基于包膜蛋白和Tat蛋白筛选HIV-1细胞融合抑制剂的高效方法

摘要

旨在建立一个细胞-细胞融合系统,高效筛选对HIV-1病毒细胞-细胞间传播有抑制作用的药物.构建了pEGFP-Tat质粒,将pEGFP-Tat质粒和HIV-1包膜质粒共转染HEK-293T细胞,成为表达Tat蛋白和包膜蛋白的效应细胞,然后与表达CD4及辅助受体和β-半乳糖苷酶、荧光素酶双报告基因的靶细胞TZM-bl融合,建立了细胞-细胞融合系统,并进行条件优化,确定了最佳的融合体系.用阳性融合抑制剂maraviroc以及没有融合抑制作用的AZT和raltegravir作用于该体系,证明该系统可以特异性有效筛选具有细胞融合抑制作用的药物.用该系统测试了8个样本,发现两种样品对融合有一定的抑制作用.该方法背景值低,特异性强,可用来高效筛选具有切断HIV-1病毒细胞-细胞间传播作用的抗病毒药物.%An HIV-1 cell-cell fusion system was developed to screen HIV-1 entry inhibitors that block cell-cell fusion.In this system,the pEGFP-Tat plasmid was constructed and cotransfected into effector cells (HEK-293T) with HIV-1 envelope plasmid.TZM-bl cell,a genetically engineered cell line that expresses CD4,CXCR4,CCR5 as well as Tat-inducible β-galactosidase and luciferase reporter gene,was used as target cell.Thus,the co-culture of target cells and effector cells allows the cell fusion via Env and the activity of the fusion inhibitor can be quantified by measuring the reporter protein expression.The experimental parameters were optimized and 11 anti-HIV-1 agents including CCR5 antagonist maraviroc,reverse transcription inhibitor zidovudine (AZT) and integrase inhibitor raltegravir were tested.The result showed that the system exhibited high specificity and sensitivity.Two of eight tested anti-HIV-1 agents were found to block the cell-cell fusion.The system is suitable for efficient screening of HIV-1 cell-cell fusion inhibitors.