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研究生:林靜宜
研究生(外文):Jing-Yi Lin
論文名稱:腸病毒71型3C蛋白質與粒線體中細胞色素氧化次單位Ⅱ蛋白質相互作用之探討
論文名稱(外文):Interaction of enterovirus 71 3C protein and the mitochondrial cytochrome c oxidase subunitⅡprotein
指導教授:張晃猷施信如施信如引用關係
指導教授(外文):Hwan-You ChangShin-Ru Shih
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:74
中文關鍵詞:腸病毒71型3C蛋白酶細胞色素氧化次單位Ⅱ蛋白質細胞凋亡粒線體
外文關鍵詞:enterovirus 713C proteasecytochrome c oxidase subunit IIapoptosismitochondria
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本篇論文主要是探討腸病毒71型之3C蛋白質可以和宿主細胞內那些蛋白質有相互作用,且進一步研究其作用機轉。利用酵母菌雙雜交系統來篩選人類腦部cDNA基因庫內那些蛋白質可以和3C蛋白質產生交互作用。我們挑選mitochondria cytochrome c oxidase subunitⅡ(COXⅡ)作為進一步研究之對象,COXⅡ為粒線體酵素複合體Ⅳ中的一個次單位,在呼吸鏈的電子傳遞中扮演著接受ferrocytochrome c傳來之電子,再將其傳給下一個接受者。將3C和會產生綠色螢光之蛋白質構築成融合蛋白質,發現其會進入粒線體中。且當3C短暫的表現在SF268細胞中48小時後會促使COXⅡ蛋白質表現量增加,cytochrome c被釋放至細胞質中,同時也會促使caspase 9、caspase 3活化,使細胞產生細胞凋亡之現象。進一步將腸病毒71型感染至SF268細胞,發現COXⅡ蛋白質表現量增加。在病毒感染6小時後,cytochrome c 釋放至細胞質中,在12小時可以測得到caspase 9及caspase 3被活化。從以上結果來看經由粒線體釋出cytochrome c,活化下游caspase 9、caspase 3最後產生細胞凋亡之現象是腸病毒71型之3C蛋白質導致細胞計畫性死亡的其中一條路徑。或許細胞走向計畫性死亡在腸病毒71型可以導致中樞神經系統的病變中扮演著重要的角色。

The goal of this study was to identify the host cellular proteins interacting with the 3C protein of enterovirus 71. 3C was found to interact with mitochondrial cytochrome c oxidase subunitⅡ(COXⅡ)in the use of yeast two hybrid system from human brain cDNA library. COXⅡ, a subunit of mitochondrial complex Ⅳ, is the acceptor of electrons from ferrocytochrome c. 3C-green fluorescent protein(GFP)fusion protein was found to localized on the mitochondria by confocal microscopy. After transient expression of 3C protein, the release of cytochrome c and the activation of caspase 9 and caspase 3 occurred. On the other hand, cytochrome c was released to the cytoplasm at 6 hour post-infection. Caspase 9 and caspase 3 were also activated after 12 hours. Our findings suggest that the 3C protein is responsible for triggering apoptosis in the SF268 cells by releasing of cytochrome c to the cytoplasm, and than activating caspase 9 and caspase 3, which may play an important role in the pathogenesis of EV71 infection.

中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅱ
第一章 前言………...…………………………..……………………….1
第二章 材料與方法 …………………………….……………………...12
第三章 實驗結果……..……………………….……………………….25
第四章 討論………….………………………….……………………..32
第五章 參考文獻……..……………………….……………………….41
圖表…..…………………………………………………………………47
附錄A 使用試劑…………………………………….……….………...66
附錄B 儀器設備……………………………………………….………72
附錄C 縮寫表………………………………………………….………73

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