一种基于EGFP的、安全的抗HIV药物评价系统

A Safe Anti-HIV Drug Evaluation System Based on EGFP Expression

目的:建立基于EGFP的、安全的抗人免疫缺陷病毒(HIV)药物评价系统。方法:用增强型绿色荧光蛋白(EGFP)基因替代HIV感染性克隆质粒pUC18-HIV-NL4-3中的部分包膜基因(env),构建重组假病毒质粒pUC18-NL4-3-EGFP,将其与水疱性口炎病毒糖蛋白(VSV-G)真核表达载体共转染人胚肾293FT细胞,观察绿色荧光蛋白的表达,同时用该细胞培养上清进一步感染其他293FT细胞培养物。为了检验该假病毒系统能否用于抗病毒药物的评价,在假病毒复制和感染过程中加入不同浓度的抗HIV药物AZT(Zidovudine),采用荧光显微镜检测和流式细胞仪定量检测,分析AZT对假病毒的抑制作用。结果:假病毒质粒pUC18-NL4-3-EGFP能够在转染细胞和再感染细胞中有效地表达绿色荧光蛋白基因,不同浓度的AZT能以剂量依赖方式抑制假病毒的感染和报告基因的表达。结论:建立了一种基于EGFP表达和检测的、安全的HIV假病毒复制和感染系统,该系统可以用于抗HIV药物的筛选和评价。

Objective： To develop a safe cell model based on the enhanced green fluorescent protein（EGFP） for anti-HIV drug evaluation. Methods： The infectious HIV-1 clone pUC18-HIV-NIA-3 was reconstructed to be the pseudovirus plasmid pUC18-NIA-3-EGFP, in which the EGFP gene replaced the HIV-1 env gene. The human embryonic kidney cell line 293FT was transfected with pUC18-NL4-3-EGFP and vesicular stomatitis virus glycoprotein（VSV-G） producing plasmids. The reporter gene expression in the eo-transfected cells was examined by detecting green fluorescence and the conditioned medium containing the pseudovirus was used to infect naive 293FF cells. To assess whether this pseudovirus production and infection system can be used as an anti-HIV drug evaluation cell model or not, the AIDS drug AZT（Zidovudine） was applied into the cell culture at different concentration, and the inhibitory effect of AZT on pseudovirus infection was analyzed via detecting EGFP expression with fluorescence microscope and fluorescent activated cell sorter. Results： The pseudovirus plasmids and VSV-G expressing plasmids co-transfected 293FT cells and the conditioned medium infected naive 293FF cells both efficiently produced EGFP. AZT inhibited the EGFP expression in the conditioned medium infected 293FF cells in a dose dependant manner. Conclusion： A safe system based on EGFP expression and detection and representing HIV pseudovirus replication and secretion was successfully established, which has the potential to be used for anti-HIV drug evaluation.