基因4型HEV上海株ORF2编码蛋白表达及其抗原性分析  

Expression and antigenic analysis of recombinant ORF_2 protein of hepatitis E virus genotype 4 from Shanghai

作　　者：牟静[1] 张文[2] 侯加法[1] 沈权[3] 杨志彪[2] 崔立[2] 华修国[2] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]上海交通大学农业与生物学院,上海200240 [3]南京师范大学生命科学学院,江苏南京210097

出　　处：《畜牧与兽医》2008年第5期32-36,共5页Animal Husbandry & Veterinary Medicine

基　　金：上海市科委登山行动计划重点项目(063919121)

摘　　要：为了表达基因4型戊型肝炎病毒(HEV)上海株ORF2编码蛋白并分析其抗原性,采用套式RT-PCR方法扩增上海猪基因4型HEV代表株结构蛋白基因ORF2部分片段,构建含有该基因片段的pET30a(+)重组表达质粒,命名为pET-E;将重组表达质粒转化大肠杆菌BL21,经1mmol/L IPTG诱导得到表达,进行SDS-PAGE分析,然后用Western blot鉴定其抗原性,最后纯化蛋白。结果:SDS-PAGE证明该片段得到融合表达,分子量为33 ku。Western blot分析表明,重组蛋白可以与HEV阳性血清反应,表明该蛋白具有良好的抗原性;该融合蛋白含有组氨酸标签,可以用MagExtractor-His-tag-kit进行纯化。用纯化的重组蛋白作为包被抗原建立的间接ELISA检测89份猪血清样品,阳性率达80.4%,与已有的试剂盒检测结果相比,阳性符合率达95%。In order to express the recombinant ORF2 protein of hepatitis E virus （HEV） genotype 4 from Shanghai and analyze its antigenicity, RT-nPCR was adopted to amplify the fragment of structure protein gene ORF2 of HEV genotype 4 from Shanghai. The fragment of ORF2 was cloned into pET 30 a （ ＋ ） , designated as pET-E. The recombinant plasmid was transformed into E. coli BL21 and the protein was expressed by the induction with IPTG. The analysis of SDS-PAGE indicated a protein band with the molecular weight of 33 ku. The recombinant protein could react with the serum against HEV in Western blot. The indirect ELISA method was developed by using the recombinant protein purified with the MagExtractor-His-tag-kit as antigen. The investigation for 89 serum samples indicated that the coincidence rate between the recombinant protein ELISA and available kit was up to 95% in detecting HEV antibodies.

关 键 词：戊型肝炎病毒 ORF2 重组质粒 原核表达 

分 类 号：S858.28[农业科学—临床兽医学]