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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨 | |
dc.contributor.author | Chiao-Ching Hsu | en |
dc.contributor.author | 許巧青 | zh_TW |
dc.date.accessioned | 2021-06-15T13:57:16Z | - |
dc.date.available | 2020-08-31 | |
dc.date.copyright | 2015-08-31 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-24 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51918 | - |
dc.description.abstract | Pichia pastoris 為嗜甲醇酵母菌,是常用的異源表達平台。藉由參數即時監測系統,可對醱酵過程作即時的判斷與調整,以避免汙染或是過多的原料浪費。然而目前尚無針對目標產物的即時監測工具,需經由取樣並進行特定的分析與定量才能確認其表現狀況。為使醱酵過程更有效率的生產目標蛋白質,本研究希望結合2A短胜肽與綠色螢光基因,將綠色螢光強度發展成醱酵製程中對目標蛋白質產量即時監控的參數。
根據實驗室早期研究的建構方式,以口足病毒 (foot and mouth disease virus) 可自我截切的2A短胜肽基因 (2A peptide gene),連接目標蛋白質基因GMI-X與不同的綠色螢光蛋白質基因 (Green fluorescent protein, GFP),其中包括常用的EGFP (Enhance green fluorescent protein, EGFP),只會形成單體的mutated EGFP (EGFP A206K胺基酸突變, mEGFP),及易降解的destabilized mEGFP (將mEGFP接上一段PEST sequence, dmEGFP)。利用胞內綠色螢光強度間接代表胞外目標蛋白質的表現量,以螢光流式細胞分選儀 (Fluorescent-activated cell sorter, FACS) 篩選高螢光強度的轉形株,並以搖瓶與醱酵槽進行甲醇誘導,分析不同綠色螢光蛋白質其螢光強度與目標蛋白質產量之間的關係。搖瓶誘導結果發現,原EGFP會因雙體化傾向,其螢光強度較形成單體的mEGFP弱,但兩者目標蛋白質產量與螢光強度變化具一致性。而dmEGFP因半衰期短,其螢光蛋白質不會過度累積於胞內,螢光強度較EGFP與mEGFP低,且無隨誘導時間增強之趨勢,但目標產物持續增加,其產量較EGFP與mEGFP的轉形株高。另外,醱酵槽結果也證實mEGFP與dmEGFP其綠色螢光強度可反映目標蛋白質表現量,且配合FACS分選得之轉形株皆為多拷貝數,因此未來即可根據不同的需求,選擇適當的建構方式作為目標蛋白質的即時監控系統。 | zh_TW |
dc.description.abstract | The production of recombinant proteins plays an important role in the application of modern biotechnology. Microbial submerged fermentation is often used to produce the recombinant proteins using computer-controlled fermenter and appropriate induction strategy. The effective fermentation processes depend on real-time parameter monitoring, especially the production of target proteins. However, the production of the target proteins would not be detected until sampling and assay in most cases. Thus, it is important to have a real-time parameter to monitor the target protein production. In this study, a method for on-line monitoring of the target protein production in the methylotrophic yeast Pichia pastoris was developed by measuring the fluorescence intensity which was highly related to the heterologous gene expression. Three different polycistronic vectors were constructed. The vectors contain the AOX promoter, a signal peptide gene, the genes of GMI-X (an immunomodulatory protein from Ganoderma microsporum), a 2A peptides and EGFP, mutant EGFP (a point mutation A206K) or destabilized mEGFP, respectively. The high-yield transformats were sorted according to their fluorescence intensity using fluorescent-activated cell sorter (FACS). After methanol induction, the GMI-X extracellular supernatant was analyzed by ELISA and the intracellular EGFPs fluorescence intensity was measured by fluorescence spectrometry. The results showed that transformants of EGFP with the dimerization tendency fluoresced had less intensity than transformants of mutant EGFP, which tends to form monomer and soluble green fluorescent protein in cells. There was positive correlation between the fluorescence intensity and target gene expression in both EGFP and mEGFP transformants. Surprisingly, destabilized mEGFP was degraded quickly after translation due to the PEST sequence, and the fluorescence intensity of dmEGFP transformant remained at a low level, whereas the production of GMI-X still increased with the time of methanol induction. The highest yield of GMI-X was found in the dmEGFP transformant among three different EGFP forms. Also, the data from fermenter showed dmEGFP transformant had higher methanol utilization efficiency and GMI-X expression than mEGFP counterpart. Furthermore, all transformants sorted by FACS contained high copy numbers. In conclusion, the EGFP and mEGFP constructions provide a real-time parameter to monitor the target protein production, while the dmEGFP construction minimizes the EGFP burden and is feasible for elongated induction. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T13:57:16Z (GMT). No. of bitstreams: 1 ntu-104-R02b22014-1.pdf: 3214307 bytes, checksum: 5d52df982dc7c1b751a14d9c0cb854fb (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 目錄
謝誌 I 摘要 III Abstract IV 目錄 VI 圖目錄 IX 表目錄 X 附圖目錄 XI 第一章、 前言 1 一、 異源表達系統 1 1. 原核系統 1 2. 真核系統 2 二、 Pichia pastoris 表達系統 5 1. 酒精氧化酶及其啟動子 5 2. 菌株與甲醇表現型 5 3. 訊息胜肽 7 4. 醣基化修飾 7 三、 醱酵製程與即時參數監控系統 8 1. 溫度 8 2. pH值 8 3. 溶氧量 (Dissolve oxygen, DO) 8 4. 細胞質量 (Cell mass) 9 5. 二氧化碳溶氧量 (Dissolved carbon dioxide) 9 6. 目標產物之光學特性 9 四、 綠色螢光蛋白質 (Green fluorescence protein, GFP) 11 1. 綠色螢光蛋白質歷史 11 2. 綠色螢光蛋白質衍生物 12 五、 螢光流式細胞分選儀篩選高螢光強度轉形株 14 1. 2A 短胜肽 (2A peptide) 14 2. 流式細胞術與螢光流式細胞分選儀 (Fluorescent-activated cell sorter, FACS) 15 3. 目標蛋白質: GMI-X 16 六、 研究動機與目的 17 第二章、 材料與方法 20 一、 培養基與藥品 20 二、 菌株與培養條件 22 1. 細菌 22 2. 真菌 22 3. 菌種保存 22 三、 質體與轉形株 23 1. 建構pPICZmα-GMI-X載體 23 2. 建構pPICZmα-GMI-X-2AEGFP載體 (65) 23 3. 建構pPICZmα-GMI-X-2AmEGFP載體 23 4. 建構pPICZmα-GMI-X-2AdmEGFP載體 24 5. 大腸桿菌轉形株篩選 24 四、 P. pastoris轉形 28 1. P. pastoris勝任細胞製備 (66) 28 2. 轉形DNA置備 28 3. 電穿孔轉形 28 五、 P. pastoris 轉形株篩選與培養 29 1. 抗藥性濃度梯度篩選 29 2. 轉形株染色體DNA分析 29 3. 搖瓶培養 30 4. 醱酵槽培養 30 六、 螢光強度測定及流式細胞分選儀分選高螢光強度轉形株 33 1. 螢光強度分析 33 2. 流式細胞分選儀篩選高螢光強度轉形株 33 七、 P. pastoris 轉形株拷貝數測定 34 1. 即時定量聚合酶鏈鎖反應 34 2. 測定方法 34 八、 異源蛋白質產物分析 36 1. 西方墨點法 36 2. 三明治酵素免疫法 36 第三章、 實驗結果 40 一、 表現載體確認 40 二、 轉形株的篩選與確認 40 1. 抗性濃度梯度培養基篩選轉形株 40 2. 染色體PCR確認目標基因插入 41 三、 綠色螢光與異源蛋白質表現分析 45 1. 螢光光譜儀確認綠色螢光蛋白質表現 45 2. 西方墨點法確認目標蛋白質順利表現 45 3. 確認pPICZmα-GMI-X-2AdmEGFP轉形株適合進行FACS分選的時間 45 四、 螢光流式細胞儀分選高螢光強度之轉形株 52 1. FACS分選高螢光強度轉形株 52 2. 染色體PCR確認分選之轉形株 52 五、 搖瓶誘導分選後之新轉形株 56 1. 異源蛋白質表現量與螢光強度 56 2. 螢光顯微鏡觀察綠色螢光表現 56 3. 誘導後上清液蛋白質電泳分析 57 六、 醱酵槽誘導分選後新轉形株之異源蛋白質表現量與螢光強度 61 1. 利用醱酵槽誘導分選後新轉形株 61 2. 誘導後上清液蛋白質電泳分析 61 七、 轉形株拷貝數分析 65 第四章、 討論 67 一、 mG-H1螢光強度趨於飽和與目標蛋白質表現量偏低之探討 67 1. 綠色螢光蛋白質累積達細胞負荷極限 67 2. 負回饋抑制 68 二、 dmG-H1高表現量之原因 70 1. 拷貝數與目標蛋白質表現量關係 70 三、 提升目標蛋白質產量 70 1. 不同甲醇誘導策略 70 2. 不同分生策略 71 第五章、 結論 73 第六章、 未來展望 75 第七章、 參考文獻 77 附圖 85 | |
dc.language.iso | zh-TW | |
dc.title | 利用2A短胜肽連接不同綠色螢光蛋白質
探討螢光強度與目標蛋白質之間的關係 | zh_TW |
dc.title | The correlation between fluorescence intensity and target protein using 2A-mediated co-expression of different EGFPs | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳浩仁,許瑞祥,李昆達,楊啟伸 | |
dc.subject.keyword | Pichia pastoris,醱酵參數即時監控系統,2A胜?,綠色螢光蛋白質,螢光流式細胞分選儀, | zh_TW |
dc.subject.keyword | Pichia pastoris,Bioprocesses real-time parameter monitoring,2A peptide,Green fluorescent protein,Fluorescent-activated cell sorter (FACS), | en |
dc.relation.page | 90 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-24 | |
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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