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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | |
| dc.contributor.author | Fang-Yin Liu | en |
| dc.contributor.author | 劉芳吟 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:33:59Z | - |
| dc.date.available | 2021-07-10T21:33:59Z | - |
| dc.date.copyright | 2017-02-21 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2017-01-25 | |
| dc.identifier.citation | Ayllon, J., Domingues, P., Rajsbaum, R., Miorin, L., Schmolke, M., Hale, B.G., and Garcia-Sastre, A. (2014). A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence. J Virol 88, 12146-12151.
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. Belser, J.A., Gustin, K.M., Pearce, M.B., Maines, T.R., Zeng, H., Pappas, C., Sun, X., Carney, P.J., Villanueva, J.M., Stevens, J., et al. (2013). Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature 501, 556-559. Bouvier, N.M., and Palese, P. (2008). The biology of influenza viruses. Vaccine 26, D49-D53. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72, 248-254. Burnette, W.N. (1981). “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical biochemistry 112, 195-203. Chang, S.-Y., Lin, P.-H., Tsai, J.-C., Hung, C.-C., and Chang, S.-C. (2013). The first case of H7N9 influenza in Taiwan. The Lancet 381, 1621. Eisfeld, A.J., Neumann, G., and Kawaoka, Y. (2015). At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol 13, 28-41. Engvall, E., and Perlmann, P. (1971). Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871-874. Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., Chen, J., Jie, Z., Qiu, H., Xu, K., et al. (2013). Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368, 1888-1897. Gong, J., Xu, W., and Zhang, J. (2007). Structure and functions of influenza virus neuraminidase. Current medicinal chemistry 14, 113-122. Hale, B.G., Randall, R.E., Ortín, J., and Jackson, D. (2008). The multifunctional NS1 protein of influenza A viruses. Journal of General Virology 89, 2359-2376. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of molecular biology 166, 557-580. Hay, A.J., and Hayden, F.G. (2013). Oseltamivir resistance during treatment of H7N9 infection. The Lancet 381, 2230-2232. Knepper, J., Schierhorn, K.L., Becher, A., Budt, M., Tonnies, M., Bauer, T.T., Schneider, P., Neudecker, J., Ruckert, J.C., Gruber, A.D., et al. (2013). The novel human influenza A(H7N9) virus is naturally adapted to efficient growth in human lung tissue. MBio 4, e00601-00613. Li, W., Zhang, H., Wang, G., Zhang, C., Zeng, X., Liu, H., Chen, X., Xu, Y., and Li, K. (2012). Heterologous interactions between NS1 proteins from different influenza A virus subtypes/strains. Sci China Life Sci 55, 507-515. Loll, M.J., and Bollag, J.-M. (1983). Protein transformation in soil. Advances in Agronomy 36, 351-382. Mänz, B., Brunotte, L., Reuther, P., and Schwemmle, M. (2012). Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nature communications 3, 802. Medina, R.A., and García-Sastre, A. (2011). Influenza A viruses: new research developments. Nature Reviews Microbiology 9, 590-603. Medina, R.A., and Garcia-Sastre, A. (2011). Influenza A viruses: new research developments. Nat Rev Microbiol 9, 590-603. Min, J.-Y., and Krug, R.M. (2006). The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2′-5′ oligo (A) synthetase/RNase L pathway. Proceedings of the National Academy of Sciences 103, 7100-7105. Min, J.-Y., Li, S., Sen, G.C., and Krug, R.M. (2007). A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363, 236-243. Nelson, M.I., and Holmes, E.C. (2007). The evolution of epidemic influenza. Nature reviews genetics 8, 196-205. Obayashi, E., Yoshida, H., Kawai, F., Shibayama, N., Kawaguchi, A., Nagata, K., Tame, J.R., and Park, S.Y. (2008). The structural basis for an essential subunit interaction in influenza virus RNA polymerase. Nature 454, 1127-1131. Palese, P., and Shaw, M.L. (2007). Orthomyxoviridae: the viruses and their replication. Fields virology 2, 1647-1689. Paterson, D., and Fodor, E. (2012). Emerging roles for the influenza A virus nuclear export protein (NEP). PLoS Pathog 8, e1003019. Robb, N.C., Smith, M., Vreede, F.T., and Fodor, E. (2009). NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. Journal of general virology 90, 1398-1407. Safo, M.K., Musayev, F.N., Mosier, P.D., Zhou, Q., Xie, H., and Desai, U.R. (2014). Crystal structures of influenza A virus matrix protein M1: variations on a theme. PloS one 9, e109510. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning, Vol 2 (Cold spring harbor laboratory press New York). Schägger, H., and Von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical biochemistry 166, 368-379. Schnell, J.R., and Chou, J.J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591-595. Shimizu, T., Takizawa, N., Watanabe, K., Nagata, K., and Kobayashi, N. (2011). Crucial role of the influenza virus NS2 (NEP) C‐terminal domain in M1 binding and nuclear export of vRNP. FEBS letters 585, 41-46. Simpson, R.J. (2007). Staining proteins in gels with coomassie blue. CSH protocols 2007, pdb. prot4719. Smith, G.E., Flyer, D.C., Raghunandan, R., Liu, Y., Wei, Z., Wu, Y., Kpamegan, E., Courbron, D., Fries, L.F., 3rd, and Glenn, G.M. (2013). Development of influenza H7N9 virus like particle (VLP) vaccine: homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus. Vaccine 31, 4305-4313. Suarez, D., and Schultz-Cherry, S. (2000). Immunology of avian influenza virus: a review. Developmental & Comparative Immunology 24, 269-283. Sui, Z., Chen, Q., Fang, F., Zheng, M., and Chen, Z. (2010). Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant. Vaccine 28, 7690-7698. Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M., Yang, H., Chen, X., Recuenco, S., and Gomez, J. (2013). New world bats harbor diverse influenza A viruses. PLoS Pathog 9, e1003657. Wakefield, L., and Brownlee, G.G. (1989). RNA-binding properties of influenza A virus matrix protein M1. Nucleic acids research 17, 8569-8580. Webster, R.G., and Rott, R. (1987). Influenza virus A pathogenicity: the pivotal role of hemagglutinin. Cell 50, 665-666. White, D.O., and Fenner, F. (1994). Medical virology (Gulf Professional Publishing). Yang, J.R., Kuo, C.Y., Huang, H.Y., Wu, F.T., Huang, Y.L., Cheng, C.Y., Su, Y.T., Wu, H.S., and Liu, M.T. (2015). Characterization of influenza A (H7N9) viruses isolated from human cases imported into Taiwan. PLoS One 10, e0119792. Yang, P., Wang, W., Gu, H., Li, Z., Zhang, K., Wang, Z., Li, R., Duan, Y., Zhang, S., and Wang, X. (2014). Protection against influenza H7N9 virus challenge with a recombinant NP-M1-HSP60 protein vaccine construct in BALB/c mice. Antiviral Res 111, 1-7. Zhirnov, O.P., Isaeva, E.I., Konakova, T.E., Thoidis, G., Piskareva, L.M., Akopova, II, Kartashov, A., Altstein, A.D., Ilyinskii, P.O., and Shneider, A.M. (2007). Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1. Influenza Other Respir Viruses 1, 71-79. Zhu, C.-y., Zheng, F.-l., She, X.-s., Zhao, D., Gu, Y., Duan, Y.-t., Chang, A.K., and Liu, H.-s. (2015). Identification of NS1 domains of avian H5N1 influenza virus which influence the interaction with the NOLC1 protein. Virus genes 50, 238-244. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76621 | - |
| dc.description.abstract | 2013年3月中國爆發全球首次由低致病性H7N9禽流感病毒經基因重組而引發高致病性的人類感染。臺灣也於同年4月宣布首例之境外移入病例。根據世界衛生組織調查，截至2016年6月，確診病例已超過700例，死亡率更高達近四成。研究指出此H7N9的基質蛋白M1已產生N30D及T215A的突變，會使病毒的病原性增強；與形成核醣核蛋白複合體有關的非結構性蛋白NS1已產生P42S的突變，會使病毒的病原性增強，顯示新型H7N9禽流感病毒對哺乳類宿主已開始具有適應性，且M1、NS1及NS2等蛋白在病毒感染宿主及複製時皆有其重要的功能，因此本研究的目標為表現並純化新型H7N9流感病毒的M1、NS1、及NS2的重組蛋白以進行專一性抗體之製備。本研究已成功表現出M1、NS1及NS2的重組蛋白質，並藉由親和層析法分別進行純化且完成量產，並將M1、NS1及NS2 分別免疫BALB/c小鼠後產生多株抗體。利用單株抗體的篩選及純化，已成功得到抗NS1的單株抗體。ELISA及免疫染色法的結果顯示此抗NS1單株抗體具有高度專一性及效價，並可利用此抗體進行NS1之免疫沉澱，顯示此抗體與抗原間具有高度結合能力。 | zh_TW |
| dc.description.abstract | Since March 2013, more than 700 laboratory-confirmed human H7N9 influenza A virus infection cases have been reported by WHO as stated in June 2016, with a case fatality rate of more than 39%. Based on genetic analysis, this avian influenza A virus H7N9 shows some extent adaptation to mammalian hosts. It is reported that the novel H7N9 M1 has N30D and T215A mutations and NS1 has P42S mutation, which lead to increased virulence in mice. Therefore, it is urgently important to strengthen basic research of H7N9 and develop rapid laboratory diagnostics and antibodies. This study focused on production of the novel H7N9 influenza virus M1, NS1 and NS2 recombinant proteins for generation of antibodies. M1, NS1 and NS2 recombinant proteins were purified by affinity chromatography, and the purified M1, NS1 and NS2 proteins were used to immunize BALB/c mice for producing polyclonal antibodies. The anti-NS1 antibodies were screened by ELISA and further analyzed by western blotting. The anti-NS1 mAbs were purified, and the specificity and binding capacity toward H7N9 NS1 were analyzed by western blotting and immunoprecipitation. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:33:59Z (GMT). No. of bitstreams: 1 ntu-105-R03b22042-1.pdf: 2442435 bytes, checksum: 8fba86a027237f526ddd2f285e5670a3 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 目錄
摘要 i Abstract ii 縮寫表 iii 目錄 iv 第一章 緒論 1 1.1 流行性感冒病毒 1 1.2 流行性感冒病毒結構 1 1.3 A型流感病毒及其基因體 2 1.4 流感病毒的生活史 5 1.5 新型H7N9流感的爆發 6 1.6 新型H7N9病毒基因親緣分析與序列特性 6 1.7 研究動機與目的 7 第二章 材料與方法 8 2.1 實驗材料 8 2.1.1 大腸桿菌 (Escherichia coli) 8 2.1.2 表現載體 8 2.2 大腸桿菌表現系統 9 2.2.1 轉型作用 (Transformation) 9 2.2.2 重組蛋白質誘導表現 9 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 10 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 (Western Blotting) 11 2.4 重組蛋白之純化 12 2.4.1 親和層析法 12 2.4.2 離子交換法 12 2.4.3 膠體過濾法 13 2.5 抗體製備 13 2.5.1 抗原製備 13 2.5.2 免疫流程 14 2.5.3 多株抗體製備 14 2.5.4 細胞培養 14 2.5.5 單株抗體製備 15 2.5.6 酵素連結免疫分析法 (ELISA) 15 2.5.7 限數稀釋 (單株化) 16 2.5.8 細胞冷凍 16 2.5.9 細胞解凍 16 2.5.10 純化單株抗體 16 2.5.11 分析抗體種類 (Antibody Isotyping) 17 2.5.12 免疫沉澱法 (Immunoprecipitation) 17 第三章 結果 19 3.1 H7N9 M1重組蛋白質之純化 19 3.2 H7N9 NS1重組蛋白質之純化 20 3.3 H7N9 NS2重組蛋白質之純化 21 3.4 H7N9 M1重組蛋白質之多株抗體效價測試 22 3.5 H7N9 NS1重組蛋白質之多株抗體效價測試 23 3.6 H7N9 NS2重組蛋白質之多株抗體效價測試 23 3.7 H7N9 NS1單株抗體篩選 23 3.8 H7N9 NS1單株抗體型態測試 25 3.9 H7N9 NS1單株抗體純化 25 3.10 H7N9 NS1單株抗體效價測試 27 3.11 H7N9 NS1蛋白質原態大小測定 27 3.12 H7N9 NS1單株抗體免疫沉澱測試 29 第四章 討論 30 4.1 H7N9 rM1, rNS1及rNS2純化條件探討 30 4.2 H7N9 rM1, rNS1及rNS2多株抗體效價探討 31 4.3 H7N9 rNS1單株抗體篩選探討 31 4.4 單株抗體純化之探討 32 4.5 單株抗體生化性質之探討 32 4.6 H7N9 NS1蛋白質原態大小測定之探討 33 參考文獻 34 圖與表 38 | |
| dc.language.iso | zh-TW | |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | 新型H7N9流感病毒 | zh_TW |
| dc.subject | 基質蛋白 | zh_TW |
| dc.subject | 非結構性蛋白 | zh_TW |
| dc.subject | Novel H7N9 influenza virus | en |
| dc.subject | Monoclonal antibodies | en |
| dc.subject | Nonstructural protein | en |
| dc.subject | Matrix protein | en |
| dc.title | 表現與純化新型H7N9流感病毒M1、NS1和NS2重組蛋白及製備鼠源單株抗體 | zh_TW |
| dc.title | Expression and Purification of Novel H7N9 Influenza Virus M1, NS1 and NS2 Recombinant Proteins for Generation of Mouse Monoclonal Antibodies | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林翰佳,張麗冠,陳慧文 | |
| dc.subject.keyword | 新型H7N9流感病毒,基質蛋白,非結構性蛋白,單株抗體, | zh_TW |
| dc.subject.keyword | Novel H7N9 influenza virus,Matrix protein,Nonstructural protein,Monoclonal antibodies, | en |
| dc.relation.page | 56 | |
| dc.identifier.doi | 10.6342/NTU201603563 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2017-01-25 | |
| 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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