丙型肝炎病毒整个开放阅读框序列的克隆及各组分蛋白的表达

Cloning of hepatitis C virus open reading frame sequence and non structural protein expressions of the virus

目的在痘苗系统中表达丙型肝炎病毒(HCV)各组分蛋白。方法将编码HCV大前体蛋白的开放阅读框(ORF)序列克隆至痘苗启动子后,构建重组质粒pVHCV。重组质粒转染BHK21细胞后,再用痘苗病毒vTF7-3株感染,培养48 h后收集细胞。Western blotting和免疫荧光技术鉴定HCV非结构蛋白NS3和NS5a的表达情况。结果Western blotting结果表明,在细胞中检测到相对分子质量为70 000 (NS3)和58 000 (Ns5a)的两条带;免疫荧光试验结果表明,无论采用抗NS3 单抗还是抗NS5a 单抗,大部分转染质粒细胞的细胞质中都具有较强的荧光反应。结论HCV非结构蛋白NS3和NS5a在BHK21细胞中得到表达。

Objective To induce the expressions of the constituent proteins of hepatitis C virus (HCV) using a vaccinia virus expression system. Methods The open reading frame (ORF) sequence encoding HCV large polyprotein precursor was cloned into a vaccinia virus promoter to construct the recombinant plasmid pVHCV, which was subsequently used, along with a control plasmid (pSC59) in a parallel experiment, to transfect BHK21 cells via Lipofectamine 2000 reagent, followed by infection of the cells with vTF7-3 vaccinia viruses. After a 48-h culture, the expressions of HCV nonstructural proteins NS 3 and NS 5a in the cells were detected using Western blotting and immunofluorescence assay. Results Western blotting analysis presented the nonstructural proteins NS 3 and NS 5a detected in pVHCV-transfected BKH21 cells as two bands with molecular weights of 70 000 and 56 000, respectively. In the immunofluorescence assay, intense granular fluorescence signal was detected in the cytoplasm of pVHCV-transfected cells, irrespective of the use of either NS 3 - or NS 5a - specific mouse monoclonal antibody. Conclusion HCV nonstructural proteins NS 3 and NS 5a can be expressed in BKH21 cells.