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研究生:劉豫寧
研究生(外文):Yu-Ning Liu
論文名稱:利用螢光共振能量轉移技術研究細胞內第七十一型腸病毒2A蛋白酵素的活性
論文名稱(外文):Use of Fluorescence Resonance Energy Transfer Technique to Investigate Enterovirus 71 2A Protease Activity In Vivo
指導教授:龔思豪
指導教授(外文):Szu-Hao Kung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:88
中文關鍵詞:腸病毒71型2A蛋白酵素螢光共振能量轉移高效能篩檢
外文關鍵詞:EV712A proteaseFREThigh-throughput screening
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  • 被引用被引用:1
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在1998年台灣地區爆發腸病毒感染,之後並有數次零星的爆發。腸病毒71型是造成這些病毒感染的主要病原;引起腦炎、肌肉麻痺及許多致死病例。目前已經證實了腸病毒71型2A蛋白酶對病毒複製及造成細胞病變都扮演著關鍵決定的角色;因此也更加彰顯了細胞內2A蛋白酶活性探討之重要性。
在用來研究細胞內蛋白質間作用的光譜學方法中,螢光共振能量移轉技術為一種極具潛力的技術。此方法已經應用在測量細胞內蛋白酶的作用。該方法的原理為被激發的螢光提供者,因為與螢光接受者的距離改變,而導致螢光接受者發射出不同波長的光譜。所以,我們擬建構帶有綠色螢光基因及紅色螢光基因的質體,兩個螢光基因間接有2A蛋白酶的切割序列,並將該質體穩定轉染一可被腸病毒71型感染的細胞,並將此穩定細胞株命名為HeLa-GsR。在488nm波長激發下可以看到HeLa-GsR表現出“橙色螢光”,這表示綠色螢光蛋白與紅色螢光蛋白之間產生了螢光共振能量轉移的現象。我們以腸病毒71型感染HeLa-GsR細胞後12小時即可觀察到受感染的細胞呈現綠色螢光,推測這可能是由於細胞內的融合螢光蛋白受到腸病毒71型2A蛋白酶的切割,因而使得兩個報告蛋白被分開同時也終止了這兩者之間的螢光共振能量轉移作用。我們也以抗綠色螢光蛋白之抗體來做西方墨點法的分析進一步地證實融合蛋白專一性的切割。而利用螢光分析儀(Victor2)來定量FRET現象顯示了當以0.5、2、4 M.O.I.之腸病毒71型感染細胞之後,FRET現象分別達到51.6%、58.4%、67.6%的減少,這指出病毒感染對細胞內FRET現象有劑量依賴效應。另外,我們選用腸病毒屬中的其它種病毒感染HeLa-GsR細胞,也可觀察到FRET現象遭到破壞;而接種與腸病毒不相關的病毒則FRET現象不會被阻斷。
此系統可用來研究在單一細胞內病毒蛋白酶的作用,也可鑑定存有一般腸病毒的檢體,並可作為高效能篩檢抗2A病毒藥的平台。
An outbreak associated with enterovirus infections occurred in Taiwan area during the year of 1997, with moderate recurrences in the following years. Enterovirus type 71 (EV71) has been shown to play a major role in the severe syndromes, including encephalitis and muscular paralysis, as well as in the fatal cases during the outbreak. The EV71 2A protease was demonstrated to be a key protease responsible for essential steps in viral replication and pathogenesis in the infected cells, highlighting the importance of studying the 2A protease activity in a cell.
The fluorescence resonance energy transfer (FRET) technology has been one of the most promising spectroscopic tools for studying protein-protein interactions in living cells. By this methodology, cellular processes can be evidenced by spectral changes of the emission signal because of distance-dependent energy transfer from an excited fluorophore donor to a long-wavelength fluorophore acceptor. Hence, a recombinant construct composed of green fluorescent protein (GFP2) and red fluorescent protein (DsRed2), with a linker peptide containing the 2A protease cleavage sequences were constructed, followed by stable transfection in a cell line susceptible to EV71 infection, resulting a stable cell line designated HeLa-GsR. The HeLa-GsR cells displayed “orange fluorescence” under the excitation wavelength at 488 nm, indicating the generation of FRET effect between the GFP2 and DsRed2. As early as 18 h following infection of HeLa-GsR cells by EV71, the infected cells exhibited green fluorescence; this is presumably due to the cleavage of the fusion protein in vivo by EV71 2A protease, thereby separating the two reporter proteins and terminating FRET between the two labels. The specific cleavage of the fusion protein was substantiated by Western blot analysis using anti-GFP antibody. Quantitative measurement of the FRET phenomenon by a fluorometer (Victor2) revealed that the FRET effects were decreased to 51.6%, 58.4% and 67.6% following infection of EV71 with the multiplicity of infection at 0.5, 2 and 4, respectively, indicating a does-dependent effect. Infection with viruses that belong to enterovirus genus also disrupted the FRET, whereas inoculation with a distantly related virus did not appear to disrupt the FRET.
The system would be a valuable tool to investigate the action of a viral protease in a single cell, to identify specimens containing enteroviruses in general, as well as to serve as a miniaturized platform for high-throughput screening for anti-2A protease compounds.
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