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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81614完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳亘承(Hsuan-Chen Wu) | |
| dc.contributor.author | Wen-Chia Chen | en |
| dc.contributor.author | 陳玟嘉 | zh_TW |
| dc.date.accessioned | 2022-11-24T09:24:46Z | - |
| dc.date.copyright | 2021-11-09 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81614 | - |
| dc.description.abstract | "基因編輯系統CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Proteins)能夠精準且有效的編輯基因組，提供未來治療疾病與細胞編輯的新方向。然而截至目前，基因編輯系統仍缺乏適當與安全的傳輸方式將CRISPR/Cas傳輸至宿主體內。常用的病毒相關CRISPR載體有外源基因隨機插入宿主基因體的風險，並且無法長期控制Cas內切脢表達量，易造成脫靶效應; 另一個方式則是直接遞送已組裝之CRISPR 核酸蛋白質至宿主體內，雖然限制蛋白質總量使其較具安全性，但組裝之CRISPR藥物難以脫離細胞之胞內體 (endosome)，導致載體運送CIRSPR藥物仍為一道難題。為了改善目前傳遞蛋白質的困境，本研究欲透過合成生物學，編輯益生菌作為一種CRISPR 核酸蛋白質之傳遞系統，除了可在益生菌內高效率的生產、組裝CRISPR核酸蛋白質，同時益生菌本身也被改造為具有保護、傳輸功能的載體。 具體來說，我們以合成生物學改造probiotic E. coli Nissle 1917 (EcN) 表達誘發細胞胞吞的invasin與脫離胞內體的listeriolysin O (LLO) ，藉以釋放細菌表達的CRISPR 核酸蛋白質進入細胞質，接著核酸蛋白質藉由核定位序列 (nucleus localization signal, NLS) 的輔助進入細胞核中編輯基因 (knockout)。目前，本研究證實EcN運輸系統可透過invasin與LLO將螢光蛋白質送入細胞的細胞質中; 同時EcN組裝之核酸蛋白質具有in vitro 與 in vivo基因剪輯能力，亦即我們以HCT116-EGFP做為剔除細胞基因模式，以EcN傳輸系統傳遞CRISPR 核酸蛋白質剔除細胞內EGFP基因之後，觀察到HCT116細胞族群的GFP螢光細胞數量降低，表達證實EcN可傳遞CRISPR 核酸蛋白質進行編輯基因。期望此系統未來可做為一種經濟、安全、有效的蛋白質傳輸載體，並提供基因編輯新的應用方式。" | zh_TW |
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| dc.description.tableofcontents | 口試委員審定書 I 致謝 II 中文摘要 III 英文摘要 IV 第1章 緒論 1 1.1. 基因編輯發展 1 1.1.1. 基因編輯工具 - CRISPR/Cas9 2 1.1.2. 基因編輯工具 - CRISPR/Cas12b 3 1.1.3. 基因編輯後細胞修復 3 1.2. CRISPR/Cas在基因治療的方式 4 1.2.1. CRISPR/Cas RNP在基因治療的方式 4 1.2.2. CRISPR/Cas 缺乏合適傳遞載體進入細胞編輯 5 1.3. 細菌作為新型運輸載體的可能性 6 1.4. 益生菌E. coli Nissle 1917 (EcN) 7 1.4.1. EcN作為傳遞藥物載體的潛力 8 1.5. 細菌載體透過細胞胞吞進入細胞內部 9 1.6. 細胞胞吞分解過程與細菌脫離機制 10 1.6.1. 目標蛋白質之細胞核運輸 10 1.7. 研究策略 12 1.8. 研究目標 14 第2章 材料與方法 15 2.1. 實驗菌株與培養方式 15 2.1.1. 細菌株 15 2.1.2. 細胞株 15 2.2. 培養基與藥品 16 2.2.1. 細菌相關培養藥品 16 2.2.2. 細胞相關培養藥品 17 2.3. 質體建構 18 2.3.1. pACYC-T5-DsRed-NLS (DsRed-NLS) 18 2.3.2. pACYC-T7-lacO-Cas9-rrnB-J23119-sgPD-L1 (Cas9-sgPD-L1) 18 2.3.3. pACYC2-T5-lacO-Cas12b-rrnB-J23119-sgPD-L1/ EGFP/ Control (Cas12b-sgPD-L1/ Cas12b-sgEGFP/ Cas12b-sgControl) 18 2.3.4. pACYC2-T5-lacO-mCherry-Cas12b-3NLS-rrnB-J23119-sg EGFP/ Control (mCherry-Cas12b-sgEGFP/ mCherry-Cas12b-sgControl) 19 2.3.5. pGB3-inv-J23102-lacO-hly’(inv-hly’) 19 2.4. 蛋白質表達與純化 22 2.4.1. Cas9/Cas12b RNP親合管住層析純化與超濾膜濃縮 22 2.4.2. 聚丙烯胺膠體電泳分析 22 2.4.3. Cas9/Cas12b目標片段剪切 23 2.5. 大腸桿菌電穿孔轉型法 24 2.5.1. 大腸桿菌電轉勝任細胞製備 24 2.6. 細菌營養缺陷實驗 24 2.7. 細菌與細胞共同培養實驗 25 2.7.1. 細胞與EcN共同培養測試 25 2.7.2. Invasin表達之細胞胞吞測試 (Invasin-mediated cellular uptak assay) 26 2.7.3. 核定為序列 (Nucleus localization signal) 差異測試 27 2.7.4. 細胞與細菌比例(MOI)差異測試 27 2.7.5. 時間差異測試 28 2.7.6. 細胞編輯測試 29 第3章 結果與討論 30 3.1. 質體建構、表現、純化和分析 30 3.1.1. Cas9-sgPD-L1之建構、純化、測試 30 3.1.2. Cas12b RNP 之建構、純化、測試 34 3.1.3. Cas12b-sgPD-L1之建構、純化、測試 34 3.1.4. Cas9與Cas12b之剪切測試 36 3.1.5. Cas9與Cas12b pH耐受性比較 38 3.1.6. Cas12b-EGFP 建構、表達與測試 40 3.1.7. Cas12b-sgRNA 功能測試 40 3.1.8. DsRed-NLS質體建立 43 3.1.9. 目標蛋白於EcN共同表達 44 3.2. 細菌營養缺陷實驗 46 3.3. 細菌與細胞共同培養實驗 48 3.3.1. 細胞與EcN共同培養測試 48 3.3.2. 細胞胞吞Invasin表達菌株測試 54 3.3.3. 細菌傳遞DsRed蛋白質實驗 56 3.3.4. 檢測NLS將螢光蛋白質送入細胞中的效果 57 3.3.5. 檢測不同MOI對DsRed-NLS傳遞至細胞內差異 58 3.3.6. 檢測DsRed-NLS在不同時間點於細胞內之位置 60 3.3.7. EcN recAdapA傳遞mCherry-Cas12b-sgEGFP RNP in vivo實驗 62 第4章 結論 66 第5章 未來展望 67 第6章 參考文獻 69 | |
| dc.language.iso | zh-TW | |
| dc.subject | CRISPR/Cas | zh_TW |
| dc.subject | 蛋白質傳遞系統 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | E. coli Nissle 1917 | zh_TW |
| dc.subject | E. coli Nissle 1917 | en |
| dc.subject | protein delivery system | en |
| dc.subject | CRISPR/Cas | en |
| dc.subject | genome editing | en |
| dc.title | 利用生物工程改造益生大腸桿菌作為CRISPR合成與傳遞之基因編輯系統 | zh_TW |
| dc.title | Bioengineered probiotic Escherichia coli as a novel CRISPR synthesis and delivery system towards eukaryotic genome editing | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃筱鈞(Hsin-Tsai Liu),李哲欣(Chih-Yang Tseng),胡育誠 | |
| dc.subject.keyword | CRISPR/Cas,蛋白質傳遞系統,基因編輯,E. coli Nissle 1917, | zh_TW |
| dc.subject.keyword | CRISPR/Cas,protein delivery system,genome editing,E. coli Nissle 1917, | en |
| dc.relation.page | 75 | |
| dc.identifier.doi | 10.6342/NTU202102588 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-08-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2021-08-27 | - |
| 顯示於系所單位： | 生化科技學系 | |
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