家禽流行性感冒(Avian Influenza, AI)是由A型流行性感冒病毒所引起之全身性或呼吸性感染的疾病。依其表面醣蛋白抗原血球凝集素(Hemagglutinin, HA)可分為15種血清亞型(H1~H15)、而依神經胺酸酶(Neuraminidase, NA)亦可分為9種血清亞型(N1~N9)。其中H5及H7可能為強毒型AI病毒，感染後可能會造成嚴重的全身症狀及高死亡率。傳統上區分AI病毒血清亞型的方法為血球凝集抑制試驗(hemagglutination inhibition test, HI)，但此方法缺乏敏感性且耗時。本研究利用H5血清亞型之HA重組蛋白，經E. coli BL21(DE3)表現後純化蛋白免疫BALA/c小鼠，製備出H5血清亞型之HA單源抗體，單源抗體經純化後進行競爭性ELISA (competitive enzyme-linked immunosorbent assay, CELISA)之試驗，以期能建立強毒型AI病毒H5血清亞型之快速診斷技術。實驗結果顯示H5血清亞型之單源抗體僅能被H5血清亞型之高免血清所競爭。且有極高的特異性。H5高免血清在512HI抗體力價與單源抗體10號競爭結果有91.5的競爭百分比。而與單源抗體11號的試驗結果，有86.1的競爭百分比。因此利用原核系統表現之H5血清亞型HA重組蛋白，配合以H5血清亞型重組蛋白製備之單源抗體所建立之競爭性ELISA套組，可用於偵測H5強毒型AI之抗血清，此一診斷技術的建立將可協助高病原性H5血清亞型AI病毒之診斷。

Avian Influenza (AI) is caused by type A influenza virus, which might cause a systemic or respiratory infection and disease. According to their surface glycoprotein, fifteen HA subtypes (HA1-HA15) and nine NA subtypes (NA1-NA9) have been identified. Avian influenza viruses of subtypes H5 and H7 might be highly pathogenic, which cause a severe systemic disease and a high mortality after infection. The conventional method for differentiating HA subtypes of AIV is hemagglutination inhibition test (HI test), which is both time-consuming and insensitive. In this study, we used E. coli BL21 (DE3) as the host to express recombinant HA protein of H5 subtype, and immunized BALB/c mice with the recombinant HA protein to prepare monoclonal antibody against the HA protein. After purification, the recombinant HA protein and the monoclonal antibody were used to develop a competitive-ELISA that could differentiate the antisera of H5 subtype from antisera other subtypes. Our results show that, for this competitive-ELISA, the binding of H5 monoclonal antibodies could be specifically competed by H5-specific serum. The H5-specific serum (512 HI), showed 91.5% and 86.1% of competition when competed with monoclonal antibody number 10 and number 11. Therefore, the competitive-ELISA developed by using recombinant HA proteins and monoclonal antibody, might serve as a tool for rapid identification of highly pathogenic influenza virus of the H5 subtype.

中文摘要…………………………………………………………………. Ⅰ
英文摘要…………………………………………………………………. Ⅱ
目次………………………………………………………………………. Ⅲ
表次………………………………………………………………………. Ⅴ
圖次………………………………………………………………………. Ⅵ
第一章 緒言……………………………………………………………. 1
第二章 文獻探討………………………………………………………. 2
2-1 歷史背景…………………………………………………... 2
2-2 病毒特徵…………………………………………………... 6
2-3 結構與功能………………………………………………... 7
2-3.1 血球凝集素蛋白…………………………………… 8
2-3.2 神經胺酸酶………………………………………… 9
2-3.3 核蛋白……………………………………………… 9
2-3.4 聚合酶蛋白………………………………………… 9
2-3.5 基質蛋白…………………………………………… 10
2-3.6 非結構蛋白………………………………………… 10
2-4 病毒複製…………………………………………………... 10
2-5 診斷方法…………………………………………………... 11
2-5.1 偵測抗原………………………………………….... 11
2-5.2 偵測抗體…………………………………………… 13
2-6 單源抗體…………………………………………………... 14
2-6.1單源抗體之歷史背景………………………………... 14
2-6.2融合瘤細胞篩選之原理……………………………... 14
2-6.3 單株抗體之特性…………………………………….. 15
第三章 材料與方法……………………………………………………. 16
3-1 病毒的增殖與純化………………………………………... 16
3-1.1 病毒來源………………………………………........ 16
3.1.2 病毒增殖………………………………………........ 16
3.1.3 病毒純化………………………………………........ 16
3.1.4 病毒抗原力價測定………………………………… 17
3-1.5 應用RT-PCR確認純化之病毒…………………… 17
3-2 重組蛋白之表現與確認…………………………………... 19
3-2.1 重組蛋白之表現…………………………………… 20
3-2.2 重組蛋白之純化…………………………………… 20
3-2.3 重組蛋白之電泳分析與確認……………………… 21
3-2.4 重組蛋白特異性分析與確認……………………… 21
3-3 單源抗體之製備………………………………………....... 22
3-3.1 小鼠免疫計畫……………………………………… 22
3-3.2 骨髓瘤細胞培養…………………………………… 22
3-3.3 細胞融合………………………………………........ 23
3-3.4 融合瘤細胞篩選…………………………………… 23
3-3.5 融合瘤細胞單株化………………………………… 24
3-3.6 融合瘤細胞之確認………………………………… 24
3-3.7 腹水之生產……………………………………….... 25
3-3.8 單源抗體之純化…………………………………… 25
3-3.9 純化之單源抗體特異性分析……………………… 26
3-4 競爭性酵素連結免疫吸附分析法之架構………………... 27
3-4.1 以棋盤式力價測定法決定單源抗體與重組HA蛋白之最佳稀釋倍數………………………………... 27
3-4.2 家禽流行性感冒病毒H5血清亞型之高免血清與單原抗體之CELISA試驗………………………... 27
3-4.3 單原抗體與15種血清亞型之家禽流行性感冒高免血清CELISA試驗……………………………... 28
3-4.4 應用於偵測野外陰性血清建立CLISA陰性競爭百分比(competition percentage)…………………… 28
第四章 結果………………………………………................................. 29
4-1 病毒純化之結果……………………………………………. 29
4-2 重組蛋白之表現與確認……………………………………. 29
4-3融合瘤細胞製備之結果…………………………………….. 29
4-4 單原抗體之生產與分析……………………………………. 30
4-5 競爭性ELISA之架構與分析……………………………… 31
4-5.1 單原抗體與重組HA蛋白之最佳稀釋倍數……… 31
4-5.2 AI病毒H5血清亞型之雞高免血清與單原抗體CELISA試驗………………………………………. 31
4-5.3 單原抗體與15種血清亞型之家禽流行性感冒高免血清CELISA試驗……………………………… 31
4.5.4 應用於偵測野外陰性血清建立CLISA陰性競爭百分比……………………………………………… 32
第五章 討論……………………………………………………………. 54
參考文獻…………………………………………………………………. 59

參考文獻
吳靜如。2001。應用大腸桿菌表現家禽流行性感冒病毒血球凝集素以區別病毒血清亞型。碩士論文。國立中興大學獸醫學系研究所。台灣省台中市。中華民國。
李敏旭。1999。應用反轉錄聚合酶反應診斷家禽流行性感冒病毒及區分血清亞型。碩士論文。國立中興大學獸醫微生物學研究所。台灣省台中市。中華民國。
候憲三。2000。家禽里奧病毒毒株S1133非結構蛋白σNS單株抗體的製備與特性分析。碩士論文。國立中興大學獸醫學系研究所。台灣省台中市。中華民國。
黃文徹。1993。應用間接酵素結合免疫吸附法 (ELISA) 測定家禽流行性感冒謝清抗體。碩士論文。國立中興大學獸醫學研究所。台灣省台中市。中華民國。
鄭明珠。1998。家禽流行性感冒血清學鑑定。畜牧半月刊。60:30-33。
Ada, G. L., Jones, P. D. 1986. The immune response to influenza inflection. Current Topics in Microbio. and Immuno. 128:1-54
Adair, B. M., D. Todd, E. R. Mckillop, and M.S. McNulty. 1989. Detection of influenza A type-specific antibodies in chicken and turkey sera by enzyme linked immunosorbent assay. Avian Pathol. 18:455-463.
Brown, E. G. 2000. Influenza virus genetics. Biomed Pharmacother. 54:196-209. Review.
Cauthen, A. N., D. E. Swayne, S. Schultz-Cherry, M. L. Perdue, and D. L. Suarez. 2000. Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans. J Virol. 74:6592-6599.
Claas, E. C., A. D. Osterhaus, R. van Beek, J. C. De Jong, G. F. Rimmelzwaan, D. A. Senne, S. Krauss, K. F. Shortridge, and R. G. Webster. 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet. 351:472-477.
Clavijo, A., M. Lin, J. Riva, and Zhou EM. 2001. Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection. : J Vet Diagn Invest 2001 Jul;13:357-60
Cooper, L.A., and K. Subbarao. 2000. A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses. J Clin Microbiol. 38:2579-2583.
Davison, S., A. F. Ziegler, and R. J. Eckroade. 1998. Comparison of an antigen-capture enzyme immunoassay with virus isolation for avian influenza from field samples. Avian Dis. 42:791-795.
De St Groth, D. R. and Scheidegger, D. 1980. Production of monoclonal antibodies:stategy and tactics. J. Immuno. Meth. 35:1-21
Dybing, J. K., S. Schultz-Cherry, D. E. Swayne, D. L. Suarez, and M. L. Perdue. 2000. Distinct pathogenesis of hong kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses. J Virol. 74:1443-1450.
Englund, L., A. F. Hard, and C. Segerstad. 1998. Two avian H10 influenza A virus strains with different pathogenicity for mink (Mustela vison). Arch Virol. 143:653-666.
Guan, Y., K. F. Shortridge, S. Krauss, and R. G. Webster. 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong? Proc Natl Acad Sci U S A. 96:9363-9367.
Guan, Y., K. F. Shortridge, S. Krauss, P. S. Chin, K. C. Dyrting, T. M. Ellis, R. G. Webster, and M. Peiris. 2000. H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China. J Virol. 74:9372-9380.
Guo, Y. J., S. Krauss, D. A. Senne, I. P. Mo, K. S. Lo, X. P. Xiong, M. Norwood, K. F. Shortridge, R. G. Webster, and Y. Guant. 2000. Characterization of the Pathogenicity of Members of the Newly Established H9N2 Influenza Virus Lineages in Asia. Viro. 267:279-288.
Hoffmann, E., J. Stech, I. Leneva, S. Krauss, C. Scholtissek, P. S. Chin, M. Peiris, K. F. Shortridge, and R. G. Webster. 2000. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J Virol. 74:6309-6315.
Horimoto, T., and Y. Kawaoka. 1995. Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds. J Clin Microbiol. 33:748-751.
Ito, T., and Y. Kawaoka. 2000. Host-range barrier of influenza A viruses. Vet Microbiol. 74:71-75. Review.
Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol. 72:7367-7373.
Katz, J. M., X. Lu, A. M. Frace, T. Morken, S. R. Zaki, and T. M. Tumpey. 2000. Pathogenesis of and immunity to avian influenza A H5 viruses. Biomed Pharmacother. 54:178-187.
Kuszewski, K., and L. Brydak. 2000. The epidemiology and history of influenza. Biomed Pharmacother. 54:188-195. Review.
Lamb, R. A., and R. M. Krug. 1996. Orthomyxoviridae: The virus and their replication. In: B.N. Fields, et al. Fundamental virology, 3rd ed. Lippincott-Raven Publisher, Philadelphia, PA. pp.605-639.
Lin, Y. P., M. Shaw, V. Gregory, K. Cameron, W. Lim, A. Kilmov, K. Subbarao, Y. Guan, S. Krauss, K. Shortridge, R. Webster, N. Cox, and A. Hay. 2000. Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates.
Lu Y. S, T. Sugimura, H. K. Shien, Y. L. Lee, and M. H. Jong. 1985. Isolation and identification of an influenza A virus in ducks in Taiwan. J. Chin. Soc.Vet. Sci. 11:23-34.
Matrosovich, M., N. Zhou, Y. Kawaoka, and R. Webster. 1999. The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol. 73:1146-115
Monika G. W., J. Liesbth. K. Hinri. and B. S. Krystyna. 2000. A highly specific and senitive sandwich blocking ELISA based on baculvirus pseudorabies virus glycoprotein B. J. Virol. Methods 88:63-71
Munch, M., L. P. Nielsen, K. J. Handberg, and P. H. Jorgensen. 2001. Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol. 146:87-97.
Olufemi, O. F., J. A. Newman, V. Sivanandan, and D. A. Halvorson. 1989. A Broad-Spectrum Avian Influenza Subtype Antigen for Indirect Enzyme-Link immunosorbent Assay. Avian Disease 33: 264-269
Perdue, M. L., and D. L. Suarez. 2000. Structural features of the avian influenza virus hemagglutinin that influence virulence. Vet Microbiol. 74:77-86.
Rebelo-de-Andrade, H., and M. C. Zambon. 2000. Different diagnostic methods for detection of influenza epidemics. Epidemiol Infect. 124:515-522.
Rowe, T., R. A. Abernathy, J. Hu-Primmer, W. W. Thompson, X. Lu, W. Lim, K. Fukuda, N. J. Cox, and J. M. Katz. 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.
Schafer, J. R. Kawaoka, Y. Bean, W. J., Senne, D., Webster, R. G. 1993. Origin of the pandemic 1957 H2 influenza A virus and the persistence of its possible progenitors in the avian reservoir. Virology 194:781-8.
Shafer, A.L., J. B. Katz, and K. A. Eernisse.1998. Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Dis. 42:28-34.
Shieh H. K. W. J. Huang, J. H. Shien, S. Y. Chiu, L. F. Lee, and Y. S. Lu. 1992. Studies on avian influenza in Taiwan, R.O.C. (III) Isolaion, identification, and pathogencity tests of the virus isolated from breeding chicken. Taiwan J Vet. Med. Ani. Husb. 59:45-55.
Snyder, D.B., W. W. Marquardt, F. S. Yancey, and P. K. Savage. 1985. An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus. Avian Dis. 29:136-144.
Suarez, D. L. 2000. Evolution of avian influenza viruses. Vet Microbiol. 74:15-27.
Suarez, D. L., M. L. Perdue, N. Cox, T. Rowe, C. Bender, J. Huang, and D. E. Swayne. 1998. Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong. J Virol. 72:6678-6688.
Vabret, A., G. Sapin, B. Lezin, A. Mosnier, J. Cohen, L. Burnouf, J. Petitjean, S. Gouarin, M. Campet, and F. Freymuth. 2000. Comparison of three non-nested RT-PCR for the detection of influenza A viruses. J Clin Virol. 17:167-175.
Walker, J. A., and Y. Kawaoka. 1993. Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. J Gen Virol. 74 ( Pt 2):311-314.
Xu, X., K.Subbarao, N. J. Cox, and Y. Guo. 1999. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology. 261:15-19.
Zambon, M.C. 1999. Epidemiology and pathogenesis of influenza. J Antimicrob Chemother. 44 Suppl B:3-9. Review.
Zhou, E.M., M. Chan, R. A. Heckert, J. Riva, and M.F. Cantin. 1998. Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein. Avian Dis. 42:517-522.
Zhou, N. N., K. F. Shortridge, E. C. Claas, S. L. Krauss, and R. G. Webster. 1999. Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong. J Virol. 73:3366-3374.
Zhou, N. N., K. F. Shortridge, E. C. J. Claas, S. L. Krauss, and R. G. Webster. 1999. Rapid Evolution of H5N1 Influenza Viruses in Chicken in Hong Kong. J. Virol. 73:3366-3374.