论文部分内容阅读
目的:对反式转录激活因子Tat第41位赖氨酸(K41)在HIV-1转录活化过程中的作用进行探讨，并对B、C亚型HIV-1中Tat K41的作用进行比较。方法:通过荧光素酶活性实验检测野生型Tat、突变型Tat K41A、Tat K41R对HIV-1转录活性的影响，并比较B、C亚型HIV-1中Tat K41作用的差异；通过免疫沉淀(immunoprecipitation, IP)实验检测B、C亚型HIV-1中Tat K41是否影响Tat与SIRT1的结合；通过Western blot检测细胞核内B、C亚型HIV-1的Tat K41是否影响p65 K310的乙酰化水平。结果:与B亚型HIV-1不同，C亚型HIV-1的Tat K41突变后显著降低荧光素酶活性，减弱C亚型Tat与SIRT1的结合，降低细胞核内p65 K310ac的水平。结论:C亚型Tat K41在HIV-1转录中发挥重要作用。“,”Objective:To investigate the effect of trans-activation of transcription (Tat) lysine 41 on HIV-1 transcription and compare the effect of HIV-1 subtypes B and C.Methods:The effects of wildtype Tat, Tat K41A and Tat K41R mutants on HIV-1 transcription were assessed by luciferase assay, and the differences of Tat K41 effect were compared in subtypes B and C viruses. Next, immunoprecipitation (IP) assay was used to detect whether Tat K41 of HIV-1 clades B and C affects the interaction between Tat and SIRT1. Finally, Western blot was used to detect whether K41 of Tat B and Tat C affects the acetylation level of p65 K310 in the nucleus.Results:Mutants of subtype C Tat K41 significantly inhibited Tat transactivation, reduced the interaction between SIRT1 and Tat C and decreased the level of p65 K310ac in the nucleus compared with wildtype of subtype C Tat, but there were no significant changes in subtype B Tat.Conclusions:Subtype C Tat K41 plays a vital role in HIV-1 transcription.