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

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.