Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 19-23, 2003.
Article
in Chinese
| WPRIM
| ID: wpr-270045
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pichia
/
Blotting, Western
/
Polymerase Chain Reaction
/
Electrophoresis, Polyacrylamide Gel
/
Allergy and Immunology
/
Single-Chain Antibodies
/
Genetics
/
Hepatitis B Surface Antigens
/
Metabolism
Type of study:
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2003
Type:
Article
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