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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張世宗(Shih-chung Chang) | |
dc.contributor.author | Kai-Ting Hsieh | en |
dc.contributor.author | 謝凱婷 | zh_TW |
dc.date.accessioned | 2021-07-11T14:44:15Z | - |
dc.date.available | 2021-10-14 | |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
dc.identifier.citation | Allison, A., and Gregoriadis, G. (1974). Liposomes as immunological adjuvants.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78161 | - |
dc.description.abstract | 於2013年3月底在中國上海和安徽兩地首次發現新型H7N9流感病毒感染人的病例,且其病發期短、重症率與死亡率均高而引起高度關注。同年4月台灣亦出現首例境外移入病例,並從檢體中驗出具有克流感抗藥性的H7N9病毒株。根據世界衛生組織WHO統計,截至2016年5月23日全球共累計786例病例,其中307例死亡,死亡率高達39%。遺傳學的分析顯示此新型H7N9病毒株已逐漸適應哺乳類的宿主,因此研發疫苗與快速檢驗及治療所需之抗體迫切重要。
本研究主要利用大腸桿菌表現系統及昆蟲細胞表現系統生產新型H7N9病毒之血球凝集素 (Hemagglutinin, HA) 的球狀頭部HA1,以提供基礎科學研究、疫苗與抗體的研發。目前已成功表現及量產岀HA1重組蛋白,經免疫小鼠後都能成功引起免疫反應,並順利取得能抑制雞血球凝集的多株抗體,尤其以昆蟲細胞表現系統所生產的HA1,其免疫效果更佳。此外,本研究也成功製備出抗H7N9 HA1之單株抗體,此單株抗體亦具有抑制雞血球凝集的能力,且將其稀釋至256,000倍時,仍可利用免疫染色法偵測岀HA1抗原。未來將以本研究之基礎與成果,進行快篩平台與次單位疫苗之研發工作。 | zh_TW |
dc.description.abstract | The novel H7N9 influenza virus emerged in March 2013 in China and continues to cause sporadic human infections. In April 2013, Taiwan CDC reported the first imported case of H7N9 infection from China, and the H7N9 virus variants isolated from this patient display Tamiflu resistance. As of May 23, 2016, the World Health Organization (WHO) had reported 783 human infections and 306 deaths with mortality rates approaching 38%. Based on genetic analysis, this novel H7N9 virus shows some extent adaptation to mammalian hosts. Therefore, it is very important to develop rapid laboratory diagnostics, antibodies and subunit vaccines for better pandemic preparedness.
This study focused on production of the globular head domain of H7N9 hemagglutinin (HA) by using E. coli expression system and baculovirus expression system for generation of antibodies. The HA1 proteins expressed in E. coli and Sf21 insect cells can induce strong immune response in mice. The purified HA1 proteins were applied for immunization of BALB/c mice to produce polyclonal and monoclonal antibodies. The monoclonal antibody generated in the present study shows high specificity to H7N9 HA1 and also has great ability for inhibiting hemagglutination of chicken red blood cells. The HA1 proteins and the specific mAb are useful tools for furthermore developing laboratory diagnostic platform and subunit vaccine candidates in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:44:15Z (GMT). No. of bitstreams: 1 ntu-105-R03b22041-1.pdf: 3194060 bytes, checksum: 0687fced6c35714b46c68d48fb4b29f9 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目錄 i
摘要 iv Abstract v 縮寫表 vi 第一章 緒論 1 1.1禽流感概述 1 1.2 A型流感病毒 1 1.3新型H7N9流感 1 1.3.1 H7N9病毒傳染途徑 2 1.3.2 H7N9感染宿主機制 2 1.4血球凝集素 2 1.5流感疫苗 3 1.6桿狀病毒表現系統 3 1.7單株抗體 4 1.8研究動機 4 第二章 材料方法 6 2.1實驗材料 6 2.1.1 H7N9 HA基因來源 6 2.1.2大腸桿菌菌株 (Escherichia coli) 6 2.1.3昆蟲細胞 6 2.2大腸桿菌表現系統 7 2.2.1大腸桿菌細胞轉形 7 2.2.2重組蛋白誘導表現 7 2.2.3重組蛋白之純化 7 2.3昆蟲細胞表現系統 8 2.3.1建構帶HA1基因之pFastBac-1質體 8 2.3.2 HA1之桿狀病毒表現系統Bacmid質體製備 8 2.3.3 抽取Bacmid 8 2.3.4 重組Bacmid轉染及病毒液放大 9 2.3.5 H7 ΔSigHA1-His重組桿狀病毒液效價測試 9 2.3.6 昆蟲細胞表現HA1最佳條件測試 9 2.3.7昆蟲細胞之外泌表現HA1 10 2.3.8 昆蟲細胞所表現HA1之純化 10 2.4 去醣實驗 10 2.5多株、單株抗體之製備 11 2.5.1小白鼠免疫 11 2.5.2多株抗體效價測試 11 2.5.3細胞融合 11 2.5.4專一性單株抗體篩選 12 2.6抗體型別鑑定 12 2.7單株抗體之純化 13 2.8 HA1紅血球凝集試驗 13 2.9膠體過濾法 13 2.10 HA1抗體紅血球凝集抑制試驗 14 第三章 結果 15 3.1 利用大腸桿菌表現HA1 15 3.1.1 HA1(E. coli) 表現及純化 15 3.1.2 HA1(E. coli) 之多株抗體效價測試 15 3.1.3 HA1(E. coli) 之多株抗體的紅血球凝集抑制試驗 16 3.1.4 HA1(E. coli) 之單株抗體效價測試 16 3.2利用昆蟲細胞表現HA1 16 3.2.1 HA1(Sf21) 重組桿狀病毒表現載體之建構 16 3.2.2 HA1(Sf21) 之重組桿狀病毒液效價測試 17 3.2.3 HA1(Sf21) 重組蛋白質表現最佳條件測試 17 3.2.4 HA1(Sf21) 重組蛋白質之純化 17 3.2.5 HA1(Sf21) 之去醣實驗 18 3.2.6 HA1(Sf21) 之紅血球凝集試驗 18 3.2.7 利用膠體過濾管柱分析HA1(Sf21) 是否形成三聚體 18 3.2.8 HA1(Sf21) 重組蛋白質之多株抗體效價測試 19 3.2.9 HA1(Sf21) 多株抗體紅血球凝集抑制試驗 19 3.2.10 HA1(Sf21) 之單株抗體效價測試 19 3.2.11 F3 mAb型別鑑定 20 3.2.12 F3 mAb純化 20 3.2.13 HA1(Sf21) 之抗體效價測試 20 3.2.14 HA1(Sf21) 之抗體紅血球凝集抑制測試 20 第四章 討論 21 4.1 大腸桿菌與昆蟲細胞表現系統表現 HA1 之探討 21 4.2 HA1(Sf21) 重組蛋白質於昆蟲細胞之最佳表現條件探討 21 4.3 HA1(E. coli)、HA1(Sf21) 之血球凝集試驗 21 4.4 本研究之 HA1 重組蛋白發展成次單位疫苗之潛力 22 參考文獻 23 圖與表 27 附錄 52 | |
dc.language.iso | zh-TW | |
dc.title | 表現與純化新型H7N9流感病毒血球凝集素球狀頭部以製備鼠源單株抗體 | zh_TW |
dc.title | Expression and Purification of the Novel Influenza A(H7N9) Virus Hemagglutinin Head Domain for Generation of Mouse Monoclonal Antibodies | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林翰佳(Han-Jia Lin),張麗冠,陳慧文 | |
dc.subject.keyword | 新型 H7N9 流感病毒,血球凝集素,單株抗體,次位疫苗, | zh_TW |
dc.subject.keyword | Novel H7N9 influenza virus,Hemagglutinin,Monoclonal antibody,Subunit vaccine, | en |
dc.relation.page | 53 | |
dc.identifier.doi | 10.6342/NTU201601871 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-05 | |
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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