利用基因工程乳酸菌產製外源性胞外超氧歧化? 王妙齡、陳小玲 ; 陳全木

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利用基因工程乳酸菌產製外源性胞外超氧歧化?

王妙齡、陳小玲 ; 陳全木

摘要

活性氧物質，如超氧陰離子，與許多疾病之生成有關。胞外超氧歧化?為胞外基質中唯一可清除超氧陰離子之酵素，能夠降低超氧陰離子對細胞或組織所引發的一連串傷害，如心血管疾病、肺部疾病、神經失調與發炎。有鑑於此，本研究旨在探討運用利用基因工程方式於乳酸桿菌中產制人類胞外超氧歧化?之可行性。首先，人類胞外超氧歧化?基因片段以聚合?鏈連鎖反應擴增後，選殖至乳酸菌表現型載體pLP3537，並且利用電穿孔方式轉型至乳酸桿菌中，經過紅黴素的篩選過程，

選取具有高套數目標基因的轉型菌株為實驗材料。進而以肝素親和性管柱配合快速蛋白質液相層析系統自乳酸桿菌轉型株中初步純化出人類胞外超氧歧化?，由硫酸十二酯鈉膠體電泳與西方吸漬法分析結果顯示，粗純化酵素之分子量約為38 與50 kDa，分別為人類胞外超氧歧化?單元體與二元體形式，經超氧歧化?活性分析試劑套組之分析試驗結果顯示，重組乳酸菌株所表現之外源性胞外超氧歧化?具有抗氧化活性。

關鍵詞 : 胞外超氧化物歧化? ; 乳酸桿菌 ; 抗氧化活性

目錄封面內頁簽名頁授權書...iii 中文摘要...iv 英文摘要...v 誌謝...vi 目錄...vii 圖目錄...viii 表目錄...ix 第一章緒言...1 第二章文獻檢討...4 2.1 超氧歧化?之分類...4 2.1.1 銅鋅型超氧歧

化?...5 2.1.2 錳型超氧歧化?...6 2.1.3 鐵型超氧歧化?...7 2.2 超氧歧化?之反應機制...9 2.3 胞外超氧歧化?之特性...12 2.3.1 胞外超氧化物歧化?之分子與生物特性...12 2.3.2 肝素結合區域...15 2.3.3 肝素結合區域之蛋白質水解作用...18 2.3.4 胞外超氧歧化?之基因多型性...20 2.4 胞外超氧歧化?與疾病之關係...21 2.4.1 心血管疾病...21 2.4.1.1 胞外超氧歧化?與一氧化氮...21 2.4.1.2 動脈粥狀硬化症...24 2.4.1.3 高血壓...28 2.4.1.4 糖尿

病...31 2.4.2 肺部疾病...34 2.4.2.1 胞外超氧歧化?於肺部之表現情形...34 2.4.2.2 急性肺損傷...35 2.4.2.3 肺部纖維化...37 2.4.2.4 肺部發炎...39 2.4.3 類風濕性關節炎...42 2.5 超氧化物歧化?之代謝...44 2.6 利用微生物系統表現外源性超氧歧化?...45 2.6.1 原核系統...45 2.6.2 真核系統...47 第三章材料與方法...49 3.1人類胞外超氧化物歧化?於乳酸菌表現型載體之建構...49 3.1.1人類胞外超氧化物歧化?之聚合?連鎖反應擴增...49 3.1.2 選殖片段與載體之接合反應...50 3.1.3 大腸桿菌之轉型作用...51 3.1.4 小量質體之DNA萃

取...53 3.1.5 核酸限制酵素之截切作用...55 3.1.6 DNA片段之回收...55 3.1.7 乳酸菌表現型載體之構築...56 3.2 重組乳酸菌菌株之轉型與篩選...60 3.2.1 乳酸菌勝任細胞之製

備...60 3.2.2 電穿孔轉型作用...60 3.2.3 重組乳酸菌菌株之篩選...61 3.3 乳酸菌轉型株之胞內蛋白質分析...62 3.3.1 乳酸菌轉型株之胞內蛋白萃取...62 3.3.2 硫酸十二酯鈉-聚丙烯醯胺凝膠電泳分析...63 3.3.3 西方吸漬法分析...65 3.3.4 重組人類胞外超氧化物歧化?之初步純化...68 3.3.5 胞外超氧化物歧化?活性測定方法...70 第四章結果與討論...74 4.1 人類胞外超氧化物歧化?基因cDNA 序列之選殖...74 4.2 人類胞外超氧化物歧化?基因於乳酸菌表現型載體之建構...74 4.3 人類胞外超氧化物歧化?重組載體之核酸定序分析...78 4.4 人類胞外超氧化物歧化?於乳酸菌轉型株之蛋白質表現分

析...81 4.5 人類胞外超氧化物歧化?於乳酸菌轉型株之蛋白質表現分析...84 4.6 重組人類胞外超氧化物歧化?之初步純化...87 4.7 重組人類胞外超氧化物歧化?之抗氧化活性分析...89 第五章結論...95 參考文獻...96 附錄...122 作者小傳...122

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