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論文中文名稱:利用基因遺傳工程優化酵母菌中的啟動子 [以論文名稱查詢館藏系統]
論文英文名稱:Optimization of Promoters in Yeast by Genetic Engineering [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:化學工程與生物科技系生化與生醫工程碩士班
畢業學年度:105
畢業學期:第二學期
出版年度:106
中文姓名:黃雅婷
英文姓名:HUANG,YA-TING
研究生學號:104688003
學位類別:碩士
語文別:中文
口試日期:2017/07/19
論文頁數:84
指導教授中文名:黃志宏
指導教授英文名:Ya-Ting Huang
口試委員中文名:陳月茸;黃姿雯
口試委員英文名:Chen, Yueh-Jung;Huang, Tzu-Wen
中文關鍵詞:酵母菌蛋白質重組啟動子
英文關鍵詞:yeastproteinrecombinant promoter
論文中文摘要:多數異源性蛋白的生產,是藉由基因轉殖到大腸桿菌中表現,但由於大部分有藥物價值的蛋白質源自於真核生物,當真核的蛋白質編碼基因以原核生物系統表現時,其生產出的蛋白質因缺乏轉譯後修飾,經常失去原有的功能,或是容易形成包涵體及蛋白質萃取破菌時會伴隨內毒素汙染等缺點,因此用真核細胞表現真核生物蛋白質的系統為目前研究的一大重要方向。而酵母菌具有真核細胞的特性,例如:轉譯後修飾的功能、培養容易、方便操作、不會形成包涵體及內毒素等優勢,目前在工業生產異源性蛋白上,備受矚目。而酵母菌中以 Yarrowia lipolytica 實用性最高,因其比傳統酵母 Saccharomyces cerevisiae有更高的蛋白質分泌量、屬於安全菌種及可利用低成本之碳源生長等特性,因此Y. lipolytica 為本研究使用之菌株做為蛋白質表現平台。本篇研究目的,建構出一套方便操作且可在Y. lipolytica高表達異源性蛋白的基因遺傳工具。本實驗的策略是,利用過去發現可以增強酵母菌表達蛋白質的啟動子,以基因工程技術,將上游活性序列啟動子(UAS1B)與核心啟動子(LEU2,TEF)做不同的排列組合置入載體上,形成重組啟動子質體,利用重組啟動子調控蛋白質的表現,並以紅色螢光蛋白(DsRed-Express2)當作蛋白質表現系統的報導基因,方便實驗觀察及檢測蛋白表現量,另外再以異源性蛋白SLEEP作為重組啟動子質體於工業發展的實用性測試。為了提高蛋白質產量,實驗策略是利用 Y. lipolytica CLIB122染色體上存在多個重複序列的特性,使用長末端重複序列的反轉錄轉座子,Ylt1,作為序列嵌入點,期望藉由多點嵌入方式來倍增蛋白產量。目前已成功建構出pYLF4、pYLF5、pYLF6、pYLF7、pYLF8、pYLF9、pYLF10這幾組重組啟動子質體,其中以pYLF5、pYLF7、pYLF10的重組啟動子強度較強。此項研究為食品業、化妝品業、生物燃料等功能性蛋白生產方面,提供了一個方便使用且在酵母菌中具有高蛋白質表現的質體系統。
論文英文摘要:In most cases, production of heterologous protein in produced in E. coli through gene transfer. However, most of the valuable drug-derived proteins are originated from eukaryoties. When the eukaryotic protein-encoding gene is expressed in the prokaryotic system, there’s a chance that target proteins may loss their functions due to the absence of proper post-translational modification. Meanwhile such as these proteins are easy to form inclusion bodies, and are contaminted by endotoxins during protein extraction also happens oftenly. Therefore, using an eukaryotic system for protein expression is the major research trend. Yeasts, has the characteristics of eukaryotic cells, such as, post-translational modification, are easy to cultivate and operate, do not form inclusion bodies and endotoxin. This orgaism is getting quite popular for heterologous protein production in the industry. Yarrowia lipolytica is one of the most potential orgamsms among yeast because it secrets higher protein than traditional- Yeast Saccharomyces cerevisiae. Y. lipolytica is classified as a safe strain and has the ability to grow with low-cost carbon sources. Therefore, Yarrowia lipolytica is used for protein expression in this study. The purpose of this study is to construct a genetic tool that is easy to operate, and overexpress heterologous proteins in Y. lipolytica. The strategy of this experiment is to using the genetic engineering technology to recombine the upstream active sequence promoter (UAS1B) and the core promoters (LEU2 & TEF) and forme the recombinant promoter, which can be used to enhance the expression of the protein in yeast. The red fluorescent protein (DsRed-Express2) served as a reporter gene in this system. We used the SLEEP a heterologous protein to test this system for the estimating the potential use in the industrial. In order to improve the protein yield, our experimental strategy was to use the multiple-copy integration sites, Ylt1 retrotransposon. Ylt1 is flanked by long terminal repeats and exist inY. lipolytica CLIB122 chromosome. It is design as multiple integration sites to enhance the heterologous protein yield. We have been successfully construncted the recombinant promoter plasmid of pYLF4、pYLF5、pYLF6、pYLF7、pYLF8、pYLF9、pYLF10. Among them, the recombinant promoters of pYLF5, pYLF7 and pYLF10 have strong intensity. This study provides a system which is easy to operate and has high protein expression in Yeast, for food industry, the cosmetic industry, and biofuels to product the functional proteins.
論文目次:中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 異源性蛋白表現系統 1
1.2 非傳統酵母菌─Yarrowia lipolytica主要特性介紹 4
1.3 調控蛋白質表達相關元件:啟動子、分泌信號 5
1.3.1 分泌系統 5
1.3.2 啟動子 6
1.4 基因嵌入工具:多重拷貝因子,Ylt1 7
1.5 本實驗目的 8
1.6 本實驗之酵母菌質體表達系統 9
第二章 材料與方法 11
2.1 菌種及質體 11
2.1.1 菌種 11
2.1.2 質體 12
2.1.3 引子 14
2.2 藥品及酵素 17
2.3 培養基及生長條件 17
2.4 菌種儲存 17
2.5 大腸桿菌質體分離 17
2.6 限制酶、連接酶、DNA純化及膠體回收 17
2.7 聚合酶連鎖反應 18
2.8 大腸桿菌轉型 18
2.9 酵母菌轉型 18
3.0 聚丙烯醯胺膠體電泳(SDS-PAGE) 18
3.1 西方點墨法(Western blot) 18
第三章 實驗結果 19
3.1重組啟動子及紅色螢光蛋白之質體建構 19
3.1.1重組啟動子質體建構 19
3.1.2 pYLF4重組啟動子質體建構 20
3.1.3 pYLF5重組啟動子質體建構 22
3.1.4 pYLF6重組啟動子質體建構 24
3.1.5 pYLF7重組啟動子質體建構 26
3.1.6 pYLF8重組啟動子質體建構 28
3.1.7 pYLF9、pYLF10重組啟動子質體建構 31
3.2利用紅色螢光蛋白分析啟動子強度 33
3.2.1酵母菌的轉型株檢測 33
3.2.2紅色螢光蛋白的分析 36
3.3酵母菌異源性蛋白的萃取分析及優化酵母菌生長條件 39
3.3.1分析酵母菌中異源性蛋白的萃取方式 41
3.3.2優化酵母菌的生長條件 43
3.4利用重組質體系統表現異源性蛋白 45
3.4.1質體構築介紹 45
3.4.2 pYLF4A、pYLF5A、pYLF7A、pYLF10A質體建構 46
3.4.3 pYLF4B、pYLF5B、pYLF7B、pYLF10B質體建構 52
3.5異源性蛋白分析 56
3.5.1酵母菌的轉型株檢測 57
3.5.2異源性蛋白的分析 58
3.6利用多重嵌入的方式增加蛋白質產量 60
第四章 討論 63
參考文獻 66
附錄
一 Plasmid Isolation Using MiniPrep Kit 72
二 E.coli competent cell preparation and transformation 73
三 Media and buffer 75
四 Nickel column 77
五 Polymerase Chain Reaction 78
六 Sodium Dodecyl Sulfate Polyacrylamide Gel Protocol 79
七 DNA Cleanup for Ligation and Transformation 81
八 Yarrowia lipolytica Transformation 82
九 SDS-PAGE ratio of 26KDa band ……………………………………………..83
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