請用此 Handle URI 來引用此文件：
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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊榮輝 | |
| dc.contributor.author | Chih-Chi Li | en |
| dc.contributor.author | 黎芷琪 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:30:42Z | - |
| dc.date.available | 2019-08-24 | |
| dc.date.copyright | 2016-08-24 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-16 | |
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PNAS, 2014. 111(42): p. 15214-15219. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49480 | - |
| dc.description.abstract | 2012年夏天，發生首例證實感染新型冠狀病毒-中東呼吸症候群冠狀病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）的個案，此種新型病毒在人類宿主具有高度傳染力，其致死率為36%，遠高於感染嚴重急性呼吸道症候群（Sever acute respiratory syndrome, SARS）之致死率。同時，2013年12月，西非地區伊波拉病毒（Ebola virus）引爆大流行疫情，為伊波拉出血熱流行病學史上最嚴重的一次疫情。上述MERS-CoV和Ebola在目前並無適合之治療藥物與疫苗，對人類威脅甚鉅，基於疫情控管與檢測考量之重要性，發展對應之中和性單株抗體已是刻不容緩。本論文針對此兩種新興病毒侵入宿主之機制，選擇具有高抗原性之膜醣蛋白（glycoprotein, GP）與spike protein（SP），開發出具有中和性效價之單株抗體。預期在篩選具有專一性及高效價之單株抗體後，將可投入Ebola與MERS-CoV之快篩檢驗技術平台開發。Ebola單株抗體製備部分如下：(1)自GP中挑選出表面抗原性高之序列，避開可能之後轉譯修飾區域，選擇在蛋白質立體結構上，位於表面之3段短鏈胜肽，人工合成抗原進行免疫；(2)參考已發表之伊波拉病毒單株抗體 KZ52做為參考，製備部分GP2重組蛋白片段做為抗原。MERS-CoV部分，分別針對侵入宿主之S1區域與宿主結合之RBD部位，使用桿狀病毒表現系統製備重組蛋白做為抗原，進行小鼠免疫，目前已取得專一性單株抗體。在獲得高度專一性之抗體後，將以表現SP之細胞株進行中和性試驗，證實單株抗體有抑制病毒感染宿主細胞受體DPP4之功能。未來將利用blocking ELISA 與 antigen-capture ELISA兩種檢驗法，應用於病毒防疫與檢驗之快篩平台。 | zh_TW |
| dc.description.abstract | The Middle East respiratory syndrome coronavirus (MERS-CoV) was first reported to infect human beings in September 2012. This virus seemed to develop into a highly contagious disease and caused high mortality rate up to 36%. Since December 2013 west Africa experienced the largest outbreak of Ebola, which was first discovered in 1976. The mortality of Ebola virus disease is relatively high, and yet the therapeutic drugs and vaccines are still under development. Researches for further understanding of the disease progression as well as accurate detection methods for these viruses are important for endemic control. Thus, the goal of this study is to develop specific antibodies against Ebola virus or MERS-CoV for the application in virus detection and clinical therapy. Two of the virus surface proteins, glycoprotein（GP）and spike protein（SP）, which were predicted as having high specificity and antigenicity were chosen as the antigens. In Ebola case, 3 peptides fragments of GP and its truncated form, constructed in refer to the published anti-GP mAb KZ52, were synthesized and expressed for mice immunization. In MERS-CoV case, S1 domain and receptor binding domain（RBD）were constructed and expressed in Bac-to-Bac Expression system for immunization. After repeat boosts, mice were sacrificed for producing hybridoma. Monoclonal antibodies（mAb） were screened by enzyme-linked immunosorbent assay（ELISA）and then identified by Western blot. Finally, neutralizing assays for MERS-CoV were used to verify the inhibition of the specific mAb which might block the virus invasion through the expressed DPP4 receptor on the cell surface. Useful mAb will be selected and used to develop the blocking ELISA or the antigen-capture ELISA, which could be utilized in clinical diagnosis or quick viral detection in the field. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:30:42Z (GMT). No. of bitstreams: 1 ntu-105-R03b22019-1.pdf: 7241085 bytes, checksum: 10f23a74b0452a90ac16f82cc912c743 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 iii
Abstract iv 縮寫表 vi 第一章 緒論 1 1.1 伊波拉病毒之解析與研究 1 1.1.1 伊波拉病毒之歷史 1 1.1.2 伊波拉病毒之型態構造 3 1.1.3 伊波拉病毒之基因體 5 1.1.4 伊波拉病毒之蛋白質功能簡介 6 1.1.5 伊波拉病毒生活史 7 1.2中東呼吸症候群冠狀病毒之探討與研究 10 1.2.1 MERS-CoV病毒之歷史 10 1.2.2 MERS-CoV病毒之構造與型態 11 1.2.3 MERS-CoV病毒之基因體 12 1.2.4 MERS-CoV病毒之蛋白功能簡介 14 1.2.5 MERS-CoV病毒生活史 15 1.3新興病毒膜上之醣蛋白研究 18 1.3.1 伊波拉病毒膜上醣蛋白之介紹 18 1.3.2 針對伊波拉之醣蛋白兩區域設計抗原 23 1.3.3 MERS-CoV膜上棘蛋白之介紹 25 1.3.4 MERS-CoV與其受體DPP4之關係 28 1.4 研究動機 30 第二章 材料與方法 32 2.1 EBOV 抗原之製備 32 2.1.1 EBOV GP抗原決定基之預測 32 2.1.2製備GP2之重組蛋白 33 2.2 MERS-CoV抗原之製備 36 2.2.1製備S1、RBD之重組蛋白 36 2.2.2 慢病毒包裝系統表現Spike全長 39 2.3傳統抗血清與單株抗體製備 42 2.3.1抗原處理-挖取SDS-PAGE膠體電泳蛋白質條帶免疫 42 2.3.2 小鼠免疫 42 2.3.3 細胞融合 42 2.3.4 免疫球蛋白初步純化 45 2.4細胞培養法 45 2.4.1 動物細胞培養 45 2.4.2 昆蟲細胞培養 46 2.4.3 細胞冷凍法 46 2.4.4 細胞解凍法 46 2.5免疫學方法 46 2.5.1 酵素連結免疫分析法 46 2.5.2 西方墨點法 48 2.6以表現Spike蛋白之細胞與帶DPP4受體宿主細胞共培養 49 2.7 蛋白質體學技術 49 2.8 共軛焦顯微鏡分析 50 第三章 結果與討論 51 3.1伊波拉病毒單株抗體製備 51 3.1.1 抗原片段之挑選 51 3.1.2 Truncated GP2重組蛋白抗原製備 60 3.1.3 單株抗體製備 69 3.2中東呼吸症候群冠狀病毒之單株抗體製備 78 3.2.1 RBD與S1重組蛋白抗原製備 78 3.2.2 單株抗體製備 85 3.3 MERS-CoV單株抗體之中和性測試 93 3.3.1以表現Spike蛋白之細胞與帶DPP4受體宿主細胞共培養 93 3.3.2 以Cell based ELISA測試單株抗體之中和效價 102 第四章 未來研究方向 104 參考文獻 106 問答錄 111 | |
| dc.language.iso | zh-TW | |
| dc.subject | 中和性單株抗體 | zh_TW |
| dc.subject | 中東呼吸症候群冠狀病毒 | zh_TW |
| dc.subject | 伊波拉病毒 | zh_TW |
| dc.subject | 醣蛋白 | zh_TW |
| dc.subject | 棘蛋白 | zh_TW |
| dc.subject | Glycoprotein | en |
| dc.subject | neutralizing monoclonal antibody | en |
| dc.subject | Spike protein | en |
| dc.subject | MERS-CoV | en |
| dc.subject | Ebola virus | en |
| dc.title | 發展新興傳染病毒膜醣蛋白之中和性單株抗體 | zh_TW |
| dc.title | Development of neutralizing antibody against the membrane glycoproteins of emerging disease viruses. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張明富,張世宗,陳翰民,陳慧文 | |
| dc.subject.keyword | 中東呼吸症候群冠狀病毒,伊波拉病毒,醣蛋白,棘蛋白,中和性單株抗體, | zh_TW |
| dc.subject.keyword | Ebola virus,MERS-CoV,Glycoprotein,Spike protein,neutralizing monoclonal antibody, | en |
| dc.relation.page | 115 | |
| dc.identifier.doi | 10.6342/NTU201602873 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-17 | |
| 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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