流感病毒血球凝集素(II)膜外區域之物理化學特性分析

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王聖銓(Sheng-Cyuan Wang) 查詢紙本館藏

畢業系所

化學學系

論文名稱

流感病毒血球凝集素(II)膜外區域之物理化學特性分析
(none.)

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摘要(中)

流行性感冒病毒時時對全球生命造成的威脅不容小覷，本文聚焦於流感病毒
表面的膜蛋白分子：血球凝集素2 (hemagglutinin2，HA2)，尤其是其膜外區域
(ectodomain)。針對膜外區域與膜的直接交互作用之探討，以及作為未來可能的
摺合與結構轉換之動力學實驗的基礎數據，本文以數種物理化學方法，嘗試對於
HA2 膜外區域的若干特性進行分析與探討。
首先筆者以衰減全反射紅外線光譜 (ATR-FTIR)、圓二色極譜 (CD)以調查HA2
膜外區域對膜的擾動及其二級結構，我們發現其具有高度α螺旋比例但對膜的本
身並無顯著擾動。接著，作為後續動力學實驗的基礎，筆者利用圓二色極譜數據
加上不同的螢光 (Rhodamine螢光標定，以及樣本本身的色胺酸螢光)作為工具，
觀察蛋白樣本在不同環境下對於變性劑的反應，並找出結構從有到無之間的臨界
點。
除此之外，本文以三種不同的摺合路徑處理樣本並進行實驗以相互比較，各
個路徑樣本雖然大致相同，然而其中表現出許多差異，值得討論比較。另外，筆
者以膠體電泳配合化學聯結劑 (chemical crosslinker)進行了蛋白樣本形成多聚體傾向的一系列調查，發現這段膜外區域蛋白是以單體為主，且形成多聚體的
傾向並不高；多聚體中傾向較高者為雙體及其倍數體，鮮有單純的三聚體存在。
本論文的結果有助於日後摺合動力學實驗的進行以及改善蛋白樣本的製備流程。此外，對於HA2 膜外區域的特性也產生若干推論，增進吾人對其認識與了解。

摘要(英)

The influenza virus have become a global threat for years. In this research, we put the focus on the influenza virus membrane protein hemagglutinin2 (HA2) ectodomain region. Several means and experiment have been executed in order to understand more about the protein, including attenuated total reflection infrared spectroscopy (ATR-FTIR), circular
dichorism (CD), several guanidine (denaturant) titration equilibrium experiments: on CD spectra, on UV spectra of rhodamine (fluorescence emitting group) labeled protein samples, and on the emission spectra of
tryptophan on the protein sample strand. And additionally, the gel electrophoresis experiment.
After the investigation and experiment around the protein sample, the nature of HA2 ectodomain, such as membrane interaction, protein structure,
difference under acidic and neutral buffers, and oligomer-formation
tendency, become more concrete, with little new inferences.
The research gives us a more clear picture of the hemagglutinin protein
ectodomain area. Furthermore, by the guanidine titration data mentioned
above, we are able to accomplish the further works such as the folding
kinetic experiment in the near future.

關鍵字(中)

★ 流行性感冒
★ 血球凝集素

關鍵字(英)

★ protein folding
★ hemagglutinin
★ H5N1

論文目次

目錄
中文摘要 -------------------------------------------------- i
英文摘要 ------------------------------------------------- ii
謝誌 --------------------------------------------------- iii
目錄 ----------------------------------------------------- v
圖目錄 -------------------------------------------------- vii
表目錄 ---------------------------------------------------- x
第一章緒論
1-1 流行性感冒之大流行病史及其潛在之威脅性---------------- 1
1-2 包膜病毒簡介------------------------------------------ 1
1-3 流感病毒之生活史簡介---------------------------------- 2
1-4 流感病毒之膜蛋白以及膜的融合-------------------------- 2
1-5 關於血球凝集素2 (HA2)，各部位簡介--------------------- 4
1-6 研究動機---------------------------------------------- 6
第二章實驗方法與理論依據
2-1 生物分子 (脂質、蛋白質)之紅外線光譜-------------------- 8
2-2 衰減全反射傅立葉轉換紅外線光譜 (Attenuated Total
Reflection Fourier Transformed infrared spectroscopy，
ATR-FTIR)--------------------------------------------- 12
2-3 圓二色極譜儀 (circular dichorism，CD spectroscopy)---- 17
第三章實驗方法
3-1 蛋白質樣品的製備------------------------------------- 23
3-2 單一薄層小囊泡 (small unilamellar visicles, SUV)的製備 29
3-3 ATR-FTIR 實驗操作------------------------------------ 29
3-4 CD 實驗操作------------------------------------------ 31
3-5 Rho-labeled HA2 stacking equilibrium experiment------ 32
3-6 蛋白質在變性劑 (denaturant)變性溶解之色胺酸螢光實驗
(tryptophan fluorescence denaturant titration)------- 33
3-7 多聚體 (oligomer)的分析方法-------------------------- 34
第四章實驗結果
4-1 HA2ΔC 與HA2 wild type 之ATR-FTIR 結果比較------------ 36
4-2 ATR-FTIR 結果---------------------------------------- 39
4-3 CD 實驗結果------------------------------------------- 42
4-4 標定樣本的螢光基團 (rhodamine)對於變性劑的堆疊平衡實驗
(rhodamine labeled samples: stacking equilibrium
experiment against GdnCl)結果------------------------- 49
4-5 HA2ΔC 螢光實驗結果----------------------------------- 51
4-6 形成多聚體的傾向調查--------------------------------- 56
4-7 綜合評述--------------------------------------------- 61
第五章實驗討論
5-1 HA2 ectodomain 對膜的擾動----------------------------- 64
5-2 二級、三級結構---------------------------------------- 66
5-3 對變性劑的耐受性------------------------------------- 67
5-4 Rhodamine 螢光實驗------------------------------------ 68
5-5 色胺酸螢光實驗--------------------------------------- 68
5-6 酸性條件--------------------------------------------- 69
5-7 Folding 路徑與蛋白結構-------------------------------- 71
5-8 形成多聚體的傾向------------------------------------- 73
5-9 流感病毒膜蛋白HA 與人類免疫缺失病毒膜蛋白穿膜次單元
(transmembrane subunit) gp41 就穿膜性質之簡單討論----- 75
5-10 總結------------------------------------------------ 77
第六章參考文獻 79
第七章附錄 83

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指導教授

張定國、謝發坤
(Ding-kuo Chang、Fa-kuen Shieh)

審核日期

2010-8-27

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