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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
dc.contributor.author | Yan-Jiun Huang | en |
dc.contributor.author | 黃彥鈞 | zh_TW |
dc.date.accessioned | 2021-06-16T05:42:02Z | - |
dc.date.available | 2019-08-17 | |
dc.date.copyright | 2014-08-17 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-11 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56684 | - |
dc.description.abstract | Pichia pastoris為嗜甲醇酵母菌的一種,是常用的異源蛋白質表達系統,具低生產成本、轉譯後修飾能力、能利用甲醇調控蛋白質生產等特色。傳統上高表現量的P. pastoris轉形株是利用抗生素濃度梯度篩選,但此方式相當地耗時費力且效果不彰。而目前使用96孔培養盤,以小量培養基同時培養多株轉形株,再分析蛋白質表現量,但此法使用的培養基體積太小,導致其中的轉形株活性容易受培養基內養份、廢物等的影響,導致其篩選的結果不一定是菌株本身能力優劣,而是因為培養基環境好壞的影響。本研究欲建立一個穩定的高通量篩選P. pastoris高表現量轉形株系統,方法為參考前人研究,建構一個多基因表現載體,利用來自口蹄疫病毒 (Foot and mouth disease virus) 中可進行自我截切的2A胜肽基因 (2A peptide gene),連接目標蛋白質基因 (使用Mouse Endostatin-IL2融合蛋白作為目標蛋白質) 與綠色螢光蛋白質基因 (Enhance green fluorescent protein, EGFP)。利用綠色螢光強度間接代表目標蛋白質的表現量,並藉由螢光流式細胞分選儀 (Fluorescent-activated cell sorter, FACS)篩選具有高螢光表現量的轉形株。此外,前人使用不須後轉譯修飾的原核細胞蛋白質作為目標,因此本研究將目標置換成需後轉譯修飾,構形較為複雜的真核細胞融合蛋白質,結果顯示,本系統能夠順利表現真核細胞蛋白質。此外,轉形株的綠色螢光強度和目標蛋白質表現量呈現正相關,但由於使用單一菌落作為篩選材料,導致藉由螢光流式細胞分選儀篩選出來的轉形株,其表現量未如預期的提升。本研究確立了綠色螢光強度能作為目標蛋白質表現量的預測指標,但欲利用此系統篩選出高表現量轉形株,則仍需繼續研究。 | zh_TW |
dc.description.abstract | The methylotrophic yeast Pichia pastoris is a generally used expression system in production of industrial and pharmaceutical proteins. Traditionally, screening high-yield candidates relies on assay transformant by transformant. However, It is labor extensive, time consuming and unstable. In this study, a high-throughput screening system in P. pastoris was developed. The polycistronic expression plasmids harboring the genes of Endostatin-IL2, 2A peptides from foot-and-mouth disease virus and EGFP were introduced into P. pastoris. The transformants and wild types were distinguished by their fluorescence. Furthermore, since previous study using prokaryotic protein as target protein, this study replace the protein to the eukaryotic fusion protein with more complex structure. The results showed this system successfully express the eukaryotic fusion protein. There is positive correlation between the production of Endo-IL2 and EGFP. Furthermore, the transformants could be isolated using fluorescent-activated cell sorter (FACS) based on their fluorescence intensity. However, because using the single colony as sample and cultivated to sort, the Endo-IL2 production of transformants sorting by FACS was not better than the original transformants. It still needed to continue study to solve this problem. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:42:02Z (GMT). No. of bitstreams: 1 ntu-103-R01b22048-1.pdf: 1867445 bytes, checksum: 90da31bd510becf8f7ed8b9742f9685c (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 摘要 I
Abstract II 目錄 III 表目錄 VI 第一章 前言 1 一、異源表達系統 1 1. 原核生物表現系統 1 2. 真核生物表現系統 2 二、Pichia pastoris表現系統 4 1. 酒精氧化酶及其啟動子 4 2. 菌種 4 3. 訊息胜肽 5 4. 醣基化修飾作用 6 5. P. pastoris轉形株之篩選 7 三、以螢光流式細胞分選儀建立高通量篩選系統 11 1. 螢光增強之綠色螢光蛋白GFP (green fluorescent protein) 11 2. 2A胜肽 (2A peptide) 12 3. 目標蛋白: 內皮抑素與白細胞介素-2複合蛋白質 13 4. 流式細胞術 (Flow cytometry) 14 五、研究動機與目的 15 第二章 材料與方法 18 一、實驗菌株與培養條件 18 1 細菌 18 2 真菌 18 二、培養基 19 三、本實驗所使用之引子 20 四、表現載體建構 22 1. yT&A-mEIL2-F2AeGFP 22 2. pPICZmα-mEIL2-F2AeGFP 22 3. 大腸桿菌轉形株篩選 24 五、電穿孔轉形 25 1. P. pastoris勝任細胞製備 25 2. 電穿孔轉形 25 六、轉形株篩選與培養 25 1. 抗藥性濃度梯度篩選 25 2. 轉形株染色體DNA分析 26 3. 三角瓶培養轉形株 26 七、螢光強度分析 27 八、螢光激活細胞分選儀分選高螢光表現轉形株 27 九、異源蛋白質產物分析 27 1. 胞內蛋白質萃取 27 2. 西方墨點法 28 3. 三明治酵素連結免疫反應 28 第三章 結果 33 一、建構表現載體 33 1. 建構yT&A-mEIL2載體 33 2. 建構yT&A-mEIL2-F2AeGFP載體 33 3. 建構pPICZmα-mEIL2-F2AeGFP載體 33 二、轉形株篩選與確認 42 1. 抗性濃度梯度培養基篩選轉形株 42 2. 染色體PCR確認目標基因插入 42 三、異源蛋白質表現分析 45 1. 螢光顯微鏡觀察確認EGFP螢光表現 45 2. 西方墨點法確認目標蛋白質順利表現 45 3. 確認目標蛋白質與螢光表現量呈正相關性 45 四、螢光流式細胞分選儀分選高螢光表現量之轉形株 49 五、分選後新轉形株之異源蛋白質表現量 52 六、螢光流式細胞分選儀二度分析之前分選後的轉形株 57 第四章 討論 59 一、經分選後菌株的蛋白質表現量探討 59 1. 異源基因拷貝數 (gene copy number) 59 2. 改善策略 60 二、異源蛋白質外泌效果不佳之探討 63 1.目標蛋白質-內皮抑素與白細胞介素-2複合蛋白質疏水性分析 63 2.外泌途徑發生問題 65 三、綠色螢光蛋白質 (EGFP) 相關探討 66 第五章 結論 67 第六章 未來展望 68 第七章 參考文獻 69 附件 73 圖目錄 圖一 本論文之研究架構圖 17 圖二 本研究所使用之表現載體圖譜 23 圖三 yT&A-mEIL2 colony PCR 34 圖四 抽取yT&A-mEIL2質體 35 圖五 yT&A-mEIL2-F2AeGFP colony PCR 36 圖六 抽取yT&A-mEIL2-F2AeGFP質體 37 圖七pPICZmα-mEIL2-F2AeGFP colony PCR 38 圖八 抽取pPICZmα-mEIL2-F2AeGFP質體 39 圖九 pPICZmα-mEIL2-F2AeGFP表現載體定序結果 41 圖十 以不同Zeocin抗性濃度篩選轉形株 43 圖十一 染色體DNA之聚合酶連鎖反應 44 圖十二 以0.5%甲醇誘導3天,螢光顯微鏡觀察EGFP螢光表現 46 圖十三 蛋白質西方墨點法分析 47 圖十四 綠色螢光強度與mouse IL2蛋白質之相關性統計圖 48 圖十五 螢光流式細胞儀分選高螢光表現量轉形株 51 圖十六 比較分選前後菌株之上清液中mouse IL2產率 53 圖十七 比較分選前後菌株之菌體螢光強度 54 圖十八 分選前後菌株上清液mouse IL2產率與菌體內螢光強度之相關性分析 54 圖十九 比較分選前後菌株之胞內中mouse IL2產率 55 圖二十 比較分選前後菌株之上清液與胞內中mouse IL2產率 55 圖二十一 比較分選前後菌株之胞內與上清液mouse IL2之產率總和 56 圖二十二 分選前後菌株胞內與上清液mouse IL2產率總和與菌體內螢光強度之 相關性分析 56 圖二十三 螢光流式細胞儀分選二度分析之前分選的轉形株 58 圖二十四 使用改善策略後的分選結果 62 圖二十五 目標蛋白質-內皮抑素與白細胞介素-2複合蛋白質斥水性分析 64 表目錄 表一 本研究所使用之引子 21 表二 破菌取胞內蛋白質所用試劑成分 29 表三 西方墨點法分析所用試劑之組合成分 30 表四 三明治酵素連結免疫反應所用試劑之組合成分 32 附件 附圖一 嗜甲醇酵母菌甲醇之利用途徑 73 附圖二 綠色螢光蛋白質 (EGFP)之結構圖 74 附圖三 2A peptide 斷裂機制 75 附圖四 螢光流式細胞分選儀示意圖 76 | |
dc.language.iso | zh-TW | |
dc.title | 利用螢光流式細胞分選儀於嗜甲醇酵母菌Pichia pastoris建立高通量篩選系統 | zh_TW |
dc.title | Establishment of High-Throughput Screening System Using Fluorescent-Activated Cell Sorter in Pichia pastoris | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李昆達(Kung-Ta Lee),楊啟伸(Chii-Shen Yang),林晉玄(Ching-Hsuan Lin) | |
dc.subject.keyword | 螢光流式細胞分選儀,2A胜?,綠色螢光蛋白,Pichia pastoris KM71H, | zh_TW |
dc.subject.keyword | FACS,2A peptide,EGFP,Pichia pastoris KM71H, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-12 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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