HIV-1 Tat通过抑制启动子活性调控MCAK基因表达

HIV-1 Tat down-regulates expression of mitotic centromere-associated kinesin through inhibiting promoter activity

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-1 Tat on mitotic centromere-associated kinesin（MCAK） expression and the related mechanism.Methods Tat-expression TE671 cell model and purified prokaryotically expressed Tat protein were used to verify the down-regulated expression of MCAK using Northern blotting and Western blotting analysis.Various DNA fragments in the promoter region of the MCAK gene were amplified with PCR,linked to the luciferase reporter plasmid,and then transferred into TE671 cells.Luciferase activity analysis was performed to measure the promoter activity of various DNA fragments,so as to determine the active promoter region of MCAK gene.Moreover,HIV-1 Tat protein was co-incubated with TE671 cells,and the promoter activity was detected to analyze the modulating effect of Tat on promoter activity in vitro.Results The results of Northern blotting and Western blotting analysis indicated that the mRNA and protein levels of MCAK were strongly decreased by Tat protein.The core region of MCAK promoter was located in-399 ～ ＋ 1 bp region,and the promoter activity was strikingly inhibited by Tat protein.Conclusion HIV-1 Tat has a marked inhibitory effect on MCAK expression,which might be related to the decreased promoter activity caused by Tat protein.