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系統識別號 U0026-2608201013420600
論文名稱(中文) 鑑定介白素二十單株抗體辨識介白素二十之抗原決定位
論文名稱(英文) Determination of Epitope on IL-20 Recognized by Anti-IL-20 monoclonal antibodies
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 徐建民
研究生(英文) Chien-Ming Hsu
學號 s1697105
學位類別 碩士
語文別 中文
論文頁數 73頁
口試委員 指導教授-張明熙
口試委員-莊偉哲
口試委員-鄭宏祺
中文關鍵字 介白素  發炎  單株抗體  抗原決定位 
英文關鍵字 interleukin  antibody  epitope 
學科別分類
中文摘要 介白素二十(Interleukin-20, IL-20)是介白素十(Interleukin-10, IL-10)家族之ㄧ的成員,IL-10家族包含了有IL-10、IL-19、IL-20、IL-22、IL-24、IL-26等成員。在IL-10家族中IL-19、IL-20、IL-24共用了相同的受體;其中IL-19、IL-20、IL-24可以透過IL-20R1/IL-20R2進行下游訊息傳遞,而IL-20、IL-24還可透過IL-22R1/IL20-R2進行傳遞。目前有許多研究顯示,在乾癬、類風濕性關節炎、腎衰竭、粥狀動脈硬化,以及缺血性中風等疾病的致病機轉中,IL-20不僅參與其中,同時也扮演著重要的角色。在細胞與動物實驗模式中,IL-20的單株抗體可中和由IL-20所引起的致病發炎反應。由這些實驗結果顯示,IL-20與其單株抗體結合的抗原決定位(epitope)對IL-20 所調控的細胞反應非常重要。因此本研究目標在於鑑定IL-20和其單株抗體的抗原決定位置。首先我們構築並表現各種不同長短序列之IL-20重組蛋白,並經由西方墨點法的實驗證實IL-20與其單株抗體的結合位置。研究結果顯示,這些單株抗體主要來自於兩株不同的母株,一個來自於7E,另一個則來自於C2,他們各自辨認不同的抗原決定位。而先前研究結果已證實7E與IL-20之確切的結合位置,且也證實它確實具有中和致病反應的活性,在此研究中,我們也鑑定出單株抗體C2與IL-20的結合位置。而與7E不同之處在於我們初步證實C2並沒有中和致病反應的作用。同時我也利用狗IL-20更進一步去確定單株抗體7E辨認IL-20的結構為何。這些研究成果對於針對IL-20所開發的單株抗體藥物會帶來莫大的助益。
英文摘要 Interleukin-20 (IL-20) belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-19, IL-20, and IL-24 share their receptor subunits; all three members are capable of signaling through IL-20R1/IL-20R2, while IL-20 and IL-24 can also use IL-22R1/IL-20R2. Previous studies showed that IL-20 is involved in several diseases, such as psoriasis, rheumatoid arthritis, renal failure, atherosclerosis, and ischemic stroke. IL-20-mediated inflammatory responses can be neutralized by its monoclonal antibody 7E in vitro. These results suggested that the binding motif of IL-20 by monoclonal antibody must be very important in the IL-20-mediated cellular response. Therefore, we aimed to determine the epitope on IL-20 recognized by 7E. We performed a serial deletion of IL-20 coding region and expressed the recombinant proteins of these deleted mutants in E. coli. We mapped the epitope of IL-20 which was recognized by 7E by using Western blot. Western blot showed that the monoclonal antibodies, 7E and C2, recognized different binding motif on IL-20. Previously, we determined the binding epitope of IL-20 by 7E and showed that 7E could neutralize IL-20-mediated cellular responses, and also determined the binding epitope of IL-20 by C2 in this study. In contrast to 7E, C2 showed no neutralization activity for IL-20-mediated cellular response. I also used Dog IL-20 as a model to further delineate the structure recognized by 7E. Our study provide information for further development of IL-20-specific targeting therapy.
論文目次 中文摘要 1
Abstract 2
致謝 3
目錄 5
圖表目錄 7
縮寫檢索表 9
第一章 緒論 11
1-1 細胞激素 (Cytokine) 11
1-2 介白素20 (Interleukin-20, IL-20) 之介紹 11
1-3 介白素20與其相關疾病之介紹 12
1-4 單株抗體之介紹 15
1-5 鑑定抗原決定位(epitope mapping)之方法 15
1-6 研究動機與目的 16
第二章 材料與方法 17
2-1 實驗材料 17
A.質體(Vectors) 17
B.菌株(host stain) 17
C.培養液及培養基 28
D.實驗溶劑 19
E.限制酶 22
2-2 實驗方法 23
(1)構築人類截短型IL-20 重組蛋白於pMAL-c2X 載體 23
(2)構築人類突變型IL-20 重組蛋白於pMAL-c2X 載體 26
(3)由E.coli 系統表現人類截短型(突變型)IL-20 重組蛋白 27
(4)利用GeneRacer Kit 解出單株抗體融合瘤細胞全長的核酸序列 27
第三章 實驗結果 34
3-1 表現截短型IL-20蛋白質及分析與IL-20單株抗體7E、C2的結合能力 34
3-2 IL-20單株抗體對其他物種IL-20的辨認性 35
3-3 構築突變型IL-20蛋白質 35
3-4 IL-20單株抗體7E對於狗(dog, Canis familiaris) IL-20之辨認性 35
3-5 構築突變型IL-20蛋白質 36
3-6 構築融合型(Chimeric) IL-20蛋白質 36
3-7 解出人類IL-20單株抗體融合瘤細胞完整的全長序列 37
3-8 製備抗體抗原複合體(antibody-antigen complex) 38
第四章 討論 39
參考文獻 41
附錄 65
自述 73
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