Li Min-min, Xia Cheng-lai, Mao Qin-chao, Jiang Shi-bo, Liu Shu-wen
Center of Clinical Medicinal Laboratory, First Affiliated Hospital of Jinan University, Guangzhou 510630, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 May;30(5):941-4.
To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.
HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.
No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-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-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.
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.
开发一种用于定量检测HIV诱导的细胞-细胞融合的客观生物测定法，以筛选HIV进入抑制剂。
将表达HIV包膜蛋白gp120/gp41、Tat及其他HIV蛋白的HL2/3细胞与表达CD4受体和HIV LTR触发报告基因β-半乳糖苷酶的HeLa-CD4-LTR-β-半乳糖苷酶细胞共培养。用显色底物氯酚红-β-吡喃半乳糖苷（CPRG）检测β-半乳糖苷酶的酶活性。使用特异性HIV进入抑制剂验证所建立的检测方法。
HL2/3细胞与HeLa-CD4-LTR-β-半乳糖苷酶细胞混合未形成多核巨细胞。然而，细胞膜可发生融合，HL2/3细胞表达的Tat可与HeLa-CD4-LTR-β-半乳糖苷酶细胞上的HIV LTR结合并触发β-半乳糖苷酶的表达。CPRG可对β-半乳糖苷酶的活性进行定量和灵敏检测。进一步研究表明，HIV进入抑制剂可呈剂量依赖性抑制β-半乳糖苷酶的活性。
我们开发了一种简单、廉价、客观且定量的非感染性细胞-细胞融合生物测定法，可用于从天然和合成化合物文库中筛选针对病毒进入的抗HIV药物。
