[Expression of human anti-HBsAg single chain Fab gene in Pichia pastoris].
OBJECTIVE
To investigate the relation of the affinity activity to the structure of recombinant Fab.


METHODS
The human anti-HBsAg single chain Fab gene was obtained by overlaping PCR from the H and L chains of Fab, and was introduced into the expression system of Pichia pastoris. The expression vector of single chain Fab was transformed into GS115 cells by the method of LiCl transformation. The transformants were obtained and cultured in shake flask. The culture supernatant of the recombinant yeast was deposited in (NH(4))(2)SO(4) and purified by affinity chromatography. The affinity of culture supernatant of recombinant yeast and purified single chain Fab was analyzed by ELISA.


RESULTS
The analysis of SDS-PAGE and Western blot showed that the single chain Fab gene was expressed successfully in Pichia pastoris, and the expression quantity was about 5-10 mg/L in shake flask. The recombinant Fab was purified by affinity chromatography and the purity reached 97.8%. The ELISA analysis showed that the recombinant single chain Fab had good HBsAg binding properties.


CONCLUSIONS
The single chain Fab gene could be expressed successfully in Pichia pastoris and the recombinant single chain Fab can efficiently bind with HBsAg.
Deng
Ning
N
Research Center of Molecular Immunology and Antibody Engineering, Life Science and Technology College, Jinan University, Guangzhou 510632, China.
Xiang
Jun-jian
JJ
Su
Kuan-yuan
KY
Wang
Hong
H
Tang
Yong
Y
D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D006514:Hepatitis B Surface Antigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains