臺灣博碩士論文加值系統

家禽流行性感冒病毒（avian influenza virus；AIV）為正黏液病毒科（Orthomyxoviridae）之A型流行性感冒病毒。高病原性家禽流行性感冒（highly pathogenic avian influenza, HPAI）俗稱雞瘟，對雞或火雞造成高度傳染及急速死亡。以酵母菌（Pichia pastoris）蛋白質表現系統表現禽流感H5N2 HA膜蛋白，經SDS-PAGE及西方墨點法分析後，在胞內及胞外皆有HA融合蛋白之存在。在酵母菌表現方面，HA蛋白似被切割為HA 1（47 kDa）及HA 2（22 kDa）。1209病毒可誘導MDCK發生凋亡之現象。為進ㄧ步探討家禽流行性感冒病毒誘發凋亡之分子機制，構築禽流感病毒H5N2 CE9毒株NA膜蛋白表現質體，短暫轉染至細胞株內以探討NA是否會藉導禽流感病毒所造成之細胞凋亡，結果顯示在相同視野下，表現GFP-NA融合蛋白之MDCK細胞，並無capsase 3活化的情形發生。完成定序之HA及NA基因與NCBI基因庫其他H5N2 HA及NA基因以MEGA 3.1軟體進行親源關係分系。結果發現就HA基因來說，台灣1209株在親源關係上與義大利株分為同一類；在NA基因親源關係分析上，台灣之1209株與CE9株同樣與義大利株分為同一類。

Avian influenza virus（AIV）belongs to orthomyxoviridae family. Highly pathogenetic avian influenza virus causes flowl plague in chickens and turkeys. The HA protein of AIV 1209 strain was expressed by Pichia pastoris yeast expression system. The results indicated that the tagged fusion protein was present both in medium and pellet. The HA protein could be cleaved into HA1（47 kDa） and HA2（22 kDa）. The AIV CE9 could induce apoptosis in MDCK cell. To further study the molecular mechanism of AIV-induced apoptosis, the NA protein of AIV H5N2 CE9 strain was cloned into pcDNA3.1(-), and the recombinant DNA transfected into MDCK cell lines. The results indicated that at the same focus, caspase 3 were not detected in the MDCK cell expressing the GFP-NA fusion protein. Phylogenesis analysis of HA and NA gene of strains Taiwanese 1209 and CE9 was done to sequence with other H5N2 viruses in the world using MEGA 3.1 software. The result indicate the 1209 and CE9 strain belong to the same group as the Italy strain.

摘要 I Abstrate Ⅲ 誌謝 Ⅳ
目錄 Ⅴ 圖表目錄 Ⅸ 第1章 緒言 1 第2章 文獻探討 2 2.1 家禽流行性感冒病毒歷史背景與流行現況 2 2.2 家禽流行性感冒病毒之特性 5
2.2.1 ㄧ般特性 5
2.2.2 物理特性 7
2.2.3 病毒蛋白特性 7
2.3 酵母菌（Pichia pastoris）表現系統 8 2.4 細胞凋亡（apoptosis） 8
2.4.1 細胞凋亡的簡介 8
2.4.2 細胞凋亡與細胞壞死 9
2.4.3 細胞凋亡之分析方法 9
2.4.4 細胞凋亡之調控 11
2.4.4.1 細胞凋亡之外在調控路徑………………………………. 11
2.4.4.2 細胞凋亡之內在調控路徑 11
2.4.5 病毒與細胞凋亡 12
2.5 演化分析方法 12
第3章 材料與方法 15
3.1 供試細胞 15
3.1.1 BHK-21及MDCK細胞株 15 3.1.2 細胞冷凍 15
3.1.3 細胞解凍 15
3.2 AIV NA及HA基因構築 16
3.2.1 病毒核酸之萃取 16
3.2.2 引子（primers）設計 16
3.2.3 反轉錄作用及聚合酶連鎖反應 16
3.2.4 PCR產物鑑定 17
3.2.5 PCR產物純化 17
3.2.6 勝任細胞之製備 17
3.2.7 選殖載體之製備 17
3.2.8 PCR產物之分子選殖 18
3.2.9 轉形作用 18
3.2.10 重組DNA之抽取與鑑定 18
3.3 重組基因之蛋白質表現 19
3.3.1 勝任酵母菌之製備 19
3.3.2 電穿孔之轉形作用 19
3.3.3 以高濃度Zeocin抗生素篩選高套數轉殖菌株 20
3.3.4 重組酵母菌之染色體DNA萃取及PCR篩選 20
3.3.5 酵母菌選殖菌株培養與甲醇誘導重組蛋白之分析 20
3.3.6 酵母菌選殖株細胞內外蛋白質萃取 21
3.3.6.1 酵母菌選殖株細胞內蛋白質萃取 21
3.3.6.2 酵母菌選殖株細胞外蛋白質萃取 21
3.4 細胞凋亡之研究……………………………………………………21

3.4.1 DNA fragmentation assay 21
3.4.2 細胞核染色（nuclear staining） 22
3.4.3 細胞轉染 22
3.4.4 細胞內活化態caspases 3、8及9之螢光染色 23
3.4.5 細胞蛋白質萃取及蛋白質之定量 23
3.4.6 蛋白質電永及冷光壓片西方轉漬分析 24
第4章 結果 25
4.1 AIV HA基因選殖及蛋白質表現 25
4.1.1 HA基因選殖 25
4.1.2 核酸定序 25
4.1.3 HA重組酵母菌胞內及胞外蛋白質表現 25 4.2 細胞凋亡之研究 26
4.2.1 DNA fragmentation assay 26
4.2.2 螢光染色分析活化態之caspase 3、8、9 26
4.2.3 細胞核染色分析 26
4.2.4 AIV神經醯胺酵素（neuraminidase；NA）誘發細胞凋亡 27
4.2.4.1 AIV NA-GFP融合蛋白之基因選殖 27
4.2.4.2 短暫轉染AIV NA-GFP融合蛋白於細胞株內之表現 27
4.2.4.3 AIV GFP-NA融合蛋白之基因選殖 27
4.2.4.4 短暫轉染AIV GFP-NA融合蛋白於細胞株內之表現
及螢光染色分析活化態之caspase 3 28
4.3 HA及NA基因親源關係分析 28
4.4 CE9 NA基因核苷酸及胺基酸序列分析 28
第5章 討論 44
參考文獻 48
附錄1. 比較家禽流行性感冒病毒分離株NA基因核酸序列 56
附錄2. 比較家禽流行性感冒病毒分離株NA胺基酸序列 71
作者簡介 76

Alexander, D. J.（1982）Ecological aspects of influenza viruses in animal and their relationship to influenza: A review. Journal of the Royal Society of Medicine 75: 799-811.
Alexander, D. J.（2000）A review of influenza in different bird species. Vet. Microbiol. 74: 3-13.
Alexander, D.J.（2001）Ecology of avian influenza in domestic birds. In B. Dodet and M. Vicari (Eds.) Proceedings of the International Symposium on Emergence and Control of Zoonotic Ortho- and Paramyxovirus Diseases. Merieux Foundation, Veyrierdu-lac, France.
Allan, W. H.（1981）Diagnostic procedures-response. Proc. 1st. Int. Symp. Avian Influenza. USA.
Ashkenazi, A., and V. M. Dixit（1998）Death receptors: singaling and mokulation. Science 281: 1305-1308.
Attenson, K.（1997）The performance of neighbor-joining algorithms of phylogeny reconstruction, p. 101-110. In Jiang, T., and Lee, D., eds., Lecture Notes in Computer Science, 1276, Springer-Verlag, Berlin. COCOON’97.
Bosh, F. X., M. Orlich, H. D. Klenk, and R. Rott（1979）The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. Virology 95: 197-207.
Capua, I., and D. J. Alexander（2004）Avian influenza: recent developments. Avian pathology 33: 393-404.
Capua, I., and S. Marangon（2000）Avian influenza in Italy (1999-2000): a review. Avian Pathology 29: 289-294.
Capua, I., C. Terregino, G. Cattoli, and A. Toffan（2004）Increased resistance of vaccinated turkeys to experimental infection with an H7N3 low-pathogenicity avian influenza virus. Avian Pathology 33: 158-163.
Capua, I., C. Terregino, G. Cattoli, F. Mutinelli, and J. F. Rodriguez（2003）Development of a DIVA-Differentiating infected from vaccinated animals - strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza. Avian Pathology 32: 47-5.Capua, I., and F. Mutinelli（2001）A Colour Atlas and Text on Avian Influenza . Casalecchio di Reno, Italy.
Capua, I., S. Marangon, L. Selli, D. J. Alexander, D. E. Swayne, M. Dalla Pozza, E. Parenti, and F. M. Cancellotti（1999）Outbreaks of highly pathogenic avian influenza (H5N2) in Italy during October 1997-January 1998. Avian Pathology 28: 455-460.
Campbell, G., and H. De Geus（1999）Non-pathogenic avian influenza in Ireland in 1998. In Proceedings of the Joint Fifth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union. Vienna, Austria.
Cregg, J. M., K. J. Barringer, A. Y. Hessler, and K. R. Madden（1985）Pichia pastoris as a host system for transformations. Molecular Cell Biology 5: 3376-3385.
Cregg, J. M., K. R. Madden, K. J. Barringer, G. Thill, and C. A. Stillman（1989）. Functional characterization of the two alcohol oxidase genes from the yeast, Pichia pastoris. Molecular Cell Biology 9: 1316-1323.
Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami, and F. Traganos（1997）Cytometry in cell necrobiology：analysis of apoptosis and accidental cell death ( necrosis ). Cytometry 27: 1-20.
Davison, S., R. J. Eckroade, and A. F. Ziegler（2003）A Review of the 1997-1998 Nonpathogenic H7N2 Avian Influenza Outbreak in Pennsylvania. In: Proceedings of the Fifth International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 823-827.
Eick, D., and H. Hermeking（1996）Viruses as pacemakers in the evolution of defence mechanisms against cacer. Trends in Genetics 12: 4-6.
Felsenstein, J.（1978）Cases in which parsimony and compatibility methods will be positively misleading. Syst. Zool 27: 401-410.
Fioretti, A., M. Calabria, A. Piccirillo, and L. F. Menna（1999）The epidemiological situation of avian influenza in Italy during 1998. In Proceedings of the Joint Fifth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union. Vienna, Austria.
Glod, R., M. Schmied, G. Giegerich, H. Breitschopf, H. P. Hartung, K. V. Toyka, and H. Lassmann（1994）Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Laboratory Investigation 71: 219-225.
Graham, D., S. McCullough, and T. Connor（1999）Avian influenza in Northern Ireland: Current situation. In Proceedings of the Joint Fifth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union. Vienna, Austria.
Green, D. R., and J. C. Reed（1998）Mitochondria and apoptosis. Science 281: 1309-1312.
Hay, H., and G. Kannourakis（2002）A time to kill: viral manipulation of the cell death program. Journal of General Virology 83: 1547-1564.
Henzler, D. J., D. C. Kradel, S. Davison, A. F. Ziegler, D. Singletary, P. DeBok, A. E. Castro, H. Lu, R. Eckroade, D. Swayne, W. Lagoda, and B. Schmucker（2003）Epidemiology, production losses and control measures associated with an outbreak of avian influenza subtype H7N2 in Pennsylvania (1996-1998) In: Proceedings of the Fifth International Symposium on Avian Influenza Athens, Georgia, April 14-17 2002. Avian Diseases 47: 1022-1036.
Kerr, J. F.（1995）Neglected opportunities in apoptosis research. Trends in Cell Biology 5: 55-57.
Kerr, J. F., A. H. Wyllie, and A. R. Currie（1972）Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239-257.
Kiechli, F. L., and X. Zhang（2002）Apoptosis: biochemical aspects and clinical implications. Clinica Chimica Acta 326: 27-45.
Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, and X. Wang（1997）Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479-489.
Lowe, S. W., and H. E. Ruley（1993）Stabilization of the p53 tumor srppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes and Development 7: 535-545.
Menno, D. de Jong, and T. H. Tran（2006）Avian influenza A (H5N1), Journal of Clinical Virology 35: 2-13.
Meulemans, G., M. Boschmans, M. Deccaesstecker, and T. Van Den Berg（2000）Isolation of a H5N2 non-pathogenic avian influenza virus in Belgium in 1999. In Proceedings of the Joint Sixth Annual Meetings of the National Newcastle Disease and Avian Influenza Laboratories of Countries of the European Union. Brussels, Belgium.
Naeem, K.（1998）The avian influenza outbreak in South Central Asia. In Proceedings of the 4th International Symposium on Avian Influenza. Athens, GA, USA.
Nicholson, D. W., A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, and Y. A. Lazebnik（1995）Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43.
O’Brien, V.（1998）Viruses and apoptosis. Journal of General Virology 79: 1833-1845.
Ogata, K., H. Nishikawa, and M. Ohsugi（1969）A yeast capable of utilizing methanol. Agriculture Biology Chemistry 33: 1519-1520.
Oldstone, M. B. A.（1997）How virus excape from cytotoxic T lyphocytes: molecular parameters and players. Virology 234: 179-185.
Pasick, J., H. Weingartl, A. Clavijo, J. Riva, H. Kehler, E. Watkins, and K. Hills（2003）Characterisation of avian influenza virus isolates submitted to the National Centre for Foreign Animal Disease between 1997 and 2001. In: Proceedings of the Fifth International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 1208-1213.
Rojas, H., R. Moreira, P. Avalos, I. Capua, and S. Marangon（2002）Avian influenza in poultry in Chile. Veterinary Record , 151, 188. Rott, R. (1992). The pathogenic determinant of influenza virus. Veterinary Microbiology 33: 303-310.
Roulston, A., R. C. Marcellus, and P. E. Branton（1999）Viruses and apoptosis. Annual review of Microbiology 53: 577-628.
Saitou, N., and M. Nei（1987）The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Evolution Biology 4: 406-425.
Schwartzman, R. A., and J. A. Cidlowski（1993）Apoptosis：the biochemistry and molecular biology of programmed cell death (Review). Endocrinology 14: 133-151.
Selleck, P. W., P. D. Kirkland, A. R. Gould, G. Arzey, R. L. Reece, P. W. Daniels, and H. A. Westury（2003）An outbreak of HPAI in Australia in 1997 caused by an H7N4 virus. In: Proceedings of the Fifth International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 806-811.
Senne, D. A.（1998）Hemagglutinin cleavage site sequences of avain influenza viruses isolated between 1992 and 1997. In Proceedings of the 4th International Symposium on Avian Influenza. Athens, GA, USA.
Senne, D.A.（2003）Avian influenza in the Western hemisphere including the Pacific Islands and Australia. Proceedings of the 5th International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 798-805.
Shortridge, K. F.（1999）Poultry and the influenza H5N1 outbreak in Hong Kong, 1997: Abridged chronology and virus isolation. Vaccine 17: 26-29.
Sims, L. D., T. M. Ellis, K. K. Liu, K. Dyrting, H. Wong, M. Peiris, Y. Guan, and K. F. Shortridge（2003）Avian influenza in Hong Kong. In: Proceedings of the Fifth International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 832-838.
Skehel, J. J., and D. C. Wiley（2000）Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annual review of biochemistry 69: 531-569.
Spackman, E., and D. L. Suarez（2003）Evaluation of molecular markers for pathogenicity in recent H7N2 avian influenza isolates from the northeastern United States. In Proceedings of the 52nd WPDC. Sacramento, CA, USA.
Suarez, D. L., D. A. Senne, J. Banks, I. H. Brown, S. C. Essen, C. W. Lee, R. J. Manvell, C. Mathieu-Benson, J. Pedersen, B. Panigrahy, E. Spackman, and D. J. Alexander（2003）A shift in virulence from low pathogenic to highly pathogenic avian influenza for the H7N3 virus responsible for outbreaks of disease in poultry in Chile appears to be the result of intergenic recombination between the haemagglutinin and NP genes. In Abstracts of the XII International Conference on Negative Strand RNA Viruses. Pisa, Italy.
Sumantran, V. N., M. W. Ealovega, G. Nunez, M. F. Clarke, and M. S. Wicha（1995）Overexpression of Bcl-XS sensitizes MCF-7 cells to chemotherapy -induced apoptosis. Cancer Research 55: 2507-2510.
Swayne, D. E., and D. L. Suarez（2001）Avian influenza in Europe, Asia and Central America during 2001. In Proceedings of the 104th annual meeting of the US Animal Health Association. Richmond, VA, GA.
Swayne, D. E., J. R. Beck, M. Garcia, and H. D. Stone（1999）Influence of virus strain and antigen mass on the efficacy of H5 avian influenza inactivated vaccines. Avian Pathology 28: 245-255.
Symonds, H., L. Krall, L. Remington, M. Saenzrobles, S. Lowe, T. Jacks, and T. Van Dyke（1994）P53-dependent apoptosis suppresses human growth and progression in vivo. Cell 78: 703-711.
Telford, W. G., L. E. King, and P. J. Fraker（1992）Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13: 137-143.
Teodoro, J. G., and P. E. Branton（1997）Regulation of apoptosis by viral gene products. Journal of Virology 71: 1739-1746.
Vermes, I., and C. Haanen（1994）Apoptosis and programmed cell death in health and disease. Advances Clinical Chemicalology 31: 177-246.
Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger（1995）A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. Journal of Immunology Methods 184: 39-51.
Villareal-Chavez, C., and E. Rivera-Cruz（2003）An update on avian influenza in Mexico. In: Proceedings of the 5th International Symposium on Avian Influenza. Georgia Center for Continuing Education, The University of Georgia, Athens, Georgia, USA, April 14-17 2002. Avian Diseases 47: 1002-1005.
Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka（1992）Evolution and ecology of influenza A viruses. Microbiology Review. 56: 152-178.
Werner, O., E. Starick, and C. H. Grund（2003）Isolation and characterization of a low pathogenic H7N7 influenza virus from a turkey in a small mixed free-range poultry flock in Germany. In: Proceeding of the Fifth International Symposium on Avian Influenza, Athens, Georgia, April 14-17 2002. Avian Diseases 47: 1104-1106.
Westbury, H. A.（1998）History of high pathogenic avian influenza in Australia and the H7N3 outbreak. In Proceedings of the 4th International Symposium Avian Influenza. Athens, GA, USA.
White, E.（1994）. Tumor biology p53 guardian of Rb. Nature 371: 21-22.
Wyllie, A. H.（1980）Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555-556.