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目的研究HIV-1 Tat对MCAK启动子活性及基因表达的抑制作用,并鉴定Tat与MCAK启动子的作用位点。方法利用原核表达的Tat蛋白处理A549细胞,通过Western印迹技术验证HIV-1Tat对MCAK基因表达的抑制作用;构建MCAK启动子-荧光素酶载体,转染A549细胞,通过荧光素酶活性分析Tat对启动子活性的调控作用;运用染色质免疫沉淀法(CHIP)分析Tat与MCAK启动区的相互作用,并进一步通过凝胶阻滞实验(EMSA)确定Tat与MCAK启动区的结合位点。结果 Western印迹实验证实Tat对MCAK表达具有强烈的抑制作用;荧光报告质粒检测系统分析表明在Tat存在的情况下,MCAK的核心启动子活性受到明显的抑制,其效应具有明显的剂量依赖性;CHIP和EMSA实验确定Tat蛋白与MCAK基因启动子区的直接相互作用,MCAK基因-337～-232区域存在Tat蛋白的结合位点。结论 HIV-1 Tat对MCAK基因表达具有强烈的抑制作用,并与Tat蛋白结合于MCAK启动区-337～-232区域,从而导致MCAK启动子活性降低有关。
Objective To study the inhibitory effect of HIV-1 Tat on MCAK promoter activity and gene expression, and to identify the site of action of Tat and MCAK promoter. Methods The prokaryotic expression of Tat protein was used to treat A549 cells. The inhibitory effect of HIV-1Tat on MCAK gene expression was verified by Western blotting. The MCAK promoter-luciferase vector was transfected into A549 cells and analyzed by luciferase activity assay. The interaction between Tat and MCAK promoter region was analyzed by chromatin immunoprecipitation (CHIP), and the binding site between Tat and MCAK promoter region was further determined by gel retardation assay (EMSA). Results Western blotting showed that Tat had a strong inhibitory effect on MCAK expression. Fluorescent reporter plasmid detection system analysis showed that in the presence of Tat, the core promoter activity of MCAK was significantly inhibited and the effect was significantly dose-dependent; CHIP And EMSA experiments to determine the direct interaction between Tat protein and MCAK gene promoter region, Tat protein binding sites exist in -337 ~ -232 region of MCAK gene. Conclusion HIV-1 Tat strongly inhibits the expression of MCAK gene and binds with Tat protein in the -337 ~ -232 region of MCAK promoter, resulting in the decrease of MCAK promoter activity.