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Bioscreening of phage display antibody library and expression of a humanized single‐chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2)
Author(s) -
Wu Guoqiu,
Fan Xiaobo,
Wu Hongbin,
Liu Naifeng,
Li Xiaofang,
Gou Lixia,
Nie Yu,
Zhao Rui,
Xi Tao
Publication year - 2010
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20100031
Subject(s) - ctgf , biopanning , phage display , microbiology and biotechnology , antibody , fusion protein , growth factor , connective tissue , single chain variable fragment , immunoglobulin light chain , biology , chemistry , monoclonal antibody , recombinant dna , peptide library , immunology , biochemistry , peptide sequence , receptor , gene , genetics
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%.