Genetic construction of HLA-A2 restricted multi-epitopes gene of hepatitis C virus,expression and purification in E.coli for antigenic analysis / 中国免疫学杂志

ABSTRACT

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 plasmid 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 column.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.