腺相关病毒Rep78蛋白对乙型肝炎病毒C基因的抑制作用  

Binding and inhibition of adeno-associated virus Rep78 protein with hepatitis B virus C promoter

作　　者：阎钟钰[1] 丛敏[1] 王萍[1] 唐淑珍[1] 王宝恩[1] 贾继东[1] 刘勇[2] 尤红[1] 

机构地区：[1]首都医科大学附属北京友谊医院肝病中心,北京100050 [2]美国阿肯色州立医科大学基因治疗中心

出　　处：《中华肝脏病杂志》2005年第3期187-189,共3页Chinese Journal of Hepatology

基　　金：北京市科技新星计划(2004B32)

摘　　要：目的 研究腺相关病毒(AAV)Rep78蛋白对乙型肝炎病毒C基因(HBV-C)的抑制作用及机制。 方法 以pEOB6(含有HBV全长的质粒)为模板,聚合酶链反应法分别扩增出HBV-C启动子基因及含有HBV-C启动子的HBV-C基因。利用pMAL-Rep78体外扩增得到Rep78蛋白。以凝胶电泳阻滞实验检测Rep78与HBV-C启动子的结合,体外转录实验观察Rep78对HBV-C转录的影响。 结果 聚合酶链反应法扩增出了的HBV-C启动子基因(182 bp)及含有HBV-C启动子的HBV-C基因(789 bp)。凝胶电泳阻滞实验结果显示Rep 78与HBV-C启动子结合,呈剂量依赖性并可以被Rep 78抗体阻滞。体外转录实验显示Rep78明显抑制HBV-C的转录。 结论 AAVRep78可以通过与HBV-C启动子结合抑制HBV-C的转录。Objective Adeno-associated virus (AAV) Rep78 is known for its inhibitory effects on replication of several viruses and oncogenes transformations. The study was to investigate the effect of Rep78 on hepatitis B virus C (HBV-C) gene and the mechanism of it. Methods HBV-C promoter and HBV-C gene with its promoter were amplified by PCR and labeled with 32P-ATP. Electrophoretic mobility shift assay (EMSA) and in vitro transcription were utilized to detect the binding of MBP-Rep78 with HBV-C promoter and the transcription of HBV-C gene. Results EMSA showed that by increasing the amount of Rep78 protein from 0.1 μg to 1.0 μg, the binding bands got stronger in a dose-dependent manner. In addition, Rep78 antibody was used to certify the specificity of this binding. The compound of Rep78, Rep78 antibody and HBV-C promoter were seen as super shift bands in EMSA. Meanwhile, HBV-C gene transcription was 'gnificantly inhibited by in vitro transcription which meant that Rep78 could not only bind with HBV-C promoter, but ilso could inhibit the transcription of HBV-C gene. Conclusion AAV Rep78 could inhibit the transcription of HBV-C gene through its binding with HBV-C promoter.

关 键 词：HBV C基因 腺相关病毒 乙型肝炎病毒 阻滞 蛋白 聚合酶链反应法 启动子 凝胶电泳 转录 

分 类 号：R346[医药卫生—基础医学]