Fusion expression of structural protein gene gag and gp120 of HIV-1_(CN) in Pichia pastoris

Aim To express fusion protein of HIV-1 CNB gag and gp120 in Pichia pastoris. Methods Using the yeast expression plasmid pPIC9 as the vector, the recombinant yeast expression plasmid pPICGP containing gag and gp120 chimeric genes of HIV-1 was constructed. After being linearized by Sac I, the plasmid pPICGP was electrotransformed into Pichia pastoris strain GS115. The positive yeast transformants were identified by PCR, and fusion protein was expressed in Pichia pastoris under the induction of methanol. The expressed products were analyzed by SDS-PAGE and Western blot, and the genetic stability of the positive strains were also studied. Results Of twelve yeast colonies, 8 transformants were positive, indicating that the integration rate was 66.7%. The SDS-PAGE and Western blot analysis showed that there was a specific protein band at the site of Mr 57 000, which reacted with anti-p24 mAb and anti-gp120 mAb. The yeast transformants still had the foreign gene after ten times of passage in YPD medium. Conclusion The chimeric gene of gag and gp120 of HIV-1 was successfully expressed in Pichia pastoris, but the relative molecular mass(Mr) of expressed protein is smaller than the expected value, suggesting partial expression of gp120 gene in the fused gene.