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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 | 劉思媛 | zh_TW |
| dc.contributor.author | Ssu-Yuan Liu | en |
| dc.date.accessioned | 2021-07-10T21:53:12Z | - |
| dc.date.available | 2024-08-14 | - |
| dc.date.copyright | 2019-08-16 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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ALTEX-Alternatives to animal experimentation 31, 37-42 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77262 | - |
| dc.description.abstract | 目前對於新型H7N9流感仍無有效的疫苗，且病毒株已對克流感產生抗藥性，造成治療的難度增加，因此本研究擬將可分別辨認H7N9 HA1及HA2的單株抗體F3-2及1C6B，製備成可同時結合上HA1及HA2之雙特異性抗體 (Bispecific antibody, BsAb)，藉此形成類似”分子鉗”的結構配置，達到抑制血球凝集素的構形改變，進而抑制H7N9病毒感染宿主細胞的功效。實驗首先擷取F3-2或1C6B之變異區 (Variable region) 以組成單鏈抗體 (scFv)，再利用不同長度的linker (例如G、(G4S)x2、(G4S)x4、(G4S)x6 或 (G4S)x8) 來連結F3-2 scFv及1C6B scFv，以增加結構的延展性與靈活性。於大腸桿菌中進行蛋白質表現時，此BsAb大部分會形成不可溶之包涵體，僅有極少量的BsAb為可溶狀態，但是此可溶狀態之BsAb具有可同時結合HA1及HA2之能力。然而，用尿素緩衝溶液所純化出之BsAb，則會喪失其抗原結合能力。因此，本論文繼續嘗試不同的表現條件以解決BsAb不可溶且產量低的問題。最後以GST-BsAb融合蛋白質的方式進行表現，可以製備出較多可溶之GST-BsAb，但是此融合蛋白質雖然仍可辨認HA1，但是卻無法辨認HA2。進一步利用TEV protease將GST-BsAb截切成free form BsAb後，仍無法使其恢復辨認HA2的能力，顯示此BsAb可能仍須直接表現為可溶形式的單體，才具有能同時結合上HA1及HA2之功能。 | zh_TW |
| dc.description.abstract | There is currently no effective vaccine for prevention of the novel H7N9 influenza pandemic, and the virus strain has developed resistance to Tamiflu, resulting in increased treatment difficulties. Thus, the present study aimed to develpe bispecific antibodies (BsAbs), composed of the single-chain variable fragments (scFvs) derived from the monoclonal antibody F3-2 and 1C6B which can respectively bind to H7N9 HA1 and HA2, to form a "molecular clamp" structural configuration for increasing efficacy of inhibiting the conformational change of hemagglutinin and the H7N9 infection in host cells. In addition, a linker with different length (i.e., G, (G4S)x2, (G4S)x4, (G4S)x6, or (G4S)x8) was also introduced in the BsAb to connect the F3-2 scFv with the 1C6B scFv for increasing the extensibility and flexibility. When performing protein expression in Escherichia coli, most of the BsAbs formed insoluble inclusion bodies, and only a very small amount of BsAb was soluble, but this soluble BsAb contained the ability to specifically bind to HA1 and HA2. However, the BsAb purified with a urea buffer lost its antigen binding ability. To reach the goal of getting large amount of soluble BsAbs, different protein expression protocols and plasmids were also applied in the experiments. The results showed that more soluble GST-BsAb can be produced by expressing it as the GST fusion protein. This GST-BsAb fusion protein can still recognize HA1, but it can not recognize HA2. Further use of TEV protease to cut the GST-BsAb into a free form BsAb still failed to restore its ability to recognize HA2, indicating that this BsAb may still need to be directly expressed as a soluble form of monomer to retain the function of simultaneously binding to HA1 and HA2. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:53:12Z (GMT). No. of bitstreams: 1 ntu-108-R06b22016-1.pdf: 2752291 bytes, checksum: 34c7e805efe3dd36937a6a8346ad96e4 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 摘要 i
Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 流行性感冒病毒簡介 1 1.1.1 流感病毒的分類 1 1.1.2 A型流感病毒的構造及組成 1 1.1.3 A型流感病毒生活週期 2 1.2 新型H7N9流感病毒簡介 2 1.2.1 新型H7N9流行病學 3 1.2.2 H7N9感染的治療方式 3 1.3 H7N9之血球凝集素 3 1.3.1 血球凝集素之結構 4 1.3.2 血球凝集素與受體結合 4 1.3.3 膜融合之構形改變 4 1.4 抗體藥物 5 1.4.1 抗體藥物發展 5 1.4.2 針對流感病毒治療之抗體藥物 6 1.4.3 單鏈抗體 6 1.4.4 雙特異性抗體 (BsAb) 7 1.5 單株抗體F3-2及1C6B介紹 7 1.6 研究動機 8 第二章 材料與方法 9 2.1 實驗材料 9 2.1.1 F3-2 scFv及1C6B scFv基因來源 9 2.1.2 H7N9病毒顆粒來源 9 2.1.3 大腸桿菌表現載體 9 2.1.4 大腸桿菌菌株 10 2.2 基因表現載體之建構 11 2.2.1 抗原基因建構 11 2.2.2 F3-2 scFv-G-1C6B scFv核酸引子設計 11 2.2.3 合成 (G4S)4 及 (G4S)8 linker 11 2.2.4 聚合酶連鎖反應 12 2.2.5 限制酶切反應 12 2.2.6 接合反應 (ligation) 12 2.2.7 質體轉形 (transformation) 12 2.2.8 質體DNA製備 12 2.3 BsAb表現及純化 13 2.3.1 測試抗體表現條件 13 2.3.2 His-tag重組蛋白質純化 13 2.3.3 GST-tag融和蛋白純化 14 2.3.4 TEV protease截切 14 2.4 蛋白質相關實驗 14 2.4.1 蛋白質定量法 14 2.4.2 蛋白質膠體電泳 15 2.4.3 蛋白質電泳膠片染色 15 2.4.4 蛋白質轉印 15 2.4.5 免疫染色 (Immunoblotting) 15 2.5.1 利用Western blotting確認抗體效價 16 2.5.2 利用ELISA確認抗體辨認病毒顆粒 16 第三章 結果 17 3.1 BsAb之表現質體建構 17 3.1.1 建構linker為G之BsAb 17 3.1.2 建構linker為(G4S)4及(G4S)8之BsAb 17 3.1.3 建構linker為(G4S)2及(G4S)6之BsAb 18 3.2 利用大腸桿菌表現並純化BsAb 18 3.2.1 純化可溶之BsAb 18 3.2.2 純化不可溶之包涵體中的BsAb 19 3.3 確認BsAb辨認重組抗原及病毒顆粒之效果 19 3.4 優化BsAb之表現條件 20 3.4.1 在不同菌株及溫度下表現BsAb 20 3.4.2 重新建構及表現加入融合蛋白MBP之BsAb 21 3.4.3 重新建構及表現不同質體中之BsAb 22 3.4.4 純化pET28a-MBP及pGEX4T-1質體表現之BsAb 22 3.5 截切GST融合蛋白並測試BsAb功能 23 3.5.1 利用TEV蛋白酶截切融合蛋白中之GST 23 3.5.2 切除GST後之BsAb的抗原辨識效果 23 第四章 討論 24 4.1. 初步表現及純化BsAb結果之探討 24 4.2. 比較可溶及不可溶之BsAb辨認抗原效果 24 4.3. 比較不同條件表現之BsAb 25 4.3.1. pET28a-BsAb在不同菌株及溫度培養之抗體表現量探討 25 4.3.2. pMAL-p2-BsAb在不同菌株及溫度培養之抗體表現量探討 25 4.3.3. 比較BsAb於不同質體下表現量及溶解狀況 26 4.3.4. 比較BsAb於pET28a-MBP及pGEX4T-1質體中純化效果 26 4.4. 比較TEV蛋白酶截切融合蛋白中GST之條件 26 4.5. 比較GST-BsAb及加入TEV截切GST後之BsAb辨認效果 27 參考文獻 28 圖與表 33 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 雙特異性抗體 | zh_TW |
| dc.subject | H7N9 流感病毒 | zh_TW |
| dc.subject | 血球凝集素 | zh_TW |
| dc.subject | Bispecific antibody | en |
| dc.subject | H7N9 influenza virus | en |
| dc.subject | Hemagglutinin | en |
| dc.title | 開發可辨認 H7N9 HA1 及 HA2 之雙特異性抗體 | zh_TW |
| dc.title | Development of the Bispecific Antibody for Binding with the H7N9 HA1 and HA2 Domains | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 廖憶純;陳慧文;游傑華 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | H7N9 流感病毒,血球凝集素,雙特異性抗體, | zh_TW |
| dc.subject.keyword | H7N9 influenza virus,Hemagglutinin,Bispecific antibody, | en |
| dc.relation.page | 48 | - |
| dc.identifier.doi | 10.6342/NTU201903209 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-13 | - |
| 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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