针对HBV X基因的siRNA抑制HepG2细胞HBV的复制和表达

目的构建能转录产生针对乙型肝炎病毒（HBV）X基因转录体的小干扰RNA（siRNA）转录载体pSIH—BV／X,研究其在体外对HBV复制和抗原表达的抑制作用。方法将构建成功的pSIHBV／X与HBV1．3倍体真核表达质粒pHBV1．3共转染HepG2细胞，转染后24、48、72h检测上清液中HBsAg、HBeAg的变化，并检测72h时HBV DNA的变化。结果成功构建了针对HBVX基因转录体的siRNA表达载体pSIHBV／X，并发现它能抑制HBsAg、HBeAg的分泌，抑制高峰在72h．抑制率分别是64％和61％，而无关对照序列的siRNA无此作用。荧光定量PCR证实HBV DNA的复制亦受到抑制，抑制率31％。结论针对HBVX基因转录体的siRNA在体外具有抑制HBV复制和抗原表达的作用。

Inhibitory effect of siRNA targeting hepatitis B viral X gene on hepatitis B viral replication and HBsAg and HBeAg expression in vitro

Objectives To establish an RNAi approach that can specifically target the X gene sequence of hepatitis B virus ( HBV ) by synthesizing short interfering RNA ( siRNA ) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication and its antigen expression. Methods A recombinant plasmid pSIHBV/X which can transcribe the siRNA targeting HBV X gene was constructed by cloning the annealed synthesized sequences into linearized pTZU6+1 vector and named as pSIHBV/X. Secondly, the eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells using GenePORTER2 transfection reagent with either the RNAi plasmid pSIHBV/X or unrelated control plasmid pSIGFP. At 24, 48 and 72 hours after transfection, the levels of HBsAg and HBeAg in the cell culture supernatant were determined with ELISA kits. The HBV DNA in the supernatant was also determined by fluorogenic quantitative PCR ( FQ-PCR ). Results A siRNA transcribing vector, pSIHBV/X, which targeted the X gene of hepatitis B virus was constructed successfully. The introduction of RNAi plasmid had shown to efficiently and specifically inhibit the synthesis of surface antigen and e antigen of HBV, with inhibitory rates of 64% and 61%, peaking at 72 hours after transfection. FQ-PCR also showed that replication of HBV DNA was reduced to lower levels, when the ratio of pSIHBV1.3 and pSIHBV/X was at 1: 20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. Conclusions Results in this experiment demonstrates that the short interfering RNA targeting HBV X gene exerts robust inhibition on HBV replication and antigen expression.