丙型肝炎病毒HLA-A2限制性复合多表位基因重组腺病毒的包装、筛选及鉴定

Packaging,selection and identification of recombinant adenoviruses carrying HCV HLA-A2 restricted compound multi-epitopes antigen

目的构建丙型肝炎病毒(HCV)HLA-A2限制性复合多表位基因的重组转移质粒pAdtrack-CMV-Ub-Mep,转染HEK293细胞,包装重组腺病毒rAd-Ub-Mep。方法复合表位Ub-Mep基因克隆到pAdTrack-CMV穿梭质粒,得到重组的穿梭质粒pAdtrack-CMV-Ub-Mep,将穿梭质粒pAdtrack-CMV-Ub-Mep和腺病毒骨架质粒pAdEasy1共转染HEK293细胞,产生重组腺病毒rAd-Ub-Mep,通过检测绿色荧光蛋白(GFP)的表达和PCR扩增目的基因的方法鉴定重组腺病毒,并测定重组腺病毒滴度。结果经酶切鉴定、PCR鉴定和荧光分析,均证实得到重组的腺病毒。测定病毒滴度为1.2×1011cfu/L。结论成功包装出重组腺病毒rAd-Ub-Mep,构建了HCV HLA-A2限制性多表位基因的重组腺病毒疫苗,为进一步研究HLA-A2限制性复合多表位基因诱导的细胞免疫应答奠定了基础。

Objective To construct recombinant shuttle plasmid pAdtrack-CMV-Ub-Mep carrying HCV HLA-A2 restricted compound multi-epitopes gene,and transfect to HEK293 cells,to obtain recombinant adenoviruses rAd-Ub-Mep.Methods The compound antigen gene Ub-Mep was cloned into shuttle plasmid pAdTrack-CMV for pAdtrack-CMV-Ub-Mep.The recombinant adenoviruses rAd-Ub-Mep was packed in HEK293 cells by co-transfection with pAdtrack-CMV-Ub-Mep and frame plasmid pAdEasy1.The recombinant adenoviruses rAd-Ub-Mep was identified by PCR and expression of GFP,and the virus titer was determined by TCID50.Results The recombinant adenoviruses rAd-Ub-Mep was confirmed by enzyme cutting,PCR and fluorometric analysis.The virus titer was 1.2×1011cfu/L.Conclusion Success to construct recombinant adenoviruses vaccine,is the base for the study of cellular immunity induced by HCV HLA-A2 restricted compound multi-epitopes gene.