Preparation and analysis of the single chain variable fragment antibodies recognizing the ovarian tumor associated antigen

• Main Authors: Pei-Hsuan Lin, 林佩璇
• Other Authors: Yi-Yuan Yang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/43346929065481500799

碩士 === 臺北醫學大學 === 醫學科學研究所 === 98 === Ovarian cancer is one in top ten female killer cancers. CA-125 protein is generally used as a clinical tumor label of ovarian cancer; however the sensitivity is not satisfied. Previous researches showed OVTA-1 might be an ovarian cancer related gene by screening patients’ serum with the peptide library. This research aimed to construct a polyclonal or monoclonal antibody which specifically binds to OVTA-1 by phage display technique. According to the gene bank of NCBI, the full length of OVTA-1 gene is 3792bp being translated into 1264 amino acids. We cloned partial OVTA-1 gene in the length of 1002bp into pET-21a vector. The OVTA-1 protein fused with His protein would be performed by E.coli then. Results of western blot showed anti-His antibody would recognize His-OVTA-1 fusion protein. Purified His-OVTA-1 fusion protein was subcutaneous injected into leghorn chicken thighs, and immunoglobin Y (IgY) would be purified from their eggs. Next, ELISA and western blot were used to demonstrate antibodies from immunized chickens could also recognize His-OVTA-1 fusion protein. To construct anti-His-OVTA-1 scFv phagemid gene bank, total RNA was first extracted from chicken’s spleen and reverse transcribed into cDNA. Next the gene fragments of chicken antibodies’ heavy chain and light chain variable regions were amplified with PCR and ligated. At last, these ligated genes were cloned into pComb3x vector and expressed. In the future, we expect this gene bank to be the antibody source for OVTA-1 researches, clinical examinations or treatments by studying its application and therapeutic potential.