腺相关病毒Rep78蛋白抑制乙型肝炎病毒复制的体外研究

目的 研究腺相关病毒(adeno-associated virus,AAV)Rep78蛋白对乙型肝炎病毒(HBV)复制的抑制作用及机制.方法 将土拨鼠肝炎病毒(WHV)全基因组DNA从质粒中酶切回收,线状DNA重新连接呈环状.用脂质体Fugene 6体外转染至HepG2细胞,同时共同转染含有Rep78的质粒AAVdl52-91.5 d后收获细胞DNA,Southern blot检测WHV DNA复制.以含有HBV全长的质粒为模板,PCR法分别扩增出HBV-S、C和X基因全长.以凝胶电泳阻滞实验(EMSA)检测Rep78与HBV-S、C和X的结合.结果 Southern blot结果表明,AAV-Rep78可以明显抑制WHV病毒在HepG2细胞中的复制,并呈剂量依赖关系.EMSA结果显示,Rep78蛋白在体外能够与HBV-S、C和X的DNA结合,其中与HBV-C的结合最强而且有剂量依赖关系.此外,这种Rep78蛋白与HBV-C DNA结合可以被特异性的Rep78抗体阻滞,形成超结合带.结论 AAV-Rep78可以抑制HBV DNA的复制,其机制可能在于Rep78蛋白结合并抑制了HBV-C基因.

Inhibition of hepatitis B virus replication by adeno-associated virus Rep78 protein in vitro

Objective Adeno-associated virus (AAV) Rep78 is known for inhibition of several viruses, such as adenovirus and human papillomavirus, replication and oncogenes transformation. This study is to investigate the effect of AAV Rep78 on hepatitis virus replication and the mechanisms. Methods Woodchuck hepatitis virus (WHV) was removed from the plasmid and recircularized. HepG2 cells were transfected with the recircularized WHV DNA and Rep78 plasmids. After 5 days, total cellular DNA was digested with DpnI, size separated, Southern blotted and probed with 32P-WHV DNA. HBV-S, C or X gene were amplified by PCR. Electrophoretic mobility shift assay (EMSA) was utilized to detect the binding of MBP-Rep78 protein with HBV-S, C or X genes.Results WHV replication was reduced by the presence of AAV Rep78 expression plasmid in a dose-dependent manner. EMSA showed all the three genes, HBV-S, C or X gene, could bind to Rep78 protein, with HBV-C DNA the strongest. Moreover, the binding of HBV-C DNA to Rep78 protein was not only in a dose-dependent manner, but also could be retarded by specific Rep78 antibody.Conclusion AAV Rep78 could inhibit the replication of hepatitis virus, this effect may be through its binding with HBV-C gene.