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

摘要

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EcoR Ⅰ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.%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 pCEM-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 EcoR I , 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 transcription 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 foundation for the research of the host range of PERV.