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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻 | |
dc.contributor.author | Ming-Hung Chen | en |
dc.contributor.author | 陳明宏 | zh_TW |
dc.date.accessioned | 2021-06-16T13:16:46Z | - |
dc.date.available | 2017-07-31 | |
dc.date.copyright | 2013-07-31 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61875 | - |
dc.description.abstract | 基因轉譯工程是近年來頗受歡迎的研究熱點之一,學者們設計出許多不同基因序列編碼,利用微生物將基因序列轉譯重組生產出特定聚胜肽或是蛋白質,並研究其相關功能性後加以應用。有學者則將聚胜肽(蛋白質)與合成高分子進行結合,創造出具有特殊功能的新穎生物高分子材料。
本研究主要目標在將聚胜肽與合成高分子進行共聚合得到由天然與人工聚合物所組成的嵌段共聚物。本實驗利用基因轉譯合成目標重組蛋白,再經由凝血酶切位作用得到6kDa目標重組聚胜肽,並再與合成高分子進行共聚合,以獲得聚胜肽高分子產物。在預聚物部分選用PEG-250 Diacid以及Jeffamine® D2000寡聚物,利用這兩個寡聚物個別帶有雙酸及雙胺官能基,進行縮合聚合反應脫水以結合形成胜肽鍵。透過80°C以及EDC兩種合成方式將6kDa聚胜肽與PEG-250 Diacid以及Jeffamine® D2000寡聚物共聚合,合成出聚胜肽-PEG-D2000多嵌段共聚物。 本篇研究結果顯示,經由西方墨點法與核磁共振儀(NMR)分析鑑定聚胜肽產物,確實得到6kDa聚胜肽;並利用FTIR確定PEG-250 Diacid與Jeffamine® D2000能夠順利反應生成胜肽鍵;最後經由His-tag螢光抗體染色,確定順利合成具有聚胜肽之多嵌段共聚物。 本研究成功將聚胜肽與高分子透過共聚合方式進行結合,獲得擁有聚胜肽的多嵌段共聚物。對於未來如何將具有功能性之聚胜肽與高分子進行結合起到了一定的幫助。 | zh_TW |
dc.description.abstract | In recent years, Translation Engineering has become popular. Many researchers have devised different genetic codes in order to use microorganisms to produce specific recombinant polypeptides or proteins for further study on their functions and application. Some researchers combined the polypeptides (proteins) and synthetic polymers together to create novel biopolymers.
The objective of this study was to copolymerize polypeptides and polymers together to obtain polypeptide-co-polymers that consisted of natural and artificial polymers. In the experiment, we used target gene translation to produce a recombinant target protein; then we used thrombin cleavage to acquire 6kDa target polypeptides. After that, we copolymerized 6kDa polypeptides with the synthetic polymer to obtain polypeptide-polymers. PEG-250 Diacid and Jeffamine® D2000 oligomers were selected as prepolymers, these two oligomers of individual diacids and diamines were then used to form peptide bonds by condensation polymerization reactions. Through 80°C and EDC two synthesis methods, 6kDa polypeptides combined with PEG-250 Diacid and Jeffamine® D2000 oligomers were polymerized into peptide-PEG-D2000 multiblock copolymers. The results showed that 6kDa polypeptides were identified by Western blot and nuclear magnetic resonance (NMR); the peptide bond, which was formed by both PEG-250 Diacid and Jeffamine® D2000, was determined by FTIR. Finally we stained a specific label (His-tag) of 6kDa polypeptides with the His-tag fluorescent antibody and determined that the synthesis of polypeptides multiblock copolymers was successfully. This study successful confirms the combination of polypeptides and polymer binding by copolymerization can obtain multiblock polypeptide-co-polymers. For the future, this study will play some useful methods to combine functional polypeptides and polymers. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T13:16:46Z (GMT). No. of bitstreams: 1 ntu-102-R00549002-1.pdf: 3545756 bytes, checksum: e9f68921626655cd81e5f73d432a8553 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 口試委員審定書 #
誌謝 i 摘要 ii Abstract iii 目錄 iv 圖目錄 vii 表目錄 ix 第一章 緒論 1 1.1 前言 1 1.2 研究目的 2 第二章 文獻回顧 4 2.1 人工設計聚胜肽 4 2.1.1 自組裝合成聚胜肽 4 2.1.2 基因轉譯重組聚胜肽 6 2.1.2.1 類彈性蛋白聚胜肽 7 2.1.2.2 人造蛋白水凝膠 9 2.2 聚胜肽與高分子之應用 11 2.2.1 聚胜肽與高分子合成嵌段共聚物 11 2.2.1.1 聚胜肽嵌段共聚物用於基因治療 11 2.2.1.2 類彈性蛋白多嵌段共聚物 13 2.2.2 聚胜肽與高分子之接枝共聚物 14 2.2.2.1 聚胜肽高分子水凝膠 14 2.2.2.2 高分子改質生物蛋白 17 第三章 實驗材料與方法 19 3.1 實驗藥品與器材 19 3.2 實驗儀器 23 3.3 實驗流程與方法 28 3.3.1 pET-32b(+)質體轉形 28 3.3.1.1 質體複製轉形 28 3.3.1.2 質體純化 28 3.3.1.3 質體表現轉形 29 3.3.2 pET-32b(+)蛋白製備 29 3.3.2.1 pET-32b(+) - 20.4kDa目標重組蛋白表現 29 3.3.2.2 pET-32b(+) - 20.4kDa目標重組蛋白量產與鑑定 30 3.3.2.3 pET-32b(+) - 20.4kDa目標重組蛋白分離純化 31 3.3.2.4 pET-32b(+) - 6kDa聚胜肽純化 32 3.3.3 聚胜肽高分子合成 33 3.3.3.1 PEG-D2000多嵌段共聚物合成 33 3.3.3.2 80°C 合成聚胜肽高分子 33 3.3.3.3 EDC合成聚胜肽高分子 34 3.3.4 十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳 (SDS-PAGE) 34 3.3.5 Coomassie Brilliant Blue 染色鑑定 35 3.3.6 西方墨點法(Western Blot) 35 3.3.7 樣品製備 36 3.3.7.1. NMR核磁共振儀樣品製備 36 3.3.7.2. FTIR樣品製備 36 3.3.7.3. 分光光譜儀穿透度樣品製備 36 3.3.7.4. 粒徑分析儀樣品製備 37 3.3.7.5. 聚胜肽高分子螢光抗體染色 37 第四章 結果與討論 38 4.1 pET-32b(+)質體序列鑑定 38 4.2 pET-32b(+) - 20.4kDa目標重組蛋白表現測試 39 4.3 pET-32b(+) - 20.4kDa目標重組蛋白可溶性確認 39 4.4 pET-32b(+) - 20.4kDa目標重組蛋白純化確認 40 4.5 pET-32b(+) - 20.4kDa目標重組蛋白切割獲得6kDa聚胜肽 41 4.5.1 Coomassie Brilliant Blue染色鑑定6kDa聚胜肽 41 4.5.2 H-NMR鑑定6kDa聚胜肽 42 4.6 PEG-D2000 多嵌段共聚物合成鑑定 42 4.6.1 FTIR合成鑑定 43 4.6.2 PEG-D2000 自組裝行為初步觀察 43 4.6.2.1. PEG-D2000溫度敏感性 43 4.6.2.2. PEG-D2000於不同pH值下對應溫度變化 45 4.7 聚胜肽高分子合成 45 4.7.1 80-6k-PEG-D2000聚胜肽高分子合成 45 4.7.2 EDC-6k-PEG-D2000聚胜肽高分子合成 46 4.8 聚胜肽高分子微胞粒徑分析 46 4.9 His-tag螢光抗體鑑定聚胜肽高分子 47 4.9.1 80-6k-PEG-D2000聚胜肽高分子螢光抗體表現 47 4.9.2 EDC-6k-PEG-D2000聚胜肽高分子螢光抗體表現 47 4.9.3 80-6k-PEG-D2000與EDC-6k-PEG-D2000聚胜肽高分子比較 48 4.9.4 EDC-6k-PEG-D2000聚胜肽高分子與EDC-PEG-D2000螢光抗體表現 48 第五章 結論 50 第六章 參考文獻 51 第七章 圖表 58 | |
dc.language.iso | zh-TW | |
dc.title | 基因轉譯合成聚胜肽及其應用之探討 | zh_TW |
dc.title | Genetically Encoded Synthesis of Polypeptides and Its Applications | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭廖平,林宏殷 | |
dc.subject.keyword | 基因序列,蛋白質純化,胜肽,合聚合,嵌段共聚物, | zh_TW |
dc.subject.keyword | DNA Sequence,rotein Purification,olypeptides,ondensation Polymerization,ultiblock Copolymers, | en |
dc.relation.page | 71 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-07-29 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 高分子科學與工程學研究所 | zh_TW |
顯示於系所單位: | 高分子科學與工程學研究所 |
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