請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77290完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | zh_TW |
| dc.contributor.author | 鄭淯宸 | zh_TW |
| dc.contributor.author | Yu-Chen Cheng | en |
| dc.date.accessioned | 2021-07-10T21:54:25Z | - |
| dc.date.available | 2024-08-13 | - |
| dc.date.copyright | 2019-08-14 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1. Ehrhardt, C., Seyer, R., Hrincius, E. R., Eierhoff, T., Wolff, T., and Ludwig, S. (2010) Interplay between influenza A virus and the innate immune signaling. Microbes Infect 12, 81-87
2. Mair, C. M., Ludwig, K., Herrmann, A., and Sieben, C. (2014) Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection. Biochimica et Biophysica Acta (BBA) - Biomembranes 1838, 1153-1168 3. Steinhauer, D. A. (1999) Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258, 1-20 4. Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A., and Klenk, H. D. (2004) Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A 101, 4620-4624 5. Shu, Y., and McCauley, J. (2017) GISAID: Global initiative on sharing all influenza data - from vision to reality. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 22, 30494 6. Zhang, Y., Sun, Y., Sun, H., Pu, J., Bi, Y., Shi, Y., Lu, X., Li, J., Zhu, Q., Gao, G. F., Yang, H., and Liu, J. (2012) A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the Pathogenicity and Neurovirulence of H5N1 Influenza Virus in Mice. Journal of Virology 86, 6924 7. Sturm-Ramirez, K. M., Hulse-Post, D. J., Govorkova, E. A., Humberd, J., Seiler, P., Puthavathana, P., Buranathai, C., Nguyen, T. D., Chaisingh, A., Long, H. T., Naipospos, T. S. P., Chen, H., Ellis, T. M., Guan, Y., Peiris, J. S. M., and Webster, R. G. (2005) Are Ducks Contributing to the Endemicity of Highly Pathogenic H5N1 Influenza Virus in Asia? Journal of Virology 79, 11269 8. Wan, X. F. (2012) Lessons from emergence of A/goose/Guangdong/1996-like H5N1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern China. Zoonoses and public health 59 Suppl 2, 32-42 9. Bender, C., Hall, H., Huang, J., Klimov, A., Cox, N., Hay, A., Gregory, V., Cameron, K., Lim, W., and Subbarao, K. (1999) Characterization of the Surface Proteins of Influenza A (H5N1) Viruses Isolated from Humans in 1997–1998. Virology 254, 115-123 10. Li, K. S., Guan, Y., Wang, J., Smith, G. J. D., Xu, K. M., Duan, L., Rahardjo, A. P., Puthavathana, P., Buranathai, C., Nguyen, T. D., Estoepangestie, A. T. S., Chaisingh, A., Auewarakul, P., Long, H. T., Hanh, N. T. H., Webby, R. J., Poon, L. L. M., Chen, H., Shortridge, K. F., Yuen, K. Y., Webster, R. G., and Peiris, J. S. M. (2004) Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430, 209-213 11. Wong, S. S. Y., and Yuen, K.-y. (2006) Avian Influenza Virus Infections in Humans. Chest 129, 156-168 12. Guan, Y., and Smith, G. J. D. (2013) The emergence and diversification of panzootic H5N1 influenza viruses. Virus research 178, 35-43 13. Spackman, E., Pantin-Jackwood, M. J., Kapczynski, D. R., Swayne, D. E., and Suarez, D. L. (2016) H5N2 Highly Pathogenic Avian Influenza Viruses from the US 2014-2015 outbreak have an unusually long pre-clinical period in turkeys. BMC veterinary research 12, 260-260 14. Rajesh Kumar, S., Chelvaretnam, S., Tan, Y., and Prabakaran, M. (2017) Broadening the H5N3 Vaccine Immunogenicity against H5N1 Virus by Modification of Neutralizing Epitopes. Viruses 10, 2 15. Bi, Y., Chen, Q., Wang, Q., Chen, J., Jin, T., Wong, G., Quan, C., Liu, J., Wu, J., Yin, R., Zhao, L., Li, M., Ding, Z., Zou, R., Xu, W., Li, H., Wang, H., Tian, K., Fu, G., Huang, Y., Shestopalov, A., Li, S., Xu, B., Yu, H., Luo, T., Lu, L., Xu, X., Luo, Y., Liu, Y., Shi, W., Liu, D., and Gao, G. F. (2016) Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China. Cell Host Microbe 20, 810-821 16. Nagarajan, S., Kumar, M., Murugkar, H. V., Tripathi, S., Shukla, S., Agarwal, S., Dubey, G., Nagi, R. S., Singh, V. P., and Tosh, C. (2017) Novel Reassortant Highly Pathogenic Avian Influenza (H5N8) Virus in Zoos, India. Emerging infectious diseases 23, 717-719 17. Lee, D.-H., Bahl, J., Torchetti, M. K., Killian, M. L., Ip, H. S., DeLiberto, T. J., and Swayne, D. E. (2016) Highly Pathogenic Avian Influenza Viruses and Generation of Novel Reassortants, United States, 2014-2015. Emerging infectious diseases 22, 1283-1285 18. Lee, M.-S., Chen, L.-H., Chen, Y.-P., Liu, Y.-P., Li, W.-C., Lin, Y.-L., and Lee, F. (2016) Highly pathogenic avian influenza viruses H5N2, H5N3, and H5N8 in Taiwan in 2015. Veterinary Microbiology 187, 50-57 19. Li, M., Liu, H., Bi, Y., Sun, J., Wong, G., Liu, D., Li, L., Liu, J., Chen, Q., Wang, H., He, Y., Shi, W., Gao, G. F., and Chen, J. (2017) Highly Pathogenic Avian Influenza A(H5N8) Virus in Wild Migratory Birds, Qinghai Lake, China. Emerging infectious diseases 23, 637-641 20. Selim, A. A., Erfan, A. M., Hagag, N., Zanaty, A., Samir, A.-H., Samy, M., Abdelhalim, A., Arafa, A.-S. A., Soliman, M. A., Shaheen, M., Ibraheem, E. M., Mahrous, I., Hassan, M. K., and Naguib, M. M. (2017) Highly Pathogenic Avian Influenza Virus (H5N8) Clade 2.3.4.4 Infection in Migratory Birds, Egypt. Emerging infectious diseases 23, 1048-1051 21. Ghafouri, S. A., GhalyanchiLangeroudi, A., Maghsoudloo, H., Kh Farahani, R., Abdollahi, H., Tehrani, F., and Fallah, M. H. (2017) Clade 2.3.4.4 avian influenza A (H5N8) outbreak in commercial poultry, Iran, 2016: the first report and update data. Tropical Animal Health and Production 49, 1089-1093 22. Pohlmann, A., Starick, E., Harder, T., Grund, C., Höper, D., Globig, A., Staubach, C., Dietze, K., Strebelow, G., Ulrich, R. G., Schinköthe, J., Teifke, J. P., Conraths, F. J., Mettenleiter, T. C., and Beer, M. (2017) Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016. Emerging infectious diseases 23, 633-636 23. Olsen, B., Munster, V. J., Wallensten, A., Waldenström, J., Osterhaus, A. D. M. E., and Fouchier, R. A. M. (2006) Global Patterns of Influenza A Virus in Wild Birds. Science 312, 384 24. Ganzenmueller, T., Kluba, J., Hilfrich, B., Puppe, W., Verhagen, W., Heim, A., Schulz, T., and Henke-Gendo, C. (2010) Comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of RT-PCR for the detection of novel 2009 influenza A (H1N1) virus in respiratory specimens. 59, 713-717 25. Leonardi, G. P., Mitrache, I., Pigal, A., and Freedman, L. (2010) Public hospital-based laboratory experience during an outbreak of pandemic influenza A (H1N1) virus infections. J Clin Microbiol 48, 1189-1194 26. Ginocchio, C. C., Zhang, F., Manji, R., Arora, S., Bornfreund, M., Falk, L., Lotlikar, M., Kowerska, M., Becker, G., Korologos, D., de Geronimo, M., and Crawford, J. M. (2009) Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak. Journal of Clinical Virology 45, 191-195 27. Landry, M. L., and Ferguson, D. (2010) Cytospin-enhanced immunofluorescence and impact of sample quality on detection of novel swine origin (H1N1) influenza virus. J Clin Microbiol 48, 957-959 28. Chartrand, C., Leeflang, M. M. G., Minion, J., Brewer, T., and Pai, M. (2012) Accuracy of Rapid Influenza Diagnostic Tests: A Meta-analysis. Annals of Internal Medicine 156, 500-511 29. Merckx, J., Wali, R., Schiller, I., Caya, C., Gore, G. C., Chartrand, C., Dendukuri, N., and Papenburg, J. (2017) Diagnostic Accuracy of Novel and Traditional Rapid Tests for Influenza Infection Compared With Reverse Transcriptase Polymerase Chain Reaction: A Systematic Review and Meta-analysisIncreased Accuracy of Novel Rapid Influenza Tests. Annals of Internal Medicine 167, 394-409 30. Chan, K.-H., To, K. K. W., Chan, J. F. W., Li, C. P. Y., Chan, K.-M., Chen, H., Ho, P.-L., and Yuen, K.-Y. (2014) Assessment of antigen and molecular tests with serial specimens from a patient with influenza A(H7N9) infection. J Clin Microbiol 52, 2272-2274 31. Chen, Y., Cui, D., Zheng, S., Yang, S., Tong, J., Yang, D., Fan, J., Zhang, J., Lou, B., Li, X., Zhuge, X., Ye, B., Chen, B., Mao, W., Tan, Y., Xu, G., Chen, Z., Chen, N., and Li, L. (2011) Simultaneous detection of influenza A, influenza B, and respiratory syncytial viruses and subtyping of influenza A H3N2 virus and H1N1 (2009) virus by multiplex real-time PCR. J Clin Microbiol 49, 1653-1656 32. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28, E63-E63 33. Kubo, T., Agoh, M., Mai, L. Q., Fukushima, K., Nishimura, H., Yamaguchi, A., Hirano, M., Yoshikawa, A., Hasebe, F., Kohno, S., and Morita, K. (2010) Development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (H1N1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings. J Clin Microbiol 48, 728-735 34. Imai, M., Ninomiya, A., Minekawa, H., Notomi, T., Ishizaki, T., Van Tu, P., Tien, N. T. K., Tashiro, M., and Odagiri, T. (2007) Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method. Journal of Virological Methods 141, 173-180 35. Nakauchi, M., Takayama, I., Takahashi, H., Tashiro, M., and Kageyama, T. (2014) Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection. Journal of Virological Methods 204, 101-104 36. Poon, L. L. M., Leung, C. S. W., Chan, K. H., Lee, J. H. C., Yuen, K. Y., Guan, Y., and Peiris, J. S. M. (2005) Detection of human influenza A viruses by loop-mediated isothermal amplification. J Clin Microbiol 43, 427-430 37. Townsend, M. B., Dawson, E. D., Mehlmann, M., Smagala, J. A., Dankbar, D. M., Moore, C. L., Smith, C. B., Cox, N. J., Kuchta, R. D., and Rowlen, K. L. (2006) Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance. J Clin Microbiol 44, 2863-2871 38. Lodes, M. J., Suciu, D., Elliott, M., Stover, A. G., Ross, M., Caraballo, M., Dix, K., Crye, J., Webby, R. J., Lyon, W. J., Danley, D. L., and McShea, A. (2006) Use of semiconductor-based oligonucleotide microarrays for influenza a virus subtype identification and sequencing. J Clin Microbiol 44, 1209-1218 39. Levine, M., Sheu, T. G., Gubareva, L. V., and Mishin, V. P. (2011) Detection of hemagglutinin variants of the pandemic influenza A (H1N1) 2009 virus by pyrosequencing. J Clin Microbiol 49, 1307-1312 40. Rutvisuttinunt, W., Chinnawirotpisan, P., Simasathien, S., Shrestha, S. K., Yoon, I.-K., Klungthong, C., and Fernandez, S. (2013) Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform. Journal of Virological Methods 193, 394-404 41. Bommakanti, G., Citron, M. P., Hepler, R. W., Callahan, C., Heidecker, G. J., Najar, T. A., Lu, X., Joyce, J. G., Shiver, J. W., Casimiro, D. R., ter Meulen, J., Liang, X., and Varadarajan, R. (2010) Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci U S A 107, 13701-13706 42. Schoemaker, J. M., Brasnett, A. H., and Marston, F. A. (1985) Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. The EMBO Journal 4, 775-780 43. He, F., Madhan, S., and Kwang, J. (2009) Baculovirus vector as a delivery vehicle for influenza vaccines. Expert Review of Vaccines 8, 455-467 44. O’Reilly, D. R. (1997) Use of Baculovirus Expression Vectors. in Recombinant Gene Expression Protocols (Tuan, R. S. ed.), Humana Press, Totowa, NJ. pp 235-246 45. 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. in Glycovirology Protocols (Sugrue, R. J. ed.), Humana Press, Totowa, NJ. pp 149-161 46. Throsby, M., van den Brink, E., Jongeneelen, M., Poon, L. L. M., Alard, P., Cornelissen, L., Bakker, A., Cox, F., van Deventer, E., Guan, Y., Cinatl, J., ter Meulen, J., Lasters, I., Carsetti, R., Peiris, M., de Kruif, J., and Goudsmit, J. (2008) Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One 3, e3942-e3942 47. Ekiert, D. C., Bhabha, G., Elsliger, M.-A., Friesen, R. H. E., Jongeneelen, M., Throsby, M., Goudsmit, J., and Wilson, I. A. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246-251 48. Henry Dunand, C. J., Leon, P. E., Huang, M., Choi, A., Chromikova, V., Ho, I. Y., Tan, G. S., Cruz, J., Hirsh, A., Zheng, N.-Y., Mullarkey, C. E., Ennis, F. A., Terajima, M., Treanor, J. J., Topham, D. J., Subbarao, K., Palese, P., Krammer, F., and Wilson, P. C. (2016) Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection. Cell Host Microbe 19, 800-813 49. Steel, J., Lowen, A. C., Wang, T. T., Yondola, M., Gao, Q., Haye, K., García-Sastre, A., and Palese, P. (2010) Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio 1, e00018-00010 50. Tan, G. S., Krammer, F., Eggink, D., Kongchanagul, A., Moran, T. M., and Palese, P. (2012) A Pan-H1 Anti-Hemagglutinin Monoclonal Antibody with Potent Broad-Spectrum Efficacy <em>In Vivo</em>. Journal of Virology 86, 6179 51. Throsby, M., van den Brink, E., Jongeneelen, M., Poon, L. L. M., Alard, P., Cornelissen, L., Bakker, A., Cox, F., van Deventer, E., Guan, Y., Cinatl, J., Meulen, J. t., Lasters, I., Carsetti, R., Peiris, M., de Kruif, J., and Goudsmit, J. (2008) Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells. PLoS One 3, e3942 52. Rowe, T., Abernathy, R. A., Hu-Primmer, J., Thompson, W. W., Lu, X., Lim, W., Fukuda, K., Cox, N. J., and Katz, J. M. (1999) Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol 37, 937-943 53. He, Q., Velumani, S., Du, Q., Lim, C. W., Ng, F. K., Donis, R., and Kwang, J. (2007) Detection of H5 avian influenza viruses by antigen-capture enzyme-linked immunosorbent assay using H5-specific monoclonal antibody. Clin Vaccine Immunol 14, 617-623 54. Chen, Y.-C., Chen, C.-H., and Wang, C.-H. (2008) H5 Antibody Detection by Blocking Enzyme-Linked Immunosorbent Assay Using a Monoclonal Antibody. 52, 124-129, 126 55. Ren, H., Wang, G., Wang, S., Chen, H., Chen, Z., Hu, H., Cheng, G., and Zhou, P. (2016) Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir. MAbs 8, 1156-1166 56. Cui, S., and Tong, G. (2008) A Chromatographic Strip Test for Rapid Detection of One Lineage of the H5 Subtype of Highly Pathogenic Avian Influenza. Journal of Veterinary Diagnostic Investigation 20, 567-571 57. Tsuda, Y., Sakoda, Y., Sakabe, S., Mochizuki, T., Namba, Y., and Kida, H. (2007) Development of an Immunochromatographic Kit for Rapid Diagnosis of H5 Avian Influenza Virus Infection. Microbiology and Immunology 51, 903-907 58. Wada, A., Sakoda, Y., Oyamada, T., and Kida, H. (2011) Development of a highly sensitive immunochromatographic detection kit for H5 influenza virus hemagglutinin using silver amplification. Journal of Virological Methods 178, 82-86 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77290 | - |
| dc.description.abstract | 2014年起在歐洲、亞洲、非洲與美洲等地的野生鳥類和家禽中，檢出了H5N8高致病性禽流感病毒（highly pathogenic avian influenza virus, HPAIV）。2015年台灣亦首次檢測到H5N8 HPAIV，自此每年都有檢出該病毒。因此，開發快速檢測試劑與中和抗體對於減少農業和經濟損失至關重要。血球凝集素（hemagglutinin, HA）是流感病毒表面的醣蛋白，是檢測感染樣品中流感病毒亞型的主要標地。本研究利用大腸桿菌表現系統，去表現並純化出H5N8 HA1和HA2的重組蛋白，以應用於單株抗體的製備，並順利篩選出7H6C和YC8兩株能分別辨識H5N8 rHA1和rHA2的單株抗體。其中7H6C可以結合H5N1和H5N8的rHA，但不能結合H1N1、H3N2和H7N9的rHA，顯示它具有辨識H5亞型的特異性。而YC8則可以結合H1N1、H5N1和H5N8的rHA，但不能結合H3N2和H7N9的rHA，顯示它具有辨識H1和H5亞型的特異性。經分析YC8之抗原結合序列後，發現其所辨認之HA2序列於H1和H5亞型中具有高度保守性的區域。此外，7H6C和YC8還具有抑制H5N8 rHA之血球凝集能力，因此具有開發成診斷試劑和中和抗體的潛力。 | zh_TW |
| dc.description.abstract | In 2014, the highly pathogenic avian influenza A (H5N8) virus (HPAIV) was detected in wild birds and poultry in Asia, Africa, Europe, and America. H5N8 HPAIV was also detected for the first time in Taiwan in 2015. Since then, the threatness caused by H5N8 HPAIV still exists. As a result, the preparedness for epidemic prevention and decreasing the agricultural and economic lost is extremely important. Hemagglutinin (HA), a glycoprotein found on the surface of influenza viruses, is considered as the major target for detection of the influenza virus subtype in the infected samples. In this study, the recombinant H5N8 HA1 and HA2 proteins were expressed in E. coli, and were utilized to generate two monoclonal antibodies, named 7H6C and YC8. 7H6C was generated by rHA1 immunization and can bind the rHA proteins of H5N1, and H5N8, but can not bind the rHA proteins of H1N1, H3N2 and H7N9, indicating that it has H5-subtype specificity. In contrast, YC8 was generated by rHA2 immunization and can bind the rHA proteins of H1N1, H5N1, and H5N8, but can not bind the rHA proteins of H3N2 and H7N9, indicating that it has H1-subtype and H5-subtype specificity. The antigen binding sequence of YC8 is highly conserved among the H1 and H5 subtypes. Both of 7H6C and YC8 have great capability to inhibit hemagglutination, therefore they can be applied in developing the diagnostict kit and neutralizing antibodies in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:54:25Z (GMT). No. of bitstreams: 1 ntu-108-R06b22047-1.pdf: 2132614 bytes, checksum: 52eaffa08d6b704f50845895e4d60b45 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 摘要 VI
Abstract VII 縮寫表 VIII 第一章 緒論 1 1.1 流感病毒之血球凝集素 1 1.2 H5亞型禽流感病毒 2 1.2.1 H5N1禽流感病毒 2 1.2.2 H5N2流感病毒 3 1.2.3 H5N3流感病毒 3 1.2.4 H5N6流感病毒 3 1.2.5 H5N8流感病毒 4 1.3 流感病毒的型別鑑定與檢測方法 5 1.3.1 病毒之分離與培養 5 1.3.2 病毒抗原檢測 6 1.3.3 流感的快速診斷檢測 7 1.3.4 實時反轉錄核酸檢測 7 1.4 研究動機與目的 10 第二章 材料與方法 11 2.1 實驗材料 11 2.1.1 大腸桿菌株 11 2.1.2 昆蟲細胞 12 2.1.3 骨髓瘤細胞 12 2.2 表現載體建構 12 2.2.1 原核表現系統載體 12 2.2.2 昆蟲細胞表現系統載體 12 2.2.3 核酸引子設計 13 2.2.4 聚合酶鏈鎖反應 13 2.2.5 限制酶切反應 13 2.2.6 DNA接合反應 14 2.2.8 重組載體之篩選 14 2.3 核酸實驗方法 15 2.3.1 洋菜膠體電泳 15 2.3.2 核酸純化 15 2.3.3 核酸定量 15 2.3.4 質體DNA製備 16 2.4 蛋白質實驗方法 16 2.4.1 蛋白質定量 16 2.4.2 聚丙烯醯胺凝膠電泳 16 2.4.3 CBR染色法 17 2.4.4 蛋白質轉印法 17 2.4.5 西方墨點法 17 2.4.6 酵素結合免疫吸附分析法 18 2.5 桿狀病毒表現系統 18 2.5.1 質體轉形作用 19 2.5.2 重組Bacmid純化 19 2.5.3 以聚合酶鏈鎖反應檢驗Bacmid 20 2.5.4 昆蟲細胞轉染作用 20 2.6 重組蛋白質表現 21 2.6.1 大腸桿菌表現系統 21 2.6.2 昆蟲細胞表現系統 22 2.7 重組蛋白純化 22 2.7.2 純化H5N8 HA重組蛋白 23 2.7.3 蛋白質脫鹽與濃縮 23 2.8 H5N8 rHA1、rHA2之單株抗體製備 23 2.8.1 小鼠免疫 23 2.8.2 抗血清製備 24 2.8.3 融合瘤細胞製備 25 2.8.4 融合瘤細胞限數稀釋法 26 2.8.5 單株抗體之純化 27 2.9 單株抗體之效價與抗原結合能力測試 27 2.10 抗體型別測定 28 2.11 單株抗體對流感病毒株之專一性測試 28 2.12 製備H5重組蛋白質片段 28 2.13 血球凝集測試 (Hemagglutination Test, HA Test) 29 2.14 血球凝集抑制測試 (Hemagglutination Inhibition Test, HI Test) 29 第三章 結果 30 3.1 製備H5N8 HA1及HA2 重組蛋白質 30 3.1.1 H5N8 HA1及HA2之表現載體建構 30 3.1.2 H5N8 rHA1及rHA2於大腸桿菌之表現 30 3.2 製備H5N8 HA重組蛋白質 31 3.2.1 H5N8 HA表現載體之建構 31 3.2.2 製備帶有H5N8 HA基因之桿狀病毒 31 3.2.3 HA重組蛋白質表現與純化 31 3.2.4 分析HA重組蛋白質 32 3.3 Anti-H5 rHA1與Anti-H5 rHA2單株抗體製備 32 3.3.1 免疫小鼠與血清效價測試 32 3.3.2 製備融合瘤細胞 33 3.3.3 純化單株抗體 33 3.4 分析單株抗體 34 3.4.1 單株抗體型別鑑定 34 3.4.2 7H6C對rHA的親和能力優於YC8 34 3.4.3 7H6C與YC8對不同亞型禽流感病毒rHA之專一性分析 34 3.4.4 7H6C與YC8之抗原結合位點分析 35 3.4.5 7H6C與YC8皆具備抑制rHA之血球凝集能力 35 第四章 討論 37 參考文獻 40 圖與表 46 圖一 H5N8 HA、HA1及HA2表現載體之建構 47 圖二 利用聚合酶鏈鎖反應確認桿狀病毒重組Bacmid 48 圖三 H5N8 HA 重組蛋白質於 Sf21 昆蟲細胞表現 49 圖四 以pET28a載體表現H5N8 HA1及HA2重組蛋白質 50 圖五 利用陰離子交換法與親和層析法純化昆蟲細胞表現之HA重組蛋白質 51 圖六 昆蟲細胞表現之HA重組蛋白質具有血球凝集能力 52 圖七 分析免疫後小鼠血清之抗體效價 53 圖八 7H6C之效價測定 54 圖九 YC8之效價測定 55 圖十 利用Protein G親和層析法純化7H6C及YC8 56 圖十一 以rHA為抗原測試7H6C及YC8之效價 57 圖十二 以rHA1及rHA2為抗原測試7H6C及YC8之效價 58 圖十三 YC8之抗體型別鑑定 59 圖十四 7H6C之抗體型別鑑定 60 圖十五 YC8能辨認H1N1、H5N1與H5N8 rHA，而7H6C則可以辨認H5N1與H5N8 rHA 61 圖十六 鑑定7H6C所辨認之HA抗原序列 63 圖十七 鑑定YC8所辨認之 HA抗原序列 65 圖十八 7H6C及YC8皆具有抑制rHA之血球凝集功能 66 圖十九 單株抗體結合於H5N8 HA之示意圖 (以PDB ID：5ykc.1為模板) 67 附錄 68 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | H5N8高致病性禽流感病毒 | zh_TW |
| dc.subject | 血球凝集素 | zh_TW |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | Highly pathogenic avian influenza A (H5N8) virus | en |
| dc.subject | Hemagglutinin | en |
| dc.subject | Monoclonal antibody | en |
| dc.title | 開發單株抗體以準確偵測H5亞型禽流感病毒血球凝集素 | zh_TW |
| dc.title | Development of monoclonal antibodies for accurate detection of the H5-subtype avian influenza virus hemagglutinins | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳慧文;游傑華;廖憶純 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | H5N8高致病性禽流感病毒,血球凝集素,單株抗體, | zh_TW |
| dc.subject.keyword | Highly pathogenic avian influenza A (H5N8) virus,Hemagglutinin,Monoclonal antibody, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU201902733 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-10 | - |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-107-2.pdf 未授權公開取用 | 2.08 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。