Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGβ-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGβ following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGβ-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGβ and hCGβ-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGβ, pCMV4-hCGβ, pcDNA3-hCGβ-C3d3 and pCMV4-hCGβ-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGβ IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGβ-C3d3-DNAs elicited the highest titer of hCGβ-specific antibody among the serial doses and the immune response induced by pCMV4-hCGβ-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGβ-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGβ at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGβ-C3d3-DNAs and the ratio of IL-4/IFN-γ showed a Th2 bias of immune response in the mice immunized with hCGβ-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGβ DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3. Copyright Sons, Ltd.