OBJECTIVETo express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen.

METHODSThe Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%.

RESULTSWestern-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual.

CONCLUSIONGag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.