Bioscreening of phage display antibody library and expression of a humanized single-chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2).
Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.
Wu
Guoqiu
G
Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, People's Republic of China. guoqiuwu@163.com
Fan
Xiaobo
X
Wu
Hongbin
H
Liu
Naifeng
N
Li
Xiaofang
X
Gou
Lixia
L
Nie
Yu
Y
Zhao
Rui
R
Xi
Tao
T
D055513:Connective Tissue Growth Factor / Q000276:immunology*; D005355:Fibrosis / Q000276:immunology; D006801:Humans; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
C526317:CCN2 protein, human; D019151:Peptide Library; D057127:Single-Chain Antibodies; D055513:Connective Tissue Growth Factor