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研究生:方銘盛
研究生(外文):Ming-Shang Fang
論文名稱:腸炎弧菌Vibrio parahaemolyticus α澱粉酶碳端區的蛋白質工程
論文名稱(外文):C-terminal Protein Engineering of α-amylase from Vibrio parahaemolyticus
指導教授:林富邦
指導教授(外文):Fu-Pang Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:100
語文別:中文
論文頁數:78
中文關鍵詞:腸炎弧菌α澱粉酶蛋白質工程碳端區Factor Xa
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由腸炎弧菌 (Vibrio parahaemolyticus) BCRC13026選殖出的α澱粉酶基因,以pET20b(+)的大腸桿菌表現系統表現出具有 α 澱粉酶活性的重組澱粉酶(VpAmy),分子量約為 53 kDa。VpAmy碳端區K424胺基酸處以PCR方式增設蛋白酶Factor Xa的切位 Ile-Glu-Gly-Arg (VpAmyK424/Xa),嘗試以in vitro方式取得VpAmyK424突變酵素,並研究其生化特性。結果未能由大腸桿菌的表現系統發現VpAmyK424/Xa的重組蛋白質,經過Peptide mass fingerprinting (PMF)分析確認重組蛋白質VpAmyK424/Xa未表現出來。
The α-amylase gene from Vibrio parahaemolyticus BCRC13026 (VpAmy) encoding a protein with MW of 53kDa was cloned and expressed in Escherichia coli-pET20b(+) expression system with α-amylase activity. Protease factor Xa cleavage site of Ile-Glu-Gly-Arg was inserted at K424 amino acid of VpAmy by PCR techniques (VpAmyK424/Xa). In order to characterize the VpAmyK424 mutant enzyme, the recombinant VpAmyK424 was attempted to be obtained by the proteolytic cleavage of VpAmyK424/Xa with protease factor Xa in vitro. Results indicated that the VpAmyK424/Xa was not expressed from the E.coli expression system and was confirmed by peptide mass finger printing analysis (PMF).
謝辭
中文摘要
英文摘要
圖目錄
表目錄
第一章 前言
1.1腸炎弧菌
1.2澱粉
1.2.1澱粉的生合成
1.2.2澱粉的水解
1.3醣類水解酵素
1.3.1澱粉酶
1.3.2 α 澱粉酶
1.4研究背景及目的
1.4.1Factor Xa
1.4.2蛋白質3D結構模擬
第二章 材料與方法
2.1 實驗材料
2.1.1 菌株與質體
2.1.2 酵素
2.1.3 純化套組與試劑材料
2.1.4 MW Marker
2.1.5 濃縮器及過濾器
2.1.6 SDS-PAGE
2.1.7 藥品
2.1.8 試劑
2.1.9 緩衝液配製
2.1.10 儀器
2.1.11 引子
2.1.12 定序
2.2 實驗方法
2.2.1 VpAmyK424/Xa基因之構築
2.2.2 聚合酶鏈鎖反應
2.2.3 洋菜膠體電泳
2.2.4 質體的小量製備
2.2.5 限制酶剪切
2.2.6 載體與目標 DNA 接合反應
2.2.7 重組基因表現載體之構築
2.2.8 受容細胞的製備
2.2.9 轉形
2.2.10 快速篩選
2.2.11 重組質體 DNA 之檢視
2.2.12 核苷酸定序
2.2.13 表現宿主大腸桿菌 Rosetta(DE3)pLysS 的轉形
2.2.14 重組澱粉酶基因表現之誘導及粗酵素液的製備
2.2.15 受質液的配製與活性檢測
2.2.16 蛋白質的定量
2.2.17 蛋白質電泳
2.2.18 重組 VpAmyK424/Xa 蛋白質純化
2.2.19 蛋白質濃縮
2.2.20 重組目標蛋白質以蛋白酶Factor Xa 進行切割
2.2.21 重組目標蛋白質身分鑑定
2.3 VpAmy胺基酸序列分析
2.4 蛋白質3D結構預測
第三章 結果
3.1 VpAmyK424/Xa基因選殖
3.2 VpAmyK424/Xa之表現、純化及其蛋白質身份鑑定
3.3 VpAmy及VpAmyK424/Xa之蛋白Factor Xa切割
第四章 討論與未來展望
第五章 參考文獻
附錄

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