Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris.
- Author:
Sheng XIONG
1
;
Xiang-Rong REN
;
Yong-Hong TANG
;
Kuan-Yuan SU
;
Zhou-Yao YU
;
Yong LUO
;
Yi-Fei WANG
;
Jiu-Xiang LI
Author Information
1. Biomedicine Research & Development Center, Jinan University, Guangzhou 510640, China.
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Electrophoresis, Polyacrylamide Gel;
Hepatitis B Surface Antigens;
immunology;
Humans;
Pichia;
genetics;
metabolism;
Polymerase Chain Reaction;
Single-Chain Antibodies;
genetics;
immunology;
metabolism
- From:
Chinese Journal of Biotechnology
2003;19(1):19-23
- CountryChina
- Language:Chinese
-
Abstract:
To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
