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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張世宗 | |
dc.contributor.author | Chia-Wei Weng | en |
dc.contributor.author | 翁家葳 | zh_TW |
dc.date.accessioned | 2021-06-15T16:48:34Z | - |
dc.date.available | 2018-08-16 | |
dc.date.copyright | 2015-08-16 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-07 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53168 | - |
dc.description.abstract | 流感是最具全球大流行潛力的病毒傳染病。2013年首次出現低致病性禽流感病毒H7N9經基因重組而成新病毒的人感染病例,且致重症的病程急速惡化與死亡率偏高,而病例無禽接觸史的比率也漸升,再加上臺灣首例境外移入的H7N9病例已具有克流感的抗藥性,且病毒蛋白也顯現可提昇人傳染力的胺基酸突變,因此發展快速檢驗與治療所需抗體及疫苗對防疫工作迫切重要。
H7N9流感病毒含有8段基因 (HA、NA、M、PB1、PB2、PA、NS、NP),屬於負股分段RNA基因的正黏液病毒 (Orthomyxoviridae)。其病毒表面結構蛋白血球凝集素Hemagglutinin (HA) 與病毒入侵細胞有關,而神經胺酸酶Neuraminidase (NA) 則與病毒離開宿主細胞有關,兩者為疫苗與抗病毒藥物的主要標的,因此本研究致力於利用桿狀病毒與昆蟲細胞表現系統製備H7N9 HA與NA之重組蛋白,以提供未來疫苗與抗體研發之龐大需求。本研究已將HA或NA基因選殖至pFastBac HT-A及pFastBac1質體,再轉形至DH10Bac勝任細胞以獲得含有HA或NA基因的重組Bacmid。經轉染至Sf9或Sf21昆蟲細胞後,已成功製備出能表現HA或NA重組蛋白之桿狀病毒,並以質譜儀確認由Sf9昆蟲細胞所表現出之NA的胺基酸序列,與H7N9之基因所推測的序列完全相符。本論文亦已進行H7N9 HA與NA最適表現條件與HA蛋白質純化層析流程之探討,成功完成了量產前的先導研究任務。 | zh_TW |
dc.description.abstract | Influenza virus is the most potential global pandemic virus. Severe and fatal human cases of the newly reasserted novel H7N9 influenza viruses have continuously occurred since the first case reported in March, 2013. Public health preparedness must be initiated after risk assessment based on recent findings, including the fast disease progression, the increasing percentages of cases without contact history of birds, the appearance of Tamiflu drug-resistant H7N9 virus strains (the first imported Taiwan case from mainland China) and the cumulative evidence of the isolated human H7N9 viruses possessing amino acid residues with aerosol transmissibility. Therefore, it is very important to develop rapid laboratory diagnostics, antibodies and vaccines for better pandemic preparedness.
H7N9, belonging to the family of Orthomyxoviridae, is an enveloped virus with a genome made up of negative sense, single-stranded and segmented RNAs, including hemagglutinin (HA), neuraminidase (NA), matrix protein (M), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), non-structural protein (NS) and nucleoprotein (NP). Virus surface structure protein HA is involved in the cell invasion, while NA is linked to the release of virus particles. HA and NA are major targets of vaccines and anti-virus drugs. The present work was aimed at studying the protein expression and purification of novel H7N9 influenza virus HA and NA by baculovirus expression system for huge demand of vaccines and antibodies in the future. The gene encoding HA or NA has been cloned into pFastBac HT-A and pFastBac1 and then transformed to DH10Bac competent cells for producing the recombinant bacmid. After transfecting the recombinant bacmid to Sf9 or Sf21 insect cells, the baculovirus that can express recombinant HA or NA protein was successfully produced. The amino acid sequence of the recombinant NA analyzed by mass spectrometry revealed that it is exactly the same as the deduced sequence from H7N9 NA gene. The optimal protein expression conditions and purification procedures of H7N9 HA and NA were also determined and investigated in the present study. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:48:34Z (GMT). No. of bitstreams: 1 ntu-104-R02b22045-1.pdf: 13158249 bytes, checksum: 9444ebb0b15a7b2878e35cd6867df3ad (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 目錄
中文摘要 iii Abstract iv 縮寫表 v 第一章 緒論 1 1.1 流行性感冒病毒 1 1.2 A型流感病毒 1 1.2.1 血球凝集素 (Hemagglutinin) 2 1.2.2 神經胺酸酶 (Neuraminidase) 3 1.3 新型H7N9禽流感起源及疫情 3 1.4 新型H7N9基因親源分析 4 1.5 桿狀病毒表現系統 4 1.6 研究動機 5 第二章 材料方法 6 2.1 實驗材料 6 2.1.1 H7N9表面結構蛋白HA及NA基因來源 6 2.1.2 昆蟲細胞 6 2.1.3 宿主菌株 6 2.2 表現載體建構 6 2.2.1 真核表現系統載體 6 2.2.2 核酸引子設計 7 2.2.3 HA及NA表現質體之製備 7 2.3 桿狀病毒表現系統 7 2.3.1 質體轉形 7 2.3.2 重組Bacmid萃取 8 2.3.3 重組Bacmid轉染 (Transfection) 及病毒液放大 8 2.3.4 重組蛋白表現條件測試 9 2.3.5 病毒液之效價檢驗及病毒感染劑量 (MOI) 9 2.3.6 重組蛋白表現 10 2.3.7 重組蛋白純化 10 第三章 結果 11 3.1 新型H7N9流感病毒之HA與NA表現載體建構 11 3.1.1 建構帶有H7N9 HA與NA基因之pFastBac HT-A 11 3.1.2 建構帶有H7N9 HA與NA基因之pFastBac 1 11 3.2 H7N9 HA與NA之桿狀病毒表現系統Bacmid質體製備 11 3.3 H7N9 HA與NA之Bacmid轉染及Baculovirus病毒液製備 12 3.3.1 NA全長於Sf9及Sf21昆蟲細胞之表現 12 3.3.2 HA-His∆TM於Sf21昆蟲細胞之表現 12 3.3.3 ∆SigHA-His∆TM於Sf21昆蟲細胞之表現 13 3.3.4 HA1-His於Sf21昆蟲細胞之表現 13 3.3.5 ∆SigHA1-His於Sf21昆蟲細胞之表現 13 3.3.6 His-NA∆TM於Sf21昆蟲細胞之表現 14 3.4 H7N9 ∆SigHA1-His重組蛋白表現條件之測試 14 3.5 H7N9 ∆SigHA1-His病毒液效價檢驗 14 3.6 H7N9 ∆SigHA1-His重組蛋白之純化 14 第四章 討論 16 4.1 蛋白質表現系統之選擇 16 4.2 新型H7N9流感病毒NA全長於Sf9及Sf21昆蟲細胞之重組蛋白質表現 16 4.2.1 新型H7N9流感病毒HA重組蛋白之表現 17 4.2.2 新型H7N9流感病毒HA1重組蛋白之表現 17 4.2.3 新型H7N9流感病毒NA重組蛋白之表現 17 4.3 新型H7N9流感病毒∆SigHA1-His重組蛋白純化條件 18 參考文獻 19 圖與表 23 | |
dc.language.iso | zh-TW | |
dc.title | 新型H7N9流感病毒血球凝集素與神經胺酸酶之蛋白質表現與純化 | zh_TW |
dc.title | Protein Expression and Purification of the Novel H7N9 Influenza Virus Hemagglutinin and Neuraminidase | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張麗冠,陳慧文,陳威戎,林翰佳 | |
dc.subject.keyword | H7N9流感病毒,血球凝集素,神經胺酸?,桿狀病毒表現系統, | zh_TW |
dc.subject.keyword | H7N9 influenza virus,Hemagglutinin,Neuraminidase,Baculovirus expression system, | en |
dc.relation.page | 48 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-07 | |
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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