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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | |
| dc.contributor.author | Yu-Fan Tu | en |
| dc.contributor.author | 杜毓凡 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:56:55Z | - |
| dc.date.available | 2025-02-21 | |
| dc.date.copyright | 2020-02-21 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-02-14 | |
| dc.identifier.citation | 1. WHO (2019). Middle East respiratory syndrome coronavirus (MERS-CoV). https://www.who.int/news-room/fact-sheets/detail/middle-east-respiratory-syndrome-coronavirus-(mers-cov)
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Du, L., Zhao, G., Kou, Z., Ma, C., Sun, S., Poon, V.K., Lu, L., Wang, L., Debnath, A.K., Zheng, B.J., et al. (2013). Identification of a receptor-binding domain in the S protein of the novel human coronavirus Middle East respiratory syndrome coronavirus as an essential target for vaccine development. J Virol 87, 9939-9942. 35. Mou, H., Raj, V.S., van Kuppeveld, F.J., Rottier, P.J., Haagmans, B.L., and Bosch, B.J. (2013). The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol 87, 9379-9383. 36. Tan, A.-J. (2018). Protien Exprssion and Isolation of the MERS-CoV Spike Protein, Membrane Protein and Envelope Protein. In Department of Biochemical Science and Technology, College of Life Science. (National Taiwan University). 37. Li, C.-C. (2016). Development of neutralizing antibodies against the membrane glycoproteins for emerging disease viruses. In Department of Biochemical Science and Technology, College of Life Science, Volume Master. (National Taiwan University). 38. O’Reilly, D.R. (1997). Use of Baculovirus Expression Vectors. In Recombinant Gene Expression Protocols, R.S. Tuan, ed. (Totowa, NJ: Humana Press), pp. 235-246. 39. Tan, B.H., Brown, G., and Sugrue, R.J. (2007). Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system. Methods Mol Biol 379, 149-161. 40. Rosano, G.L., and Ceccarelli, E.A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5, 172. 41. Tomita, M., and Tsumoto, K. (2011). Hybridoma technologies for antibody production. Immunotherapy 3, 371-380. 42. Yuan, Y., Cao, D., Zhang, Y., Ma, J., Qi, J., Wang, Q., Lu, G., Wu, Y., Yan, J., Shi, Y., et al. (2017). Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nat Commun 8, 15092. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78436 | - |
| dc.description.abstract | 2012年，在沙烏地阿拉伯通報了首例新型中東呼吸症候群冠狀病毒 (MERS-CoV) 感染人的病例，並開始在全球散播。MERS-CoV的致死率高達36%，高於曾經在2002年造成全球性傳染的SARS-CoV的9%致死率。因此，為了預防疫情發生，開發出有效的診斷方法和治療藥物為當務之急。MERS-CoV棘蛋白 (Spike protein) 是病毒表面的重要抗原，棘蛋白根據其不同的功能被分S1和S2兩段次單元，其S1含有一段受體結合域 (receptor binding domain, RBD)，可介導病毒與宿主細胞的DPP4 (dipeptidyl peptidase receptor 4) 表面受體結合。本論文成功開發出可結合於MERS-CoV Spike RBD之C端的兩株單株抗體A8和G2，經比較之後發現與實驗室已開發的可結合在RBD 之N端的單株抗體 6-9具有不同之抗原決定基 (epitope)。單株抗體型別鑑定結果顯示A8、G2和6-9 皆屬於IgG1並帶有kappa 輕鏈。Sandwich ELISA 的實驗結果也揭示了G2和6-9能同時結合至RBD上。這些單株抗體將可開發成能準確診斷MERS-CoV的酵素連結免疫吸附分析法及快速免疫色譜分析試片。而這些單株抗體是否能阻斷棘蛋白與DPP4結合的能力仍需進一步測試。 | zh_TW |
| dc.description.abstract | In 2012, a novel coronavirus was reported in Saudi Arabia and spread out worldwide. The novel coronavirus was then officially named as Middle East respiratory syndrome coronavirus (MERS-CoV) by WHO. The mortality of MERS-CoV reaches 36%, much higher than 9.6% for SARS-CoV which caused worldwide pandemic in 2002. Thus, an effective diagnostic tool and medical treatment would be urgently needed to prevent worse pandemic. The MERS-CoV spike protein is an important surface antigen known to mediate host-receptor binding interaction and virus entry. This spike protein can be divided to two functional subunits. The S1 subunit, including receptor binding domain (RBD), can bind with dipeptidyl peptidase 4 (DPP4) on host cells. In this research, two clones of monoclonal antibodies (mAbs) A8 and G2 against MERS-CoV RBD were successfully produced. The epitope mapping experiments reveal that A8, G2, and another 6-9 mAbs previously developed in our laboratory have different binding epitopes. The results of antibody isotyping exhibit that A8, G2 and 6-9 mAbs are all IgG1 with kappa light chains. Results of sandwich ELISA (enzyme-linked immunosorbent assay) illustrated that G2 and 6-9 mAbs can simultaneously bind on RBD. G2 and 6-9 mAbs would be worth to develop into ELISA and rapid immunochromatographic strip test that accurately diagnose MERS-CoV. The capabilities of mAbs for blocking spike protein binding to DPP4 shall be further characterized. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:56:55Z (GMT). No. of bitstreams: 1 ntu-109-R06b22064-1.pdf: 11327323 bytes, checksum: 4b440b204419b95eaeb23d2d5597da95 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 中文摘要 i
ABSTRACT ii CONTENTS iii LIST OF TABLES AND FIGURES vi ABBREVIATION TABLE viii Chapter 1 Introduction 1 1.1 Middle East respiratory syndrome coronavirus 1 1.1.1 History and epidemiology of MERS-CoV 1 1.1.2 Genomics of MERS-CoV 2 1.1.3 Structure of MERS-CoV 3 1.1.4 MERS-CoV viral entry to host cells 4 1.1.5 Therapeutic and diagnostic for MERS-CoV 4 Chapter 2 Rationale and Specific aims 6 Chapter 3 Materials and Methods 7 3.1 Baculovirus expression system 7 3.1.1 Generation of pFastBacTM expression vectors 7 3.1.2 Generation of expression bacmids 9 3.1.3 Expression of recombinant protein in insect cell 9 3.2 E. coli expression system 10 3.2.1 Generation of pET-28a (+) expression vectors 10 3.2.2 Expression of target genes in E. coli BL21 (DE3) 11 3.2.3 Purification of recombinant proteins by Ni-NTA Affinity Chromatography 12 3.2.4 Desalting and buffer exchange for recombinant proteins 13 3.3 Hybridoma technology 14 3.3.1 Antigen immunization 14 3.3.2 Mice serum collection 15 3.3.3 Mice B-lymphocytes fusion with myeloma cells 15 3.3.4 Monoclonal antibody screening 17 3.4 Monoclonal antibody characterization 18 3.4.1 Epitope mapping 18 3.4.2 Antibody isotyping 19 3.4.3 Sandwich ELISA 19 Chapter 4 Results 21 4.1 Production of MERS-CoV RBD and HR1-HR2 recombinant proteins in baculovirus expression system 21 4.1.1 Plasmid construction of MERS-CoV RBD and HR1-HR2 for baculovirus expression system 21 4.1.2 Protein expression of recombinant MERS-CoV RBD and HR1-HR2 in baculovirus expression system 22 4.2 Production of MERS-CoV RBD and HR1-HR2 recombinant proteins in E. coli expression system 23 4.2.1 Plasmid construction of MERS-CoV RBD and HR1-HR2 for E. coli expression system 23 4.2.2 Protein purification of recombinant MERS-CoV RBD and HR1-HR2 in E. coli expression system 24 4.2.3 Recombinant MERS-CoV RBD and HR1-HR2 antigen preparation for monoclonal antibody production 25 4.3 Production of anti-RBD and anti-HR1-HR2 monoclonal antibodies by hybridoma technology 26 4.3.1 Titer confirmation of serum antibody for antigens immunized mice 26 4.3.2 Screening of monoclonal antibody recognizing recombinant MERS-CoV RBD and HR1-HR2 27 4.4 Characteristics of anti-RBD monoclonal antibodies 28 4.4.1 Binding specificity of anti-RBD monoclonal antibodies 28 4.4.2 Binding epitopes for anti-RBD monoclonal antibodies 29 4.4.3 Isotypes of anti-RBD monoclonal antibodies 31 4.4.4 Capability of G2 and 6-9 mAbs simultaneously binding on MERS-CoV RBD 31 Chapter 5 Discussion 33 REFERENCE 37 TABLES AND FIGURES 44 | |
| dc.language.iso | en | |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | 中東呼吸症候群冠狀病毒 | zh_TW |
| dc.subject | 棘蛋白 | zh_TW |
| dc.subject | 受體結合域 | zh_TW |
| dc.subject | DPP4 | zh_TW |
| dc.subject | monoclonal antibody | en |
| dc.subject | spike protein | en |
| dc.subject | MERS-CoV | en |
| dc.subject | dipeptidyl peptidase 4 (DPP4) | en |
| dc.subject | receptor binding domain (RBD) | en |
| dc.title | 開發抗中東呼吸症候群冠狀病毒棘蛋白之單株抗體 | zh_TW |
| dc.title | Development of Monoclonal Antibodies Targeting the MERS-CoV Spike Protein | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 廖憶純,吳?承,陳慧文 | |
| dc.subject.keyword | 中東呼吸症候群冠狀病毒,棘蛋白,受體結合域,單株抗體,DPP4, | zh_TW |
| dc.subject.keyword | MERS-CoV,spike protein,receptor binding domain (RBD),monoclonal antibody,dipeptidyl peptidase 4 (DPP4), | en |
| dc.relation.page | 70 | |
| dc.identifier.doi | 10.6342/NTU201904163 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-02-14 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2025-02-21 | - |
| 顯示於系所單位： | 生化科技學系 | |
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