Abstract: Objective To observe the inhibitory effect of targeted ribonuclease delivered via adenovirus against HBV replication in vitro.Methods The shuttle plasmids pDC316 were constructed on the basis of the previous plasmids pcDNA3.1(-)/TRL,pcDNA3.1(-)/TR,pcDNA3.1(-)/TRmut,pcDNA3.1(-)/HBVc,and pcDNA3.1(-)/hEDN by subcloning the target gene sequences of TRL,TR,HBVc,and hEDN,respectively.HEK 293 cells were cotransfected with the pDC316 plasmids respectively in the presence of the rescue plasmid pBHGlox(delta)E1,3Cre to yield the recombinant adenoviral vectors which comprised the above genes.After transfection of HepG2.2.15 cells with the vectors,RAd/TRL expression was detecetd by indirect immunoflurescence staining and RT-PCR.Radioimmunoassay was used to analyse anti-HBV activity of RAd/TRL.Results Recombinant RAd vectors were prepared successfully.Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of HBsAg and HBeAg concentration in comparison with the controls.Conclusion Adenoviral vector-mediated targeted ribonuclease can effectively inhibit HBV replication.