請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69975
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭益謙(Ivan-Chen Cheng) | |
dc.contributor.author | Rung-Rung Kao | en |
dc.contributor.author | 高蓉榕 | zh_TW |
dc.date.accessioned | 2021-06-17T03:36:53Z | - |
dc.date.available | 2018-03-01 | |
dc.date.copyright | 2018-03-01 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-02-09 | |
dc.identifier.citation | 行政院農業委員會動植物防疫檢疫局:http://www.baphiq.gov.tw/。
李光琇。以抗HA單源抗體建立阻斷型ELISA區別抗禽流感病毒H5或H6亞型之抗體。國立台灣大學碩士論文,台北,2015。 Adair, B. M., McNulty, M. S., Todd, D., Connor, T. J., & Burns, K. (1989). Quantitative estimation of Newcastle disease virus antibody levels in chickens and turkeys by ELISA. Avian Pathol, 18(1), 175-192. doi:10.1080/03079458908418589 AlKurashi, M., Eastick, F. A., Kuchipudi, S. V., Rauch, C., Madouasse, A., Zhu, X. Q., & Elsheikha, H. M. (2011). Influence of culture medium pH on internalization, growth and phenotypic plasticity of Neospora caninum. Vet Parasitol, 177(3-4), 267-274. doi:10.1016/j.vetpar.2010.11.053 Blissard, G. W., & Rohrmann, G. F. (1990). Baculovirus diversity and molecular biology. Annu Rev Entomol, 35, 127-155. doi:10.1146/annurev.en.35.010190.001015 Chandrasekaran, A., Srinivasan, A., Raman, R., Viswanathan, K., Raguram, S., Tumpey, T. M., . . . Sasisekharan, R. (2008). Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol, 26(1), 107-113. doi:10.1038/nbt1375 Chang, D. K., Cheng, S. F., Kantchev, E. A., Lin, C. H., & Liu, Y. T. (2008). Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex. BMC Biol, 6, 2. doi:10.1186/1741-7007-6-2 Chen, Y. C., Chen, C. H., & Wang, C. H. (2008). H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody. Avian Dis, 52(1), 124-129. doi:10.1637/8076-071807-Reg Cheng, M. C., Soda, K., Lee, M. S., Lee, S. H., Sakoda, Y., Kida, H., & Wang, C. H. (2010). Isolation and Characterization of Potentially Pathogenic H5N2 Influenza Virus from a Chicken in Taiwan in 2008. Avian Diseases, 54(2), 885-893. Coombs, P. J., Taylor, M. E., & Drickamer, K. (2006). Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling. Glycobiology, 16(8), 1c-7c. doi:10.1093/glycob/cwj126 Couceiro, J. N., Paulson, J. C., & Baum, L. G. (1993). Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res, 29(2), 155-165. Crowther, J. R. (2001). The ELISA guidebook. Totowa, NJ: Humana Press. Davidson, D. J., & Castellino, F. J. (1991). Asparagine-linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells. Biochemistry, 30(25), 6165-6174. de Vries, R. P., Smit, C. H., de Bruin, E., Rigter, A., de Vries, E., Cornelissen, L. A., . . . de Haan, C. A. (2012). Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin. J Virol, 86(21), 11735-11744. doi:10.1128/jvi.01084-12 Emery, V. C. (1992). Baculovirus expression vectors : choice of expression vector. Methods Mol Biol, 8, 287-307. doi:10.1385/0-89603-191-8:287 Fiebig, P., Shehata, A. A., & Liebert, U. G. (2015). Generation of monoclonal antibodies reactive against subtype specific conserved B-cell epitopes on haemagglutinin protein of influenza virus H5N1. Virus Res, 199, 46-55. doi:10.1016/j.virusres.2015.01.006 Gething, M. J., McCammon, K., & Sambrook, J. (1986). Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell, 46(6), 939-950. Hanson, S. R., Culyba, E. K., Hsu, T. L., Wong, C. H., Kelly, J. W., & Powers, E. T. (2009). The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability. Proc Natl Acad Sci U S A, 106(9), 3131-3136. doi:10.1073/pnas.0810318105 He, Q., Velumani, S., Du, Q., Lim, C. W., Ng, F. K., Donis, R., & 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(5), 617-623. doi:10.1128/cvi.00444-06 Hirschberg, C. B., & Snider, M. D. (1987). Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem, 56, 63-87. doi:10.1146/annurev.bi.56.070187.000431 Hornbeck, P. V. (1991). Enzyme‐linked immunosorbent assays (Current protocols in immunology, 2.1. 1-2.1. 23 %@ 0471142735. Hu, Y. C., Luo, Y. L., Ji, W. T., Chulu, J. L., Chang, P. C., Shieh, H., . . . Liu, H. J. (2006). Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system. J Virol Methods, 135(1), 43-48. doi:10.1016/j.jviromet.2006.01.023 Hurt, A. C., Alexander, R., Hibbert, J., Deed, N., & Barr, I. G. (2007). Performance of six influenza rapid tests in detecting human influenza in clinical specimens. J Clin Virol, 39(2), 132-135. doi:10.1016/j.jcv.2007.03.002 Isin, B., Doruker, P., & Bahar, I. (2002). Functional motions of influenza virus hemagglutinin: a structure-based analytical approach. Biophys J, 82(2), 569-581. doi:10.1016/s0006-3495(02)75422-2 Ito, T., Couceiro, J. N., Kelm, S., Baum, L. G., Krauss, S., Castrucci, M. R., . . . Kawaoka, Y. (1998). Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol, 72(9), 7367-7373. Killian, M. L. (2008). Hemagglutination assay for the avian influenza virus. Methods Mol Biol, 436, 47-52. doi:10.1007/978-1-59745-279-3_7 Klenk, H. D., Wagner, R., Heuer, D., & Wolff, T. (2002). Importance of hemagglutinin glycosylation for the biological functions of influenza virus. Virus Res, 82(1-2), 73-75. Lai, A. L., Park, H., White, J. M., & Tamm, L. K. (2006). Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity. J Biol Chem, 281(9), 5760-5770. doi:10.1074/jbc.M512280200 Lee, C. C., Zhu, H., Huang, P. Y., Peng, L., Chang, Y. C., Yip, C. H., . . . Guan, Y. (2014). Emergence and evolution of avian H5N2 influenza viruses in chickens in Taiwan. J Virol, 88(10), 5677-5686. doi:10.1128/jvi.00139-14 Lee, C. W., Lee, Y. J., Senne, D. A., & Suarez, D. L. (2006). Pathogenic potential of North American H7N2 avian influenza virus: a mutagenesis study using reverse genetics. Virology, 353(2), 388-395. doi:10.1016/j.virol.2006.06.003 Legastelois, I., Chevalier, M., Bernard, M. C., de Montfort, A., Fouque, M., Pilloud, A., . . . Moste, C. (2011). Avian glycan-specific IgM monoclonal antibodies for the detection and quantitation of type A and B haemagglutinins in egg-derived influenza vaccines. J Virol Methods, 178(1-2), 129-136. doi:10.1016/j.jviromet.2011.08.027 Lin, S. C., Jan, J. T., Dionne, B., Butler, M., Huang, M. H., Wu, C. Y., . . . Wu, S. C. (2013). Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans. PLoS One, 8(6), e66719. doi:10.1371/journal.pone.0066719 McDougal, V. V., & Guarino, L. A. (2001). DNA and ATP binding activities of the baculovirus DNA helicase P143. J Virol, 75(15), 7206-7209. doi:10.1128/jvi.75.15.7206-7209.2001 Mullberg, J., Schooltink, H., Stoyan, T., Gunther, M., Graeve, L., Buse, G., . . . Rose-John, S. (1993). The soluble interleukin-6 receptor is generated by shedding. Eur J Immunol, 23(2), 473-480. doi:10.1002/eji.1830230226 Murphy, C. I., Piwnica-Worms, H., Grunwald, S., Romanow, W. G., Francis, N., & Fan, H. Y. (2004). Overview of the baculovirus expression system. Curr Protoc Mol Biol, Chapter 16, Unit 16.19. doi:10.1002/0471142727.mb1609s65 OIE. Word Organization for Animal Health. Avian influenza. In: Manual of diagnositc tests and vaccines for terrestrial animals. 2015 Available from: http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/ Reid, A. H., Fanning, T. G., Hultin, J. V., & Taubenberger, J. K. (1999). Origin and evolution of the 1918 'Spanish' influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A, 96(4), 1651-1656. Rogers, G. N., Paulson, J. C., Daniels, R. S., Skehel, J. J., Wilson, I. A., & Wiley, D. C. (1983). Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature, 304(5921), 76-78. Rohrmann, G. F. (2013). Structural proteins of baculovirus occlusion bodies and virions. Rott, R. (1992). The pathogenic determinant of influenza virus. Vet Microbiol, 33(1-4), 303-310. Russell, R. J., Haire, L. F., Stevens, D. J., Collins, P. J., Lin, Y. P., Blackburn, G. M., . . . Skehel, J. J. (2006). The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature, 443(7107), 45-49. doi:10.1038/nature05114 Schrader, C., Schielke, A., Ellerbroek, L., & Johne, R. (2012). PCR inhibitors - occurrence, properties and removal. J Appl Microbiol, 113(5), 1014-1026. doi:10.1111/j.1365-2672.2012.05384.x Shan, S., Ko, L. S., Collins, R. A., Wu, Z., Chen, J., Chan, K. Y., . . . Yu, A. C. (2003). Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation. Biochem Biophys Res Commun, 302(2), 377-383. Shih, A. C., Hsiao, T. C., Ho, M. S., & Li, W. H. (2007). Simultaneous amino acid substitutions at antigenic sites drive influenza A hemagglutinin evolution. Proc Natl Acad Sci U S A, 104(15), 6283-6288. doi:10.1073/pnas.0701396104 Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N., & Kawaoka, Y. (2006). Avian flu: influenza virus receptors in the human airway. Nature, 440(7083), 435-436. doi:10.1038/440435a Six, D. A., & Dennis, E. A. (2000). The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim Biophys Acta, 1488(1-2), 1-19. Skehel, J. J., & Wiley, D. C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem, 69, 531-569. doi:10.1146/annurev.biochem.69.1.531 Sriwilaijaroen, N., & Suzuki, Y. (2012). Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc Jpn Acad Ser B Phys Biol Sci, 88(6), 226-249. Starick, E., Romer-Oberdorfer, A., & Werner, O. (2000). Type- and subtype-specific RT-PCR assays for avian influenza A viruses (AIV). J Vet Med B Infect Dis Vet Public Health, 47(4), 295-301. Starick, E., Werner, O., Schirrmeier, H., Kollner, B., Riebe, R., & Mundt, E. (2006). Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species. J Vet Med B Infect Dis Vet Public Health, 53(8), 370-375. doi:10.1111/j.1439-0450.2006.01007.x Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., & Wilson, I. A. (2006). Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol, 355(5), 1143-1155. doi:10.1016/j.jmb.2005.11.002 Stieneke-Grober, A., Vey, M., Angliker, H., Shaw, E., Thomas, G., Roberts, C., . . . Garten, W. (1992). Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. Embo j, 11(7), 2407-2414. Summers, M. (1977). Baculoviruses (baculoviridae). The Atlas of Insect and Plant Viruses, 3-27. Summers, M. D., & Smith, G. E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures. Sun, S., Wang, Q., Zhao, F., Chen, W., & Li, Z. (2011). Glycosylation site alteration in the evolution of influenza A (H1N1) viruses. PLoS One, 6(7), e22844. doi:10.1371/journal.pone.0022844 Swenson, K. I., Piwnica-Worms, H., McNamee, H., & Paul, D. L. (1990). Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul, 1(13), 989-1002. Taubenberger, J. K., & Kash, J. C. (2010). Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe, 7(6), 440-451. doi:10.1016/j.chom.2010.05.009 Wang, C. C., Chen, J. R., Tseng, Y. C., Hsu, C. H., Hung, Y. F., Chen, S. W., . . . Wong, C. H. (2009). Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci U S A, 106(43), 18137-18142. doi:10.1073/pnas.0909696106 Wang, R., Zhao, J., Jiang, T., Kwon, Y. M., Lu, H., Jiao, P., . . . Li, Y. (2013). Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J Virol Methods, 189(2), 362-369. doi:10.1016/j.jviromet.2013.03.006 Wang, T. T., Tan, G. S., Hai, R., Pica, N., Petersen, E., Moran, T. M., & Palese, P. (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog, 6(2), e1000796. doi:10.1371/journal.ppat.1000796 Wei, C. J., Xu, L., Kong, W. P., Shi, W., Canis, K., Stevens, J., . . . Nabel, G. J. (2008). Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol, 82(13), 6200-6208. doi:10.1128/jvi.00187-08 Widjaja, L., Krauss, S. L., Webby, R. J., Xie, T., & Webster, R. G. (2004). Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses. J Virol, 78(16), 8771-8779. doi:10.1128/jvi.78.16.8771-8779.2004 Wood, G. W., McCauley, J. W., Bashiruddin, J. B., & Alexander, D. J. (1993). Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes. Arch Virol, 130(1-2), 209-217. Yang, D. G., Chung, Y. C., Lai, Y. K., Lai, C. W., Liu, H. J., & Hu, Y. C. (2007). Avian influenza virus hemagglutinin display on baculovirus envelope: cytoplasmic domain affects virus properties and vaccine potential. Mol Ther, 15(5), 989-996. doi:10.1038/mt.sj.6300131 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69975 | - |
dc.description.abstract | 禽流感病毒可以根據病毒表面上的血球凝集素(Hemagglutinin, HA)與神經氨酸酶(Neuraminidase, NA)分類成不同亞型,HA是一種三聚體的病毒外膜醣蛋白,能與宿主細胞表面的唾液酸(sialic acid)受體結合進入細胞後造成感染。然而,台灣在2003年分離到低病原性(Low pathogenic avian influenza virus, LPAIV)的H5N2禽流感病毒,並首次於2008年分離到具有高病原性(Highly pathogenic avian influenza virus, HPAIV)潛力的H5N2毒株,表示病毒已經由LPAIV演化為HPAIV之傾向。本實驗使用(A/chicken/Taiwan/1209/03(H5N2))福馬林不活化全病毒免疫BALB/c小鼠,以融合瘤技術製備單源抗體(αHA MAb),並分別使用全病毒與真核表現系統抗原盤透過免疫螢光染色(immunofluorescent assay, IFA)篩選出12株抗HA的單源抗體。除此之外,利用桿狀昆蟲表現系統表現出具有醣基化的重組HA(rHA△TM)蛋白作為抗原,透過條件最優化下建立1209/H5N2 bELISA及rHA△TM/H5N2 bELISA不同抗原之阻斷型酵素免疫吸附法(blocking ELISA, bELISA)。共有265個雞血清樣品使用兩種bELISA檢測,1209/H5N2 bELISA敏感性為88.3%(76/86)、特異性為98.8%(168/170); rHA△TM/H5N2 bELISA敏感性為93.0%(80/86)、特異性為98.8%(168/170)並以血球凝集抑制法(HI test)當作檢測的金標準。總結上述結果,建立之兩種bELISA可檢測雞隻血清樣品中抗H5的抗體,敏感性雖稍有不足但具有高特異性。 | zh_TW |
dc.description.abstract | Avian influenza virus (AIV) is classified into subtypes based on the hemagglutinin (HA) and neuraminidase (NA) expressed on viral surfaces. HA is a viral coat glycoprotein as trimetric forms coded from a gene segment, can attach to sialic acid receptors on the host cell, and cause infection. Moreover, Low pathogenic avian influenza virus (LPAIV) H5N2 have been isolated in Taiwan since 2003. Also, the highly pathogenic avian influenza virus (HPAIV) H5N2 was first isolated in 2008. Indicating that the virus has been mutated from LPAIV to HPAIV. In this study, BALB/c mice were immunized with (A/chicken/Taiwan/1209/03(H5N2) AIV for generating anti-HA monoclonal antibodies (αHA MAb). We have obtained 12 MAbs against HA H5 by immunofluorescent assay (IFA) screening with whole-virus and HTK-H5HA antigen plates. Furthermore, rHA△TM/H5N2 was cloned and constructed into pBacPAK8 to prepare glycosylated HA antigen by the baculovirus expression system. After we optimizing the blocking ELISA (bELISA) condition, two bELISA with 1209/H5N2 and rHA△TM/H5N2 were established. The HI test was taken as the golden-standard of detecting αH5 antibodies in chicken sera. 265 chicken sera were tested by these two bELISA. The sensitivity and specificity of 1209/H5N2 bELISA were 88.3%(76/86) and 98.8%(168/170). The sensitivity and specificity of rHA△TM/H5N2 bELISA were 93.0%(80/86) and 98.8%(168/170)。The preliminary results show that 1209/H5N2 or rHA△TM/H5N2 bELISA has relatively low sensitivity but has high specificity for detecting anit-H5 antibodies in chicken sera. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:36:53Z (GMT). No. of bitstreams: 1 ntu-107-R04629013-1.pdf: 12764848 bytes, checksum: 67db73f0efc61bc9cf686b93462459c1 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 目錄
口試委員審定書 i 致謝 ii 中文摘要 iv 英文摘要 v 目錄 vi 圖目錄 xii 表目錄 xiii 第一章 序言 1 第二章文獻回顧 3 第一節禽流感之起源背景 3 第二節流感病毒介紹 3 第三節流感病毒複製 4 第四節 H5N2禽流感疫情 5 第五節 血球凝集素(HA)介紹 5 2-5.1 亞型分類 6 2-5.2 HA0之結構與功能 6 2-5.3 HA1之結構與功能 7 2-5.4 HA2之結構與功能 8 2-5.5 HA醣基化之構造與功能 8 第六節AIV診斷方式 10 2-6.1 AIV抗原診斷 10 2-6.1 AIV抗體診斷 10 第七節 酵素連結免疫吸附法(Enzyme-linked immunosorbent assay, ELISA 11 2-7.2競爭型或阻斷型酵素連結免疫吸附法(cELISA/bELISA) 11 第八節 桿狀病毒-昆蟲細胞表現系統(Baculovirus expression system, BEVS 12 第三章 材料與方法 14 第一節 細胞培養 14 3-1.1 MDCK細胞 14 3-1.2 HTK細胞 14 3-1.3 Sf9細胞 15 3-1.4 HEK-293細胞 15 第二節 抗禽流感病毒結構蛋白HA之融合瘤單源抗體製備 15 3-2.1免疫計劃 15 3-2.2骨髓瘤細胞培養 16 3-2.3融合瘤製備 16 3-2.4親代融合瘤細胞篩選 17 3-2.5融合瘤細胞單株化 18 第三節 αHA單源抗體之特性鑑定與製備 19 3-3.1免疫螢光染色法(IFA) 19 3-3.2血球凝集抑制試驗(HI test) 19 3-3.3中和試驗(Neutralization test) 19 3-3.4真核表現系統(Eukaryotic expression system, EES) 19 3-3.5單源抗體之亞型分析 (Isotyping) 20 3-3.6腹水製備 20 3-3.7單源抗體純化與定量 21 3-3.8單源抗體之酵素標示(HRP-conjugation) 21 第四節 重組rHA∆TM/H5N2抗原製備 22 3-4.1重組rHA∆TM/H5N2基因選殖 22 3-4.1.1 RNA萃取 22 3-4.1.2反轉錄作用(Reverse transcription, RT) 23 3-4.1.3聚合酶連鎖反應(PCR)增幅1209/H5N2 full-length HA gene 23 3-4.1.4建構重組轉移載體 (Transfer vector) 24 3-4.1.5重組轉移載體PAK8-rHA∆TM之轉型作用 (Transformation) 25 3-4.1.6重組轉移載體PAK8-rHA∆TM之確認 26 3-4.1.7重組轉移載體PAK8-rHA∆TM之定序 (Sequencing) 26 3-4.1.8重組轉移載體pcDNA-rHA∆TM之轉型作用 (Transformation) 27 3-4.1.9重組轉移載體pcDNA-rHA∆TM之確認 27 3-4.1.10重組轉移載體pcDNA-rHA∆TM之定序 (Sequencing) 27 3-4.1.10轉殖質體之保存 27 3-4.1.11重組轉移載體PAK8-rHA∆TM之萃取 27 3-4.2建構重組桿狀病毒 28 3-4.2.1共轉移感染 (Co-transfection) 28 3-4.2.2基因轉殖成功之重組病毒增殖 (Amplification) 29 3-4.2.3測定桿狀病毒力價(50% Tissue Culture Infective Dose, TCID50) 30 3-4.3重組蛋白rHA∆TM製備、純化與定量 30 3-4.3.1 rHA∆TM/H5N2蛋白表現 30 3-4.3.2 rHA∆TM/H5N2蛋白濃縮 30 3-4.3.3 rHA∆TM/H5N2蛋白純化 31 3-4.3.4 rHA∆TM/H5N2蛋白定量 31 3-4.3.5 rHA∆TM/H5N2蛋白確認 32 3-4.4建立stable cell line試驗 33 3-4.4.1 rHA∆TM/H5N2蛋白純化 33 3-4.4.2 rHA∆TM/H5N2蛋白確認 33 3-4.4.3 rHA∆TM/H5N2蛋白定量 33 第五節 建立αH5HA MAbs-based blocking ELISA 34 3-5.1以indirect ELISA測試αH5HA MAbs之特異性及敏感性 34 3-5.2最佳化αH5HA MAbs-based blocking ELISA 34 3-5.2.1最佳化tracer Ab與rH5HA之比例 34 3-5.2.2最佳化docking MAb、rH5HA與tracer Ab之比例 35 3-5.3 血清檢體檢測 35 3-5.3.1 rHA∆TM/H5N2 與1209/H5N2 blocking ELISA檢測血清檢體 35 3-5.3.2 rHA∆TM/H5N2與1209/H5N2 bELISA cut-off value、敏感性及特異性分析 36 第四章 結果 37 第一節 抗禽流感病毒H5HA之融合瘤單源抗體製備 37 4-1.1 1209/H5N2免疫小鼠之血清力價 37 4-2.1 H5親代融合瘤細胞篩選 37 第二節 αH5HA單源抗體之特性鑑定與製備 38 4-2.1免疫螢光染色(IFA) 38 4-2.2血球凝集抑制試驗(HI test) 38 4-2.3中和試驗(Neutralization) 38 4-2.4 單源抗體之亞型分析(Isotyping) 38 4-2.5單源抗體之純化、定量與酵素標示(HRP-conjugation) 39 第三節 rHA∆TM/H5N2抗原之製備與確認 39 4-3.1重組rHA∆TM/H5N2基因選殖 39 4-3.1.1 PCR增幅1209/H5N2 full length HA gene 39 4-3.1.2建構重組轉移載體 39 4-3.1.3重組載體pBacPAK8-HA∆TM、pcDNA-HA∆TM之確認 39 4-3.1.4 重組載體pBacPAK8-HA∆TM、pcDNA-HA∆TM之定序 40 4-3.2桿狀病毒力價測定(50% Tissue Culture Infective Dose, TCID50) 40 4-3.3 Bac-H5抗原盤之免疫螢光染色 40 4-3.4 rHA∆TM/H5N2蛋白濃縮、純化與定量 40 4-3.5 rHA∆TM/H5N2西方墨點法(western blot) 41 4-3.4建立stable cell line 試驗 42 4-3.4.1 H5HA/HEK 之免疫螢光染色 42 4-3.4.2 rHA∆TM /HEK蛋白純化與確認 42 第四節 建立αHA MAbs-based blocking ELISA 43 4-4.1以indirect ELISA測試αHA MAbs 特異性與敏感性 43 4-4.2最佳化αHA MAbs-based blocking ELISA 43 4-4.3 篩選最佳抗體組合(MAbs pairs) 44 4-4.3.1挑選OD值較高的MAbs pairs 44 4-4.3.2 以少量雞血清最佳化αH5 MAbs-based blocking ELISA 44 4-4.3.3 以敏感性與特異性篩選出最佳組合抗體 44 4-4.4 血清檢體之檢測 45 4-4.4.1 rHA∆TM/H5N2 與1209/H5N2 blocking ELISAcut-off value、敏感性特異性分析 45 第五章 討論 47 第一節抗體製備與特性鑑定 47 5-1.1 αH5 MAbs特型鑑定 48 第二節 rHA∆TM/H5N2蛋白表現 49 第三節MAbs-based blocking ELISA建立與測試 50 | |
dc.language.iso | zh-TW | |
dc.title | 應用桿狀病毒表現HA以阻斷型ELISA檢測抗H5抗體 | zh_TW |
dc.title | H5 antibody detection by blocking ELISA using baculovirus expression HA | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王金和(Ching-Ho Wang),鄭明珠(Ming-Chu Cheng) | |
dc.subject.keyword | 禽流感病毒,血球凝集素,桿狀病毒,單源抗體,阻斷型酵素免疫吸附法, | zh_TW |
dc.subject.keyword | Avian influenza virus,hemagglutinin,baculovirus,monoclonal antibody,blocking ELISA, | en |
dc.relation.page | 91 | |
dc.identifier.doi | 10.6342/NTU201800156 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-02-12 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-1.pdf 目前未授權公開取用 | 12.47 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。