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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭益謙 | |
| dc.contributor.author | Chung-Kung Chen | en |
| dc.contributor.author | 陳俊光 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:52:08Z | - |
| dc.date.available | 2013-02-20 | |
| dc.date.copyright | 2011-02-20 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-02-14 | |
| dc.identifier.citation | Air, G.M., 1981. Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. Proc Natl Acad Sci U S A 78(12), 7639-7643.
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Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351(9101), 467-471. Zambon, M., 1999. Cell culture for surveillance on influenza. Dev Biol Stand 98, 65-71; discussion 73-64. Zanella, A., 2003. Avian influenza attributable to serovar H7N1 in light layers in Italy. Avian Dis 47(3 Suppl), 1177-1180. Zhang, A., Jin, M., Liu, F., Guo, X., Hu, Q., Han, L., Tan, Y., Chen, H., 2006. Development and evaluation of a DAS-ELISA for rapid detection of avian influenza viruses. Avian Dis 50(3), 325-330. Ziegler, A.F., Davison, S., Acland, H., Eckroade, R.J., 1999. Characteristics of H7N2 (nonpathogenic) avian influenza virus infections in commercial layers, in Pennsylvania, 1997-98. Avian Dis 43(1), 142-149. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48311 | - |
| dc.description.abstract | 家禽流行性感冒病毒 (Avian influenza virus, AIV) 除了造成養禽場重大經濟損失外，因其具人畜共通之風險，近年來為世界所關注，快速、敏感性及特異性高的診斷方法或檢測工具便有迫切需求，而單源抗體 (MAb) 憑著對抗原的高特異性及專一性，乃為生醫領域所廣泛應用。本研究室利用2003年台灣分離到的病毒株A/chicken/Taiwan/1209/03 (H5N2)，製備出抗AIV的MAb共3株，以血球凝集抑制試驗 (hemagglutination inhibition test, HI test) 及病毒中和試驗 (virus neutralization test, VN test) 發現，其中兩株UB-4a及YB-4a具有HI力價及病毒中和能力，且西方墨點法 (western blot assay) 結果顯示具有辨識病毒表面醣蛋白血球凝集素 (hemagglutinin, HA, 75 kDa) 的能力。另一株單源抗體WF-4b利用西方墨點法發現可辨識病毒核蛋白 (nucleoprotein, NP, 56 kDa)，且與10種HA亞型病毒感染的狗腎細胞 (Madin-Darby canine kidney, MDCK) 之免疫螢光染色(immunofluorescence assay, IFA)，均呈陽性反應，將其應用於實驗感染AIV雞隻免疫組織化學染色 (immunohistochemistry, IHC)，及原核系統 (prokaryotic) 所表現AIV重組NP蛋白質等皆可辨識，希望未來能利用WF-4b之特性，發展敏感性與特異性均高之診斷工具與方法，如ELISA、免疫組織化學染色等，或是應用於病毒學的相關研究。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:52:08Z (GMT). No. of bitstreams: 1 ntu-100-R97629031-1.pdf: 2119368 bytes, checksum: 9746d4f3348107138561f3b0aa876d34 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii Abstract iv 第一章 序言 8 第二章 文獻回顧 10 第一節 歷史背景 10 第二節 病毒之特性 12 2-2.1 病毒特徵 12 2-2.2 病毒型態、結構及功能 13 2-2.2.1 血球凝集素 (hemagglutinin, HA) 13 2-2.2.2 神經胺酸酶 (neuraminidase, NA) 14 2-2.2.3 核蛋白 (nucleoprotein, NP) 14 2-2.2.4 基質蛋白 (matrix protein, M) 15 2-2.2.5 非結構性蛋白 (nonstructural protein, NS) 15 2-2.2.6 聚合酶蛋白 (polymerase protein) 16 2-2.3 病毒複製 16 2-2.4 病毒的物理化學特性 17 2-2.5 病毒抗原變異性 17 第三節 實驗室診斷 18 2-3.1 偵測抗原 18 2-3.1.1 雞胚胎蛋增殖病毒 18 2-3.1.2 細胞培養 18 2-3.1.3 血球凝集試驗 (hemagglutination test, HA test) 19 2-3.1.4 反轉錄聚合酶鏈反應 (reverse transcription-polymerase chain reaction, RT-PCR) 19 2-3.1.5 抗原免疫偵測套組 (antigen detection immunoassay kit) 20 2-3.2 偵測抗體 20 2-3.2.1 瓊脂膠體免疫擴散法 (agar gel immunodiffusion, AGID) 20 2-3.2.2 血球凝集抑制試驗 (hemagglutination inhibition test, HI test) 20 2-3.2.3 神經胺酸酶抑制試驗 (neuraminidase inhibition test, NI test) 21 2-3.2.4 酵素連結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA) 21 第四節 單源抗體 22 2-4.1 歷史背景 22 2-4.2 篩選融合瘤細胞原理 22 2-4.3 單源抗體之應用 23 第三章 材料方法 24 第一節 家禽流行性感冒病毒 24 3-1.1 家禽流行性感冒病毒毒株 24 3-1.2 家禽流行性感冒病毒增殖及濃縮 24 3-1.3 反向遺傳技術 (reverse genetic technique) 製備家禽流行性感冒病毒 25 第二節 抗家禽流行性感冒病毒之單源抗體製備 25 3-2.1 實驗動物 25 3-2.2 免疫計畫 25 3-2.3 骨髓瘤細胞培養 26 3-2.4 細胞融合 26 3-2.5 融合瘤細胞培養 27 3-2.6 陽性親代融合瘤細胞篩選 27 3-2.6.1 免疫螢光染色法（IFA） 27 3-2.7 陽性融合瘤細胞單株化 28 3-2.8 小鼠腹水之生產 28 第三節 單源抗體 29 3-3.1 單源抗體之亞型分析 29 3-3.2 單源抗體之特性分析 29 3-3.2.1 病毒中和試驗 (virus neutralization test, VN test) 29 3-3.2.2 紅血球凝集素抑制試驗 (hemagglutination inhibition test, HI test) 30 3-3.2.3 免疫螢光染色法（IFA） 30 3-3.2.4 聚丙烯醯胺膠片電泳 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 及西方墨點法 (western blot assay) 30 第四節 MAb辨識家禽流行性感冒病毒核蛋白 (neucleoprotein, NP) 之確認 32 3-4.1 免疫組織化學染色 ( IHC) 32 3-4.2 家禽流行性感冒病毒NP蛋白基因選殖 33 3-4.2.1 增幅基因 33 3-4.2.1.1 引子 (primer) 設計 33 3-4.2.1.2 質體之萃取 33 3-4.2.1.3 PCR 增幅NP蛋白基因 34 3-4.2.1.4 NP蛋白基因之確認 34 3-4.2.1.5 NP蛋白基因之純化 35 3-4.2.2 重組質體之構築 35 3-4.2.2.1 限制酶酵素切割 (digestion) 35 3-4.2.2.2 接合反應 (ligation) 36 3-4.2.2.3 轉型作用（transformation） 36 3-4.2.2.4 確認重組質體 36 3-4.2.2.5 重組質體之定序 37 3-4.3 重組NP蛋白質之分析 38 3-4.3.1 重組NP蛋白質之表現 38 3-4.3.2 重組NP蛋白之純化 38 3-4.3.3 重組NP蛋白質之電泳分析與確認 39 第四章 結果 40 第一節 H5亞型家禽流行性感冒病毒單源抗體製備 40 4-1.1 H5病毒濃縮之結果 40 4-1.2 融合瘤細胞之篩選 40 第二節 H5亞型家禽流行性感冒病毒單源抗體特性分析 40 4-2.1 單源抗體亞型分析 40 4-2.2 單源抗體中和能力之分析 41 4-2.3 單源抗體血球凝集抑制能力之分析 41 4-2.4 免疫螢光染色法 ( IFA) 41 4-2.5 SDS-PAGE及西方墨點法 41 第三節 抗家禽流行性感冒病毒核蛋白之確認 42 4-3.1 NP蛋白基因之確認 42 4-3.2 重組質體之構築 42 4-3.3 重組NP蛋白質之表現與純化 42 4-3.4 重組NP蛋白質之SDS-PAGE及西方墨點法 43 第四節 單源抗體WF-4b應用於IHC 43 第五章 討論 44 參考文獻 61 附錄 74 | |
| dc.language.iso | zh-TW | |
| dc.title | 抗家禽流行性感冒病毒單源抗體之特性分析及應用 | zh_TW |
| dc.title | Characterization and Application of Monoclonal Antibodies against Avian Influenza Virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王金和,蔡向榮,李淑慧,陳啟銘 | |
| dc.subject.keyword | 家禽流行性感冒病毒,單源抗體,核蛋白,免疫螢光染色法,免疫組織化學染色, | zh_TW |
| dc.subject.keyword | Avian infl,uenza virus (AIV),monoclonal antibody (MAb),nucleoprotein (NP),immunofluorescence assay (IFA),immunohistochemistry (IHC), | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-02-14 | |
| 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 | |
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