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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85232完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陶秘華(Mi-Hua Tao) | |
| dc.contributor.author | Chi-Wen Chiu | en |
| dc.contributor.author | 邱琪雯 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:51:51Z | - |
| dc.date.copyright | 2022-10-04 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-08-02 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85232 | - |
| dc.description.abstract | SARS相關病毒至今仍持續威脅人類的健康，繼2003年SARS大流行後，2019年出現的新冠病毒因突變株的產生令疫情至今仍無法趨緩。現有的SARS-CoV-2相關疫苗和單株抗體藥物，主要是以病毒表面的棘蛋白為目標，防止病毒和血管收縮素轉換酶II（hACE2）結合而進入人體感染。但是最近被WHO列為高度關注的突變株，其中包含alpha, beta, gamma, delta, 和omicron等，在棘蛋白上都有不少突變位點，研究顯示這些突變位點除了增加病毒和ACE2的結合能力外，更大幅降低現有疫苗和抗體的保護效果，因此，研發能夠廣泛預防和治療COVID-19的方法仍有迫切需要。於本篇論文中我們探討anti-hACE2單株抗體是否可以廣效預防SARS-CoV-2的感染？因為SARS-CoV-2是透過呼吸道感染，我們模擬黏膜抗體的組成，產生anti-hACE2 IgG、IgA、和 IgM三種不同isotype的單株抗體，利用腺相關病毒載體AAV6遞送抗體基因至小鼠呼吸道表現，達到長期的抗病毒效果。先前我們實驗室利用融合瘤技術分離出一株和hACE2高結合的單株抗體，命名為2H2，並且從偽病毒中和實驗證實此抗體對不同的突變株皆具中和效果。我們透過基因工程的方式，將2H2抗體可變區分別接到human IgG1、human IgA1、和human IgM的骨架上，並利用CHO cell產生ch2H2-IgG、ch2H2-IgA、和ch2H2-IgM三種抗體。Western Blot確認三種抗體在輕鏈、重鏈或J chain的表現符合預期。接下來我們分別用protein A、peptide M和ion exchange的方式，純化這些嵌合抗體。中和試驗證實， ch2H2-IgG、ch2H2-IgA或ch2H2-IgM對不同突變株皆具中和效果，但三種抗體的中和能力沒有明顯差異。在動物實驗方面，我們先使用帶有報導基因的AAV來優化氣管注射的遞送條件，確認AAV6可以使目標基因在肺部表現長達至少28週。接著製造AAV6分別表現ch2H2-IgG、ch2H2-IgA或ch2H2-IgM。接下來利用AAV遞送抗體至BALB/c的呼吸道表現，在此我們發現IgG抗體在的第七天會達到最高點，隨後抗體表現量會下降，而在AAV/ch2H2-IgA感染的小鼠中則是沒有測到IgA抗體表現，AAV/ch2H2-IgM感染的小鼠中三隻只有一隻有測到抗體表現。我們利用ELISA與NSG小鼠進行測試，發現抗體下降的原因是AAV/ch2H2-IgA和AAV/ch2H2-IgM誘發小鼠產生高濃度anti-antibody抗體使得目標抗體被中和。本篇論文證明ch2H2-IgG, IgA和IgM抗體確實可以中和SARS-CoV-2的不同突變株，並發展了AAV遞送系統在小鼠肺部表現ch2H2抗體，未來將進行小鼠攻毒試驗，驗證是否可以有效保護小鼠免於受到病毒感染，本研究成果將成為預防SARS-CoV-2突變株以及未來新興病毒的另一種策略。 | zh_TW |
| dc.description.abstract | The COVID-19 pandemic caused by SARS-CoV-2 has threatened human life around the world. New SARS-CoV-2 variants that are resistant to existing antiviral therapies continue to emerge. Therefore, there is an urgent need to develop broadly effective interventions to limit the transmission and disease severity caused by SARS-CoV-2. Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for SARS-CoV-2 and SARS-related coronaviruses, whose interaction with viral spike protein plays a crucial role in the infection. We hypothesize that blocking the binding event or reducing the accessibility of virus to hACE2 receptor may be an effective strategy to prevent SARS-CoV-2 infection. In this study, we generated a hACE2-specific monoclonal antibody (mAb) 2H2, which exhibited potent inhibitory activity against SARS-CoV-2 infection. To reduce the immunogenicity for clinical use, we generated chimeric mAb by grafting the variable regions of the heavy chain (VH) and light chain (VL) onto human IgG1 and kappa backbones (ch2H2-IgG). Because IgM and IgA, the two mucosal multivalent antibodies, play a key role in the first line defense against SARS-CoV-2, we also constructed human IgM and IgA chimeric 2H2 mAbs (ch2H2-IgM, ch2H2-IgA). The CHO cell system was used to produce chimeric antibodies. Western Blot analysis showed the presence of human heavy chain ( and ) and light chain in the corresponding chimeric antibodies, and in the case of IgM and IgA, the presence of an additional J chain. We also confirmed the broad inhibitory activity of the three chimeric antibodies against six SARS-CoV-2 variants (D614G, alpha, beta, gamma, delta, omicron) by in vitro pseudovirus neutralization assay. We first used AAV expressing luciferase to optimize delivery protocols for intratracheal injection, demonstrating that AAV6 can express the target gene in the lung for at least 28 weeks. Next, AAV was used to deliver chimeric antibody expression in BALB/c mice. We found that all mice produced chimeric IgG antibody which peaked at day 7, followed by a decline, whereas no chimeric IgA antibody was detected, and only one out of three mice produced detectable chimeric IgM antibody. Using ELISA assay and immunodeficient NSG mice we found high levels of anti-human IgA and IgM antibodies in the AAV-injected mice. We are now testing whether AAV6/ch2H2-IgG-treated K18 hACE-2 transgenic mice can be protected from SARS-CoV-2 challenge. Our research may provide an opportunity to develop a broadly effective antiviral drugs against emergence of SARS-CoV-2 variants and other SARS-related coronaviruses. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:51:51Z (GMT). No. of bitstreams: 1 U0001-3107202210321500.pdf: 5324507 bytes, checksum: 140a0abeb4824894087e39c4d3a269d3 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 論文口試委員審定書…………………………………………………………………....i 謝辭 ii 摘要 iii Abstract v 目錄 vii 圖目錄 vii 表目錄 viii 第一章、緒論 1 第一節、新型冠狀病毒 1 1.1 新型冠狀病毒簡介 1 1.2 新型冠狀病毒進入人體的途徑 1 1.3 目前新型冠狀病毒預防和治療的限制 2 1.4 使用靶向病毒接受器的單株抗體預防與治療SARS-CoV-2 3 第二節、黏膜免疫系統 3 2.1 黏膜免疫系統簡介 3 2.2 黏膜抗體對於預防與治療新型冠狀病毒的重要性 4 第三節、腺相關病毒 (Adeno-associated virus) 5 3.1 腺相關病毒簡介 5 3.2 腺相關病毒遞送單株抗體的治療 6 第二章、實驗目的與設計 7 第三章、實驗方法與材料 8 1. 重組腺相關病毒質體製備 8 1.1 pAAV/ch2H2-IgG,A,M質體製備 8 1.2 病毒製備 9 1.3 病毒純化 9 1.4 病毒定量 10 2. 細胞培養 10 2.1 HEK293T-hACE2 10 2.2 293N 10 2.3 3T3 10 3. 體外病毒感染 11 4. 流式細胞儀分析 11 4.1 抗體與hACE2結合能力分析 11 4.2 分析抗體與病毒棘蛋白競爭結合hACE2能力 11 5. 抗體純化 11 5.1 ch2H2-IgG純化 11 6.1 ch2H2-IgA純化 12 6.2 ch2H2-IgM純化 12 6. 西方墨點法 12 6.1 確認2H2融合瘤細胞株產生anti-hACE2 抗體 12 6.2 確認抗體輕鏈、重鏈、J chain的表現 13 7. SDS-PAGE 13 7.1 Reducing SDS-PAGE 13 7.2 Non-reducing SDS-PAGE 13 8. 中和試驗 13 8.1 Pseudovirus neutralization assay 13 8.2 Authentic SARS-CoV-2 virus neutralization assay 14 9. 實驗小鼠 14 10. 遞送重組腺相關病毒至小鼠的方式 14 11. 活體冷光影像系統 14 12. 採集肺部沖洗液與鼻腔沖洗液 15 12.1 肺部沖洗液 (Bronchoalveolar lavage fluid, BALF) 15 12.2 鼻腔抽取液 (Nasal lavage fluid, NLF) 15 13. 酵素免疫分析法 15 13.1 Hybridoma的抗體篩選 15 13.2 ch2H2-IgG、ch2H2-chIgA、ch2H2-chIgM抗體定量 15 13.3 Mouse anti-transgene antibody的檢測 16 第四章、實驗結果 17 第一節、產生和人類ACE2高結合力的抗體 17 1.1 使用AAV/hACE2與人類ACE2蛋白進行小鼠免疫 17 1.2 利用融合瘤技術製作anti-hACE2單株抗體 17 1.3 2H2抗體的特性分析 18 1.4 2H2 抗體對hACE2的結合能力 18 1.5 2H2阻斷hACE2和病毒棘蛋白結合的能力 19 第二節、人源化抗體 19 2.1 製備人源化抗體 19 2.2 ch2H2 抗體的特性分析 19 2.3 ch2H2抗體對hACE2的結合能力 20 2.4 ch2H2對於偽病毒的中和效果 20 2.5 ch2H2對於authentic SARS-CoV-2的中和效果 20 第三節、構築表現ch2H2-Ig的腺相關病毒載體 21 3.1 構築表現ch2H2-IgG、A、M的腺相關病毒載體的實驗設計 21 3.2 構築表現ch2H2-IgG、A、M的腺相關病毒載體的實驗流程 21 3.3 構築表現ch2H2-IgG、A、M的腺相關病毒載體的PCR結果 22 第四節、生產AAV/ch2H2-IgG、A、M病毒載體 23 4.1 製作AAV/ch2H2-IgG、A、M病毒載體 23 4.2 確認腺相關病毒載體感染細胞後抗體的表現 23 第五節、生產ch2H2-IgG、A、M抗體 24 5.1 生產ch2H2-IgG、A、M抗體 24 5.2 純化ch2H2-IgG、A、M抗體 25 5.3 確認ch2H2-IgG、A、M對於SARS-CoV-2 pseudovirus的中和效…………………………………………………………………………….26 5.4 確認ch2H2-IgG、A、M對於authentic SARS-CoV-2的中和效果 26 第六節、遞送AAV/ch2H2-IgG、A、M到小鼠呼吸道上皮細胞 26 6.1 利用AAV6/CB-luc-EF1a-EGFP優化遞送條件 26 6.2 遞送AAV/ch2H2-IgG、A、M到BALB/c小鼠進行抗體表現 27 6.3 檢測BALB/c小鼠是否產生抗體反應 27 6.4 遞送AAV/ch2H2-IgG到NSG進行抗體表現 28 第五章、討論 29 1. 比較不同的共表達轉譯策略 29 2. ch2H2-Ig三種抗體對SARS-CoV-2中和能力沒有差異的原因 30 3. ch2H2-Ig對authentic SARS-CoV-2的中和效果較差可能原因 30 4. 應用anti-hACE2抗體於人體上安全的疑慮 30 5. 比較不同的遞送方式 31 6. 使用AAV遞送抗體可能會遇到的問題 31 7. AAV/ch2H2-Ig於臨床上的可應用性 32 8. 結論 32 第六章、參考文獻 33 | |
| dc.language.iso | zh-TW | |
| dc.subject | 腺相關病毒 | zh_TW |
| dc.subject | 新冠病毒 | zh_TW |
| dc.subject | 人類ACE2抗體 | zh_TW |
| dc.subject | 黏膜抗體 | zh_TW |
| dc.subject | Anti-human ACE2 antibody | en |
| dc.subject | mucosal antibody | en |
| dc.subject | Adeno-associated virus (AAV) | en |
| dc.subject | SARS-CoV-2 | en |
| dc.title | 利用腺相關病毒載體表現抗人類ACE2抗體對抗新冠病毒變異株 | zh_TW |
| dc.title | Development of AAV-delivered neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 王宜萱(I-Hsuan Wang) | |
| dc.contributor.oralexamcommittee | 林靜宜(Jing-Yi Lin),莊雅惠(Ya-Hui Chuang) | |
| dc.subject.keyword | 新冠病毒,人類ACE2抗體,黏膜抗體,腺相關病毒, | zh_TW |
| dc.subject.keyword | SARS-CoV-2,Anti-human ACE2 antibody,mucosal antibody,Adeno-associated virus (AAV), | en |
| dc.relation.page | 77 | |
| dc.identifier.doi | 10.6342/NTU202201906 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-08-02 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-02 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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