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論文中文名稱:構築用於枯草桿菌孢子外套蛋白展示法之質體 [以論文名稱查詢館藏系統]
論文英文名稱:Plasmid Construction for Bacillus subtilis spore display [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生化與生醫工程研究所
畢業學年度:105
畢業學期:第一學期
中文姓名:葉昱緯
英文姓名:YEH, YU-WEI
研究生學號:103688005
學位類別:碩士
語文別:中文
口試日期:2017/01/16
論文頁數:71
指導教授中文名:侯劭毅
口試委員中文名:許哲奇;劉正哲;黃光策;王勝仕
中文關鍵詞:重組蛋白枯草桿菌孢子枯草桿菌孢子外套蛋白表面展示法質體β-內醯胺酶甘油脫氫酶
英文關鍵詞:recombinant proteinsBacillus subtilissporeBacillus subtilis spore surface displayplasmidbeta-lactamaseglycerol dehydrogenase
論文中文摘要:近年來重組蛋白(Recombinant protein)被用於許多生化工程領域中,其生產過程中,主要的成分在於純化及分離。由於,枯草桿菌孢子具有良好的環境耐受性、純化費用低廉且操作簡易,再加上枯草桿菌不具致病力,用於食品工業上較為安全。因此,以枯草桿菌孢子外套蛋白表面展示法(Bacillus subtilis spore surface display)生產重組蛋白被視為新穎且具潛力的生產方式。
本研究構築了用於枯草桿菌孢子外套蛋白展示法之質體。該質體含有枯草桿菌外套蛋白B(cotB)基因,其中cotB之終止密碼子被去除且於其3’端加入數個可供選殖基因插入的限制酶切點。於本研究中我們以外套蛋白B基因作為攜帶蛋白(Carrier protein)並將β-內醯胺酶(Beta-lactamase, bla)基因與甘油脫氫酶(Glycerol dehydrogenase, gldA)基因分別接至cotB之3’端使之形成融合蛋白(Fusion protein)基因。質體構築完成後,將上述質體轉殖至枯草桿菌細胞中。我們測試孢子外套蛋白展示法並量測枯草桿菌孢子上重組蛋白之活性。
論文英文摘要:Recently, recombinant proteins have been used in the biochemistry engineering. The main production cost of recombinant proteins is purification and separation. Bacillus subtilis spore (endospore) has several features such as good environmental tolerance, low purification cost and easy operation. Furthermore, Bacillus subtilis is quite safe for the food industry owing to its non-pathogenicity. Therefore, producing recombinant proteins using Bacillus subtilis spore surface display has been considered a novel and potential method.
In this study, we constructed a plasmid for Bacillus subtilis spore display. The plasmid contains the coat protein gene cotB whose stop codon was removed and several restriction sites at 3’ end of cotB for cloning. We constructed two fusion protein genes that contain beta-lactamase gene bla and glycerol dehydrogenase gene gldA at the 3’end of the cotB gene. The plasmids were used for Bacillus subtilis spore display and the activity of recombinant proteins displayed on Bacillus subtilis spore was measured.
論文目次:目錄
摘 要 I
誌 謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
1.1枯草桿菌(Bacillus subtilis) 1
1.2枯草桿菌之轉型方法 4
1.3現行single-step重組蛋白分離、純化及固定化之方法 5
1.4枯草桿菌外套蛋白表面展示法(B.subtilis spore display) 8
1.5β-内醯胺酶(beta-lactamase) 10
1.6甘油脫氫酶(glycerol dehydrogenase) 11
1.7穿梭載體pMK4 12
1.8研究目的與策略 14
第二章 實驗方法與流程 15
2.1實驗架構 15
2.2實驗儀器與設備 16
2.3實驗藥品 20
2.4實驗材料 24
2.4.1菌株 24
2.4.2質體 25
2.4.3培養基 26
2.5實驗流程 28
2.5.1質體構築 30
2.5.1.1引子設計 30
2.5.1.2聚合酶鏈式反應(Polymerase chain reaction,PCR) 32
2.5.1.3質體純化 33
2.5.1.4染色體萃取 34
2.5.1.5瓊脂糖膠電泳 35
2.5.1.6限制酶酶切 36
2.5.1.7膠體純化 37
2.5.1.8連結酶接合反應(Ligation) 38
2.5.1.9電轉殖勝任細胞的製備(E. coli) 38
2.5.1.10電轉殖(Transformation for E. coli) 39
2.5.2枯草桿菌外套蛋白表面展示法實驗 40
2.5.2.1電轉殖勝任細胞的製備(B. subtilis) 40
2.5.2.2電轉殖(Transformation for B. subtilis) 41
2.5.2.3產孢(Sporulation) 42
2.5.3 Nitrocefin assay 43
2.5.4 Bradford protein assay 44
第三章 結果與討論 45
3.1質體構築 46
3.1.1質體pYU-2-CotB的構築 46
3.1.2質體pYU-2-CotB-L7-Bla的構築 49
3.1.3質體pYU-2-CotB-L12-Bla的構築 51
3.1.4質體pYU-2-CotB-L12-GldA的構築 53
3.1.5質體pYU-3-CotB的構築 55
3.2枯草桿菌孢子外套蛋白量與孢子量之關係 57
3.2.1 Bradford protein assay測試 57
3.2.2孢子再發芽測試 58
3.3不同長度Linker DNA質體產出蛋白質之活性比較 59
第四章 結論 60
參考文獻 61
附錄一.pYU-2-CotB(僅定序CotB基因) 64
附錄二.pYU-2-CotB-L7-Bla(僅定序Bla基因) 66
附錄三.pYU-2-CotB-L12-Bla(僅定序Bla基因) 68
附錄四.pYU-2-CotB-L12-GldA(僅定序GldA基因) 70
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