针对HBV—P基因的RNA干扰抑制乙型肝炎病毒的研究

Specific anti-viral effects of HBV-P gene targeted RNA interference on replication of hepatitis B virus

目的构建针对乙型肝炎病毒P基因编码区、能在体内转录产生发夹状小干扰RNA（siRNA）的表达载体psiHBV／p，观察RNA干扰对HBsAg、HBeAg及乙型肝炎病毒DNA复制的抑制作用。方法针对HBV—P基因区特异序列，构建siRNA的表达载体psiHBV／p。采用脂质体介导方法将其与1．3倍HBV真核表达质粒pHBV1．3共转染HepG2细胞。分别于转染后24小时、48小时、72小时用ELISA法对HepG2细胞培养上清液进行HBsAg、HBeAg的检测；于转染后72小时通过FQ-PCR法分析RNA干扰作用对HBVDNA的抑制效果。结果成功构建了针对HBV—P基因区的siRNA的真核表达重组体psiHBV／p，并发现它能明显抑制HBsAg及HBeAg的分泌，转染后第二天抑制率达高峰，分别为84％、65％。FQ—PCR结果也证实了转染72小时后，随psiHBV／p比例的升高，其对HBV DNA的抑制作用也随之增加。结论成功构建的psiHBV／p，它能在体内持续转录产生针对P基因转录体的发夹状siRNA；在细胞水平上，体内转录产生的、针对乙型肝炎病毒P基因区特异序列的siRNA对共转染的重组载体pHBV1．3有显著和特异的抑制作用。

Objective To develop an RNA interference（RNAi） approach that specifically targets the P gene sequence of HBV by short interfering RNA（siRNA） ,and to assess the inhibitory effect of this siRNA on HBV replication. Methods A siRNA targeting HBV-P gene sequence was constructed based on pTZU6 ＋ 1 vector. The eukaryotic expression plasmid pHBV1. 3 and psiHBV/p were cotransfected into HepG2 ceUs to assess the inhibitory effect of RNAi on HBV-P gene expression. At 24 h,48 h,72 h post transfection,the levels of HBsAg and HBeAg secretec into the cell culture medium were determined with ELISA. Fluorogenic quantitative PCR（FQ-PCR） was used to detect HBV DNA at 72 hours post transfection. Results The expressing siRNA vector psiHBV/p which targeting P gene of Hepatitis B virus was successfully constructed. The introducing of RNAi plasmid efficiently inhibited the synthesis of surface and e antigen of HBV,with inhibitory rates at 84% and 65% respectively at 24 hours post transfected. FQ-PCR showed that HBV DNA in the cell culture medium was sharply reduced. Conclusions Results in this experiment demonstrate that the short RNA targeting HBV-P gene exerts robust inhibition on HBV viral replication and expression, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.