Inhibition of HbsAg and HbeAg expression of recombinant adeno-associated virus encoding HBsAg-shRNA in HepG2.215 cells in vitro

Objective To construct the recombinant adeno-associated virus(rAAV)encoding HBsAg-shRNA and to observe its effect on HBsAg and HBeAg expression in HepG2215 cells in vitro. Methods pAAV-shHBs-hrGFP expressing plasmid was constructed by molecular biological techniques. The recombinants were cotransfected with p-RC and p-Helper into AAV-293 cells mediated by calcium acid phosphate. The rAAVs encoding HBsAg-shRNA (rAAV-shHBs-hrGFP)were harvested and infected HepG2215 cells. The silencing effect of this virus on HbsAg and HBeAg gene expression was assessed by ELISA. Results The plasmid of pAAV-shHBs-hrGFP was successfully constructed by identification of restrict enzyme and DNA sequencing. GFP expression was observed about 80% in AAV-293 cells. After cotransfection,the recombinant AAV-shHBs-hrGFP was harvested and the virus titer was tested. The HBsAg and HBeAg expression in HepG2.215 cells was down-regulated markedly after infection of rAAV -shHBs -hrGFP. Conclusion The recombinant AAV -shHBs -hrGFP vector is successfully constructed and can be used as a potential antivirus agent to inhibit HBV replication.