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研究生:黃鈺宏
研究生(外文):Yu Hung Huang
論文名稱:人類多瘤性病毒JCV主要及次要殼體蛋白於病毒顆粒組裝之研究
論文名稱(外文):Investigation of the major and minor structural proteins, VP1 and VP2, of human JC virus in virus particle assembly
指導教授:李宣佑李宣佑引用關係張德卿
指導教授(外文):Shuan Yow LiDeching Chang
學位類別:碩士
校院名稱:中山醫學院
系所名稱:醫學研究所
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:2001
畢業學年度:89
語文別:中文
論文頁數:54
中文關鍵詞:多瘤性病毒結構蛋白組裝
外文關鍵詞:polyomavirusstructure proteinassembly
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中文摘要
在人類多瘤性病毒JCV的病毒殼體內,包含有結構蛋白VP1、VP2、VP3和病毒的DNA。目前仍不清楚次要結構蛋白VP2和VP3在病毒殼體扮演什麼角色。在我們的研究中,JC病毒主要殼体蛋白VP1和次要殼体蛋白VP2基因己被選殖到酵母菌細胞,且VP1和VP2蛋白質同時在酵母菌細胞內表達後可自我組裝為類似殼体構造(capsid-like particle),可與人類O型紅血球細胞產生凝集。利用病毒顆粒特定沉降系數的特性,以10-50%蔗糖梯度離心可純化出此病毒顆粒。在電子顯微鏡觀察下,其外形與真實的病毒顆粒virion相似。在比較EGTA及DTT同時處理單獨VP1與VP1VP2所組成的殼体時,VP1殼体會快速被瓦解為capsomere,而VP1VP2的殼體則有抵抗被瓦解的能力,推測VP2可能參與穩定病毒殼體的功能。另外,以pseudocapsid攜帶外生性DNA到猴子腎臟細胞,也發現有VP2存在時,可提高殼體運送DNA的能力。未來我們可藉由此酵母菌的表達系統,進一步探討結構蛋白之間與結構蛋白和病毒DNA之間的關係。
Abstract
The human polyomavirus, JC virus, contains three capsid proteins, VP1, VP2, VP3 and a viral minichromosome. Interactions of these three capsid proteins for virus assembly are not well understood. In the current study, the major capsid protein VP1 and minor capsid protein VP2 of JC virus, have been cloned and expressed in yeast cells. Yeast expression system was employed to co-express VP1 and VP2 proteins for facilitating understanding the function of minor structural protein VP2. When VP1 and VP2 gene were co-transformed into yeast cells, both proteins were expressed and detected by their own monospecific antibodies. VP1 and VP2 proteins co-expressed in yeast were able to cause hemagglutination. Self-assembled capsid-like particles were purified by 10-50% sucrose gradient centrifugation and morphology of the particles were observed by electron microscopy. The VP1 capsid could be disrupted into pentameric capsomeres in the presence of both EGTA and DTT but the VP1VP2 capsid was more resistant to the disruption at the same conditions. These findings indicate that VP2 may play a role in stablizing capsid structure. Furthermore, the efficiency of DNA packaging and delivery into monkey kidney COS-7 cell of VP1VP2 capsid-like particles was increased. The yeast co-expression system will be further employed to investigate protein-protein and DNA-protein interactions during virus maturation.
目 錄
頁次
誌謝 1
中文摘要 2
英文摘要 3
人類多瘤性病毒背景介紹 4
本研究相關背景介紹 9
材料與方法 11
結果 22
討論 28
參考文獻 32
圖表 42
參考文獻
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