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

韦三华; 任继鸿; 刘丽; 贾战生; 董轲; 林芳; 王希; 张利军; 刘昕阳; 张惠中

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

1. 第四军医大学唐都医院临床实验与检验输血科
2. 第四军医大学唐都医院全军感染病诊疗中心

基金项目:

国家自然科学基金项目(30700760)；国家“863”计划专题(2007AA02Z441)；

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中图分类号: R392
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出版历程
- 出版日期: 2011-01-20

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

Research funding:

摘要

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。
- 肝炎病毒属 /
- 表位 /
- 腺病毒,人
Abstract:
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.
- hepacivirus /
- epitopes /
- adenoviruses

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参考文献(12)

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