CN108707589B

CN108707589B - 一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株及其应用

Info

Publication number: CN108707589B
Application number: CN201810581460.6A
Authority: CN
Inventors: 张斌; 阮文强; 汤承; 岳华; 王远微; 张丹丹
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2021-11-26
Anticipated expiration: 2038-06-07
Also published as: CN108707589A

Abstract

本发明公开了一种牛病毒性腹泻病毒SMU‑Z6/1a/SC/2016分离株及其应用。其中牛病毒性腹泻病毒的保藏号为CCTCC NO:V201813，保藏于中国典型培养物保藏中心，该牛病毒性腹泻病毒具有较高的毒价，而且该病毒株免疫原性良好，将该病毒灭活后与201佐剂制备成灭活疫苗免疫犊牛能产生较高的抗体，为一株良好的牛病毒性腹泻病毒疫苗候选毒株。因此，以该病毒为基础制备牛病毒性腹泻病毒相关的诊断试剂，灭活疫苗、弱毒疫苗，用于牛病毒性腹泻病毒地区性流行的诊断及疫苗预防控制具有广阔的市场应用前景。

Description

一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株及其应用

技术领域

本发明属于生物工程技术领域，具体地说，涉及一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株及其应用。

背景技术

牛病毒性腹泻病毒(Bovine virus diarrhea virus，BVDV)是导致牛腹泻的重要病原之一，牛感染BVDV后可导致产奶量下降，繁殖力降低，生长迟缓和继发感染其他疾病等，是牛的一种急性、热性、接触性传染病，该病主要以腹泻及消化道粘膜坏死、糜烂或溃疡为特征。BVDV感染牛后可表现为亚临床感染、急性感染、黏膜病和持续性感染等临床类型。世界动物卫生组织(OIE)将牛病毒性腹泻粘膜病定位B类传染病，给世界养牛业造成了不可估量的经济损失。由于没有很好的免疫防护措施，该病在我国大部分地区广泛存在。对牛群的育肥、产奶和繁殖影响极大，给养牛业造成了巨大的经济损失。

目前，国际上用于BVD的防控措施主要有两个，一是鉴别和扑杀持续性感染动物，其次是疫苗免疫。其中灭活疫苗因在使用过程中具有较好的安全性，在犊牛以及具有较高经济价值的奶牛群中得到广泛使用，对牛病毒性腹泻病毒的流行起到了很好的控制作用。然而，目前我国投入使用的商品化BVDV疫苗较少，更重要的是我国BVDV毒株的基因型和基因亚型等呈现逐渐增多趋势。

因此，迫切需要分离我国的流行毒株，筛选免疫原性强的BVDV典型毒株，研制疫苗来预防该病的发生，减少该病毒对养牛业造成的经济损失。

发明内容

有鉴于此，本发明提供了一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株及其应用，本发明对我国不同地区的流行毒株进行分离、筛选，获得了合适的疫苗候选毒株，使牛病毒性腹泻病毒的防控更具有针对性和有效性，在牛病毒性腹泻病毒灭活疫苗、弱毒疫苗的应用研究方面具有重要的价值。同时，本发明还为牛病毒性腹泻病毒诊断试剂的研发奠定了基础。

为了解决上述技术问题，本发明公开了一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株，系从牛病毒性腹泻病毒流行地区分离获得，于2018年3月28日保藏于中国典型培养物保藏中心地址为：湖北省武汉市武昌区八一路299号武汉大学校内(武汉大学第一附小对面)，保藏号CCTCC NO:V201813。

优选的，其核苷酸如SEQ ID NO.1所示。

本发明公开了一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株的分离方法，包括如下步骤：

步骤1、采集发病牛腹泻粪便，处理后接种牛肾脏上皮细胞，分离获得致细胞病变毒株；

步骤2、以BVDV-F和BVDV-R为特异性引物，用PCR方法进行检测扩增，分离株中扩增出牛病毒性腹泻病毒特异性片段；

步骤3、将分离毒株进行蚀斑纯化，制备得到牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株。

优选地，所述的BVDV-F的核苷酸序列如SEQ ID NO.3所示；BVDV-R的核苷酸序列如SEQ ID NO.4所示。

本发明公开了一种上述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株在制备牛病毒性腹泻病毒疫苗中的应用。

优选地，所述牛病毒性腹泻病毒疫苗为全病毒灭活疫苗、弱毒疫苗。

优选地，所述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株的毒价为10^-8.57/0.1mL。

本发明还公开了一种牛病毒性腹泻病毒灭活疫苗的制备方法，包括以下步骤：将分离纯化的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016病毒大量培养，之后病毒液中加入0.5％β-丙内酯于37℃灭活6h，灭活完成后进行无菌、灭活检验，然后将灭活的病毒液与201佐剂等体积充分混合，制成水包油包水的灭活疫苗。

本发明还公开了一种上述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株在制备牛病毒性腹泻病毒诊断试剂中的应用。

优选地，所述牛病毒性腹泻病毒与致病性病原体抗原、抗体以及核酸检测相关的试剂。

与现有技术相比，本发明可以获得包括以下技术效果：

1)本发明筛选出的BVDV临床分离株具有较高的滴度，且具有很好的免疫原性。

2)本发明以该病毒为基础制备诊断抗原、牛病毒性腹泻病毒灭活疫苗、弱毒疫苗，对牛免疫和诊断有很强的针对性，具有广阔的市场应用前景。

当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有技术效果。

附图说明

此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：

图1是本发明SMU-Z6/1a/SC/2016毒株致MDBK细胞病变；其中，A：产生细胞病变的MDBK细胞；B：蚀斑纯化产生病变的MDBK细胞；C：正常MDBK细胞；

图2是本发明分离毒株PCR检测；其中，凝胶电泳加样顺序依次为：泳道1：阳性对照:；泳道2：SMU-Z6/1a/SC/2016:；泳道3：阴性对照；M：MarkerⅡ，由上到下依次为1200bp、900bp、700bp、500bp、300bp、100bp；

图3是本发明SMU-Z6/1a/SC/2016特异性、纯净性检测；其中，泳道1：BVDV阳性对照；泳道2：BVDV阴性对照；泳道3：SMU-Z6/1a/SC/2016检测；泳道4：BRV阳性对照；泳道5：BRV阴性对照；泳道6：SMU-Z6/1a/SC/2016BRV检测；泳道7：BEV阳性对照；泳道8：BEV阴性对照；泳道9：SMU-Z6/1a/SC/2016BEV检测；泳道10：BcoV阳性对照；泳道11：BcoV阴性对照；泳道12：SMU-Z6/1a/SC/2016BcoV检测；M：MarkⅡ，由上到下依次为1200bp、900bp、700bp、500bp、300bp、100bp；

图4是本发明分离株SMU-Z6/1a/SC/2016灭活疫苗免疫犊牛后中和抗体水平的测定。

具体实施方式

以下将配合实施例来详细说明本发明的实施方式，藉此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。

本发明所应用的方法可以采用病毒研究领域中常用的方法，而不仅限于本发明实施例的具体记载。下述实施例中所用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径获得。

下述实施例中所用试剂和材料如下：恒温培养箱和恒温振荡培养箱购自Thermoforma公司；高速离心机5804购自Eppendorf公司；凝胶成像系统VersaDoc2000、普通PCR仪、核酸蛋白电泳仪powerpacuniversal^TM购自Bio-Rad公司；Quick Taq HS DyeMix购自TOYOBO东洋纺生物科技有限公司；DNA MarkerⅡ、Prime Script^TMRT试剂盒购自大连宝生物工程有限公司；Tirzol试剂盒(RNAiso Plus)购于Takara公司；MDBK(Madin-Darby bovinekidney，MDBK)细胞购自博士德生物有限公司；DMEM培养基和0.25％Trypsin-EDTA(1×)胰酶购自Gibco公司；胎牛血清购自BI公司；商品化灭活疫苗(BVDV&IBRV二联苗)购自扬州优邦生物药品有限公司；MONTANIDE ISA 201VG佐剂购自赛彼科(上海)特殊化学品有限公司。

实施例1牛病毒性腹泻病毒的分离鉴定

1.1牛病毒性腹泻病毒的分离

首先观察牛群发病情况，发明人采集初步诊断为腹泻的的牛粪便样本。将粪便样本用PBS(100U/mL青霉素，100g/mL链霉素)1：5稀释。3000r/min离心10min，得到上清液经0.22μm滤膜过滤除菌后得到病料处理液。

将上述病料处理液接种于6孔培养板上生长旺盛的单层MDBK细胞，每孔接种0.5mL，同时设立阴性对照孔，37℃含5％CO₂培养箱内感作1h，弃去感作液，加入2mL维持液(1％胎牛血清的DMEM)，于37℃含5％CO₂培养箱内培养，每隔12h观察细胞病变，培养72h后收毒，细胞毒反复冻融3次之后再次接种MDBK细胞，如此传代，直至出现典型的细胞病变。

病料处理液接种MDBK单层细胞后，前两代未出现细胞病变，病毒盲传至第7代，腹泻粪便样本SMU-Z6/1a/SC/2016开始出现局部细胞病变，收获细胞培养物，再次接种MDBK细胞21h后产生典型的细胞病变现象，主要表现为感染细胞发生颗粒性变形，细胞聚集成堆，细胞圆缩等(见图1)，完全病变后则表现为大部分细胞脱落，其他未出现细胞病变，对照组细胞生长良好。

1.2牛病毒性腹泻病毒的RT-PCR鉴定

1.2.1病毒PCR检测

参照文献中已发表的BVDV检测引物，并送至上海生工生物技术有限公司合成，引物序列如下。

表1 BVDV检测引物

去400μL上述分离毒株的MDBK细胞培养物作为病毒RNA的提取样品，参照上海英俊生物科技有限公司Trizol试剂盒(RNAisoPlus)说明书提取粪便的RNA，取400μL上清液加入700μL的Trizol，室温静置10min；加入200μL氯仿，剧烈震荡15s，取上清液；加入等体积的异丙醇充分颠倒混匀，室温静置10min；4℃、12000r/min离心15min，弃上清液，加入1mL750mL/L(经DEPC处理过的灭菌水配置)乙醇；4℃、12000r/min离心5min，弃上清液，用15μL经过DEPC处理过的无菌水溶解沉淀，置-80℃冰箱备用。利用Prime Script TMRT试剂盒将RNA反转录成cDNA。以cDNA为模板，所述RT-PCR扩增的反应体系如下：模板cDNA 2μL，QuickTaq HS DyeMix 10μL，上游引物和下游引物各1.0μL，余下的由ddH₂O补足，总量为20μL。反应程序如下：94℃预变性2min；94℃变性30s，52℃退火30s，68℃延伸1min，共35个循环；72℃延伸8min。扩增产物经琼脂糖凝胶电泳进行检测，结果显示分离株得到一条大小约为232bp的特异性条带(见图2)，并将PCR产物送至上海生工生物技术有限公司进行测序分析，其核苷酸序列如SEQ ID NO.1所示，其编码蛋白的氨基酸序列如SEQ ID NO.2所示，测定结果与NCBI已发表的BVDV序列进行基因进化树分型分析，结果显示分离株SMU-Z6/1a/SC/2016属于BVDV1a亚型。

1.2.2纯净性检测

取400μLMDBK细胞病毒液作为RNA提取样品，参照上海英俊生物科技有限公司Trizol试剂盒(RNAisoPlus)说明书提取粪便的RNA，并利用Prime Script TMRT试剂盒将RNA反转录成cDNA。以合成的cDNA为模板，应用本实验室建立的RT-PCR检测方法，对牛轮状病毒(BRV)、牛冠状病毒(BcoV)、牛肠道病毒(BEV)进行外源病毒检测。

表2外源病毒检测引物序列

所述RT-PCR扩增的反应体系如下：模板cDNA 2μL，Quick Taq HS DyeMix 10μL，上游引物和下游引物各1μL，余下的由ddH₂O补足，总量为20μL。反应程序如下：94℃预变性2min；94℃变性30s，52℃退火30s，68℃延伸1min，共35个循环；72℃延伸8min。经RT-PCR检测，BVDV分离株经外源病毒检测BEV、BcoV、BRV均为阴性。

1.3病毒蚀斑纯化

将病毒液做10倍系列稀释到10^-6，将其接种到生长良好的单层MDBK细胞6孔培养板中，每孔接种100μL病毒稀释液，每个稀释度接种2孔，同时设立空白对照孔，置细胞培养箱中吸附1h，每隔20min摇晃一次使病毒均匀分布。吸附后弃去6孔板中的病毒液，然后每孔加入含4％FBS的2％甲基纤维素培养基2mL，使其平铺在细胞表面，室温凝固后置于37℃含5％CO₂的细胞培养箱中继续培养。待适当稀释度度出现单个蚀斑后，用无菌枪头挑选典型的蚀斑重复性克隆，接种单层MDBK细胞继续培养增殖，按照上述步骤重复3次。纯化3次之后大批量扩繁病毒液作为种毒。

1.4病毒滴度的测定

将生长状态良好的MDBK细胞悬浮液铺满96孔板，然后对细胞病毒培养液做连续10倍系列稀释后，加入96孔细胞板内，每一个稀释度8个孔，每孔100μL，置于37℃含5％CO细胞培养箱内感作1h，弃去病毒液，每孔加入含1％的胎牛血清DMEM培养液，37℃含5％CO₂细胞培养箱培养，2～3日观察细胞的病变情况，按照Reed-Muench计算法对分离株进行TCID₅₀测定，结果为SMU-Z6/1a/SC/2016＝10^-8.57/0.1mL。

实施例2BVDV灭活疫苗的研制

2.1病毒的繁殖

取生长状态良好的MDBK单层细胞，用Hanks清洗2遍，将上述免疫原性良好的疫苗候选毒株SMU-Z6/1a/SC/2016细胞培养液接种到MDBK细胞，加入含1％胎牛血清的DMEM细胞维持液，置于37℃含5％CO₂的培养箱内培养，待细胞病变为80％左右时，收获病毒培养液，反复冻融3次，4℃高速离心，获得病毒上清液作为疫苗用病毒。

2.2病毒灭活疫苗的制备

将上述免疫原性良好的疫苗候选株SMU-Z6/1a/SC/2016细胞培养上清液，利用终浓度为0.5％的β-丙内酯对BVDV病毒液进行充分混匀，在37℃摇床内，灭活处理6h。各取两份灭活之后的病毒液分别接种到MDBK细胞进行灭活检验并经MDBK细胞盲传两代，每代培养48～72h，观察细胞病变情况，同时设立阳性病毒对照和空白对照。接种灭活病毒液的检测组和阴性对照组细胞无CPE，而阳性对照组出现典型的BVDV细胞病变，对检测组进行2代盲传，仍未见CPE，表明所检测灭活病毒液灭活完全。最后将灭活的BVDV与201佐剂等体积混合，搅拌后充分混匀，在乳化机内乳化5min，制成水包油包水的灭活疫苗，分装于透明玻璃瓶内，观察疫苗为乳白色均匀混悬液，用注射器吸一滴灭活疫苗于盛水的烧杯中，疫苗均匀的分散在整个烧杯内，并将灭活疫苗在3000r/min离心5min，未见分层现象，疫苗检验合格，可用于后续实验。

表3

选取3～6月龄BVDV抗原、抗体阴性健康犊牛21头。每次接种3～5日，每日观察有无临床异常，所有动物采血，用于病毒抗体水平检测。首次免疫通过颈部肌肉接种灭活疫苗(SMU-Z6/1a/SC/2016)和商品化灭活疫苗(BVDV&IBRV二联苗)(购自扬州优邦生物药品有限公司，生产批号：17192003)，接种剂量2mL/头；空白对照组接种相同剂量不含病毒的细胞液。疫苗接种后21日进行二免，接种方式和剂量同前所述。疫苗接种后不同时间采血，利用抗体中和试验测定犊牛免疫后的中和抗体水平(图4)。不同动物的免疫试验结果表明，SMU-Z6/1a/SC/2016毒株可诱导产生高水平的抗体，可以作为疫苗研制的候选毒株。

上述说明示出并描述了发明的若干优选实施例，但如前所述，应当理解发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离发明的精神和范围，则都应在发明所附权利要求的保护范围内。

序列表

<110> 西南民族大学

<120> 一种牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株及其应用

<130> 2018

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11703

<212> DNA

<213> 牛病毒性腹泻病毒(Bovine virus diarrhea virus，BVDV)

<400> 1

atggaattga ttgcaaatga actcttatac aaaacataca agcaaaaacc cgtcggggtg 60

gaggaacctg tttacgatca ggcaggtaaa cctttgtttg gtgaaagagg agcaatccac 120

cctcaatcga cgctaaagct cccacacaag agaggggaac gcgatgttcc cacaaatctg 180

gcgtccctgc caaaaagagg tgactgcagg tcaggtaata gcaaaggacc tgtgagtgga 240

atctacctaa agccagggcc actatttttt caggactata agggccccgt ctaccacaga 300

gccccacttg aactcttcga ggagagtttt atgtgtgaaa caacaaagcg gatagggaga 360

gtgactggca gtgacggaaa gctataccac atttatgtat gtatagatgg atgtataata 420

ataaaaagtg ccacaagaag tcaccaaagg gtgcttaggt gggtccataa taggctcgac 480

tgccctctat gggtcacaag ctgctcagat gcaaaagaag aagggggcgc aaaaaagaag 540

caacaaaaac ccgacagact agagaagggg aggatgaaaa tagtgcccaa agagactgag 600

aaagacagca agactaaacc accggatgct acgatagtag tagatggagt taaataccag 660

gtaaagaaga agggaaaagt caagagtaaa aacacccagg acggtttata tcataacaaa 720

aataagccac cagaatcacg aaagaaactt gagaaagcac tattggcatg ggcaatattg 780

gctatggtct tgatccaagt tacaatgggt gaaaacataa cacagtggaa cccacaagat 840

aatggaacag aagggataca acgagcaatg ttccaaaggg gggtgaacag aagtctacac 900

ggtatctggc cagagaaaat ctgtacaggc gtcccctccc atctagccac cgatgtggaa 960

ctaaaaacaa tccatggcat gatggacgca agtgaaaaga ccaactatac gtgttgcaga 1020

ccccaacgcc atgagtggaa caagcacggt tggtgcaact ggtacaatat cgagccttgg 1080

attttgatca tgaataaaac ccaggccaat ctttctgagg gtcaaccacc aagagaatgc 1140

gcagtcactt gccgctatga tagggatagt gacctaaatg tggtaacaca agctagagat 1200

agccctacac cattgacagg ctgtaagaaa gggaaaaatt tttcctttgc aggcatattg 1260

atgaggggcc cctgcaactt tgaaatagca gcgagtgatg tgttgttcaa ggagcacgac 1320

tgcaccagtg tgttgcagga tactgctcat taccttgttg acgggatgac caattccttg 1380

gaaagtgcca gacaagggac cgctaaactg acaacctggt taggcaagca acttggaaaa 1440

ctaggaaaaa aattggaaaa aaagagtaag acgtggttcg gagcatatgc ggcttcccct 1500

tactgtgatg ttgatcgaaa acttggctac atatggtaca caaaaaattg tacccctgcc 1560

tgcttaccca aaaacacaaa aattattggc cctggcaagt ttgacaccaa tgcagaggat 1620

ggcaagatat tgcacgagat ggggggccac ttgtcgggag gtactactac tttcttagta 1680

gtagtgttgt ctgactttgc accagaaaca gctagtgcaa tgtatatagt tctacatttc 1740

tctatcccgc agagtcacgt tgatatagtg gattgtgaca agacccaatt gaatctcact 1800

gtggagctaa caacagcaga tgtaatacca gggtcggtct ggaatctagg cagatatgtc 1860

tgcataagac cggattggtg gccttatgag acaactgcag tgctggcatt cgaaagtggt 1920

gggtcagtga taaacgaata tgtctgtggc actgagagat tcgacagcat ctggaacgct 1980

gccacgacta ctgcattctt cagtatgccg tggttcaaga tggtcagagg gcgaaatagt 2040

acagggtcat ccgatggcta ctactgttat tcaggcgatg caacggcaca taagtttcaa 2100

actggaatac tcatatgcca tagcaaagag tgttatgaat cgaatcacta tgagttgaag 2160

acgtcagcac tgtttggaag gataactcac acgaaatgcc acttgtacac acaatggtca 2220

tcgtttggtg catggatggt aaaatttgta tatctcgcaa ggtgcacaag agaaaccaga 2280

tatcttgcaa ttttgcattc aagagcgtta cataccagag tggtatttga aaaattgttt 2340

gatgggttaa ggcagtggga cacagttgaa atggatgaca attttgaatt tggactttgc 2400

ccatgtgacg ccaaacccat agtaagaggg aaatttaaca caactctggt gaacggacgg 2460

gcgttccaga tggtatgccc tataggatgg acagggactg tgagttgtat gctggctaac 2520

agggataccc tagacacagc agttgggcgg agttatagga ggtccaaacc attcctttac 2580

aggcaaggct gtatcaccca aaaaatactg ggggaggatc tctataactg tgatcttgga 2640

ggaaattgga cttgcgtgac tggggaccaa ctacaataca caggaggctc tattgaatcc 2700

tgcaagtggt gtggttttaa attccagaaa agtgagggat taccacacta ccccattggc 2760

aagtgtaggt tggagaatga gactggctac agatttgtag acgacacctc ttgcaacaga 2820

gaaggtgtgg ccatagtacc aacagggcag ataaagtgta agataggaga cacaattgta 2880

caggtcttag ctcttgacac caaactcgga cctatgcctt gcaagccaca tgagatcata 2940

tcaagtgagg ggcctgtaga aaagacggca tgtaccttca actacacgaa gacattgaaa 3000

aataaatatt ttgagcccag agacagttac ttccagcaat atatgctaaa aggagagtat 3060

caatactggt ttgacctgga ggtcaccgac catcatcggg attactttgc cgagtccata 3120

ttactggtgg tggtacccct cctgggtggt agatacgtac tttggttact actcacatat 3180

atggtcttat cagaacaccg ggcctcaggg actcaatatg gagcaggaga agtggtgatg 3240

atgggcaact tgctaacaca tgatagcatc gaagtggtga catatttcct gttgctatac 3300

ctactgctca gagaggaaaa tgtaaagaaa tgggtcttac ttatatacca catcttagtg 3360

gcacacccat taaaatcagt aacagtgatc ttattgatga ttggggatgt ggtgaaggcc 3420

gatccagggg gccaagggta ctatgggcag atagatctct gttttacaat gattgtagtg 3480

atcgttatag gtttaatcat agccaggcgt gccccgacta tagtgccact ggtaacaata 3540

gttgcatcac tgagggtcac cggattgacc tacggacctg gggttgccgt cgatatggca 3600

gtcatgacca caaccctgct gatggttact tatgtgacag attacttcag atatacaaga 3660

tggctgcagt gcatactcag cctggtgtca ggggtgttct tgataagaag cctgatacat 3720

ctgggtacag ttgcggtgcc agaggtaacc atcccaaact ggagaccact aactttaatt 3780

ctgttatact tgatttcaac aacagttgtt acaatgtgga aggttgcctt ggctggccta 3840

ctgttgcaat gcctgcctat cttattgatg gttgcaacct tgtgggccga cttcttaact 3900

cttatactaa tcctgcctac ctatgcattg gttacattat actacctgaa ggctgttaag 3960

actgatatag aaagaagttg gctagggggg gttagactat acaagagtta ctccatctac 4020

gatgttgatg agagtggaga gggcgtgtat ctttttccat caaggcagaa ggcacagaaa 4080

aacttctcca tactcttgcc ccttgtcaga gctacactga taagttgtgt tagtagtaaa 4140

tggcagctaa tatacatggc ttacctaact gtggacttta tatactacat gcacaggaaa 4200

gttatagaag agatctcagg aggtaccaac atgatatcca ggttagtggc agcacttata 4260

gagctggact ggtccataga agaagaggag agcaaaggct taaagaagtt ttatctattg 4320

tctggaaggt tgagaggcct aataataaaa cataaagtta agaatgagac cgtggcttct 4380

tggtccgggg aggaggaagt ctacggtatg ccaaagatca tggcaatact caaggccagt 4440

acgctgggta agagcaagca ctgcatgcta tgcacggtat gtgagagccg agagtggaaa 4500

ggcggcacct gcccaaaatg tggacgccat gggaagccga tactgtgtgg gatgtcgcta 4560

ccggattttg aagaaagaca ctataaaaga atctttatac gggaaggtaa ctttgagggt 4620

cctttcaggc aagaatacaa tggctttgta caatataccg ctagggggca attacttgtg 4680

cgaaacttgc ccgtactggc aactaaagta caaatgctca tggtacgcaa ccttggagaa 4740

gaaattggtg atctggaaca tcttgggtgg atcctacggg ggcctgccgt gtgtaagaag 4800

atcacagagc acgaaaaatg ccacatcaat atactggata aactacctgc attttttggg 4860

atcatgccga gggggactac acccagagcc ccggtgcggt tccctacgag cttactacaa 4920

gtgcggggcc tggagactgg ctgggcttac acacaccaag gtgggatacg ttcagtcgac 4980

catgtacccg ctggcaaaga tctattggtc tgtgccagca tggggaggac tacggtggtc 5040

tgccagagca acaacatgtt gaccgatgca acagagtacg gtgtcaagac tgcctcagga 5100

tgcccagacg gagccagatg ttatgtgctc aatccggagg cagtcaacat atcagggtcc 5160

aagggggcag tcgtccacct ccaaaagaca ggcggggaat tcacgtgtgt cactgcatca 5220

ggtacaccgg ctttcttcga tctacagaac ttgcagggat ggtcgggcct gcccattttt 5280

ggagcctcca gtggaagggt ggttggcagg gttacagtac ggaagaacga agagtctaca 5340

cccacaaaga tactgcgtgg catccagacc gtctcaaaga acacagcaga tctgcctgcg 5400

atggtcaaga agatcaccag tatgcacagg ggggacttca agcagatacc tttaccgaca 5460

ggagcaggaa aaaccacaga acttccaaaa gcagtggtgg aggagatacg aagacacaag 5520

cgagtgttac tccttatacc actgcgggca gcggcggagt cagtctacca ttatatgcga 5580

ttgcaacacc ctattatatc cttcaacctg cggatacggg acatgcaaga gggggacatg 5640

gcaaccggga taccttatgc atcatacggg tacttctgtc agatgcctca accaaagctt 5700

acagcagcca tggtggaata ctcatacata ttcttacacg aataccactg tgccaccccc 5760

gaacaattgg cgatcattgg aaaaatccat acattctcag agagtatacg agtggttgcc 5820

atgcctgcca cccccgcagg gtcagtaccc acaacaggac aaaagcaccc aatacaggag 5880

ttcatacccc ctgtggtgct gcaaggagaa gacctacgca gccagttcct tgctataccg 5940

gggctacaaa tccccgtggc tgcgatgcaa ggtaccatgc tggtttttgt acccacgagg 6000

aacatggctg ttgtggtggc aaagaagcta caggccaagg gctacaattc tgggtactac 6060

tacagcggag aggatccagc tattctacga gttgtgccat cacagtcccc ttatgtactt 6120

gtggctacga atgccattgc atcaggagta ccattaccag atttggacac ggtcgtggac 6180

acagggttgc aatgtgcaaa gagggtgcgg atatcatcaa aagtaccctt cattgtacca 6240

ggccttaaga ggatggccgt cactgtgggt gaacaggctc aacgtagagg cagagtacgt 6300

agggtgcagc ccgggaaata ctacaggagt caggaaacag cgactgggtc aaaggactac 6360

cactatgacc tcttgcaggc acaaagatac ggcattgagg atggaatcaa cgtaccgaag 6420

tcctttaggg agatgcatta cgattggagc ctatacgagg aggacagcct actactatcc 6480

cagttggaaa tactacataa tctactcatc tcagaagact tgccagccgc tgttaagaat 6540

atactggcca ggacagatca cccagagcca atccaacttg catacaacag ctatgaagtc 6600

caggttccag tcctgtttcc caaaataagg aatggagaag tcacagacac ctacgaaaac 6660

tactcatttc taaatgctag aaagttggga gaggatgtgc ctgtgtatat ctacgccact 6720

gaagatgagg acctggcagt tgacctccta gggctagact ggcccgatcc tgggaaccaa 6780

caggtcgtgg agactagcaa agcactgaag caagtggccg ggttgtcttc agctgcgaat 6840

gctttactcg tggctttatt tgggtatgta ggttaccaaa ccctatcgaa gaggcatgtc 6900

ccaatgatca cagacatata cactatagag gatcagaggt tagaagatac cacccacctt 6960

cagtatgcgc ccaacgccat aaaaactgcg gggacagata ccgaattgaa ggaattggca 7020

tcgggtgacg tggaaaaaat tatgggagct atctcggatt atgcagctgg aggactggag 7080

tttgtgaaat ctcaagcgga aaggataaag acagcccctt cgttcaagga aaatgtagaa 7140

gctgcaaagg ggtacgtcca aaaattaatt gactcattaa ttgaaaataa agatgtaata 7200

atcagatatg gtctatgggg aacacataca gcactctata aaagcatagc tgcaaggcta 7260

gggcatgaaa cagcgtttgc cacactagtg ctgaaatggc tggctttcgg aggggaaaca 7320

gtgtcagacc acgtcagaca ggcggcggtt gatctagtgg tctattatgt gatgaacaag 7380

ccctccttcc caggtgacac tgaaacacag caggaaggga ggcgattcgt cgctagccta 7440

ttcatctccg cattggcgac ttacacatac aaaacctgga attatcacaa tctctcaaaa 7500

gtggtggaac cagccctagc gtacctccct tatgccacca gcgcatataa atgttcaccc 7560

caacgaagat tggaaagcgt ggtgatactg agcactacaa tatacaaaac atacctctcc 7620

ataaggaaag ggaagagtgc tggattgctg ggtacgggga tcagtgcagc catggagatc 7680

ctgtcacaaa acccagtatc ggtaggtata tccgtgatgc tgggggtagg agctatcgct 7740

gcgcataccg ccattgcatc cagcgaacag aaaaggaccc tgcttatgaa ggtgtttgta 7800

aagaactttt tagatcaggc tgcaccggat gcgctggtta cagaaaaccc agaaaaaatc 7860

ataatggctt tatttgcagc agtccagaca attggcaacc ccctaagatt aatataccac 7920

ctgtacgggg tctactacaa gggctgggag gccaaggagt tatcagagag gacagcaggc 7980

agaaacttat tcacactgat aatgtttgcg gccttcgaat tgctagggat ggattcagaa 8040

gggaaaataa gaaatctgtc tggaaactac atattagact taatctacgg attacatacg 8100

cagatcaaca gagggctaaa gaagatagtg ttggggtggg cccctgcacc cttcagctgt 8160

gcctggaccc ctaccgatgc gaggatcaga ttaccaacgg acaactactt acgagtggaa 8220

acaaggtgcc cgtgcggcta tcagatgaga gctatcaaaa acgtaggtgg tacgctcacc 8280

aaggtggagg agagtggacc tttcctatgt acgaacagac ctggtacggg gccagtcaat 8340

cacagagtca ccaagtacta cgatgccaat ctcagagaga taaaaccagt ggcgaggtta 8400

gaaggacagg tggagcacta ctacaaaggg gtcacggcaa aaattgacta cagtaaagga 8460

aaaacactct tagctactga taaatgggag gtggaacatg gtaccattac caggttggct 8520

aagagataca ctggggtcgg gttcaacggt gcgtacctag gcgaccaacc caatcaccgt 8580

gatctagtgg agagggtttg tgcaactata actaaaaaca cagtgcagtt tctaaaaatg 8640

aagaaagggt gtgcttttac ctacgacctg accatctcca atttgaccag gctcatcgaa 8700

ctagtgcaca ggaacaacct tgaagagaag gaaatacccg tcgccacagt caccacatgg 8760

ctagcttata ccttcgtgaa cgaagacgtg gggactataa aaccagtact aggggagagg 8820

gttatccctg accctgtaga tgatgtcaat ttacaaccag aggtccaggt ggacacatca 8880

gaggttggga tcacaataat tgggcgggga gcctcgatga caacaggagt gacgcccgtt 8940

gtggaaaagg tagagcccga cgctaatggc agccaaaacc cagtgaagat cgggttggat 9000

gaaggtaatt acccagggct tggtgtgcag acacgcacac tagtagaaga aatacacaac 9060

agggacgcga gaccctttat catggtctta ggctcaaaga gttccatgtc aaacagagca 9120

aggacagcta gaaatataaa cctgtataca ggaaatgacc ccagggaggt aagagacttg 9180

atggcagaag ggcgtatgtt agtggtggca ttgagggaca tcggccctga tctacaagaa 9240

ctggttgact tcaaagggac ttttttagat agggaagcct tggaagctct aagtcttggg 9300

cagccaaaac cgaagcgggc taccaaagca gcaattaggg aattgttgaa agaggaaaga 9360

caggtggaga tccctaactg gtttacatca gatgatccag tattcttgga ggtggccata 9420

aataaagata agtaccactt agtaggagat gttggagagg taaaagatca agctaaaaca 9480

cttggggcac ccgatcagac aagaattgta aaggaggtag gccgtaccgt accagccaag 9540

gacgtatacc atgaagctcc ctacttggtt cctccaatca tcacacaaca gatcagttta 9600

actccactgt ttgaggaact gctgctacgg tgtccacctg caactaagag cactaagggg 9660

cacatggcat cagcttacca attggcacag ggtaactggg agcccctcgg ttgtggggtg 9720

cacctaggta ccataccagc cagaagggtg aagatacacc catatgaagc ttacctaaag 9780

tttaaaaagc ttcatagaag aagaagaaga gaaaccaagg atcaaggata cagtaatgag 9840

agagcacaac aaatggatac ttacaaaaat aaggtttcaa ggaaacctca ggaccaagaa 9900

aatactcaac cccggaaaac tatctgcaca gctggacagg gaggggcgca aaagaaacat 9960

ctacaacaac cagatcggta ccataatgtc cagtgcaggc ataaggctgg aaaaattgcc 10020

aatagtaagg gcccaaaccg acactataac ctttcacgag gcaataagag atatgataga 10080

catgaatatg agaaccggca aaatccagaa ttgcacaaca aattgttgga aatctttcac 10140

acaatagccc accccaccct aaaacacacc tacggtgagg tgacgtggga gcaacttgag 10200

gcagggataa ataggaaagg ggctgcaggc ttcctggaga agaagaacat cggggaagta 10260

ttggattcag aaaaacacct ggtggaacaa ttgatcaggg acctgaaggc cgggagaaag 10320

gtgaaatatt atgaaactgc aataccaaaa aatgagaaaa gagatgtcag cgatgactgg 10380

caggcagggg acctggtgga tgagaagaag ccaagagtga ttcaataccc tgaagccaag 10440

ataaggttag ccatcactaa ggtcatgtat aactgggtaa aacagcagcc cgttgtaatt 10500

ccaggatatg agggaaagac ccccttgttc aacatcttta ataaagtgag aaaggaatgg 10560

gacttgttca atgagccagt ggccgtaagc tttgatacca aagcttggga cacacaagtg 10620

actagtaggg atctacaact tatcggagaa atccagaaat attactatag gaaggaatgg 10680

cacaagttca ttgacaccat caccgaccac atgatagaag tgccagttat aacagcagat 10740

ggtgaagtat atataagaaa tgggcagaga gggagtggcc agccagacac aagtgcgggc 10800

aacagcatgt taaatgtcct aacaatgatg tacgcttttt gcgaaagcac aggggtcccg 10860

tacaagagtt tcaacagggt ggcaaggatc catgtctgtg gggatgatgg cttcttaata 10920

actgaaaaag ggttaggact gaaatttgcc aacaagggga tgcaaatcct tcacgaagca 10980

ggcaagcccc agaaaataac ggaaggggaa aagatgaaag ttgcctatag attcgaggac 11040

atagagtttt gctcccatac cccagtccct gttaggtggt ctgataatac cagtagtcac 11100

atggccggca gagacacccg ctgtgatact atcaaagatg gctacaagat tggattcaag 11160

tggggaaagg ggtaccacag catatgcaaa agcggtagcc ttcagtttct tgctgatgta 11220

ttcctggaac ccgcttgtta cgatttgcct gttggtactc tcgcagcagc cagagctaga 11280

cccatccaca cagaccactt attattatac atgatgatcc acttacgacc tatacagatg 11340

tgcacctata gagaacaggc ttcgagaaat tggccaatct acacctacgc ctgtccacat 11400

tacggatctg gactaaacac acaagcaaaa gaatactcaa tgactgtgtt gccattggga 11460

aaggaagagg gcaactggct agttaacgcc gacaggttga tatccagcaa aactggccac 11520

ttatacatac ctgataaggg ctttacatta caaggaaggc attatgagca actacagcta 11580

ggaacagaga caaacccggc tacgggtgtc gggactgaga gatacaagtt aggtcccata 11640

gtcaatctgc tgctgagaag gttgaaggtc ctgctcatga cggccgtcgg tgccagctgc 11700

tga 11703

<210> 2

<211> 3900

<212> PRT

<213> 牛病毒性腹泻病毒(Bovine virus diarrhea virus，BVDV)

<400> 2

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

50 55 60

Lys Arg Gly Asp Cys Arg Ser Gly Asn Ser Lys Gly Pro Val Ser Gly

65 70 75 80

Ile Tyr Leu Lys Pro Gly Pro Leu Phe Phe Gln Asp Tyr Lys Gly Pro

85 90 95

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

165 170 175

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

180 185 190

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

260 265 270

Ile Thr Gln Trp Asn Pro 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 Pro 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 Lys Thr Gln

355 360 365

Ala Asn Leu Ser 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 Ile Leu Met Arg Gly Pro Cys Asn Phe Glu Ile Ala Ala Ser

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Ala Ala Ser Pro Tyr Cys Asp Val Asp Arg Lys Leu 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

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 Gly Gly Thr Thr Thr Phe Leu Val

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Gly Ser Val Ile Asn Glu Tyr Val Cys Gly Thr Glu Arg Phe Asp Ser

645 650 655

Ile Trp Asn Ala Ala Thr Thr Thr Ala Phe Phe Ser Met Pro Trp Phe

660 665 670

Lys Met Val Arg Gly Arg Asn Ser Thr Gly Ser Ser Asp Gly Tyr Tyr

675 680 685

Cys Tyr Ser Gly Asp Ala Thr Ala His Lys Phe Gln Thr Gly Ile Leu

690 695 700

Ile Cys His Ser Lys Glu Cys Tyr Glu Ser Asn His Tyr Glu Leu Lys

705 710 715 720

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

725 730 735

Thr Gln Trp Ser Ser Phe Gly Ala Trp Met Val Lys Phe Val Tyr Leu

740 745 750

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

755 760 765

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

770 775 780

Gln Trp Asp Thr Val Glu Met Asp Asp Asn Phe Glu Phe Gly Leu Cys

785 790 795 800

Pro Cys Asp Ala Lys Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu

805 810 815

Val Asn Gly Arg Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly

820 825 830

Thr Val Ser Cys Met Leu Ala Asn Arg Asp Thr Leu Asp Thr Ala Val

835 840 845

Gly Arg Ser Tyr Arg Arg Ser Lys Pro Phe Leu Tyr Arg Gln Gly Cys

850 855 860

Ile Thr Gln Lys Ile Leu Gly Glu Asp Leu Tyr Asn Cys Asp Leu Gly

865 870 875 880

Gly Asn Trp Thr Cys Val Thr Gly Asp Gln Leu Gln Tyr Thr Gly Gly

885 890 895

Ser Ile Glu Ser Cys Lys Trp Cys Gly Phe Lys Phe Gln Lys Ser Glu

900 905 910

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

915 920 925

Gly Tyr Arg Phe Val Asp Asp Thr Ser Cys Asn Arg Glu Gly Val Ala

930 935 940

Ile Val Pro Thr Gly Gln Ile Lys Cys Lys Ile Gly Asp Thr Ile Val

945 950 955 960

Gln Val Leu Ala Leu Asp Thr Lys Leu Gly Pro Met Pro Cys Lys Pro

965 970 975

His Glu Ile Ile Ser Ser Glu Gly Pro Val Glu Lys Thr Ala Cys Thr

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Leu Leu Val Val Val Pro Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu

1045 1050 1055

Leu Leu Thr Tyr Met Val Leu Ser Glu His Arg Ala Ser Gly Thr Gln

1060 1065 1070

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

1075 1080 1085

Ser Ile Glu Val Val Thr Tyr Phe Leu Leu Leu Tyr Leu Leu Leu Arg

1090 1095 1100

Glu Glu Asn Val Lys Lys Trp Val Leu Leu Ile Tyr His Ile Leu Val

1105 1110 1115 1120

Ala His Pro Leu Lys Ser Val Thr Val Ile Leu Leu Met Ile Gly Asp

1125 1130 1135

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

1140 1145 1150

Leu Cys Phe Thr Met Ile Val Val Ile Val Ile Gly Leu Ile Ile Ala

1155 1160 1165

Arg Arg Ala Pro Thr Ile Val Pro Leu Val Thr Ile Val Ala Ser Leu

1170 1175 1180

Arg Val Thr Gly Leu Thr Tyr Gly Pro Gly Val Ala Val Asp Met Ala

1185 1190 1195 1200

Val Met Thr Thr Thr Leu Leu Met Val Thr Tyr Val Thr Asp Tyr Phe

1205 1210 1215

Arg Tyr Thr Arg Trp Leu Gln Cys Ile Leu Ser Leu Val Ser Gly Val

1220 1225 1230

Phe Leu Ile Arg Ser Leu Ile His Leu Gly Thr Val Ala Val Pro Glu

1235 1240 1245

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

1250 1255 1260

Ile Ser Thr Thr Val Val Thr Met Trp Lys Val Ala Leu Ala Gly Leu

1265 1270 1275 1280

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

1285 1290 1295

Asp Phe Leu Thr Leu Ile Leu Ile Leu Pro Thr Tyr Ala Leu Val Thr

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Asn Phe Ser Ile Leu Leu Pro Leu Val Arg Ala Thr Leu Ile Ser Cys

1365 1370 1375

Val Ser Ser Lys Trp Gln Leu Ile Tyr Met Ala Tyr Leu Thr Val Asp

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

Ser Ile Glu Glu Glu Glu Ser Lys Gly Leu Lys Lys Phe Tyr Leu Leu

1425 1430 1435 1440

Ser Gly Arg Leu Arg Gly Leu Ile Ile Lys His Lys Val Lys Asn Glu

1445 1450 1455

Thr Val Ala Ser Trp Ser Gly Glu Glu Glu Val Tyr Gly Met Pro Lys

1460 1465 1470

Ile Met Ala Ile Leu Lys Ala Ser Thr Leu Gly Lys Ser Lys His Cys

1475 1480 1485

Met Leu Cys Thr Val Cys Glu Ser Arg Glu Trp Lys Gly Gly Thr Cys

1490 1495 1500

Pro Lys Cys Gly Arg His Gly Lys Pro Ile Leu Cys Gly Met Ser Leu

1505 1510 1515 1520

Pro Asp Phe Glu Glu Arg His Tyr Lys Arg Ile Phe Ile Arg Glu Gly

1525 1530 1535

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

1540 1545 1550

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

1555 1560 1565

Lys Val Gln Met Leu Met Val Arg Asn Leu Gly Glu Glu Ile Gly Asp

1570 1575 1580

Leu Glu His Leu Gly Trp Ile Leu Arg Gly Pro Ala Val Cys Lys Lys

1585 1590 1595 1600

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

1605 1610 1615

Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro Arg Ala Pro Val

1620 1625 1630

Arg Phe Pro Thr Ser Leu Leu Gln Val Arg Gly Leu Glu Thr Gly Trp

1635 1640 1645

Ala Tyr Thr His Gln Gly Gly Ile Arg Ser Val Asp His Val Pro Ala

1650 1655 1660

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

1665 1670 1675 1680

Cys Gln Ser Asn Asn Met Leu Thr Asp Ala Thr Glu Tyr Gly Val Lys

1685 1690 1695

Thr Ala Ser Gly Cys Pro Asp Gly Ala Arg Cys Tyr Val Leu Asn Pro

1700 1705 1710

Glu Ala Val Asn Ile Ser Gly Ser Lys Gly Ala Val Val His Leu Gln

1715 1720 1725

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

1730 1735 1740

Phe Phe Asp Leu Gln Asn Leu Gln Gly Trp Ser Gly Leu Pro Ile Phe

1745 1750 1755 1760

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

1765 1770 1775

Glu Glu Ser Thr Pro Thr Lys Ile Leu Arg Gly Ile Gln Thr Val Ser

1780 1785 1790

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

1795 1800 1805

His Arg Gly Asp Phe Lys Gln Ile Pro Leu Pro Thr Gly Ala Gly Lys

1810 1815 1820

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

1825 1830 1835 1840

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

1845 1850 1855

His Tyr Met Arg Leu Gln His Pro Ile Ile Ser Phe Asn Leu Arg Ile

1860 1865 1870

Arg Asp Met Gln Glu Gly Asp Met Ala Thr Gly Ile Pro Tyr Ala Ser

1875 1880 1885

Tyr Gly Tyr Phe Cys Gln Met Pro Gln Pro Lys Leu Thr Ala Ala Met

1890 1895 1900

Val Glu Tyr Ser Tyr Ile Phe Leu His Glu Tyr His Cys Ala Thr Pro

1905 1910 1915 1920

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

1925 1930 1935

Arg Val Val Ala Met Pro Ala Thr Pro Ala Gly Ser Val Pro Thr Thr

1940 1945 1950

Gly Gln Lys His Pro Ile Gln Glu Phe Ile Pro Pro Val Val Leu Gln

1955 1960 1965

Gly Glu Asp Leu Arg Ser Gln Phe Leu Ala Ile Pro Gly Leu Gln Ile

1970 1975 1980

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

1985 1990 1995 2000

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

2005 2010 2015

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

2020 2025 2030

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

2035 2040 2045

Gly Val Pro Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly Leu Gln

2050 2055 2060

Cys Ala Lys Arg Val Arg Ile Ser Ser Lys Val Pro Phe Ile Val Pro

2065 2070 2075 2080

Gly Leu Lys Arg Met Ala Val Thr Val Gly Glu Gln Ala Gln Arg Arg

2085 2090 2095

Gly Arg Val Arg Arg Val Gln Pro Gly Lys Tyr Tyr Arg Ser Gln Glu

2100 2105 2110

Thr Ala Thr Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gln Ala Gln

2115 2120 2125

Arg Tyr Gly Ile Glu Asp Gly Ile Asn Val Pro Lys Ser Phe Arg Glu

2130 2135 2140

Met His Tyr Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu Leu Leu Ser

2145 2150 2155 2160

Gln Leu Glu Ile Leu His Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala

2165 2170 2175

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

2180 2185 2190

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

2195 2200 2205

Ile Arg Asn Gly Glu Val Thr Asp Thr Tyr Glu Asn Tyr Ser Phe Leu

2210 2215 2220

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

2225 2230 2235 2240

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

2245 2250 2255

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

2260 2265 2270

Ala Gly Leu Ser Ser Ala Ala Asn Ala Leu Leu Val Ala Leu Phe Gly

2275 2280 2285

Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Val Pro Met Ile Thr

2290 2295 2300

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

2305 2310 2315 2320

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

2325 2330 2335

Lys Glu Leu Ala Ser Gly Asp Val Glu Lys Ile Met Gly Ala Ile Ser

2340 2345 2350

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

2355 2360 2365

Ile Lys Thr Ala Pro Ser Phe Lys Glu Asn Val Glu Ala Ala Lys Gly

2370 2375 2380

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

2385 2390 2395 2400

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

2405 2410 2415

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

2420 2425 2430

Trp Leu Ala Phe Gly Gly Glu Thr Val Ser Asp His Val Arg Gln Ala

2435 2440 2445

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

2450 2455 2460

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

2465 2470 2475 2480

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

2485 2490 2495

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

2500 2505 2510

Thr Ser Ala Tyr Lys Cys Ser Pro Gln Arg Arg Leu Glu Ser Val Val

2515 2520 2525

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

2530 2535 2540

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

2545 2550 2555 2560

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

2565 2570 2575

Gly Ala Ile Ala Ala His Thr Ala Ile Ala Ser Ser Glu Gln Lys Arg

2580 2585 2590

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

2595 2600 2605

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

2610 2615 2620

Phe Ala Ala Val Gln Thr Ile Gly Asn Pro Leu Arg Leu Ile Tyr His

2625 2630 2635 2640

Leu Tyr Gly Val Tyr Tyr Lys Gly Trp Glu Ala Lys Glu Leu Ser Glu

2645 2650 2655

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

2660 2665 2670

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

2675 2680 2685

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

2690 2695 2700

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

2705 2710 2715 2720

Ala Trp Thr Pro Thr Asp Ala Arg Ile Arg Leu Pro Thr Asp Asn Tyr

2725 2730 2735

Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Gln Met Arg Ala Ile

2740 2745 2750

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

2755 2760 2765

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

2770 2775 2780

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

2785 2790 2795 2800

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

2805 2810 2815

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

2820 2825 2830

His Gly Thr Ile Thr Arg Leu Ala Lys Arg Tyr Thr Gly Val Gly Phe

2835 2840 2845

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

2850 2855 2860

Arg Val Cys Ala Thr Ile Thr Lys Asn Thr Val Gln Phe Leu Lys Met

2865 2870 2875 2880

Lys Lys Gly Cys Ala Phe Thr Tyr Asp Leu Thr Ile Ser Asn Leu Thr

2885 2890 2895

Arg Leu Ile Glu Leu Val His Arg Asn Asn Leu Glu Glu Lys Glu Ile

2900 2905 2910

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

2915 2920 2925

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

2930 2935 2940

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

2945 2950 2955 2960

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

2965 2970 2975

Val Thr Pro Val Val Glu Lys Val Glu Pro Asp Ala Asn Gly Ser Gln

2980 2985 2990

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

2995 3000 3005

Val Gln Thr Arg Thr Leu Val Glu Glu Ile His Asn Arg Asp Ala Arg

3010 3015 3020

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

3025 3030 3035 3040

Arg Thr Ala Arg Asn Ile Asn Leu Tyr Thr Gly Asn Asp Pro Arg Glu

3045 3050 3055

Val Arg Asp Leu Met Ala Glu Gly Arg Met Leu Val Val Ala Leu Arg

3060 3065 3070

Asp Ile Gly Pro Asp Leu Gln Glu Leu Val Asp Phe Lys Gly Thr Phe

3075 3080 3085

Leu Asp Arg Glu Ala Leu Glu Ala Leu Ser Leu Gly Gln Pro Lys Pro

3090 3095 3100

Lys Arg Ala Thr Lys Ala Ala Ile Arg Glu Leu Leu Lys Glu Glu Arg

3105 3110 3115 3120

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

3125 3130 3135

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

3140 3145 3150

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

3155 3160 3165

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

3170 3175 3180

Glu Ala Pro Tyr Leu Val Pro Pro Ile Ile Thr Gln Gln Ile Ser Leu

3185 3190 3195 3200

Thr Pro Leu Phe Glu Glu Leu Leu Leu Arg Cys Pro Pro Ala Thr Lys

3205 3210 3215

Ser Thr Lys Gly His Met Ala Ser Ala Tyr Gln Leu Ala Gln Gly Asn

3220 3225 3230

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

3235 3240 3245

Arg Val Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Phe Lys Lys Leu

3250 3255 3260

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

3265 3270 3275 3280

Arg Ala Gln Gln Met Asp Thr Tyr Lys Asn Lys Val Ser Arg Lys Pro

3285 3290 3295

Gln Asp Gln Glu Asn Thr Gln Pro Arg Lys Thr Ile Cys Thr Ala Gly

3300 3305 3310

Gln Gly Gly Ala Gln Lys Lys His Leu Gln Gln Pro Asp Arg Tyr His

3315 3320 3325

Asn Val Gln Cys Arg His Lys Ala Gly Lys Ile Ala Asn Ser Lys Gly

3330 3335 3340

Pro Asn Arg His Tyr Asn Leu Ser Arg Gly Asn Lys Arg Tyr Asp Arg

3345 3350 3355 3360

His Glu Tyr Glu Asn Arg Gln Asn Pro Glu Leu His Asn Lys Leu Leu

3365 3370 3375

Glu Ile Phe His Thr Ile Ala His Pro Thr Leu Lys His Thr Tyr Gly

3380 3385 3390

Glu Val Thr Trp Glu Gln Leu Glu Ala Gly Ile Asn Arg Lys Gly Ala

3395 3400 3405

Ala Gly Phe Leu Glu Lys Lys Asn Ile Gly Glu Val Leu Asp Ser Glu

3410 3415 3420

Lys His Leu Val Glu Gln Leu Ile Arg Asp Leu Lys Ala Gly Arg Lys

3425 3430 3435 3440

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

3445 3450 3455

Ser Asp Asp Trp Gln Ala Gly Asp Leu Val Asp Glu Lys Lys Pro Arg

3460 3465 3470

Val Ile Gln Tyr Pro Glu Ala Lys Ile Arg Leu Ala Ile Thr Lys Val

3475 3480 3485

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

3490 3495 3500

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

3505 3510 3515 3520

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

3525 3530 3535

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

3540 3545 3550

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

3555 3560 3565

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

3570 3575 3580

Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp Thr Ser Ala Gly

3585 3590 3595 3600

Asn Ser Met Leu Asn Val Leu Thr Met Met Tyr Ala Phe Cys Glu Ser

3605 3610 3615

Thr Gly Val Pro Tyr Lys Ser Phe Asn Arg Val Ala Arg Ile His Val

3620 3625 3630

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

3635 3640 3645

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

3650 3655 3660

Lys Ile Thr Glu Gly Glu Lys Met Lys Val Ala Tyr Arg Phe Glu Asp

3665 3670 3675 3680

Ile Glu Phe Cys Ser His Thr Pro Val Pro Val Arg Trp Ser Asp Asn

3685 3690 3695

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

3700 3705 3710

Asp Gly Tyr Lys Ile Gly Phe Lys Trp Gly Lys Gly Tyr His Ser Ile

3715 3720 3725

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

3730 3735 3740

Ala Cys Tyr Asp Leu Pro Val Gly Thr Leu Ala Ala Ala Arg Ala Arg

3745 3750 3755 3760

Pro Ile His Thr Asp His Leu Leu Leu Tyr Met Met Ile His Leu Arg

3765 3770 3775

Pro Ile Gln Met Cys Thr Tyr Arg Glu Gln Ala Ser Arg Asn Trp Pro

3780 3785 3790

Ile Tyr Thr Tyr Ala Cys Pro His Tyr Gly Ser Gly Leu Asn Thr Gln

3795 3800 3805

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

3810 3815 3820

Asn Trp Leu Val Asn Ala Asp Arg Leu Ile Ser Ser Lys Thr Gly His

3825 3830 3835 3840

Leu Tyr Ile Pro Asp Lys Gly Phe Thr Leu Gln Gly Arg His Tyr Glu

3845 3850 3855

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

3860 3865 3870

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

3875 3880 3885

Lys Val Leu Leu Met Thr Ala Val Gly Ala Ser Cys

3890 3895 3900

<210> 3

<211> 20

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 3

gccatgccct tagtaggact 20

<210> 4

<211> 20

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 4

caccctatca ggctgtrtyc 20

<210> 5

<211> 20

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 5

ccaccaggta tgaattggac 20

<210> 6

<211> 20

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 6

cgccatctga gtgattactc 20

<210> 7

<211> 18

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 7

cgagttgaac acccagat 18

<210> 8

<211> 18

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 8

gagacgggca tctacact 18

<210> 9

<211> 19

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 9

agcaacactg gattgtgcc 19

<210> 10

<211> 18

<212> DNA

<213> 人工序列(Artificial sequence)

<400> 10

ggagtagtcc gactccgc 18

Claims

1.一株牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株，其特征在于，系从牛病毒性腹泻病毒流行地区分离获得，于2018年3月28日保藏于中国典型培养物保藏中心，保藏号CCTCCNO:V201813。

2.权利要求1所述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株在制备牛病毒性腹泻病毒疫苗中的应用。

3.根据权利要求2所述的应用，其特征在于，所述牛病毒性腹泻病毒疫苗为全病毒灭活疫苗或弱毒疫苗。

4.一种牛病毒性腹泻病毒灭活疫苗的制备方法，其特征在于，包括以下步骤：将权利要求1所述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016大量培养，之后病毒液中加入0.5% β-丙内酯于37℃灭活6h，灭活完成后进行无菌，灭活检验，然后将灭活的病毒液与201佐剂等体积充分混合，制成水包油包水的灭活疫苗。

5.权利要求1所述的牛病毒性腹泻病毒SMU-Z6/1a/SC/2016分离株在制备牛病毒性腹泻病毒诊断试剂中的应用。