HIV NL4-3病毒株非感染性细胞-细胞融合模型的构建

Construction of non-infectious HIV cell-cell fusion model of the NL4-3strain

目的构建一种基于HIV实验室株NL4-3包膜蛋白env的非感染性、可视化细胞-细胞融合模型,用于检测HIV-1进入抑制剂的抑制活性。方法构建可同时表达HIV NL4-3 env及GFP的真核表达质粒pAAV-IRES-GFP-NL4-3,瞬时转染293T细胞,使293T细胞同时表达2种蛋白(293T/NL4-3/GFP)后,与靶细胞TZM-b1细胞共同培养,观察细胞融合情况及合胞体的形成。同时,在该模型上测试HIV进入抑制剂C34和T1144的抑制活性。结果293T/NL4-3/GFP和TZM-b1细胞混合2h后,可在荧光显微镜下发生明显融合;24h后可形成大面积融合。HIV-1进入抑制剂C34、T1144能够抑制细胞融合,其半抑制浓度(IC50)分别为(72±6.24)nmol/L和(15±6.94)nmol/L。结论成功构建了一种可在普通实验室完成的,可视化且无感染性的HIV-1进入抑制剂药物检测筛选模型。

NL4-3is a widely used HIV-1laboratory strain.In order to screen HIV-1entry inhibitors in ordinary laboratories,a non-infectious and visual cell-cell fusion model based on the NL4-3envelope protein env was constructed.In this study,a eukaryotic expression plasmid pAAV-IRES-GFP-NL4-3that can express GFP and HIV-1NL4-3 env at the same time was constructed,and transiently transfected into 293Tcells.The 293Tcells expressing both NL4-3 env and GFP(293T/NL4-3/GFP)were used to replace HIV-1NL4-3to fuse with target cells TZM-b1.After 293T/NL4-3/GFP was co-cultured with the TZM-b1,obvious cell fusion could be observed after 2hours under a fluorescence microscope,and large-area fusion and syncytia formation could be observed after 24hours.This study further tested the inhibitory activity of HIV-1entry inhibitors C34 and T1144on this model,and their IC50reached(72±6.24)nmol/L and(15±6.94)nmol/L,respectively.In conclusion,this cell-cell fusion detection model can be experimented on in ordinary laboratories,and is a visual and non-infectious model for HIV-1entry inhibitor detection and screening.