Abstract:［Objective］We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV ) replication and gene expression in vitro． ［Methods］ siRNA targeting HBV Sgene (siHBS) was designed ，synthesized and cloned into a minicircle DNA vector pMC． BESPX-MCS2. After sequencing，we transformed the recombinant pMC-H1-siHBS-U6 into E.coli ZYCY10P3S2T， and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium． As expected，a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette． Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1. 3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells． ［Results］We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6，which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks． Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%． ［Conclusion］ The minicircle DNAbased RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically，efficiently and steadily． Thus，this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection．