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目的验证HIV-1Tat对有丝分裂着丝点关联驱动蛋白MCAK基因表达的调节作用并探讨其分子机制。方法利用表达Tat的人横纹肌肉瘤细胞(TE671)模型及原核表达纯化的Tat蛋白,通过Northern印迹法和蛋白印迹技术验证HIV-1 Tat对MCAK基因表达的抑制作用。PCR扩增MCAK基因各启动区片段,与荧光素酶报告质粒相连接,并转染到TE671细胞,通过荧光素活性分析法检测DNA片段启动活性,确定MCAK基因的活性启动区域。进一步通过HIV-1Tat与MCAK启动区作用,分析Tat对启动子活性的调控作用。结果Northern印迹和蛋白印迹结果证实Tat蛋白对MCAK转录、翻译水平的表达都有强烈的抑制作用;荧光报告质粒检测系统分析表明MCAK的核心启动区存在于-399～+1bp区域,且其启动子活性在Tat存在的情况下受到明显的抑制。结论HIV-1 Tat能作用于MCAK基因启动区-399～+1bp区域,导致MCAK启动子活性降低,从而抑制MCAK基因的表达。
Objective To investigate the regulatory effect of HIV-1Tat on the expression of MCAK gene in mitotic centromere and its molecular mechanism. Methods Tat Tat-positive human rhabdomyosarcoma cells (TE671) model and purified Tat protein were expressed in vitro. The inhibitory effect of HIV-1 Tat on MCAK gene expression was verified by Northern blotting and Western blotting. The promoter region of MCAK gene was amplified by PCR, ligated with luciferase reporter plasmid and transfected into TE671 cells. The promoter activity of DNA fragment was detected by luciferase activity assay, and the activation region of MCAK gene was identified. Further, through the action of HIV-1Tat and MCAK promoter, the regulatory effect of Tat on promoter activity was analyzed. Results Northern blotting and Western blotting results showed that Tat protein strongly inhibited the transcription and translation of MCAK. The results of fluorescent reporter plasmid assay showed that the core promoter region of MCAK existed in the -399 ~ +1 bp region, and its promoter Activity was significantly inhibited in the presence of Tat. Conclusion HIV-1 Tat can act on the -399 ~ + 1bp region of MCAK gene promoter, leading to the decrease of MCAK promoter activity and the inhibition of MCAK gene expression.