双位点核酶细胞内抑制乙型肝炎病毒基因的表达

摘要

目的:构建针对乙型肝炎病毒(HBV)C区基因(2029-31及2063-65位点)的双位点核酶的真核表达载体,观察其在细胞内对HBV基因表达的抑制作用.方法:利用亚克隆技术,从质粒pGEMRz123切下EcoR I-BamH I片段, 双粘端克隆于真核质粒pBBS212中,将该质粒与p1.2Ⅱ(含HBV全序列)共转染HHCC细胞(人肝癌细胞株),观察该核酶对HBV基因表达的抑制作用.结果:在HHCC细胞中p1.2Ⅱ可表达出HBsAg及HBeAg.该核酶对HBeAg的合成抑制率达65%.结论:该双位点核酶可能通过针对HBV C区mRNA的剪切作用,阻断C区mRNA的表达,抑制了HBeAg的合成.%AIM: To construct a eukaryotic expression vector with dual-target ribozyme against hepatitis B virus and to observe the inhibition of HBeAg expressed in the HHCC cell (human hepatic carcinoma cell line) transfected by HBV genome and dual-target ribozyme against HBV. METHODS: Using subclone technique, the dual-target ribozyme gene cut from pGEM-Rz 123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid pBBS212-Rz and p1.2Ⅱ carrying genome of adr-subtype HBV were cotransfected into the HHCC cell by using lipofectamine. The intracellular e/c antigen of HBV was detected by ELISA and immunohistochemistry. RESULTS: The cotransfected cells did express HBV e antigen and ribozyme RNA, and the dual-target ribozyme inhibited the level of HBeAg expressed on cotransfected cells by up to 65%. CONCLUSION: The dual-target ribozyme can cleave the HBV core region mRNA and inhibit HBV the expression of HBV core region gene.