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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張世宗 | |
dc.contributor.author | Yi-Hao Lin | en |
dc.contributor.author | 林一豪 | zh_TW |
dc.date.accessioned | 2021-07-11T14:38:15Z | - |
dc.date.available | 2022-08-29 | |
dc.date.copyright | 2017-08-29 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-07-27 | |
dc.identifier.citation | 1. Barik, S. (2012) New treatments for influenza. BMC Med 10, 104
2. Kilbourne, E. D. (2006) Influenza pandemics of the 20th century. Emerg Infect Dis 12, 9-14 3. Li, K. S., Guan, Y., Wang, J., Smith, G. J., Xu, K. M., Duan, L., Rahardjo, A. P., Puthavathana, P., Buranathai, C., Nguyen, T. D., Estoepangestie, A. T., Chaisingh, A., Auewarakul, P., Long, H. T., Hanh, N. T., Webby, R. J., Poon, L. L., Chen, H., Shortridge, K. F., Yuen, K. Y., Webster, R. G., and Peiris, J. S. (2004) Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430, 209-213 4. Bui, C. M., Gardner, L., MacIntyre, R., and Sarkar, S. (2017) Influenza A H5N1 and H7N9 in China: A spatial risk analysis. PLoS One 12, e0174980 5. Cordova-Villalobos, J. A., Macias, A. E., Hernandez-Avila, M., Dominguez-Cherit, G., Lopez-Gatell, H., Alpuche-Aranda, C., and Ponce de Leon-Rosales, S. (2017) The 2009 pandemic in Mexico: Experience and lessons regarding national preparedness policies for seasonal and epidemic influenza. Gac Med Mex 153, 102-110 6. Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M., Yang, H., Chen, X., Recuenco, S., Gomez, J., Chen, L. M., Johnson, A., Tao, Y., Dreyfus, C., Yu, W., McBride, R., Carney, P. J., Gilbert, A. T., Chang, J., Guo, Z., Davis, C. T., Paulson, J. C., Stevens, J., Rupprecht, C. E., Holmes, E. C., Wilson, I. A., and Donis, R. O. (2013) New world bats harbor diverse influenza A viruses. PLoS Pathog 9, e1003657 7. Taubenberger, J. K., and Kash, J. C. (2010) Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7, 440-451 8. Samji, T. (2009) Influenza A: understanding the viral life cycle. Yale J Biol Med 82, 153-159 9. Watanabe, T., Watanabe, S., Maher, E. A., Neumann, G., and Kawaoka, Y. (2014) Pandemic potential of avian influenza A (H7N9) viruses. Trends Microbiol 22, 623-631 10. Centers for Disease, C., and Prevention. (2013) Emergence of avian influenza A(H7N9) virus causing severe human illness - China, February-April 2013. MMWR Morb Mortal Wkly Rep 62, 366-371 11. Liu, D., Shi, W., Shi, Y., Wang, D., Xiao, H., Li, W., Bi, Y., Wu, Y., Li, X., Yan, J., Liu, W., Zhao, G., Yang, W., Wang, Y., Ma, J., Shu, Y., Lei, F., and Gao, G. F. (2013) Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 381, 1926-1932 12. Kageyama, T., Fujisaki, S., Takashita, E., Xu, H., Yamada, S., Uchida, Y., Neumann, G., Saito, T., Kawaoka, Y., and Tashiro, M. (2013) Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013. Euro Surveill 18, 20453 13. Lamb, R. A., Choppin, P. W., Chanock, R. M., and Lai, C. J. (1980) Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. Proc Natl Acad Sci U S A 77, 1857-1861 14. Lamb, R. A., and Choppin, P. W. (1979) Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proc Natl Acad Sci U S A 76, 4908-4912 15. Richardson, J. C., and Akkina, R. K. (1991) NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Arch Virol 116, 69-80 16. Ward, A. C., Castelli, L. A., Lucantoni, A. C., White, J. F., Azad, A. A., and Macreadie, I. G. (1995) Expression and analysis of the NS2 protein of influenza A virus. Arch Virol 140, 2067-2073 17. Yasuda, J., Nakada, S., Kato, A., Toyoda, T., and Ishihama, A. (1993) Molecular assembly of influenza virus: association of the NS2 protein with virion matrix. Virology 196, 249-255 18. Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Muller, C. W., Ruigrok, R. W., and Baudin, F. (2003) Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J 22, 4646-4655 19. Iwatsuki-Horimoto, K., Horimoto, T., Fujii, Y., and Kawaoka, Y. (2004) Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. J Virol 78, 10149-10155 20. Neumann, G., Hughes, M. T., and Kawaoka, Y. (2000) Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J 19, 6751-6758 21. Hutchinson, E. C., Denham, E. M., Thomas, B., Trudgian, D. C., Hester, S. S., Ridlova, G., York, A., Turrell, L., and Fodor, E. (2012) Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog 8, e1002993 22. Darapaneni, V., Prabhaker, V. K., and Kukol, A. (2009) Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins. J Gen Virol 90, 2124-2133 23. Pal, S., Santos, A., Rosas, J. M., Ortiz-Guzman, J., and Rosas-Acosta, G. (2011) Influenza A virus interacts extensively with the cellular SUMOylation system during infection. Virus Res 158, 12-27 24. Paterson, D., and Fodor, E. (2012) Emerging roles for the influenza A virus nuclear export protein (NEP). PLoS Pathog 8, e1003019 25. O'Neill, R. E., Talon, J., and Palese, P. (1998) The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288-296 26. Nguyen, K. T., Holloway, M. P., and Altura, R. A. (2012) The CRM1 nuclear export protein in normal development and disease. Int J Biochem Mol Biol 3, 137-151 27. Ma, K., Roy, A. M., and Whittaker, G. R. (2001) Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclear periphery. Virology 282, 215-220 28. Elton, D., Simpson-Holley, M., Archer, K., Medcalf, L., Hallam, R., McCauley, J., and Digard, P. (2001) Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway. J Virol 75, 408-419 29. Dong, X., Biswas, A., Suel, K. E., Jackson, L. K., Martinez, R., Gu, H., and Chook, Y. M. (2009) Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 458, 1136-1141 30. Baudin, F., Petit, I., Weissenhorn, W., and Ruigrok, R. W. (2001) In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281, 102-108 31. Gorai, T., Goto, H., Noda, T., Watanabe, T., Kozuka-Hata, H., Oyama, M., Takano, R., Neumann, G., Watanabe, S., and Kawaoka, Y. (2012) F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding. Proc Natl Acad Sci U S A 109, 4615-4620 32. 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. J Gen Virol 90, 1398-1407 33. Nayak, D. P., Balogun, R. A., Yamada, H., Zhou, Z. H., and Barman, S. (2009) Influenza virus morphogenesis and budding. Virus Res 143, 147-161 34. Rossman, J. S., and Lamb, R. A. (2011) Influenza virus assembly and budding. Virology 411, 229-236 35. Shtykova, E. V., Baratova, L. A., Fedorova, N. V., Radyukhin, V. A., Ksenofontov, A. L., Volkov, V. V., Shishkov, A. V., Dolgov, A. A., Shilova, L. A., Batishchev, O. V., Jeffries, C. M., and Svergun, D. I. (2013) Structural analysis of influenza A virus matrix protein M1 and its self-assemblies at low pH. PLoS One 8, e82431 36. Arzt, S., Petit, I., Burmeister, W. P., Ruigrok, R. W., and Baudin, F. (2004) Structure of a knockout mutant of influenza virus M1 protein that has altered activities in membrane binding, oligomerisation and binding to NEP (NS2). Virus Res 99, 115-119 37. 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 38. Boulo, S., Akarsu, H., Ruigrok, R. W., and Baudin, F. (2007) Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124, 12-21 39. Radiukhin, V. A. (2009) The concept of transmembrane asymmetry of lateral domains in biomemranes and influenza virus envelope fine structure. Mol Biol (Mosk) 43, 579-589 40. Lee, K. K. (2010) Architecture of a nascent viral fusion pore. EMBO J 29, 1299-1311 41. Martin, K., and Helenius, A. (1991) Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol 65, 232-244 42. Bui, M., Whittaker, G., and Helenius, A. (1996) Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol 70, 8391-8401 43. Hu, Y., Liu, X., Zhang, A., Zhou, H., Liu, Z., Chen, H., and Jin, M. (2015) CHD3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2. Cell Mol Life Sci 75, 971-982 44. Gao, S., Wu, J., Liu, R. Y., Li, J., Song, L., Teng, Y., Sheng, C., Liu, D., Yao, C., Chen, H., Jiang, W., Chen, S., and Huang, W. (2015) Interaction of NS2 with AIMP2 facilitates the switch from ubiquitination to SUMOylation of M1 in influenza A virus-infected cells. J Virol 89, 300-311 45. Taylor, R. G., Walker, D. C., and McInnes, R. R. (1993) E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res 21, 1677-1678 46. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254 47. Georgiou, C. D., Grintzalis, K., Zervoudakis, G., and Papapostolou, I. (2008) Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins. Anal Bioanal Chem 391, 391-403 48. Sato, A. K., Zarutskie, J. A., Rushe, M. M., Lomakin, A., Natarajan, S. K., Sadegh-Nasseri, S., Benedek, G. B., and Stern, L. J. (2000) Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1. J Biol Chem 275, 2165-2173 49. Cerpa, R., Cohen, F. E., and Kuntz, I. D. (1996) Conformational switching in designed peptides: the helix/sheet transition. Fold Des 1, 91-101 50. Wallace, B. A., and Janes, R. W. (2003) Circular dichroism and synchrotron radiation circular dichroism spectroscopy: tools for drug discovery. Biochem Soc Trans 31, 631-633 51. Kuroda, M., Kohira, Y., and Sasaki, M. (1994) Conformational change of skeletal muscle alpha-actinin induced by salt. Biochim Biophys Acta 1205, 97-104 52. Arzt, S., Baudin, F., Barge, A., Timmins, P., Burmeister, W. P., and Ruigrok, R. W. (2001) Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer. Virology 279, 439-446 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77956 | - |
dc.description.abstract | 研究發現禽流感病毒的NEP (nuclear export protein) 扮演許多重要生理角色,包括運輸、調控病毒基因以及幫助病毒離開宿主細胞。NEP透過與M1交互作用幫助複製完成病毒基因運送出宿主細胞核以利病毒組裝。然而目前尚未有研究探討pH改變對於NEP與M1交互作用的影響。本研究在進行膠體過濾實驗時,意外發現H7N9病毒之NEP會隨著使用不同pH值之緩衝液,而使其流洗出的位置不同,因此環境pH的改變似乎能使NEP之分子大小或構形隨之改變。此外,當NaCl濃度改變時,pH對於NEP流洗體積影響趨勢仍相同,但是隨著鹽濃度提升,pH改變對於NEP影響漸弱。由於已知NEP能夠透過與M1交互作用幫助複製完成的vRNP運送離開宿主細胞核,以利病毒組裝,因此推測若pH造成NEP的構形改變或許會影響NEP與M1交互作用力改變,造成生理功能上的影響。然而在pH 7至pH 9的實驗條件下,經過crosslinking以及CD光譜分析,發現NEP聚體之產生或其二級結構並未受到此pH改變而影響;反之H7N9病毒之基質蛋白M1卻會隨著pH升高而產生較多的多聚體。透過pulldown實驗,的確發現M1與NEP之結合現象隨著pH值提升而減少。由於M1-M1間交互作用的區域與M1-NEP交互作用的位置有所重疊,因此當pH提升後,M1傾向形成多聚體,而阻擋了M1與NEP之結合,進而使M1與NEP形成複合體之現象減少。然而此推論尚需更充足的實驗證據佐證。 | zh_TW |
dc.description.abstract | Studies showed that NEP has many important biological functions, including transportation and regulation of viral genome and virus budding from host cells. However, the effect of pH change on the interaction between M1 and NEP is unknown.
In this study, I unexpectedly found that the retention volume of NEP would change while performing gel filtration in different pH conditions. In addition, the elution volume of NEP would also change by using buffers with different NaCl concentration at the same pH. It seemed that change of pH might alter the molecular size or conformation of NEP. Besides, it was known that M1-NEP interaction would help export replicated vRNP from nucleus for assembly of viral particles. Hence, it was hypothesized that if the conformation of NEP would change at different pH, it might interrupt M1-NEP interaction and affect its biological function. However, the results of crosslinking and CD experiment showed that the polymerization and secondary structure of NEP were not different between pH 7 and pH 9. In contrast, the matrix protein M1 of H7N9 tended to undergo polymerization as pH increases. Moreover, the interaction between M1 and NEP decreased as pH increases according to the result of pulldown assay. Previous studies have shown that the regions of M1 for interacting with M1 and NEP were overlapped for some extent. Therefore, the formation of the M1-NEP complex was reduced due to the polymerization of M1 and blocking the interaction site between M1 and NEP upon pH change. More investigation should be conducted for elucidating this assumption. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:38:15Z (GMT). No. of bitstreams: 1 ntu-106-R04b22021-1.pdf: 1943447 bytes, checksum: 35ed53036f5aa49bb0946413c7da3fd9 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 摘要 I
Abstract II 縮寫表 III 第一章 前言 1 1.1 流行性感冒 1 1.2 A型病毒流感及其生活史 2 1.3 新型H7N9流感病毒 3 1.4 H7N9病毒親緣分析 4 1.5 核輸出蛋白 (Nuclear export protein, NEP) 4 1.5.1 NEP基因表現與命名 4 1.5.2 NEP的結構 4 1.5.3 NEP運輸vRNP的功能 5 1.5.4 NEP其它生理功能 6 1.6 基質蛋白1 (Matrix protein 1, M1) 6 1.6.1 M1基因表現及其結構 6 1.6.2 M1功能 6 1.7 研究動機 7 第二章 材料與方法 9 2.1 新型H7N9流感病毒之M1與NEP基因 9 2.2 大腸桿菌 (Escherichia coli) 9 2.3 質體 9 2.4 大腸桿菌表現系統 10 2.4.1 質體轉型作用 10 2.4.2 重組蛋白質表現 10 2.5 重組蛋白質純化 10 2.5.1 His-M1以及His-NEP重組蛋白純化 11 2.5.2 GST-NEP重組蛋白純化 11 2.6 蛋白質定量 12 2.7 蛋白質膠體電泳 12 2.8 西方墨點法 (Western blotting) 12 2.9 膠體過濾 13 2.10 圓二色光譜 13 2.11 Pulldown assay 14 2.12 Crosslinking 14 第三章 結果 16 3.1 重組蛋白His-M1、His-NEP及GST-NEP之純化 16 3.2 不同pH環境對NEP結構之影響探討 16 3.2.1 膠體過濾法分析NEP構型改變 16 3.2.2 圓二色光譜分析NEP構型改變 17 3.3 pH改變影響NEP與M1之間的交互作用 18 3.4 pH改變影響NEP與M1多聚體形成 18 第四章 討論 21 參考文獻 24 圖與表 29 圖一 以HisTrap管柱純化帶有His-tag的H7N9 NEP 29 圖二 以HisTrap管柱純化帶有His-tag的H7N9 M1 30 圖三 以GSTrap管柱純化帶有GST-tag的H7N9 NEP 31 圖四 His-NEP進行膠體過濾 32 圖五 NEP進行crosslinking探討不同pH下多聚體的產生 33 圖六 His-NEP於不同pH緩衝液進行膠體過濾 34 圖七 His-NEP於不同pH緩衝液進行圓二色光譜分析 35 圖八 以pulldown assay確認GST-NEP與His-M1間的交互作用 36 圖九 pulldown assay探討不同pH是否影響GST-NEP與His-M1之間的交互作用 37 圖十 NEP以及M1在不同pH條件下進行crosslinking,探討兩者多聚體的產生 38 圖十一 探討飽和濃度下His-NEP聚體的產生 39 附錄 40 附錄一 NEP胺基酸序列以及結構示意圖 40 附錄二 M1胺基酸序列以及結構示意圖 41 | |
dc.language.iso | zh-TW | |
dc.title | 探討pH改變對於新型H7N9流感病毒NEP與M1蛋白質交互作用之影響 | zh_TW |
dc.title | Investigation of the Interaction between the Novel Avian Influenza A (H7N9) Virus NEP and M1 Proteins Affected by pH change | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 廖憶純,陳慧文,鄭貽生 | |
dc.subject.keyword | 流感病毒,基質蛋白1,核輸出蛋白, | zh_TW |
dc.subject.keyword | H7N9,M1,NEP, | en |
dc.relation.page | 41 | |
dc.identifier.doi | 10.6342/NTU201702069 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-07-27 | |
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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