[Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris].
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.
Xiong
Sheng
S
Biomedicine Research & Development Center, Jinan University, Guangzhou 510640, China.
Ren
Xiang-Rong
XR
Tang
Yong-Hong
YH
Su
Kuan-Yuan
KY
Yu
Zhou-Yao
ZY
Luo
Yong
Y
Wang
Yi-Fei
YF
Li
Jiu-Xiang
JX
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D006514:Hepatitis B Surface Antigens; D057127:Single-Chain Antibodies