丙型肝炎病毒NS3基因真核质粒的构建及其在人肝细胞中的诱导表达

目的：进行丙型肝炎病毒非结构蛋白3基因真核表达载体的构建，并分析其在体外培养的人肝细胞中的表达。方法：从含有丙肝病毒全长基因的重组质粒pBRTM／HCV1-3011表达载体中PCR扩增出HCVNS3基因片段，将其与表达载体pcDNA3．1（-）重组，得到重组的真核表达载体pcDNA3．1（-）／NS3。然后采用阳离子多聚体将其转染人肝细胞QSG7701，以免疫组织化学SP法及Western Blotting检测HCVNS3蛋白的表达。结果：所得到的NS3片段正确，序列正确，所构建的真核质粒成功转染QSG7701细胞并表达蛋白，表达的NS3蛋白相对分子质量为70000。结论：成功构建了丙型肝炎病毒非结构蛋白3基因的真核表达载体pcDNA3．1（-）／NS3，并且该载体在体外培养的人肝细胞中能有效表达特异性HCVNS3蛋白。

Construction of eukaryotic plasmid including HCV NS3 gene and expression of NS3 protein in human hepatocytes

Aim: To construct eukaryotic plasmid which includes HCV NS3 gene, and induce the expression of NS3 protein in human hepatocytes. Methods: HCV NS3 gene was cloned by using PCR from plasmid pBRTM/HCV1-3011 which includes the full length of HCV gene. We recombined the NS3 segment with expression plasmid pcDNA3.1(-) to construct eukaryotic plasmid pcDNA3.1(-)/NS3. Then plasmid pcDNA3.1(-)/NS3 was transfected into human hepatocytes by using poly-cation. The expression of NS3 protein was detected by immunohistochemical staining method and Western Blotting. Results: The cloned NS3 segment had correct length and sequence. The QSG7701 cells were successfully transfected and expressed NS3 protein. Conclusion: Eukaryotic expression plasmid pcDNA3.1(-)/NS3 incluing HCV NS3 gene was successfully constructed and expressed in human hepatocytes.