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研究生:江晨暐
研究生(外文):Chen-Wei Chiang
論文名稱:探討抗α-異烯醇酶單株抗體抑制白色念珠菌之作用機制
論文名稱(外文):Investigation of Monoclonal Antibody against Candida albicans Alpha-Enolase
指導教授:呂思潔呂思潔引用關係
指導教授(外文):Sy-Jye Leu
口試委員:蘇慶華李雨青林良宗
口試委員(外文):Su, Ching-HuaYu-Ching LeeLiang-Tzung Lin
口試日期:2015-07-14
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:93
中文關鍵詞:α-異烯醇酶白色念珠菌抗體
外文關鍵詞:Alpha-EnolaseCandida albicansAntibodies
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白色念珠菌(Candida albicans)對人類是一種伺機性病原,喜愛酸性環境並常存於人體的口腔、皮膚、消化道、陰道等黏膜組織和臟器中。一般情況下,寄生於人體的念珠菌成酵母細胞型,不會引起疾病。但若宿主處在某些病理或生理因素影響下,導致免疫力降低時,白色念珠菌即大量繁殖成假菌絲型,侵犯人體組織,引起其他的併發症及造成高死亡率。近數十年來,患有免疫功能有缺陷病人族群如器官移植、血液腫瘤、愛滋病、接受免疫抑制劑或化學治療等病人的增加,C. albicans已是國內外院內感染最重要的致病菌之一。在美國,念珠菌菌屬為造成院內血液感染的第四名,也造成了49%的死亡率,在台灣更是蟬連四年(2011-2014) 院內感染菌種的第一名。烯醇酶(Enolase),也被稱為磷酸丙酮酸水合酶(phosphopyruvate hydratase),大量存在於人體組織器官中,在糖化和發酵反應中都扮演了重要的角色。此外,Alpha-enolase (ENO1)在細胞表面上也會和plasminogen交互作用,加強tissure-plasminogen activator的表現和保護plasminogen不被α2-plasmin inhibitor抑制。 近年來累積的學術文件中也表明ENO1與病原菌感染宿主有關聯。可能機制為ENO1在許多病原體中扮演胞漿素原受器(plasminogen receptor) 的角色,並利用位於細胞表面的ENO1 蛋白去捕捉宿主的plasminogen,而病原體細胞表面的tissue plasminogen activator (tPA) 或urokinase plasminogen activator (uPA) 會將plasminogen活化成plasmin,進而降解宿主細胞胞外基質(Extracellular matrix, ECM),幫助病原體侵入細胞並感染宿主。
在實驗室先前的研究中,用噬菌體展現技術成功篩選出對白色念珠菌的α-異烯醇酶(CaENO1) 具有特異性的單株抗體(CaS1 scFv),並證實CaS1 scFv可降低CaENO1與plasminogen的結合力,進而減少C. albicans的感染力(Po-Yen Liao, unpublished data)。但CaS1 scFv與CaENO1之間,還有CaENO1與plasminogen之間的交互作用還不明瞭。本計畫運用不同的分子生物實驗進一步探討CaS1 scFv對CaENO1的結合能力,計算出CaS1 scFv的EC50 (KD)為1.88 × 10-8 M 。接著也經由實驗證實CaS1 scFv可以抑制C. albicans菌絲的生長。再來進行CaS1 scFv與 CaENO1結合的抗原決定位分析,找出抗原決定位為240KGKVGIAMDV249 和278PQLADLYEQLISEYP292。此外,我們也將CaENO1和plasminogen之間的結合區域範圍縮小至300AEDDWD-------VGLRSG437 (137個胺基酸),並堆測CaS1 scFv在其中所扮演的角色。最後也成功將CaS1 scFv人源化(humanization) 並表現純化出hzCaS1 V1 and V3,實驗結果證實humanized CaS1 scFv保有對CaENO1的特異性結合能力。進而計算出hzCaS1 V1 and V3 scFv的EC50 (KD)分別為4.6 × 10-8 M 和8.4 × 10-8 M 。最後我們以matrix-gel degradation assay 證實CaS1、hzCaS1 V1 and V3 scFv確實可以抑制CaENO1和plasminogen的結合,並可能經由此路徑,降低C. albicans的感染能力。綜合以上結果顯示,此單株抗體有發展成為臨床抗體治療藥物和檢驗試劑的淺力。並當作日後進一步發展此單株抗體運用於對病原體檢測或標靶治療等臨床應用價值的參考。
Candida albicans is an opportunistic human pathogen, which colonizes at several sites including skin, oral, gastrointestinal track, vagina, mucosa and organ. C. albicans is also a major pathogen causing clinical candidemia. Candidemia is a fungal infection that can occur when Candida yeasts enter the bloodstream, and it is extremely rare in healthy individuals. In recent decades, due to a defective immune patient population such as organ transplantation, leukemia, receiving immunosuppressive, or chemotherapy and AIDS patients increasing, Candidemia becomes an important issue. Candidemia has a very significant morbidity and mortality. In the United States, Candida species are the fourth leading cause of of nosocomial bloodstream infection (BSI) in US hospitals. In Taiwan, Candida species are also a very important sourse of nosocomial bloodstream infection in hospitals.
Enolase, is glycolytic enzyme expressed in most tissues, one of the isozymes of enolase. Alpha-Enolase (ENO1) is a key glycolytic enzyme that acts as a 2-phospho-D-glycerate hydrolase in the cytoplasm of prokaryotic and eukaryotic cells. On the cell surface, interaction of plasminogen with ENO1 enhances its activation by tissue-plasminogen activator and protects plasminogen from inhibition by α2-plasmin inhibitor (α2-PI). In recent study, many pathogens use extracellular ENO1 to capture plasminogen, potentially allowing bacteria to acquire surface-associated proteolytic activity. Consequently, bacterial invasion and dissemination in the infected host is markedly facilitated.
In previous study, anti-Candada albicans ENO1 scFv antibody (CaS1) was isolated by phage display, and confirmed that CaS1 scFv could inhibit interaction of Candada albicans ENO1 (CaENO1) with plasminogen (Po-Yen Liao unpublished data), but the interaction (epitope) of CaENO1 with CaS1 scFv is not clear. In this study, we focus on understanding the role of CaS1 scFv interaction between CaENO1 and plasminogen. CaS1 scFv was characterized and the binding affinity (EC50) of CaS1 scFv is 1.31 × 10-8 M. We also confirmed that CaS1 scFv has influence on C. albicans infection through inhibition of hyphae growth. The epitope of CaENO1 recognized by CaS1 scFv was mapped to 240KGKVGIAMDV249 and 278PQLADLYEQLISEYP292. Moreover, the binding site of CaENO1 with plasminogen was mapped to location 300AEDDWD-------VGLRSG437 (137 amino acids). Furthermore, humanized CaS1 scFv (V1 and V3) were constructed and expressed successfully. Both of humanized CaS1 scFv (V1 and V3) have binding ability against CaENO1. Binding affinity (EC50) of hzCaS1 V1 and V3 scFv were 4.56 × 10-8 M and 3.8 × 10-8 M, respectively. Finally, we confirmed hzCaS1 V1, V3 and CaS1 scFv could inhibit CaENO1 binding to plasminogen by matrix-gel degradation assay.
According to the results of this study, CaS1 scFv could possess application to detect and treat C. albicans infection in the future.
目錄 I
圖目錄 IV
表目錄 VI
中文摘要 1
Abstract 3
壹、緒論 5
一、白色念珠菌(Candida albicans)及其臨床上的意義 5
二、Alpha-異烯醇酶 (Alpha-Enolase) 蛋白 6
三、抗體與免疫反應 7
四、來亨雞動物模式與其抗體IgY 8
五、單株抗體 8
六、噬菌體展現(Phage display)技術 9
七、抗Candida albicans ENO1 (CaENO1)多株與單株抗體的製備 10
貳、研究目的 11
參、實驗材料 12
肆、實驗方法 18
一、表現及純化recombinant CaENO1和CaS1 scFv 18
二、酵素免疫分析法分析CaS1 scFv與CaENO1的結合能力(EC50) 19
三、CaS1 scFv結合CaENO1的競爭性酵素免疫反應分析 20
四、競爭性酵素免疫反應分析CaS1 scFv與CaENO1的結合能力(EC50) 20
五、CaS1 scFv對白色念珠菌菌絲生長抑制實驗 21
六、CaENO1片段蛋白建構與表現 21
七、利用CaENO1片段蛋白對CaS1 scFv進行抗原決定位分析 22
八、分析CaS1 scFv 對不同種系的ENO1結合能力 23
九、利用直接性位點突變方式Site directed mutagenesis (Kunkel method) 建構特定胜肽表現噬菌體 23
十、利用特定胜肽表現噬菌體對CaS1 scFv進行抗原決定位分析 25
十一、CaS1 scFv- CaENO1複合體結構模擬分析 25
十二、分析CaENO1與plasminogen的作用位點 26
十四、分析hzCaS1 V1 and V3 scFv與CaENO1結合能力 27
十五、分析hzCaS1 V1 and V3 scFv與C. albicans ENO1結合能力 27
十六、酵素免疫分析法分析hzCaS1 V1 and V3 scFv的結合能力(EC50) 28
十七、分析CaS1, hzCaS1 V1 and V3 scFv能否抑制CaENO1和plasminogen結合 28
伍、實驗結果 30
一、表現及純化recombinant CaENO1和CaS1 scFv 30
二、酵素免疫分析法分析CaS1 scFv與CaENO1的結合能力 30
三、CaS1 scFv結合CaENO1的競爭性酵素免疫反應分析 31
四、CaS1 scFv的結合能力測定(EC50) 31
五、CaS1 scFv可以抑制白色念珠菌菌絲生長 32
六、CaENO1片段蛋白建構與表現 32
七、利用CaENO1 片段蛋白對CaS1 scFv進行抗原決定位分析 33
八、分析CaS1 scFv 對不同種系的ENO1結合能力 34
九、利用直接性位點突變方式site directed mutagenesis (Kunkel method) 建構特定胜肽表現噬菌體 35
十、利用特定胜肽表現噬菌體對CaS1 scFv進行抗原決定位分析 35
十一、CaS1 scFv- CaENO1複合體結構模擬分析 36
十二、分析CaENO1與plasminogen的作用位點 36
十三、設計與表現人源化 (humanized) CaS1 scFv 37
十四、分析hzCaS1 V1 and V3 scFv與CaENO1結合能力 38
十五、分析hzCaS1 V1 and V3 scFv與C. albicans ENO1結合能力 39
十六、hzCaS1 V1 and V3 scFv的結合能力測定(EC50) 39
十七、CaS1、hzCaS1 V1 and V3 scFv可抑制CaENO1和plasminogen 結合 40
陸、討論 41
捌、圖表 47
玖、附錄 75
拾、參考資料 78
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