A novel peptide isolated from phage display peptides library recognized by an antibody against connective tissue growth factor (CTGF).
The aim of this study was to isolate a peptide binding to an antibody against CTGF C-terminal domain from the peptide library and to evaluate its immunological and biological activities. A phage display 12-mer peptide library was screened using anti-CTGF/C antibody as the target. Ten of the positive clones were sequenced after three rounds bio-panning. The DNA encoding peptide ZD521 was cloned and expressed as the fusion protein(TrxA-ZD521). The specificity of ZD521 to anti-CTGF/C antibody was determined by competitive inhibition assay. Mice were immunized with purified fusion protein(TrxA-ZD521) and the anti-peptide or anti-CTGF response of antiserum was also tested by enzyme-linked immunoabsorbent assay (ELISA) and Western blot. The inhibition effect of anti-serum on proliferation of kidney mesangial cells was evaluated by MTT. A peptide ZD521(GEPQTKLFSFPL) that could specifically recognize anti-CTGF/C antibody was isolated. No sequence homology was found between ZD521 and CTGF/C. The purified TrxA-ZD521 could specifically bind to anti-CTGF/C antibody and block the binding of anti-CTGF/C antibody to CTGF/C and native CTGF(mesangial cell lysate). Moreover, the antiserum from mice immunized with TrxA-ZD521 could also bind to CTGF/C recombinant protein and native CTGF, as well as significantly inhibit the proliferation of kidney mesangial cells induced by CTGF/C. Therefore, ZD521 might be a conformational epitope of CTGF which is potentially useful to be developed as a vaccine for prevention and treatment of fibrosis disorders.
Liu
Naifeng
N
Institute of Cardiovascular disease of Zhongda Hospital, Southeast University, Nanjing 210009, China. liunf@seu.edu.cn
Wu
Guoqiu
G
Li
Hui
H
Li
Linxian
L
Xing
Honglei
H
Zhang
Cheng
C
Lu
Huixia
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D055513:Connective Tissue Growth Factor / Q000276:immunology* / Q000302:isolation & purification; D000939:Epitopes / Q000276:immunology; D005355:Fibrosis / Q000517:prevention & control; D006801:Humans; D050527:Mesangial Cells / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification; D013879:Thioredoxins / Q000378:metabolism; D014612:Vaccines / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D055513:Connective Tissue Growth Factor; D013879:Thioredoxins