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研究生:夏吉美
研究生(外文):chi-mei Shia
論文名稱: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
論文名稱(外文):Chicken single chain variable fragment recognizing OVTA-2 tumor associated antigen using phage display antibody technology
指導教授:楊沂淵楊沂淵引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:56
中文關鍵詞:抗體片段噬菌體
外文關鍵詞:phage display
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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。
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.
目錄.............................................................................................................Ⅰ
圖表目錄...................................................................................................III
中文摘要.....................................................................................................IV
英文摘要....................................................................................................................V
壹、緒論......................................................................................................1
一、 OVTA-2蛋白簡介...............................................................................1
二、 蛋黃免疫球蛋白 (immunoglobulin Y; IgY) 簡介.............................1
三、 單株抗體簡介及應用.........................................................................2
四、 噬菌體技術重組基因體庫選殖單株抗體簡介與應用……..............3
貳、實驗目的..............................................................................................5
參、實驗材料與方法..................................................................................6
ㄧ、實驗材料........................................................................................6
1. 化學藥品...................................................................................................6
2. 酵素類..........................................................................................7
3. 抗體類.......................................................................................................7
4. 其他...........................................................................................................8
5. 各式溶液.......................................................................................8
二、實驗方法..........................................................................................12
1. 表現以及純化GST-OVTA-2蛋白片段...............................................12
2. 聚丙烯氨膠體(SDS-PAGE)內電泳法.....................................................12
3. 西方墨點法(Western blot).......................................................................13
4. 免疫注射雞隻..........................................................................................13
5. 純化蛋黃內免疫球蛋白(IgY) ................................................................13
6. 檢測 IgY與蛋白結合反應.....................................................................14
7. 雞隻 cDNA 資料庫建構........................................................................14
8. 抗GST-OVTA-2片段蛋白的scFv基因庫的建立..................................15
9. 凝血蛋白酶切除麩胱胺酸-S-轉移酵素蛋白(glutathione S-transferase
protein, GST protein) ..............................................................................16
10. 抗GST-OVTA-2蛋白重組基因庫的篩選.............................................17
11. 帶有Anti-GST-OVTA-2 scFv 蛋白的噬菌體與GST-OVTA-2片段蛋白的ELISA結合反應.............................................................................18
12. 利用Western blot 分析anti-OVTA-2 scFv 和OVTA-2蛋白的結合反應.............................................................................................................18
肆、實驗結果.............................................................................................................19
1. GST-OVTA-2蛋白片段表現以及純化...........................................19
2. 偵測雞隻血清中抗體..............................................................................19
3. 純化IgY抗體..................................................................................... 19
4. ELISA偵測IgY與GST-OVTA-2蛋白特異性結合反應.......................19
5. 分離脾臟並抽取RNA.............................................................................19
6. 抗GST-OVTA-2蛋白的scFv基因庫的建立.........................................20
7. 確認重組DNA送入XL-1 E.coli 中...........................................20
8. 利用限制酶sfi 1確認重組DNA後送入的基因片段............................20
9. 分析凝血蛋白酶作用後GST-OVTA-2融合蛋白..................................20
10. 抗GST-OVTA-2蛋白重組基因庫的篩選.............................................20
11. 抗GST-OVTA-2重組基因庫篩選評估.................................................21
12. 帶有ScFv的噬菌體與GST-OVTA-2蛋白的ELISA結合反應............21
13. 利用西方墨點法偵測scFv和OVTA-2蛋白片段的結合反應.............21
伍、討論.....................................................................................................................22
1. 表現GST-OVTA-2蛋白…...……………........................………....22
2. 免疫注射動物...............................................................................22
3. IgY的純化....................................................................................23
4. 建構scFv重組抗體......................................................................23
5. 抗體篩選分析...............................................................................24
6. ScFv與GST-OVTA-2蛋白的結合反應..................................................24
陸、未來展望............................................................................................26
柒、圖表....................................................................................................27
捌、參考文獻............................................................................................48
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