成分還包括但不限于，產生于下列病原菌的抗原，例如，肺炎支原體(Mycoplasmahyopneumonia)、睡眠嗜血桿菌(Haemophilus somnus)、副豬嗜血桿菌(Haemophilus parasuis)、支氣管敗血波氏桿菌(Bordetella bronchiseptica)、炭疽芽胞桿菌(Bacillus anthracis)、豬胸膜肺炎放線桿菌(Actinobacilluspleuropneumonie)、多殺性巴氏桿菌(Pasteurella multocida)、Mannhemiahaemolytica、牛支原體(Mycoplasma bovis)、Mycoplasma galanacieum、雞毒支原體(Mycoplasma gallisepticum)、牛分枝桿菌(Mycobacterium bovis)、副結核分支桿菌(Mycobacterium paratuberculosis)、梭狀芽孢桿菌屬的種(Clostridial spp.)、乳房鏈球菌(Streptococcus uberis)、豬鏈球菌(Streptococcus suis)、金黃色葡萄球菌、Erysipelothrix rhusopathiae、彎曲桿菌屬的種(Campylobacter spp.)、壞死梭桿菌(Fusobacterium necrophorum)、埃希氏大腸桿菌、細胞內勞森菌(Lawsonia intracellularis)、單核細胞增生性李斯特菌(Listeria monocytogenes)、立氏立克次體(Rickettsia rickettsii)、疏螺旋體屬的種(Borrelia spp.)、艾利希氏體屬的種(Ehrlichia spp.)、衣原體屬的種(Chlamydia spp.)、布氏桿菌屬的種(Brucella spp.)、弧菌屬的種(Vibriospp.)、Salmonella enterica serovars、鉤端螺旋體屬的種(Leptospira spp.)；致病的真菌類如念珠菌屬，原生動物如小隱孢子蟲(Cryptosporidium parvum)、Neospora canium、剛地弓形蟲(Toxoplasma gondii)、愛美爾球蟲(Eimeriaspp)、梨漿蟲屬(Babesia spp)、賈第蟲屬(Giardia spp)；寄生蟲如奧斯特線蟲屬(Ostertagia)、古柏線蟲屬(Cooperia)、血矛線蟲(Haemonchus)、吸蟲(Fasciola)；它們以一種非活性的整個或部分細胞制劑的形式、或者以一種由遺傳工程技術或化學合成法獲得的抗原分子的形式存在。其他的抗原還包括了致病病毒例如馬立克氏病病毒(Marek’s disease virus)、傳染性法氏囊病病毒(Infectious bursal disease virus)、新城疫病毒(Newcastle’sdisease virus)、雞貧血病病毒(chicken anemia virus)、禽痘病毒(fowlpox virus)、禽白血病病毒(avian leukosis virus)、傳染性喉氣管炎病毒(infectiouslaryngotracheitis virus)、網(wǎng)狀內皮病毒(reticuloendothelial virus)、犬小病毒(canine parvovirus)、犬瘟病毒(canine distemper virus)、犬皰疹病毒(canineherpesvirus)、犬冠狀病毒(canine coronavirus)、犬副流感病毒-5(canineparainfluenza-5)、貓泛白細胞減少癥病毒(feline panleukopenia virus)、貓皰疹病毒(feline herpes virus)、貓卡性病毒(feline calicivirus)、貓免疫缺乏病毒、貓傳染性腹膜炎病毒(feline infectious peritonitis virus)、馬皰疹病毒、馬動脈炎病毒、馬傳染性貧血病毒、東部馬腦炎病毒、西部馬腦炎病毒、委內瑞拉馬腦炎病毒、西尼羅河病毒、傳染性胃腸炎病毒、牛冠狀病毒、牛皰疹病毒-1，3，6、牛副流行性感冒病毒、牛呼吸道合胞體病毒、牛白血病病毒、牛疫病毒(rinderpest virus)、口蹄疫病毒、狂犬病病毒、非洲豬瘟病毒(African swine fever virus)、豬細小病毒(Porcine parvovirus)、豬呼吸道冠狀病毒(PRRS virus)、豬圓環(huán)病毒(Porcine circovirus)、流感病毒、豬水皰病病毒、Techen出血熱病毒(Techen frver virus)、偽狂犬病病毒(Pseudorabiesvirus)，它們以一種改良的活的(減毒的)病毒制劑形式、一種非活性的全部或部分病毒制劑形式，或者以一種由遺傳工程技術或化學合成法獲得的抗原分子的形式存在。當使用其他減毒活病毒時，這些其他病毒優(yōu)選來自與兩種主要的減毒病毒不同的科，如上所述。
在一個優(yōu)選的實施例中，本發(fā)明提供了疫苗組合物，其含有源自BVDV-1NADL株的減毒致細胞病變BVDV-1，和源自BVDV-253637株的減毒致細胞病變BVDV-2，其中兩致細胞病變分離株在同一基因組位點均含有與致細胞病變生物型相關的突變，而且還含有至少一個(即一個或多個)下列處于非活性或改良活性形式的抗原成分牛皰疹病毒-1、牛呼吸道合胞病毒、副流感病毒-3、胚胎彎曲柑桔、犬鉤端螺旋體(Leptospiracanicola)、流感傷寒鉤端螺旋體(Leptospira grippotyphosa)、哈德焦鉤端螺旋體(Leptospira hardjo)、出血性黃疸鉤端螺旋體(Leptospiraicterohaemorrhagiae)、波摩那鉤端螺旋體(Leptospira pomona)、或者Mannhemia haemolytica。
此外，本發(fā)明的疫苗組合物還可以包括一個或多個獸醫(yī)學可接受的載體。此處所用的“一種獸醫(yī)學可接受的載體”包括任何和所有溶劑、分散介質、包覆層、佐劑、穩(wěn)定劑、稀釋劑、防腐劑、抗細菌以及抗真菌藥、等滲劑、吸附延遲劑、以及類似物質。稀釋劑可以包括水、生理鹽水、葡萄糖、乙醇、甘油、以及類似物質。等滲劑可以包括氯化鈉、葡萄糖、甘露醇、山梨糖醇、以及乳糖，及其他。穩(wěn)定劑包括白蛋白，及其他。該疫苗組合物還可以進一步的包括一種或多種其他免疫調節(jié)劑例如白細胞介素、干擾素、或其他細胞活素。
適用于該疫苗組合物的佐劑包括但不限于RIBI佐劑系統(tǒng)(Ribi Inc.)、明礬、氫氧化鋁凝膠、水包油乳劑、油包水乳劑例如完全以及不完全弗氏佐劑、Block共聚物(CytRx，Atlanta GA)，SAF-M(Chiron，Emeryville CA)，AMPHIGEN佐劑、皂甙、Quil A、膽固醇、QS-21(Cambridge Biotech Inc.，Cambridge MA)、或其他皂甙片段、單磷酰類脂A、Avridine脂類胺(lipid-amine)佐劑、來自于大腸桿菌的熱不穩(wěn)定性腸毒素(重組或別的方式)、霍亂毒素、或胞壁酰二肽，以及許多其他佐劑。
通常情況下，存在于疫苗中的活突變病毒的數(shù)量在每劑量大約1×106到大約1×108病毒粒子，配以一種獸醫(yī)學可接受的載體，體積大約在0.5到5毫升之間。在疫苗組合物中能夠有效提供保護效應的病毒的精確的量可以由熟練的獸醫(yī)來決定。當病毒的DNA或RNA分子用于該疫苗時，核酸的量一般應該在大約0.1μg/ml到大約5.0mg/ml之間。
本發(fā)明的疫苗組合物可以根據(jù)給藥途徑制成多種形式。例如，該疫苗組合物可以制成適用于注射用途的無菌水溶液或分散系的形式，或者利用制冷干燥技術制成凍干形式。凍干組合物一般保存在4℃左右，而且可以在配以或不配以佐劑的情況下在穩(wěn)定溶液例如生理鹽水或和HEPES中還原。
本發(fā)明的疫苗組合物可以給動物施用以治療或預防由疫苗組合物中病毒的致病類型所引起的疾病。因此，接種動物以抵抗由病毒所引起疾病的方法也提供于本發(fā)明中。
在實施該方法的過程中，本發(fā)明疫苗組合物給動物施用優(yōu)選是通過腸道外途徑給藥，雖然其他的給藥途徑也可以同樣適用，例如通過口服、鼻腔內、肌肉內、淋巴結內、真皮下、腹膜內、皮下、直腸或陰道給藥、或通過聯(lián)合的途徑。為了提供最適宜的疫苗接種，可能需要追加劑量方案，而且還可以調整劑量方案。
本發(fā)明通過如下的實施例進行進一步的闡述，但并不意味著限制于此。
實施例實施例1定位BVDV2 53637株中的細胞嵌入位置確定來自于BVDV2-53637的NS2-3區(qū)域的序列的一部分，用于識別和定位在此區(qū)域中的任何細胞插入的位置。使用前向引物53637U1(5’-CGTCCACAGATGGTTTGGT-3’；SEQ ID NO1)和逆向引物53637L(5’-GGCTATGTATTGGACGTAACCC-3’；SEQ ID NO2)從病毒RNA擴增670堿基的RT-PCR產物。純化該RT-PCR產物和進行序列分析(SEQ ID NO3)。當與BVDV1-NADL(Genbank入藏號M31182，SEQ ID NO4)進行比對時，觀察到了顯著的相似性(圖1)。兩種病毒都含有由Bos taurus DnaJ1基因派生出來的框內插入。就NADL來說，插入物的長度為90個氨基酸(270個核苷酸)，定位在NADL多蛋白質中1536位甘氨酸和1627位脯氨酸之間。這些坐標相應于非致細胞病變BVDV1株例如SD-1(Genbank入藏號AAA42860，SEQ ID NO6)中的1536位甘氨酸和1537位脯氨酸，說明NADL中的基因組改造是簡單的嵌入，并不伴有側翼病毒序列的缺失或復制。與BVDV1-NADL類似，BVDV2-53637中有Bos taurus DnaJ1基因的一部分的插入。細胞插入物會更長(131個氨基酸，393個核苷酸)，相對于BVDV1-NADL中的插入在兩個方向上均有延長。在兩病毒中NS2-3區(qū)域內細胞插入的位置是相同的。不同于BVDV1-NADL，BVDV2-53637的插入伴有側翼病毒序列的5個氨基酸(15核苷酸)的缺失。在插入物的5’末端側翼有三個氨基酸殘基的缺失，同時在插入物的3’末端側翼有兩個氨基酸殘基的缺失。由于該細胞插入在兩疫苗病毒中的同一基因組位點上，所以它們無法經(jīng)過同源重組消除插入來產生非致細胞病變的嵌合病毒。
實施例2在共傳代的BVDV1-NADL/BVDV2-53637培養(yǎng)基中試圖檢測非細胞病變BVDV病毒為了確定兩疫苗病毒是否能夠通過重組來產生可觀測水平的非致細胞病變BVDV，在易感細胞上共同培養(yǎng)病毒，而且使用敏感的半嵌套RT-PCR試驗從過量的仍含有細胞插入的較長期致細胞病變產物中來檢測潛在的非致細胞病變病毒。為了增加體外類型間重組的可能性，每個病毒都在6孔板中以感染復數(shù)2-4同時接種于滿板BK-6細胞(每次實驗12次重復)。2-3天共同培養(yǎng)以后對細胞進行兩次冷凍和解凍，然后通過低速離心清除細胞碎片。將得到的上清液作為下一次傳代的接種物。在幾次研究中總共進行了七次連續(xù)的傳代。在傳代過程中，BVDV1-NADL遠比BVDV2-53637生長得快，但是七次傳代以后利用嵌套RT-PCR仍然可以檢測出II型病毒。使用敏感的半嵌套RT-PCR試驗來檢測出任何非致細胞病變病毒的存在。
在第一輪RT-PCR中，將前向引物53637U1(SEQ ID NO1)或者NADL4744(5’-CGTGGCTTCTTGGTACGGG-3’，SEQ ID NO7)協(xié)同逆向引物53637L(SEQ ID NO2)或者NADL5305(5’-AGCGGTATATTGTACAAAG CCA-3’，SEQ IDNO8)一起使用。前向和逆向引物的所有四個組合都是為了檢測BVDV1、BVDV2、以及類型間重組的。所需的RT-PCR產物大小對致細胞病變BVDV1-NADL來說是562bp，而對致細胞病變BVDV2-53637來說是670bp。如果存在可觀測水平的非致細胞病變病毒，預期應生成第一輪產物292bp(BVDV1-NADL)或者277bp(BVDV2-53637)。類型間重組體的大小應當與其中一個親本相似，或者介于中間的長度，這取決于重組位點的位置。第一輪RT-PCR后沒有檢測出非致細胞病變BVDV。
在有過量的致細胞病變BVDV存在情況下為了增加檢測非致細胞病變BVDV的敏感度，在嵌套PCR之前還包括了限制性內切酶消化的步驟用以破壞派生于致細胞病變病毒的較大的NS2-3模板。在觀察到MspI和DraI切割Bos taurus DnaJ1插入物而并不切割側翼病毒序列的基礎上，選擇了MspI和DraI的組合。在第二輪(半嵌套)PCR中，將前向引物53637U2(5’-TGCACGATCTGTGAAGGGAAAGAA-3’，SEQ ID NO9)或者NADL4844(5’-TGCACTGTATGTGAGGGCCGAGAG-3’，SEQ ID NO10)協(xié)同相同的兩個逆向引物53637L或者NADL5305一起使用。使用適當?shù)囊锝M合來試圖檢測類型間重組體和BVDV1和BVDV2。所需的RT-PCR產物大小對致細胞病變BVDV1-NADL來說是462bp，而對致細胞病變BVDV2-53637來說是570bp(由于致細胞病變BVDV RT-PCR產物的不完全消化以低水平存在)。如果存在可觀測水平的非致細胞病變病毒，預期應產生192bp(BVDV1-NADL)或者177bp(BVDV2-53637)的第二輪產物。類型間重組體的大小應當與其中一個親本相似，或者介于中間的長度，這取決于重組位點的位置。第二輪RT-PCR后沒有檢測出非致細胞病變BVDV。在一些個別的反應中，看到了各種大小的異常條帶。所有介于100到300bp之間的條帶都被認為是潛在的非致細胞病變產物并提交了DNA序列分析。每種情況下異常條帶都是PCR過程中錯誤引發(fā)的結果。在所有研究中并沒有非致細胞病變病毒存在的證據(jù)。
序列表<110>輝瑞產品公司(Pfizer Products Inc)Welch，Siao-Kun WanCalvert，Jay GregoryO’Hara，Michael K.
Cao，Xuemei K.
<120>安全的突變病毒疫苗<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>1cgtccacaga tggtttggt 19<210>2<211>22<212>DNA<213>人工序列<220>
<223>合成逆向引物53637L<400>2ggctatgtat tggacgtaac cc 22<210>3<211>670<212>DNA<213>牛病毒性腹瀉病毒253637株<400>3cgtccacaga tggtttggtg aggaggaaat atatggggca cccaaggtga tcaccatcat 60aaaagctagt accctaagta aaaacaggca ctgcataatc tgcacgatct gtgaagggaa 120agaatggaac ggagccaact gcccaaagtg tggaagacaa ggaaagccca taacatgtgg 180aatgacactc gcagactttg aggagaaaca ttacaaaaag atatttataa gagaaggacg 240ccaagaagca atgaatacga tgatgtgcag ccgatgccag ggaaagcata ggaggtttga 300
aacggaccgg gaacctaaga gtgccagata ctgtgctgag tgtaataggc tgcatcctgc 360tgaggaaggt gacttttggg cagagtcaag catgttgggc ctcaaaatca cctactttgc 420gctgatggat ggaaaggtgt atgatatcac agagtgggct ggatgccagc gtgtgggaat 480ctccccagat acccacagag tcccttgtca catctcattt ggttcacgga tgccaggcac 540cagtgggcgg cagagagcta ctccagatgc ccctcctgct gaccttcagg atttcttgag 600ccggatcttt caagtacccc caggccagat gtccagggaa gagtataagg gttacgtcca 660atacatagcc670<210>4<211>12573<212>DNA<213>牛病毒性腹瀉病毒NADL 1株<400>4gtatacgaga attagaaaag gcactcgtat acgtattggg caattaaaaa taataattag 60gcctagggaa caaatccctc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcata atgagggggg tagcaacagt ggtgagttcg ttggatggct taagccctga 180gtacagggta gtcgtcagtg gttcgacgcc ttggaataaa ggtctcgaga tgccacgtgg 240acgagggcat gcccaaagca catcttaacc tgagcggggg tcgcccaggt aaaagcagtt 300ttaaccgact gttacgaata cagcctgata gggtgctgca gaggcccact gtattgctac 360taaaaatctc tgctgtacat ggcacatgga gttgatcaca aatgaacttt tatacaaaac 420atacaaacaa aaacccgtcg gggtggagga acctgtttat gatcaggcag gtgatccctt 480atttggtgaa aggggagcag tccaccctca atcgacgcta aagctcccac acaagagagg 540ggaacgcgat gttccaacca acttggcatc cttaccaaaa agaggtgact gcaggtcggg 600taatagcaga ggacctgtga gcgggatcta cctgaagcca gggccactat tttaccagga 660ctataaaggt cccgtctatc acagggcccc gctggagctc tttgaggagg gatccatgtg 720tgaaacgact aaacggatag ggagagtaac tggaagtgac ggaaagctgt accacattta 780tgtgtgtata gatggatgta taataataaa aagtgccacg agaagttacc aaagggtgtt 840caggtgggtc cataataggc ttgactgccc tctatgggtc acaacttgct cagacacgaa 900agaagaggga gcaacaaaaa agaaaacaca gaaacccgac agactagaaa gggggaaaat 960gaaaatagtg cccaaagaat ctgaaaaaga cagcaaaact aaacctccgg atgctacaat1020agtggtggaa ggagtcaaat accaggtgag gaagaaggga aaaaccaaga gtaaaaacac1080tcaggacggc ttgtaccata acaaaaacaa acctcaggaa tcacgcaaga aactggaaaa1140
agcattgttg gcgtgggcaa taatagctat agttttgttt caagttacaa tgggagaaaa1200cataacacag tggaacctac aagataatgg gacggaaggg atacaacggg caatgttcca1260aaggggtgtg aatagaagtt tacatggaat ctggccagag aaaatctgta ctggcgtccc1320ttcccatcta gccaccgata tagaactaaa aacaattcat ggtatgatgg atgcaagtga1380gaagaccaac tacacgtgtt gcagacttca acgccatgag tggaacaagc atggttggtg1440caactggtac aatattgaac cctggattct agtcatgaat agaacccaag ccaatctcac1500tgagggacaa ccaccaaggg agtgcgcagt cacttgtagg tatgataggg ctagtgactt1560aaacgtggta acacaagcta gagatagccc cacaccctta acaggttgca agaaaggaaa1620gaacttctcc tttgcaggca tattgatgcg gggcccctgc aactttgaaa tagctgcaag1680tgatgtatta ttcaaagaac atgaacgcat tagtatgttc caggatacca ctctttacct1740tgttgacggg ttgaccaact ccttagaagg tgccagacaa ggaaccgcta aactgacaac1800ctggttaggc aagcagctcg ggatactagg aaaaaagttg gaaaacaaga gtaagacgtg1860gtttggagca tacgctgctt ccccttactg tgatgtcgat cgcaaaattg gctacatatg1920gtatacaaaa aattgcaccc ctgcctgctt acccaagaac acaaaaattg tcggccctgg1980gaaatttggc accaatgcag aggacggcaa gatattacat gagatggggg gtcacttgtc2040ggaggtacta ctactttctt tagtggtgct gtccgacttc gcaccggaaa cagctagtgt2100aatgtaccta atcctacatt tttccatccc acaaagtcac gttgatgtaa tggattgtga2160taagacccag ttgaacctca cagtggagct gacaacagct gaagtaatac cagggtcggt2220ctggaatcta ggcaaatatg tatgtataag accaaattgg tggccttatg agacaactgt2280agtgttggca tttgaagagg tgagccaggt ggtgaagtta gtgttgaggg cactcagaga2340tttaacacgc atttggaacg ctgcaacaac tactgctttt ttagtatgcc ttgttaagat2400agtcaggggc cagatggtac agggcattct gtggctacta ttgataacag gggtacaagg2460gcacttggat tgcaaacctg aattctcgta tgccatagca aaggacgaaa gaattggtca2520actgggggct gaaggcctta ccaccacttg gaaggaatac tcacctggaa tgaagctgga2580agacacaatg gtcattgctt ggtgcgaaga tgggaagtta atgtacctcc aaagatgcac2640gagagaaacc agatatctcg caatcttgca tacaagagcc ttgccgacca gtgtggtatt2700caaaaaactc tttgatgggc gaaagcaaga ggatgtagtc gaaatgaacg acaactttga2760atttggactc tgcccatgtg atgccaaacc catagtaaga gggaagttca atacaacgct2820
gctgaacgga ccggccttcc agatggtatg ccccatagga tggacaggga ctgtaagctg2880tacgtcattc aatatggaca ccttagccac aactgtggta cggacatata gaaggtctaa2940accattccct cataggcaag gctgtatcac ccaaaagaat ctgggggagg atctccataa3000ctgcatcctt ggaggaaatt ggacttgtgt gcctggagac caactactat acaaaggggg3060ctctattgaa tcttgcaagt ggtgtggcta tcaatttaaa gagagtgagg gactaccaca3120ctaccccatt ggcaagtgta aattggagaa cgagactggt tacaggctag tagacagtac3180ctcttgcaat agagaaggtg tggccatagt accacaaggg acattaaagt gcaagatagg3240aaaaacaact gtacaggtca tagctatgga taccaaactc ggacctatgc cttgcagacc3300atatgaaatc atatcaagtg aggggcctgt agaaaagaca gcgtgtactt tcaactacac3360taagacatta aaaaataagt attttgagcc cagagacagc tactttcagc aatacatgct3420aaaaggagag tatcaatact ggtttgacct ggaggtgact gaccatcacc gggattactt3480cgctgagtcc atattagtgg tggtagtagc cctcttgggt ggcagatatg tactttggtt3540actggttaca tacatggtct tatcagaaca gaaggcctta gggattcagt atggatcagg3600ggaagtggtg atgatgggca acttgctaac ccataacaat attgaagtgg tgacatactt3660cttgctgctg tacctactgc tgagggagga gagcgtaaag aagtgggtct tactcttata3720ccacatctta gtggtacacc caatcaaatc tgtaattgtg atcctactga tgattgggga3780tgtggtaaag gccgattcag ggggccaaga gtacttgggg aaaatagacc tctgttttac3840aacagtagta ctaatcgtca taggtttaat catagctagg cgtgacccaa ctatagtgcc3900actggtaaca ataatggcag cactgagggt cactgaactg acccaccagc ctggagttga3960catcgctgtg gcggtcatga ctataaccct actgatggtt agctatgtga cagattattt4020tagatataaa aaatggttac agtgcattct cagcctggta tctgcggtgt tcttgataag4080aagcctaata tacctaggta gaatcgagat gccagaggta actatcccaa actggagacc4140actaacttta atactattat atttgatctc aacaacaatt gtaacgaggt ggaaggttga4200cgtggctggc ctattgttgc aatgtgtgcc tatcttattg ctggtcacaa ccttgtgggc4260cgacttctta accctaatac tgatcctgcc tacctatgaa ttggttaaat tatactatct4320gaaaactgtt aggactgata cagaaagaag ttggctaggg gggatagact atacaagagt4380tgactccatc tacgacgttg atgagagtgg agagggcgta tatctttttc catcaaggca4440gaaagcacag gggaattttt ctatactctt gccccttatc aaagcaacac tgataagttg4500cgtcagcagt aaatggcagc taatatacat gagttactta actttggact ttatgtacta4560
catgcacagg aaagttatag aagagatctc aggaggtacc aacataatat ccaggttagt4620ggcagcactc atagagctga actggtccat ggaagaagag gagagcaaag gcttaaagaa4680gttttatcta ttgtctggaa ggttgagaaa cctaataata aaacataagg taaggaatga4740gaccgtggct tcttggtacg gggaggagga agtctacggt atgccaaaga tcatgactat4800aatcaaggcc agtacactga gtaagagcag gcactgcata atatgcactg tatgtgaggg4860ccgagagtgg aaaggtggca cctgcccaaa atgtggacgc catgggaagc cgataacgtg4920tgggatgtcg ctagcagatt ttgaagaaag acactataaa agaatcttta taagggaagg4980caactttgag ggtatgtgca gccgatgcca gggaaagcat aggaggtttg aaatggaccg5040ggaacctaag agtgccagat actgtgctga gtgtaatagg ctgcatcctg ctgaggaagg5100tgacttttgg gcagagtcga gcatgttggg cctcaaaatc acctactttg cgctgatgga5160tggaaaggtg tatgatatca cagagtgggc tggatgccag cgtgtgggaa tctccccaga5220tacccacaga gtcccttgtc acatctcatt tggttcacgg atgcctttca ggcaggaata5280caatggcttt gtacaatata ccgctagggg gcaactattt ctgagaaact tgcccgtact5340ggcaactaaa gtaaaaatgc tcatggtagg caaccttgga gaagaaattg gtaatctgga5400acatcttggg tggatcctaa gggggcctgc cgtgtgtaag aagatcacag agcacgaaaa5460atgccacatt aatatactgg ataaactaac cgcatttttc gggatcatgc caagggggac5520tacacccaga gccccggtga ggttccctac gagcttacta aaagtgagga ggggtctgga5580gactgcctgg gcttacacac accaaggcgg gataagttca gtcgaccatg taaccgccgg5640aaaagatcta ctggtctgtg acagcatggg acgaactaga gtggtttgcc aaagcaacaa5700caggttgacc gatgagacag agtatggcgt caagactgac tcagggtgcc cagacggtgc5760cagatgttat gtgttaaatc cagaggccgt taacatatca ggatccaaag gggcagtcgt5820tcacctccaa aagacaggtg gagaattcac gtgtgtcacc gcatcaggca caccggcttt5880cttcgaccta aaaaacttga aaggatggtc aggcttgcct atatttgaag cctccagcgg5940gagggtggtt ggcagagtca aagtagggaa gaatgaagag tctaaaccta caaaaataat6000gagtggaatc cagaccgtct caaaaaacag agcagacctg accgagatgg tcaagaagat6060aaccagcatg aacaggggag acttcaagca gattactttg gcaacagggg caggcaaaac6120cacagaactc ccaaaagcag ttatagagga gataggaaga cacaagagag tattagttct6180tataccatta agggcagcgg cagagtcagt ctaccagtat atgagattga aacacccaag6240catctctttt aacctaagga taggggacat gaaagagggg gacatggcaa ccgggataac 6300
ctatgcatca tacgggtact tctgccaaat gcctcaacca aagctcagag ctgctatggt6360agaatactca tacatattct tagatgaata ccattgtgcc actcctgaac aactggcaat6420tatcgggaag atccacagat tttcagagag tataagggtt gtcgccatga ctgccacgcc6480agcagggtcg gtgaccacaa caggtcaaaa gcacccaata gaggaattca tagcccccga6540ggtaatgaaa ggggaggatc ttggtagtca gttccttgat atagcagggt taaaaatacc6600agtggatgag atgaaaggca atatgttggt ttttgtacca acgagaaaca tggcagtaga6660ggtagcaaag aagctaaaag ctaagggcta taactctgga tactattaca gtggagagga6720tccagccaat ctgagagttg tgacatcaca atccccctat gtaatcgtgg ctacaaatgc6780tattgaatca ggagtgacac taccagattt ggacacggtt atagacacgg ggttgaaatg6840tgaaaagagg gtgagggtat catcaaagat acccttcatc gtaacaggcc ttaagaggat6900ggccgtgact gtgggtgagc aggcgcagcg taggggcaga gtaggtagag tgaaacccgg6960gaggtattat aggagccagg aaacagcaac agggtcaaag gactaccact atgacctctt7020gcaggcacaa agatacggga ttgaggatgg aatcaacgtg acgaaatcct ttagggagat7080gaattacgat tggagcctat acgaggagga cagcctacta ataacccagc tggaaatact7140aaataatcta ctcatctcag aagacttgcc agccgctgtt aagaacataa tggccaggac7200tgatcaccca gagccaatcc aacttgcata caacagctat gaagtccagg tcccggtcct7260attcccaaaa ataaggaatg gagaagtcac agacacctac gaaaattact cgtttctaaa7320tgccagaaag ttaggggagg atgtgcccgt gtatatctac gctactgaag atgaggatct7380ggcagttgac ctcttagggc tagactggcc tgatcctggg aaccagcagg tagtggagac7440tggtaaagca ctgaagcaag tgaccgggtt gtcctcggct gaaaatgccc tactagtggc7500tttatttggg tatgtgggtt accaggctct ctcaaagagg catgtcccaa tgataacaga7560catatatacc atcgaggacc agagactaga agacaccacc cacctccagt atgcacccaa7620cgccataaaa accgatggga cagagactga actgaaagaa ctggcgtcgg gtgacgtgga7680aaaaatcatg ggagccattt cagattatgc agctggggga ctggagtttg ttaaatccca7740agcagaaaag ataaaaacag ctcctttgtt taaagaaaac gcagaagccg caaaagggta7800tgtccaaaaa ttcattgact cattaattga aaataaagaa gaaataatca gatatggttt7860gtggggaaca cacacagcac tatacaaaag catagctgca agactggggc atgaaacagc7920gtttgccaca ctagtgttaa agtggctagc ttttggaggg gaatcagtgt cagaccacgt7980caagcaggcg gcagttgatt tagtggtcta ttatgtgatg aataagcctt ccttcccagg8040
tgactccgag acacagcaag aagggaggcg attcgtcgca agcctgttca tctccgcact8100ggcaacctac acatacaaaa cttggaatta ccacaatctc tctaaagtgg tggaaccagc8160cctggcttac ctcccctatg ctaccagcgc attaaaaatg ttcaccccaa cgcggctgga8220gagcgtggtg atactgagca ccacgatata taaaacatac ctctctataa ggaaggggaa8280gagtgatgga ttgctgggta cggggataag tgcagccatg gaaatcctgt cacaaaaccc8340agtatcggta ggtatatctg tgatgttggg ggtaggggca atcgctgcgc acaacgctat8400tgagtccagt gaacagaaaa ggaccctact tatgaaggtg tttgtaaaga acttcttgga8460tcaggctgca acagatgagc tggtaaaaga aaacccagaa aaaattataa tggccttatt8520tgaagcagtc cagacaattg gtaaccccct gagactaata taccacctgt atggggttta8580ctacaaaggt tgggaggcca aggaactatc tgagaggaca gcaggcagaa acttattcac8640attgataatg tttgaagcct tcgagttatt agggatggac tcacaaggga aaataaggaa8700cctgtccgga aattacattt tggatttgat atacggccta cacaagcaaa tcaacagagg8760gctgaagaaa atggtactgg ggtgggcccc tgcacccttt agttgtgact ggacccctag8820tgacgagagg atcagattgc caacagacaa ctatttgagg gtagaaacca ggtgcccatg8880tggctatgag atgaaagctt tcaaaaatgt aggtggcaaa cttaccaaag tggaggagag8940cgggcctttc ctatgtagaa acagacctgg taggggacca gtcaactaca gagtcaccaa9000gtattacgat gacaacctca gagagataaa accagtagca aagttggaag gacaggtaga9060gcactactac aaaggggtca cagcaaaaat tgactacagt aaaggaaaaa tgctcttggc9120cactgacaag tgggaggtgg aacatggtgt cataaccagg ttagctaaga gatatactgg9180ggtcgggttc aatggtgcat acttaggtga cgagcccaat caccgtgctc tagtggagag9240ggactgtgca actataacca aaaacacagt acagtttcta aaaatgaaga aggggtgtgc9300gttcacctat gacctgacca tctccaatct gaccaggctc atcgaactag tacacaggaa9360caatcttgaa gagaaggaaa tacccaccgc tacggtcacc acatggctag cttacacctt9420cgtgaatgaa gacgtaggga ctataaaacc agtactagga gagagagtaa tccccgaccc9480tgtagttgat atcaatttac aaccagaggt gcaagtggac acgtcagagg ttgggatcac9540aataattgga agggaaaccc tgatgacaac gggagtgaca cctgtcttgg aaaaagtaga9600gcctgacgcc agcgacaacc aaaactcggt gaagatcggg ttggatgagg gtaattaccc9660agggcctgga atacagacac atacactaac agaagaaata cacaacaggg atgcgaggcc9720cttcatcatg atcctgggct caaggaattc catatcaaat agggcaaaga ctgctagaaa9780
tataaatctg tacacaggaa atgaccccag ggaaatacga gacttgatgg ctgcagggcg 9840catgttagta gtagcactga gggatgtcga ccctgagctg tctgaaatgg tcgatttcaa 9900ggggactttt ttagataggg aggccctgga ggctctaagt ctcgggcaac ctaaaccgaa 9960gcaggttacc aaggaagctg ttaggaattt gatagaacag aaaaaagatg tggagatccc10020taactggttt gcatcagatg acccagtatt tctggaagtg gccttaaaaa atgataagta10080ctacttagta ggagatgttg gagagctaaa agatcaagct aaagcacttg gggccacgga10140tcagacaaga attataaagg aggtaggctc aaggacgtat gccatgaagc tatctagctg10200gttcctcaag gcatcaaaca aacagatgag tttaactcca ctgtttgagg aattgttgct10260acggtgccca cctgcaacta agagcaataa ggggcacatg gcatcagctt accaattggc10320acagggtaac tgggagcccc tcggttgcgg ggtgcaccta ggtacaatac cagccagaag10380ggtgaagata cacccatatg aagcttacct gaagttgaaa gatttcatag aagaagaaga10440gaagaaacct agggttaagg atacagtaat aagagagcac aacaaatgga tacttaaaaa10500aataaggttt caaggaaacc tcaacaccaa gaaaatgctc aacccaggga aactatctga10560acagttggac agggaggggc gcaagaggaa catctacaac caccagattg gtactataat10620gtcaagtgca ggcataaggc tggagaaatt gccaatagtg agggcccaaa ccgacaccaa10680aacctttcat gaggcaataa gagataagat agacaagagt gaaaaccggc aaaatccaga10740attgcacaac aaattgttgg agattttcca cacgatagcc caacccaccc tgaaacacac10800ctacggtgag gtgacgtggg agcaacttga ggcgggggta aatagaaagg gggcagcagg10860cttcctggag aagaagaaca tcggagaagt attggattca gaaaagcacc tggtagaaca10920attggtcagg gatctgaagg ccgggagaaa gataaaatat tatgaaactg caataccaaa10980aaatgagaag agagatgtca gtgatgactg gcaggcaggg gacctggtgg ttgagaagag11040gccaagagtt atccaatacc ctgaagccaa gacaaggcta gccatcacta aggtcatgta11100taactgggtg aaacagcagc ccgttgtgat tccaggatat gaaggaaaga cccccttgtt11160caacatcttt gataaagtga gaaaggaatg ggactcgttc aatgagccag tggccgtaag11220ttttgacacc aaagcctggg acactcaagt gactagtaag gatctgcaac ttattggaga11280aatccagaaa tattactata agaaggagtg gcacaagttc attgacacca tcaccgacca11340catgacagaa gtaccagtta taacagcaga tggtgaagta tatataagaa atgggcagag11400agggagcggc cagccagaca caagtgctgg caacagcatg ttaaatgtcc tgacaatgat11460
gtacggcttc tgcgaaagca caggggtacc gtacaagagt ttcaacaggg tggcaaggat11520ccacgtctgt ggggatgatg gcttcttaat aactgaaaaa gggttagggc tgaaatttgc11580taacaaaggg atgcagattc ttcatgaagc aggcaaacct cagaagataa cggaagggga11640aaagatgaaa gttgcctata gatttgagga tatagagttc tgttctcata ccccagtccc11700tgttaggtgg tccgacaaca ccagtagtca catggccggg agagacaccg ctgtgatact11760atcaaagatg gcaacaagat tggattcaag tggagagagg ggtaccacag catatgaaaa11820agcggtagcc ttcagtttct tgctgatgta ttcctggaac ccgcttgtta ggaggatttg11880cctgttggtc ctttcgcaac agccagagac agacccatca aaacatgcca cttattatta11940caaaggtgat ccaatagggg cctataaaga tgtaataggt cggaatctaa gtgaactgaa12000gagaacaggc tttgagaaat tggcaaatct aaacctaagc ctgtccacgt tgggggtctg12060gactaagcac acaagcaaaa gaataattca ggactgtgtt gccattggga aagaagaggg12120caactggcta gttaagcccg acaggctgat atccagcaaa actggccact tatacatacc12180tgataaaggc tttacattac aaggaaagca ttatgagcaa ctgcagctaa gaacagagac12240aaacccggtc atgggggttg ggactgagag atacaagtta ggtcccatag tcaatctgct12300gctgagaagg ttgaaaattc tgctcatgac ggccgtcggc gtcagcagct gagacaaaat12360gtatatattg taaataaatt aatccatgta catagtgtat ataaatatag ttgggaccgt12420ccacctcaag aagacgacac gcccaacacg cacagctaaa cagtagtcaa gattatctac12480ctcaagataa cactacattt aatgcacaca gcactttagc tgtatgagga tacgcccgac12540gtctatagtt ggactaggga agacctctaa cag 12573<210>5<211>3988<212>PRT<213>牛病毒性腹瀉病毒1NADL株<400>5Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys1 5 10 15Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asp Pro Leu20 25 30Phe Gly Glu Arg Gly Ala Val His Pro Gln Ser Thr Leu Lys Leu Pro35 40 45His Lys Arg Gly Glu Arg Asp Val Pro Thr Asn Leu Ala Ser Leu Pro50 55 60
Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Arg Gly Pro Val Ser Gly65 70 75 80Ile Tyr Leu Lys Pro Gly Pro Leu Phe Tyr Gln Asp Tyr Lys Gly Pro85 90 95Val Tyr His Arg Ala Pro Leu Glu Leu Phe Glu Glu Gly Ser Met Cys100 105 110Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu115 120 125Tyr His Ile Tyr Val Cys Ile Asp Gly Cys Ile Ile Ile Lys Ser Ala130 135 140Thr Arg Ser Tyr Gln Arg Val Phe Arg Trp Val His Asn Arg Leu Asp145 150 155 160Cys Pro Leu Trp Val Thr Thr Cys Ser Asp Thr Lys Glu Glu Gly Ala165 170 175Thr Lys Lys Lys Thr Gln Lys Pro Asp Arg Leu Glu Arg Gly Lys Met180 185 190Lys Ile Val Pro Lys Glu Ser Glu Lys Asp Ser Lys Thr Lys Pro Pro195 200 205Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Arg Lys Lys210 215 220Gly Lys Thr Lys Ser Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys225 230 235 240Asn Lys Pro Gln Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala245 250 255Trp Ala Ile Ile Ala Ile Val Leu Phe Gln Val Thr Met Gly Glu Asn260 265 270Ile Thr Gln Trp Asn Leu Gln Asp Asn Gly Thr Glu Gly Ile Gln Arg275 280 285Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro290 295 300Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr Asp Ile Glu305 310 315 320Leu Lys Thr Ile His Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr325 330 335Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys340 345 350Asn Trp Tyr Asn Ile Glu Pro Trp Ile Leu Val Met Asn Arg Thr Gln355 360 365Ala Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys Ala Val Thr Cys
370 375 380Arg Tyr Asp Arg Ala Ser Asp Leu Asn Val Val Thr Gln Ala Arg Asp385 390 395 400Ser Pro Thr Pro Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe405 410 415Ala Gly Ile Leu Met Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser420 425 430Asp Val Leu Phe Lys Glu His Glu Arg Ile Ser Met Phe Gln Asp Thr435 440 445Thr Leu Tyr Leu Val Asp Gly Leu Thr Asn Ser Leu Glu Gly Ala Arg450 455 460Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile465 470 475 480Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Thr Trp Phe Gly Ala Tyr485 490 495Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Ile Gly Tyr Ile Trp500 505 510Tyr Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile515 520 525Val Gly Pro Gly Lys Phe Gly Thr Asn Ala Glu Asp Gly Lys Ile Leu530 535 540His Glu Met Gly Gly His Leu Ser Glu Val Leu Leu Leu Ser Leu Val545 550 555 560Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Val Met Tyr Leu Ile565 570 575Leu His Phe Ser Ile Pro Gln Ser His Val Asp Val Met Asp Cys Asp580 585 590Lys Thr Gln Leu Asn Leu Thr Val Glu Leu Thr Thr Ala Glu Val Ile595 600 605Pro Gly Ser Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asn610 615 620Trp Trp Pro Tyr Glu Thr Thr Val Val Leu Ala Phe Glu Glu Val Ser625 630 635 640Gln Val Val Lys Leu Val Leu Arg Ala Leu Arg Asp Leu Thr Arg Ile645 650 655Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Val Cys Leu Val Lys Ile660 665 670Val Arg Gly Gln Met Val Gln Gly Ile Leu Trp Leu Leu Leu Ile Thr675 680 685
Gly Val Gln Gly His Leu Asp Cys Lys Pro Glu Phe Ser Tyr Ala Ile690 695 700Ala Lys Asp Glu Arg Ile Gly Gln Leu Gly Ala Glu Gly Leu Thr Thr705 710 715 720Thr Trp Lys Glu Tyr Ser Pro Gly Met Lys Leu Glu Asp Thr Met Val725 730 735Ile Ala Trp Cys Glu Asp Gly Lys Leu Met Tyr Leu Gln Arg Cys Thr740 745 750Arg Glu Thr Arg Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr755 760 765Ser Val Val Phe Lys Lys Leu Phe Asp Gly Arg Lys Gln Glu Asp Val770 775 780Val Glu Met Asn Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala785 790 795 800Lys Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro805 810 815Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys820 825 830Thr Ser Phe Asn Met Asp Thr Leu Ala Thr Thr Val Val Arg Thr Tyr835 840 845Arg Arg Ser Lys Pro Phe Pro His Arg Gln Gly Cys Ile Thr Gln Lys850 855 860Asn Leu Gly Glu Asp Leu His Asn Cys Ile Leu Gly Gly Asn Trp Thr865 870 875 880Cys Val Pro Gly Asp Gln Leu Leu Tyr Lys Gly Gly Ser Ile Glu Ser885 890 895Cys Lys Trp Cys Gly Tyr Gln Phe Lys Glu Ser Glu Gly Leu Pro His900 905 910Tyr Pro Ile Gly Lys Cys Lys Leu Glu Asn Glu Thr Gly Tyr Arg Leu915 920 925Val Asp Ser Thr Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro Gln930 935 940Gly Thr Leu Lys Cys Lys Ile Gly Lys Thr Thr Val Gln Val Ile Ala945 950 955 960Met Asp Thr Lys Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile965 970 975Ser Ser Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr980 985 990
Lys Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln995 10001005Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu101010151020Val Thr Asp His His Arg Asp Tyr Phe Ala Glu Ser Ile Leu Val102510301035Val Val Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu Leu104010451050Val Thr Tyr Met Val Leu Ser Glu Gln Lys Ala Leu Gly Ile Gln105510601065Tyr Gly Ser Gly Glu Val Val Met Met Gly Asn Leu Leu Thr His107010751080Asn Asn Ile Glu Val Val Thr Tyr Phe Leu Leu Leu Tyr Leu Leu108510901095Leu Arg Glu Glu Ser Val Lys Lys Trp Val Leu Leu Leu Tyr His110011051110Ile Leu Val Val His Pro Ile Lys Ser Val Ile Val Ile Leu Leu111511201125Met Ile Gly Asp Val Val Lys Ala Asp Ser Gly Gly Gln Glu Tyr113011351140Leu Gly Lys Ile Asp Leu Cys Phe Thr Thr Val Val Leu Ile Val114511501155Ile Gly Leu Ile Ile Ala Arg Arg Asp Pro Thr Ile Val Pro Leu116011651170Val Thr Ile Met Ala Ala Leu Arg Val Thr Glu Leu Thr His Gln117511801185Pro Gly Val Asp Ile Ala Val Ala Val Met Thr Ile Thr Leu Leu119011951200Met Val Ser Tyr Val Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu120512101215Gln Cys Ile Leu Ser Leu Val Ser Ala Val Phe Leu Ile Arg Ser122012251230Leu Ile Tyr Leu Gly Arg Ile Glu Met Pro Glu Val Thr Ile Pro123512401245Asn Trp Arg Pro Leu Thr Leu Ile Leu Leu Tyr Leu Ile Ser Thr125012551260Thr Ile Val Thr Arg Trp Lys Val Asp Val Ala Gly Leu Leu Leu126512701275Gln Cys Val Pro Ile Leu Leu Leu Val Thr Thr Leu Trp Ala Asp
1280 1285 1290Phe Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Val Lys129513001305Leu Tyr Tyr Leu Lys Thr Val Arg Thr Asp Thr Glu Arg Ser Trp131013151320Leu Gly Gly Ile Asp Tyr Thr Arg Val Asp Ser Ile Tyr Asp Val132513301335Asp Glu Ser Gly Glu Gly Val Tyr Leu Phe Pro Ser Arg Gln Lys134013451350Ala Gln Gly Asn Phe Ser Ile Leu Leu Pro Leu Ile Lys Ala Thr135513601365Leu Ile Ser Cys Val Ser Ser Lys Trp Gln Leu Ile Tyr Met Ser137013751380Tyr Leu Thr Leu Asp Phe Met Tyr Tyr Met His Arg Lys Val Ile138513901395Glu Glu Ile Ser Gly Gly Thr Asn Ile Ile Ser Arg Leu Val Ala140014051410Ala Leu Ile Glu Leu Asn Trp Ser Met Glu Glu Glu Glu Ser Lys141514201425Gly Leu Lys Lys Phe Tyr Leu Leu Ser Gly Arg Leu Arg Asn Leu143014351440Ile Ile Lys His Lys Val Arg Asn Glu Thr Val Ala Ser Trp Tyr144514501455Gly Glu Glu Glu Val Tyr Gly Met Pro Lys Ile Met Thr Ile Ile146014651470Lys Ala Ser Thr Leu Ser Lys Ser Arg His Cys Ile Ile Cys Thr147514801485Val Cys Glu Gly Arg Glu Trp Lys Gly Gly Thr Cys Pro Lys Cys149014951500Gly Arg His Gly Lys Pro Ile Thr Cys Gly Met Ser Leu Ala Asp150515101515Phe Glu Glu Arg His Tyr Lys Arg Ile Phe Ile Arg Glu Gly Asn152015251530Phe Glu Gly Met Cys Ser Arg Cys Gln Gly Lys His Arg Arg Phe153515401545Glu Met Asp Arg Glu Pro Lys Ser Ala Arg Tyr Cys Ala Glu Cys155015551560Asn Arg Leu His Pro Ala Glu Glu Gly Asp Phe Trp Ala Glu Ser
156515701575Ser Met Leu Gly Leu Lys Ile Thr Tyr Phe Ala Leu Met Asp Gly158015851590Lys Val Tyr Asp Ile Thr Glu Trp Ala Gly Cys Gln Arg Val Gly159516001605Ile Ser Pro Asp Thr His Arg Val Pro Cys His Ile Ser Phe Gly161016151620Ser Arg Met Pro Phe Arg Gln Glu Tyr Asn Gly Phe Val Gln Tyr162516301635Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Val Leu Ala164016451650Thr Lys Val Lys Met Leu Met ValGly Asn Leu Gly Glu Glu Ile165516601665Gly Asn Leu Glu His Leu Gly Trp Ile Leu Arg Gly Pro Ala Val167016751680Cys Lys Lys Ile Thr Glu His Glu Lys Cys His Ile Asn Ile Leu168516901695Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr170017051710Pro Arg Ala Pro Val Arg Phe Pro Thr Ser Leu Leu Lys Val Arg171517201725Arg Gly Leu Glu Thr Ala Trp Ala Tyr Thr His Gln Gly Gly Ile173017351740Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys174517501755Asp Ser Met Gly Arg Thr Arg Val Val Cys Gln Ser Asn Asn Arg176017651770Leu Thr Asp Glu Thr Glu Tyr Gly Val Lys Thr Asp Ser Gly Cys177517801785Pro Asp Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn179017951800Ile Ser Gly Ser Lys Gly Ala Val Val His Leu Gln Lys Thr Gly180518101815Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe182018251830Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu183518401845Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn185018551860
Glu Glu Ser Lys Pro Thr Lys Ile Met Ser Gly Ile Gln Thr Val186518701875Ser Lys Asn Arg Ala Asp Leu Thr Glu Met Val Lys Lys Ile Thr188018851890Ser Met Asn Arg Gly Asp Phe Lys Gln Ile Thr Leu Ala Thr Gly189519001905Ala Gly Lys Thr Thr Glu Leu Pro Lys Ala Val Ile Glu Glu Ile191019151920Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala192519301935Ala Glu Ser Val Tyr Gln Tyr Met Arg Leu Lys His Pro Ser Ile194019451950Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala195519601965Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Met Pro197019751980Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe198519901995Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile200020052010Gly Lys Ile His Arg Phe Ser Glu Ser Ile Arg Val Val Ala Met201520202025Thr Ala Thr Pro Ala Gly Ser Val Thr Thr Thr Gly Gln Lys His203020352040Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp204520502055Leu Gly Ser Gln Phe Leu Asp Ile Ala Gly Leu Lys Ile Pro Val206020652070Asp Glu Met Lys Gly Asn Met Leu Val Phe Val Pro Thr Arg Asn207520802085Met Ala Val Glu Val Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn209020952100Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ala Asn Leu Arg Val210521102115Val Thr Ser Gln Ser Pro Tyr Val Ile Val Ala Thr Asn Ala Ile212021252130Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Ile Asp Thr213521402145
Gly Leu Lys Cys Glu Lys Arg Val Arg Val Ser Ser Lys Ile Pro215021552160Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Val Gly Glu216521702175Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg218021852190Tyr Tyr Arg Ser Gln Glu Thr Ala Thr Gly Ser Lys Asp Tyr His219522002205Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile221022152220Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu222522302235Tyr Glu Glu Asp Ser Leu Leu Ile Thr Gln Leu Glu Ile Leu Asn224022452250Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile225522602265Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn227022752280Ser Tyr Glu Val Gln Val Pro Val Leu Phe Pro Lys Ile Arg Asn228522902295Gly Glu Val Thr Asp Thr Tyr Glu Asn Tyr Ser Phe Leu Asn Ala230023052310Arg Lys Leu Gly Glu Asp Val Pro Val Tyr Ile Tyr Ala Thr Glu231523202325Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Leu Asp Trp Pro Asp233023352340Pro Gly Asn Gln Gln Val Val Glu Thr Gly Lys Ala Leu Lys Gln234523502355Val Thr Gly Leu Ser Ser Ala Glu Asn Ala Leu Leu Val Ala Leu236023652370Phe Gly Tyr Val Gly Tyr Gln Ala Leu Ser Lys Arg His Val Pro237523802385Met Ile Thr Asp Ile Tyr Thr Ile Glu Asp Gln Arg Leu Glu Asp239023952400Thr Thr His Leu Gln Tyr Ala Pro Asn Ala Ile Lys Thr Asp Gly240524102415Thr Glu Thr Glu Leu Lys Glu Leu Ala Ser Gly Asp Val Glu Lys242024252430
Ile Met Gly Ala Ile Ser Asp Tyr Ala Ala Gly Gly Leu Glu Phe243524402445Val Lys Ser Gln Ala Glu Lys Ile Lys Thr Ala Pro Leu Phe Lys245024552460Glu Asn Ala Glu Ala Ala Lys Gly Tyr Val Gln Lys Phe Ile Asp246524702475Ser Leu Ile Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp248024852490Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Ala Ala Arg Leu Gly249525002505His Glu Thr Ala Phe Ala Thr Leu Val Leu Lys Trp Leu Ala Phe251025152520Gly Gly Glu Ser Val Ser Asp His Val Lys Gln Ala Ala Val Asp252525302535Leu Val Val Tyr Tyr Val Met Asn Lys Pro Ser Phe Pro Gly Asp254025452550Ser Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe255525602565Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr His257025752580Asn Leu Ser Lys Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr258525902595Ala Thr Ser Ala Leu Lys Met Phe Thr Pro Thr Arg Leu Glu Ser260026052610Val Val Ile Leu Ser Thr Thr Ile Tyr Lys Thr Tyr Leu Ser Ile261526202625Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala263026352640Ala Met Glu Ile Leu Ser Gln Asn Pro Val Ser Val Gly Ile Ser264526502655Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu266026652670Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys267526802685Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn269026952700Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile270527102715Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr
272027252730Lys Gly Trp Glu Ala Lys Glu Leu Ser Glu Arg Thr Ala Gly Arg273527402745Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly275027552760Met Asp Ser Gln Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile276527702775Leu Asp Leu Ile Tyr Gly Leu His Lys Gln Ile Asn Arg Gly Leu278027852790Lys Lys Met Val Leu Gly Trp Ala Pro Ala Pro Phe Ser Cys Asp279528002805Trp Thr Pro Ser Asp Glu Arg Ile Arg Leu Pro Thr Asp Asn Tyr281028152820Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala282528302835Phe Lys Asn Val Gly Gly Lys Leu Thr Lys Val Glu Glu Ser Gly284028452850Pro Phe Leu Cys Arg Asn Arg Pro Gly Arg Gly Pro Val Asn Tyr285528602865Arg Val Thr Lys Tyr Tyr Asp Asp Asn Leu Arg Glu Ile Lys Pro287028752880Val Ala Lys Leu Glu Gly Gln Val Glu His Tyr Tyr Lys Gly Val288528902895Thr Ala Lys Ile Asp Tyr Ser Lys Gly Lys Met Leu Leu Ala Thr290029052910Asp Lys Trp Glu Val Glu His Gly Val Ile Thr Arg Leu Ala Lys291529202925Arg Tyr Thr Gly Val Gly Phe Asn Gly Ala Tyr Leu Gly Asp Glu293029352940Pro Asn His Arg Ala Leu Val Glu Arg Asp Cys Ala Thr Ile Thr294529502955Lys Asn Thr Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe296029652970Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr Arg Leu Ile Glu Leu297529802985Val His Arg Asn Asn Leu Glu Glu Lys Glu Ile Pro Thr Ala Thr299029953000Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Val Gly300530103015
Thr Ile Lys Pro Val Leu Gly Glu Arg Val Ile Pro Asp Pro Val302030253030Val Asp Ile Asn Leu Gln Pro Glu Val Gln Val Asp Thr Ser Glu303530403045Val Gly Ile Thr Ile Ile Gly Arg Glu Thr Leu Met Thr Thr Gly305030553060Val Thr Pro Val Leu Glu Lys Val Glu Pro Asp Ala Ser Asp Asn306530703075Gln Asn Ser Val Lys Ile Gly Leu Asp Glu Gly Asn Tyr Pro Gly308030853090Pro Gly Ile Gln Thr His Thr Leu Thr Glu Glu Ile His Asn Arg309531003105Asp Ala Arg Pro Phe Ile Met Ile Leu Gly Ser Arg Asn Ser Ile311031153120Ser Asn Arg Ala Lys Thr Ala Arg Asn Ile Asn Leu Tyr Thr Gly312531303135Asn Asp Pro Arg Glu Ile Arg Asp Leu Met Ala Ala Gly Arg Met314031453150Leu Val Val Ala Leu Arg Asp Val Asp Pro Glu Leu Ser Glu Met315531603165Val Asp Phe Lys Gly Thr Phe Leu Asp Arg Glu Ala Leu Glu Ala317031753180Leu Ser Leu Gly Gln Pro Lys Pro Lys Gln Val Thr Lys Glu Ala318531903195Val Arg Asn Leu Ile Glu Gln Lys Lys Asp Val Glu Ile Pro Asn320032053210Trp Phe Ala Ser Asp Asp Pro Val Phe Leu Glu Val Ala Leu Lys321532203225Asn Asp Lys Tyr Tyr Leu Val Gly Asp Val Gly Glu Leu Lys Asp323032353240Gln Ala Lys Ala Leu Gly Ala Thr Asp Gln Thr Arg Ile Ile Lys324532503255Glu Val Gly Ser Arg Thr Tyr Ala Met Lys Leu Ser Ser Trp Phe326032653270Leu Lys Ala Ser Asn Lys Gln Met Ser Leu Thr Pro Leu Phe Glu327532803285Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys Ser Asn Lys Gly329032953300
His Met Ala Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro330533103315Leu Gly Cys Gly Val His Leu Gly Thr Ile Pro Ala Arg Arg Val332033253330Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Asp Phe Ile333533403345Glu Glu Glu Glu Lys Lys Pro Arg Val Lys Asp Thr Val Ile Arg335033553360Glu His Asn Lys Trp Ile Leu Lys Lys Ile Arg Phe Gln Gly Asn336533703375Leu Asn Thr Lys Lys Met LeuAsn Pro Gly Lys Leu Ser Glu Gln338033853390Leu Asp Arg Glu Gly Arg Lys Arg Asn Ile Tyr Asn His Gln Ile339534003405Gly Thr Ile Met Ser Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro341034153420Ile Val Arg Ala Gln Thr Asp Thr Lys Thr Phe His Glu Ala Ile342534303435Arg Asp Lys Ile Asp Lys Ser Glu Asn Arg Gln Asn Pro Glu Leu344034453450His Asn Lys Leu Leu Glu Ile Phe His Thr Ile Ala Gln Pro Thr345534603465Leu Lys His Thr Tyr Gly Glu Val Thr Trp Glu Gln Leu Glu Ala347034753480Gly Val Asn Arg Lys Gly Ala Ala Gly Phe Leu Glu Lys Lys Asn348534903495Ile Gly Glu Val Leu Asp Ser Glu Lys His Leu Val Glu Gln Leu350035053510Val Arg Asp Leu Lys Ala Gly Arg Lys Ile Lys Tyr Tyr Glu Thr351535203525Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Gln353035353540Ala Gly Asp Leu Val Val Glu Lys Arg Pro Arg Val Ile Gln Tyr354535503555Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Asn356035653570Trp Val Lys Gln Gln Pro Val Val Ile Pro Gly Tyr Glu Gly Lys357535803585Thr Pro Leu Phe Asn Ile Phe Asp Lys Val Arg Lys Glu Trp Asp
359035953600Ser Phe Asn Glu Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp360536103615Asp Thr Gln Val Thr Ser Lys Asp Leu Gln Leu Ile Gly Glu Ile362036253630Gln Lys Tyr Tyr Tyr Lys Lys Glu Trp His Lys Phe Ile Asp Thr363536403645Ile Thr Asp His Met Thr Glu Val Pro Val Ile Thr Ala Asp Gly365036553660Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp366536703675Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Met Tyr368036853690Gly Phe Cys Glu Ser Thr Gly Val ProTyr Lys Ser Phe Asn Arg369537003705Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr371037153720Glu Lys Gly Leu Gly Leu Lys Phe Ala Asn Lys Gly Met Gln Ile372537303735Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Glu Lys374037453750Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His375537603765Thr Pro Val Pro Val Arg Trp Ser Asp Asn Thr Ser Ser His Met377037753780Ala Gly Arg Asp Thr Ala Val Ile Leu Ser Lys Met Ala Thr Arg378537903795Leu Asp Ser Ser Gly Glu Arg Gly Thr Thr Ala Tyr Glu Lys Ala380038053810Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Val381538203825Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Gln Pro Glu Thr Asp383038353840Pro Ser Lys His Ala Thr Tyr Tyr Tyr Lys Gly Asp Pro Ile Gly384538503855Ala Tyr Lys Asp Val Ile Gly Arg Asn Leu Ser Glu Leu Lys Arg386038653870Thr Gly Phe Glu Lys Leu Ala Asn Leu Asn Leu Ser Leu Ser Thr387538803885
Leu Gly Val Trp Thr Lys His Thr Ser Lys Arg Ile Ile Gln Asp389038953900Cys Val Ala Ile Gly Lys Glu Glu Gly Asn Trp Leu Val Lys Pro390539103915Asp Arg Leu Ile Ser Ser Lys Thr Gly His Leu Tyr Ile Pro Asp392039253930Lys Gly Phe Thr Leu Gln Gly Lys His Tyr Glu Gln Leu Gln Leu393539403945Arg Thr Glu Thr Asn Pro Val Met Gly Val Gly Thr Glu Arg Tyr395039553960Lys Leu Gly Pro Ile Val Asn Leu Leu Leu Arg Arg Leu Lys Ile396539703975Leu Leu Met Thr Ala Val Gly Val Ser Ser39803985<210>6<211>3898<212>PRT<213>牛病毒性腹瀉病毒1<400>6Met Glu Leu Ile Thr Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys1 5 10 15Pro Val Gly Val Glu Glu Pro Val Tyr Asp Gln Ala Gly Asn Pro Leu20 25 30Phe Gly Glu Arg Gly Ala Ile His Pro Gln Ser Thr Leu Lys Leu Pro35 40 45His Lys Arg Gly Glu Arg Asn Val Pro Thr Ser Leu Ala Ser Leu Pro50 55 60Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Lys Gly Pro Val Ser Gly65 70 75 80Ile Tyr Leu Lys Pro Gly Pro Leu Phe Tyr Gln Asp Tyr Lys Gly Pro85 90 95Val Tyr His Arg Ala Pro Leu Glu Leu Phe Glu Glu Gly Ser Met Cys100 105 110Glu Thr Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Lys Leu115 120 125Tyr His lle Tyr Ile Cys lle Asp Gly Cys Ile Thr Val Lys Ser Ala130 135 140Thr Arg Ser His Gln Arg Val Leu Arg Trp Val His Asn Arg Leu Asp145 150 155 160
Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Thr Lys Glu Glu Gly Ala165 170 175Thr Lys Lys Lys Gln Gln Lys Pro Asp Arg Leu Glu Lys Gly Arg Met180 185 190Lys Ile Val Pro Lys Glu Ser Glu Lys Asp Ser Lys Thr Lys Pro Pro195 200 205Asp Ala Thr Ile Val Val Asp Gly Val Lys Tyr Gln Val Lys Lys Lys210 215 220Gly Lys Val Lys Ser Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys225 230 235 240Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala245 250 255Trp Ala Ile Leu Ala Val Val Leu Ile Glu Val Thr Met Gly Glu Asn260 265 270Ile Thr Gln Trp Asn Leu Gln Asp Asn Gly Thr Glu Gly Ile Gln Arg275 280 285Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro290 295 300Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr Asp Val Glu305 310 315 320Leu Lys Thr Ile His Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr325 330 335Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys340 345 350Asn Trp Tyr Asn Ile Glu Pro Trp Ile Leu Ile Met Asn Arg Thr Gln355 360 365Ala Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys Ala Val Thr Cys370 375 380Arg Tyr Asp Arg Asp Ser Asp Leu Asn Val Val Thr Gln Ala Arg Asp385 390 395 400Ser Pro Thr Pro Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe405 410 415Ala Gly Val Leu Thr Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser420 425 430Asp Val Leu Phe Lys Glu His Glu Cys Thr Gly Val Phe Gln Asp Thr435 440 445Ala His Tyr Leu Val Asp Gly Val Thr Asn Ser Leu Glu Ser Ala Arg450 455 460
Gln Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile465 470 475480Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Thr Trp Phe Gly Ala Tyr485 490 495Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Ile Gly Tyr Ile Trp500 505 510Phe Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile515 520 525Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu530 535 540His Glu Met Gly Gly His Leu Ser Glu Val Leu Leu Leu Ser Leu Val545 550 555 560Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Ala Met Tyr Leu Ile565 570 575Leu His Phe Ser Ile Pro Gln Ser His Val Asp Ile Thr Asp Cys Asp580 585 590Lys Thr Gln Leu Asn Leu Thr Ile Glu Leu Thr Thr Ala Asp Val Ile595 600 605Pro Gly Ser Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp610 615 620Trp Trp Pro Tyr Glu Thr Ala Ala Val Leu Ala Phe Glu Glu Val Gly625 630 635 640Gln Val Val Lys Ile Val Leu Arg Ala Leu Arg Asp Leu Thr Arg Ile645 650 655Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Val Cys Leu Ile Lys Met660 665 670Val Arg Gly Gln Val Val Gln Gly Ile Leu Trp Leu Leu Leu Ile Thr675 680 685Gly Val Gln Gly His Leu Asp Cys Lys Pro Glu Tyr Ser Tyr Ala Ile690 695 700Ala Lys Asn Asp Arg Val Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr705 710 715 720Val Trp Lys Asp Tyr Ser His Glu Met Lys Leu Glu Asp Thr Met Val725 730 735Ile Ala Trp Cys Lys Gly Gly Lys Phe Thr Tyr Leu Ser Arg Cys Thr740 745 750Arg Glu Thr Arg Tyr Leu Ala Ile Leu His Ser Arg Ala Leu Pro Thr755 760 765Ser Val Val Phe Lys Lys Leu Phe Glu Gly Gln Lys Gln Glu Asp Thr
770 775 780Val Glu Met Asp Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala785 790 795 800Lys Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro805 810 815Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys820 825 830Met Leu Ala Asn Arg Asp Thr Leu Asp Thr Ala Val Val Arg Thr Tyr835 840 845Arg Arg Ser Val Pro Phe Pro Tyr Arg Gln Gly Cys Ile Thr Gln Lys850 855 860Thr Leu Gly Glu Asp Leu Tyr Asp Cys Ala Leu Gly Gly Asn Trp Thr865 870 875 880Cys Val Thr Gly Asp Gln Ser Arg Tyr Thr Gly Gly Leu Ile Glu Ser885 890 895Cys Lys Trp Cys Gly Tyr Lys Phe Gln Lys Ser Glu Gly Leu Pro His900 905 910Tyr Pro Ile Gly Lys Cys Arg Leu Asn Asn Glu Thr Gly Tyr Arg Leu915 920 925Val Asp Asp Thr Ser Cys Asp Arg Glu Gly Val Ala Ile Val Pro His930 935 940Gly Leu Val Lys Cys Lys Ile Gly Asp Thr Thr Val Gln Val Ile Ala945 950 955 960Thr Asp Thr Lys Leu Gly Pro Met Pro Cys Lys Pro His Glu Ile Ile965 970 975Ser Ser Glu Gly Pro Ile Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr980 985 990Arg Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln995 10001005Gln Tyr Met Leu Lys Gly Asp Tyr Gln Tyr Trp Phe Asp Leu Glu101010151020Val Thr Asp His His Arg Asp Tyr Phe Ala Glu Ser Ile Leu Val102510301035Val Val Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu Leu104010451050Val Thr Tyr Met Val Leu Ser Glu Gln Lys Ala Ser Gly Ala Gln105510601065Tyr Gly Ala Gly Glu Val Val Met Met Gly Asn Leu Leu Thr His107010751080
Asp Asn Val Glu Val Val Thr Tyr Phe Phe Leu Leu Tyr Leu Leu108510901095Leu Arg Glu Glu Ser Val Lys Lys Trp Val Leu Leu Leu Tyr His110011051110Ile Leu Val Ala His Pro Leu Lys Ser Val Ile Val Ile Leu Leu111511201125Met Ile Gly Asp Val Val Lys Ala Asp Pro Gly Gly Gln Gly Tyr113011351140Leu Gly Gln Ile Asp Val Cys Phe Thr Met Val Val Ile Ile Ile114511501155Ile Gly Leu Ile Ile Ala Arg Arg Asp Pro Thr Ile Val Pro Leu116011651170Ile Thr Ile Val Ala Ser Leu Arg Val Thr Gly Leu Thr Tyr Ser117511801185Pro Gly Val Asp Ala Ala Met Ala Val Ile Thr Ile Thr Leu Leu119011951200Met Val Ser Tyr Val Thr Asp Tyr Phe Arg Tyr Lys Arg Trp Leu120512101215Gln Cys Ile Leu Ser Leu Val Ser Gly Val Phe Leu Ile Arg Cys122012251230Leu Ile His Leu Gly Arg Ile Glu Thr Pro Glu Val Thr Ile Pro123512401245Asn Trp Arg Pro Leu Thr Leu Ile Leu Phe Tyr Leu Ile Ser Thr125012551260Thr Va1 Val Thr Met Trp Lys Ile Asp Leu Ala Gly Leu Leu Leu126512701275Gln Gly Val Pro Ile Leu Leu Leu Ile Thr Thr Leu Trp Ala Asp128012851290Phe Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Val Lys129513001305Leu Tyr Tyr Leu Lys Thr Ile Lys Thr Asp Ile Glu Lys Ser Trp131013151320Leu Gly Gly Leu Asp Tyr Lys Arg Val Asp Ser Ile Tyr Asp Val132513301335Asp Glu Ser Gly Glu Gly Val Tyr Leu Phe Pro Ser Arg Gln Lys134013451350Ala Gln Lys Asn Phe Ser Met Leu Leu Pro Leu Val Arg Ala Thr135513601365
Leu Ile Ser Cys Val Ser Ser Lys Trp Gln Leu Ile Tyr Met Ala137013751380Tyr Leu Ser Val Asp Phe Met Tyr Tyr Met His Arg Lys Val Ile138513901395Glu Glu Ile Ser Gly Gly Thr Asn Met Ile Ser Arg Ile Val Ala140014051410Ala Leu Ile Glu Leu Asn Trp Ser Met Glu Glu Glu Glu Ser Lys141514201425Gly Leu Lys Lys Phe Tyr Leu Leu Ser Gly Arg Leu Arg Asn Leu143014351440Ile Ile Lys His Lys Val Arg Asn Glu Thr Val Ala Gly Trp Tyr144514501455Gly Glu Glu Glu Val Tyr Gly Met Pro Lys Ile Met Thr Ile Ile146014651470Lys Ala Ser Thr Leu Asn Lys Asn Lys His Cys Ile Ile Cys Thr147514801485Val Cys Glu Gly Arg Lys Trp Lys Gly Gly Thr Cys Pro Lys Cys149014951500Gly Arg His Gly Lys ProIle Thr Cys Gly Met Ser Leu Ala Asp150515101515Phe Glu Glu Arg His Tyr Lys Arg Ile Phe Ile Arg Glu Gly Asn152015251530Phe Glu Gly Pro Phe Arg Gln Glu Tyr Asn Gly Phe Ile Gln Tyr153515401545Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala155015551560Thr Lys Val Lys Met Leu Met Val Gly Asn Leu Gly Glu Glu Val1565l5701575Gly Asp Leu Glu His Leu Gly Trp Ile Leu Arg Gly Pro Ala Val158015851590Cys Lys Lys Ile Thr Glu His Glu Arg Cys His Ile Asn Ile Leu159516001605Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr161016151620Pro Arg Ala Pro Val Arg Phe Pro Thr Ser Leu Leu Lys Val Arg162516301635Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile164016451650Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys
165516601665Asp Ser Met Gly Arg Thr Arg Val Val Cys Gln Ser Asn Asn Lys167016751680Leu Thr Asp Glu Thr Glu Tyr Gly Val Lys Thr Asp Ser Gly Cys168516901695Pro Asp Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn170017051710Ile Ser Gly Ser Lys Gly Ala Val Val His Leu Gln Lys Thr Gly171517201725Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe173017351740Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu174517501755Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn176017651770Glu Glu Ser Lys Pro Thr Lys Ile Met Ser Gly Ile Gln Thr Val177517801785Ser Lys Asn Thr Ala Asp Leu Thr Glu Met Val Lys Lys Ile Thr179017951800Ser Met Asn Arg Gly Asp Phe Lys Gln Ile Thr Leu Ala Thr Gly180518101815Ala Gly Lys Thr Thr Glu Leu Pro Lys Ala Val Ile Glu Glu Ile182018251830Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala183518401845Ala Glu Ser Val Tyr Gln Tyr Met Arg Leu Lys His Pro Ser Ile185018551860Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala186518701875Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Met Pro188018851890Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe189519001905Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile191019151920Gly Lys Ile His Arg Phe Ser Glu Ser Ile Arg Val Val Ala Met192519301935Thr Ala Thr Pro Ala Gly Ser Val Thr Thr Thr Gly Gln Lys His194019451950
Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Glu Gly Glu Asp195519601965Leu Gly Ser Gln Phe Leu Asp Ile Ala Gly Leu Lys Ile Pro Val197019751980Asp Glu Met Lys Gly Asn Met Leu Val Phe Val Pro Thr Arg Asn198519901995Met Ala Val Glu Val Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn200020052010Ser Gly Tyr Tyr Tyr Ser Gly Glu Asp Pro Ala Asn Leu Arg Val201520202025Val Thr Ser Gln Ser Pro Tyr Val Ile Val Ala Thr Asn Ala Ile203020352040Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr204520502055Gly Leu Lys Cys Glu Lys Arg Val Arg Val Ser Ser Lys Ile Pro206020652070Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Val Gly Glu207520802085Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg209020952100Tyr Tyr Arg Ser Gln Glu Thr Ala Thr Gly Ser Lys Asp Tyr His210521102115Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile212021252130Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu213521402145Tyr Glu Glu Asp Ser Leu Leu Ile Thr Gln Leu Glu Ile Leu Asn215021552160Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile216521702175Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn218021852190Ser Tyr Glu Val Gln Val Pro Val Leu Phe Pro Lys Ile Arg Asn219522002205Gly Glu Val Thr Asp Thr Tyr Glu Asn Tyr Ser Phe Leu Asn Ala221022152220Arg Lys Leu Gly Glu Asp Val Pro Val Tyr Ile Tyr Ala Thr Glu222522302235
Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Leu Asp Trp Pro Asp224022452250Pro Gly Asn Gln Gln Val Val Glu Thr Gly Lys Ala Leu Lys Gln225522602265Val Ala Gly Leu Ser Ser Ala Glu Asn Ala Leu Leu Val Ala Leu227022752280Phe Gly Tyr Val Gly Tyr Gln Ala Leu Ser Lys Arg His Val Pro228522902295Met Ile Thr Asp Ile Tyr Thr Ile Glu Asp Gln Arg Leu Glu Asp230023052310Thr Thr His Leu Gln Tyr Ala Pro Asn Ala Ile Lys Thr Glu Gly231523202325Thr Glu Thr Glu Leu Lys Glu Leu Ala Ser Gly Asp Val Glu Lys233023352340Ile Met Gly Ala Ile Ser Asp Tyr Ala Ala Gly Gly Leu Asp Phe234523502355Val Lys Ser Gln Ala Glu Lys Ile Lys Thr Ala Pro Leu Phe Lys236023652370Glu Asn Val Glu Ala Ala Arg Gly Tyr Val Gln Lys Leu Ile Asp237523802385Ser Leu Ile Glu Asp Lys Asp Val Ile Ile Arg Tyr Gly Leu Trp239023952400Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Ala Ala Arg Leu Gly240524102415His Glu Thr Ala Phe Ala Thr Leu Val Leu Lys Trp Leu Ala Phe242024252430Gly Gly Glu Thr Val Ser Asp His Ile Arg Gln Ala Ala Val Asp243524402445Leu Val Val Tyr Tyr Val Met Asn Lys Pro Ser Phe Pro Gly Asp245024552460Thr Glu Thr Gln Gln Glu Gly Arg Arg Phe Va1 Ala Ser Leu Phe246524702475Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn248024852490Asn Leu Ser Lys Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr249525002505Ala Thr Ser Ala Leu Lys Met Phe Thr Pro Thr Arg Leu Glu Ser251025152520Val Val Ile Leu Ser Thr Thr Ile Tyr Lys Thr Tyr Leu Ser Ile
252525302535Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala254025452550Ala Met Glu Ile Leu Ser Gln Asn Pro Val Ser Val Gly Ile Ser255525602565Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu257025752580Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys258525902595Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn260026052610Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile261526202625Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr263026352640Lys Gly Trp Glu Ala Lys Glu Leu Ser Glu Arg Thr Ala Gly Arg264526502655Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly266026652670Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile267526802685Leu Asp Leu Ile His Gly Leu His Lys Gln Ile Asn Arg Gly Leu269026952700Lys Lys Ile Val Leu Gly Trp Ala Pro Ala Pro Phe Ser Cys Asp270527102715Trp Thr Pro Ser Asp Glu Arg Ile Arg Leu Pro Thr Asp Ser Tyr272027252730Leu Arg Val Glu Thr Lys Cys Pro Cys Gly Tyr Glu Met Lys Ala273527402745Leu Lys Asn Val Ser Gly Lys Leu Thr Lys Val Glu Glu Ser Gly275027552760Pro Phe Leu Cys Arg Asn Arg Pro Gly Arg Gly Pro Val Asn Tyr276527702775Arg Val Thr Lys Tyr Tyr Asp Asp Asn Leu Arg Glu Ile Arg Pro278027852790Val Ala Lys Leu Glu Gly Gln Val Glu His Tyr Tyr Lys Gly Val279528002805Thr Ala Arg Ile Asp Tyr Ser Lys Gly Lys Thr Leu Leu Ala Thr281028152820
Asp Lys Trp Glu Val Glu His Gly Thr Leu Thr Arg Leu Thr Lys282528302835Arg Tyr Thr Gly Val Gly Phe Arg Gly Ala Tyr Leu Gly Asp Glu284028452850Pro Asn His Arg Asp Leu Val Glu Arg Asp Cys Ala Thr Ile Thr285528602865Lys Asn Thr Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe287028752880Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr Arg Leu Ile Glu Leu288528902895Val His Arg Asn Asn Leu Glu Glu Lys Glu Ile Pro Thr Ala Thr290029052910Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Val Gly291529202925Thr Ile Lys Pro Val Leu Gly Glu Arg Val Ile Pro Asp Pro Val293029352940Val Asp Ile Asn Leu Gln Pro Glu Val Gln Val Asp Thr Ser Glu294529502955Val Gly Ile Thr Ile Ile Gly Lys Glu Ala Val Met Thr Thr Gly296029652970Val Thr Pro Val Met Glu Lys Val Glu Pro Asp Thr Asp Asn Asn297529802985Gln Ser Ser Val Lys Ile Gly Leu Asp Glu Gly Asn Tyr Pro Gly299029953000Pro Gly Val Gln Thr His Thr Leu Val Glu Glu Ile His Asn Lys300530103015Asp Ala Arg Pro Phe Ile Met Val Leu Gly Ser Lys Ser Ser Met302030253030Ser Asn Arg Ala Lys Thr Ala Arg Asn Ile Asn Leu Tyr Thr Gly303530403045Asn Asp Pro Arg Glu Ile Arg Asp Leu Met Ala Glu Gly Arg Ile305030553060Leu Val Val Ala Leu Arg Asp Ile Asp Pro Asp Leu Ser Glu Leu306530703075Val Asp Phe Lys Gly Thr Phe Leu Asp Arg Glu Ala Leu Glu Ala308030853090Leu Ser Leu Gly Gln Pro Lys Pro Lys Gln Val Thr Lys Ala Ala309531003105
Ile Arg Asp Leu Leu Lys Glu Glu Arg Gln Val Glu Ile Pro Asp311031153120Trp Phe Thr Ser Asp Asp Pro Val Phe Leu Asp Ile Ala Met Lys312531303135Lys Asp Lys Tyr His Leu Ile Gly Asp Val Val Glu Val Lys Asp314031453150Gln Ala Lys Ala Leu Gly Ala Thr Asp Gln Thr Arg Ile Val Lys315531603165Glu Val Gly Ser Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Phe317031753180Leu Gln Ala Ser Ser Lys Gln Met Ser Leu Thr Pro Leu Phe Glu318531903195Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys Ser Asn Lys Gly320032053210His Met Ala Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro321532203225Leu Gly Cys Gly Val His Leu Gly Thr Val Pro Ala Arg Arg Val323032353240Lys Met His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Asp Leu Val324532503255Glu Glu Glu Glu Lys Lys Pro Arg Ile Arg Asp Thr Val Ile Arg326032653270Glu His Asn Lys Trp Ile Leu Lys Lys Ile Lys Phe Gln Gly Asn327532803285Leu Asn Thr Lys Lys Met Leu Asn Pro Gly Lys Leu Ser Glu Gln329032953300Leu Asp Arg Glu Gly His Lys Arg Asn Ile Tyr Asn Asn Gln Ile330533103315Ser Thr Val Met Ser Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro332033253330Ile Val Arg Ala Gln Thr Asp Thr Lys Ser Phe His Glu Ala Ile333533403345Arg Asp Lys Ile Asp Lys Asn Glu Asn Arg Gln Asn Pro Glu Leu335033553360His Asn Lys Leu Leu Glu Ile Phe His Thr Ile Ala Asp Pro Ser336533703375Leu Lys His Thr Tyr Gly Glu Val Thr Trp Glu Gln Leu Glu Ala338033853390Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe Leu Glu Lys Lys Asn
339534003405Ile Gly Glu Val Leu Asp Ser Glu Lys His Leu Val Glu Gln Leu341034153420Val Arg Asp Leu Lys Ala Gly Arg Lys Ile Arg Tyr Tyr Glu Thr342534303435Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Gln344034453450Ala Gly Asp Leu Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr345534603465Pro Glu Ala Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Asn347034753480Trp Val Lys Gln Gln Pro Val Val Ile Pro Gly Tyr Glu Gly Lys348534903495Thr Pro Leu Phe Asn Ile Phe Asn Lys Val Arg Lys Glu Trp Asp350035053510Leu Phe Asn Glu Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp351535203525Asp Thr Gln Val Thr Ser Arg Asp Leu His Leu Ile Gly Glu Ile353035353540Gln Lys Tyr Tyr Tyr Arg Lys Glu Trp His Lys Phe Ile Asp Thr354535503555Ile Thr Asp His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly356035653570Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp357535803585Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr359035953600Ala Phe Cys Glu Ser Thr Gly Val Pro Tyr Lys Ser Phe Asn Arg360536103615Val Ala Lys Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr362036253630Glu Lys Gly Leu Gly Leu Lys Phe Ser Asn Lys Gly Met GlnIle363536403645Leu His Glu Ala Gly Lys Pro Gln Lys Leu Thr Glu Gly Glu Lys365036553660Met Lys Val Ala Tyr Lys Phe Glu Asp Ile Glu Phe Cys Ser His366536703675Thr Pro Val Pro Val Arg Trp Ser Asp Asn Thr Ser Ser Tyr Met368036853690
Ala Gly Arg Asp Thr Ala Val Ile Leu Ser Lys Met Ala Thr Arg369537003705Leu Asp Ser Ser Gly Glu Arg Gly Thr Thr Ala Tyr Glu Lys Ala371037153720Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Val372537303735Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Arg Pro Glu Thr Ala374037453750Pro Ser Thr Gln Thr Thr Tyr Tyr Tyr Lys Gly Asp Pro Ile Gly375537603765Ala Tyr Lys Asp Val Ile Gly Arg Asn Leu Ser Glu Leu Lys Arg377037753780Thr Gly Phe Glu Lys Leu Ala Asn Leu Asn Leu Ser Leu Ser Thr378537903795Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Ile Ile Gln Asp380038053810Cys Val Ala Ile Gly Lys Glu Glu Gly Asn Trp Leu Val Asn Ala381538203825Asp Arg Leu Ile Ser Ser Lys Thr Gly His Leu Tyr Ile Pro Asp383038353840Lys Gly Phe Thr Leu Gln Gly Lys His Tyr Glu Gln Leu Gln Leu384538503855Gly Ala Glu Thr Asn Pro Val Met Gly Val Gly Thr Glu Arg Tyr386038653870Lys Leu Gly Pro Ile Val Asn Leu Leu Leu Arg Arg Leu Lys Val387538803885Leu Leu Met Ala Ala Val Gly Ala Ser Ser38903895<210>7<211>19<212>DNA<213>人工序列<220>
<223>合成寡核苷酸引物NADL4744<400>7cgtggcttct tggtacggg 19<210>8<211>22
<212>DNA<213>人工序列<220>
<223>合成寡核苷酸引物NADL5305<400>8agcggtatat tgtacaaagc ca 22<210>9<211>24<212>DNA<213>人工序列<220>
<223>合成寡核苷酸引物53637U2<400>9tgcacgatct gtgaagggaa agaa 24<210>10<211>24<212>DNA<213>人工序列<220>
<223>合成寡核苷酸引物NADL4844<400>10tgcactgtat gtgagggccg agag 2權利要求
1.一種疫苗組合物，所述組合物包含同一科的至少兩種活的突變病毒，其中每一種病毒在病毒基因組中都包含突變，而且病毒中的突變存在于同一基因組位點上從而使突變病毒無法相互重組來抵消突變。
2.如權利要求1所述的疫苗組合物，其中所述的科是黃病毒科。
3.如權利要求1所述的疫苗組合物，其中所述的兩種活的突變病毒都來自于疫病毒屬。
4.如權利要求1所述的疫苗組合物，其中所述的兩種突變的活病毒由突變BVDV-1和突變BVDV-2組成。
5.如權利要求4所述的疫苗組合物，其中所述的兩種突變的活病毒由致細胞病變(cp)BVDV-1和致細胞病變BVDV-2組成，而且它們都是被減毒的。
6.如權利要求5所述的疫苗組合物，其中致細胞病變BVDV-1和致細胞病變BVDV-2在NS2-3區(qū)域都含有導致致細胞病變生物型的突變。
7.如權利要求6所述的疫苗組合物，其中致細胞病變BVDV-1是BVDV-1 NADL，而致細胞病變BVDV-2是BVDV-2 53637。
8.如權利要求4所述的疫苗組合物，進一步含有下列的至少一種牛皰疹病毒-1、牛呼吸道合胞體病毒、副流感病毒-3、胚胎彎曲桿菌、犬鉤端螺旋體、流感傷寒鉤端螺旋體、哈德焦鉤端螺旋體、黃疸出血鉤端螺旋體、波摩那鉤端螺旋體或者Mannhemia haemolytica。
9.如權利要求1所述的疫苗組合物，進一步含有獸醫(yī)學上可接受的載體。
10.一種制備安全病毒疫苗的方法，包括選擇或構建同一科的兩種活的突變病毒，其中每一種病毒都包含突變，而且病毒中的突變都存在于同一基因組位點上從而使突變病毒無法進行同源重組來抵消突變。
11.如權利要求10所述的方法，其中所述的突變選自于缺失、插入、取代或者其組合。
12.如權利要求10所述的方法，其中所述突變賦予下列表型，所述表型選自減毒作用、細胞趨向性或生物型的改造、物種趨向性的改造、外源基因盒的表達或者其組合。
13.如權利要求10所述的方法，其中所述的科是黃病毒科。
14.如權利要求10所述的方法，其中兩種活的突變病毒都來自于疫病毒屬。
15.如權利要求10所述的方法，其中所述的兩種突變的活病毒由突變BVDV-1和突變BVDV-2組成。
全文摘要
本發(fā)明提供了一種安全疫苗以及制備此種疫苗的方法。本發(fā)明的疫苗中包含至少兩種活的同一科的突變病毒或是編碼所述病毒的核酸分子，其中兩種中的每一種病毒或其編碼核酸分子都包含能夠賦予所需表型的突變，而且病毒中的突變都存在于同一基因組位點上從而使突變病毒無法相互重組來抵消突變。
文檔編號A61K39/295GK1976717SQ200480028362
公開日2007年6月6日 申請日期2004年7月26日 優(yōu)先權日2003年7月29日
發(fā)明者萬小坤, 杰伊·G·卡爾弗特, 邁克爾·K·奧哈拉, 曹雪梅 申請人:輝瑞產品公司