应用RNA干扰技术抑制乙型肝炎病毒抗原表达的实验研究

Potent and specific inhibition of hepatitis B virus antigen expression by RNA interference

目的 构建针对乙型肝炎病毒 (HBV)核心区的siRNA表达载体pSIHBV/C ,观察其对HBV复制和表达的影响。方法 将构建成功的 pSIHBV/C与 1 3倍HBV真核表达质粒pHBV1 3共转染HepG2细胞 ,转染后 2 4、4 8、72h ,用Abbott试剂检测细胞上清中HBsAg和HBeAg ,并用免疫荧光染色法观察细胞内病毒核心抗原和表面抗原的表达。结果 成功构建了针对HBV核心区的siRNA表达载体 pSIHBV/C ,并发现它能明显抑制HBsAg和HBeAg的分泌 ,转染后第 2天抑制率达高峰 ,分别为 92 %、85 % ,而随机序列的siRNA无此作用。免疫荧光染色结果也证实转染 2 4h后 ,随pSIHBV/C比例的升高 ,其对HBV抗原表达的抑制作用也随之增加 ,当pSIRNA/C与 pHBV1 3比例为 1∶2 0时 ,pSIHBV/C对细胞内HBsAg和HBcAg表达的抑制作用最强。 结论 乙型肝炎病毒核心区的siRNA具有显著和特异的抗HBV复制和表达的作用。

Objective To develop an RNAi approach that specifically targets the core gene sequence of hepatitis B virus by synthesizing short interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication. Methods The eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells with either the RNAi plasmid pSIHBV/C or unrelated control plasmid pSI. At 24, 48, 72 hours post transfection (p.t.), the levels of HBsAg and HBeAg in the cell culture medium were determined with Abbott MEIA Kits. The expression of intracellular viral proteins was also determined by immunofluorescence staining. Results Successfully constructed expressing siRNA vector pSIHBV which targeted the core gene of Hepatitis B virus.The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of surface and e antigen of HBV by Abbot MEIA kits, with inhibitory rates at 92%?85% peaking at 24 hours p.t. Immunofluorescence staining showed that intracellular synthesis of viral antigen was sharply reduced to nearly background levels when the ratio of pSIHBV/C and pHBV1.3 was at 1∶20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. Conclusion Our results demonstrate that the short interfering RNA targeting HBV core gene exerts robust inhibition on HBV viral replication, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.