Reconstruction and
Analysis of A Human Small Molecular Antibody to Tumor Necrosis Factor Alpha
CHEN Ping1, CHEN Chang-Qing2*, YAO
Li-Bo1, HAN Hua1,SU Cheng-Zhi1
( 1Department of Biochemistry and Molecular Biology, The Fourth
Military Medical University, Xi’an 710032, China；2Shanghai Research Center of
Biotechnology, Chinese Academy of Science, Shanghai 200233, China )
Abstract A Fab
antibody gene was constructed on the basis of the reconstruction of the linker
of a human anti-TNF-α 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-α. The
ScFv containing GGGGS had an affinity constant of 6.70×104(mol/L)-1,
and the ScFv containing (GGGGS)3 had an affinity constant of 7.27×105(mol/L)-1.The
affinity constant of Fab was 7.61×105(mol/L)-1. The Fab
and reconstructed ScFv was indifferent in affinity activity； 3) ScFv and Fab neutralized
the cytotoxicity of rhTNF-α. The neutralizing ability of Fab was the same as
the reconstructed ScFv, but lower than a mouse anti-TNF-α mAb. These data may
be helpful for using human anti-TNF-α small molecular Ab in antagonizing the
activity of TNF-α in therapy.
Key words TNF-α；human ScFv；human Fab；expression；affinity
*Corresponding author：Tel,86-21-64700892-306；Fax,86-21-64700244；e-mail,[email protected]