腺相关病毒载体介导siRNA抑制HBV表达和复制的研究

Inhibition effect of adeno-associated virus vector mediated siRNA on the replication and expression of hepatitis B virus

目的构建能抑制HBVm RNA表达的siRNA-重组腺相关病毒(rAAV)表达载体,观察重组病毒对HBV的抑制作用。方法将设计好的siRNA克隆到骨架质粒pAAV-MC中,将该质粒与pAAV-RC和pHelper质粒共同转染人胚肾293T细胞,进行病毒包装。收获并纯化病毒,感染模型细胞HepG2.215,采用ELISA法和荧光定量PCR法检测包装的rAAV-shRNA-GFP病毒抑制HBV情况。结果经酶切及测序证实,rAAV载体质粒pAAV-shRNA-GFP构建成功,包装的rAAV-shRNA-GFP病毒感染HepG2.215细胞后,可以抑制HBsAg、HBeAg的表达和HBV的复制。结论成功制备出rAAV-shRNA-GFP病毒,其能够抑制HBV的表达和复制。

Objective To construct the recombinant adeno-associated virus( rAAV) vectors for siRNA targeting hepatitis B virus( HBV) mRNA and to observe the inhibitive effect on HBsAg and HbeAg expression and replication of HBV in vitro. Methods The pAAV-shRNA-GFP expressing plasmid was constructed by molecular biological techniques. This plasmid combined with pAAV-RC and pHelper were transfected into human embryonic kidney 293T cells using LipofecLamineTM 2000. The rAAV-shRNA-GFP was packaged,amplified and purified,which was used to infect HepG 2. 2. 15 cells. The inhibitory effect of this recombinant on HBV was analyzed by ELISA and fluorescence quantitative RCR. Results The result of restriction enzyme digestion and sequencing showed that the recombinant adeno-associated virus vector plasmid pAAV-shRNA-GFP was successfully constructed. After the HepG2. 2. 15 cells were infected by purified rAAVshRNA-GFP virus,the copies of HBV DNA and RNA and HBsAg and HBeAg expression were significantly inhibited. Conclusion The rAAV-shRNA-GFP virus which is successfully constructed can effectively inhibit the replication and expression of HBV in vitro.