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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳俊任(Chun-Jen Chen) | |
dc.contributor.author | Tsung-Kai Huang | en |
dc.contributor.author | 黃宗凱 | zh_TW |
dc.date.accessioned | 2021-06-15T01:51:25Z | - |
dc.date.available | 2016-08-20 | |
dc.date.copyright | 2011-08-20 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43353 | - |
dc.description.abstract | 腸病毒七十一型感染嬰幼兒後,會造成手足口症,甚至導致中樞神經系統癱瘓及死亡。早在 1998 年,就在臺灣爆發首次的大流行,但至今卻沒有疫苗供給預防。在此篇研究中,我們嘗試在嗜甲醇酵母菌及啤酒酵母中生產腸病毒七十一型類病毒顆粒作為疫苗的主要成份。在嗜甲醇酵母菌的表現系統中,所轉殖的基因位於 AOX1 promoter 下游,因此當以甲醇進行誘導時,基因得以表現。相對的,在啤酒酵母的表現系統中,轉基因則是持續地表現,並且在銅離子誘導後可使表現量提昇。共轉形結構性蛋白 (P1) 及病毒蛋白酶 (3C/3CD) 於此兩種系統後,產生了 VP0、VP1 及 VP3,意謂 P1 可以在酵母菌中成功地被 3C/3CD 截切。在破細胞後,以不連續式之蔗糖梯度離心以及免疫沉澱法純化,並以免疫金進行標定,類病毒顆粒可以在穿透式電子顯微鏡下被觀察到,此實驗所生產之類病毒顆粒將可合併之後生產的cholera toxin B (CTB),更進一步於動物實驗上進行免疫評估。 | zh_TW |
dc.description.abstract | Enterovirus-71 (EV-71) infection causes hand, foot and mouth disease (HFMD) in children, and in some cases, it may also cause severe central nerve system collapse and even death. Since 1998, EV-71 has become an emerging infectious agent in Taiwan; however, there is currently no vaccine available to prevent the infection. In this study, we attempt to produce EV-71 virus-like particles (VLPs) in two yeast expression systems, including Pichia pastoris and Saccharomyces cerevisiae, and use the VLPs as a vaccine candidate. In the P. pastoris expression system, transgenes are placed under the AOX1 promoter which is activated upon methanol induction. In contrast, transgenes are constitutively expressed in the S. cerevisiae expression system and protein expression is enhanced after Cu2+ added. Co-transformation of genes encoding the structural protein P1 and the viral protease 3C/3CD leads to the production of VP0, VP1 and VP3 in both P. pastoris and S. cerevisiae expression systems, indicating that P1 can be successfully processed by 3C or 3CD in yeast cells. After cell lysis, discontinuous sucrose gradient centrifugation, and affinity purification with antibodies, VLPs can be detected under transmission electron microscopy (TEM) by immunogold staining. The EV-71 VLPs generated in this study will be used as a vaccine antigen to immunize animals with CTB in future experiments. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:51:25Z (GMT). No. of bitstreams: 1 ntu-100-R98b47101-1.pdf: 4308379 bytes, checksum: cff7ce21e49164cd731e98553cc3376f (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要 I
Abstract II 目錄 III 圖表目錄 VI 壹、前言 1 一、腸病毒七十一型 (Enterovirus 71, EV-71) 1 1.1 源起 1 1.2 分類 1 1.3 病毒結構特性與基因體組成 2 1.4 病毒生活史 3 1.5 病毒感染途徑、臨床症狀 3 1.6 預防方法 4 二、疫苗與類病毒顆粒 4 2.1 疫苗 4 2.2 類病毒顆粒 (Virus like particles, VLPs) 5 2.3 腸病毒疫苗 7 三、黏膜免疫及佐劑 8 3.1 黏膜免疫系統 8 3.2黏膜佐劑 9 3.3霍亂毒素及 B 次單位 10 四、酵母菌表現系統 11 4.1 Sacchromyces cerevisiae (S. cerevisiae) 11 4.2 Pichia pastoris (P. pastoris) 13 4.3 酵母菌生產之優勢 16 貳、實驗目的與架構 17 參、材料與方法 18 一、材料: 18 1.1 病毒株 18 1.2 細胞株 18 1.3 菌株 18 1.4 載體 18 二、病毒實驗 18 2.1 Virus Propagation 18 2.2 Plaque assay 19 三、建構質體 19 3.1 Flow chart 19 3.2 核酸萃取 19 3.3 Reverse transcription – polymerase chain reaction (RT-PCR) 20 3.4 Polymerase chain reaction (PCR) 20 3.5 限制酶截切 20 3.6 Alkaline Phosphatase, Calf Intestinal (CIP) 21 3.7 DNA 純化 21 3.8 接合反應 (ligation) 21 3.9 轉型作用 (Transformation) 21 3.10 質體抽取及限制酶截切確認 21 3.11 DNA 定序 21 四、酵母菌轉型作用 22 4.1 大量質體抽取 22 4.2 酵母菌勝任細胞製備 22 4.3 酵母菌之電穿孔轉型法 22 4.4 Colony PCR 確認 23 五、蛋白質表現 23 5.1 蛋白質誘導 23 5.2 萃取蛋白 24 5.3 蛋白質電泳與西方點墨法 (SDS-PAGE & Western blotting) 24 六、類病毒蛋白純化 25 6.1 粗蛋白濃縮 25 6.2 蔗糖梯度離心 25 6.3 VLPs 濃縮 (一):超高速離心 25 6.4 VLPs 濃縮 (二):免疫沉澱法 25 6.5 病毒大量製備 25 七、Transmission Electron Microscopy (TEM) 26 7.1 TEM 觀察 26 7.2 Immunogold labeling 26 肆、結果 27 一、建構重組質體 27 二、外殼前驅蛋白及蛋白酶表現 28 三、VLPs 蛋白之表現 29 四、VLPs 之純化 29 五、Immunogold staining & transmission electron microscopy 30 六、佐劑 CTB 之表現 32 伍、討論 33 陸、結論 39 柒、參考文獻 40 捌、表 46 玖、圖 48 拾、附錄 68 | |
dc.language.iso | zh-TW | |
dc.title | 利用酵母菌生產腸病毒七十一型類病毒顆粒 | zh_TW |
dc.title | Production of enterovirus-71 virus-like particles in yeast | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 江伯倫(Bor-Luen Chiang),龔思豪(Szu-Hao Kung) | |
dc.subject.keyword | 腸病毒七十一型,類病毒顆粒,嗜甲醇酵母菌,啤酒酵母菌, | zh_TW |
dc.subject.keyword | EV-71,VLPs,P. pastoris,S. cerevisiae, | en |
dc.relation.page | 70 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-15 | |
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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