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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王金和 | |
dc.contributor.author | Wen-Tzu Tsai | en |
dc.contributor.author | 蔡文慈 | zh_TW |
dc.date.accessioned | 2021-06-15T05:15:05Z | - |
dc.date.available | 2015-07-22 | |
dc.date.copyright | 2010-07-22 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-21 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46550 | - |
dc.description.abstract | 多種家禽對網狀內皮增生病毒(Reticuloendotheliosis virus, REV) 具有感受性,其臨床症狀大多輕微主要造成雞隻免疫抑制,生長不良等,少部分會有慢性淋巴腫瘤,其中以runting syndrome對家禽產業造成的經濟損害最大。過去資料顯示台灣地區REV感染情形相當普遍,然而缺乏長期之REV陽性率監測資料。因此本研究之目的為調查台灣土雞REV的盛行率及建立兩種塗鍍重組蛋白質以檢測REV抗體的ELISA。在調查台灣土雞REV的盛行率方面,利用偵測前病毒核酸方式了解台灣土雞REV的陽性率,共計239個屠宰場來源樣本及170個台灣種土雞樣本前病毒核酸偵測結果顯示,屠宰場樣本的REV前病毒場陽性率均高於75%。而三種台灣種土雞品系之REV前病毒檢測結果顯示名古屋系之REV前病毒陽性率(11.1%)低於S系(38.5%)。在兩種檢測抗體的ELISA方面,塗鍍重組蛋白質p30的間接型(indirect) ELISA,與重組封套蛋白質env的阻斷型(blocking) ELISA,以中和試驗結果當作金標準(gold standard)時,計算出此p30 indirect ELISA之敏感性為92% (46/50),特異性為94.8% (91/96)。對照以塗鍍重組蛋白質env做為抗原的阻斷型ELISA敏感性為90% (45/50)特異性為94.8% (91/96)。相較於商品化之ELISA kit敏感性為94% (47/50)、特異性為93.7% (90/96)。由於三種ELISA判讀結果與中和試驗均具有高相關性(p30 indirect ELISA 80.3%, env blocking ELISA 80.1%, I牌商業化套組80.5%), 故我們推論此兩種塗鍍重組蛋白質以檢測REV抗體之ELISA可做為未來家禽田間調查及快速診斷工具之選擇。 | zh_TW |
dc.description.abstract | Naturally occurring avian reticuloendotheliosis virus (REV) infections is common in many domestic fowls. REV causes immunosupression, runting disease, and chronic lymphoma, resulting in a great economic loss. Although REV infection was common, no monitoring data were reported in Taiwan. Thus, the aims of this study were to investigate the REV infection in Taiwan Country chickens and to establish ELISAs for REV antibody detection. For REV monitoring in Taiwan Country chickens, 239 blood samples from a slaughter house and 170 blood samples from a breeder flock were tested for the presence of REV provirus by PCR. The results revealed that REV prevalence in Taiwan Country chicken flocks ranged from 75% to 100% in different kinds of chickens from a slaughterhouse. In the breeder flock, Nogoya breed had a prevalence of 11.1% (4/36) and S breed 38.5% (27/70). Two ELISAs were developed for REV antibody detection. One of the ELISAs was an indirect ELISA coating with recombinant protein p30 and the other was a blocking ELISA with expression env protein. The sensitivity and specificity of p30 ELISA were 92% (46/50) and 94.8% (91/96) and those of blocking ELISA were 90% (45/50) and 94.8% (91/96), respectively. Comparing with a commercial ELISA kit, the three ELISAs were correlated with neutralization result(p30 indirect ELISA 80.3%, env blocking ELISA 80.1%, and a commercial ELISA 80.5%). In conclusion, these two new ELISAs can be applied to detect anti-REV antibody in the field. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:15:05Z (GMT). No. of bitstreams: 1 ntu-99-R97629022-1.pdf: 864118 bytes, checksum: b97f0fd43b6b5afc4835e28a9c8fdb52 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目錄
誌謝........…………………………………………………………………………………i 目錄…………………………………………………………………………………… iii 表次…………………………………………………………………………………......ix 圖次…………………………………………………………………………………….. x 中文摘要………………………………………………………………………………..xi 英文摘要…………………………………………………………………………….....xii 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 第三節 病毒結構與功能 ………………………………………………………….4 2-3.1 病毒基因體…………………………………………………………..........4 2-3.2 病毒蛋白質…………………………………………………………..........5 第四節 病毒複製…………………………………………………………………...6 2-4.1 形成provirus……………………………………………………………. ..6 2-4.2 病毒的轉錄與轉譯………………………………………………………..6 第五節 病毒物理化學特性………………………………………………………...7 第六節 宿主………………………………………………………………………...7 2-6.1 自然與實驗感染宿主……………………………………………………..7 2-6.2 病毒複製之宿主特異性…………………………………………………..7 第七節 傳播途徑…………………………………………………………………..8 第八節 臨床症狀與病理變化……………………………………………………..9 2-8.1 潛伏期……………………………………………………………………..9 2-8.2 臨床症狀…………………………………………………………………10 2-8.3 病理變化…………………………………………………………………10 第九節 診斷方式………………………………………………………………….11 2-9.1 病毒分離…………………………………………………………………11 2-9.2 分子生物學診斷…………………………………………………………12 2-9.3 血清學診斷………………………………………………………………12 2-9.3.1 單株抗體……………………………………………………………….12 2-9.3.2 酵素連結免疫吸附法(ELISA)……………………………………..13 第十節 流行病學 ………………………………………………………………...14 2-10.1 台灣地區情形…………………………………………………………..14 第三章 材料方法…………………………………………………………………….15 第一節 以聚合鏈鎖反應(PCR)偵測REV前病毒(provirus)核酸………....15 3-1.1 材料來源…………………………………………………………………15 3-1.1.1 部分台灣種土雞品系REV感染之檢測………………………………15 3-1.1.2 屠宰場樣本…………………………………………………………….15 3-1.1.3 自屠宰場採集血液樣本……………………………………………….15 3-1.1.4 樣材處理……………………………………………………………….16 3-1.2 DNA萃取………………………………………………………………...16 3-1.3 以PCR增幅REV provirus核酸…………………………………………16 3-1.4 洋菜膠電泳分析…………………………………………………………17 第二節 REV病毒中和試驗………………………………………………………17 3-2.1 材料來源…………………………………………………………………17 3-2.1.1 血液樣本採樣………………………………………………………….17 3-2.2 病毒力價測定……………………………………………………………18 3-2.2.1 細胞培養……………………………………………………………….18 3-2.2.2 計算細胞數目………………………………………………………….19 3-2.2.3 病毒增殖……………………………………………………………….19 3-2.2.4 接種病毒……………………………………………………………….19 3-2.2.5 聚合酶鍊反應(PCR)……………………………………………….20 3-2.2.6 計算病毒力價………………………………………………………….20 3-2.3 血清中和試驗……………………………………………………………20 3-2.3.1 中和反應……………………………………………………………….21 第三節 單株抗體………………………………………………………………….21 3-3.1 抗原製備…………………………………………………………………21 3-3.1.1 重組蛋白鞘蛋白(capsid protein; p30)的表現與確認………………21 3-3.1.1.1 重組質體之抽取……………………………………………………..22 3-3.1.1.2 重組質體之確認與定序……………………………………………..22 3-3.1.1.3 重組質體之序列分析………………………………………………..22 3-3.1.1.4 以原核表現系統表現重組蛋白……………………………………..23 3-3.1.1.5 重組質體之抽取……………………………………………………..23 3-3.1.1.6 重組質體之轉型作用(transformation) …………………………23 3-3.1.1.7 重組質體之表現 ……………………………………………………23 3-3.1.1.8 重組蛋白之電泳分析與確認 ………………………………………24 3-3.1.2 重組蛋白之純化……………………………………………………….25 3-3.1.3 蛋白質定量…………………………………………………………….25 3-3.1.4 純化之p30 重組蛋白電泳分析與確認………………………………26 3-3.2 單株抗體特性分析………………………………………………………26 3-3.2.1 單株抗體製備過程…………………………………………………….26 3-3.2.2 檢測單株抗體辨認全病毒能力……………………………………….27 3-3.2.2.1 病毒增殖……………………………………………………………..27 3-3.2.2.2 病毒濃縮與純化……………………………………………………..27 3-3.2.2.3 以西方墨點法檢測單株抗體辨認全病毒能力……………………..27 3-3.3 單株抗體純化……………………………………………………………28 3-3.3.1 以蛋白質A瓊脂糖凝膠(Protein A Sepharose)純化單株抗體 ……28 3-3.4 透析純化之單株抗體……………………………………………………29 3-3.5 蛋白質定量………………………………………………………………29 第四節 酵素連結免疫吸附分析法……………………………………………….29 3-4.1 介紹偵測抗體的indirect ELISA ………………………………………..29 3-4.1.1 塗鍍重組鞘蛋白(capsid protein; p30)之間接型 (indirect)ELISA….30 3-4.1.1.1 檢視商品化多株抗體與重組蛋白結合能力………………………..30 3-4.1.1.2 以棋盤方格法找出indirect ELISA之最佳化條件 ………………..31 3-4.2 偵測抗體的阻斷型(blocking) ELISA…………………………………...31 3-4.2.1 塗鍍p30 重組蛋白之間接阻斷型(indirect blocking)ELISA ………31 3-4.2.1.1 以棋盤格方法決定p30 indirect blocking ELISA最佳塗鍍條件…..31 3-4.2.1.2 間接阻斷型ELISA 最佳化條件……………………………………32 3-4.2.2 塗鍍重組封套蛋白(env)重組蛋白之阻斷型(blocking)ELISA……..33 3-4.2.2.1 重組封套蛋白(env)的表現與確認……………………………….33 3-4.2.2.1.1 重組質體之確認與定序 ………………………………………….33 3-4.2.2.1.2 重組質體之序列分析……………………………………………...33 3-4.2.2.1.3 以原核表現系統表現重組封套蛋白(env)………………………34 3-4.2.2.1.3.1 重組質體之抽取…………………………………………………..34 3-4.2.2.1.4 重組質體之轉型作用(transformation)………………………….34 3-4.2.2.1.5 重組質體之表現 ………………………………………………….34 3-4.2.2.1.6 重組蛋白之電泳分析與確認…………….………………………..35 3-4.2.2.2 恢復重組蛋白構型(refolding)…………………………………….36 3-4.2.2.3 檢視商品化多株抗體與重組蛋白結合能力………………………..36 3-4.2.2.4 阻斷型ELISA 最佳化條件 ………………………………………..37 3-4.3 分析塗鍍重組蛋白p30 之 indirect ELISA及env之bELISA 之敏感性 特異性…………………………………………………………………….37 3-4.3.1 雞隻田間血清樣本收集……………………………………………….38 3-4.3.1.1 indirect ELISA操作流程…………………………………………….38 3-4.3.1.2 A450 值轉換成S/P ratio……………………………………...………38 3-4.3.1.3 indirect ELISA cut-off值分析……………………………………….38 3-4.3.2.1 env bELISA操作流程………………………………………………..39 3-4.3.2.2 env bELISA 之cut-off值分析………………………………………39 3-4.3.2.3 A450 值轉換成血清抑制百分比…………………………………….39 3-4.3.3.1 商品化REV抗體檢測套組 ………………………………………...39 3-4.3.3.2 A450 值轉換成S/P ratio……………………………………………...40 3-5 Receiver operating characteristic curves決定最佳敏感度及特異度之方 法…………………………………………………………………………….40 3-6 敏感度及特異度之計算…………………………………………………...40 第四章 結果………………………………………………………………………….41 第一節 病毒核酸之檢測………………………………………………………….41 4-1.1 偵測前病毒核酸…………………………………………………………..41 第二節 REV病毒中和試驗……………………………………………………42 4-2.1 病毒力價測定……………………………………………………………42 4-2.2 以田間血清進行中和試驗………………………………………………42 第三節 單株抗體………………………………………………………………….43 4-3.1 抗原製備…………………………………………………………………43 4-3.1.1 以原核系統表現REV部分鞘蛋白(p30)………………………….43 4-3.1.2 重組蛋白p30之純化與確認…………………………………………..44 4-3.2 病毒濃縮與純化…………………………………………………………44 4-3.3 以西方墨點法測定單株抗體辨認全病毒之能力………………………44 4-3.4 以塗鍍不同抗原之indirect ELISA測定單株抗體特異度……………..45 4-3.5 單株抗體純化與定量……………………………………………………45 第四節 偵測REV 抗體之ELISA………………………………………………..45 4-4.1 間接型ELISA……………………………………………………………45 4-4.1.1 塗鍍重組鞘蛋白p30 之間接型(indirect)ELISA…………………..45 4-4.1.2 檢視商品化多株抗體與重組蛋白結合能力………………………….45 4-4.2 阻斷型ELISA……………………………………………………………45 4-4.2.1 塗鍍p30 重組蛋白之間接阻斷型(indirect blocking)ELISA ………45 4-4.2.1.1 以棋盤方格法找出間接阻斷型ELISA最佳化條件……………….46 4-4.2.2 塗鍍重組封套蛋白(env)重組蛋白之阻斷型(blocking)ELISA……46 4-4.2.2.1 以原核系統表現REV封套蛋白(env)之表現與確認……………46 4-4.2.2.2 恢復重組蛋白構型(refolding)…………………………………….47 4-4.2.2.3 檢視商品化多株抗體與重組蛋白結合能力. ………………………47 4-4.3 以中和試驗做為金標準比較三種不同ELISA之敏感性、特異性與一致 性………………………………………………………………………….47 4-4.3.1 塗鍍重組鞘蛋白p30 之間接型(indirect)ELISA…………………..47 4-4.3.1.1 p30 indirect ELISA cut-off值之計算……………………………….47 4-4.3.1.2 以Receiver operating characteristic curve (ROC Curve)畫出p30 indirect ELISA最佳敏感性及特異性……………………………….48 4-4.3.1.3 p30 indirect ELISA之敏感性、特異性及一致性之分析………….48 4-4.3.2 塗鍍重組封套蛋白(env)重組蛋白之阻斷型(blocking)ELISA…..49 4-4.3.2.1 env bELISA cut-off值之計算……………………………………...49 4-4.3.2.2 以Receiver operating characteristic curve (ROC Curve)畫出env bELISA最佳敏感性及特異性……………………………………….49 4-4.3.2.3 env bELISA之敏感性、特異性及一致性之分析…………………..49 4-4.3.3 商品化REV抗體檢測套組之敏感性、特異性及一致性之分析……50 第五章 討論………………………………………………………………………….51 第六章 參考文獻…………………………………………………………………….58 表次 Table1. 本論文所使用之特異性引子列表…………………………………………...71 Table.2 以146個血清樣本畫出ROC curve並計算p30 indirect ELISA 之cut-off value………………………………………………………………………….72 Table 3. 以146個血清樣本畫出ROC curve並計算env bELISA之cut-off value…..74 Table 4. 比較兩種不同計算間接型ELISA cut-off值差異…………………………..76 Table 5. 比較兩種不同計算阻斷型ELISA cut-off值差異…………………………..76 Table 6. 比較三種ELISA與SN test之相關性……………………………………….76 Table 7. 以Spearman's Rank Correlation Coefficient方法比較三種ELISA間相關 性…………………………………………………………..………………….77 圖次 Figure 1. REV strain A 及strain T 基因結構示意圖。……………………………….78 Figure 2. p30重組蛋白表現之確認……………………………………………….......79 Figure 3. p30重組蛋白之純化……………………………………………...................81 Figure 4. 利用Western blot分析三株腹水(2C5、C10及7E8) 對重組蛋白、濃縮 純化後REV病毒株3410以及沒有insert之載體pET32b表現蛋白結合 特異度..…………………………………………….......................................83 Figure 5. 以塗鍍重組蛋白p30、全病毒REV 3410以及沒有insert之空pET32b 表現蛋白之indirect ELISA檢測三株單株抗體辨識病毒之能力...………84 Figure 6. 以rProtein A純化單株抗體之SDS-Page圖..…………………………….85 Figure 7. 以棋盤格方法找出最適合之p30 indirect ELISA條件……………………86 Figure 8. 以Receiver operating characteristic curves (ROC) 畫出p30 indirect ELISA 最佳敏感性及特異性……………………………………………………….86 Figure 9. 以棋盤格方法找出最適合之p30 bELISA條件...…………………………87 Figure 10. env重組蛋白表現之確認..…………………………………………...........88 Figure 11. env重組蛋白以不同濃度urea回溶之確認..……………………………...89 Figure 12. 以棋盤格方法檢測配合env bELISA最佳之tracer條件..………………90 Figure 13. 以Receiver operating characteristic curves (ROC) 畫出env bELISA最佳敏感性及特異性…………………………………………………………...90 Figure 14. p30 indirect ELISA之敏感性分析..……………….………………………91 Figure 15. p30 indirect ELISA之特異性分析………………………………………...91 Figure 16. env bELISA之敏感性分析…………………………………………….......92 Figure 17. env bELISA之特異性分析…………………………………………….......92 附錄 Appendix 1. 雲林縣屠宰場樣本資料…………………………………………….....93 | |
dc.language.iso | zh-TW | |
dc.title | 以酵素連結免疫吸附法偵測雞隻網狀內皮增生病抗體方法之確立 | zh_TW |
dc.title | Evaluation of Enzyme-Linked ImmunoSorbent Assays (ELISA) for Detecting Anti- Reticuloendotheliosis Antibody in Chickens | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 謝快樂,陳秋麟,沈瑞鴻 | |
dc.subject.keyword | 家禽,網狀內皮增生病毒,病毒中和試驗,病毒力價,單株抗體,間接型酵素連結免疫吸附法,間接阻斷型酵素連結免疫吸附法,阻斷型酵素連結免疫吸附法, | zh_TW |
dc.subject.keyword | Avian reticuloendotheliosis virus,Taiwan Country chicken,monoclonal antibodies,serum neutralization test,indirect ELISA,indirect blocking ELISA,blocking ELISA, | en |
dc.relation.page | 95 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-22 | |
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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