Screening Antigen Epitope Of Connective Tissue Growth Factor By Phage Display

Aim: 1. To prepare the connective tissue growth factor(CTGF) antigen; 2. To evaluate properties of monoclonal antibody against CTGF produced in our laboratory by Western blotting and ELISA; 3. Screening antigenic/ functional epitope of CTGF by phage display. Methods: 1. Total RNA was exracted from human endothelial cells and reverse transcripted into cDNA. The gene encoded CTGF C region 242-349 residues was obtained by PCR with designed primers and inserted to upstream of the gene of phage coat protein III.The CTGF protein was expressed and was examined by the method of ELISA; 2. Human mesangial cells were cultured in DuLbecco Modified Eagle Medium(DMEM) containing 30mmol/L glucose for 2 days. Total CTGF protein was exracted from the cells, which is used in the method of Westen blotting by which the monoclonal antibody against CTGF and CTGF standard material produced by our research group were identified; 3. The phage display 12 peptides library was screened with monoclonal antibody against CTGF by increased selective pressure. After three circles screening, ten isolated phage clones specially inhibited CTGF binding to its antibody. The sequence of the positive clones had been obtained by sequencing. The specificity of positive clones was determined by indirected ELISA and competitive inhibitive ELISA.Results: 1. The gene of CTGF C region was successfully cloned and inserted into expressive vector. The required protain of 25 ku was obtain by IPTG induced and purification;2. The result of Western blotting shows that the monoclonal antibody against CTGF produced by our research group can bind to the CTGF natural protain and the recombinant protain as well; 3. All the sequence analysis of the isolated clones showed that the epitopes shared a highly consensus sequence. A sequence of 12 peptide(GEPQTKLFSFPL) was obtained, and named ZD 521. The results of indirected ELISA and competitive inhibition ELISA were in accordance with the expected. Conclusion: A peptide specially combined with anti-CTGF antibody was obtained successfully, and it is valuable for the studies of CTGF function and vaccine design in the future.