猪内源性反转录病毒囊膜基因env真核表达质粒的构建及鉴定

目的:构建猪内源性反转录病毒(PERV)囊膜基因env的真核表达质粒pHCMV-env并加以鉴定,为研究PERV的细胞嗜性和宿主范围奠定基础。方法:用RT-PCR方法扩增五指山猪来源PERV的env基因,将其插入pGEM-T easy载体中,构建重组质粒pGEM-T-env,酶切鉴定正确后,将pGEM-T-env与pHCMV-VSV-G表达质粒同时经EcoRⅠ酶切消化后连接,构建重组表达质粒pHCMV-env,并进行酶切、测序鉴定;将鉴定正确的质粒pHCMV-env转染HEK293T细胞,采用PCR、RT-PCR检测转染后env基因的整合和转录情况。结果:扩增得到五指山猪来源PERV的env基因,并构建了pHCMV-env真核表达质粒,转染HEK293T细胞系后,该细胞系中有目的基因的整合和转录。结论:构建了真核表达质粒pHCMV-env,并且在HEK293T细胞中能够整合并转录,为研究PERV的细胞嗜性和宿主范围奠定了基础。

Construction and Identification of Eukaryotic Expression Plasmid of Porcine Endogenous Retrovirus env Gene

Objective： To construct and identify an eukaryotic expression plasmid of porcine endogenous retrovirus（PERV） env gene.Methods： The env gene of PERV was amplified by RT-PCR from total RNA of mononuclear cells in peripheral blood of Wuzhishan miniatuer pigs and inserted into pGEM-T easy vector to construct a recom-binant plasmid pGEM-T-env,which was identified by enzyme digesting.The eukaryotic expression plasmid pHCMV-VSV-G and pGEM-T-env were digested with EcoRⅠ,and then linked together to construct the recombi-nant eukaryotic expression plasmid pHCMV-env,which was identified by enzyme digesting and sequencing analysis.The plasmid pHCMV-env was extracted and transfected into the HEK293T cells,while its integration and transcrip-tion levels were identified by PCR and RT-PCR.Results Conclusion： The recombinant eukaryotic expression plasmid pHCMV-env has been constructed and transfected into HEK293T cells successfully,which has laid a foun-dation for the research of the host range of PERV.