【摘要】 目的:构建丙型肝炎病毒(HCV)非结构基因NS3的真核表达载体,并分析其在HeLa细胞中的表达。方法:以HCV全长cDNA质粒为模板进行PCR扩增,获得全长NS3基因,将其插入克隆载体pMD18-T中,鉴定后与真核表达载体pcDNA3.1(+)重组,用脂质体介导法转染HeLa细胞,间接免疫荧光法及Western blot鉴定转染后HCVNS3蛋白的表达。结果:PCR扩增的NS3序列正确,构建的真核表达载体成功转染HeLa细胞,表达的NS3蛋白相对分子量为70kD。结论:成功构建了真核表达载体pcDNA3.1(+)/NS3,并在HeLa细胞中获得表达。更多还原

【Abstract】 Objects:To construct eukaryotic plasmid which includes HCV NS3 gene,and induce the expression of NS3 protein in cells of HeLa.Methods:HCV NS3 gene was amplified with PCR and inserted into the pMD 18-T vector.We recombined the NS3 segment with expression plasmid pcDNA3.1(+) to construct eukaryotic plasmid pcDNA3.1(+)/NS3.Then the plasmid pcDNA3.1(+)/NS3 was transfected into cells of HeLa with Lipofectamine 2000 transfection reagent.The expression of NS3 protein was detected by Immunofluorescent staining method and Western Blot.Results:The cloned NS3 segment had correct length and sequence.The cells of HeLa were successfully transfected and expressed NS3 protein.Conclusion:Eukaryotic expression plasmid pcDNA3.1(+)/NS3 including HCV NS3 gene is successfully constructed and expressed in cells of HeLa.更多还原