草鱼呼肠孤病毒HZ08株S10编码蛋白多克隆抗体制备及其特性分析  

Preparation and characteristics of polyclonal antibody against S10 encoded protein of grass carp reovirus HZ08 strain

作　　者：李永刚[1,2] 曾伟伟[1] 王庆[1] 殷亮[1,2] 王英英[1] 梁红茹[1] 刘春[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔药创制重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2014年第3期449-456,共8页Journal of Fisheries of China

基　　金：国家自然科学基金项目(31202026);国家科技支撑计划(2012BAD25B02);现代农业产业技术体系建设专项(CARS-46)

摘　　要：为研究草鱼呼肠孤病毒(GCRV)HZ08株S10基因节段编码蛋白的可能功能,采用PCR方法扩增草鱼呼肠孤病毒HZ08株S10基因节段,并把该基因节段克隆至表达载体pET-32a(+),获得的重组表达载体pET-32a-S10转化到大肠杆菌BL21(DE3)菌株,用IPTG诱导表达,表达产物通过SDS-PAGE分析鉴定后,再通过变性、过Ni柱纯化、透析复性纯化获得目的蛋白。然后用纯化的重组蛋白免疫昆明小白鼠,制得多克隆抗体,用间接ELISA方法测定抗体效价,用Western blot和IFA(间接免疫荧光试验)鉴定抗体特异性。结果表明,SDS-PAGE分析表达的重组蛋白约为53 ku,大小与预期相符,目的蛋白主要存在于包涵体中;过Ni柱纯化、透析复性纯化后的重组蛋白纯度可达97.4%;间接ELISA测得制备的多克隆抗体效价约为1∶106,Western blot和IFA结果显示,制备的多克隆抗体能识别HZ08毒株,表明S10编码蛋白为GCRV-HZ08株的结构蛋白。To explore the probable function of segment 10 （S10）encoded protein of grass carp reovirus （GCRV） HZ08 strain,the ORF of S10 gene was amplified and cloned into the prokaryotic expression vector pET-32a（ ＋ ）, and the obtained recombinant expression vector was named pET32a-S10. The recombinant vectors were transformed into BL21 competent cells. The Escherichia coli containing recombinant vector expressed fusion protein of approximately 53 ku after induction by IPTG. The recombinant protein was purified through Ni-chelating affinity chromatography,and the purity was above 97.4% explained by SDS- PAGE gel scan analysis. Polyclonal antibody against S10 encoded protein was generated by immunization of female Kunming mice with purified recombined protein. The antibody titers in sera of the immunized mice were detected by ELISA and the specificity of the antibody was identified by Western blot and indirect immunefluorescence assay （IFA）. The results showed that the antibody titer cloning is 1 ： 10^6 and can specially identify the GCRV-HZ08. This means the S10 encoded protein is one of the structural protein of GCRV-HZ08.

关 键 词：草鱼呼肠孤病毒 HZ08株 S10编码蛋白 多克隆抗体 

分 类 号：Q785[生物学—分子生物学] S965.112[农业科学—水产养殖]