具有免疫反应性的猪瘟病毒多表位基因在大肠杆菌中的克隆表达和鉴定  

Cloning expression and characterization of the immuoreactive multiepitope gene of CSFV in E.coli

作　　者：侯相民[1,2] 田宏[2] 尚佑军[2] 田永强[1] 刘湘涛[2] 

机构地区：[1]兰州交通大学化学与生物工程学院,兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《生物学杂志》2012年第5期6-10,共5页Journal of Biology

基　　金：国家高技术研究发展计划(863)项目(2006AA10A204)

摘　　要：将猪瘟病毒不同抗原表位基因串联构成重组基因BT21,化学合成后克隆至pMD18-T载体中,然后再将BT21串联基因片段插入原核表达载体pGEX-6P-1。经酶切和测序鉴定后,构建的重组质粒pGEX-BT21转化大肠杆菌BL21(DE3),通过IPTG诱导表达,表达产物进行SDS-PAGE分析,用Glutathione Sepharose 4B亲和层析法纯化目的蛋白和Western blot分析其免疫学活性。结果表明:融合蛋白GST-BT21以可溶形式表达,分子量约为33kDa,与预期大小相符,纯化的重组蛋白可被猪瘟病毒阳性血清所识别,具有良好的免疫学活性。从而为进一步研究该融合蛋白的免疫特性和功能奠定了基础。Different epitopes genes of classical swine fever virus （CSFV） were constituted a recombinant gene BT21 in series synthesized by chemical method and subcloned into pMD18-T vector. Then the BT21was inserted into prokaryotic expression vector pGEX-6P-1. After digested and sequenced analyses, the constructing pGEX-BT21 was transformed into E. coli BI21 （DE3） , GST- BT21 fusion protein expression was induced by IPTG and analyzed by SDS-PAGE. After that, it was purified with Glutathione Sepharose 4B and by performed to confirm its characterization Western-blotting. The results showed that the target protein was mainly expressed in soluble form and the molecular weight was about 33kDa, which is consistent with the expected molecular weight of interest protein. The purified recombinant protein could specifically react with antiserum against classical swine fever virus. Thus, it can be used for the further investigation on immunogenicity and function of the recombinant protein.

关 键 词：猪瘟病毒 E2结构糖蛋白 多表位抗原 抗原活性 

分 类 号：Q786[生物学—分子生物学]