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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝 | |
dc.contributor.author | AN-JIE TAN | en |
dc.contributor.author | 譚安絜 | zh_TW |
dc.date.accessioned | 2021-06-08T03:49:06Z | - |
dc.date.copyright | 2018-12-17 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-12-11 | |
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Liu F, Wu X, Li L, Liu Z, & Wang Z (2013) Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expr Purif 90(2):104-116. 48. Wang C, et al. (2017) MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques. Oncotarget 8(8):12686-12694. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21838 | - |
dc.description.abstract | 中東呼吸道症候群冠狀病毒 (Middle East Respiratory Syndrome Coronavirus ; MERS-CoV) 於2012年首度在沙烏地阿拉伯被發現,之後也陸續傳播到歐洲、亞洲、非洲及北美洲。截至2018年9月為止已經有2249例確診病例,其中有798名病患死亡,死亡率高達36%。然而目前尚無有效的疫苗與藥物可供治療,因此發展出有效預防的疫苗與治療的抗體對於防疫工作迫切重要。
MERS-CoV為含有單股正向RNA的冠狀病毒,其外套膜上具有三種結構蛋白spike (S)、membrane (M)及envelope (E),從感染時辨識宿主與受體結合,到病毒出芽時的組裝與結構穩定都和這三種結構蛋白息息相關,也是近年來重要疫苗候選蛋白與抗病毒藥物的主要標的。本研究致力於利用桿狀病毒與昆蟲細胞表現系統製備MERS-CoV的三種結構蛋白,以提供未來疫苗與抗體之研發需求。將含有S、M及E基因的重組Bacmid經轉染至Sf21昆蟲細胞後,已成功獲得能表現S、M及E重組蛋白之桿狀病毒,並進行了最適表現條件探討,成功分離各重組蛋白後再以質譜儀分析確認S、M及E重組蛋白之的胺基酸序列與由基因所推測之序列完全相符,完成製備疫苗與抗體前的抗原表現先導實驗。本研究亦利用大腸桿菌表現系統生產S蛋白質上負責與受體 dipeptidyl peptidase 4 (DPP4) 結合之receptor binding domain (RBD),並以實驗室先前製備之Anti-RBD單株抗體 (6-9B及2-11A) 來進行分析,實驗結果顯示RBD於大腸桿菌表現時會形成inclusion bodies。 | zh_TW |
dc.description.abstract | MERS-CoV was first identified in Saudi Arabia in 2012 and has spread to Europe, Asia, Africa and North America. A total of 2,249 MERS-CoV-infected cases with a mortality of 36% was reported in September 2018. WHO declares that there are no vaccines or specific treatment available now. Thus, there is an urgent need for the development of vaccines and theranostic antibodies for better pandemic preparedness.
MERS-CoV has a positive sense, single-stranded RNAs, including the spike (S), membrane (M) and envelope (E) genes. These three viral surface structure proteins are involved in the cell invasion, structural stability and the release of the viral particles, and can become potential vaccines or be major targets of anti-virus drugs. The present work was aimed to establish the MERS-CoV S, M, and E recombinant protein expression system for the demand of preparing vaccines and antibodies in the future. The recombinant baculovirus bacmids containing S, M, and E genes of MERS-CoV has been tansfected to the Sf21 cells, and the baculoviruses that can individually express recombinant S, M and E proteins were successfully produced. The amino acid sequences of the recombinant S, M and E proteins analyzed by mass spectrometry revealed that they are exactly the same as the sequences deduced from MERS-CoV S, M and E genes. The optimal protein expression conditions and isolationn procedures of MERS-CoV S, M and E proteins were also determined and investigated in the present study. In addition, the receptor binding domain (RBD) of S protein, which is responsible for binding to the receptor dipeptidyl peptidase 4 (DPP4), was expressed in Escherichia coli and was analyzed by western blotting using the previously prepared anti-RBD monoclonal antibody (6-9B and 2-11A) in the laboratory. The experimental results showed that RBD formed inclusion bodies while expressed in E. coli. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:49:06Z (GMT). No. of bitstreams: 1 ntu-107-R05b22046-1.pdf: 3034281 bytes, checksum: 33e8aa6d99ce21311bfdd5715143bb7a (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 國立臺灣大學碩士學位論文 口試委員審定書 i
謝辭 ii 中文摘要 iii Abstract iv 縮寫表 v 第一章 緒論 1 1.1 中東呼吸道症候群冠狀病毒之探討與研究 1 1.1.1 MERS-CoV病毒之歷史與來源 1 1.1.2 MERS-CoV病毒感染之臨床症狀與傳播途徑 1 1.1.3 MERS-CoV病毒之構造與型態 2 1.1.4 MERS-CoV病毒之基因體 2 1.1.5 MERS-CoV病毒之結構蛋白質功能簡介 4 Spike protein (S): 4 Membrane protein (M): 5 Envelope protein (E): 5 1.1.6 MERS-CoV病毒之生活史(5) 6 1.2 MERS-CoV抗體之相關研究 7 1.3 桿狀病毒表現系統 8 1.4 研究動機 8 第二章 材料與方法 10 2.1 實驗材料 10 2.1.1 大腸桿菌 (Escherichia coli) 10 2.1.2 昆蟲細胞 (Sf21) 10 2.2 表現載體之建構 11 2.2.1 表現系統載體之構築 11 2.2.2 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 11 2.2.3 限制酶切反應 (Restriction Enzyme Digestion) 12 2.2.4 接合反應 (Ligation) 12 2.2.5 轉型作用(Transformation) 13 2.2.6 抗生素篩選 13 2.3 核酸實驗方法 14 2.3.1 質體DNA抽取 (Plasmid Miniprep) 14 2.3.2 洋菜膠體電泳 (Agarose Gel Electrophoresis) 14 2.3.3 膠體純化 (Gel Extraction) 15 2.3.4 核酸定量 15 2.4 蛋白質實驗方法 15 2.4.1 蛋白質定量 15 2.4.2 蛋白質電泳法 16 2.4.3 蛋白質電泳膠片染色法 16 2.4.4 蛋白質膠片電泳轉印法 17 2.4.5 免疫染色法 (Western Blotting) 17 2.5 細胞培養 18 2.5.1 昆蟲細胞培養 18 2.5.2 細胞解凍 18 2.5.3 細胞冷凍 18 2.6 桿狀病毒表現系統 18 2.6.1 質體轉型作用 19 2.6.2 重組Bacmid純化 19 2.6.3 昆蟲細胞之轉染作用 20 2.6.4 病毒最佳感染比例檢測 20 2.6.5 感染後最佳培養天數 21 2.6.6 重組蛋白表現 21 2.6.7 重組蛋白分離 21 2.7 原核表現系統 24 2.7.1 質體轉型作用 24 2.7.2 重組蛋白質誘導表現 25 2.7.3 重組蛋白純化 25 第三章 結果 28 3.1 MERS-CoV之S、M及E protein表現載體建構 28 3.1.1 建構帶有S、M及E基因之pFastBac Dual 28 3.1.2 建構帶有RBD基因之pFastBac HT-A-gp67 28 3.1.3 建構帶有RBD基因之pGEX-4T-1和pET28a (+) 29 3.2 帶有MERS-CoV S、M、E基因之Bacmid製備 29 3.3 帶有MERS-CoV S、M、E基因之Baculovirus病毒液製備 29 3.3.1 於Sf21昆蟲細胞表現MERS-CoV S基因 30 3.3.2 於Sf21昆蟲細胞表現MERS-CoV M基因 30 3.3.3 於Sf21昆蟲細胞表現MERS-CoV E基因 30 3.3.4 於Sf21昆蟲細胞表現MERS-CoV S、M與E基因 30 3.4 MERS-CoV S、M與E重組蛋白表現條件測試 31 3.4.1 表現MERS-CoV S重組蛋白之表現條件測試 32 3.4.2 表現MERS-CoV M重組蛋白之表現條件測試 32 3.4.3 表現MERS-CoV E重組蛋白之表現條件測試 33 3.5 MERS-CoV S、M與E重組蛋白之分離 33 3.5.1 MERS-CoV S重組蛋白質之分離: 33 3.5.2 MERS-CoV M重組蛋白質之分離: 34 3.5.3 MERS-CoV E重組蛋白質之分離: 34 3.6 MERS-CoV RBD重組蛋白之表現與純化 35 第四章 討論 36 4.1 MERS-CoV S重組蛋白質之表現與分離探討 36 4.2 MERS-CoV M重組蛋白質之表現與分離探討 37 4.3 MERS-CoV E重組蛋白質之表現與分離探討 38 4.4 同時表現MERS-CoV S、M與E重組蛋白質之探討 39 圖與表 41 參考文獻 72 | |
dc.language.iso | zh-TW | |
dc.title | 中東呼吸道症候群冠狀病毒之棘蛋白、膜蛋白與包膜蛋白的表現與分離 | zh_TW |
dc.title | Protein Expression and Isolation of the MERS-CoV
Spike Protein, Membrane Protein and Envelope Protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 張世宗 | |
dc.contributor.oralexamcommittee | 楊健志,陳翰民 | |
dc.subject.keyword | MERS-CoV冠狀病毒,Spike protein (S),Membrane protein (M),Envelope protein (E),桿狀病毒表現系統, | zh_TW |
dc.subject.keyword | Middle East respiratory syndrome coronavirus,Spike protein,Membrane protein,Envelope protein,Baculovirus expression system, | en |
dc.relation.page | 75 | |
dc.identifier.doi | 10.6342/NTU201804332 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2018-12-11 | |
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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