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研究生:江曉玲
研究生(外文):Chiang, Hsiao-Ling
論文名稱:開發增強醣類免疫抗原性之載體
論文名稱(外文):Development of the carrier for the preparation of potent carbohydrate immunogen
指導教授:黃昭蓮施子弼
指導教授(外文):Jaulang HwangDr.Daniel Tzu-Bi Shih
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:細胞及分子生物研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:56
中文關鍵詞:醣類免疫抗原蛋白質載體
外文關鍵詞:carbohydrate immunogenprotein carrier
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醣類在疾病診斷上被認為是重要的標的,大部份細菌或病毒表面之醣類抗原被證明是感染性疾病中最具臨床意義的抗原;而醣化程度的不同,也被視為正常細胞是否轉型成腫瘤細胞的一個指標。因此,腫瘤相關之醣類抗原便相繼被選擇用來作為開發抗癌免疫療法及癌症早期診斷的主要標的。由於醣類抗原的抗原性極低,需要結合至蛋白質載體,以增加其抗原性,但就目前所使用的蛋白質載體中,增強該醣類抗原所引發免疫反應的效益並不理想,使得以醣類抗原為主的抗癌疫苗開發一直難以突破。故此篇論文探討,如何設計一可供醣類抗原有效結合之蛋白質載體,並順利送至抗原呈現細胞產生免疫反應。為達此目的,我們利用線性重覆抗體辨認區段 (Linear Array Epitope, LAE ) 的概念,合成一段可轉譯出富含半胱氨酸 (Cysteine) 的DNA序列,希望能增加醣類在載體上結合的效率,並構築成包含綠膿桿菌(Pseudomonas aeruginosa) 外毒素A之接受器結合區及富含半胱氨酸片段的蛋白質載體。實驗的結果,我們設計的蛋白質載體,確實能讓醣類抗原高效結合,且在西方點墨法及圓點點墨法的實驗中顯示,用此抗原所免疫的老鼠血清,可辨認所結合的醣類抗原。因此,可預期的,我們所設計的抗原載體應可提供一醣類相關疾病研究之良好平台,並解決因醣類低抗原性,而難以生產醣類抗體的研究困難。
Changes in glycosylation are often a hallmark of disease states. Recently, carbohydrates have been proved to the most clinical relevant antigens of the many well-defined antigens for vaccines against infectious diseases. In fact, cancer cells also display glycans at different levels than normal cells. This also leads tumor-associated carbohydrate antigens as one of the targets for immunotherapy and diagnosis of human cancer. Because the immunogenicity of carbohydrate is very weak, it is essential for elevating immune response by conjugating carbohydrate to a protein carrier. Unfortunately, the protein carriers used so far have low conjugating efficiency. It leads the development of carbohydrate-based cancer vaccine becomes a very difficult job. In this study, we design and construct an antigen carrier for carbohydrate conjugating efficiently and delivering into antigen presenting cells. For this propose, we used the idea of Linear-Array-Epitope (LAE) to construct DNA fragments encoding cysteine rich peptide for carbohydrate conjugation, followed by subcloning it into a protein expression vector. The final carrier protein is a fusion protein containing the receptor binding domain of pseudomonas aeruginosa exotoxin A and cysteine rich peptide. Our preliminary study showed that the ratio of carbohydrate conjugated to the protein carrier is great increased when compared to commercial available carbohydrate carrier. We then apply it in animal study. After immunizing the BALB/c mice with this carbohydrate immunogen, the induced antibodies against carbohydrate were examined by western blotting and dot blotting. In this study, we demonstrate that our new designed carrier can offer high conjugating efficiency for various carbohydrates to solve the low immunogenicity of carbohydrate in the generation of anti-carbohydrate
antibody.
中文摘要……………………………………………………… 第i頁
英文摘要……………………………………………………… 第ii頁
目次…………………………………………………………… 第iv頁
圖目錄………………………………………………………… 第vi頁


第壹章 緒論
第一節 醣纇抗原………………………………………… 第1頁
第二節 醣類抗原之蛋白質載體設計概念
一、 抗體產生的機轉………………………………… 第4頁
二、 綠膿桿菌外毒素A與其於疫苗之應用………… 第6頁
三、 線性重複排列抗原 (linear array epitope; LAE)… 第9頁


第貳章 研究材料與方法
第一節 材料與試劑…………………………………… 第12頁
第二節 試劑配方……………………………………… 第14頁
第三節 實驗方法……………………………………… 第18頁


第參章 實驗結果與分析
第一節 重複模板聚合酶鏈鎖反應與轉接端聚合酶鏈鎖反應
…………………………………………………… 第 27頁
第二節 蛋白質載體的產生與純化……………………… 第 28頁
第三節 醣類結合反應效率分析………………………… 第 29頁
第四節 動物免疫實驗與抗體分析……………………… 第 30頁


第肆章 討論
第一節 毒素遞送系統結合線性重覆排列ploycysteine做為醣抗原 之載體…………………………………………… 第 31頁

第二節 醣類結合效率的分析…………………………… 第 32頁
第三節 免疫動物的效益………………………………… 第 34頁
第四節 應用…………………………………………… 第 35頁


第伍章 參考文獻………………………………………… 第 37頁







圖目錄

圖1、PEIa-polycysteine與醣類抗原結合之示意圖
圖2、PEIa-polycysteine-carbohydrate LAE 抗原活化B細胞之機制
圖3、綠膿桿菌外毒素A 的三度空間結構圖及功能區
圖4、綠膿桿菌外毒素A (PE) 的細胞毒化過程
圖5、重複模板聚合酶鏈鎖反應(TR-PCR)的示意圖
圖6、轉接端聚合酶鏈鎖反應(Adaptor PCR)示意圖
圖7、The product of TR-PCR and Adaptor-PCR
圖8、融合蛋白質PEIa-(polycysteine)7 純化結果
圖9、醣類結合反應效率分析
圖10、使用BSA-GM3作西方點墨法分析老鼠血清
圖11、使用GM3作圓點點墨法分析老鼠血清
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