Generation and characterization of chicken antibody fragments against potential tumor-associated antigen, OVTA-1, by phage display technology

• Main Authors: Chien-Hsiu Weng, 翁乾修
• Other Authors: 楊沂淵
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/83756395969725936932

碩士 === 臺北醫學大學 === 細胞及分子生物研究所 === 95 === In previous studies, OVTA-1 was identified as a tumor-associated antigen identified in ovarian cancer patient. In this study, we aimed to generate polyclonal and monoclonal chicken antibodies with high affinity against OVTA-1 protein by using phage display technology. The full length of OVTA-1 DNA is 6072 bp whose 520 bp DNA fragment was cloned into pGEX vector and was expressed as GST-OVTA-1 fusion protein. In western blot analysis, the result showed that the GST-OVTA-1 fusion protein can be detected by goat anti-GST antibody. Besides, both SDS-PAGE and western blot analysis indicated that OVTA-1 protein separated from GST is about 19 kDa. The result demonstrated that this E.coli-expressed protein is partial OVTA-1 protein. The Leghorn chicken was immunized by intramuscular injection with purified GST-OVTA-1 fusion protein. Polyclonal IgY antibodies were isolated from the collected eggs and were estimated to be 50-100 mg per egg by SDS-PAGE analysis. Moreover, GST-OVTA-1 fusion protein can be recognized by these polyclonal antibodies in western blot and ELISA analysis. To construct OVTA-1-specifc antibody libraries, overlap PCR was performed to generate a variety of 750 bp DNA fragments containing IgY heavy and light chain variable genes. These scFv DNA fragments were cloned into pComb3X vectors and then infected the XL1-Blue E.coli with VCSM13 phage. Several antibody libraries against OVTA-1 protein were obtained and one has 1.1x105 clones. The antibody library was performed with four rounds of phage panning in order to isolate antibody fragments against OVTA-1 protein. After panning, total phagemid DNA was introduced into TOP10F’ E.coli and ten clones were selected for antibody expression. Our result indicated that the protein size of E.coli-expressed scFv antibody is about 28 kDa. These clones containing scFv antibody fragments were examined for their binding ability to OVTA-1 protein by ELISA and western blot analysis. Our result showed that several clones containing scFv can specifically bind to OVTA-1 protein. Besides, sequence analysis of these antibody fragments indicated that two particular clones were enriched through phage panning process. These antibody fragments with specific binding ability to tumor-associated antigens can be applied in the development of diagnostic or therapeutic agents in the future.