流行性乙型脑炎病毒E基因的克隆、表达及初步应用  

Cloning and Expression of E Gene of Japanese Encephalitis Virus and Preliminary application

作　　者：张雪寒[1] 何孔旺[1] 郭容利[1] 倪艳秀[1] 王芳[1] 俞正玉[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部畜禽疫病诊断重点开放实验室,江苏南京210014

出　　处：《中国病毒学》2005年第5期494-497,共4页Virologica Sinica

基　　金："863"项目资助(2001AA249012)

摘　　要：参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a(+)中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。The whole cDNA of E gene was amplified by RT-PCR from JEV strain SA14-14, and cloned into the pMD18-T vector. The fragment was identified by restriction enzymes digestion with EcoR Ⅰ and Xho Ⅰ,cloned into the pET32a（＋） vector. The recombinant plasmid was transformed into BL21 and the recombinant bacteria was induced by optimal concentration of IPTG. SDS-PAGE and Western blotting were performed to detect the E fusion protein. Our result indicates the molecular weight of E protein is of E protein 73kDa and its antigenicity is good. Indirect ELISA procedure has been developed with the purified E protein as antigen for detection of JEV.

关 键 词：乙型脑炎病毒 E基因 表达 间接ELISA 

分 类 号：R373[医药卫生—病原生物学]