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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 蔡向榮 | |
| dc.contributor.author | Chih-Ming Hsu | en |
| dc.contributor.author | 許志明 | zh_TW |
| dc.date.accessioned | 2021-06-13T05:50:22Z | - |
| dc.date.available | 2006-09-20 | |
| dc.date.copyright | 2006-09-20 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-07 | |
| dc.identifier.citation | 第六章 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33971 | - |
| dc.description.abstract | Avian polyomavirus (APV)及Psittacine beak and feather disease virus (PBFDV)感染症，二者皆為鸚鵡類常見病毒性疾病。鳥多瘤病毒感染症 (APV)，又稱為小鸚哥病 (budgerigar fledgling disease; BFD)，首度於1981年被發現，屬於乳多瘤病毒科 (Papovaviridae)之鳥多瘤性病毒 (Avipolyomavirus)，主要引起年幼鸚哥之急性致死性疾病，其死亡率可高達100%。鸚鵡喙羽病毒感染症(PBFDV)，屬於環狀病毒科 (Circoviridae)之環狀病毒 (Circovirus)，可感染約六十種的野生及寵物鸚鵡。本研究使用聚合酶連鎖反應檢測台灣鸚鵡感染APV和PBFDV之情形，調查時間是從2002年至2005年，共檢驗包含22屬品種165件送檢樣本，結果APV檢測陽性率為15.2%，PBFDV檢測陽性率為41.2%，而混合感染陽性率為10.3%。將部份核酸產物經定序後，與基因庫 (GenBank)之序列比對，APV之VP1基因比對結果相似度為97.5-100%，APV另一個T antigen coding region片段核酸序列與基因庫之APV核酸序列比對，則相似度97.6-100%。PBFDV之ORF V1與基因庫核酸序列比對結果相似度為92.2-100%，另一個ORF C1與基因庫之比對結果相似度為83.3-100%。另以PCR與重組DNA技術，選擇APV-VP1之結構蛋白基因，以原核 (E. coli.)系統成功表現出與預期大小相當之重組蛋白(45 kDa)，此蛋白皆可被特異性之his-tag與抗APV抗體所辨識。利用此APV-VP1重組蛋白作為抗原，免疫BALB/c小鼠，並製備出具分泌抗APV-VP1重組蛋白抗體之融合瘤細胞，經由ELISA、Western blot篩選分析確認，共得8株融合瘤細胞的單株抗體。利用D10-6單株抗體製備Sandwich ELISA套組，用於檢測人工感染虎皮鸚鵡羽囊之APV抗原，並以PCR檢測比較其專一性及敏感性。以二種方法檢測之結果，並無統計學上的差異，証實此Sandwich ELISA可以用於感染APV的虎皮鸚鵡之診斷，並可進一步應用於快速免疫分析試紙。 | zh_TW |
| dc.description.abstract | Avian polyomavirus (APV) infection and psittacine beak and feather disease (PBFD) are the most common viral diseases of psittacine birds. APV was first isolated from budgerigars in the early 1980s. APV belongs to genes Avipolyomavirus of the family Papovaviridae and causes acute fatal disease in young budgerigars with 100% mortality rate. APV infection is also known as budgerigar fledgling disease. PBFDV, categorized into genes Circovirus of the family Circoviridae, affects over 60 species of wild and captive psittacine birds. In this study polymerase chain reaction (PCR) was applied to diagnosis either APV or PBFDV infections in psittacine birds of Taiwan. From 2002 to 2005, the positive rate of APV, PBFDV, and APV/PBFDV infection over 165 cases were 15.2%, 41.2%, and 10.3% respectively. APVs indicated over 97% nucleotide identity in VP1 and T antigen coding regions. PBFDVs had over 92.2% and 83.3% nucleotide identity in ORF V1 and ORF C1 sequences. Another aim of this study is to prepare Sandwich ELISA for detection of APV and to analyze the specificity and sensitivity of Sandwich ELISA. First of all, we applied prokaryotic (E. coli.) system to express the viral structural protein VP1. The expressed VP1 were specifically recognized by his-tag and anti-APV antibodies. The recombinant protein was used as an immunogen to inject BALB/c mice for preparation of hybridomas which produced anti-VP1 antibodies. Eight monoclonal antibodies secreting hybridomas were obtained as determined by ELISA and Western blot. After screening of these 8 monoclonal antibodies, one monoclonal antibody, D10-6, is used to develop the Sandwich ELISA for the detection of APV. No significant difference was formed between PCR and Sandwich ELISA at the sensitivity. The Sandwich ELISA could be applied for diagnosis of APV infected budgerigars, and could further be applied to immunoassay strip for the rapid diagnosis of APV infection in budgerigars. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T05:50:22Z (GMT). No. of bitstreams: 1 ntu-95-R93629040-1.pdf: 2081488 bytes, checksum: a70fab64704f417e6276ea54edcce680 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目錄
中文摘要 I 英文摘要 II 目錄 III 表次 VII 圖次 VIII 第一章 緒言 1 第二章 文獻探討 3 第一節 病毒特性及分類 3 第二節 致病性及感染途徑 6 第三節 臨床症狀 8 第四節 組織病理學變化 10 第五節 流行病學 11 第六節 診斷及治療 13 第七節 預防及控制 14 第八節 原核表現系統與其應用 16 第九節 細胞融合之原理 17 第十節 單株抗體的特性與應用 18 第三章 材料與方法 20 第一節 病材來源 20 1.1 病毒及細胞培養與病毒增殖 20 第二節 病毒核酸的萃取 20 2.1 核酸(DNA)之萃取 20 2.2 引子(primer)之選用 21 2.3基因序列分析 23 第三節 重組APV-VP1表現載體之構築 23 3.1基因增幅 23 3.1.1引子的設計 23 3.1.2聚合酶連鎖反應(PCR) 24 3.1.3 APV之PCR產物之純化 24 3.2構築含PCR產物的重組質體 24 3.2.1 Insert及載體的製備 24 3.2.2接合作用(ligation) 25 3.2.3重組質體之轉型作用(transformation) 25 3.2.4小量質體的製備 25 3.2.5質體確認 26 3.2.6製備完成載體送入表現宿主 26 第四節 病毒蛋白的表現及確認 26 4.1勝任細胞的製備 27 4.2病毒蛋白的表現 27 4.3表現蛋白質之純化 28 4.3.1以Ni2+-NTA親和管柱純化重組蛋白質 28 4.3.2純化APV-VP1重組蛋白濃度測定 29 4.4表現蛋白之確認 29 4.4.1表現蛋白之電泳分析與確認 29 4.4.2 His-Tag Western blot 30 4.4.3西方墨點法(Western blot) 30 4.4.4冷光檢測(Enhanced chemiluminescence detection；ECL) 31 4.4.5 高免血清的置備 31 第五節 單株抗體 31 5.1 BALB/c小鼠免疫計畫 31 5.1.1血清抗體檢測 32 5.2 細胞融合 32 5.2.1 骨髓癌細胞之培養 32 5.2.2 免疫小鼠之脾細胞收集 32 5.2.3 HAT及ELISA篩選 33 5.2.4 融合瘤細胞擴大培養 34 5.2.5 單株化 34 5.2.6 酵素連結免疫吸附試驗 (Enzyme-linked immunosorbent assay; ELISA) 34 5.3 單株抗體大量生產 35 5.3.1小鼠腹水產生法 35 5.3.2 Protein A 親和性管柱(Protein A affinity column)之純化抗體 35 5.3.3 雞血清抗體純化 36 5.4 單株抗體亞型分析 36 5.4.1 單株抗體同種型(isotypes)測定 36 5.4.2 過氧化氫酶標定單株抗體(Horseradish peroxidase conjugated MAbs) 36 5.4.3 三明治酵素連結免疫吸附試驗(Sandwich Enzyme-linked immunosorbent assay)之分析 37 5.4.4 定出cut off value 37 第六節 APV動物接種試驗 38 第四章 結果 40 第一節 聚合酶連鎖反應檢測結果 40 第二節 PCR產物定序與核酸序列及胺基酸序列比對 42 2.1 Avian polyomavirus 基因序列分析及胺基酸 序列分析 42 2.2 Psittacine beak and feather disease virus基因 序列及胺基酸序列分析 42 第三節 大腸桿菌表現系統之構築APV-VP1全長基因 43 3.1 APV-VP1 基因之選殖及表現載體的構築 43 3.2 重組APV-VP1蛋白之純化與確認 44 3.3 抗APV-VP1之抗血清製備結果 44 第四節 單株抗體的製備 45 4.1 融合瘤細胞篩選與單株化效價測定 45 4.2 單株抗體生產與純化 45 第五節 ELISA架構與分析 46 5.1 ELISA架構與分析 46 5.2 建構Sandwich ELISA以檢測APV-VP1 46 5.3 Sandwich ELISA應用於檢測APV之敏感度 47 5.4 應用於感染鸚鵡之羽毛檢體檢測 48 5.5 應用於人工感染鸚鵡之臟器檢測 48 第五章 討論 75 第六章 參考文獻 82 | |
| dc.language.iso | zh-TW | |
| dc.title | APV及PBFDV的分子分析與鸚鵡APV酵素免疫分析
套組之開發 | zh_TW |
| dc.title | Molecular analysis of APV and PBFDV and the development of a sandwich ELISA for the detection of APV infections in psittacine birds | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 郭應誠 | |
| dc.contributor.oralexamcommittee | 王汎熒,張紹光,蔡信雄 | |
| dc.subject.keyword | 鸚鵡, | zh_TW |
| dc.subject.keyword | APV,PBFDV, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-07 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| Appears in Collections: | 獸醫學系 | |
Files in This Item:
| File | Size | Format | |
|---|---|---|---|
| ntu-95-1.pdf Restricted Access | 2.03 MB | Adobe PDF |
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