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

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

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HCV NS3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HCV NS3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70 000。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HCV NS3蛋白。

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. Tnen 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 Westem Blotting. Results： Tne clonedNS3 segmnent had correct length and sequence. The QSG7701 cells were successfully transfeeted and expressed NS3 protein. Conclusion： Eukaryotic expression plasmid peDNA3.1 （ - ）/ NS3 incluing HCV NS3 gene was successfully constructed and expressed in human hepatoeytes.