摘要
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。
This study aimed to investigate the inhibitory effect of Tat on the promoter activity of MCAK gene and investigate the interactive sites.Tat protein was expressed using prokaryotic expression system and used to treat to A549 cells.Then we used Western blotting to verify the effects of Tat on the expression of MCAK.Furthermore,we employed fluorescence reporting plasmid system to clarify the effect of Tat on MCAK promoter activity.And,the interaction between the Tat protein and the MCAK promoter region was identified with Chromatin immunoprecipitation（CHIP） test and EMSA assay.We found the protein level of MCAK was strongly decreased in the presence of Tat protein;the MCAK promoter activity was strikingly inhibited by Tat protein in a dose-dependent manner.CHIP and EMSA results showed that there existed direct interaction between Tat and MCAK promoter region,and the-337^-232 bp region may be a potential binding site.These results indicate that HIV-1 Tat has a markedly inhibitory effect on MCAK expression,which is related to the decreased promoter activity caused by Tat protein binding to the-337^-232 region of MCAK gene.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2011年第3期211-214,共4页
Immunological Journal
基金
国际原子能机构合作项目(12510/RO)
国家自然科学基金项目(30400120)
四川省卫生厅科研课题(090175)
