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論文中文名稱:以線型轉殖改善細菌生產重組蛋白質時之基因穩定性 [以論文名稱查詢館藏系統]
論文英文名稱:Improving Genetic Stability by Utilizing Bacteria with Linear Transformation during Recombinant Protein Overexpression [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:化學工程所
中文姓名:陳幸達
英文姓名:Hsing-Ta Chen
研究生學號:92328007
學位類別:碩士
語文別:中文
口試日期:2005-07-11
論文頁數:96
指導教授中文名:侯劭毅
指導教授英文名:Shao-Yi Hou
口試委員中文名:顏宏偉;王勝仕
口試委員英文名:Hong-Wei Yen;Sheng-Shih Wang
中文關鍵詞:線型轉殖質體不穩定同源序列營養缺陷
英文關鍵詞:linear transformationplasmid instabilityhomologous sequenceauxotrophic defect
論文中文摘要:本研究以線型轉殖的方法,利用線性DNA兩端與宿主細胞E.coli上的同源序列,將目標基因直接轉移到大腸桿菌的染色體上。由於無須使用質體,且能持續表達重組蛋白基因,故可有效的解決質體不穩定的問題。
在實際操作上,利用一具多個營養缺陷的大腸桿菌ZSC114,其無法代謝葡萄糖、甘露糖及乳糖等醣類,我們將各個彌補營養缺陷的基因與lacZ基因聯合,製備成線性DNA,並以線型轉殖的方式構築在ZSC114的染色體上,因此可彌補原菌株的營養缺陷,並藉著較多lacZ基因的表達,而增進重組蛋白質β-galactosidase的生產。
在結果分析方面,我們是利用蛋白質電泳及半乳糖苷酶活性測驗來比較以線型轉殖所構築的各株新菌種其表達目標蛋白LacZ的產量及活性表現;我們發現隨著細菌染色體中重組蛋白基因lacZ份數的增加,其基因產物LacZ的產量及活性亦有所提升。
另一方面,我們利用連續稀釋培養的方法,探討本研究構築含有三份lacZ基因的新菌種ZSCGMtZ與一般以質體表達重組蛋白基因的系統-本實驗中以ZSC114/pJL為代表-在持續表達重組蛋白基因時,其基因穩定性的表現。我們發現以IPTG誘導且初始的誘導量相同的條件下,ZSC114/pJL因質體pJL會在生產過程中流失,使其在連續培養約35個小時後就沒有LacZ活性表達;而新菌株ZSCGMtZ即使連續培養60小時仍能維持相當高的重組蛋白表達,其LacZ活性值保持在6000-8000個Miller Unit,具備良好的基因穩定性。
論文英文摘要:In this research, we use linear transformation that takes advantage of the homologous sequence between two ends of linear DNA and the host cell E.coli, to transfer the target genes directly into the chromosome of the strain. Because this method enables expressing recombinant proteins continuously without plasmid, the problem of plasmid instability is solved.
In practice, we use ZSC114, a multi-auxotrophic E.coli strain that cannot metabolize glucose, mannose and lactose, as our host cell. First, we unite each of the three metabolism-related genes with lacZ gene, to prepare for a functional linear DNA. Then we put these linear substrates into the chromosome of ZSC114. We can make up for the auxotrophic phenotype of the original strain, and with expression of more lacZ genes, enhance the production of recombinant protein β-galactosidase.
Protein gel electrophoresis andβ-galactosidase activity assay are executed to measure the production of recombinant protein LacZ among our newly constructed strains. We find out that the production and activity of the genetic product increase while the copy number of lacZ gene rises in the bacterial chromosome.
We use the method of serial dilution culture to investigate genetic stability between the three-lacZ gene-carrying strain, ZSCGMtZ, and the plasmid expression system, ZSC114/pJL. We find out that in serial dilution culture, the ZSC114/pJL system would loss LacZ expression at 35th hour, but new strain ZSCGMtZ still keep quite high recombinant protein expression after 60 hours. The result indicates the genetic stability of new strain is better.
論文目次:      目 錄
中文摘要………………………………………………………………………………i
英文摘要………………………………………………………………………………ii
誌謝 iv
目錄…………………………………………………………………………………... v
表目錄……………………………………………………………………………….. vii
圖目錄………………………………………………………………………………. viii
第一章 緒論 1
1.1 重組基因工程 1
1.2 質體不穩定 3
1.3 同源重組 5
1.4 λ Red 線型轉殖系統 6
1.5 本研究策略簡介 8
1.6 研究動機與目的 9
第二章 文獻回顧 10
2.1 質體不穩定 10
2.1.1 影響質體穩定性的因素 10
2.1.2 改善質體穩定性的研究 11
2.2 同源重組 12
2.3 λ Red 線型轉殖系統 14
2.4 本研究策略 17
第三章 實驗材料與方法 19
3.1 實驗儀器與設備 19
3.2 實驗藥品 20
3.3 菌種與質體 22
3.3.1 本研究使用菌種 22
3.3.2 本研究使用質體 24
3.4 培養基與抗生素 25
3.5 實驗方法 27
3.5.1 lacZ基因選殖 29
3.5.2 glk基因選殖 30
3.5.3 manXYZ基因選殖 31
3.6 重組質體構築 32
3.6.1 PCR引子設計 32
3.6.2 染色體純化 32
3.6.3 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 33
3.6.4 瓊脂糖膠電泳 (agarose gel electrophoresis) 35
3.6.4.1 膠體製備 35
3.6.4.2 凝膠電泳分離 36
3.6.4.3影像分析 36
3.6.5 膠體純化 (gel extraction) 37
3.6.6 質體純化 37
3.6.6.1 鹼性質體純化法 38
3.6.6.2 質體純化組 (kit) 純化法 38
3.6.7 限制酶酶切 (restriction enzyme cut) 39
3.6.8 連接酶接合反應 39
3.6.9 轉型作用(transformation) 40
3.6.9.1 化學法 40
3.6.9.2 電轉殖法 41
3.7 線型轉殖 41
3.7.1製備線性DNA 42
3.7.1.1 同源序列PCR引子設計 42
3.7.1.2 以PCR擴增線性DNA片段 43
3.7.2 λ Red線型轉殖 43
3.8 重組蛋白質分析 44
3.8.1 蛋白質電泳 44
3.8.1.1 SDS-PAGE之製備 44
3.8.1.2 SDS-PAGE電泳分析 45
3.8.2半乳糖苷酶活性測驗 45
3.9 前培養 46
3.10 批次培養 46
3.11 生產重組蛋白質之批次培養 46
3.12 基因穩定性分析 47
第四章 結果與討論 48
4.1 重組質體構築 50
4.1.1 質體pMIL構築 50
4.1.2 質體pTGL構築 55
4.1.3 質體pTML構築 60
4.2 以線型轉殖構築重組菌株 65
4.2.1 各段線性DNA的製備 65
4.2.2 以線型轉殖構築菌株ZSCtZ 68
4.2.3 以線型轉殖構築菌株ZSCGtZ 70
4.2.4 以線型轉殖構築菌株ZSCGMtZ 72
4.3 重組蛋白質分析 74
4.3.1 SDS-PAGE 分析 74
4.3.2 半乳糖苷酶活性測驗 76
4.4 基因穩定性分析 78
4.4.1 以300μM IPTG誘導時各新菌株的LacZ活性表達 78
4.4.2 不同IPTG濃度下ZSCGMtZ之LacZ活性的表達 80
4.4.3 不同IPTG濃度下ZSC114/pJL之LacZ活性的表達 82
4.4.4 連續稀釋培養下ZSCGMtZ的基因穩定性 84
第五章 結論 91
參考文獻 92
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