- 學位論文
製備及分析可辨認卵巢癌相關抗原的單鏈變異片段抗體
Preparation and analysis of the single chain variable fragment antibodies recognizing the ovarian tumor associated antigen
摘要
卵巢癌是女性十大癌症之ㄧ，目前臨床大都以「CA-125」作為卵巢癌表現的腫瘤標記，但是靈敏度並不令人滿意。根據先前的研究利用卵巢癌病人血清中的抗體從peptide library進行篩選顯示OVTA-1可能是卵巢癌的腫瘤相關蛋白基因。本篇研究目的希望藉由噬菌體展現技術（phage display technique）製作並篩選出具有OVTA-1特異性結合反應的多株抗體與單株抗體。根據Gene Bank所公布OVTA-1的基因序列全長為3792 base pairs (bps)，可轉譯出1264個胺基酸。本實驗重組其中長度為1002bp片段DNA，選殖入pET-21a質體，再利用大腸桿菌表現出與His融合的OVTA-1蛋白。西方墨點法實驗結果顯示抗His的抗體可辨識到His-OVTA-1融合蛋白。純化後的His-OVTA-1融合蛋白，以皮下注射方式注射來亨雞大腿部位，由雞蛋中純化出免疫球蛋白(IgY)，再利用酵素連結免疫吸附分析法(Enzyme-Linked Immunosorbent Assay, ELISA)以及西方墨點法證實免疫過後雞隻所誘發出來的抗體可辨認His-OVTA-1片段蛋白。為了建構出可與His-OVTA-1蛋白結合的scFv噬菌體基因庫，首先自雞脾臟中萃取出total RNA反轉錄成cDNA後利用聚合酶鏈鎖反應(PCR)，做出雞隻抗體的重鏈和輕鏈變異區基因片段，最後將這些基因片段連結後接入pComb3x載體中。未來希望能利用此基因庫作為OVTA-1研究和檢驗治療用之抗體來源，並探討其應用於預防及治療之潛力。
關鍵字
卵巢癌並列摘要
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.