Reconstruction and Analysis of A Human Small Molecular Antibody to Tumor Necrosis Factor Alpha.
A Fab antibody gene was constructed on the basis of the reconstruction of the linker of a human anti-TNF-alpha ScFv gene. The two ScFvs before and after reconstruction were cloned into expression vector pBV220. About 30 kD recombinant proteins were expressed by induction and they constituted 6.5% and 13.8% of the total bacterial protein, respectively. A soluble Fab expression vector was constructed and transformed into E.coli HB2151.After induction by IPTG, a new protein band about 50 kD appeared on SDS-PAGE. The expressed ScFv and Fab were purified from E.coli lysates, and further experiments showed that 1) the expression amount of reconstructed ScFv was increased distinctly 2) ScFv and Fab could bind rhTNF-alpha. The ScFv containing GGGGS had an affinity constant of 6.70x10(4)(mol/L)(-1), and the ScFv containing (GGGGS) (3) had an affinity constant of 7.27x10(5) (mol/L) (-1).The affinity constant of Fab was 7.61x10(5) (mol/L) (-1). The Fab and reconstructed ScFv was indifferent in affinity activity 3) ScFv and Fab neutralized the cytotoxicity of rhTNF-alpha. The neutralizing ability of Fab was the same as the reconstructed ScFv, but lower than a mouse anti-TNF-alpha mAb. These data may be helpful for using human anti-TNF-alpha small molecular Ab in antagonizing the activity of TNF-alpha in therapy.
Chen
Ping
P
Shanghai Research Center of Biotechnology, Chinese Academy of Science, Shanghai 200233, China. cqchen@srcb.ac.cn
Chen
Chang-Qing
CQ
Yao
Li-Bo
LB
Han
Hua
H
Su
Cheng-Zhi
CZ