利用噬菌體重組基因體庫製造 OVTA-2 腫瘤相關抗原之雞抗體片段
Chicken single chain variable fragment recognizing OVTA-2 tumor associated antigen using phage display antibody technology
中文摘要
卵巢癌的死亡率始終居高不下的原因，是由於大部份病患在癌症發生的早期沒有 明顯症狀也缺乏適當的腫瘤標記來做判斷。在先前的實驗中發現到卵巢癌病人相 較於正常人體內會產生一些特異性抗體，而也找到了其中的一個相對應蛋白基因 OVTA-2。因此本篇的研究目標是利用重組表現片段的 OVTA-2 蛋白來免疫雞 隻，並藉由噬菌體展現技術 (phage display technique) 來建立重組抗體基因庫 (antibody library) 篩選對於 OVTA-2 蛋白具有特異性結合力的單株抗體片段 (scFv, single-chain variable fragment) 。OVTA-2 基因全長 4150 bp，由 872 胺基酸 組成。重組的 GST-OVTA-2 融合蛋白為 35 KDa，經由大腸桿菌大量表現後，利 用 GST Sepharose 4B 做純化後，可以利用 coomassie blue 染色的 SDS-PAGE 以及 西方墨點法 (western blot) 來辨認。將純化後的 GST-OVTA-2 片段蛋白混合適當 的佐劑，以肌肉注射方式注入實驗雞隻體大腿部位，每週一次、連續四週。純化 雞蛋內的多株抗體 (poly-IgY)，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的抗 體可辨認 GST-OVTA-2 片段蛋白。萃取出雞脾臟內的 RNA，反轉錄成 cDNA 後 利用多鏈聚合酶反應方法 (PCR) ，做出雞隻抗體的重鏈及輕鏈變異區基因片 段，將這些基因片段連結後接入 pComb3X 載體建構出 3.2×103 免疫雞隻抗體基 因庫，並將這些基因庫將 scFv 表現在噬菌體上。經過 4 次 panning 步驟，隨機 篩選出 14 個帶有 scFv 噬菌體，利用 ELISA 分析這些帶有 scFv 基因的噬菌體對 於 OVTA-2 蛋白的結合能力，結果得知噬菌體表面帶有的 scFv 抗體片段，對於 OVTA-2 蛋白具有特異性結合能力。進一步利用西方墨點法分析 scFv 抗體片段 對於 OVTA-2 蛋白結合能力，發現這些 scFv 抗體片段也能特異性的辨認 OVTA-2 蛋白。未來希望能進一步運用這些 scFv 抗體片段應用到臨床及實驗研究上，並 成為專一性的診斷試劑。
英文摘要
Ovarian cancer has a high death rate because most patients do not have obvious symptom and lack the proper tumor marker for detection. Previous experiment showed that OVTA-2 protein can be recognized by antibodies of ovary cancer patients so we believe this protein is an ovarian tumor associated antigen. In this research, our aim is to use a recombinant OVTA-2 protein fragment to immunize chicken and constructed an antibody library by phage display technology. By screening of a scFv (single-chain variable fragment) phage library, the OVTA-2
protein specific scFv antibodies can be isolated. The full length OVTA-2 gene is 4150 bp but in this study, we cloned 600 bp DNA fragment into pGEX vector and the recombinant GST-OVTA-2 protein fragment is 35 kDa. After expression in a large amount in E.coli, we used GST Sepharose 4B to purify GST-OVTA-2 protein than use coomassie blue stain and western blot to detect the purified GST-OVTA-2 fusion protein. These purified GST-OVTA-2 fusion protein was mixed with adjuvant and the mixture was injected intramuscularly into Leghorn chicken. Polyclonal IgY
antibodies were purified from the immunized chicken and examined by
enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The result indicated that IgY polyclonal antibodies can recognize GST-OVTA-2 protein
fragment. Chicken''s spleen mRNA was isolated and chicken heavy chain and light chain antibody fragments were generated by use PCR. Antibody fragments were cloned into pComb3X vector and a chicken antibody library was constructed (3.2×103 library size). These antibody fragments were displayed at the phage tail and they were used for phage panning. Fourteen recombinant phages were randomly selected after 4 times panning steps. These phages were analyzed for their binding ability to OVTA-2 protein by ELISA test. Our results showed that several isolated recombinant phages have particular binding ability to GST-OVTA-2 protein. Further scfv was identified binding ability by western blot. We found scFv can recognize OVTA-2 protein fragment specifically. We hope to use these scFv antibody molecule apply to clinic and experiment and study in the future.
