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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | |
| dc.contributor.author | Tung-Lin Chou | en |
| dc.contributor.author | 周東霖 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:45:12Z | - |
| dc.date.available | 2021-08-24 | |
| dc.date.copyright | 2016-08-24 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-25 | |
| dc.identifier.citation | Almond, J., and Felsenreich, V. (1982). Phosphorylation of the nucleoprotein of an avian influenza virus. Journal of General Virology 60, 295-305.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50539 | - |
| dc.description.abstract | 2013年3月，中國大陸爆發人類感染H7N9禽流感之疫情，同年4月，台灣也宣布第一起境外移入之H7N9病例。此為全球首次出現低致病性H7N9亞型病毒經基因重組引發人類感染，且某些病毒株也被驗出具有克流感之抗藥性。至2016年5月為止，全球已超過786個確定病例，死亡人數為307位，致死率高達39%。因此加強對H7N9禽流感病毒之基礎研究及研發快速篩檢與治療所需之抗體迫切重要。NP、PA、PB1、PB2及RNA組成核醣核蛋白複合體 (Ribonucleoprotein, RNP) 結構，扮演流感病毒遺傳物質RNA複製與轉錄的重要角色。其中NP會與病毒RNA纏繞，並且不僅能與病毒自身蛋白質有交互作用，在宿主體內更和多種蛋白質有結合現象，為一種多功能蛋白質，因此已成為目前研究抗流感病毒的標的之一。本論文的研究目標為表現與純化H7N9病毒NP、PA、PB1及PB2的重組蛋白質，並進行單株抗體的製備。本研究已成功利用昆蟲細胞穩定表現這四個蛋白質，並以親和層析法純化到NP，亦成功篩選出抗NP之單株抗體。其型別為IgG1 kappa，且該抗體具有可免疫沉澱NP的能力。此外，本研究發現PA、PB1及PB2之N端若含有鞘膜表面醣蛋白67 (envelope surface glycoprotein, GP67) 之signal peptide，能增加其蛋白質之表現量。然而，GP67-PA及GP67-PB1易形成不溶體，因此未能順利以層析法將其進行有效地純化出來。 | zh_TW |
| dc.description.abstract | In March 2013, the first human case infected by a novel avian influenza H7N9 virus subtype was reported in China. In April 2013, Taiwan CDC confirmed the first imported case of H7N9 infected patient from China. It is the first time that a low pathogenic avian influenza virus has led to human infection on account of genetic reassortment. Moreover, some isolated H7N9 viruses have shown Tamiflu drug-resistant. As of May, 2016, 786 documented human cases were confirmed by WHO and the mortality rate was as high as 39%. Therefore, it is urgently important to strengthen basic research of H7N9 and develop rapid laboratory diagnostics and antibodies. Influenza virus ribonucleoproteins (RNP), composed of NP, PA, PB1, PB2, and viral RNA, play important roles in viral gene transcription and replication. NP is a multifunction protein, which not only interacts with viral proteins but also interacts with host cell proteins. Therefore, NP is a potential antiviral target. The present study is aimed to express NP, PA, PB1 and PB2 recombinant proteins by for production of monoclonal antibodies. These four proteins have been stably generated in Sf21 insect cells and the NP recombinant proteins were purified by affinity chromatography. The anti-NP monoclonal antibody 2-4-7B showed great specificity and can be applied for NP immunoprecipition. In addition, this study found that the expression levels of PA, PB1 and PB2 proteins were increased by fusion of GP67 signal peptide at the protein N-terminus. However, PA, PB1 and PB2 were expressed as inclusion bodies, therefore the protein purification strategy should be further investigated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:45:12Z (GMT). No. of bitstreams: 1 ntu-105-R03b22004-1.pdf: 2661236 bytes, checksum: bd18020ce3d36cab54c381849bf17d98 (MD5) Previous issue date: 2016 | 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流感病毒分類 1 1.1.2流感病毒之構造 2 1.1.3流感病毒之基因體及蛋白質體 2 1.1.4核糖核蛋白之功能及重要性 4 1.2新型H7N9流感病毒簡介 6 1.2.1新型H7N9流感之疫情 6 1.2.2新型H7N9流感之序列特性 6 1.3 桿狀病毒表現系統 7 1.4 研究動機 8 第二章 材料與方法 9 2.1 實驗材料 9 2.1.1 H7N9 NP, PA, PB1, PB2基因來源 9 2.1.2 大腸桿菌菌株 9 2.1.3 昆蟲細胞 9 2.2基因表現載體之建構 10 2.2.1 核酸引子設計 10 2.2.2 聚合酶鏈鎖反應 10 2.2.3 限制酶切反應 11 2.2.4 DNA膠體電泳及萃取 11 2.2.5 接合反應 (ligation) 11 2.2.6 質體轉型 (Transformation) 12 2.3 桿狀病毒表現系統 12 2.3.1 DH10Bac大腸桿菌轉型及藍白篩選 12 2.3.2 重組Bacmid之純化 13 2.3.3 聚合酶鏈鎖反應檢驗法 13 2.3.4 重組Bacmid轉染及病毒液放大 14 2.3.5 病毒液效價分析 (Plaque Assay) 14 2.3.6 蛋白質最佳表現條件測試 15 2.3.7 重組蛋白之放大培養 15 2.4 重組蛋白質之純化 15 2.4.1 His-tag重組蛋白質親和性層析法 16 2.4.2 不可溶蛋白質之純化 16 2.4.3 膠體過濾法 (Gel filtration) 17 2.4.4 蛋白質脫鹽及濃縮 17 2.5 蛋白質相關實驗 18 2.5.1 蛋白質定量法 18 2.5.2 蛋白質膠體電泳 18 2.5.3 蛋白質電泳膠片染色 18 2.5.4 蛋白質轉印 18 2.5.5免疫染色 (Immunobloting) 19 2.6 單株抗體製備 19 2.6.1 小鼠免疫 19 2.6.2 小鼠抗體免疫效價測定 20 2.6.3 細胞融合 20 2.6.4 酵素連結免疫分析法 21 2.6.5 細胞單株化篩選 22 2.6.6 單株抗體之純化 22 2.6.7 單株抗體效價測試 (Titer Test) 23 2.6.8 單株抗體分型鑑定 (Isotyping) 24 2.6.9 單株抗體免疫沉澱 (Immunoprecipitaton) 24 2.7 細胞培養 25 2.7.1 昆蟲細胞培養 25 2.7.2 骨髓瘤細胞及融合瘤細胞培養 25 2.7.3 細胞凍存 25 2.7.4 細胞解凍 26 第三章 結果 27 3.1 H7N9 NP, PA, PB1, PB2桿狀病毒重組Bacmid之建構 27 3.2利用昆蟲細胞表現H7N9 NP, PA, PB1與PB2重組蛋白質 27 3.3 H7N9 NP重組蛋白質表現條件測試 28 3.4 H7N9 NP重組蛋白質之純化 28 3.5 H7N9 NP重組蛋白質之小鼠免疫及抗體測試 29 3.6 細胞融合及抗體單株化篩選 30 3.7 NP單株抗體之純化 30 3.8 NP單株抗體之生化性質研究及免疫沉澱 31 3.9 建構含有GP67 signal peptide之H7N9 PA, PB1, PB2重組Bacmid 32 3.10 GP67-PA, GP67-PB1與GP67-PB2於昆蟲細胞之表現 33 3.11 GP67-PA, GP67-PB1與GP67-PB2於不同酸鹼值緩衝溶液中之溶解度測試 34 3.12 GP67-PA, GP67-PB1與GP67-PB2之純化 34 第四章 討論 36 4.1 H7N9 NP、PA、PB1、PB2重組蛋白質之表現探討 36 4.2 H7N9 NP重組蛋白質之單株抗體製備 36 4.3 H7N9 NP重組蛋白質之生化性質研究 37 4.4含有GP67之PA、PB1與PB2重組蛋白質之表現探討 38 4.5含有GP67之PA、PB1與PB2重組蛋白質之純化條件探討 38 參考文獻 40 表與圖 45 附錄 68 | |
| dc.language.iso | zh-TW | |
| dc.subject | 新型H7N9流感病毒 | zh_TW |
| dc.subject | 核蛋白 | zh_TW |
| dc.subject | 核蛋白單株抗體 | zh_TW |
| dc.subject | 核糖核蛋白 | zh_TW |
| dc.subject | Anti-nucleoprotein monoclonal antibodies | en |
| dc.subject | Ribonucleoproteins | en |
| dc.subject | Nucleoprotein | en |
| dc.subject | Novel influenza A(H7N9) virus | en |
| dc.title | 新型H7N9流感病毒重組核糖核蛋白與核蛋白鼠源單株抗體之開發 | zh_TW |
| dc.title | Production of Novel Influenza A(H7N9) Virus Recombinant Ribonucleoproteins and Anti-Nucleoprotein Monoclonal Antibodies | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張麗冠,陳慧文,林翰佳 | |
| dc.subject.keyword | 新型H7N9流感病毒,核糖核蛋白,核蛋白,核蛋白單株抗體, | zh_TW |
| dc.subject.keyword | Novel influenza A(H7N9) virus,Ribonucleoproteins,Nucleoprotein,Anti-nucleoprotein monoclonal antibodies, | en |
| dc.relation.page | 73 | |
| dc.identifier.doi | 10.6342/NTU201601289 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-26 | |
| 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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