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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗(Shih-Chung Chang) | |
| dc.contributor.author | Shiau-Yu Lin | en |
| dc.contributor.author | 林筱渝 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:48:58Z | - |
| dc.date.copyright | 2022-08-31 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-26 | |
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Antiviral Res 200, 105290. 36. Cao, Y., Yisimayi, A., Jian, F., Song, W., Xiao, T., Wang, L., Du, S., Wang, J., Li, Q., Chen, X., et al. (2022). BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature 608, 593-602. 37. Li, C.J., Chao, T.L., Chang, T.Y., Hsiao, C.C., Lu, D.C., Chiang, Y.W., Lai, G.C., Tsai, Y.M., Fang, J.T., Ieong, S., et al. (2022). Neutralizing Monoclonal Antibodies Inhibit SARS-CoV-2 Infection through Blocking Membrane Fusion. Microbiol Spectr 10, e0181421. 38. Wang, C., Li, W., Drabek, D., Okba, N.M.A., van Haperen, R., Osterhaus, A., van Kuppeveld, F.J.M., Haagmans, B.L., Grosveld, F., and Bosch, B.J. (2020). A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun 11, 2251. 39. Almagro, J.C., and Fransson, J. (2008). Humanization of antibodies. Front Biosci 13, 1619-1633. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86321 | - |
| dc.description.abstract | 已肆虐全球兩年的SARS-CoV-2病毒，其表面的棘蛋白在病毒入侵宿主細胞上扮演重要角色，尤其是棘蛋白上的受體結合區域 (receptor-binding domain, RBD) 是病毒與人類細胞的血管收縮素轉化酶2 (angiotensin-converting enzyme 2, ACE2) 結合的位置，因此RBD成為中和性抗體的主要標的。但因為RBD為病毒的高度變異區，這類抗體遇到新的突變株時，容易發生免疫逃脫的現象。故本研究以RBD作為標的，開發出六個對於RBD以及棘蛋白都具有高度辨識力的抗體，分別是2A11、2D5、2F9、3B3、3D10和3F4。針對病毒各個變異株進行中和性測試，2F9和3B3對於SARS-CoV-2武漢株具有中和能力；2F9、3B3、3D10和3F4對於Alpha變異株具有中和能力；2A11、2D5、2F9、3B3和3D10則對於Delta變異株具有中和能力；3F4則是唯一對於Omicron BA.1突變株具有中和能力的抗體。本研究使用丙胺酸定點突變的方式，鑑定出單株抗體對RBD的確切結合位。由3F4或2A11以及S2-4A或S2-8D所組成的抗體雞尾酒，對於Omicron BA.2突變株具有加乘成的中和能力。本研究顯示搭配使用RBD抗體與S2 抗體的抗體雞尾酒療法，具有潛力開發成對付新冠病毒感染之預防與治療藥物。 | zh_TW |
| dc.description.abstract | The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARA-CoV-2) is an important viral surface protein for receptor binding and virus-host cell membrane fusion. The receptor-binding domain (RBD) of the S1 subunit of S protein mediates viral binding to human angiotensin-converting enzyme 2 (ACE2). Thus, RBD is considered as the main target of the neutralizing antibodies. Recently, many SARS-CoV-2 variants appear sequence mutations at RBD. Therefore, antibody cocktail is believed to be a better therapeutics for preventing the SARS-CoV-2 immune escape. In the study, monoclonal antibodies (mAbs) 2A11, 2D5, 2F9, 3B3, 3D10 and 3F4 show great specificity to SARS-CoV-2 RBD and S protein. 2F9 and 3B3 exhibit neutralizing activities against SARS-CoV-2 Wuhan strain. 2F9, 3B3, 3D10, and 3F4 exhibit neutralizing activities against SARS-CoV-2 Alpha variant. 2A11, 2D5, 2F9, 3B3, and 3D10 exhibit neutralizing activities against SARS-CoV-2 Delta variant. Only 3F4 exhibits neutralizing activities against SARS-CoV-2 Omicron sublineage BA.1. The binding epitopes of these RBD-specific mAbs were determined by using the alanine-scanning mutagenesis. The antibody cocktail, composed of the RBD-specific mAb 3F4 or 2A11 and the S2-specific mAb S2-4A or S2-8D, exhibits synergistic neutralizing activities against SARS-CoV-2 Omicron sublineage BA.2. The results suggest that the combination of the RBD-specific mAb and the S2-specific mAb are potential candidates for development as prophylactic or therapeutic agents against SARS-CoV-2 infection. | en |
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| dc.description.tableofcontents | 目錄 I 摘要 VI ABSTRACT VII 縮寫表 VIII 第一章 緒論 1 1.1 新型冠狀病毒簡介 1 1.1.1 SARS-CoV-2 1 1.1.2 SARS-CoV-2基因體組成 1 1.1.3 SARS-CoV-2結構蛋白 2 1.1.4 SARS-CoV-2進入宿主細胞之方式 3 1.2 SARS-CoV-2突變株介紹及突變位點 4 1.2.1 Alpha突變株 4 1.2.2 Beta突變株 4 1.2.3 Gamma突變株 4 1.2.4 Delta突變株 5 1.2.5 Omicron突變株 5 1.3 SARS-CoV-2的治療策略 6 1.3.1 阻止病毒進入宿主細胞 6 1.3.2 抑制病毒複製 6 1.3.3 調節免疫系統 6 1.4 Anti-RBD 抗體療法 7 1.4.1 現今RBD抗體 7 1.4.2免疫逃脫 7 1.4.3雞尾酒療法 7 1.5 研究動機與目的 8 第二章 材料與方法 9 2.1 實驗材料 9 2.1.1 大腸桿菌 9 2.1.2 骨髓瘤細胞 9 2.2核酸實驗方法 9 2.2.1 原核表現載體 9 2.2.2 核酸引子設計 10 2.2.3 聚合酶連鎖反應 10 2.2.4 限制酶切反應 11 2.2.5 接合反應 11 2.2.6 質體轉形 11 2.2.7 重組載體之篩選 12 2.2.8 製備質體DNA 12 2.2.9 瓊脂膠體電泳 12 2.2.10 核酸純化 13 2.2.11 核酸定量 13 2.4 蛋白質實驗方法 13 2.4.1 蛋白質定量 13 2.4.2. 聚丙烯醯胺凝膠電泳 14 2.4.3 CBR染色法 14 2.4.4西方點墨法 15 2.4.5 酵素免疫分析法 15 2.5重組蛋白質之表現 16 2.6 重組蛋白質純化 16 2.6.1 純化RBM、RBM-A、RBM-B 16 2.6.2 蛋白質脫鹽 17 2.7 SARS-CoV-2單株抗體製備 17 2.7.1 小鼠免疫 17 2.7.2 融合瘤細胞製備 18 2.7.3 限數稀釋法 19 2.7.4 單株抗體純化 19 2.8 抗體類別測試 20 2.9 抗體結合位測試 20 2.10 RBD定位點突變 20 2.11 病毒溶斑抑制中和試驗 21 2.12 雞尾酒療法之抗體挑選 21 第三章 結果 22 3.1 置備Anti-SARS-CoV-RBD 單株抗體 22 3.1.1 篩選單株抗體 22 3.1.2 純化單株抗體 22 3.2 RBD單株抗體樣態分析與專一性分析 22 3.2.1 RBD單株抗體類別分析 22 3.2.2 RBD單株抗體結合片段分析 23 3.2.3 RBD單株抗體位於RBM的關鍵結合位點分析 23 3.3 RBD單株抗體的中和能力 24 3.3.1 單株抗體對武漢病毒株之中和能力 24 3.3.2 單株抗體對Alpha 突變株之中和能力 24 3.3.3 單株抗體對Delta 突變株之中和能力 25 3.3.4 單株抗體對Omicron 突變株之中和能力 25 3.4抗體雞尾酒之中和性測試 26 3.4.1 抗體雞尾酒對武漢病毒株之中和能力 26 3.4.2 抗體雞尾酒對Omicron BA.2突變株之中和能力 27 第四章 討論 28 4.1 單株抗體結合於RBD之位點探討 28 4.2 單株抗體對於不同SARS-CoV-2病毒株中和能力之探討 29 4.3 單株抗體所組成抗體雞尾酒療法之探討 30 參考文獻 31 圖與表 35 圖一 2A11、2D5、2F9、3B3、3D10及3F4專一性測定 36 圖二 利用Protein G 管柱純化單株抗體2A11、2D5、2F9、3B3、3D10及3F4 37 圖三 2A11、2D5、2F9、3B3、3D10及3F4 抗體類型測定 38 圖四 分析2A11、2D5、2F9、3B3、3D10及3F4辨識的RBD片段 39 圖五 分析2A11、2F9、3B3、3D10及3F4辨識的RBM片段 40 圖六 2A11及3F4位於RBM-A的關鍵結合位點分析 42 圖七 2F9及3B3位於RBM-B的關鍵結合位點分析 43 圖八 以病毒溶斑抑制中和試驗分析RBD單株抗體對武漢株、Alpha突變株、Delta 突變株的中和能力 44 圖九 分析2A11與3F4對於與病毒株相同的突變RBM之結合能力 46 圖十 以病毒溶斑抑制中和試驗分析RBD單株抗體對Omicron突變株的中和能力 47 圖十一 分析Anti-RBD 與Anti-S2 所組成之抗體雞尾酒對武漢病毒株的中和能力 49 圖十二 分析Anti-RBD 與Anti-S2 所組成之抗體雞尾酒對Omicron BA.2 突變株的中和能力 51 圖十三 SARS-CoV-2 RBD 與ACE2結合之結構圖 52 圖十四 2A11及3F4位於RBM-A的關鍵結合位點之3D結構示意圖 53 圖十五 2F9及3B3位於RBM-B的關鍵結合位點之3D結構示意圖以PDB ID 6LZG作為模板，藍點為RBM-B中會與ACE2結合的關鍵位點 54 附錄 55 附錄表一 製備RBM-N439K、RBM-L454R、RBM-Y435F、RBM-E484K 及RBM-N501Y蛋白質表現質體所使用的引子 56 附錄表二 製備RBM-A及RBM-B蛋白質表現質體所使用的引子 57 附錄表三 製備定位點突變RBD之蛋白質表現質體所使用的引子 58 | |
| dc.language.iso | zh-TW | |
| dc.subject | Spike protein | zh_TW |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | 抗體雞尾酒療法 | zh_TW |
| dc.subject | Receptor-binding domain (RBD) | zh_TW |
| dc.subject | SARS-CoV-2 | zh_TW |
| dc.subject | Antibody cocktail | en |
| dc.subject | SARS-CoV-2 | en |
| dc.subject | Spike protein | en |
| dc.subject | Receptor-binding domain | en |
| dc.subject | Monoclonal antibody | en |
| dc.title | 開發新冠病毒中和性抗體雞尾酒療法 | zh_TW |
| dc.title | Development of the Neutralizing Antibody Cocktail against SARS-CoV-2 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 廖憶純(Yi-Chun Liao),黃楓婷(Feng-Ting Huang),林翰佳(Han-Jia Lin),陳威戎(Wei-Jung Chen) | |
| dc.subject.keyword | SARS-CoV-2,Spike protein,Receptor-binding domain (RBD),單株抗體,抗體雞尾酒療法, | zh_TW |
| dc.subject.keyword | SARS-CoV-2,Spike protein,Receptor-binding domain,Monoclonal antibody,Antibody cocktail, | en |
| dc.relation.page | 60 | |
| dc.identifier.doi | 10.6342/NTU202202835 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-08-26 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2027-08-24 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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