CN108456663B - 一种1型牛病毒性腹泻病毒样颗粒及其制备与应用 - Google Patents
一种1型牛病毒性腹泻病毒样颗粒及其制备与应用 Download PDFInfo
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Abstract
本发明公开一种1型牛病毒性腹泻病毒（BVDV‑1）病毒样颗粒及其制备方法和用途。通过将BVDV‑1的结构蛋白C、Erns、E1、E2编码基因克隆至pFastBacDual构建pFBD‑BVDV‑1重组杆状病毒转移载体，在大肠杆菌DH10Bac中转座获得重组杆状病毒载体Bac‑BVDV‑1，转染昆虫细胞，获得重组杆状病毒Baculo‑BVDV‑1，感染昆虫细胞表达BVDV‑1的结构蛋白C、Erns、E1、E2，在细胞内自动组装为BVDV‑1病毒样颗粒（VLP）。本发明获得的病毒样颗粒更接近天然BVDV病毒粒子的形态结构，接种动物后可刺激机体产生较好的免疫反应，可达到更好的免疫效果。所获的病毒样颗粒可利用蔗糖密度梯度离心直接纯化，与其它表达系统相比，避免了病毒结构蛋白分别纯化然后在细胞外组装的繁琐过程，有利于提高效率和节约成本，而且在生产过程中不用感染性的BVDV活病毒，提高了安全性。
Description
技术领域
本发明涉及一种重组杆状病毒、一种病毒的病毒样颗粒及其制备与应用，确切讲本发明涉及一种1型牛病毒性腹泻病毒基因重组的杆状病毒、一种1型牛病毒性腹泻病毒样颗粒，这种病毒样颗粒的制备方法和用途。
背景技术
牛病毒性腹泻—黏膜病（Bovine Viral Diarrhea - Mucosal Disease，BVD-MD）是由牛病毒性腹泻病毒（BVDV）引起的一种严重危害养牛业的传染病。该病由Olafson 1946年首次在美国纽约州汤普金斯县首府伊萨卡的牛群中发现。在1957年和1960年，研究人员分离获得了非细胞致病变性毒株NY-1和致细胞病变毒株Oregon C24V，利用病毒中和试验证实牛病毒性腹泻和黏膜病是同一种病原导致的不同临床症状，这一疾病也随后被官方定为牛病毒性腹泻—黏膜病，简称为牛病毒性腹泻。1980年，李佑民等从牛的流产胎儿脾脏分离获得BVDV，首次证实了该病在我国的存在。在20世纪90年代初，我国二十几个省市和自治区上万份血清中和抗体检测，发现牛抗体阳性率为19.15% （郑志刚等，动物检疫，1991, 5:42-44），在20世纪90年代末，感染率明显上升。在甘肃、陕西、青海、宁夏和四川五省，黄牛感染率为46.15%，牦牛感染率为28.0%～38.46%（高双娣等，中国兽医科技，1999, 27 (7):17-18）。2012～2013年，该病在我国奶牛、黄牛、牦牛、水牛的抗体阳性率总体达58.09%，分别为奶牛89.49%、肉牛63.27%、牦牛45.38%、水牛14.18%（Deng et al., Plos One. 2015,10(7):e0134777）。目前，我国除云南、贵州、重庆、广东、海南、香港和台湾外，其余各地均有BVD的报道。
BVDV不同毒株之间存在较大的抗原变异，目前可将其分为三个基因型，即BVDV-1、BVDV-2和BVDV-3，基因型又可进一步分为不同的基因亚型（BVDV-1a～1u；BVDV-2a，-2b）等。BVDV-1是世界各地广泛流行的病毒基因型（Brownlie et al. Ann Rech Vet. 1987, 18(2):157-166.）BVDV-2最早被发现于北美，欧洲、亚洲、南美洲也有报道，在我国的新疆、青海、山东等省区也有发生。BVDV-3最早报道在巴西发现并且在欧洲牛群感染导致了严重的呼吸道疾病（Decaro et al. Emerg Infect Dis. 2011, 17(8):1549-1552.），近期在我国河南省也有报道（Shi et al., Transbound Emerg Dis. 2016,63, 480-484.）
疫苗免疫可提高动物对BVDV的抵抗力、减少持续感染牛的存在从而降低由于其持续排毒带来的威胁。弱毒活疫苗最早在美国应用至今已有50余年的历史，但这类疫苗免疫后可能导致牛发生黏膜病，也可通过胎盘屏障感染胎儿，导致流产或胎儿先天性缺陷。目前国内用于预防BVD的疫苗主要有牛病毒性腹泻/黏膜病灭活疫苗和牛病毒性腹泻/黏膜病和传染性鼻气管炎二联灭活疫苗。灭活疫苗相对安全，但存在免疫活性较低、免疫持续期较短的问题。因此开发可靠、高效的新一代疫苗，是未来重要的方向。
BVDV是黄病毒科瘟病毒属的单股正链RNA病毒，病毒粒子为含有囊膜的球型粒子，直径约40～60nm，其核衣壳位于病毒粒子的中央，由脂质双层包裹C蛋白和病毒基因组RNA构成，直径约为30nm，其余3种病毒结构蛋白包括Erns、E1、E2镶嵌在病毒囊膜上，为囊膜蛋白。病毒基因组为单股正链RNA，在无外源基因插入的情况下长约12.4 Kb，编码一个约4000个氨基酸组成的多聚蛋白，然后在病毒蛋白水解酶（参与病毒非结构蛋白切割）与宿主细胞蛋白水解酶（参与病毒结构蛋白切割）共同作用下，产生成熟的病毒蛋白，其中E2是BVDV最主要的抗原蛋白，在不同亚型毒株之间、田间流行毒株与疫苗株间存在较高的变异时可以导致疫苗免疫的失败（Ridpath et al.，Vet Microbiol. 2000, 77(1-2):145-155）。在新型疫苗开发发面，Bolin等利用杆状病毒表达系统表达了BVDV Singer毒株的E2亚单位，将其免疫动物发现可对免疫动物产生有效的保护，通过增加E2蛋白的免疫剂量、引入病毒其他结构蛋白、优化佐剂、利用细胞因子作为佐剂等方式可提高E2亚单位疫苗的效力（Bolinet al., Arch Virol. 1996, 141(8):1463-1477）。Harpin 等将表达BVDV E2蛋白的真核表达载体通过肌注和静脉注射BALB/c小鼠，免疫小鼠可产生针对BVDV-1和BVDV-2毒株的中和抗体，其中BVDV-1抗体在免疫后6个月内还可检出，该疫苗在免疫牛16周后可使动物得到部分保护，并且诱导动物产生了针对BVDV-1和BVDV-2毒株的免疫记忆以及黏膜免疫（Harpin et al., FEMS Microbiol Lett. 1997, 46(2):229-234; Harpin et al., JGen Virol. 1999, 80 (12):3137-3144）。Liang等构建了表达BVDV-1和BVDV-2 E2蛋白的真核表达质粒，在接种小牛后经E2蛋白加强免疫，接种动物产生了体液免疫和细胞免疫，能够在一定程度上抵抗BVDV-2病毒的攻击（Liang et al., J Gen Virol. 2008, 89(2):453-466）。Ferrer等制备了可表达E2蛋白的重组杆状病毒，在Rachiplusia nuperos中表达E2蛋白亚单位，制备疫苗免疫小鼠，可诱导中和抗体的产生（Ferrer et al., J VirolMethods. 2007, 146(1-2): 424-7.）。中国发明专利（申请号：CN201610517156.6）也公布了一种牛病毒性腹泻病毒E2蛋白亚单位疫苗制备方法，表明利用杆状病毒表达系统制备的E2蛋白亚单位具有良好的免疫原性。另外，制备对动物无感染性的病毒粒子模拟物，在BVDV疫苗开发中也展现出良好的前景，Reimann等构建了缺失衣壳蛋白基因、Erns基因以及E1基因缺失的BVDV复制子，在牛源辅助细胞中包装获得非感染性的BVDV复制子，以衣壳蛋白基因缺失的BVDV复制子免疫牛获得了针对BVDV强毒的完全保护（Reimann et al.,Virology.2007, 366(2):377-386）。
发明内容
本发明提供一种重组杆状病毒，本发明同时提供这种重组杆状病毒可高效表达的并能产生可靠免疫反应的病毒样颗粒，本发明还提供该病毒样颗粒的制备及其应用。
本发明的一种具有免疫原性的BVDV-1病毒样颗粒是由1型BVDV 的4种结构蛋白C、Erns、E1、E2组成。
本发明所涉及的重组杆状病毒命名为苜蓿银纹夜蛾核型多角体病毒Baculo-BVDV-1（Autographa californica multiple nucleopolyhedrovirus Baculo-BVDV-1），已于2018年1月28日送交中国典型培养物保藏中心保藏，保藏号为：CCTCC No.V201803，培养物名称为苜蓿银纹夜蛾核型多角体病毒Baculo-BVDV-1，保藏地址为中国武汉 武汉大学。
本发明所述的重组杆状病毒制备方法是：将BVDV-1的结构蛋白C、Erns、E1、E2编码基因克隆至pFastBacDual构建pFBD-BVDV-1重组杆状病毒转移载体，在大肠杆菌DH10Bac中转座获得重组杆状病毒载体Bac-BVDV-1；将获得的重组杆状病毒载体Bac-BVDV-1转染昆虫细胞，培养后收获得到重组杆状病毒Baculo-BVDV-1。
本发明的重组杆状病毒在制备防治牛病毒性腹泻病毒免疫疫苗或药物中的应用。
本发明的具有免疫原性的BVDV-1病毒样颗粒是采用前述的重组杆状病毒Baculo-BVDV-1制备。
优选地，本发明的具有免疫原性的BVDV-1病毒样颗粒的制备方法是用所述的重组杆状病毒感染昆虫细胞，进而从昆虫细胞裂解物中分离获得病毒样颗粒。
本发明的具有免疫原性的BVDV-1病毒样颗粒可在制备预防牛病毒性腹泻病毒感染的疫苗中的应用。
本发明提供一种重组杆状病毒转移载体pFBD-BVDV-1，具有1个表达盒，含BVDV-1结构蛋白（C-Erns-E1-E2）的编码序列SEQ ID No.1，其编码的氨基酸序列为SEQ ID No.2。进一步的，所述的杆状病毒转移载体pFBD-BVDV-1是通过将1型BVDV结构蛋白（C-Erns-E1-E2）的编码序列利用Bam HI和Hind III酶切位点插入载体pFastBacDaul（pFBD）上获得的。
利用本发明获得的1型牛病毒性腹泻病毒的重组杆状病毒，可用于感染昆虫细胞而获得BVDV-1重组蛋白C、Erns、E1、E2，所述的重组杆状病毒的基因组中至少含有1个上述表达盒。同时利用这一重组病毒表达的重组蛋白得到了病毒样颗粒。
病毒样颗粒（virus like particles，VLPs）是由病毒衣壳蛋白自主组装而成的空衣壳结构，结构与形态与天然病毒相似但不含病毒的遗传物质，能有效刺激机体产生体液免疫和细胞免疫应答，免疫原性及安全性好。目前研究者已经对多种感染人和动物的病毒研制出了相应的VLPs，其中乙型肝炎、人乳头瘤病毒、戊型肝炎病毒的VLPs疫苗已经获得卫生部分的批准应用，另外，蓝舌病毒、非洲马瘟病毒、猪细小病毒、猪圆环病毒、禽流感病毒、脑心肌炎病毒、口蹄疫病毒的VLPs 已成为预防病毒感染的潜在候选疫苗（Liu et al.,Res Vet Sci. 2012, 93(2):553-559）。近期我国农业部已批准猪圆环病毒2型的病毒样颗粒为新兽药（中华人民共和国农业部公告259号）。由于本发明的病毒样颗粒具有良好的免疫原性，可以预期本发明的病毒样颗粒将可成为一类很有前景的疫苗。
本发明具有的优点和效果包括：
（1）本发明采用杆状病毒和昆虫细胞系统以多聚蛋白形式表达BVDV-1结构蛋白，在细胞内实现多聚蛋白的切割形成4种结构蛋白，进而自动组装获得具有较好空间结构的病毒样颗粒。与基因工程亚单位疫苗相比，获得的病毒样颗粒更接近天然BVDV病毒粒子的形态结构，接种动物后可刺激机体产生较好的免疫反应，达到更好的免疫效果。所获的病毒样颗粒可利用蔗糖密度梯度离心直接纯化，与其它表达系统相比，避免了病毒结构蛋白分别纯化然后在细胞外组装的繁琐过程，有利于提高效率和节约成本。
（2）与弱毒疫苗和灭活疫苗相比，所述的1型BVDV病毒样颗粒疫苗不含病毒基因组，在接种动物体内不存在与野生型BVDV基因组重组，而且在生产过程中不用感染性的BVDV活病毒，提高了安全性。
（3）所述1型BVDV病毒样颗粒疫苗不含BVDV非结构蛋白，在接种的动物不会产生BVDV非结构蛋白的抗体，与现有的BVDV抗体检测试剂盒兼容性好，可区分疫苗接种动物和病毒感染的动物，有利于BVDV的净化。
附图说明
图1重组杆状病毒转移载体pFBD-BVDV-1的PCR鉴定电泳，
其中M为DNA分子量标准DL10000；1、2、3为pFBD-BVDV-1重组载体1号、2号、3号PCR结果。
图2重组杆状病毒载体Bac-BVDV-1的PCR鉴定电泳，
其中M为DNA分子量标准DL5000；1、2、3为Bac-BVDV-1重组杆状病毒载体1号、2号、3号PCR结果。
图3杆粒Bac-BVDV-1转染Sf9细胞产生的细胞病变照片。
图4 Western Blot分析杆状病毒外源蛋白表达，
其中M为预染彩虹蛋白Marker （10-170KD）；1为Baculo-BVDV-1感染sf9细胞Erns蛋白鉴定；2为sf9对照细胞Erns蛋白鉴定，无表达；3为Baculo-BVDV-1感染sf9细胞E2蛋白鉴定；4为sf9对照细胞E2蛋白鉴定，无表达。
图5 BVDV病毒样颗粒透射电镜鉴定图。
图6 BVDV病毒样颗粒免疫牛和兔21天的E2抗体测定图。
图7 BVDV病毒样颗粒在豚鼠二免后攻毒后血液中BVDV核酸含量图，星号表示差异极显著。
具体实施方式
本发明以下结合实施例进行解说。
发明人依据BVDV病毒粒子具有的较高的pH耐受范围（在pH5.7～9.3范围内稳定）的特点，结合宿主细胞的蛋白水解酶加工切割BVDV 4种结构蛋白的特性，发现以杆状病毒昆虫细胞系统表达1型BVDV结构蛋白基因可在细胞内自动组装成稳定的BVDV病毒样颗粒，在此基础上完成了本发明。
实施例1：一种BVDV-1结构蛋白基因重组杆状病毒的构建
1.pFBD-BVDV-1重组杆状病毒转移载体的构建
根据测定获得的1型BVDV毒株GS4的结构蛋白编码基因序列（部分提交GenBank，登陆号为：KC700344），设计带酶切位点的上游引物（SEQ ID No.3）5’-GCCGGATCC BamHI ATGTCCGACACAAATACAGAAGG-3’和下游引物（SEQ ID No.4）5’-CTCAAGCTT HindIII CTAACCCGAGGTCATTTGTTCTG-3’，在上游引物和下游引物分别加上了起始密码子和终止密码子（斜体），用于1型BVDV结构蛋白C-Erns-E1-E2编码基因的扩增。通过反转录聚合酶链式反应(RT-PCR)，以1型BVDV病毒RNA为模板，利用上游引物（SEQ ID No.3）和下游引物（SEQ ID No.4）扩增C-Erns-E1-E2编码基因，回收目的片段，利用BamHI和HindIII双酶切后与同样酶切处理的pFastBacDaul载体（简称pFBD，购自Invitrogen公司）用DNA连接酶在16℃连接，转化大肠杆菌DH5α感受态细胞，涂布含100μg/mL氨苄青霉素的LB固体培养基平板，37℃培养15h，挑取单个克隆于含60 μg/mL氨苄青霉素的LB液体培养基摇培10h，利用上游引物（SEQ ID No.3）和下游引物（SEQ ID No.4）进行PCR鉴定，扩增获得2694 bp的条带，参见附图1，提取阳性质粒测序鉴定获得重组杆状病毒转移载体pFBD-BVDV-1。
2. 重组杆状病毒载体Bac-BVDV-1的构建
取测序正确的重组杆状病毒转移载体pFBD-BVDV-1 1.0 μL，转化大肠杆菌DH10Bac 感受态细胞（Invitrogen公司）1支，置于冰上30 min，于42℃热激90 s，冰浴2min，加入500 μL LB液体培养基，37℃振荡培养4h，取200μL涂布含50 μg/mL卡那霉素、7 μg/mL庆大霉素、10 μg/mL四环素、100 μg/mL Bluo-gal、40 μg/mL IPTG的三抗高盐固体LB平板，37℃培养48h，挑选白色克隆，利用含同样浓度抗生素的LB液体培养基于37℃摇培24h，提取重组杆粒Bac-BVDV-1，利用引物pUC/M13F（SEQ ID No.5）5’-CCCAGTCACGACGTTGTAAAACG-3’、pUC/M13R（SEQ ID No.6）5’-AGCGGATAACAATTTCACACAGG-3’引物进行鉴定，反应体系为ExTaqVersion 2.0 plus dye 25μL、杆粒4μL、上游引物1μL、下游引物1μL、H2O 19 μL，反应条件为94℃ 2min预变性，然后94℃ 30s、55℃ 30s，72℃ 5min 30s，40个循环。扩增产物利用1%琼脂糖进行电泳分析，扩增获得约5200bp的预期条带，参见附图2，表明BVDV-1结构蛋白基因重组成功。根据PureLink™ HiPurePlasmid DNA Midiprep提取试剂盒（Invitrogen公司）说明书，摇培50 mLBac-BVDV-1阳性菌，在对数期收集以6000g离心10 min，利用4 mLR3液重悬菌体，4 mL L7液室温裂解15 min，加入4 mL N3液混匀，15000 g室温离心10 min，将上清加入预先用10 mL EQ1液平衡处理的DNA纯化柱进行吸附，之后利用10mL W8液体洗剂2次，以5 mL E4液洗脱。向洗脱产物中加入3.5 mL 异丙醇，混匀，15000 g， 30 min，向DNA 沉淀中加 3 mL70% 乙醇，15000 g， 4℃离心 5min，最后风干所得 DNA 沉淀 10min，然后加入 100 µL TE Buffer溶解，利用Nanodrop2000定量后置于4℃保存备用。
3. 重组杆状病毒Baculo-BVDV-1获取和鉴定
按Bac-to-Bac baculovirus Expression System操作说明，利用Cellfectin II（Invitrogen公司）将重组杆粒Bac-BVDV-1转染Sf9细胞，在28℃培养72h，产生细胞病变后，参见附图3，收集重组杆状病毒Baculo-BVDV-1（标记为F1stock），继续扩大培养至第二代（F2stock）和第三代（F3stock），将F1-F3代病毒作为种毒，测定毒价后保存于-80℃。将sf-9细胞接种于5 cm2细胞培养瓶，在细胞长成90%单层时，用1个MOI的F3stock种毒接种，在接种72h后收集病变细胞，同时收集未接种Baculo-BVDV-1的正常细胞，分别利用500μL1XSDS-PAGE buffer裂解，100℃加热处理10 min，之后进行SDS-PAGE并转印至PVDF膜，分别利用实验室制备的兔抗BVDV Erns多克隆抗体为一抗，碱性磷酸酶标记的山羊抗兔IgG（Abcam公司）为二抗进行Western Blot检测Erns的表达；同时利用抗1型BVDV E2蛋白的单克隆抗体（IgG2a Isotype, VMRD公司），碱性磷酸酶标记的驴抗小鼠多克隆抗体（Abcam公司）为二抗进行Western Blot检测E2蛋白的表达。结果显示重组杆状病毒Baculo-BVDV-1感染细胞样品可检出Erns蛋白和E2蛋白，而未接种病毒的sf9细胞无特异性条带检出，见附图4。
重组杆状病毒Baculo-BVDV-1经测序得到核苷酸序列为SEQ ID No.1，其编码的氨基酸序列为SEQ ID No.2。对所得重组杆状病毒Baculo-BVDV-1送武汉大学进行了保藏，保藏编号为CCTCC NO: V201803。
实施例2：在昆虫细胞内表达制备1型BVDV VLPs
将sf-9细胞接种于5 cm2细胞培养瓶，在细胞长成90%单层时，用1个MOI的F3stock种毒接种，在72h时收集上清和细胞超声裂解，在4℃条件下4000g离心30min去除细胞碎片，利用Amicon Ultra-15超滤离心管将上清浓缩至2 mL，然后加入10-60% 蔗糖密度梯度在Beckman超速离心机（SW41 rotor）以4°C，35000 rpm离心2h，取1mL分装并标记次序，制样进行E2蛋白Western检测。对检测含有E2蛋白的样品，取10μL加到滴在覆有支持膜和碳膜的200目铜网，室温吸附3min，干燥后用3%磷钨酸染色，用FEI Tecnai G2 F20 透射电子显微镜进行观察，结果可见约50nm的病毒样颗粒，参见附图5，说明本发明在sf9细胞内成功制备了1型BVDV 病毒样颗粒。
实施例3：1型牛病毒性腹泻病毒样颗粒疫苗制备及免疫效力测定
利用实施例1中F3stock种毒以1个MOI接种sf9细胞，在接种72h后收集病变细胞，超声破碎并于4℃，6000g离心10 min除去细胞碎片，利用BCA蛋白浓度测定试剂盒（碧云天生物技术公司）测定粗提抗原的蛋白浓度，之后利用10mM的二乙烯亚胺（BEI)灭活48h，加入终浓度15mM的硫代硫酸钠中和，与MerckinadeSDA-201双相油佐剂充分乳化至少量滴于冷水中呈油滴状不扩散。将制备的1型牛病毒性腹泻病毒样颗粒疫苗无菌定量分装，4℃保存。
利用制备的1型牛病毒性腹泻病毒样颗粒疫苗和等量sf9细胞对照抗原分别接种BVDV抗体阴性的成年牛两头（500μg/头）、3月龄新西兰大耳白兔2只（100μg/只），在接种21天后采集全血，分离血清，用本实验原核表达纯化的BVDV E2蛋白（博士论文：高闪电：我国西北地区牛病毒性腹泻病毒的基因亚型多样性及其E2基因嵌合猪瘟病毒的构建）进行间接ELISA测定抗体滴度，同时根据中华人民共和国出入境检验检疫行业标准（SN/T 1129-2007）《牛病毒性腹泻/粘膜病检疫规范》中的病毒中和试验方法，用细胞病变型毒株AV69（购自中国兽医药品监察所）进行微量中和试验测定免疫牛的抗体中和效价。结果间接ELISA测定表明疫苗免疫组牛和兔血清E2抗体呈强阳性，sf9细胞对照抗原接种牛和兔血清E2抗体呈阴性，参见图6；牛在首免后抗体中和效价均达1:64以上，而sf9细胞对照抗原接种牛血清无中和活性。这些结果显示制备的牛病毒性腹泻病毒样颗粒疫苗具有很好的免疫效果。
利用制备的1型牛病毒性腹泻病毒样颗粒疫苗和等量sf9细胞对照抗原分别免疫2月龄豚鼠各5只（50 μg/只），在21天采血利用病毒中和试验测定抗体滴度，结果免疫组豚鼠血清中和抗体均达1:64以上，而sf9细胞抗原对照组豚鼠血清抗体无中和活性。在首免21天利用等量抗原进行二免，在二免20天利用106TCID50 BVDV经典毒株AV69（购自中国兽医药品监察所）进行攻毒，采集第3天、第4天、第5天、第6天、第9天的全血各1mL，利用荧光定量RT-PCR（Baxi et al.Vet Microbiol. 2006, 116(1-3):37-44.）检测病毒核酸，设计豚鼠GAPDH荧光定量上游引物GAPDHF （SEQ ID No.7 5’-AACATCATCCCCGCATC-3’）和下游引物GAPDHR（SEQ ID No.8 5’-CCTCGGTGTAGCCCAAG-3’）对豚鼠GAPDH mRNA检测，以GAPDH为内参，采用2－ΔΔCt进行数据分析，对对照组和免疫组豚鼠的BVDV 核酸进行相对定量，结果免疫组的病毒核酸在第3～5天均低于对照组，在第6天与对照组存在显著差异，参见图7，说明制备的1型牛病毒性腹泻病毒样颗粒疫苗具有较好的保护作用。
序列表
<110> 中国农业科学院兰州兽医研究所
<120> 一种1型牛病毒性腹泻病毒样颗粒及其制备与应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2694
<212> DNA
<213> 1型牛病毒性腹泻重组病毒结构蛋白的编码核苷酸序列(BVDV-1)
<400> 1
tccgacacaa atacagaagg ggcgacagga aagaaacaac aaaaaccaga taggttggaa 60
agggggagaa tgaagataac ccctaaagag tcggaaaaag atagtaagac caaaccgcca 120
gatgccacta tagtggtaga tggagtcaaa tatcaggtaa agaaaaaagg gaaagtcaag 180
agcaagaaca cccaggacgg cttgtatcac aacaaaaaca aacctcaaga gtcacgcaag 240
aaactagaga aagccctgtt agcatgggca ataataggct tagttttgtt tcaagtcgca 300
gcgggggaga acataacaca atggaactta caagataatg ggacggaagg aatacaacgg 360
gccatgttcc aaagaggagt aaatagaagt ctgcatggga tttggccaga gaagatctgt 420
acaggggtcc cctcccactt agccactgac acagaactga aggcaattca tggtatgatg 480
gatgcaagtg agaagacaaa ttacacatgt tgcaggctcc aacgccatga gtggaacaag 540
catggttggt gcaattggta taatatagaa ccttggattc ttctcatgaa taaaacccaa 600
gccaacctta ctgagggtca gccaccaagg gagtgtgccg tcacatgccg gtatgaccga 660
gatagtgacc taaatgtagt gacacaagct agggacagcc ccacaccatt gacaggctgc 720
aagaaaggta agaacttttc ctttgcaggt gtgttggtgc aaggaccttg caactttgaa 780
atagctgtaa gtgatgtgct gtttaaagaa cacgattgca ccagcatgat acaagacacg 840
gctcactatc tggtagacgg gatgaccaac tcactggaaa aggctaggca agggactgcg 900
aaactaacaa cttggttggg taggcagctt gggatactgg ggaagaagtt ggaaaacaag 960
agtaagacat ggtttggagc atatgcagcc tctccctact gtgaggtaga acggaggctt 1020
ggttacatct ggcatacaaa gaattgtact cccgcctgtt tgccaaaaaa cacaaagatt 1080
gttggccccg gtagatttga caccaatgcg gaggatggta aaatactgca cgagatggga 1140
ggccacttgt cggaggtatt actactctca ctagtggtgc tttccgattt tgctccagag 1200
acagccagtg tggtatactt aattctacat ttctccatcc cacaaggaca cactgatata 1260
catgactgtg acaagaacca gttaaacctc accgtagaac tcacagcagc tgaagtaata 1320
ccagggtcag tctggaatct tggcaaatac gtttgcataa ggccagactg gtggccttat 1380
gagacagcca cggtcctagt attcgaagaa gtgggtcagg tgatcaagat agtcttaagg 1440
gcactaaggg atctaacgcg catttggacc gctgctacga ccactgcgtt cctggtgtgt 1500
ctggtgaagg tggtgagagg ccaagtgttg caaggcgtac tatggttgtt actcataaca 1560
ggggcacaag ggctaccagc ctgcaaaccc gactttcggt acgccatatc caaaaataat 1620
gaggtcggcc ctctcggagc tacgggcctc accactcagt ggtatggata ctcggatggg 1680
atgcggctgc aagacacggc agttgtagtg tggtgtaaag aaggagagat gagacatctt 1740
actacatgtg agagggaagc ccggtacttg gccgtcctac acacgagagc tctgccgaca 1800
tctgtggtat ttgaaaaaat catagatggg gaaaaacacg aggaagtagt agaaatggat 1860
gatgactttg aattcggtct ttgcccgtgt gatgctaaac ccttggtaag aggtaaattc 1920
aatacaacac ttctaaatgg gccagccttc cagatggttt gccctattgg atggacagga 1980
actgtgagct gtgcactggc caacaaggat acgttggatg tgaccattgt aagaacatac 2040
atgaggctca agcctttccc ctataggcaa ggctgtacta cccagaaaac catcggggaa 2100
gacctctaca actgtctctt gggagggaac tggacttgta taccggggga cgtattacga 2160
tatgtagacg ggcctgttga gtcttgcaag tggtgtggtt acaagttcct taaagatgag 2220
ggtctgccac acttcccaat tggcaagtgc aagctgaaga atgaaagtgg ctacagacta 2280
gtagatgaga cctcttgcaa cagagacggt gtggccatag ttccaagtgg tatggtgaaa 2340
tgcaagatag gggacacagt ggtgcaagtc atagcaatgg atgaaaagct agggcctatg 2400
ccttgcagac catatgaaat cttttccagt gaggggccgg tggaaaagac ggcatgtacc 2460
ttcaactaca caaagacatt aaagaacaag tattatgagc ccagggataa ttatttccaa 2520
caatacatgc taaaggggga gtaccaatac tggtttgacc tggagatcac tgaccaccac 2580
cgggactact tcgctgagtc cctactggtg atagtggttg cactcctggg tggtaggtac 2640
gtgctctggt tactggttac gtacatgatc ctatcagaac aaatgacctc gggt 2694
<210> 2
<211> 898
<212> PRT
<213> 1型牛病毒性腹泻病毒结构蛋白的氨基酸序列(BVDV-1)
<400> 2
Ser Asp Thr Asn Thr Glu Gly Ala Thr Gly Lys Lys Gln Gln Lys Pro
1 5 10 15
Asp Arg Leu Glu Arg Gly Arg Met Lys Ile Thr Pro Lys Glu Ser Glu
20 25 30
Lys Asp Ser Lys Thr Lys Pro Pro Asp Ala Thr Ile Val Val Asp Gly
35 40 45
Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Lys Ser Lys Asn Thr
50 55 60
Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro Gln Glu Ser Arg Lys
65 70 75 80
Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Ile Gly Leu Val Leu
85 90 95
Phe Gln Val Ala Ala Gly Glu Asn Ile Thr Gln Trp Asn Leu Gln Asp
100 105 110
Asn Gly Thr Glu Gly Ile Gln Arg Ala Met Phe Gln Arg Gly Val Asn
115 120 125
Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys Thr Gly Val Pro
130 135 140
Ser His Leu Ala Thr Asp Thr Glu Leu Lys Ala Ile His Gly Met Met
145 150 155 160
Asp Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg Leu Gln Arg His
165 170 175
Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr Asn Ile Glu Pro Trp
180 185 190
Ile Leu Leu Met Asn Lys Thr Gln Ala Asn Leu Thr Glu Gly Gln Pro
195 200 205
Pro Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Arg Asp Ser Asp Leu
210 215 220
Asn Val Val Thr Gln Ala Arg Asp Ser Pro Thr Pro Leu Thr Gly Cys
225 230 235 240
Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Leu Val Gln Gly Pro
245 250 255
Cys Asn Phe Glu Ile Ala Val Ser Asp Val Leu Phe Lys Glu His Asp
260 265 270
Cys Thr Ser Met Ile Gln Asp Thr Ala His Tyr Leu Val Asp Gly Met
275 280 285
Thr Asn Ser Leu Glu Lys Ala Arg Gln Gly Thr Ala Lys Leu Thr Thr
290 295 300
Trp Leu Gly Arg Gln Leu Gly Ile Leu Gly Lys Lys Leu Glu Asn Lys
305 310 315 320
Ser Lys Thr Trp Phe Gly Ala Tyr Ala Ala Ser Pro Tyr Cys Glu Val
325 330 335
Glu Arg Arg Leu Gly Tyr Ile Trp His Thr Lys Asn Cys Thr Pro Ala
340 345 350
Cys Leu Pro Lys Asn Thr Lys Ile Val Gly Pro Gly Arg Phe Asp Thr
355 360 365
Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met Gly Gly His Leu Ser
370 375 380
Glu Val Leu Leu Leu Ser Leu Val Val Leu Ser Asp Phe Ala Pro Glu
385 390 395 400
Thr Ala Ser Val Val Tyr Leu Ile Leu His Phe Ser Ile Pro Gln Gly
405 410 415
His Thr Asp Ile His Asp Cys Asp Lys Asn Gln Leu Asn Leu Thr Val
420 425 430
Glu Leu Thr Ala Ala Glu Val Ile Pro Gly Ser Val Trp Asn Leu Gly
435 440 445
Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr Glu Thr Ala Thr
450 455 460
Val Leu Val Phe Glu Glu Val Gly Gln Val Ile Lys Ile Val Leu Arg
465 470 475 480
Ala Leu Arg Asp Leu Thr Arg Ile Trp Thr Ala Ala Thr Thr Thr Ala
485 490 495
Phe Leu Val Cys Leu Val Lys Val Val Arg Gly Gln Val Leu Gln Gly
500 505 510
Val Leu Trp Leu Leu Leu Ile Thr Gly Ala Gln Gly Leu Pro Ala Cys
515 520 525
Lys Pro Asp Phe Arg Tyr Ala Ile Ser Lys Asn Asn Glu Val Gly Pro
530 535 540
Leu Gly Ala Thr Gly Leu Thr Thr Gln Trp Tyr Gly Tyr Ser Asp Gly
545 550 555 560
Met Arg Leu Gln Asp Thr Ala Val Val Val Trp Cys Lys Glu Gly Glu
565 570 575
Met Arg His Leu Thr Thr Cys Glu Arg Glu Ala Arg Tyr Leu Ala Val
580 585 590
Leu His Thr Arg Ala Leu Pro Thr Ser Val Val Phe Glu Lys Ile Ile
595 600 605
Asp Gly Glu Lys His Glu Glu Val Val Glu Met Asp Asp Asp Phe Glu
610 615 620
Phe Gly Leu Cys Pro Cys Asp Ala Lys Pro Leu Val Arg Gly Lys Phe
625 630 635 640
Asn Thr Thr Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys Pro Ile
645 650 655
Gly Trp Thr Gly Thr Val Ser Cys Ala Leu Ala Asn Lys Asp Thr Leu
660 665 670
Asp Val Thr Ile Val Arg Thr Tyr Met Arg Leu Lys Pro Phe Pro Tyr
675 680 685
Arg Gln Gly Cys Thr Thr Gln Lys Thr Ile Gly Glu Asp Leu Tyr Asn
690 695 700
Cys Leu Leu Gly Gly Asn Trp Thr Cys Ile Pro Gly Asp Val Leu Arg
705 710 715 720
Tyr Val Asp Gly Pro Val Glu Ser Cys Lys Trp Cys Gly Tyr Lys Phe
725 730 735
Leu Lys Asp Glu Gly Leu Pro His Phe Pro Ile Gly Lys Cys Lys Leu
740 745 750
Lys Asn Glu Ser Gly Tyr Arg Leu Val Asp Glu Thr Ser Cys Asn Arg
755 760 765
Asp Gly Val Ala Ile Val Pro Ser Gly Met Val Lys Cys Lys Ile Gly
770 775 780
Asp Thr Val Val Gln Val Ile Ala Met Asp Glu Lys Leu Gly Pro Met
785 790 795 800
Pro Cys Arg Pro Tyr Glu Ile Phe Ser Ser Glu Gly Pro Val Glu Lys
805 810 815
Thr Ala Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys Asn Lys Tyr Tyr
820 825 830
Glu Pro Arg Asp Asn Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr
835 840 845
Gln Tyr Trp Phe Asp Leu Glu Ile Thr Asp His His Arg Asp Tyr Phe
850 855 860
Ala Glu Ser Leu Leu Val Ile Val Val Ala Leu Leu Gly Gly Arg Tyr
865 870 875 880
Val Leu Trp Leu Leu Val Thr Tyr Met Ile Leu Ser Glu Gln Met Thr
885 890 895
Ser Gly
<210> 3
<211> 31
<212> DNA
<213> 人工序列(上游引物)
<400> 3
gccggatccb gtccgacaca aatacagaag g 31
<210> 4
<211> 33
<212> DNA
<213> 人工序列(下游引物)
<400> 4
ctcaagctth ctaacccgag gtcatttgtt ctg 33
<210> 5
<211> 23
<212> DNA
<213> 人工序列(鉴定用上游引物)
<400> 5
cccagtcacg acgttgtaaa acg 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列(鉴定用下游引物)
<400> 6
agcggataac aatttcacac agg 23
<210> 7
<211> 17
<212> DNA
<213> 人工序列(荧光定量上游引物GAPDHF)
<400> 7
aacatcatcc ccgcatc 17
<210> 8
<211> 17
<212> DNA
<213> 人工序列(荧光定量下游引物GAPDHR)
<400> 8
cctcggtgta gcccaag 17
Claims (6)
1.一种重组杆状病毒，该重组杆状病毒命名为苜蓿银纹夜蛾核型多角体病毒Baculo-BVDV-1（Autographa californica multiple nucleopolyhedrovirus Baculo-BVDV-1），已于2018年1月28日送交中国典型培养物保藏中心保藏，保藏号为：CCTCC No.V201803，培养物名称为苜蓿银纹夜蛾核型多角体病毒Baculo-BVDV-1，保藏地址为中国武汉 武汉大学。
2.权利要求1所述的重组杆状病毒的制备方法，其特征在于：将BVDV-1的结构蛋白C、Erns、E1、E2编码基因克隆至pFastBacDual构建pFBD-BVDV-1重组杆状病毒转移载体，在大肠杆菌DH10Bac中转座获得重组杆状病毒载体Bac-BVDV-1；将获得的重组杆状病毒载体Bac-BVDV-1转染昆虫细胞，培养后收获得到重组杆状病毒Baculo-BVDV-1。
3.权利要求1所述的重组杆状病毒在制备防治牛病毒性腹泻病毒免疫疫苗或药物中的应用。
4.一种具有免疫原性的BVDV-1病毒样颗粒，其特征在于采用权利要求1所述的重组杆状病毒Baculo-BVDV-1制备。
5.权利要求4所述的具有免疫原性的BVDV-1病毒样颗粒的制备方法，其特征在于用权利要求1所述的重组杆状病毒感染昆虫细胞，进而从昆虫细胞裂解物中分离获得病毒样颗粒。
6.权利要求1所述的重组杆状病毒或权利要求4所述的具有免疫原性的BVDV-1病毒样颗粒在制备预防牛病毒性腹泻病毒感染的疫苗中的应用。
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| CN110004178A (zh) * | 2019-04-02 | 2019-07-12 | 石河子大学 | 一种牛病毒性腹泻病毒样颗粒的制备的制备方法 |
| CN112646840B (zh) * | 2020-12-25 | 2022-11-18 | 石河子大学 | 一种杆状病毒表达系统制备牛病毒性腹泻病毒e2蛋白的方法及应用 |
| CN112961224B (zh) * | 2021-03-26 | 2022-09-27 | 中国农业大学 | 牛病毒性腹泻病毒1型病毒样颗粒的制备及应用 |
| CN114921495A (zh) * | 2022-06-15 | 2022-08-19 | 中国农业科学院兰州兽医研究所 | 一种猪瘟病毒病毒样颗粒疫苗的制备方法及应用 |
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