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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 | Chin-Fang Ho | en |
dc.contributor.author | 何金芳 | zh_TW |
dc.date.accessioned | 2021-06-15T06:52:35Z | - |
dc.date.available | 2014-02-20 | |
dc.date.copyright | 2011-02-20 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-02-13 | |
dc.identifier.citation | Aldo D, Juan L, and Luis R. 2006. Vesicular diseases. In: Straw BE, Zimmerman JJ, D’ Allaire S, and Taylor DJ, editors. Diseases of swine. 9 ed. Iowa: Blackwell. p 528-531.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48324 | - |
dc.description.abstract | 豬水疱病 (swine vesicular disease, SVD) 是由豬水疱病病毒 (SVDV) 所引起的疾病。常見的臨床症狀以鼻吻部、唇部、舌頭以及四肢皮膚出現水疱為特徵,其臨床症狀與其它豬水疱性疾病難以辨別,如口蹄疫 (foot and mouth, FMD)、水疱性口炎 (vesicular stomatitis, VS) 及水疱疹 (vesicular exanthema, VE)。由於豬水疱病病灶與口蹄疫非常相似,被世界動物衛生組織 (Office International des Epizooties, World Organisation for Animal Health, OIE) 列為須通報之疾病,而我國列為甲類動物傳染病。此病毒於環境中十分穩定,且部分SVDV所造成的臨床症狀相當輕微而不易查覺,一旦爆發此疾病,容易造成嚴重的經濟損失,因此能夠正確且迅速的診斷對於疾病的控制十分重要。本研究主要目的是製備出抗SVDV單源抗體,以便進行快速診斷。利用台灣豬水疱病病毒分離株 (TW/99) 免疫小鼠,經免疫螢光抗體染色法 (IFA) 篩選後共製備出七株單源抗體,進行西方墨點法分析,其中一株單源抗體 (CH9-6b) 可特異性辨識以E. coli原核表現的豬水疱病病毒VP1。以病毒中和試驗檢測此單源抗體,結果發現單源抗體 (CH9-6b) 並無中和能力,推測其辨識的位置並非在病毒的中和決定位上。由於單源抗體對於抗原具有很高的特異性,利用所製備的單源抗體可檢測SVDV並得知其病毒特性,並可開發快速診斷SVDV之試劑,將快速診斷試劑應用於SVDV血清學調查。其餘六株單源 (CG12-1a, FG10-1a, FE5-4a, CD5-5b, HB3-1b, HB3-2b) 抗體則具有中和能力,這些抗SVDV單源抗體對於研究SVDV抗原結構、病毒的致病機制、流行病學、疾病診斷及研發疫苗皆有極大的助益。 | zh_TW |
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dc.description.tableofcontents | 目錄
口試委員會審定書 i 致謝 ii 中文摘要 iii Abstract iv 圖目錄 x 表目錄 xi 第一章 序言 1 第二章 文獻回顧 3 第一節 歷史背景與簡介 3 第二節 病毒特性與型態 3 2-2.1 病毒型態與結構 3 2-2.2 病毒血清學特性 4 2-2.3 病毒之增殖 5 2-2.4 病毒之抗原構造 5 2-2.5 病毒之RNA genome特性 6 2-2.6 病毒物理化學特性 7 第三節 流行病學 7 第四節 宿主 8 第五節 致病機轉 9 第六節 傳播途徑 9 第七節 臨床症狀 10 第八節 病變 10 第九節 診斷 11 第十節 控制與預防 11 第十一節 單源抗體 12 第三章 材料與方法 14 第一節 豬水疱病病毒製備 14 3-1.1 豬水疱病病毒株 14 3-1.2 細胞培養與繼代 14 3-1.3 豬水疱病病毒增殖 15 3-1.4 豬水疱病病毒力價測定 15 3-1.5 豬水疱病病毒濃縮 15 3-1.6 豬水疱病病毒純化 16 第二節 抗豬水疱病病毒之單源抗體製備 16 3-2.1 實驗動物 17 3-2.2 免疫計畫 17 3-2.3 骨髓瘤細胞培養 17 3-2.4 融合瘤製備 17 3-2.5 融合瘤篩選 18 3-2.5.1 HMT篩選 (Hypoxanthine methotrexate thymidine) 19 3-2.5.2 IFA篩選 (Immunofluorescence assay) 19 3-2.6 融合瘤細胞單株化 19 3-2.7 腹水之生產 20 第三節 單源抗體的特性分析 20 3-3.1 單源抗體亞型分析 (isotyping) 20 3-3.2 病毒中和試驗 (virus neutralization test, VN test) 21 3-3.3 免疫螢光抗體染色法 (immunofluorescent assay) 21 3-3.4 西方墨點法 (western blotting assay) 22 第四節 重組結構蛋白 (VP1) 之表現與確認 23 3-4.1 增幅VP1基因 23 3-4.1.1 病毒RNA萃取 23 3-4.1.2 primer之設計 24 3-4.1.3 RT-PCR (reverse transcriptase polymerase chain reaction) 25 3-4.1.4 PCR產物電泳分析 26 3-4.1.5 基因片段之純化 27 3-4.1.6 TA plasmid cloning 27 3-4.2 重組TA plasmid之選殖 28 3-4.2.1 重組TA plasmid之colony PCR 28 3-4.2.2 重組TA plasmid萃取 29 3-4.2.3 重組TA plasmid基因核酸定序 31 3-4.3 pET-32b表現載體基因選殖 31 3-4.3.1 限制酶酵素作用 31 3-4.3.2 pET-32b vector接合反應 33 3-4.3.3 重組pET-32b vector之轉型反應 (transformation) 33 3-4.3.4 重組pET-32b vector檢定 33 3-4.4 重組質體之表現與確認 34 3-4.4.1 重組質體之表現 34 3-4.4.2 重組蛋白之電泳分析與確認 35 3-4.4.3 檢測單源抗體與重組蛋白結合能力 36 3-4.4.4 重組蛋白之特異性分析與確認 36 3-4.4.5 重組蛋白之純化 37 3-4.4.6 重組蛋白之定量 37 第四章 結果 38 第一節 豬水疱病病毒製備 38 4-1.1 豬水疱病病毒力價測定 38 4-1.2 豬水疱病病毒純化與蛋白質定量 38 4-1.3 豬水疱病全病毒之電泳圖譜 38 第二節 抗豬水疱病病毒之單源抗體 39 4-2.1 單源抗體篩選 39 4-2.2 病毒中和試驗 39 4-2.3 單源抗體辨認全病毒之能力 39 4-2.3.1 西方墨點法 39 4-2.3.2 免疫螢光抗體染色法 40 4-2.4 單源抗體亞型分析 40 第三節 重組蛋白 (VP1) 之表現與確認 40 4-3.1 基因片段選殖與重組TA質體之確認 40 4-3.2 限制酶酵素作用 41 4-3.3 重組pET-32b vector檢定 41 4-3.4 重組蛋白 (VP1) 表現 41 4-3.5 重組蛋白之純化與定量 42 4-3.6 西方墨點法檢測重組蛋白與單源抗體之結合能力 42 4-3.7 重組蛋白之特異性分析與確認 42 第五章 討論 44 第六章 參考文獻 49 附錄 78 | |
dc.language.iso | zh-TW | |
dc.title | 抗豬水疱病病毒單源抗體的特性分析及其應用 | zh_TW |
dc.title | Characterization and Application of Monoclonal Antibodies against Swine Vesicular Disease Virus | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王金和,蔡向榮,陳啟銘,李淑慧 | |
dc.subject.keyword | 豬水疱,病,水疱,單源抗體,結構蛋白VP1,表現蛋白, | zh_TW |
dc.subject.keyword | Swine vesicular disease (SVD),vesicles,monoclonal antibodies,VP1,protein expression, | en |
dc.relation.page | 79 | |
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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