CN114480308A - 一种重组杆状病毒及其制备方法和应用 - Google Patents
一种重组杆状病毒及其制备方法和应用 Download PDFInfo
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- CN114480308A CN114480308A CN202210049785.6A CN202210049785A CN114480308A CN 114480308 A CN114480308 A CN 114480308A CN 202210049785 A CN202210049785 A CN 202210049785A CN 114480308 A CN114480308 A CN 114480308A
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- recombinant baculovirus
- bovine viral
- viral diarrhea
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Abstract
本发明实施例涉及一种表达牛病毒性腹泻病毒E2基因的重组杆状病毒及其制备方法和应用，属于生物工程领域，重组杆状病毒包含一个或多个拷贝的牛病毒性腹泻病毒E2蛋白的编码基因，所述E2蛋白的氨基酸序列如SEQ ID NO.1所示。本发明通过E2蛋白基因序列进行碱基修饰和优化，使蛋白的免疫原性更好。通过与专利的605佐剂混合配制疫苗，更增强了疫苗的免疫效果。
Description
技术领域
本发明涉及兽用生物制品领域，具体为，一种表达牛病毒性腹泻病毒E2蛋白的重组杆状病毒、重组杆状病毒的制备方法、一种重组杆状病毒在制备牛病毒性腹泻病毒的抗原、抗体或亚单位疫苗中的应用、一种牛病毒性腹泻病毒亚单位疫苗、一种牛病毒性腹泻病毒亚单位疫苗的制备方法。
背景技术
牛病毒性腹泻病毒(Bovine Viral Diarrhea Virus，BVDV)是引起的牛的粘膜发炎、糜烂、坏死和腹泻为特征的传染性疾病的病原，是黄病毒科、瘟病毒毒属的代表种。牛病毒性腹泻病毒可感染各种年龄的牛，以幼龄牛易感性最高，主要侵害6～18月龄的幼牛。传染来源主要是病畜，以直接接触或间接接触方式进行传播。患牛表现为发病急，体温突然升高至40～42℃，食欲废绝，消化道粘膜损伤严重，最初常表现为水样腹泻，后期便中带血和粘膜，病牛的死亡率可高达90％。怀孕母牛感染后，可造成流产、早产或死胎。如足月生产，犊牛可表现为先天性缺陷，小脑发育不全，共济失调，或不能站立、发育不良、生长缓慢、饲养困难。因此养殖场必须防控牛病毒性腹泻病毒。
BVDV感染情况复杂，持续性感染个体症状隐秘，即使BVDV感染90％情况下也很难引起注意，因此防控BVDV十分必要。另外，目前由牛病毒性腹泻病毒2型引起的犊牛腹泻，感染更为严重。目前为止，牛病毒性腹泻病毒疫苗的使用和试剂盒辅助检测是预防本病既简单又有效的方法。但由于疫苗毒株基因型差异、母源抗体、多次免疫和自然感染等因素的影响使得BVDV感染后血清型变得复杂。目前，全球商品化的BVD疫苗有许多种，但大多数疫苗都是针对BVDV1型毒株的，专门针对预防BVDV2型流行毒株的疫苗尚未见报道。减少牛病毒性腹泻病毒2型感染对养牛业造成的损失，是本领域亟需解决的技术问题。
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本发明的目的在于提供在于提供一种重组杆状病毒、重组杆状病毒的制备方法、一种重组杆状病毒在制备牛病毒性腹泻病毒的抗原、抗体或亚单位疫苗中的应用、一种牛病毒性腹泻病毒亚单位疫苗、一种牛病毒性腹泻病毒亚单位疫苗的制备方法。
本发明将2型BVDV病毒结构蛋白E2基因与Erns基因经过碱基修饰后克隆至pFastBacDual载体，构建pFBD-BVDV2-E2重组杆状病毒载体，在大肠杆菌中转座，获得重组杆状病毒载体Bacmid-BVDV2-E2，再转染昆虫细胞sf9进行表达，收获即得；能够稳定表达具有良好免疫原性的BVDV2-E2蛋白。
本发明的重组杆状病毒，属于杆状病毒科，核型多角体病毒属，拉丁文学名为Autographa californica multiple nucleopolyhedrovirus，该重组杆状病毒命名为苜蓿银纹夜蛾核型多角体病毒(简称杆状病毒)Baculo-BVDV2-E2。
解决方案
为实现本发明目的，提供如下技术方案：
一方面，本发明提供一种重组杆状病毒，其包含一个或多个拷贝的牛病毒性腹泻病毒E2蛋白的编码基因，所述E2蛋白的氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1序列为优化后的氨基酸序列：
FPECKEGFQYAISKDKKIGPLGPESLTTTWHLPTKKIVDSMVQVWCDGKDLKILKTCTKEERYLVAVHERALSTSAEFMQISSGTKGPEVIDMPDDFEFGLCPCDSKPVIRGKFNASLLNGPAFQMVCPQGWTGTIECILANQDTLDTTVVRTYRRTTPFQRRKWCTYEKIIGEDIHECILGGNWTCITGDHSKLKGGPIKKCKWCSYDFFNSEGLPHYPIGKCMLINESGYRYVDDTSCDRGGVAIVQTGTVKCRIGNTTVQVIATNTDLGPMPCSPAEVIASEGPVEKTACTFNYSETLPNKYYEPRDQYFQQYMLKGKWQYWFDLDSIDHHKDYFSEFI
未优化前的氨基酸序列如SEQ ID NO.2所示：
FPECKEGFQYAISKDKKIGPLGPESLTTTWHLPTKKIVDSMVQVWCDGKDLKILKTCTKEERYLVAVHERALSTSAEFMQISSGTKGPEVIDMPDDFEFGLCPCDSKPVIRGKFNASLLNGPAFQMVCPQGWTGTIECILANQDTLDTTVVRTYRRTTPFQRRKWCTYEKIIGEDIHECILGGNWTCITGDHSKLKGGPIKKCKWCSYDFFNSEGLPHYPIGKCMLINESGYRYVDDTSCDRGGVAIVQTGTVKCRIGNTTVQVIATNTDLGPMPCSPAEVIASEGPVEKTACTFNYSETLPNKYYEPRDQYFQQYMLKGKWQYWFDLDSIDHHKDYFSEFIVIAVVALLGGKYVLWLLVTYMILSEQMAMGAG
进一步地，如SEQ ID NO.1所示的E2蛋白的编码基因的序列如SEQ ID NO.3所示。
SEQ ID NO.3序列为优化密码子后的基因序列：
进一步地，所述牛病毒性腹泻病毒E2蛋白的编码基因的5’端连接有BVDV2-Erns基因的编码序列，可选地，所述BVDV2-Erns基因的编码序列如SEQ ID NO.5所示。
BVDV2-Erns基因碱基序列如SEQ ID NO.5所示(优化后的序列)：
BVDV2-Erns基因氨基酸序列(如SEQ ID NO.9所示)：
ENITQWNLMDNGTEGIQQAMFLRGVNRSLHGIWPEKICTGVPTHLATDYELKEIVGMMDASEKTNYTCCRLQRHEWNKHGWCNWFHIEPWIWLMNKTQSNLTEGQPPRECAVTCRYDRETELNIVTQARDRPTTLTGCKKGKKFSFAGVVLDGPCNFKVSVEDVLFKEHDCGNMLQETAIQLLDGATNTIEGARAGTAKLTTWLGKQLRTLGRKLENKSKAWFGAHA
进一步地，所述重组杆状病毒含有如SEQ ID NO.7所示的序列。SEQ ID NO.7序列为SEQ ID NO 3序列和SEQ ID NO.5序列串联在一起的序列。
进一步地，所述E2蛋白的氨基酸序列如SEQ ID NO.2所示。可选地，如SEQ ID NO.2所示的E2蛋白的编码基因的序列可以如SEQ ID NO.4所示。
进一步地，所述牛病毒性腹泻病毒E2蛋白的编码基因在P10启动子的控制下转录；
进一步地，用于构建所述重组杆状病毒的基础转移载体为pFastBacDual。
第二方面，提供一种重组杆状病毒的制备方法，包括如下步骤：
(1)密码子优化：对编码牛病毒性腹泻病毒E2蛋白的基因进行密码子优化，优化后的E2蛋白的编码基因序列如SEQ ID NO.3所示；可选地，在如SEQ ID NO.3所示的序列的5’端连接BVDV2-Erns基因的编码序列，获得如SEQ ID NO.7所示的BVDV2-Erns-E2基因序列；
(2)载体构建：将如SEQ ID NO.3或SEQ ID NO.7所示的序列与基础转移载体连接，构建含有一个或多个拷贝的E2蛋白编码基因的重组转移载体；可选地，基础转移载体为pFastBacDual；
(3)将步骤(2)重组转移载体转入大肠杆菌DH10Bac，获得携带一个或多个拷贝的BVDV2-Erns-E2蛋白编码基因的重组质粒；
(4)将步骤(3)的重组质粒导入昆虫细胞，获得重组杆状病毒。
第三方面，提供一种第一方面所述的重组杆状病毒或采用第二方面所述的制备方法制备得到的重组杆状病毒在制备牛病毒性腹泻病毒的抗原、抗体或亚单位疫苗中的应用。
第四方面，提供一种牛病毒性腹泻病毒亚单位疫苗，其包含牛病毒性腹泻病毒E2蛋白，所述E2蛋白的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
进一步地，所述E2蛋白为采用第一方面所述的重组杆状病毒或采用第二方面所述的制备方法制备得到的重组杆状病毒表达得到。
第五方面，提供一种牛病毒性腹泻病毒亚单位疫苗的制备方法，采用重组杆状病毒表达牛病毒性腹泻病毒E2蛋白，所述重组杆状病毒为第一方面所述的重组杆状病毒或采用第二方面所述的制备方法制备得到的重组杆状病毒；
进一步地，所述制备方法包括如下步骤：
(1)将所述重组杆状病毒导入昆虫细胞，培养，表达牛病毒性腹泻病毒E2蛋白；
(2)分离和纯化E2蛋白；
(3)将经纯化的E2蛋白与佐剂混合，制备亚单位疫苗；可选地，所述佐剂包括605佐剂，可选地，纯化的E2蛋白与佐剂605的重量比为1：(0.5～2)，可选地为1:1。
第六方面，提供一种疫苗组合物，含有第一方面所述的重组杆状病毒或采用第二方面所述的制备方法制备得到的重组杆状病毒、或第四方面所述的牛病毒性腹泻病毒亚单位疫苗、或第五方面所述的制备方法制备的牛病毒性腹泻病毒亚单位疫苗。
第七方面，提供一种检测2型牛病毒性腹泻病毒病的诊断试剂盒，含有第一方面所述的重组杆状病毒或采用第二方面所述的制备方法制备得到的重组杆状病毒、或第四方面所述的牛病毒性腹泻病毒亚单位疫苗、或第五方面所述的制备方法制备的牛病毒性腹泻病毒亚单位疫苗。
优选的，所述重组杆状病毒表达的BVDV2-E2蛋白与605佐剂混合制备疫苗对牛施用两剂，免疫剂量为2ml/头份，可对牛病毒性腹泻产生很好的免疫保护作用。
优选的，采用所述重组杆状病毒表达的BVDV2-E2蛋白可用作牛病毒性腹泻病毒2型抗体ELISA检测试剂盒的制备，用于牛病毒性腹泻2型病毒病的实验室诊断检测。
有益效果
(1)本发明可高效稳定的表达牛病毒性腹泻病毒2型E2蛋白，制备预防牛病毒性腹泻2型病的疫苗制剂和用于制备检测2型牛病毒性腹泻病毒的诊断试剂盒，减少牛病毒性腹泻2型病对养牛业造成的损失。
(2)本发明通过E2蛋白基因序列进行碱基修饰和优化，使蛋白的免疫原性更好。通过与专利的605佐剂混合配制疫苗，更增强了疫苗的免疫效果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
下述实施方案中，605佐剂为北京华夏兴洋生物科技有限公司申请的专利佐剂(专利公开号CN103083659A)，并且已经应用到疫苗的研究和生产中，利用605佐剂研发注册的新兽药制品牛曼氏杆菌灭活疫苗和牛传染性鼻气管炎灭活疫苗已经获得国家新兽药注册证书。
质粒pFastBacDual购自武汉淼灵生物科技有限公司。
实施例1本发明重组杆状病毒Baculo-BVDV2的构建
1.1牛病毒性腹泻病毒Baculo-BVDV2 E2基因克隆载体的构建
1.1.1从牛病毒性腹泻病毒2型流行毒株XJ株中获得牛病毒性腹泻病毒E2基因，其基因序列如为SEQ ID NO.4所示。
1.1.2牛病毒性腹泻病毒E2基因的密码子优化为了提高目的蛋白在昆虫细胞中的表达量，优化后的核苷酸序列如SEQ ID NO.3所示。
将BVDV2-Erns基因序列添加到E2基因序列的5’端：
将经密码子优化的BVDV2-Erns基因序列(如SEQ ID NO.5所示)添加到如SEQ IDNO.3所示的E2基因序列的5’端，得到经密码子优化的如SEQ ID NO.7所示的串联基因BVDV2-Erns-E2-1；
将未经密码子优化的BVDV2-Erns基因序列(如SEQ ID NO.6所示)添加到如SEQ IDNO.4所示的E2基因序列的5’端，得到未经密码子优化的如SEQ ID NO.8所示的串联基因BVDV2-Erns-E2-2。
SEQ ID NO.3、SEQ ID NO.4可PCR获得，也可以人工合成。
1.1.3BVDV2-E2亚克隆至pFastBacDual载体
1.1.3.1质粒提取与酶切按照质粒提取试剂盒的方法提取质粒，并将连接产物和pFastBacDual分别使用SalⅠ和HindⅢ进行双酶切。
1.1.3.2目的片段与载体pFastBacDual连接，胶回收1％琼脂糖凝胶电泳鉴定双酶切产物获取线性载体pFastBacDual分别和BVDV2-E2基因优化前后的SEQ ID NO.8(优化前的串联基因BVDV2-Erns-E2-2)、SEQ ID NO.7(优化后的串联基因BVDV2-Erns-E2-1)序列连接，将双酶切并纯化回收后的两个目的基因分别与质粒pFastBacDual混合后，用T4连接酶进行连接。连接体系的混合物离心混匀后，25℃连接1～2h。并将连接好的重组质粒命名为pFBD-BVDV2-E2-1(连接密码子优化前序列SEQ ID NO.8的质粒)和pFBD-BVDV2-E2-2(连接密码子优化后序列SEQ ID NO.7的质粒)。
1.1.3.3连接产物转化取100μl感受态细胞DH5α置冰浴，加入10μl连接产物，充分混匀，冰浴30min；42℃热休克90s，冰浴1min；加入890μl 37℃预热的LB培养液于37℃振摇45min，以复苏抗性；4000rpm离心5min，弃去部分上清，剩20μl左右全部均匀涂布于带有X-Gal、IPTG、Amp抗生素(50μg/mL)的LB平板，倒置于37℃培养12～18h。无菌挑取白色单菌落至4mL LB(Amp+)液体培养中37℃振摇培养10～14h。
1.1.3.4阳性转化子的筛选与鉴定
1.1.3.4.1菌液PCR鉴定挑取培养板上的单克隆接种于含Amp的LB培养基，37℃震荡培养10～14h，取培养菌液为模板进行菌液PCR鉴定，初步筛选出阳性克隆，送至测序。
1.1.3.4.2酶切鉴定按照质粒提取试剂盒的方法提取质粒，将pFBD-BVDV2-E2-1、pFBD-BVDV2-E2-2使用SalⅠ和HindⅢ进行双酶切鉴定。
1.2重组Bacmid DNA的获得
1.2.1质粒提取将PCR鉴定和双酶切鉴定为阳性的菌液按照质粒提取试剂盒的方法提取质粒。
1.2.2重组供体质粒pFBD-BVDV2-E2-1、pFBD-BVDV2-E2-2分别转化取100μl感受态细胞DH10Bac置冰浴，加入10μl pFBD-BVDV2-E2质粒，充分混匀，冰浴30min；42℃热休克90s，冰浴1min；加入890μl 37℃预热的LB培养液于37℃振摇培养4h后；取100μl菌液用LB培养基连续10倍稀释(10-1～10-3)，每个稀释度的菌液取取100μl均勾涂布于带有X-Gal、IPTG、卡那霉素(50μg/ml)、庆大霉素(7μg/ml)、四环素(10μg/ml)的LB平板，倒置于37℃培养48h。无菌挑取白色单菌落至4mL LB(Amp+)液体培养中37℃振摇(200-250rpm)培养24小时。
1.2.3重组子Bacmid-BVDV2-E2-1、pFBD-BVDV2-E2-2的PCR鉴定挑取培养板上的单克隆接种于含抗生素(Amp+)的LB液体培养基，37℃震荡培养10～14h，取培养菌液为模板进行菌液PCR鉴定。
1.2.4重组Bacmid-BVDV2-E2-1、pFBD-BVDV2-E2-2质粒的提取将PCR鉴定为阳性的菌液按照去内毒素质粒提取试剂盒的方法提取质粒。
1.3重组质粒转染Sf9昆虫细胞
1.3.1重组质粒转染Sf9昆虫细胞转染前24h按照0.5×106/well的密度接种Sf9细胞至6孔板；将细胞培养至对数期，进行转染，27～28℃培养72h后，取培养基上清1500rpm离心5min，得到Baculo-BVDV2病毒，-80℃保存，得到P1代病毒。
1.3.2重组杆状病毒扩增将第P1代重组杆状病毒以1％的量进行接种，接种重组杆状病毒后，置于恒温细胞培养箱中培养约72～96h左右，待细胞产生明显病变，且大部分细胞脱落时，收集细胞培养上清为第P2代毒种。
重组质粒Bacmid-BVDV2-E2-1、pFBD-BVDV2-E2-2分别按1.3.1、1.3.2的方法转染培养得到P2-1病毒、P2-2病毒。
实施例2 BVDV2-E2蛋白纯化
2.1感染前24h按照5.0×106/皿的密度接种sf9细胞至100mm培养皿；按500μl病毒粒子/100mm皿比例接种P2代病毒粒子，27～28℃培养3d；取培养基上清1500rpm离心5min收集P3代病毒粒子。以PBS洗涤细胞3遍收集细胞(P3C)，以300μL P3代病毒粒子感染1皿sf9细胞收集P4代病毒粒子及细胞(P4C)。
2.2取收集的P3C、P4C细胞，并吹打细胞，4000rpm离心10min，去上清，加入裂解液4℃裂解30min，11000rpm离心20min，分离上清及沉淀，并对裂解上清和沉淀进行WB检测，检测结果合格。
2.3重组蛋白大量表达感染前24h按照5.0×105/mL的密度接种昆虫细胞至培养瓶，150rpm培养。按1.5mL病毒粒子/50mL细胞悬液的比例接种P3代病毒粒子，27℃培养3d，4000rpm离心10min，去上清，加PBS洗涤细胞三遍。加入裂解液4℃裂解30min，11000rpm离心20min，分离上清及沉淀。
2.4重组蛋白纯化用浓HCl调节上清pH至8.0，准备纯化柱，以0.5mL/min的流速上样上清蛋白溶液，以NTA-0缓冲液(pH8.0)洗柱至流出液不含蛋白(G250检测液不变色)。分别以不同浓度的咪唑洗脱，分段收集洗脱液至G250检测液不变色，以3倍柱体积去离子水洗涤柱料，以20％乙醇封柱。对收集的洗脱液进行SDS-PAGE和WB检测，检测结果合格。
将实施例1的P2-1病毒、P2-2病毒分别按2.1～2.4的方法培养、表达、纯化，分别获得BVDV2-E2-1蛋白(密码子优化前表达的蛋白)、BVDV2-E2-2蛋白(密码子优化后表达的蛋白)。
实施例3 2型牛病毒性腹泻病毒E2蛋白应用于疫苗制备研究
3.1疫苗配制取已纯化的BVDV2-E2-1、BVDV2-E2-2蛋白，分别进行疫苗配制：用琼扩试验检测蛋白效价，当测得效价达到1:128以上时，将蛋白溶液按比例用生理盐水稀释至蛋白效价1:128，加入1％的甲醛溶液进行4℃灭活24小时后进行检验，检验合格后方可配制疫苗。将检验合格的灭活抗原与605佐剂1:1比例进行混合，PH值至7.0～7.6，2～8℃缓慢搅拌过夜，使抗原与佐剂充分结合，混合均匀后，分装，密闭保存。分别得到BVDV2-E2-1疫苗(对应密码子未优化获得的疫苗)、BVDV2-E2-2疫苗(对应密码子优化获得的疫苗)。
3.2疫苗效力研究研究所用动物为牛病毒性腹泻抗体阴性犊牛和怀孕母牛，如表1～2所示，分为BVDV2-E2-1疫苗、BVDV2-E2-2疫苗、已有专利疫苗组(牛病毒性腹泻病毒E2蛋白亚单位疫苗制备方法，专利申请号：201610517156.6)、商品疫苗组(牛病毒性腹泻/黏膜病、传染性鼻气管炎二联灭活疫苗(NMG株+LY株)，由金宇保灵生物药品有限公司生产)、健康对照组。同时对3组实验动物进行免疫，皮下注射2ml，免疫21天后进行二次免疫。该研究进行35天后，颈静脉采血后，运用牛病毒性腹泻免疫荧光测抗体试验进行抗体效价测定。同时进行攻毒试验，攻毒后连续观察14日，观察是否出现发烧，水样腹泻，后期便中带血和粘膜等症状，并采集攻毒试验动物的分泌物、排泄物、血液和脾脏等进行PCR检查是否含有病毒。
表1牛病毒性腹泻病毒(BVDV2)亚单位苗效力研究免疫分组
3.3结果
3.3.1免疫前后采血测抗体比对结果
免疫前后采血测抗体比对结果如表2，所述疫苗免疫组(BVDV2-E2-1疫苗、BVDV2-E2-2疫苗)和已有专利疫苗对照、某商品疫苗的免疫组抗体均有所上升，但与所述本专利疫苗免疫组抗体水平上升差异显著，商品疫苗免疫组和已有专利疫苗对照的免疫组抗体水平上升不显著且对照组抗体水平不变，说明本发明所述的疫苗对2型牛病毒性腹泻病毒有很好的免疫保护效果，而且本发明经密码子优化表达的BVDV2-E2-2疫苗相对未密码子优化表达的BVDV2-E2-1疫苗，抗体效价有显著提升，说明本发明经密码子优化表达的疫苗具有良好的抗体效价，而专利对照疫苗和某商品疫苗对2型牛病毒性腹泻疫苗免疫保护效果不明显。
表2靶动物牛免疫后血清中和抗体测定结果
3.3.2攻毒保护效果
对实验动物进行攻毒后观察14日，观察每日临床症状和采集发病牛病料，并进行RT-PCR检测，检测结果如表3，说明本发明所述疫苗对2型牛病毒性腹泻病毒有良好的保护作用，免疫后可有效减少感染BVDV后的排毒，从而避免感染其它健康动物，而且本发明经密码子优化表达的BVDV2-E2-2疫苗相对未密码子优化表达的BVDV2-E2-1疫苗，保护效果更显著，说明本发明经密码子优化表达的疫苗能够更好的减少BVDV的感染。
表3靶动物牛免疫接种后攻毒保护效果
以上结果证明，攻毒后的各组的保护效果，BVDV2-E2-2蛋白作为疫苗抗原配制的2型牛病毒性腹病毒亚单位疫苗，对2型牛病毒性腹泻病毒引起的疾病有非常好的保护作用，与市场上其他疫苗相比，针对2型牛病毒性腹泻病毒引起的疾病效果明显，差异显著。
实施例4 2型牛病毒性腹泻病毒E2蛋白应用于ELISA抗体检测试剂盒研究
4.1试剂盒抗原包被板制备使用Brodford蛋白含量检测试剂盒(购自北京百奥莱博科技有限公司)检测纯化的BVDV2 E2蛋白溶液中的蛋白含量，用包被缓冲液稀释蛋白浓度达到2μg/ml时，每孔100μl加入酶联稀释板中进行4℃过夜包被，弃去包被液，2％明胶溶液37℃封闭1小时后PBST洗涤一遍，塑封保存于4℃作为牛病毒性腹泻病毒(BVDV2)ELISA抗体检测试剂盒抗原包被板。
4.2该试剂盒操作步骤方法包括如下：将BVDV-E2-2蛋白(0.2μg/孔)的ELISA抗原包被板，加入稀释后样本各100μl/孔，室温孵育1h，PBST洗涤3-6次，加入HRP标记的羊抗牛抗体，100μl/孔，室温孵育1h，PBST洗涤6次，加底物显色液100μl，室温反应10分钟，各反应孔中加2mol/L硫酸50μl终止反应。反应板置于酶标仪中检测各反应孔的OD450nm的读值。
判断标准如下：
(1)阴性对照的OD平均值≤0.200，阳性对照的OD平均值≥0.600则检测结果成立；
(2)运用S/N值大小计算样本阴阳性：当S/N值≥2.1时，样本检测为阳性；S/N值≤2.1时，样本判断为阴性。
4.3牛病毒性腹泻病毒(BVDV2)ELISA试剂盒应用于临床诊断研究
4.3.1辅助疫苗免疫研究对检测为BVDV抗体阴性犊牛进行分组，分别进行疫苗免疫，如表4，隔离后饲养35天后进行采血处理，并用制备的牛病毒性腹泻病毒(BVDV2)ELISA抗体检测试剂盒进行检测。
表4牛病毒性腹泻病毒(BVDV2)ELISA试剂盒检测疫苗免疫效果
注：编号①：牛病毒性腹泻病毒(BVDV2)亚单位疫苗(BVDV2-E2-2疫苗)；编号②：商品化牛病毒性腹泻病毒(BVDV1)疫苗；编号③：实验室制备的牛病毒性腹泻病毒(BVDV2)全病毒疫苗(按照常规全病毒疫苗制备方法制备)。
4.3.2辅助临床样品检测研究选择不同地区的三家牛场采集样品，分别为150份、200份、350份。运用牛病毒性腹泻(BVDV2)ELISA抗体试剂盒对样品进行检测，进行临床诊断研究，判断牛场的感染2型牛病毒性腹泻病毒的感染率，结果见表5。
表5临床样品检测研究结果
以上结果表明：BVDV2-E2-2蛋白不仅能用于研制牛病毒性腹泻病毒2型基因工程疫苗，又能用于牛病毒性腹泻病毒(BVDV2)ELISA抗体检测试剂盒的研制，为帮助养殖户预防和检测由牛病毒性腹泻病毒2型感染引起的疫病提供有效的技术支持，减少疫病导致的经济损失。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
SEQUENCE LISTING
<110> 北京华夏兴洋生物科技有限公司
华夏兴洋（江苏）生物科技有限公司
北京生泰尔科技股份有限公司
<120> 一种重组杆状病毒及其制备方法和应用
<130> 1103-200416F
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 342
<212> PRT
<213> Artificial Sequence
<220>
<223> 密码子优化后的E2蛋白氨基酸序列
<220>
<221> DOMAIN
<222> (1)..(342)
<400> 1
Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser Lys Asp Lys
1 5 10 15
Lys Ile Gly Pro Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu
20 25 30
Pro Thr Lys Lys Ile Val Asp Ser Met Val Gln Val Trp Cys Asp Gly
35 40 45
Lys Asp Leu Lys Ile Leu Lys Thr Cys Thr Lys Glu Glu Arg Tyr Leu
50 55 60
Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln
65 70 75 80
Ile Ser Ser Gly Thr Lys Gly Pro Glu Val Ile Asp Met Pro Asp Asp
85 90 95
Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Arg Gly
100 105 110
Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys
115 120 125
Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Ile Leu Ala Asn Gln Asp
130 135 140
Thr Leu Asp Thr Thr Val Val Arg Thr Tyr Arg Arg Thr Thr Pro Phe
145 150 155 160
Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile
165 170 175
His Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His
180 185 190
Ser Lys Leu Lys Gly Gly Pro Ile Lys Lys Cys Lys Trp Cys Ser Tyr
195 200 205
Asp Phe Phe Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys
210 215 220
Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys
225 230 235 240
Asp Arg Gly Gly Val Ala Ile Val Gln Thr Gly Thr Val Lys Cys Arg
245 250 255
Ile Gly Asn Thr Thr Val Gln Val Ile Ala Thr Asn Thr Asp Leu Gly
260 265 270
Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val
275 280 285
Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Glu Thr Leu Pro Asn Lys
290 295 300
Tyr Tyr Glu Pro Arg Asp Gln Tyr Phe Gln Gln Tyr Met Leu Lys Gly
305 310 315 320
Lys Trp Gln Tyr Trp Phe Asp Leu Asp Ser Ile Asp His His Lys Asp
325 330 335
Tyr Phe Ser Glu Phe Ile
340
<210> 2
<211> 374
<212> PRT
<213> Artificial Sequence
<220>
<223> 密码子优化前的E2蛋白氨基酸序列
<220>
<221> DOMAIN
<222> (1)..(374)
<400> 2
Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser Lys Asp Lys
1 5 10 15
Lys Ile Gly Pro Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu
20 25 30
Pro Thr Lys Lys Ile Val Asp Ser Met Val Gln Val Trp Cys Asp Gly
35 40 45
Lys Asp Leu Lys Ile Leu Lys Thr Cys Thr Lys Glu Glu Arg Tyr Leu
50 55 60
Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln
65 70 75 80
Ile Ser Ser Gly Thr Lys Gly Pro Glu Val Ile Asp Met Pro Asp Asp
85 90 95
Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Arg Gly
100 105 110
Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys
115 120 125
Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Ile Leu Ala Asn Gln Asp
130 135 140
Thr Leu Asp Thr Thr Val Val Arg Thr Tyr Arg Arg Thr Thr Pro Phe
145 150 155 160
Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile
165 170 175
His Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His
180 185 190
Ser Lys Leu Lys Gly Gly Pro Ile Lys Lys Cys Lys Trp Cys Ser Tyr
195 200 205
Asp Phe Phe Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys
210 215 220
Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys
225 230 235 240
Asp Arg Gly Gly Val Ala Ile Val Gln Thr Gly Thr Val Lys Cys Arg
245 250 255
Ile Gly Asn Thr Thr Val Gln Val Ile Ala Thr Asn Thr Asp Leu Gly
260 265 270
Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val
275 280 285
Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Glu Thr Leu Pro Asn Lys
290 295 300
Tyr Tyr Glu Pro Arg Asp Gln Tyr Phe Gln Gln Tyr Met Leu Lys Gly
305 310 315 320
Lys Trp Gln Tyr Trp Phe Asp Leu Asp Ser Ile Asp His His Lys Asp
325 330 335
Tyr Phe Ser Glu Phe Ile Val Ile Ala Val Val Ala Leu Leu Gly Gly
340 345 350
Lys Tyr Val Leu Trp Leu Leu Val Thr Tyr Met Ile Leu Ser Glu Gln
355 360 365
Met Ala Met Gly Ala Gly
370
<210> 3
<211> 1026
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化后的E2蛋白编码基因序列
<220>
<221> misc_feature
<222> (1)..(1026)
<400> 3
ttccctgagt gcaaggaggg tttccagtac gctatctcca aggacaagaa gatcggtcca 60
ctgggccctg agagcctgac taccacctgg cacctgccta ctaagaagat cgtggactcc 120
atggtgcagg tctggtgcga tggaaaggac ctcaagatcc tgaagacttg caccaaggaa 180
gagaggtatc tggtggccgt gcacgagcgc gcactctcca cctcagcgga gttcatgcaa 240
atcagttctg gcactaaggg accagaggtg atcgacatgc ctgacgactt cgaattcggc 300
ctgtgcccct gcgacagcaa gcccgtgatc cgcggcaagt tcaatgctag ccttctcaac 360
ggtcccgctt tccagatggt gtgccctcag ggttggaccg gcactatcga atgcatcttg 420
gccaaccagg acacgctgga cactaccgtg gtgcgtacct accgtcgcac gacccccttc 480
caacgtcgta agtggtgcac ttacgagaag atcatcggtg aggacatcca cgagtgcatc 540
ctgggcggaa actggacctg catcaccggt gaccacagta agttgaaggg tggacccatc 600
aagaagtgca agtggtgcag ttacgatttc ttcaactctg agggtctgcc acactacccg 660
atcggtaagt gcatgctcat aaacgagtca ggctacaggt acgtcgatga cacctcctgc 720
gatcgcggcg gtgtggcgat cgtgcagacg ggcacagtca agtgcagaat cggtaacacc 780
actgtccaag tgatcgccac taacaccgac ctgggcccta tgccatgctc gcccgctgag 840
gtcatcgctt cagagggtcc tgtcgaaaag acagcttgca ctttcaacta ctcggagact 900
ctccctaaca agtactacga accccgtgat cagtacttcc agcagtacat gttgaaggga 960
aaatggcagt actggttcga cctggactct atcgaccacc acaaggacta cttctcagag 1020
ttcatc 1026
<210> 4
<211> 1122
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化前的E2蛋白编码基因序列
<220>
<221> misc_feature
<222> (1)..(1122)
<400> 4
ttccctgaat gcaaagaggg cttccaatat gccatatcaa aagacaaaaa aataggacca 60
ctggggccag agagtttaac tacaacatgg caccttccta ccaaaaaaat agtggactct 120
atggtacagg tgtggtgtga tggaaaagac ttgaaaatat taaaaacgtg cacaaaggaa 180
gagaggtact tagtggctgt gcacgaaaga gccctgtcaa ccagtgctga gttcatgcag 240
atcagtagtg ggacaaaagg cccagaagtg atagatatgc ctgatgactt tgaatttggg 300
ctctgccctt gtgattcaaa accggtaata agggggaagt tcaatgccag cctattgaac 360
ggaccagctt tccagatggt atgcccacag gggtggactg gtacaataga atgcatcctg 420
gcgaaccaag acaccttgga cacaactgtc gttaggacat atagaagaac tactccattt 480
cagcggagaa aatggtgtac ctatgaaaag ataatagggg aagatatcca tgaatgcatt 540
ctaggaggaa actggacatg cataactggt gatcatagca agttgaaagg tgggcctatc 600
aagaagtgta agtggtgcag ctacgacttc ttcaattcag aaggactgcc acactaccca 660
ataggtaagt gcatgctcat caatgagagt gggtacaggt atgtagatga cacctcttgt 720
gataggggtg gtgtagccat agttcaaaca ggtactgtaa agtgtagaat aggcaacacc 780
acggtgcagg ttatcgctac taacactgac ctgggaccca tgccctgcag cccagctgag 840
gtgatagcaa gtgaaggacc agtggaaaag acggcgtgca cgtttaacta ttcagagaca 900
ctacctaata agtattatga gccaagggac cagtacttcc aacaatacat gttaaaaggg 960
aagtggcaat attggtttga cctggattct atagaccacc acaaagacta cttttcagag 1020
ttcatagtca tagcagtggt agccttgcta ggtggtaagt atgtactgtg gctcttagta 1080
acatatatga tactgtctga gcagatggct atgggtgctg ga 1122
<210> 5
<211> 681
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化后的Erns基因序列
<220>
<221> misc_feature
<222> (1)..(681)
<400> 5
gaaaatatta cccaatggaa ccttatggat aacggcaccg agggtatcca gcaagctatg 60
ttcctgcgcg gtgtcaaccg ttcgttgcac ggaatctggc cagaaaagat ttgcactgga 120
gtccccactc atctggccac cgactacgag ctcaaagaaa ttgtgggcat gatggatgcg 180
tccgagaaga cgaactacac ttgttgccgt ttgcaacgtc atgagtggaa caagcacgga 240
tggtgcaatt ggttccacat tgagccttgg atctggctga tgaacaagac tcagtctaat 300
ctgaccgagg gacagcctcc ccgtgagtgc gctgtcacat gtcgttacga tcgcgaaact 360
gaactgaaca ttgttacgca ggctagggac agaccaacaa ctttgacagg ttgtaagaaa 420
ggtaagaaat tctctttcgc tggtgtggtg ctcgatggtc catgcaactt caaggtctca 480
gtcgaggatg tgctcttcaa agagcacgac tgtggaaata tgctgcagga gactgcgatc 540
caactgttgg acggcgccac aaacactatc gaaggagcca gagcaggaac tgccaagctc 600
acaacttggt tgggaaagca gcttcgtact ctcggccgta agctggagaa caaaagcaaa 660
gcatggttcg gtgcacacgc t 681
<210> 6
<211> 681
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化前的Erns基因序列
<400> 6
gagaacatta cccagtggaa cttgatggac aacggcaccg aaggtataca gcaagctatg 60
ttcctgagag gggtgaacag gagtctacat ggaatttggc ccgagaagat ttgcaccgga 120
gtaccaactc acttagcaac agactacgag ctcaaggaga tagtgggaat gatggacgcg 180
agtgagaaga ccaactacac atgttgcagg ttgcaaaggc atgagtggaa caaacatggt 240
tggtgcaatt ggtttcatat agaaccgtgg atatggctga tgaacaagac ccaaagcaac 300
ttgactgaag gacagccacc tagggagtgt gctgtaactt gtaggtatga cagggaaaca 360
gaattgaaca tcgtaacaca ggctagggac aggcctacaa ctctgacagg ctgcaagaaa 420
ggcaagaaat tttcctttgc gggggttgta ctggatgggc cctgcaactt taaagtatca 480
gttgaagacg tgctgttcaa ggaacacgat tgtggcaaca tgctacagga gaccgcgata 540
cagctactcg atggggcaac caacaccatt gagggagcaa gggtagggac ggccaagttg 600
acaacctggt tagggaagca attacggacc cttggtagga agttggagaa taaaagcaaa 660
gcatggtttg gtgcacacgc a 681
<210> 7
<211> 1707
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化后的BVDV2-Erns-E2基因序列
<220>
<221> misc_feature
<222> (1)..(1707)
<400> 7
gaaaatatta cccaatggaa ccttatggat aacggcaccg agggtatcca gcaagctatg 60
ttcctgcgcg gtgtcaaccg ttcgttgcac ggaatctggc cagaaaagat ttgcactgga 120
gtccccactc atctggccac cgactacgag ctcaaagaaa ttgtgggcat gatggatgcg 180
tccgagaaga cgaactacac ttgttgccgt ttgcaacgtc atgagtggaa caagcacgga 240
tggtgcaatt ggttccacat tgagccttgg atctggctga tgaacaagac tcagtctaat 300
ctgaccgagg gacagcctcc ccgtgagtgc gctgtcacat gtcgttacga tcgcgaaact 360
gaactgaaca ttgttacgca ggctagggac agaccaacaa ctttgacagg ttgtaagaaa 420
ggtaagaaat tctctttcgc tggtgtggtg ctcgatggtc catgcaactt caaggtctca 480
gtcgaggatg tgctcttcaa agagcacgac tgtggaaata tgctgcagga gactgcgatc 540
caactgttgg acggcgccac aaacactatc gaaggagcca gagcaggaac tgccaagctc 600
acaacttggt tgggaaagca gcttcgtact ctcggccgta agctggagaa caaaagcaaa 660
gcatggttcg gtgcacacgc tttccctgag tgcaaggagg gtttccagta cgctatctcc 720
aaggacaaga agatcggtcc actgggccct gagagcctga ctaccacctg gcacctgcct 780
actaagaaga tcgtggactc catggtgcag gtctggtgcg atggaaagga cctcaagatc 840
ctgaagactt gcaccaagga agagaggtat ctggtggccg tgcacgagcg cgcactctcc 900
acctcagcgg agttcatgca aatcagttct ggcactaagg gaccagaggt gatcgacatg 960
cctgacgact tcgaattcgg cctgtgcccc tgcgacagca agcccgtgat ccgcggcaag 1020
ttcaatgcta gccttctcaa cggtcccgct ttccagatgg tgtgccctca gggttggacc 1080
ggcactatcg aatgcatctt ggccaaccag gacacgctgg acactaccgt ggtgcgtacc 1140
taccgtcgca cgaccccctt ccaacgtcgt aagtggtgca cttacgagaa gatcatcggt 1200
gaggacatcc acgagtgcat cctgggcgga aactggacct gcatcaccgg tgaccacagt 1260
aagttgaagg gtggacccat caagaagtgc aagtggtgca gttacgattt cttcaactct 1320
gagggtctgc cacactaccc gatcggtaag tgcatgctca taaacgagtc aggctacagg 1380
tacgtcgatg acacctcctg cgatcgcggc ggtgtggcga tcgtgcagac gggcacagtc 1440
aagtgcagaa tcggtaacac cactgtccaa gtgatcgcca ctaacaccga cctgggccct 1500
atgccatgct cgcccgctga ggtcatcgct tcagagggtc ctgtcgaaaa gacagcttgc 1560
actttcaact actcggagac tctccctaac aagtactacg aaccccgtga tcagtacttc 1620
cagcagtaca tgttgaaggg aaaatggcag tactggttcg acctggactc tatcgaccac 1680
cacaaggact acttctcaga gttcatc 1707
<210> 8
<211> 1803
<212> DNA
<213> Artificial Sequence
<220>
<223> 密码子优化前的BVDV2-Erns-E2基因序列
<220>
<221> misc_feature
<222> (1)..(1803)
<400> 8
gagaacatta cccagtggaa cttgatggac aacggcaccg aaggtataca gcaagctatg 60
ttcctgagag gggtgaacag gagtctacat ggaatttggc ccgagaagat ttgcaccgga 120
gtaccaactc acttagcaac agactacgag ctcaaggaga tagtgggaat gatggacgcg 180
agtgagaaga ccaactacac atgttgcagg ttgcaaaggc atgagtggaa caaacatggt 240
tggtgcaatt ggtttcatat agaaccgtgg atatggctga tgaacaagac ccaaagcaac 300
ttgactgaag gacagccacc tagggagtgt gctgtaactt gtaggtatga cagggaaaca 360
gaattgaaca tcgtaacaca ggctagggac aggcctacaa ctctgacagg ctgcaagaaa 420
ggcaagaaat tttcctttgc gggggttgta ctggatgggc cctgcaactt taaagtatca 480
gttgaagacg tgctgttcaa ggaacacgat tgtggcaaca tgctacagga gaccgcgata 540
cagctactcg atggggcaac caacaccatt gagggagcaa gggtagggac ggccaagttg 600
acaacctggt tagggaagca attacggacc cttggtagga agttggagaa taaaagcaaa 660
gcatggtttg gtgcacacgc attccctgaa tgcaaagagg gcttccaata tgccatatca 720
aaagacaaaa aaataggacc actggggcca gagagtttaa ctacaacatg gcaccttcct 780
accaaaaaaa tagtggactc tatggtacag gtgtggtgtg atggaaaaga cttgaaaata 840
ttaaaaacgt gcacaaagga agagaggtac ttagtggctg tgcacgaaag agccctgtca 900
accagtgctg agttcatgca gatcagtagt gggacaaaag gcccagaagt gatagatatg 960
cctgatgact ttgaatttgg gctctgccct tgtgattcaa aaccggtaat aagggggaag 1020
ttcaatgcca gcctattgaa cggaccagct ttccagatgg tatgcccaca ggggtggact 1080
ggtacaatag aatgcatcct ggcgaaccaa gacaccttgg acacaactgt cgttaggaca 1140
tatagaagaa ctactccatt tcagcggaga aaatggtgta cctatgaaaa gataataggg 1200
gaagatatcc atgaatgcat tctaggagga aactggacat gcataactgg tgatcatagc 1260
aagttgaaag gtgggcctat caagaagtgt aagtggtgca gctacgactt cttcaattca 1320
gaaggactgc cacactaccc aataggtaag tgcatgctca tcaatgagag tgggtacagg 1380
tatgtagatg acacctcttg tgataggggt ggtgtagcca tagttcaaac aggtactgta 1440
aagtgtagaa taggcaacac cacggtgcag gttatcgcta ctaacactga cctgggaccc 1500
atgccctgca gcccagctga ggtgatagca agtgaaggac cagtggaaaa gacggcgtgc 1560
acgtttaact attcagagac actacctaat aagtattatg agccaaggga ccagtacttc 1620
caacaataca tgttaaaagg gaagtggcaa tattggtttg acctggattc tatagaccac 1680
cacaaagact acttttcaga gttcatagtc atagcagtgg tagccttgct aggtggtaag 1740
tatgtactgt ggctcttagt aacatatatg atactgtctg agcagatggc tatgggtgct 1800
gga 1803
<210> 9
<211> 227
<212> PRT
<213> Artificial Sequence
<220>
<223> Erns蛋白的氨基酸序列
<220>
<221> DOMAIN
<222> (1)..(227)
<400> 9
Glu Asn Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile
1 5 10 15
Gln Gln Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile
20 25 30
Trp Pro Glu Lys Ile Cys Thr Gly Val Pro Thr His Leu Ala Thr Asp
35 40 45
Tyr Glu Leu Lys Glu Ile Val Gly Met Met Asp Ala Ser Glu Lys Thr
50 55 60
Asn Tyr Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly
65 70 75 80
Trp Cys Asn Trp Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys
85 90 95
Thr Gln Ser Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys Ala Val
100 105 110
Thr Cys Arg Tyr Asp Arg Glu Thr Glu Leu Asn Ile Val Thr Gln Ala
115 120 125
Arg Asp Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Lys Phe
130 135 140
Ser Phe Ala Gly Val Val Leu Asp Gly Pro Cys Asn Phe Lys Val Ser
145 150 155 160
Val Glu Asp Val Leu Phe Lys Glu His Asp Cys Gly Asn Met Leu Gln
165 170 175
Glu Thr Ala Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly
180 185 190
Ala Arg Ala Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu
195 200 205
Arg Thr Leu Gly Arg Lys Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly
210 215 220
Ala His Ala
225
Claims (10)
1.一种重组杆状病毒，其特征在于，其包含一个或多个拷贝的牛病毒性腹泻病毒E2蛋白的编码基因，所述E2蛋白的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的重组杆状病毒，其特征在于，如SEQ ID NO.1所示的E2蛋白的编码基因的序列如SEQ ID NO.3所示；
可选地，所述牛病毒性腹泻病毒E2蛋白的编码基因的5’端连接有BVDV2-Erns基因的编码序列，可选地，所述BVDV2-Erns基因的编码序列如SEQ ID NO.5所示；
可选地，所述重组杆状病毒含有如SEQ ID NO.7所示的序列；
可选地，所述E2蛋白的氨基酸序列如SEQ ID NO.2所示，可选地，如SEQ ID NO.2所示的E2蛋白的编码基因的序列如SEQ ID NO.4所示。
3.根据权利要求1或2所述的重组杆状病毒，其特征在于，所述牛病毒性腹泻病毒E2蛋白的编码基因在P10启动子的控制下转录；
可选地，用于构建所述重组杆状病毒的基础转移载体为pFastBacDual。
4.一种重组杆状病毒的制备方法，其特征在于，包括如下步骤：
(1)密码子优化：对编码牛病毒性腹泻病毒E2蛋白的基因进行密码子优化，优化后的E2蛋白的编码基因序列如SEQ ID NO.3所示；可选地，在如SEQ ID NO.3所示的序列的5’端连接BVDV2-Erns基因的编码序列，获得如SEQ ID NO.7所示的BVDV2-Erns-E2基因序列；
(2)载体构建：将如SEQ ID NO.3或SEQ ID NO.7所示的序列与基础转移载体连接，构建含有一个或多个拷贝的E2蛋白编码基因的重组转移载体；可选地，基础转移载体为pFastBacDual；
(3)将步骤(2)重组转移载体转入大肠杆菌DH10Bac，获得携带一个或多个拷贝的E2蛋白编码基因的重组杆粒；
(4)将步骤(3)的重组质粒导入昆虫细胞，获得重组杆状病毒。
5.权利要求1至3任一所述的重组杆状病毒或采用权利要求4所述的制备方法制备得到的重组杆状病毒在制备牛病毒性腹泻病毒的抗原、抗体或亚单位疫苗中的应用。
6.一种牛病毒性腹泻病毒亚单位疫苗，其特征在于，其包含牛病毒性腹泻病毒E2蛋白，所述E2蛋白的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
7.根据权利要求6所述的牛病毒性腹泻病毒亚单位疫苗，其特征在于，所述E2蛋白为采用权利要求1至3任一所述的重组杆状病毒或采用权利要求4所述的制备方法制备的重组杆状病毒表达得到。
8.一种牛病毒性腹泻病毒亚单位疫苗的制备方法，其特征在于，采用重组杆状病毒表达牛病毒性腹泻病毒E2蛋白，所述重组杆状病毒为权利要求1至3任一所述的重组杆状病毒或采用权利要求4所述的制备方法制备得到的重组杆状病毒；
可选地，包括如下步骤：
1)将所述重组杆状病毒导入昆虫细胞，培养，表达牛病毒性腹泻病毒E2蛋白；
2)分离和纯化E2蛋白；
3)将经纯化的E2蛋白与佐剂混合，制备亚单位疫苗；可选地，所述佐剂包括605佐剂，可选地，纯化的E2蛋白与佐剂605的重量比为1∶(0.8～1.5)。
9.一种疫苗组合物，其特征在于，含有权利要求1至3任一所述的重组杆状病毒、或采用权利要求4所述的制备方法制备得到的重组杆状病毒、或权利要求6或7所述的牛病毒性腹泻病毒亚单位疫苗、或权利要求8所述的制备方法制备的牛病毒性腹泻病毒亚单位疫苗。
10.一种检测2型牛病毒性腹泻病毒病的诊断试剂盒，其特征在于，含有权利要求1至3任一所述的重组杆状病毒、或采用权利要求4所述的制备方法制备得到的重组杆状病毒、或权利要求6或7所述的牛病毒性腹泻病毒亚单位疫苗、或权利要求8所述的制备方法制备的牛病毒性腹泻病毒亚单位疫苗。
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