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研究生:韓家偉
研究生(外文):Hon, Kar-Wai
論文名稱:研發對具D-甘露醣基辨識位置的物種之親和抓取方法
論文名稱(外文):Affinity Capture Approaches for the Species with D-Mannose Binding Sites
指導教授:陳月枝陳月枝引用關係
指導教授(外文):Chen, Yu-Chie
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:149
中文關鍵詞:lectinmannosemultivalentEscherichia coliconcanavalin A
外文關鍵詞:凝集素甘露醣多價效應大腸桿菌刀豆蛋白 A
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醣化學在生物系統中具有相當重要的作用,舉凡細胞表面辨識與細菌黏附作用都牽涉到醣的參與。舉例而言,尿道感染是一種相當常見而由大腸桿菌所造成的腸道外感染,致泌尿道感染大腸桿菌會藉由本身的纖毛與宿主細胞表面進行黏附,大約80%的大腸桿菌能表達對甘露醣具辨識能力的Type-I纖毛;除此之外,刀豆蛋白A也具有D-甘露醣基可辨識的位置。在本論文中將發展官能化奈米粒子與官能化染料分子為此類物種之探針,藉此發展新的親和辨識法。
本論文首先討論不同酸根官能基與氧化鋁螯和鍵結的強弱,之後將具磷酸根的雙甘露醣基分子修飾在表面修飾有氧化鋁的磁性奈米粒子上做為親和抓取探針,此親和探針可將稀釋樣品中的目標物進行濃縮,但仍會與非目標物有親和作用,此非特異性吸附之問題尚待克服。在論文第二部分則以多價效應為基礎探討刀豆蛋白A與具有甘露醣基的螢光染料分子之間的辨識反應,實驗結果顯示此合成之染料分子對刀豆蛋白A及大腸桿菌J96具有高度專一辨識性,且同時能夠以肉眼直接觀察目標物之存在,偵測極限可達62.5 pmol左右。
第一章、 緒論……………………………………………… 1
1-1前言…………………………………………………………… 1
1-2研究背景與文獻回顧………………………………………… 3
1-2-1致泌尿道感染菌簡介……………………………………… 3
1-2-1-1泌尿道感染流行病學………………………………… 3
1-2-1-2大腸桿菌引發泌尿道感染之致病機制……………… 4
1-2-1-3影響致泌尿道大腸桿菌Type-I 纖毛表達的因素… 6
1-2-1-4醣類分子在細菌黏附作用及生物分子辨識的簡介… 7
1-2-1-5多價性效應在細菌黏附作用的影響………………… 11
1-2-1-6現行檢測診斷尿道感染菌方法的簡介……………… 13
1-2-2金屬氧化物親和磷酸化分子之機制與應用…………… 17
1-2-2-1金屬氧化物與磷酸根之間的親和作用力……… 17
1-2-2-2溶液酸鹼值的影響……………………………… 20
1-2-2-3鍵結常數與Langmuir-isothermal吸附模型之簡介23
1-2-2-4氧化鋁基材結合磷酸根分子之應用…………… 26
1-2-3親和質譜法簡介………………………………………… 27
1-2-3-1親和質譜法發展歷程簡介……………………… 27
1-2-3-2超順磁性材料之簡介及在生化分析之應用…… 30
1-3論文目標……………………………………………………… 36
第二章、實驗……………………………………………………… 37
2-1實驗器材與藥品……………………………………………… 37
2-1-1藥品……………………………………………………… 37
2-1-2實驗菌種與醣基化有機小分子來源…………………… 38
2-1-3實驗儀器………………………………………………… 39
2-2實驗步驟……………………………………………………… 41
2-2-1磁性奈米粒子之合成…………………………………… 41
2-2-1-1 Fe3O4奈米粒子的合成………………………… 41
2-2-1-2 Fe3O4@SiO2奈米粒子的合成…………………… 41
2-2-1-3 Fe3O4@SiO2@Al2O3奈米粒子的合成…………… 42
2-2-2磁性奈米粒子物理性質之鑑定………………………… 43
2-2-2-1以穿透式電子顯微鏡(TEM)觀察微細結構……… 43
2-2-2-2以超導量子干涉元件磁量儀(SQUID)進行磁性分析43
2-2-2-3以掃描式電子顯微鏡(SEM)觀察微細結構……… 44
2-2-3醣類分子修飾磁性奈米粒子條件優化之探討………… 44
2-2-3-1修飾反應溶劑酸鹼值之挑選…………………… 44
2-3-2-2製作醣基分子檢量線…………………………… 44
2-2-3-3反應平衡時間之測量…………………………… 45
2-3-3-4氧化鋁磁性奈米粒子與醣基分子的鍵結常數之估算45
2-3-3-5修飾磷酸化醣類小分子在氧化鋁磁性奈米粒子上46
2-2-4含甘露醣基之染料對Con A的辨識性之探討………… 46
2-2-4-1不同醣基染料與Con A反應……………………… 46
2-2-4-2甘露醣基染料與Con A之專一性測試…………… 46
2-2-4-2-1以單醣分子對MMF與Con A之反應進行抑制 46
2-2-4-2-2以不同蛋白質與甘露醣基染料反應……… 47
2-2-4-3不同溶液酸鹼值下進行Con A與染料之辨識反應 48
2-2-4-3-1在pH 4.0下進行測試……………………… 48
2-2-4-3-2在pH 7.4下進行測試……………………… 49
2-2-4-4以含不同甘露醣基數量之染料與Con A作用…… 49
2-2-4-5以含甘露醣基染料對E. coli J96進行辨識抓取 50
2-2-4-6以TMF辨識蛋白質混合物後進行膠體電泳分離… 50
2-2-4-6-1 SDS-PAGE膠片之製作(12 %分離膠體)… 51
2-2-4-6-2 SDS-PAGE膠片之製作(4%聚焦膠體)…… 52
2-2-4-6-3蛋白質電泳樣品之製備…………………… 52
2-2-4-6-4蛋白質膠體電泳分析……………………… 53
2-2-4-6-5電泳膠片染色……………………………… 53
2-2-5細菌樣品之萃取………………………………………… 54
2-2-5-1培養基之製備…………………………………… 54
2-2-5-2細菌之保存與活化……………………………… 55
2-2-5-2-1細菌保存…………………………………… 55
2-2-5-2-2細菌活化…………………………………… 55
2-2-5-3酵母菌凝聚反應………………………………… 56
2-2-5-4製作細菌檢量線………………………………… 56
2-2-5-5 DMMP修飾在MNPs對非目標菌抓取能力之測試… 57
2-2-5-6混合菌液配製…………………………………… 57
2-2-5-7 MALDI-TOF MS 的操作條件…………………… 58
2-2-5-7-1 以有機小分子為分析樣品……………… 58
2-2-5-7-2 以細菌為分析樣品……………………… 59
第三章、結果與討論……………………………………………… 60
3-1氧化鋁磁性奈米粒子的物理特性分析……………………… 60
3-1-1穿透式電子顯微鏡影像分析結果……………………… 60
3-1-2以超導量子干涉元件磁量儀進行磁性分析…………… 61
3-1-3以X光能量散佈分析儀進行元素分析………………… 63
3-1-4萃取蛋白質酵素消化之磷酸化胜肽片段測試氧化鋁之包覆63
3-2醣基分子修飾磁性奈米粒子條件優化之探討……………… 65
3-2-1醣基分子之質譜鑑定…………………………………… 65
3-2-2溶液酸鹼值對醣基化磁性奈米粒子修飾最佳化之影響 68
3-2-3醣基分子的檢量線……………………………………… 70
3-2-4醣基化磁性奈米粒子修飾反應平衡時間探討………… 72
3-2-5探討含不同磷酸根數目之醣類小分子和氧化鋁之鍵結常數73
3-3以醣基化氧化鋁磁性奈米粒子為細菌抓取探針…………… 79
3-3-1細菌檢量線之製作……………………………………… 79
3-3-2親和抓取細菌之平衡時間……………………………… 80
3-3-3酵母菌凝聚反應檢測大腸桿菌type-I纖毛的表達…… 82
3-3-4細菌之MALDI-MS指紋質譜圖…………………………… 85
3-3-5親和抓取細菌結果……………………………………… 86
3-3-5-1以單一菌種為分析樣品………………………… 86
3-3-5-2以混合菌液為分析樣品………………………… 89
3-3-6以質譜分析確認DMMP分子量…………………………… 96
3-4含甘露醣基之染料對Con A辨識性之探討………………… 100
3-4-1不同醣基化染料分子對Con A之辨識性……………… 100
3-4-2甘露醣基染料與Con A專一性測試…………………… 103
3-4-3溶液酸鹼值對染料與Con A辨識反應之影響………… 109
3-4-4探討含不同數目甘露醣基染料分子和Con A作用效應 114
3-4-5以醣基化染料純化分離蛋白質混合物中之Con A…… 116
3-4-6甘露醣基染料對Con A之偵測極限…………………… 121
3-4-7以醣基化染料對表達Type-I纖毛之E.coli J96進行辨識125
第四章、結論…………………………………………………… 132
第五章、參考文獻……………………………………………… 133

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