Expression and preliminary applications of hepatitis B surface antigen mutants

AIM:To clone and express HBsAg mutant in the Pichia pastoris.METHODS:The cloned wild type pGAP-S was used as the DNA template to generate mutant type pGAP-MS with a single or double nucleotide changes incorporated in complementary oligonucleotide primers.The product was linearized with BspH I and transformed into Pichia pastoris strain GS115,and stable multicopy integrants were screened in medium containing different concentrations of Zeocin.RESULTS:The pGAP-MS expression vector was successfully constructed and stable numbers integrated strains with high copy number were obtained.The expression of HBsAg mutant protein was identified by SDS-PAGE and Western blot with specific polyclonal antibody.The molecular weight of recombinant HBsAg mutant was 38 kDa.AxSYM HBsAg V2(Abbott)assays demonstrated all 10 HBsAg mutants were reactive.CONCLUSION:The recombinant HBsAg mutant with immunoreactivity was successfully expressed in Pichia pastoris,and it was of practical value on the quality control and clinical applications of commercial assays.