Investigation of replication capacity and antiviral agents susceptibility for clinical hepatitis B virus (HBV) isolates have been hampered by the limitations of available in vitro culture systems.The plasmids containing over-length HBV genome can mediate HBV replication and expression by transfecting these plasmids into liver cell lines.In this study we established a method for rapidly and effectively selecting anti-HBV drugs by a novel expression vector of HBV.The full-length hepatitis B virus (HBV) genome from eight patients with chronic hepatitis B (CHB) were generated by polymerase chain reaction (PCR).All patients failed to lamivudine therapy after more than seven months therapy.The amplified HBV DNA fragments were inserted into Sap I site of pHY106 expression vector respectively.The recombinant plasmids containing 1.1 copies of HBV genome were transfected into Huh7 cell line and the levels of HBsAg, HBeAg, HBV DNA and intercellular HBV replicative intermediates were determined by ELISA, quantitative PCR and Southern blot analysis respectively, and antiviral susceptibility of lamivudine and adefovir were analyzed in these vitro culture system.Furthermore, antiviral susceptibility of adefovir in vivo were observed subsequently.A total of 25 independent HBV clones were obtained from 8 patient sera and every patient had at least two clones.8 clinical HBV isolates form different individual were subcloned into pHY106 vector.Of the 8 clinical HBV isolates, 5 isolates were polymerase YVDD mutation and 3 isolates were polymerase YIDD mutation.All recombinant plasmids containing clinical HBV genome were transfected into Huh7 cells, the results shown that clinical HBV isolate could effectively replicate and express in vitro.Adefovir but not lamivudine inhibited HBV replication both in vitro and in vivo, and in vitro inhibition effect of adefovir depended upon its concentration.The novel method described here enables rapidly screening of anti-HBV agents in clinic and will be useful in future studies of antiviral therapy for CHB.