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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | zh_TW |
| dc.contributor.advisor | Shih-Chung Chang | en |
| dc.contributor.author | 何儀君 | zh_TW |
| dc.contributor.author | Yi-Jun He | en |
| dc.date.accessioned | 2021-07-10T21:52:56Z | - |
| dc.date.available | 2024-08-20 | - |
| dc.date.copyright | 2019-08-23 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77255 | - |
| dc.description.abstract | 能專一性鑑別新型H7N9流感病毒血球凝集素之單株抗體1C6B的單鏈抗體，於大腸桿菌中表現時，幾乎都形成了不溶的包涵體。相反的，表現1C6B-scFv與麥芽糖結合蛋白之融合基因時，則可形成可溶性之融合蛋白，但它卻喪失了與H7N9 HA結合的能力。因此，本研究旨在生產可溶形式的1C6B-scFv，並且使其仍具有正常之功能和專一性，並藉此建立生產可溶性scFv之蛋白質表現與純化系統。實驗結果顯示，從MBP融合蛋白切割出之1C6B-scFv可溶性佳，並且可以利用Ni-NTA及DEAE層析管柱進行純化。更重要的是，可溶形式的1C6B-scFv保留了與H7N9 HA結合的能力。本研究亦利用此步驟表現及純化出可溶形式之anti-NP-scFv與anti-NS1-scFv，並且使其仍保有辨認NS1與NP之專一性結合能力。因此，本研究利用MBP表現系統生產MBP-scFv，並進一步以TEV protease剪切及管柱層析法來純化可溶形式之scFv，成功建立了可快速獲得可溶性且具有與其原始全長抗體相同專一性之單鏈抗體的表現及純化系統。 | zh_TW |
| dc.description.abstract | The single-chain variable fragment (scFv) of the monoclonal antibody 1C6B, which is specific for recognition of the novel H7N9 influenza virus hemagglutinin (HA), was mainly expressed as inclusion bodies in Escherichia coli. In contrast, the maltose binding protein (MBP)-fused 1C6B-scFv was expressed as a soluble form, but it lost the capability for binding with H7N9 HA. Thus, the present study aims to produce the soluble form of 1C6B-scFv for characterization of the function and specificity of 1C6B-scFv, and establish a general protein expression and purification system which can be applied in generating soluble scFvs. The results showed that the 1C6B-scFv cleaved from the MBP fusion protein becomes soluble and can be easily purified by Ni-NTA and DEAE agarose chromatography columns. More importantly, the soluble form of 1C6B-scFv retains its capability for binding with H7N9 HA. By using the same procedure, the soluble form of anti-NP-scFv and anti-NS1-scFv were also purified, and they retained the capability for binding with NP and NS1, respectively. This study successfully established a protocol, composed of the MBP-scFv expression system, the TEV protease cleavage step, and the column chromatography, to produce the soluble scFv, which performs the same antigen binding specificity as the original full-length antibody does. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:52:56Z (GMT). No. of bitstreams: 1 ntu-108-R06b22009-1.pdf: 4644109 bytes, checksum: 824c9d9eb56b98fd1c99a9a9611a1ed3 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄 i
中文摘要 iv Abstract v 縮寫表 vi 圖目錄 viii 第一章 緒論 1 1.1 抗體 1 1.1.1抗體的結構 1 1.1.2 單鏈抗體之結構 2 1.1.3 單鏈抗體之應用 2 1.2 單鏈抗體生產 3 1.2.1 人類與哺乳類細胞表現系統 3 1.2.2昆蟲細胞表現系統 4 1.2.3 酵母菌表現系統 4 1.2.4 大腸桿菌表現系統 5 1.3以融合蛋白質形式表現可溶性單鏈抗體 5 1.3.1 以大腸桿菌素融合蛋白質表現可溶性單鏈抗體 6 1.3.2 以雙硫鍵氧化還原酶融合蛋白質表現可溶性單鏈抗體 6 1.3.3 以Cellulose binding domain融合蛋白質表現可溶性單鏈抗體 7 1.3.4 以GFP融合蛋白質表現可溶性單鏈抗體 7 1.3.5 以GST融合蛋白質表現可溶性單鏈抗體 8 1.3.6 以Maltose-binding protein融合蛋白質表現可溶性單鏈抗體 8 1.4 研究動機與目標 9 第二章 材料與方法 11 2.1大腸桿菌菌株 11 2.1.1大腸桿菌DH5α 11 2.1.2大腸桿菌BL21(DE3) 11 2.1.3大腸桿菌Rosetta(DE3) 11 2.1.4大腸桿菌ARP 11 2.2蛋白質表現質體建構 12 2.2.1 引子設計 12 2.2.2聚合酶鏈鎖反應 12 2.2.3限制酶切割反應 13 2.2.4接合反應 13 2.2.5質體純化 13 2.2.6核酸片段純化 14 2.2.7核酸定量 14 2.3質體轉形作用 14 2.4蛋白質表現 15 2.5蛋白質粗萃液製備 15 2.6重組單鏈抗體純化 15 2.6.1 純化MBP-scFv融合蛋白質 15 2.6.2 將MBP-scFv與TEV protease混合進行反應 16 2.6.3 純化單體形式之單鏈抗體 16 2.6.4蛋白質脫鹽與濃縮 17 2.7 Bradford 蛋白質定量法 17 2.8聚丙烯醯胺膠體電泳 17 2.9 CBR 染色法 18 2.10西方墨點法 18 2.11抗體結合能力分析 19 2.12製備GST-HA抗原片段 20 第三章 結果 21 3.1大腸桿菌表現質體建構 21 3.1.1 MBP-1C6B-scFv表現質體建構 21 3.1.2 MBP-anti-NP-scFv與MBP-anti-NS1-scFv表現質體建構 21 3.1.3 TEV protease表現質體建構 22 3.2 1C6B-scFv在不同大腸桿菌中的表現與純化 22 3.3 MBP-1C6B-scFv在不同MBP系統中的表現與純化 23 3.4 MBP-1C6B-scFv在不同大腸桿菌中的表現 23 3.5 MBP-scFv在不同溫度下的表現與純化 24 3.6 將MBP-scFv與TEV protease混合進行反應 25 3.7 純化1C6B-scFv 26 3.8 純化anti-NS1-scFv 26 3.9 純化anti-NP-scFv 27 3.10單鏈抗體專一性結合能力分析 27 3.10.1 1C6B-scFv專一性結合能力分析 27 3.10.2 anti-NS1-scFv專一性結合能力分析 28 3.10.3 anti-NP-scFv專一性結合能力分析 29 第四章 討論 30 參考文獻 35 圖與表 44 附錄 67 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | H7N9流感病毒 | zh_TW |
| dc.subject | 單鏈抗體 | zh_TW |
| dc.subject | 麥芽糖結合蛋白質 | zh_TW |
| dc.subject | 非結構蛋白NS1 | zh_TW |
| dc.subject | 血球凝集素HA | zh_TW |
| dc.subject | 核蛋白NP | zh_TW |
| dc.subject | H7N9 influenza virus | en |
| dc.subject | Hemagglutinin | en |
| dc.subject | Nucleoprotein | en |
| dc.subject | Non-structural protein 1 | en |
| dc.subject | Single-chain variable fragment | en |
| dc.subject | maltose binding protein | en |
| dc.title | 建立可溶性單鏈抗體表現與純化系統 | zh_TW |
| dc.title | Establishment of the Protein Expression and Purification System for Generating the Soluble Single-Chain Variable Fragment Antibody | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林翰佳;陳威戎;黃純芳 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | H7N9流感病毒,血球凝集素HA,非結構蛋白NS1,核蛋白NP,單鏈抗體,麥芽糖結合蛋白質, | zh_TW |
| dc.subject.keyword | H7N9 influenza virus,Hemagglutinin,Nucleoprotein,Non-structural protein 1,Single-chain variable fragment,maltose binding protein, | en |
| dc.relation.page | 68 | - |
| dc.identifier.doi | 10.6342/NTU201903138 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-14 | - |
| 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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| ntu-107-2.pdf 未授權公開取用 | 4.54 MB | Adobe PDF |
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