【摘要】 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。更多还原

【Abstract】 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.更多还原