Gene fusion of molecular adjuvant C3d to hCGbeta enhances the anti-hCGbeta antibody response in DNA immunization.

OBJECTIVE

To express the hCGbeta-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination.

METHOD

We constructed a plasmid pcDNA3-hCGbeta-C3d3 which contains three copies of murine C3d cDNA and the hCGbeta gene by cloning the chimerical hCGbeta-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGbeta in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6-8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGbeta and pcDNA3-hCGbeta-C3d3, and ELISA was used to assess anti-hCGbeta IgG antibody in serum.

RESULTS

In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGbeta-C3d3, 1.0x10(5) cells could secrete 152 ng of the recombinant protein (calculated by hCGbeta contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/10(6) cells/48 h. The hCGbeta-C3d3 protein was purified by anti-hCGbeta immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGbeta and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGbeta IgG antibody titer in the pcDNA3-hCGbeta-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGbeta immunized group.

CONCLUSION

We have expressed the hCGbeta-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGbeta antigen in DNA immunization.