Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081, China.
Biochem Biophys Res Commun. 2010 Jan 22;391(4):1780-4. doi: 10.1016/j.bbrc.2009.12.155. Epub 2010 Jan 5.
To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.
为了评估 HIV-1 包膜糖蛋白（Env）与靶细胞受体之间的相互作用，已经开发了各种细胞-细胞融合测定法。在本研究中，我们建立了一种新的融合系统。在该系统中，使用靶细胞中敏感报告基因萤火虫荧光素酶（FL）基因的表达来评估细胞融合事件。同时，使用组成型表达的海肾荧光素酶（RL）基因来监测效应细胞的数量和活力。FL 比其他已知的报告基因具有更宽的动态范围，而 RL 的引入使测定更加准确和可重复。该系统特别有利于研究潜在的进入影响因子，因为它能够排除由于人为细胞数量变化引起的假抑制或增强。作为一个案例研究，我们将该融合系统应用于观察丝氨酸蛋白酶凝血酶对 HIV Env 介导的细胞-细胞融合的影响，并发现凝血酶对两种 R5 嗜性 HIV 株的融合增强活性。
