臺灣博碩士論文加值系統

胃幽門螺旋菌(Helicobacter pylori)為微需氧的革蘭氏陰性菌，可抵擋胃酸的環境，其感染會導致慢性胃炎，並可能演變成胃潰瘍(gastric ulcer)、十二指腸潰瘍(duodenal ulcer)及胃癌(gastric cancer)。在先前的研究指出，膽固醇對於胃幽門螺旋菌的存活扮演著相當重要的角色，HP0421基因所合成的膽固醇糖化轉移酵素(cholesterol-α-glucosyltransferase, αCGase)，可以從宿主表皮細胞所吸收的膽固醇加上葡萄糖，在細菌細胞壁脂質層上形成cholesterol glucosides，然後呈現在胃幽門螺旋菌的細胞膜上，重要是它可避免巨噬細胞的吞噬作用、T細胞的活化及細菌的生長，這個反應動作在胃幽門螺旋菌去躲避人體的免疫反應有相當大的關聯性。胃幽門螺旋菌在感染細胞時較傾向結合於富含膽固醇之脂筏(lipid rafts)，透過脂筏的形成而結合上胃表皮細胞，通常在脂筏上富含膽固醇和磷脂質。研究發現缺乏αCGase酵素的胃幽門螺旋菌之菌株在胃部不易形成脂筏，而較難感染胃部的細胞，這表示脂筏對於胃幽門螺旋菌在感染機制上扮演著重要的角色。本研究想探討在人工脂質膜上，αCG的存在是否會影響到脂質膜的流動性。我們利用重組蛋白質αCGase與螢光標記的膽固醇去合成含有螢光的αCG。藉由含有膽固醇的微脂體，我們發現膽固醇可以促使相位分離(phase separation)導致形成Lo與Ld區域。共軛聚焦顯微鏡觀察含有螢光αCG的微脂體發現，在Lo區域比Ld區域含有更多的螢光αCG之分佈。螢光相關光譜分析發現DiIC18的擴散係數(diffusion coefficient)是沒有差異性的。為了更深入探討αCGase酵素活性，我們純化出不同缺失片段的αCGase蛋白質，發現均沒有酵素活性反應。猜測完整的胺基酸序列對於αCGase酵素反應功能上是相對重要的。而螢光αCG大部分均分佈在Lo區域，在這區域可能影響脂質膜的流動性。

目錄

中文摘要 Ⅰ
英文摘要(Abstract) Ⅱ
目錄 Ⅲ
圖目錄 Ⅶ
表目錄 Ⅷ

Part-Ⅰ、糖化膽固醇影響微脂體膜流動性之研究
第一章、前言
1.1、胃幽門螺旋菌的發現及特徵 1
1.2、流行病學 1
1.3、感染途徑及其疾病之相關性 2
1.4、胃幽門螺旋菌之治療 2
1.5、胃幽門螺旋菌之膽固醇糖化轉移酵素(Cholesterol-α-glucosyltransferase) 2
1.6、微脂體(Giant unilamellar vesicles, GUVs)的生成與性質 4
1.7、生物細胞膜 4
1.8、研究目的 5

第二章、材料與方法
2.1、微脂體製備 7
2.1.1、實驗藥品 7
2.1.2、樣品製備流程 7
2.1.3、微脂體電製法(Liposome electroformation) 8
2.1.4、清潔ITO導電玻璃 8
2.2、蛋白質的表現與純化 9
2.2.1、重組蛋白質的表現 9
2.2.2、重組蛋白質的純化 9
2.2.3、重組蛋白質的濃縮 10
2.2.4、重組蛋白質的分析 10
2.3、分離且純化之螢光含糖膽固醇衍生物 10
2.3.1、利用重組蛋白αCGase合成NBD-αCG 11
2.3.2、利用薄層色層分析法分離及純化NBD-αCG 11
2.4、利用薄層色層分析法分析胃幽門螺旋菌之αCGase活性 11
2.5、螢光相關光譜測量微脂體 12

第三章、結果
3.1、重組之糖化膽固醇轉移酵素的活性測試 14
3.2、重組蛋白質αCGase合成與純化NBD-αCG 14
3.3、胃幽門螺旋菌合成之NBD-αCGs 14
3.4、微脂體(Giant unilamellar vesicles, GUVs)的生成 15
3.5、不同濃度的膽固醇促使細胞膜形成相位分離(phase separation)的變化 15
3.6、利用螢光相關光譜測量不同濃度的膽固醇及糖化膽固醇之膜的流動性 16
3.7、不同αCGase缺失片段之重組蛋白質純化分析 16
3.8、不同αCGase缺失片段之重組蛋白質活性分析 17

第四章、討論
4.1、糖化膽固醇轉移酵素之活性探討 18
4.2、膽固醇促使微脂體的相位分離 18
4.3、螢光相關光譜的測定 20

第五章、參考文獻

Part-Ⅱ、離胺酸消旋酵素之結晶及繞射分析

中文摘要 38
英文摘要(Abstract) 39

第一章、前言
1.1、土壤多元體基因庫(Soil metagenome library) 40
1.2、基因庫之篩選 40
1.3、Lysine之簡介 40
1.4、Lysine racemase 41
1.5、研究目的 41

第二章、材料與方法
2.1、質體與菌株 43
2.2、蛋白質表現 43
2.3、蛋白質純化 43
2.4、蛋白質濃縮 44
2.5、蛋白質濃度測定 44
2.6、晶體培養 45
2.7、X-ray繞射數據的收集 45
2.8、X-ray繞射數據的處理 46

第三章、結果
3.1、蛋白質序列比對 47
3.2、蛋白質表現 47
3.3、晶體培養 47
3.4、X-ray晶體繞射實驗 48

第四章、討論
4.1、Lysine racemase蛋白質序列之探討 50

第五章、參考文獻













圖目錄

Part-Ⅰ
圖一、胃幽門螺旋菌之膽固醇糖化轉移酵素(αCGase)催化反應及其衍生物 25
圖二、脂質與螢光膽固醇分子結構圖 26
圖三、微脂體製備流程示意圖 27
圖四、薄層色層分析法(TLC)分析重組蛋白質αCGase的活性 28
圖五、薄層色層分析法(TLC)分析重組蛋白質αCGase合成之NBD-αCG 29
圖六、薄層色層分析法(TLC)分析胃幽門螺旋菌合成NBD-αCGs 30
圖七、以共軛焦微鏡觀察不同成分膽固醇之GUVs的位相(phase)變化 31
圖八、利用螢光相關光譜(FCS)觀察GUVs之不同濃度膽固醇膜及糖化膽固醇的流動性 32
圖九、不同αCGase缺失片段蛋白質序列比對與SDS-PAGE蛋白質電泳分析 33
圖十、薄層色層分析法(TLC)分析不同αCGase缺失片段之活性 35

Part-Ⅱ
圖一、Lysine racemase與Deinococcus radiodurans的NAAAR之蛋白質序列比對 53
圖二、Lysine racemase與E. coli K12的NAGPR之蛋白質序列比對 54
圖三、Lyr蛋白質電泳分析 55
圖四、初步篩選所得到的Lyr晶體 56
圖五、Lyr晶體及X光繞射圖 57



表目錄
Part-Ⅰ
表一、微脂體的成分比例與其擴散係數 36

Part-Ⅱ
表一、Lysine rasemase晶體繞射資料（Crystallographic statistics） 58

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