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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 a prokaryotic expression vector for HLA-A2-restricted multi-epitope gene of hepatitis C virus (HCV), express and purify it, and observe its immunogenicity. Methods: The HCV HLA-A2-restricted multi-epitope gene and human ubiquitin gene were synthesized respectively, and the fusion gene Ub-Mep was obtained after tandem. The prokaryotic expression plasmid pRSET-A was cloned into E.coli BL21 and induced by IPTG. The protein composition of the expressed protein was analyzed by layer scanning. The soluble protein was purified by Ni2 + -NTA gel electrophoresis and the fusion protein was purified by dialysis and concentration. The specificity and antigenicity of the purified protein were analyzed by Western blot. Immunogenicity of the purified protein was analyzed by immunocytochemistry. Results: The prokaryotic expression plasmid pRSET-Ub-Mep was successfully constructed. The target gene was expressed efficiently. The expressed product was mainly in the form of inclusion body. Purified protein could be obtained by Ni2 + -NTA purification. The purified protein had good antigen Sexual and immunogenicity. CONCLUSION: The HCV HLA-A2 restricted multi-epitope gene was successfully constructed and prokaryotic expressed. The expressed multi-epitope antigens have good immunogenicity, which is the cellular immune response induced by further HCV A2-restricted multi-epitopes Research laid the foundation.