Related Experiment Video
Updated: Jun 12, 2026
Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library
Published on: September 30, 2019
Bioscreening of phage display antibody library and expression of a humanized single-chain variable fragment antibody
Guoqiu Wu1, Xiaobo Fan, Hongbin Wu
1Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, People's Republic of China. guoqiuwu@163.com
Abstract:
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%.
Insights
Researchers developed a novel antibody fragment targeting connective tissue growth factor (CTGF/CCN2) to combat fibrotic diseases. This new single-chain variable fragment (scFv) effectively inhibited cell migration, showing promise for fibrosis treatment.
Area of Science:
- Biotechnology
- Immunology
- Fibrosis Research
Background:
- Connective tissue growth factor (CTGF/CCN2) is overexpressed in fibrotic diseases.
- Antibody-based inhibition of CTGF/CCN2 is a potential therapeutic strategy for fibrosis management.
Purpose of the Study:
- To develop and characterize a humanized single-chain variable fragment (scFv) antibody targeting the C-terminal domain of CTGF/CCN2.
- To evaluate the efficacy of the developed scFv in inhibiting CTGF/CCN2-induced cell migration.
Main Methods:
- Screening of a phage display humanized scFv antibody library against CTGF/C.
- Expression and purification of the target scFv as a fusion protein (TrxA-scFv).
- Immunological assays and cell migration assays using human umbilical vein endothelial cells (HUVEC).
Main Results:
- A specific scFv targeting CTGF/CCN2 was successfully generated and purified.
- The purified scFv demonstrated concentration-dependent binding to CTGF/CCN2.
- The scFv significantly inhibited CTGF/CCN2-induced HUVEC migration by 90.5% at 100 ng/ml.
Conclusions:
- The novel humanized scFv effectively neutralizes CTGF/CCN2 activity.
- This scFv holds significant potential as a therapeutic agent for treating fibrotic diseases.
