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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳?承 | |
| dc.contributor.author | Shang-Ru Wu | en |
| dc.contributor.author | 吳尚儒 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:20:09Z | - |
| dc.date.available | 2023-08-21 | |
| dc.date.copyright | 2018-08-21 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72035 | - |
| dc.description.abstract | 近年來，蠶絲蛋白（silk fibroin）常被用於生醫材料應用，其特點包含良好的機械強度、生物相容性、延展性，並具有良好的材料可修飾性進而可增加生物功能性。本研究以蠶絲蛋白作為生物材料支架，並經由生物製造之酵素修飾方式交聯可吸附多種病原菌之功能性分子，進而建立一個病原菌快速捕捉平台，可望作為新型的病原菌分離與篩選的裝置。具體來說，在蠶絲支架的製備上，我們首先將蠶絲經由冷凍乾燥法製成具多孔狀的蠶絲海綿，使得可比一般平面基質具有較大的孔隙度與作用面積。接著我們進一步調整蠶絲海綿的結構穩定性，經由乙醇處理後，能提升結構的結晶度，增強蠶絲海綿的的穩定性。在功能性修飾部分，我們進一步藉由酵素horseradish peroxidase (HRP)將基因改造之功能性蛋白質apolipoprotein H (ApoH)以二聚化形成雙酪氨酸共價鍵的方式鑲嵌在絲蛋白上。ApoH 為一種人體在免疫急性期會分泌出的載脂蛋白，具有體內先天免疫系功能，能以電荷吸引的特性吸附許多表面帶有負電荷的病原菌，像是病毒、革蘭氏陰性菌，甚至是寄生蟲等等。本研究所建構的病原菌捕捉功能性海綿已經證實可以捕捉大腸桿菌與諾羅病毒，將來將進一步改良此技術平台，預期在未來能夠運用於許多環境或生醫檢測，以及血液透析的病原菌分離醫材等開發。 | zh_TW |
| dc.description.abstract | Silk fibroin produced from silkworms has been widely used as biomaterials owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered the silk fibroin as a novel platform capable of capturing various species of pathogens by functionalizing pathogen targeting moiety. Specifically, we first generated the silk material into a porous silk sponge through the lyophilization procedure, yielding enhanced porosity and accessible surface area. The structural stability of our engineered sponge was further improved with ethanol treatment. Second, the sponge was functionalized with engineered apolipoprotein H (ApoH) by forming di-tyrosine crosslinking through the catalyzation of horseradish peroxidase (HRP). ApoH, an immune acute phase human plasma protein, serves as an innate immune defense system against infection and exhibits broad spectrum of pathogen binding ability, including viruses, Gram-negative bacteria and parasites. Finally, the platform was demonstrated to be effective in E. coli and norovirus binding. We envision the device we fabricated, yet to be tuned up, can potentially as a device for rapid isolation and high throughput screening of pathogens. Additionally, the technique could be further harnessed in hemodialysis for reducing the risk of infection in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T06:20:09Z (GMT). No. of bitstreams: 1 ntu-107-R05b22057-1.pdf: 4014001 bytes, checksum: 902f7670d35ae0929f2048919e2127fe (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 摘要 2
Abstract 3 第一章前言 9 1.1 生醫材料 9 1.2 研究動機 10 1.3 蠶絲蛋白 11 1.3.1 結構與性質 11 1.3.2 蠶絲蛋白支架 12 1.3.3 蠶絲蛋白支架及處理 12 1.4 載脂蛋白 H (Apolipoprotein H, ApoH) 13 1.4.1 載脂蛋白H生理功能 13 1.4.2 載脂蛋白H結構 13 1.5 辣根過氧化酵素（Horseradish peroxidase, HRP） 13 1.5.1 HRP 的結構與催化反應機制 13 1.6 辣根過氧化酵素（Horseradish peroxidase, HRP） 14 1.6.1 HRP 的結構與催化反應機制 14 1.6.2 HRP 應用 16 1.7 實驗設計 17 1.7.1 研究架構 17 1.7.2 欲達成之目標 17 第二章 材料與方法 18 2.1 實驗菌株與培養條件 18 2.1.1 細菌 18 2.1.2 細胞株 18 2.2 培養基 18 2.3 質體建構與轉型株 19 2.3.1 短鏈核酸 (oligo DNA) 設計與合成 19 2.3.2 聚合酶連鎖反應反應試劑、溶液 19 2.3.3 核酸瓊脂膠體電泳分析 20 2.3.4 DNA純化 21 2.3.5 質體DNA之萃取 21 2.3.6 質體與目標基因切割(Digestion) 22 2.3.7 接合(Ligation)反應 22 2.3.8 質體建構 22 2.3.9 勝任細胞製備 24 2.3.10質體轉型反應(Transformation) 25 2.3.11核酸定序 26 2.3.12質體DNA之萃取 26 2.4 多孔性蠶絲海綿支架製備 26 2.4.1蠶絲蛋白水溶液製備 26 2.4.2製備多孔性蠶絲海綿支架 26 2.4.3溶解度實驗 26 2.4.4 X光繞射儀 (X-ray diffraction, XRD) 27 2.4.5掃描式電子顯微鏡觀測 (Scanning Electron Microscope, SEM) 27 2.4.6 FTIR (Fourier Transform Infrared Spectroscopy) 27 2.4.7製備材料 28 2.5 融合蛋白質表現及純化 28 2.5.1 蛋白質表現 28 2.5.2 蛋白質定量 29 2.5.3 反應試劑、溶液 29 2.5.4 融合蛋白純化及分析 30 2.6 蛋白質功能檢測 31 2.7 接合作用測試 32 2.7.1 大腸桿菌抓取能力測試 32 2.7.2 海綿與 ApoH 接合效果測試 32 2.7.3 諾羅病毒抓取能力測試 32 2.7.4 海綿抓取諾羅病毒測試 33 第三章實驗結果 34 3.1 製備及分析多孔性海綿支架結構特性 34 3.1.1以不同濃度乙醇進行處理之海綿支架 34 3.1.2 XRD分析海綿支架之結晶程度 34 3.1.2 ATR-FTIR分析海綿支架之二級結構組成 34 3.1.3分析海綿支架在水溶液中之穩定性 34 3.2 以 HEK-293T 細胞表現融合蛋白 35 3.2.1突變質體構築 35 3.2.2表現細胞株建構 35 3.2.3蛋白質表現、純化和分析 35 3.3 目標蛋白活性測試 36 3.3.1測試載脂蛋白H功能 36 3.4 將目標蛋白以HRP結合至多孔性蠶絲支架上 36 3.5 多孔性蠶絲支架抓取大腸桿菌之效果測試 36 3.6 多孔性蠶絲支架抓取諾羅病毒顆粒之效果測試 37 第四章討論 38 4.1載脂蛋白H ( ApoH )表現 38 4.2 Escherichia coli表達系統 38 4.3 Pichia pastoris表達系統 39 4.4載脂蛋白H ( ApoH )在E.coli BL21DE3及Pichia pastoris表現系統比較 39 4.5接合作用實驗 40 第五章結論 69 第六章未來展望 70 第七章、參考文獻 71 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蠶絲海綿 | zh_TW |
| dc.subject | 載脂蛋白H | zh_TW |
| dc.subject | 病原菌 | zh_TW |
| dc.subject | silk fibroin | en |
| dc.subject | sponge | en |
| dc.subject | Apolipoprotein H | en |
| dc.subject | HRP | en |
| dc.subject | pathogens | en |
| dc.title | 以功能性蠶絲海綿作為捕捉病原菌的平台 | zh_TW |
| dc.title | Developing a platform for pathogen capturing by biofunctionalized silk sponges | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊振昌,楊大毅 | |
| dc.subject.keyword | 蠶絲海綿,載脂蛋白H,病原菌, | zh_TW |
| dc.subject.keyword | silk fibroin,sponge,Apolipoprotein H,HRP,pathogens, | en |
| dc.relation.page | 74 | |
| dc.identifier.doi | 10.6342/NTU201803957 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-20 | |
| 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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