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標題: | 幽門螺旋桿菌基因 hp0499 製備與性質探討 Preparation and characterization of Helicobacter pylori gene hp0499 |
作者: | Chih-Chia Chang 張志嘉 |
指導教授: | 林俊宏 |
關鍵字: | 幽門螺旋桿菌,細胞毒素相關蛋白A,膽固醇-6-O-醯基-α-D-口比喃葡萄糖?,磷酸脂?A1, Helicobacter pylori,Cag A,CAG,phospholipaseA1, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 幽門螺旋桿菌主要生存在於人體胃部及十二指腸,主要會引起胃部的發炎、潰瘍、癌化,以及十二指腸的潰瘍等。當細菌附著於胃部上皮細胞時會利用第四型分泌系統將細胞毒素相關蛋白A ( Cytotoxin associated gene A, Cag A)送入宿主上皮細胞內,並被宿主細胞內的Src激酶在蛋白進行磷酸化作用,經過一系列下游之訊息傳遞作用促使細胞轉變成癌化細胞。
由於缺乏相關的生合成基因,當幽門螺旋桿菌會由環境中攝取膽固醇,並利用自身膽固醇-a-葡萄糖轉移酶 ( Cholesterol-α-glucosyltransferase )將膽固醇的第三位置羥基醣化轉變為膽固醇-α-D-口比喃葡糖苷 ( Cholesteryl-α-D-glucopyranoside,CG ),之後經過進一步修飾作用轉變為膽固醇-6-O-醯基-α-D-口比喃葡糖苷( Cholesteryl-6-O-acyl-α-D-glucopyranoside,CAG )、膽固醇-6-O-磷脂-α-D-口比喃葡糖苷 (Cholesteryl-6-O-phosphatidyl-α-D-glucopyranoside,CPG ),這些膽固醇糖苷類衍生物經過證實可使幽門螺旋桿菌避免被巨噬細胞吞噬,但是如何將CG乙醯化成為CAG的酵素並不清楚。 幽門螺旋桿菌hp0499基因在早期文獻中透過基因序列比對以及基因剔除實驗間接證實HP0499擁有磷酸脂酶A 1活性,該蛋白為一膜蛋白白可能鑲嵌在幽門螺旋桿菌外膜上。本論文中著重在測試不同的條件純化HP0499,最後利用界面活性劑純化出該蛋白,並利用帶螢光標定之CG受質,將此受質配合不同的磷酸脂類進行活性測試或催化反應,並利用LC/MS/MS及HPLC偵測產物生成,直接證實HP0499為擁有外膜磷酸脂酶A1及CAG合成酶之雙功能酵素。隨後進行一系列生化特性分析:包括酵素穩定度、溫度、pH、金屬離子、受質專一性,最終利用最佳條件進行酵素動力學探討,這些分析希望可以找出HP0499最適當的反應條件,也希望利用其反應條件產生擁有不同飽和程度及碳鏈長之CAG。 Helicobacter pylori is a Gram-negative, microaerophilic bacterium present in the human stomach. H pylori infection has been largely associated to the development of duodenal ulcer and stomach cancer. Long-term H. pylori infection of the gastric mucosa might cause gastric cancer via severe inflammation, but no direct molecular link was demonstrated until Hatakeyama reported that the transfer of H. pylori-derived Cytotoxin associated gene A (Cag A) to epithelial cells through a bacterial type IV secretion system promote an early event of gastric carcinogenesis. This pathogen hijacks cholesterol from the host and biosynthesis cholesteryl glucoside derivatives (CGds). In general, CGds are composed of cholesteryl α-D-glucopyranoside (CG), cholesteryl 6-O-phosphatidyl-α- D-glucopyranoside (CPG) and cholesteryl 6-O-acyl-α-D-glucopyranoside (CAG). Previous reports indicated that CGds were essential for impeding H. pylori phagocytosis and T cell activation. Our lab has recently discovered that CAG enhances the translocation of the CagA protein in to the host cell. The known enzyme cholesterol glucosyltrasferase catalyzes the formation of CG that is then converted to CAG by a possible enzyme, HP0499. In order to determine and characterize the function of HP0499, it is essential to prepare the recombinant HP0499 in E. coli. However, the major challenge was mainly attributed to poor purification. The successful expression and purification of HP0499 by detergent allowed us to obtain more pure recombinant protein with an improved yield. An HPLC and LC/MS/MS assay was established to use fluorophore-containing CG analogue, to study the enzyme function, activity, and substrate specificity. Finally, we identified that HP0499 has both phospholipaseA1 and CAG synthase activities. The standardized conditions can be further used to prepare CAG with different fatty acid chain lengths and various saturations. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52216 |
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顯示於系所單位: | 生化科學研究所 |
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