HP-PRRSV HuN4株ORF7原核表达载体的构建及表达  

Construction and Expression of Prokaryotic Expression Vector of ORF7 from HP-PRRSV Hu N4 Strain

作　　者：刘洪斌 孙心 李静 李琳 

机构地区：[1]哈药集团生物疫苗有限公司,黑龙江哈尔滨150069

出　　处：《中国动物保健》2015年第6期66-68,70,共4页China Animal Health

摘　　要：目的:构建HP-PRRSV Hu N4株ORF7原核表达载体,诱导表达重组N蛋白。方法:本实验选取HP-PRRSV经典株Hu N4株,根据ORF7序列(EF635006)及原核表达载体PGEX-6p-1中的多克隆位点设计引物,进行ORF7基因的克隆和原核表达载体的构建;将重组载体在原核表达体系中进行表达,通过SDS-PAGE电泳鉴定蛋白表达情况。结果:以HP-PRRSV Hu N4株c DNA为模板,PCR扩增出约为372 bp片段,经插入原核表达载体PGEX-6p-1,构建原核表达质粒PGEX-ORF7,鉴定后经1mmol/L IPTG在37℃下诱导,3 h后蛋白表达量最大;通过SDS-PAGE鉴定蛋白表达成功。结论:成功构建了HP-PRRS Hu N4株ORF7基因原核表达载体,以及进一步表达重组融合N蛋白;为进一步研究病毒的结构及功能奠定基础。Objective：Construction and expression of prokaryotic expression vector of HP-PRRSV HuN4 strain ORF7 gene. Method： A pair of primers were designed according to multiple clone sites in prokaryotie expression vector pGEX-6p-1 and HP-PRRSV ORF7 gene. ORF7 gene fragment was amplified with PCR and subcloned into a pGEX-6p-1 avector. The pGEX- ORF7 plasmid was taken and transformed into BL21 （DE3） for expres- sion. Induced by IPTG at 37 ,the expression product was identified by SDS-PAG.Resuh：The PCR product of ORF7 was 372 bp in length, and the prokaryotic expression vector pGEX-ORF7 was successfully constructed. The expected protein N had been expressed successfully in the form of inclusion bodies. The molecular weight of protein was39 kDa, and the quantity produced reached the highest when it was induced with 1 mmol· L-1 IPTG for 3 h. Conclusion：The prokaryotic expression vector pGEX-ORF7 was successfully expressed in E.coli, thus laying a foundation for the study of its function.

关 键 词：HP-PRRSV 原核表达 N蛋白 

分 类 号：S852.65[农业科学—基础兽医学]