The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines. The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg), and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N- terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs- MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB / c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3- [4, 5- dimethylthiazol-2-yl] -5- [3-carboxymeth The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB / c The highest titer of antibody against HCV (MFC) was 1:1280, and the ratio of IgG2a / IgG1 was 1.50 ± 0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two-part HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ± 0.07, and the IFN-γ level was 287 ± 6? pg / ml at week 21 after first immunization. The highest titer of the antibody in control BALB / c mice immunized with pVAX1-MFC was 1: 320, and the ratio of IgG2a / IgG1 was 1.33 ± 0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes to MFC was 1.52 ± 0.06, and the IFN-γ level was 225 ± 9.3? pg / ml at week 21 post-immunization The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be potential employment in the development of HCV vaccines.