HCV HLA-A2限制性复合多表位基因的构建、克隆表达及其免疫特性分析  

Genetic construction of HLA-A2 restricted multi-epitopes gene of hepatitis C virus,expression and purification in E.coli for antigenic analysis

作　　者：韦三华[1] 董轲[1] 林芳[1] 王希[1] 李斌[1] 沈建军[1] 张利军[1] 刘昕阳[1] 张惠中[1] 

机构地区：[1]西安第四军医大学唐都医院临床实验与检验输血科,西安710038

出　　处：《中国免疫学杂志》2010年第3期201-204,209,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(No.C0700760)

摘　　要：目的:构建丙型肝炎病毒(HCV)HLA-A2限制性复合多表位基因的原核表达载体,表达纯化,并观察其免疫原性。方法:分别合成HCV HLA-A2限制性多表位基因、人泛素基因,串联后得到融合基因Ub-Mep,克隆入原核表达质粒pRSET-A,转化E.coliBL21,IPTG诱导融合蛋白表达,薄层扫描分析表达蛋白组成;可溶性分析后用Ni2+-NTA凝胶亲和层析柱纯化、透析并浓缩融合蛋白;Western blot分析纯化蛋白的特异性和抗原性;免疫小鼠分析其免疫原性。结果:成功构建复合多表位抗原基因的原核表达质粒pRSET-Ub-Mep,目的基因可高效表达,表达产物主要以包涵体形式存在,Ni2+-NTA纯化可获得目的蛋白,纯化蛋白具有良好的抗原性和免疫原性。结论:成功构建HCV HLA-A2限制性复合多表位基因并进行原核表达,表达的多表位基因抗原具良好的免疫原性,为进一步的HCV A2限制性复合多表位诱导的细胞免疫应答研究奠定基础。Objective：To construct the recombinant prokaryotic plasmid to express HCV HLA-A2 restricted multi-CTL epitopes and to purify the fused protein for antigenic analysis. Methods：The human ubiquitin gene and multi-CTL epitopes gene was synthesized respectively, and digested by restrict enzyme before being cloned into pRSET-A. Then it was transformed into E. coli DH5α and the positive recombinant plasmid named pRSET-Ub-Mep was sequenced. Target protein was distinctly expressed after transformed into E. coli BL21 and induced with IPTG. Thus the protein was scanned and purified on Ni^2＋ -NTA column as well as Western blot performed after solubility analysis. Results： The recombinant plasroid pRSET-Ub-Mep was successfully constructed and it could efficiently express the target gene. Protein production was mainly in inclusion body and could be purified through Ni^2＋ -NTA colmnn. The purified protein kept the antigen activity. Conclusion：The gene encoding for HCV HLA-A2-restricted multi-CTL epitopes is efficiently expressed and the target protein is purified, which establishes a foundation of further research to evaluate the cellular immune response induced by the target gene.

关 键 词：丙型肝炎病毒(HCV) CTL 表位 表达 

分 类 号：R373.21[医药卫生—病原生物学]