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研究生: 柯嘉玲
論文名稱: 具α-2,3-Sialyltransferase功能性之磁性奈米粒子及其應用
α-2,3-Sialyltransferase Functionalized Magnetic Nanoparticle and its Application
指導教授: 陳焜銘
Chen, Kwun-Min
林俊成
Lin, Chun-Cheng
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2006
畢業學年度: 95
語文別: 中文
中文關鍵詞: α-2,3-sialyltransferasemagnetic nanoparticleintein
論文種類: 學術論文
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  • 本論文中為發展蛋白質的快速純化和酵素的重複使用,以pTYB1及pTXB1此intein系列之載體分別表達,eGFP-intein及α-2,3-sialyltransferase-intein這兩種融合蛋白,利用intein 之特性,分別在eGFP及α-2,3-sialyltransferase之C端進行修飾,以利其專一位相固化於磁性奈米粒子上。透過磁鐵與磁性奈米粒子之間的磁力吸引,可從表現完之混合蛋白質,快速的純化出eGFP及α-2,3-sialyltransferase。不需要繁瑣的更換緩衝溶液系統,和長時間的純化步驟,目標蛋白質可快速地固化於磁性奈米粒子上,並可透過磁鐵純化。我們亦證明固化後之酵素活性並無改變,且在酵素反應完之後,可快速將酵素與反應物分離,回收的酵素可再利用,更加符合經濟效應。

    目錄...........................I 圖表目錄.........................IV 簡寫表.......................... VII 摘要...........................IX 第一章 序論....................... 01 1-1磁性粒子於生物科技應用................01 1-2醣類與細胞間之辨識..................02 1-3唾液酸 (sialic acid)................... 05 1-4唾液酸轉移酶 (α-2,3-sialytransferase ,α-2,3-SiaT)應用..... 06 1-5蛋白質專一性 (site-specific)的固定............08 1-6 intein於生物科技應用..................11 1-7唾液酸轉移酶 (α-2,3-sialytransferase )的固定........13 第二章 結果與討論....................15 2-1合成6-(Dansyl)-aminohexyl O-β-D-galactopyranosyl- (1,4)-β-D-glucopyranoside................15 2-2建構含α-2,3 SiaT 酵素基因的重組載體 (recombinate DNA) .16 2-2-1引子設計......................17 2-2-2聚合酶鏈反應 (polymerase chain reaction,PCR)的條件...19 2-2-3 T-A cloning.....................23 2-2-4確定DNA序列使用之方法...............28 2-2-5 α-2,3 SiaT-pTXB1建立重組載體............29 2-3 α-2,3-sialytransferase (α-2,3-SiaT)的表現與純化.......33 2-3-1 α-2,3-SiaT於pTXB1及pTYB1的表現差異........37 2-3-2 IPTG 調控蛋白質的表現................39 2-4 α-2,3 SiaT活性測試..................40 2-4-1 α-2,3 SiaT酵素反應的pH值最佳化...........43 2-5目標蛋白固化 (the immobilization of target protein) .....44 2-5-1 eGFP重組載體建構..................44 2-5-2 eGFP的表現與純化..................47 2-5-3螢光蛋白的固化...................51 2-5-4固化於磁性奈米粒子的eGFP定量........... 52 2-5-5 α-2,3 SiaT的固化...................53 2-5-6固化於磁性奈米粒子的α-2,3 SiaT定量..........56 2-5-7固化前與固化後的α-2,3 SiaT活性比較..........57 2-5-8固化於磁性奈米粒子的α-2,3 SiaT的重複使用性......60 2-6結論.........................62 第三章 實驗材料與方法..................63 3-1一般實驗方法.....................63 3-2實驗步驟及光譜資料..................64 3-3 pH值最佳化之酵素反應.................66 3-4固化前與固化後的α-2,3 SiaT活性比較..........67 3-5固化後的α-2,3 SiaT重複使用..............68 3-6菌株、質體與培養基..................68 3-7 E.coli 質體DNA抽取..................69 3-8洋菜膠體上的DNA片段回收與純化...........71 3-9 DNA黏接反應 (ligation) ................72 3-10質體轉形作用.....................73 3-10-1勝任細胞 (competent cell)之製備............73 3-10-2轉型作用 (Transformation) ..............73 3-11 DNA 膠體電泳....................74 3-12融合酵素蛋白的表現及純化分析.............76 3-13 SDS-PAGE膠體電泳..................79 3-14蛋白質的定量分析...................81 參考文獻.........................82 附錄...........................90

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