Résumé
Objectives To synthesize hepatitis B virus e gene which was suitable for yeast protein expression and express hepatitis B virus e antigen (HBeAg) in Pichia pastoris.Methods Using synonymous codons preferred by yeast usage on protein expression to replace some native codons of wild-type HBV e gene,the synthetic e gene (syneAg-gene) was achieved by a recursive PCR (rPCR).The syneAg- gene was inserted into the yeast expression vector pPICZ?A.The recombinant plasmid was transformed into GS115 yeast by electroporation.The yeast transformant induced by methanol expressed the HBeAg.Results The restriction analysis and DNA sequencing confirmed that the syneAg-gene was inserted to yeast pPICZ?A in correct orientation.SDS-PAGE,immunoblot and ELISA indicated that the secreted form of HBeAg was expressed by the yeast transformant.The expression level of HBeAg in the strain containing syneAg-gene was 63 mg/L.The titer of the recombinant HBeAg in culture superuatant was 1 :81 920 and the maximum OD (optical density) value of absorbance was 2.8 by ELISA.No cross reactivity between Pichia pastoris-derived HBeAg and anti-HBc antibody was found.Conclusion The recombinant HBeAg with high degree of specificity and immunoreactivity is expressed efficiently in Pichia pastoris.