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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨 | zh_TW |
dc.contributor.advisor | Ching-Tsan Huang | en |
dc.contributor.author | 曹詠晴 | zh_TW |
dc.contributor.author | Yong-Cing Tsao | en |
dc.date.accessioned | 2023-10-03T17:09:24Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-10-03 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-11 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90680 | - |
dc.description.abstract | 諾羅病毒因基因多樣性和實驗困難度高,對於其預防及治療並無專一性的方法。近期研究顯示諾羅病毒奈米抗體有廣泛結合性及中和諾羅病毒效果,這兩項特性使其被認為有潛力成為抗諾羅病毒的專一性藥物,因此具有大量生產的價值。本篇研究使用Pichia pastoris生產諾羅病毒奈米抗體,P. pastoris是常見的異源基因表達系統,主要使用甲醇進行誘導並生產目標蛋白質,目前研究都針對不同生產階段進行優化,以提升P. pastoris表達系統的應用性。P. pastoris表達系統常用的AOX1啟動子屬於完全抑制型的強啟動子,無法在非甲醇碳源存在時生產目標蛋白質,像是甘油及葡萄糖。前人研究利用P. pastoris內源性啟動子pAOX2額外表現促進因子Mxr1的再程序化策略,可成功於甘油受限誘導下生產目標蛋白質,但其產量還有提升空間,所以本研究將原本的AOX2啟動子置換成來自Hansenula polymorpha異源性啟動子pMOX和pFMD表達促進因子Mxr1。藉由異源性啟動子的不完全抑制活性,表現更多促進因子Mxr1並協助AOX1啟動子更早突破完全抑制的限制,提升在不同碳源誘導下目標蛋白質產量。在本篇論文中,證實由P. pastoris產生的諾羅病毒奈米抗體仍保有與諾羅病毒P蛋白質的專一性結合及中和病毒效果。而甲醇誘導第96小時,MMON (利用H. polymorphap的pMOX表達Mxr1) 的諾羅病毒奈米抗體產量是AMON (利用P. pastoris的pAOX2表達Mxr1) 的148%;甘油受限誘導下是AMON的308%;且MMON及FMON (利用H. polymorphap的pFMD表達Mxr1) 都能夠在抑制性碳源葡萄糖誘導下成功生產諾羅病毒奈米抗體。另外本研究探討AOX1啟動子、Mxr1及目標蛋白質產量這三者之間的相互關係,得知Mxr1累積到特定量時就可開啟AOX1啟動子活性,且發現產量越多的轉形株其AOX1啟動子越早被開啟。異源性啟動子pMOX和pFMD除不受非甲醇碳源抑制外,還能夠快速累積Mxr1並提早開啟AOX1啟動子活性,所以可以提升諾羅病毒奈米抗體產量。總而言之,本研究策略不論於甲醇或非甲醇誘導都能提升目標蛋白質產量改善前人策略的缺失,並使P. pastoris表達系統的誘導策略有更多的應用彈性,未來能夠以安全及成本低的方式得到大量具中和能力的諾羅病毒奈米抗體。 | zh_TW |
dc.description.abstract | Norovirus, due to its genetic diversity and experimental challenges, lacks specific methods for prevention and treatment. Recent studies show that norovirus nanobodies exhibit broad binding activity and virus neutralization, making them potential specific drugs for norovirus infection. Therefore, there is value in their large-scale production. This study produced norovirus nanobodies using Pichia pastoris. P. pastoris is a common heterologous gene expression system, and normally uses methanol induction to produce recombinant proteins. Currently, system optimization is performed at different production stages to enhance system applicability. AOX1 promoter is commonly used in the P. pastoris expression system. It is also known for its promoter repression, meaning it cannot produce protein with existence of repressive carbon source, such as glycerol or glucose. Previous study employed a reprogramming strategy of AOX2 promoter with the additional expression of the coactivator—Mxr1, and successfully produced recombinant protein under glycerol induction. However, there is still potential for improving the yield of production. Therefore, this study replaced AOX2 promoter in reprogramming strategy by heterologous promoters pMOX and pFMD from Hansenula polymorpha. Due to the benefits of derepressed activity, the higher production of Mxr1 helps early overcome repression of the AOX1 promoter. This study confirms that norovirus nanobodies produced by P. pastoris expression system retain their specific binding and virus-neutralizing abilities. The induction results demonstrate that MMON (Mxr1 drived by pMOX from H. polymorpha) achieved 1.48 times higher expression than AMON (Mxr1 drived by pAOX2 from P. pastoris) under methanol induction and 3.08 times increase in expression under glycerol starvation induction. Moreover, both MMON and FMON (Mxr1 drived by pFMD from H. polymorpha) successfully produced norovirus nanobodies under glucose induction. In addition, this study shows that when Mxr1 accumulates to a certain level, it activated the AOX1 promoter. The results indicated that strains with higher yields exhibited earlier activation of the AOX1 promoter. In addition to derepressed activity in heterologous promoter, higher production in the strategy was attributed to the rapid accumulation of Mxr1 and the early activation of the AOX1 promoter under different carbon source induction. The strategy in this study increased productions and overcomed the limitations of previous strategies, whether under methanol or non-methanol carbon sources. It demonstrates more flexible applicability in terms of the induction strategy of the P. pastoris expression system. In the future, it exits tremendous potential to obtain norovirus nanobodies with neutralizing capabilities in a safe and cost-effective manner at large scale. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T17:09:24Z No. of bitstreams: 0 | en |
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dc.description.tableofcontents | 口試委員會審定書 I
謝辭 II 摘要 III Abstract V 圖目錄 XII 表目錄 XIV 第一章 前言 1 一、異源基因表達系統 1 1. Komagataella phaffii (P. pastoris) 2 2. P. pastoris表達系統優勢 3 三、提升目標蛋白質產量策略 7 1. 額外表現活化子Mxr1 7 2. Hansenula polymorpha啟動子 9 四、諾羅病毒及其奈米抗體 10 1. 諾羅病毒 10 2. 奈米抗體 12 3. 諾羅病毒奈米抗體 13 五、研究動機 14 1. 目的 14 2. 策略 15 3. 目標 16 第二章 材料與方法 17 一、實驗菌株與培養條件 17 1. 細菌 17 2. 真菌 17 3. 菌株保存 17 二、培養基 18 三、表現載體建構 20 1. pre-ost1-pro-⍺-nanobody 20 2. pAOX2-Mxr1 20 3. pMOX- Mxr1 20 4. pFMD- Mxr1 20 四、嗜甲醇酵母菌電穿孔轉形 25 1. P. pastoris勝任細胞製備 25 2. 電穿孔轉形 25 五、轉形株抗性篩選 25 六、轉形株培養與分析 26 1. 96深孔盤誘導 (96 DWP) 26 2. 搖瓶誘導 26 3. 轉形株染色體分析 26 七、蛋白質分析 27 1. Bradford蛋白質定量法 27 2. 十二烷基硫酸鈉聚丙烯胺膠體電泳 27 3. 西方點墨法 (Western blotting) 27 4. 胞內AOX活性分析 28 八、mRNA表現量分析 29 九、奈米抗體功能性測試 30 1. His-tag蛋白質純化 30 2. 酵素結合免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA) 31 第三章 結果 35 一、利用P. pastoris生產諾羅病毒奈米抗體 35 1. 建構具外泌訊號之諾羅病毒奈米抗體轉形株 35 2. 確認P. pastoris產出之諾羅病毒奈米抗體的功能性 40 二、建構不同啟動子額外表達Mxr1之轉形株 44 1. AOX2啟動子額外表達Mxr1轉形株建構及篩選 44 2. MOX啟動子額外表達Mxr1轉形株建構及篩選 49 3. FMD啟動子額外表達Mxr1轉形株建構及篩選 54 三、以甲醇誘導的搖瓶測試與分析 59 1. 1%甲醇誘導之奈米抗體產量比較 59 2. 1%甲醇誘導之Mxr1 mRNA與AOX1啟動子活性分析 60 四、非甲醇誘導的搖瓶測試與分析 64 1. 0.33%甘油受限誘導之諾羅病毒奈米抗體產量比較 64 2. 0.33%甘油受限誘導之Mxr1 mRNA與AOX1啟動子活性分析 65 3. 2%葡萄糖誘導之奈米抗體濃度比較 70 4. 2%葡萄糖誘導之Mxr1 mRNA與AOX1啟動子活性分析 70 第四章 討論 75 一、利用P. pastoris生產具功能性諾羅病毒奈米抗體 75 二、異源性啟動子策略使諾羅病毒奈米抗體產量提升的原因 75 1. 額外表達Mxr1可使AOX1啟動子突破碳源抑制 76 2. Mxr1 mRNA與AOX1啟動子開始活化關係 76 3. AOX1啟動子開啟時間與諾羅病毒奈米抗體產量的相關性 78 三、異源性啟動子額外表達Mxr1的策略優缺點 80 第五章 結論 81 第六章 未來展望 82 第七章 參考文獻 83 | - |
dc.language.iso | zh_TW | - |
dc.title | 利用Hansenula polymorpha 啟動子表現Mxr1提升Pichia pastoris諾羅病毒奈米抗體產量 | zh_TW |
dc.title | Production enhancement of Norovirus nanobody in Pichia pastoris by overexpression of Mxr1 using Hansenula polymorpha promoters | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 傅煦媛;林玉儒 | zh_TW |
dc.contributor.oralexamcommittee | Hsu-Yuan Fu;Yu-Ju Lin | en |
dc.subject.keyword | Pichia pastoris,Hansenula polymorpha,異源性啟動子,pMOX,pFMD,非甲醇誘導,Mxr1, | zh_TW |
dc.subject.keyword | Pichia pastoris,Hansenula polymorpha,heterologous promoter,pMOX,pFMD,Methanol-free,Mxr1, | en |
dc.relation.page | 90 | - |
dc.identifier.doi | 10.6342/NTU202303539 | - |
dc.rights.note | 同意授權(全球公開) | - |
dc.date.accepted | 2023-08-12 | - |
dc.contributor.author-college | 生命科學院 | - |
dc.contributor.author-dept | 生化科技學系 | - |
顯示於系所單位: | 生化科技學系 |
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