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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Hao-An Hsiung | en |
| dc.contributor.author | 熊浩安 | zh_TW |
| dc.date.accessioned | 2021-06-17T07:36:45Z | - |
| dc.date.available | 2022-04-11 | |
| dc.date.copyright | 2019-04-11 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2019-04-01 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73470 | - |
| dc.description.abstract | Pichia pastoris 為嗜甲醇酵母菌的一種，兼具微生物與真核系統的優勢，如培養成本低廉、生長快速，並具有轉譯後修飾、蛋白質外泌能力，利用甲醇誘導的AOX1啟動子，可大量生產重組蛋白質，是極具潛力的異源蛋白質表達系統。然而，生產異源外泌蛋白質時，大量的蛋白質常因內質網摺疊效率不足而導致未摺疊蛋白質的累積，限制蛋白質的外泌，並在細胞內產生內質網壓力 (ER stress) 進而引發未摺疊蛋白質反應 (unfolded protein response, UPR)。P. pastoris的未摺疊蛋白質反應主要由內質網內的Ire1感應，並藉由切除轉錄因子HAC1 mRNA的內含子使之轉譯出有功能的蛋白質，藉此調控下游協助蛋白質摺疊及將蛋白質降解等反應以解除內質網壓力。
本論文以P. pastoris生產表皮生長因子受體 (epidermal growth factor receptor, EGFR) 之單鏈抗體 (single-chain variable fragment, scFv) 時，發現以前人論文中的轉錄因子Mxr1再程序化策略表現目標蛋白質時，外泌效率可能是生產的瓶頸。推測P. pastoris內生的未摺疊蛋白質反應不足以疏通外泌路徑的阻塞。因此本論文希望透過一個同樣需甲醇誘導但強度較低的AOX2啟動子額外提升Ire1的表現，增強細胞內的未摺疊蛋白反應，以更有效率地處理過多的目標蛋白質累積，增加蛋白質正確摺疊及外泌效率。並研究額外表現Ire1對細胞的影響，包含目標蛋白質的表現量、未摺疊蛋白質反應的下游路徑調控等。結果顯示，額外表現Ire1同時提升了協助蛋白質摺疊及將蛋白質降解兩種反應，使胞內累積的目標蛋白質明顯減少，但胞外蛋白質產量並無顯著提升，顯示Ire1可能還有其他未被發現的調控路徑，期望透過此研究更加釐清P. pastoris未摺疊蛋白質反應的調控路徑，未來有機會將P. pastoris發展為更具外泌生產效率的蛋白質生產系統，增進其應用性。 | zh_TW |
| dc.description.abstract | Pichia pastoris has been successfully applied in the production of many recombinant proteins because of its distinguished features that combine the advantages of both microbial and eukaryotic system. Moreover, recombinant proteins can be secreted to the medium through addition of a secretory signal sequence, which simplifies the purification process. In P. pastoris, recombinant proteins production was usually driven by methanol-induced AOX1 promoter because of its strong characteristics. However, owing to the high-level expression of recombinant proteins, secretory process such as protein folding within the ER tends to be the most important limitation which reduce the productivity. The accumulation of unfolded proteins in the ER will induce a process known as unfolded protein response (UPR) that can help reduce the stress. UPR is activated by Inositol-requiring enzyme–1 (Ire1) through the translation of transcription factor Hac1. This response can relieve the ER stress by the expression of genes involved in protein folding, modification, translocation. In this research, anti-epidermal growth factor receptor (EGFR) scFv is produced in P. pastoris. Although the efficiency of AOX1 promoter was enhanced by reprogramming the transcription factor Mxr1, the recombinant proteins could not be secreted efficiently. Therefore, additional Ire1 expression driven by AOX2 promoter was applied to enhance the secretory capacity by UPR activation. Both UPR-responsive genes and genes related to ER-associated degradation were upregulated by extra Ire1. Besides, intracellular scFv was eliminated. These results suggested that there might be other signaling pathway regulated by Ire1. Further experiments are needed to demonstrate the feasibility of additional Ire1 expression in the production of recombinant proteins in Pichia pastoris. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T07:36:45Z (GMT). No. of bitstreams: 1 ntu-107-R05B22010-1.pdf: 2486454 bytes, checksum: 591e44bce9739feccf9ec9fe3efe7633 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 謝誌 I
摘要 II Abstract III 目錄 IV 表目錄 VII 圖目錄 VIII 第一章 前言 1 一、Pichia pastoris表達系統 1 1. 強力且可被甲醇調控的PAOX1啟動子 1 2. 兼具微生物及真核生物系統之優勢 1 3. 外泌系統 2 二、以轉錄調控策略提升Pichia pastoris表達系統之生產效率 2 三、Pichia pastoris表達系統之外泌瓶頸 3 1. Pichia pastoris外泌系統 3 2. Pichia pastoris外泌效率不足影響蛋白質產量 4 3. 未摺疊蛋白質反應 4 四、抗體及單鏈抗體之重要性 5 1. 抗體 5 2. 單株抗體之重要性 5 3. 單株抗體之缺點 6 4. 單鏈抗體 6 五、研究動機 7 1. 目的 7 2. 策略 8 第二章 材料與方法 11 一、微生物培養與分析 11 1. 細菌 11 2. 酵母菌 11 3. 微生物培養基與藥品 11 二、表現載體建構 13 1. pPICZαA-scFv 13 2. pAOX2KH-MXR1 14 3. pAOX2KH-IRE1 14 4. pPICHB 14 5. pAOX2HH-IRE1 15 三、P. pastoris電穿孔轉形法 19 1. P. pastoris勝任細胞之製備 19 2. 以電穿孔方式轉入質體 19 四、P. pastoris轉形株之基因分析 19 1. 轉形株之抗性篩選 19 2. 轉形株染色體簡易分析 20 3. 轉形株目標基因拷貝數分析 20 五、P. pastoris轉形株之搖瓶培養及分析 21 六、P. pastoris醱酵槽培養及分析 21 1. 種菌培養 21 2. 批次培養 (Batch) 21 3. 批次饋料培養 (Fed-batch) 22 4. 誘導培養 (Induction) 22 七、P. pastoris mRNA表現量分析 23 八、蛋白質產物分析 25 1. 胞內蛋白質萃取 25 2. 聚丙烯醯胺膠體電泳 25 3. 西方墨點法 25 4. 蛋白質純化與濃縮 26 5. 蛋白質定量 26 九、細胞培養與分析 30 1. 細胞株與培養條件 30 2. 細胞酵素免疫分析法 (Cell ELISA) 30 第三章 實驗結果 33 一、抗EGFR單鏈抗體生產菌株建立與分析 33 二、抗EGFR單鏈抗體純化與濃縮 37 三、抗EGFR單鏈抗體之細胞酵素免疫分析法 39 四、額外表現Mxr1對抗EGFR單鏈抗體之外泌產量的影響 41 1. 額外表現Mxr1顯著提升抗EGFR單鏈抗體mRNA表現量 41 2. 額外表現Mxr1顯著提升抗EGFR單鏈抗體之外泌產量 41 3. 以甘油受限方式誘導抗EGFR單鏈抗體之生產 42 4. 額外表現Mxr1提升胞內抗EGFR單鏈抗體累積量 42 五、額外表現Ire1對抗EGFR單鏈抗體之外泌產量的影響 48 1. 額外表現Ire1對生產菌株SV之影響 48 2. 額外表現Ire1對生產菌株SM之影響 49 第四章 討論 55 一、P. pastoris外泌效率之瓶頸 55 二、額外表現Ire1對P. pastoris未摺疊蛋白質反應之影響 56 1. Kar2 56 2. Hrd1、Ubc1 56 三、額外表現Ire1對P. pastoris外泌生產目標蛋白質之影響 57 第五章 結論 59 第六章 未來展望 60 第七章 參考資料 62 | |
| dc.language.iso | zh-TW | |
| dc.subject | Pichia pastoris | zh_TW |
| dc.subject | 未摺疊蛋白質反應 | zh_TW |
| dc.subject | Mxr1 | zh_TW |
| dc.subject | Ire1 | zh_TW |
| dc.subject | 單鏈抗體 | zh_TW |
| dc.subject | Ire1 | en |
| dc.subject | unfolded protein response | en |
| dc.subject | Mxr1 | en |
| dc.subject | scFv | en |
| dc.subject | Pichia pastoris | en |
| dc.title | 額外表現Ire1對Pichia pastoris外泌生產抗EGFR單鏈抗體之影響 | zh_TW |
| dc.title | Effects of Additional Ire1 Expression on Secretory Efficiency of Anti-EGFR ScFv in Pichia pastoris | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張世宗,林晉玄,陳浩仁,傅煦媛 | |
| dc.subject.keyword | Pichia pastoris,未摺疊蛋白質反應,Mxr1,Ire1,單鏈抗體, | zh_TW |
| dc.subject.keyword | Pichia pastoris,unfolded protein response,Mxr1,Ire1,scFv, | en |
| dc.relation.page | 67 | |
| dc.identifier.doi | 10.6342/NTU201804143 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-04-01 | |
| 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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