CAJ | 학술논문

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.coli BL21,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Ni~(2+)-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,Ni~(2+)-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.
목적:구건병형간염병독(HCV)HLA-A2한제성복합다표위기인적원핵표체재체,표체순화,병관찰기면역원성.방법:분별합성HCV HLA-A2한제성다표위기인、인범소기인,천련후득도융합기인Ub-Mep,극륭입원핵표체질립pRSET-A,전화E.coli BL21,IPTG유도융합단백표체,박층소묘분석표체단백조성;가용성분석후용Ni~(2+)-NTA응효친화층석주순화、투석병농축융합단백;Western blot분석순화단백적특이성화항원성;면역소서분석기면역원성.결과:성공구건복합다표위항원기인적원핵표체질립pRSET-Ub-Mep,목적기인가고효표체,표체산물주요이포함체형식존재,Ni~(2+)-NTA 순화가획득목적단백,순화단백구유량호적항원성화면역원성.결론:성공구건HCV HLA-A2한제성복합다표위기인병진행원핵표체,표체적다표위기인항원구량호적면역원성,위진일보적HCV A2한제성복합다표위유도적세포면역응답연구전정기출.
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.