臺灣博碩士論文加值系統

神經胺酸甘脢(sialidase) 是一種可以切除醣類分子上的唾液酸之水解酵素，主要分佈在高等動物、細菌、病毒和原生生物中。在受到Clostridium perfringens 感染的病人，其血漿和創傷區域的神經胺酸甘脢;濃度會升高。文獻報告顯示，將神經胺酸甘脢;加入全血中，其平均的凝血時間由原來的15.45分鐘縮短為7.17分鐘。本篇論文預期探討的問題是：當開放性的傷口受到Clostridium perfringens感染時，它會釋放大型神經胺酸甘脢;到血液中，神經胺酸甘脢;可能會和血液中的蛋白發生交互作用，而影響血液凝固。所以利用酵母菌雜交 (yeast two-hybrid) 方法來尋找和Clostridium perfringens大型神經胺酸甘脢;有交互作用的蛋白，希望找出它對凝血之影響及機制。我們以大型神經胺酸甘脢;當作釣餌來篩選人類肝臟cDNA library，結果得到167個陽性菌株，經由分析除去偽陽性菌株，最後得到的結果其中有3個蛋白與血液凝固有關，它們分別是X、Y、Z。為了更進一步證明蛋白之間的交互作用，我們採用表面電漿共振技術。以細菌表現系統純化大型神經胺酸甘脢;野生型和缺失型 (Δ1-216) 作為SPR之ligand固定在感應片上，之後分別加入這3種蛋白到BIAcore儀器分析。初步實驗結果證明這3種蛋白與野生型的大型神經胺酸甘脢;有交互作用，與缺失型則無，表示它們發生交互作用的區域位於大型神經胺酸甘脢;N端前216個胺基酸所構成之區域內，至於最小作用區域，則有待進一步實驗來證明。

Sialidases (nanH) are a superfamily of N-acylneuraminate-releasing exoglycosidases found mainly in higher eukaryotes and in some virus, bacteria and protozoans. High sialidase concentrations were found in sera and in tissues of wounded regions of patients infected with Clostridium perfringens. The previous study had shown that sialidase could shorten the blood clotting time, in this study, the mechanism of sialidase action involved in shortening clotting time was investigated. We screened human liver cDNA library with yeast two-hybrid system using large sialidase as a bait. Three of 167 putative positive clones are relevant to blood clotting. They are X, Y, Z. The protein-protein interaction between large sialidase and each of the three proteins was confirmed with surface plasmon resonance (SPR). We purified large sialidase wild type and deletion clone (Δ1-216) as SPR ligands and the three proteins as analytes. The preliminary results demonstrated that each of the three proteins may associate with wild type large sialidase, but didn’t associate with truncated large sialidase (Δ1-216). The interaction region of large sialidase was contributed from the domain formed by the N-terminal 216 amino acids.

中文摘要…………………………… 1
英文摘要…………………………… 3
前 言…………………………… 4
材料與方法…………………………13
結 果……………………………35
討 論……………………………45
參考文獻……………………………53
圖 表……………………………59
附 錄……………………………85

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