丙型肝炎病毒NS5A对p53活性调控机制研究  

Regulation mechanism of HCV NS5A on p53 protein transactivity

作　　者：龚国忠[1] 蒋永芳[1] 朱映华[1] 苏先狮[1] 

机构地区：[1]中南大学湘雅二医院肝病研究中心,长沙410011

出　　处：《中华肝脏病杂志》2003年第3期162-165,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金(3967067)

摘　　要：目的研究HCV NS5A对抑癌蛋白p5 3活性的抑制作用及其作用机制.方法采用荧光素酶报告基因系统观察pwwp-luc,pwwp-mut-luc,pC53-NS3及pCNS5A分别转染或共同转染HepG2、Huh7细胞的荧光素酶活性.应用凝胶滞留试验观察HCV NS5A是否影响p5 3与其特异DNA序列的结合.结果内源性p5 3能激活p21启动子转录功能,使荧光素酶活性明显增加(3.49×105与0.60×105,t=5.92,P＜0.01).外源性p 5 3也能激活p21启动子转录功能,荧光素酶活性为5.6 3×105,与对照组(0.47×105)比较差异具有显著性(t=10.12,P＜0.01).HCV NS5A能抑制内源性和外源性p5 3对p21启动子的激活作用,并呈剂量依赖性(F≥20.71,P＜0.01).凝胶滞留试验显示HCV NS5A能阻碍p5 3与其特异DNA序列的结合.结论HCV NS5A能抑制p5 3的反式激活功能使其目的基因p21启动子的转录功能下降,其机制是HCV NS5A能阻碍p 5 3与其特异DNA序列的结合.Objective To study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism. Methods Luciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence. Results Endogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49×10~5 vs 0.60×10~5, t = 5.92, P < 0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47×10~5), the relative luciferase activity increased (5.63×10~5) obviously (t = 10.12, P < 0.01) . HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F≥20.71, P < 0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased. Conclusion HCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

关 键 词：丙型肝炎病毒 NS5A P53蛋白 荧光素酶活性 

分 类 号：R373.2[医药卫生—病原生物学]