HIV-1包膜蛋白ENV慢病毒载体的构建及表达

Lentivirus construction and expression in outer membrane protein ENV of HIV-1

目的:构建表达HIV-1包膜蛋白ENV的慢病毒载体,感染人胚肾细胞HEK293T,观察env基因在HEK293T中的表达。方法:通过点突变获得HIV-1 env完整基因,将env基因亚克隆至慢病毒穿梭载体pLVX-IRES-ZsGreen1的EcoRⅠ、XhoⅠ位点,构建重组质粒pLV-env,采用脂质体转染法转染HEK293T,经RT-PCR、Western blot检测目的基因表达,同时利用激光共聚焦技术对env基因的表达进行了定位。结果:成功获得了HIV-1 env基因,构建了重组慢病毒质粒pLV-env,RT-PCR、Western blot检测均表明外源基因能够表达,并具有抗原性,同时env基因表达后可以分泌到细胞膜表面膜上。结论:成功构建了含有HIV-1包膜蛋白env基因的重组慢病毒质粒,并验证了其表达,为下一步慢病毒的包装以及细胞模型和动物模型的构建奠定了基础。

Objective：To construct lentiviral vector including HIV-1 envelope protein ENV and observe the env gene expression in the infected human embryonic kidney cells HEK293T.Methods：A complete HIV-1 env gene was obtained by point mutations.The env gene was subcloned to the EcoR Ⅰand Xho Ⅰ sites of pLVX-IRES-ZsGreen1,the recombinant plasmid pLV-env was constructed and transfected into HEK293T by the lipofectamine transfection method.The target gene expression was detected by RT-PCR and Western blot.Simultaneously,the env gene expression was located with the technology of laser scanning confocal microscope.Results：HIV-1 env gene was obtained;the recombinant lentiviral plasmid pLV-env was constructed;the exogenous genes was expressed by detection of RT-PCR and Western blot.Meanwhile,the env gene could be secreted onto the cell membrane after expression.Conclusion：The recombinant lentiviral plasmids including HIV-1 envelope protein env gene was constructed successfully,and its expression was verified.It laid foundation for the next lentiviral packaging and the construction of cell and animal models.