[Secretion expression in Pichia pastoris and immunogenicity comparison of two forms of HBsAg/GM-CSF fusion proteins].

AIM

To express two fusion forms of hepatitis B virus surface antigen (HBsAg-s and s-HBsAg) in the Pichia pastoris expression system, and compare immunogenicity of the two fusion proteins.

METHODS

Was fused GM-CSF to 5' or 3' terminal of HBsAg by inserting the gene fragment of connecting peptide (Gly(4)Ser)(3) to linker gene of GM-CSF and HBsAg. The two fusion proteins were expressed by secreting type expression plasmid pPIC9K in the Pichia pastoris, then the expressed products were detected by SDS-PAGE, Western blot and purified by DEAE-Sepharose Fast Flow ion exchange columns. Mice were inoculated with the two purified HBsAg/GM-CSF fusion proteins and HBsAg respectively in each, and the levels of anti-HBsAg in mice sera were tested by ELISA.

RESULTS

Two HBsAg/GM-CSF fusion proteins were successfully expressed in the form of secretion in Pichia pastoris strain GS115, and exhibited specific reaction with both anti-HBsAg and anti-GM-CSF antibodies in Western blot. ELISA results showed after the inoculation the levels of anti-HBsAg induced by the two HBsAg/GM-CSF fusion proteins was higher than by HBsAg alone (P<0.05). Furthermore, the effect by fusing GM-CSF to C terminal of HBsAg was better than by fusing GM-CSF to N terminal of HBsAg.

CONCLUSION

The immunogenicity of HBsAg could be enhanced by fusing GM-CSF.