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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | zh_TW |
| dc.contributor.author | 陳一鳴 | zh_TW |
| dc.contributor.author | Yi-Ming Chen | en |
| dc.date.accessioned | 2021-07-10T21:54:27Z | - |
| dc.date.available | 2024-08-13 | - |
| dc.date.copyright | 2019-08-14 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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Portela, A., and Digard, P. (2002) The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83, 723-734 8. Tu, Y. C., Chen, K. Y., Chen, C. K., Cheng, M. C., Lee, S. H., and Cheng, I. C. (2019) Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying. J Vet Sci 20, 51-57 9. Liu, J. K. (2014) The history of monoclonal antibody development - Progress, remaining challenges and future innovations. Ann Med Surg (Lond) 3, 113-116 10. Skerra, A., and Pluckthun, A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041 11. Harding, F. A., Stickler, M. M., Razo, J., and DuBridge, R. B. (2010) The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2, 256-265 12. Glockshuber, R., Malia, M., Pfitzinger, I., and Pluckthun, A. (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362-1367 13. Marasco, W. A., and Dana Jones, S. (1998) Antibodies for targeted gene therapy: extracellular gene targeting and intracellular expression. Adv Drug Deliv Rev 31, 153-170 14. Emanuel, P. A., Dang, J., Gebhardt, J. S., Aldrich, J., Garber, E. A., Kulaga, H., Stopa, P., Valdes, J. J., and Dion-Schultz, A. (2000) Recombinant antibodies: a new reagent for biological agent detection. Biosens Bioelectron 14, 751-759 15. Barnwal, B., Mok, C. K., Wu, J., Diwakar, M. K., Gupta, G., Zeng, Q., Chow, V. T., Song, J., Yuan, Y. A., and Tan, Y. J. (2015) A monoclonal antibody binds to threonine 49 in the non-structural 1 protein of influenza A virus and interferes with its ability to modulate viral replication. Antiviral Res 116, 55-61 16. Yodsheewan, R., Maneewatch, S., Srimanote, P., Thueng-In, K., Songserm, T., Dong-Din-On, F., Bangphoomi, K., Sookrung, N., Choowongkomon, K., and Chaicumpa, W. (2013) Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes. Antiviral Res 100, 226-237 17. Guidotti, G., Brambilla, L., and Rossi, D. (2017) Cell-Penetrating Peptides: From Basic Research to Clinics. Trends Pharmacol Sci 38, 406-424 18. Turner, J. J., Arzumanov, A. A., and Gait, M. J. (2005) Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides. Nucleic Acids Res 33, 27-42 19. Dong-din-on, F., Songserm, T., Pissawong, T., Srimanote, P., Thanongsaksrikul, J., Thueng-in, K., Moonjit, P., Lertwatcharasarakul, P., Seesuay, W., and Chaicumpa, W. (2015) Cell penetrable human scFv specific to middle domain of matrix protein-1 protects mice from lethal influenza. Viruses 7, 154-179 20. Rouet, R., Jackson, K. J. L., Langley, D. B., and Christ, D. (2018) Next-Generation Sequencing of Antibody Display Repertoires. Front Immunol 9, 118 21. Yin, C. F., Khan, J. A., Swapna, G. V. T., Ertekin, A., Krug, R. M., Tong, L., and Montelione, G. T. (2007) Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses. J Biol Chem 282, 20584-20592 22. Sharma, S. K., Suresh, M. R., and Wuest, F. R. (2014) Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer. Protein Expr Purif 102, 27-37 23. Salehinia, J., Sadeghi, H. M. M., Abedi, D., and Akbari, V. (2018) Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies. Res Pharm Sci 13, 566-574 24. Peternel, S., Grdadolnik, J., Gaberc-Porekar, V., and Komel, R. (2008) Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact 7, 34 25. Tao, H., Liu, W., Simmons, B. N., Harris, H. K., Cox, T. C., and Massiah, M. A. (2010) Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques 48, 61-64 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77291 | - |
| dc.description.abstract | 根據世界衛生組織統計，截至2019年4月全球有1568個人感染新型H7N9流感病毒的確診病例，且死亡率高達40%，為了治療及預防病毒大流行，開發能有效中和病毒的抗體是當務之急。A型流感病毒之非結構性蛋白(non-structural protein 1, NS1) 能抑制受感染宿主的免疫系統，且能增加病毒感染力；而核蛋白(nucleoprotein, NP) 的主要功能為包裹病毒的染色體，進而影響病毒的轉錄、複製及病毒顆粒包裝。因此若能抑制NS1與NP之功能，應可達到抑制病毒感染之功效。本研究的主要目標是將抗H7N9 NS1及NP之單株抗體製備成單鏈抗體之形式，並在單鏈抗體之N端加上穿膜序列，再進一步確認單鏈抗體之功能及穿膜效果。未來希望此單鏈抗體可以有效降低H7N9的病毒複製及感染能力。 | zh_TW |
| dc.description.abstract | The novel avian-origin H7N9 influenza virus has caused 1,568 laboratory-confirmed human cases with a fatality rate approaching 40% according to WHO’s reports. It is in an urgent need for developing neutralizing antibodies for disease treatment and control. The influenza non-structural protein 1 (NS1) can inhibit host cell immune response to improve virus infection, and nucleoprotein (NP) can encapsulate the virus genome for transcription, replication and packaging. The present study aims to utilize the anti-NS1 and anti-NP antibodies for inhibiting H7N9 viral infection and replication. However, the large molecular size of the antibody is not able to directly penetrate through the membranes of the infected host cells for interacting with NS1 and NP. Therefore, the single-chain variable fragment (scFv) antibodies against H7N9 NS1 and NP have been constructed with an N-terminal penetratin peptide (PEN) for enhancing membrane permeability. In the future, the membrane permeability of the scFvs and their capability for inhibiting H7N9 virus replication and infection in MDCK cells will be further characterized. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:54:27Z (GMT). No. of bitstreams: 1 ntu-108-R06b22024-1.pdf: 2145479 bytes, checksum: c4565fe20e7397e426691758305a65d0 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
摘要 i Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 流行性感冒病毒簡介 1 1.1.1 流感病毒的構造 1 1.1.2 非結構性蛋白質Non-structural protein 1 (NS1) 2 1.1.3 核蛋白Nucleoprotein (NP) 3 1.2 抗體 3 1.2.1 抗體構造及分類 3 1.2.2 單株抗體 4 1.2.3 抗體基因工程 4 1.2.4 單鏈抗體 (Single-chain variable fragment, scFv) 4 1.2.5 scFv的應用 5 1.3 研究動機 5 第二章 材料與方法 7 2.1 實驗材料 7 2.1.1新型H7N9流感病毒之NS1及NP基因 7 2.1.2 大腸桿菌 7 2.2 DNA實驗方法 7 2.2.1 表現載體之建構 7 2.2.2 聚合酶連鎖反應 8 2.2.3 限制酶切反應 8 2.2.4 DNA接合反應 8 2.2.5 質體轉形作用 9 2.2.6 質體DNA製備 9 2.2.7 洋菜膠體電泳 9 2.2.8 核酸純化 10 2.2.9 核酸定量 10 2.3 蛋白質實驗方法 10 2.3.1 蛋白質定量 10 2.3.2 蛋白質電泳法 11 2.3.3 蛋白質電泳膠片染色法 11 2.3.4 蛋白質電泳轉印法 11 2.3.5 免疫染色法 12 2.3.6 酵素連結免疫分析法 12 2.4 蛋白質純化 13 2.4.1 親和層析法 13 2.4.2 離子交換法 13 2.5 細胞實驗 14 2.5.1 細胞解凍 14 2.5.2 純化單株抗體 14 2.6 抗體基因選殖 15 2.6.1 Total RNA 抽取 15 2.6.2 次世代基因定序 15 第三章 結果 17 3.1 NS1單株抗體純化 17 3.2 NP單株抗體純化 17 3.3 Anti-NS1單株抗體結合位點測試 18 3.4 可溶scFv之純化 18 3.4.1 αNS1-scFv之純化 18 3.4.2 αNP-scFv之純化 19 3.5 不可溶αNS1-scFv及αNP-scFv之純化 19 3.6 以urea 回溶αNS1-scFv抗原結合力測試 20 3.7 以urea回溶αNP-scFv之抗原結合力測試 20 3.8 不可溶PEN-αNS1-scFv及PEN-αNP-scFv之純化 21 3.9 以urea回溶PEN-αNS1-scFv之抗原結合測試 21 3.10 以界面活性劑增加破菌效果 22 3.11 以界面活性劑回溶之PEN-αNS1-scFv的抗原結合測試 22 第四章 討論 24 4.1 以NGS取得單株抗體基因 24 4.2 Anti-NS1單株抗體結合位點討論 24 4.3 αNS1-scFv及αNP-scFv純化條件探討 24 4.4 αNS1-scFv及αNP-scFv抗原結合力探討 25 參考文獻 27 表與圖 30 附錄 43 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | NS1 | zh_TW |
| dc.subject | NP | zh_TW |
| dc.subject | 單鏈抗體 | zh_TW |
| dc.subject | 新型H7N9流感病毒 | zh_TW |
| dc.subject | NP | en |
| dc.subject | scFv | en |
| dc.subject | NS1 | en |
| dc.subject | Novel H7N9 influenza virus | en |
| dc.title | 製備抗H7N9流感病毒非結構蛋白NS1與核蛋白NP之單鏈抗體 | zh_TW |
| dc.title | Preparation of the anti-H7N9 NS1 and NP single-chain variable fragment antibodies | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳威戎;林翰佳;黃純芳 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | 新型H7N9流感病毒,NS1,NP,單鏈抗體, | zh_TW |
| dc.subject.keyword | Novel H7N9 influenza virus,NS1,NP,scFv, | en |
| dc.relation.page | 45 | - |
| dc.identifier.doi | 10.6342/NTU201902728 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-08 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| 顯示於系所單位： | 生化科技學系 | |
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