利用原核及真核表达系统体外表达草鱼Ⅱ型呼肠孤病毒外突VP56蛋白的研究  

Prokaryotic and eukaryotic expression analysis of outer capsid VP56 protein encoded by type Ⅱ reovirus of grass carp

作　　者：盛佳璐 宗乾坤[1] 喻飞[1] 王浩[1,2,3] 吕利群[1,2,3] SHENG Jialu;ZONG Qiankun;YU Fei;WANG Hao;LU Liqun(National Pathogen Collection Center for Aquatic Animals,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Agriculture Ministry for Freshwater Aquatic Genetic Resources,Shanghai Ocean University,Shanghai 201306,China;National Experimental Teaching Demonstration Center for Fishery Sciences,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学国家水生动物病原库,上海201306 [2]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《上海海洋大学学报》2018年第4期469-476,共8页Journal of Shanghai Ocean University

基　　金：国家现代农业产业技术体系建设专项(CARS-46-12);国家自然科学基金(31672690;31072244)

摘　　要：草鱼Ⅱ型呼肠孤病毒是当前引起我国草鱼出血病的主要流行毒株,VP56是其外层衣壳上的唯一突起蛋白并有可能在病毒入侵阶段发挥核心作用,但该蛋白的具体功能尚不清楚。为了探讨VP56的生物学功能,分别利用大肠杆菌原核表达系统和杆状病毒真核表达系统研究了VP56的体外表达特性。首先从GCRVJX02W株的细胞感染混合物中抽提总RNA,并通过RT-PCR方法获得目的基因VP56,分别克隆至pGEX-4T-3及pFastBacHTA载体上,将质粒pGEX-4T-3-VP56转化至BL21(DE3),经IPTG诱导后,成功表达融合蛋白GST-VP56,大小为83ku,以包涵体形式存在;蛋白经纯化后免疫老鼠,制备了VP56的多克隆抗体,可以和rVP56产生特异性免疫结合。将pFastBacHTA-VP56转化至DH10Bac,成功转座后,提取重组杆粒DNA并鉴定且测序正确后转染SF9昆虫细胞。结果表明,自转染96h起,转染杆粒的细胞生长停滞,不断裂解。Westernblot结果显示,细胞表达重组蛋白His-VP56,大小约为62ku,为可溶蛋白。本研究利用原核表达系统和真核表达系统体外表达了VP56蛋白,制备了抗VP56多克隆抗体,为深入研究VP56蛋白在病毒入侵时的生物学功能奠定了基础。Type Ⅱ reovirus of grass carp represents the pandemic strain for grass carp hemorrahagic disease in China. VP56 is the outer most protein on the virion of type Ⅱ reovirus,and is suggested to play a key role during viral entry. To facilitate the study on biological functions of VP56, here, we investigated the prokaryotic and eukaryotic expression of VP56 gene of type Ⅱ reovirus. The VP56 ORF encoded by the S7 genomic fragment was amplified by RT-PCR technology from the c DNA of GCRV-JX02 W infected CIK cells,and it was cloned into the p GEX-4 T-3 and p Fast Bac HTA,respectively. After confirmation of the clones by sequencing analysis,the p GEX4 T-3-VP56 was transformed into E. coli BL21（DE3） strain to express fusion protein GST-VP56. SDS-PAGE and Western Blot assays indicated that GST-VP56 was successfully expressed in E. coli BL21（DE3） strain with the molecular weight of about 83 ku. The recombinant protein mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified recombinant VP56 proteins and the specificity of antibodies was determined by Western Blot.p Fast Bac HTA-VP56 was transformed into E. coli DH10 Bac strain to get recombinant bacmid DNA that was analyzed by PCR. Then,the recombinant bacmid DNA was transfected into SF9 cells. Positive SF9 cells transfected with bacmid DNA stopped growth and lysed at 96 h post transfection. Western Blot results showed that the His-tag antibody could be specifically bound to His-VP56 fusion protein with the molecular weight of about 62 ku, which was soluble protein. In this paper, recombinant VP56 proteins were successfully expressed by both prokarotic and eukaryotic expression systems,and polyclonal antiserum against VP56 was generated,which laid a foundation for characterizing the function of VP56 protein during viral infection.

关 键 词：草鱼Ⅱ型呼肠孤病毒 VP56 原核表达 真核表达 

分 类 号：S917[农业科学—水产科学]