嵌合HCV串联多表位的乙肝S基因真核载体构建及表达  

Construction and expression of eukaryotic vector for chimeric gene of hepatitis C virus series multi-epitopes with hepatitis B virus S

作　　者：王晓岩[1] 雷迎峰[2] 李翠[3] 林芳[1] 崔颖[1] 舒放[1] 张惠中[1] 韦三华[1] 

机构地区：[1]第四军医大学唐都医院临床实验与检验科,陕西西安710038 [2]第四军医大学基础部微生物学教研室,陕西西安710032 [3]西安交通大学第一附属医院转化医学中心,陕西西安710061

出　　处：《生物技术》2016年第1期12-17,共6页Biotechnology

基　　金：国家自然科学基金项目("HCV中和抗原表位嵌合病毒样颗粒诱导的中和抗体应答研究";No.81072500;"乙肝S抗原为载体的HCV嵌合病毒样颗粒预防疫苗的初步实验研究";No.31370924)资助

摘　　要：[目的]构建丙型肝炎病毒(hepatitis C virus,HCV)串联多中和抗原表位与乙型肝炎病毒(hepatitis B virus,HBV)S抗原嵌合基因真核表达质粒,并在293T细胞中进行表达。[方法]将HCV基因中高度保守且具有广谱交叉中和活性的三个表位和一个HVR1模拟表位串连。串联表位嵌合于HBV S基因的氨基端胞外区,形成嵌合基因MEpS;将基因克隆至真核表达载体pCI-neo中,构建重组质粒pCI-MEpS。将质粒转染至293T细胞中,间接免疫荧光及Western Blot检测嵌合基因的表达情况。[结果]重组质粒经双酶切证实构建正确;pCI-MEpS转染的293T细胞胞浆内可见较强的绿色荧光,Western Blot显示pCI-MEpS在相对分子量约38 kDa处可见蛋白条带。[结论]构建了HBV S抗原嵌合HCV串联多中和抗原表位的重组质粒,成功在293T细胞中表达,为制备嵌合HCV串联多中和抗原表位的HBV S抗原VLPs,研究嵌合VLPs免疫动物后产生的中和抗体的保护作用奠定了实验基础。[ Object] To construct a eukaryotic expression vector for chimeric gene of hepatitis C virus（HCV） series multi - neutralizing epitopes with hepatitis B virus（HBV） S antigen and express in 293T cells. [ Methods] Select several confirmed HCV cross - neutralizing epitopes were selected and tandem of these multi - epitopes was jointed to extracellular region of HBV S gene. Then the chimeric gene of HBV S antigen with HCV series multi - neutralizing epitopes were cloned into eukaryotie ex- pression vector pCI - neo. 293T cells were transfected with the recombinant plasmids pCI - MEpS and analyzed for expression of chimeric gene by iIFA and Western Blot. [ Results] Recombinant plasmids pCI - MEpS were constructed correctly confirmed by restriction analysis. Green fluorescence can be visualized intracytoplasm and 38 kDa protein can be detected by Western Blot. [ Conclusion] pCI - MEpS was successfully constructed and effectively expressed in 293T cells,which is the experimental foundation for further preparation of chimeric HBV S antigen VLPs and protection evaluation for the neutralizing antibody induced by VLPs after immunization.

关 键 词：丙型肝炎病毒 中和抗原表位 乙型肝炎病毒 S抗原 基因表达 

分 类 号：R373.21[医药卫生—病原生物学]