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目的制备携带增强绿色荧光蛋白(EGFP)基因和ENV包膜蛋白的HIV-1 B亚型假病毒用于感染研究,并建立可行的鉴定方法。方法双质粒共转染HEK293T细胞后收获病毒上清,TRIzol法提取病毒基因组并采用RT-PCR进行报告基因扩增,Western印迹和ELISA法检测病毒P24抗原。假病毒感染HIV-1允许细胞,进行报告基因检测、病毒滴度测定以及单轮感染活性实验研究。结果与结论建立了HIV-1假病毒制备与鉴定方法,制备获得了B型HIV-1假病毒,经鉴定具备感染SupT1和TZM-bl细胞能力,为HIV-1与宿主细胞相互作用研究奠定了基础。
Objective To prepare HIV-1 B pseudotyped virus carrying enhanced green fluorescent protein (EGFP) gene and ENV envelope protein for infection research and to establish a feasible identification method. Methods Double plasmids were co-transfected into HEK293T cells and the supernatant was harvested. The viral genome was extracted by TRIzol method and amplified by RT-PCR. Western blot and ELISA were used to detect the virus P24 antigen. Pseudovirus-infected HIV-1 cells were allowed to undergo reporter assay, virus titer assay, and single-pass infection. RESULTS AND CONCLUSION: The HIV-1 pseudovirus was prepared and identified, and the HIV-1 pseudotype B virus was obtained. The virus was identified as having the ability to infect SupT1 and TZM-bl cells, which laid the foundation for the study on the interaction between HIV-1 and host cells basis.