人CD81真核表达载体的构建及在COS-7细胞中的表达

Construction of human CD81 eukaryotic expression vector and expression of the gene segment in COS-7 cell line

目的:构建含人CD81基因的真核表达载体,探讨CD81在COS-7细胞的表达,为研究丙型肝炎病毒(HCV)与CD81 相互作用奠定基础. 方法:从我们构建的含人CD81全编码基因载体pMD18-T- CD81,应用双酶切回收基因片段,定向克隆入真核表达载体pVAXl;通过脂质体介导的基因转染技术将pVAXl- CD81和空载体转入COS-7细胞;应用抗CD81单克隆抗体通过间接免疫荧光法检测COS-7细胞的表达产物. 结果:重组的含CD81基因的真核表达载体pVAXl-CD81 经酶切和PCR鉴定分析正确,并证明在COS-7细胞表面有效地表达CD81蛋白. 结论:成功构建含CD81全编码基因的真核表达载体pVAXl-CD81,并在COS-7细胞表面有效表达CD81分子, 该转染细胞可作为研究CD81在HCV感染中的作用提供模型.

AIM: To construct a human CD81 eukaryotic expression vector and to analyze the expression of CD81 in COS-7 cells. METHODS: CD81 gene from the pMD18-T-CD81 vector with double-enzyme digestion was cloned into the pVAX1 eukaryotic expression vector, named pVAX1-CD81. The recombinant vector pVAXl-CD81 and pVAX1 as controls were transfected into COS-7 cells by lipofectamine, and the transient expression product on the transfected cells was analyzed with anti-CD81 monoclonal antibody by indirect immunofluorescence assay (IFA). RESULTS: The identification of the eukaryotic expression vector pVAXl-CD81 by PCR and restriction enzyme analysis showed that CD81 gene was rightly inserted into the vector; and the product of the CD81 gene was successfully expressed on surface of COS-7 cells. CONCLUSION: The eukaryotic expression vector with CD81 gene is constructed and efficiently expressed in COS-7 cells. The results indicate that the transfected CD81 cells will need to further studies on the roles of CD81 in the process of HCV infection and entrance to cells.