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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 a lentiviral vector expressing envelope protein ENV-1 and to infect human embryonic kidney cell line HEK293T to observe the expression of env gene in HEK293T. Methods: The full-length HIV-1 env gene was obtained by point mutation. The env gene was subcloned into the EcoRI and XhoⅠ sites of lentiviral vector pLVX-IRES-ZsGreen1 to construct recombinant plasmid pLV-env. The HEK293T cells were stained with HEK293T. The expression of target gene was detected by RT-PCR and Western blot. The expression of env gene was detected by laser scanning confocal microscopy. Results: The HIV-1 env gene was successfully obtained and the recombinant lentiviral plasmid pLV-env was constructed. The results of RT-PCR and Western blot showed that the exogenous gene was expressed and antigenic. At the same time, env gene was secreted into the cell membrane Surface film. CONCLUSION: The recombinant lentiviral plasmid containing env gene of HIV-1 envelope protein was successfully constructed and its expression confirmed. This laid the foundation for further packaging of lentivirus and construction of cell model and animal model.