靶向包膜蛋白的HIV进入抑制剂非感染性定量筛选方法的研究

A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins

目的 HIV包膜蛋白能介导细胞与细胞之间的融合,本项目旨在建立一种靶向包膜蛋白的HIV进入抑制剂非感染性定量筛选方法。方法将表达包膜蛋白gp120/gp41和Tat等HIV蛋白的HL2/3细胞与表达CD4和由HIV长末端重复片段(LTR)启动的β-半乳糖苷酶的HeLa-CD4-LTR-β-gal细胞混合,采用氯酚红β半乳糖苷(CPRG)为显色底物检测β-半乳糖苷酶的表达。用特异性的HIV进入抑制剂对检测方法进行验证。结果 HL2/3细胞与HeLa-CD4-LTR-β-gal细胞共育不能形成合胞体,但二者能发生膜融合,导致效应细胞表达的Tat蛋白与靶细胞中LTR结合,启动β-半乳糖苷酶的表达。CPRG方法能灵敏地检测细胞中β-半乳糖苷酶的含量。特异性的HIV进入抑制剂能够抑制β-半乳糖苷酶的表达,且具有剂量依赖性。结论这一非感染性定量的方法能客观定量地筛选作用于HIV进入阶段的抗HIV药物,且操作方便、廉价、无感染性,用于天然和合成来源的小分子抗艾滋病药物的筛选。

Objective To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors. Methods HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-β-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene β-galactosidase. The enzyme activities of β-galactosidase were detected by a chromogenic substrate, chlorophenol red-β-galactopyranoside （CPRG）. Specific HIV entry inhibitors were used to validate the established detecting method. Results No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-β-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-β-gal cells and trigger the expression of β-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of β-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of β-galactosidase in a dose-dependent manner. Conclusion We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.