[Expression and preliminary applications of hepatitis B surface antigen mutants].
OBJECTIVE
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.


CONCLUSIONS
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.
Biochemistry and Molecular Biology, East China University of Science and Technology, Shanghai 200237, China. changchengzh@163.com
Chang-Cheng
CC
Zhu
Chun-Zhen
CZ
Luo
Man
M
Wang
D019943:Amino Acid Substitution; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D015870:Gene Expression; D006511:Hepatitis B Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D013997:Time Factors
D000954:Antigens, Surface; D006511:Hepatitis B Antigens; D050505:Mutant Proteins; D011994:Recombinant Proteins