探討登革熱病毒套膜蛋白上單醣體GlcNAc對其分子結構及溶解度之影響

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姓名

陳憲璋(Hsien-Chang Chen) 查詢紙本館藏

畢業系所

化學學系

論文名稱

探討登革熱病毒套膜蛋白上單醣體GlcNAc對其分子結構及溶解度之影響
(The investigation of the GlcNAc glycosylation effect on molecular structure and solubility property in type 2 Dengue virus envelop protein)

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摘要(中)

中文摘要
登革熱病毒是黃病毒之一，可以區分為四種血清型，是現今世界經由節肢動物感染人類當中最嚴重之病毒性疾病，是一種急性但會自行痊癒的疾病。全球每年感染登革熱之病例約一億人，其中出血性登革熱之病歷約50萬人。出血性登革熱之死亡率為10-50％，可以說是極可怕的傳染病之一。登革熱病毒利用套膜蛋白的侵蝕作用感染細胞。套膜蛋白為一個醣蛋白，對第二型的登革熱病毒而言，其上有兩個醣基化的天門冬醯氨酸-67與天門冬醯氨酸-153，天門冬醯氨酸-67的醣基為單醣體：N-乙醯葡萄醣氨，這在大多數的醣蛋白是相當大的一個差異。因此對於此單醣體在套膜蛋白所扮演的腳色相當感興趣。在本篇論文中，我們截選了包含天門冬醯氨酸-67這個氨基酸的氨基酸片段，合成出具有醣基化氨基酸片段，與不具醣基化的氨基酸片段，利用液態核磁共振來解出其結構、及觀察醣基化後其水溶性的改變，進而探討此單一醣基登革熱套膜蛋白上的功能。根據二維核磁共振頻譜顯示醣基化之後氨基酸片段會有顯著的影響此胜肽在水溶液中的二級結構，由傾向α-螺旋轉變為傾向β-strain。且根據溶解度的測試發現原本溶解度相當低的胜肽片段醣基化後溶解度大大的升高。

摘要(英)

Dengue virus contains envelope protein that infects cell by membrane fusion. Envelope protein is a glycoprotein. For type 2 Dengue virus, the envelope protein has two glycosylation sites：Asn-67 and Asn-153. The glycan of Asn-67 is monosaccharide, N-Acetyl-glucosamine, that is unique among other glycoproteins. Therefore, we were focusing on the studies of specific role of this monosaccharide in the envelope protein.
In the thesis, we synthesized a number of peptide that covers Asn-67 with glycosylation and without glycosylation. We characterized the protein secondary structure by means of chemical shift index analyses using liquid-state NMR, and to detect the solubility before and after glycosylation. We found that glycosylation serves two functions in the peptide we studied. First, the glycosylation is able to stabilize the peptide secondary structure in a preference of forming β-strain. Secondly, solubility of the peptide is significantly enhanced after glycosylation. This study might help to better understand the biological role of glycosylation in the envelope protein of type 2 Dengue virus.

關鍵字(中)

★ 登革熱
★ 結構
★ 套膜蛋白

關鍵字(英)

★ dengue
★ envelop protein
★ structure

論文目次

章節目錄
中文摘要……………………….………………………..……….……..Ⅰ
英文摘要…………………………….…………………..……….……..Ⅱ
縮寫索引…………………………….………………...……..……….. Ⅳ
圖表目錄……………………………………………..………..…………………..Ⅶ
第一章緒論
1.1 登革熱的歷史…………………………………...…...…..01
1.2 登革熱病毒概論…………………………...……….……02
1.3 研究動機與實驗設計…………………...……….………06
第二章原理簡介
2.1 核磁共振原理 …………………………………………..07
2.2 化學位移…………………………………...……...……..09
2.3 光譜脈衝程序原理……………………………..…………11
2.4 光譜譜峰的指認…………………………………………15
2.5 胺基酸的化學位移…………………………...…………17
2.6 胜肽合成……………………………….…...……………19
2.7蛋白質定量分析：ELISA………………………....………………20
第三章材料與方法
3.1試劑藥品…………………………………………………. 22
3.2 有機實驗方法………………...…………………………..24
3.3 胜肽醣化製備…………………………………………….25
3.4 胜肽樣品製備…………………………………………….29
3.5核磁共振實驗……………………………………………..33 3.6 ELISA定量實驗…………………………………………..34
第四章結果與討論（Ⅰ）胜肽樣品純化與鑑定
4.1 HPLC純化與LCQ MASS鑑定.……………..……………….36
4.2 雙硫鍵鑑定…………………………………...…………..37
第五章結果與討論（Ⅱ）核磁共振實驗結果及溶解度分析
5.1 化學位移標定及分子結構分析……………..……………….49
5.2 溶解度分析……………………………………..……………….51
5.3 醣基化在登革熱病毒套膜蛋白上所扮演的角色………....52
結論…………………………………...……………………………….………….90
參考文獻…………………………………...………………………….91

參考文獻

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指導教授

高憲明、鄒德里
(Hsien-Ming Kao、Der-Lii Tzou)

審核日期

2004-6-21

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