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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 獸醫專業學院
  4. 獸醫學系
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57202
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???org.dspace.app.webui.jsptag.ItemTag.dcfield???ValueLanguage
dc.contributor.advisor王金和(Ching-Ho Wang)
dc.contributor.authorTing-Hsiang Hsiaoen
dc.contributor.author蕭庭翔zh_TW
dc.date.accessioned2021-06-16T06:37:43Z-
dc.date.available2016-08-01
dc.date.copyright2014-08-01
dc.date.issued2014
dc.date.submitted2014-07-31
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鄭宛芯。2005。台灣家禽網狀內皮症病毒分離鑑定與序列分析。國立台灣大學獸醫學研究所碩士論文。pp. 1-103。
陳秋麟及謝快樂。1987。台灣鵝的網狀內皮增殖病及其病原性研究。台灣省畜牧獸醫學會會報。49:1-19。
吳乃慧。2009。家禽網狀內皮增生症病毒酵素連結免疫吸附法診斷試劑之開發。國立台灣大學獸醫學研究所碩士論文。pp. 1-146。
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57202-
dc.description.abstract家禽網狀內皮增生症病毒(avian reticuloendotheliosis virus, REV),可感染多種鳥禽類,包括雞、鴨、鵝、雉及孔雀等。被感染之家禽並未出現明顯病徵,故於台灣家禽疾病中較不被重視,但其卻會引起飼養場重大的經濟損失,於現場尚未有有效預防及控制之方法,為此需開發有效檢測REV之方法。本研究之目的為利用桿狀病毒/昆蟲表現系統進行REV封套蛋白質之真核表現,並評估其重組蛋白質應用於抗體之酵素連結免疫吸附法之發展潛力。以台灣病毒株goose/3410/06之封套序列作為模板,以此建構帶有封套基因之baculovirus-env重組桿狀病毒並進一步感染Sf9昆蟲細胞,收取受感染之昆蟲細胞,進行西方點墨法分析可檢測到符合預期大小約62 kDa位置之重組封套蛋白質,並通過鎳離子親和力管柱進行純化。將重組封套蛋白質塗鍍作為抗原,以棋盤格方式獲得間接型ELISA之最佳化條件,並以血清中和試驗作為抗體檢測之金標準,比較182個田間樣本血清,其中種雞138隻及台灣土雞44隻,結果顯示重組封套蛋白質用於間接型ELISA之敏感性為88.5%及特異性為97.7%。其於敏感性及特異性皆有相當不錯之結果,表示若應用於現場初步診斷使用,應具有良好之發展潛力。額外與實驗室先前所研究以大腸桿菌表現重組封套蛋白質之阻斷行ELISA進行比較,以相同樣本進行測試,結果顯示敏感性為84.6%及特異性為95.3%。此兩種方式所檢測結果相似,但本實驗所發展之間接型ELISA具有所需使用樣本量低、二抗取得容易及成本低等優點。zh_TW
dc.description.abstractReticuloendotheliosis virus (REV) infects variety animals including chickens, ducks, geese, pheasants, peafowl and other birds. REV is ignored in Taiwan because the infected poultry without obvious clinical signs. REV causes severe economic losses in commercial poultry, such as runting disease syndrome and immunosuppression. Currently, the disease lacks effective prevention and control in the field. The purpose of this study is to develop a rapid, reliable, convenient and economic method for detecting REV antibody. We use baculovirus/insect cells expression system to express REV envelope protein, and to evaluate the potential of the recombinant protein to be used as the antigen in enzyme linked-immunosorbent assay (ELISA). To use the laboratory isolated Taiwan strain goose/3410/06 as the cloning template. The full-length of REV envelope was cloned into bacmid vector and then the constructed recombinant baculoviruse-env was infected the Sf9 insect cells. The infected insect cells showed expected recombinant envelope protein in western blotting which size was about 62 kDa. The expressed recombinant envelope protein was purified by Ni-NTA (nitrilotriacetic acid) column. The purified recombinant envelope protein used as the coated antigen. Then, we test optimization condition of indirect ELISA by checkerboard method. Using 182 field samples (138 broilers and 44 Taiwan Country Chickens) to compare virus neutralization tested as the gold standard with the indirect ELISA. The result showed the sensitivity and the specificity of the indirect ELISA were 88.5% and 97.7%, respectively. Both values were good enough to screen detection in the field. The indirect ELISA of recombinant envelope protein possesses potential of development. Additional, we used blocking ELISA which envelope protein was expressed by E. coli to compare with the result of indirect ELISA. The result showed the sensitivity and the specificity were 84.6% and 95.3%, respectively. The two kind of methods displayed similar result, but the indirect ELISA possess advantages of lower usage amount of samples, easier obtainable secondary antibody and cheaper.en
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dc.description.tableofcontents口試委員會審定書 i
致謝 ii
摘要 iii
Abstract iv
目錄 vi
表次 xii
圖次 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 病原學 3
2.2.1 病毒型態與特性 3
2.2.2 病毒分類 4
2.2.3 病毒基因體 4
2.2.4 致癌基因 5
2.2.5 病毒蛋白質 5
2.2.6 病毒複製 6
第三節 宿主 7
2.3.1 宿主範圍 7
2.3.2 自然宿主 8
第四節 流行病學 8
2.4.1 傳播途徑 8
2.4.1.1 水平傳播 8
2.4.1.2 垂直傳播 9
2.4.1.3 受汙染之生物材料 10
2.4.2 台灣狀況 10
第五節 致病機轉與病理變化 11
2.5.1 潛伏期 11
2.5.2 臨床症狀 11
2.5.3 病理變化 12
第六節 實驗室診斷 13
2.6.1 病毒分離 13
2.6.2 分子生物學診斷 13
2.6.3 血清學診斷 14
2.6.4 病毒之預防與控制 14
第七節 桿狀病毒表現系統 15
2.7.1 蛋白質表現系統 15
2.7.2 桿狀病毒 16
2.7.3 桿狀病毒表現系統 16
2.7.4 轉譯後修飾作用 18
第八節 單株抗體 18
第九節 酵素連結免疫吸附法 19
2.9.1 酵素連結免疫吸附法介紹 19
2.9.2 本研究相關ELISA 20
2.9.2.1 間接型酵素連結免疫吸附分析法(indirect ELISA) 20
2.9.2.2 阻斷型酵素連結免疫吸附分析法(blocking ELISA) 20
第三章 材料與方法 21
第一節 REV病毒增殖及力價測定 21
3.1.1 病毒來源 21
3.1.2 病毒增殖 21
3.1.2.1 細胞培養 21
3.1.2.2 病毒增殖 22
3.1.3 病毒力價測定 22
3.1.3.1 病毒接種 22
3.1.3.2 計算細胞數目 23
3.1.3.3 計算病毒力價 23
3.1.4 血清中和試驗 23
3.1.4.1 樣本來源 23
3.1.4.2 血清中和試驗 23
第二節 重組病毒蛋白質之建構與表現 24
3.2.1 病毒封套基因之bacmid-env建構 24
3.2.2 Bacmid-env序列確認 26
3.2.2.1 抽取重組Bacmid 26
3.2.2.2 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 26
3.2.2.3 電泳分析 27
3.2.2.4 封套基因產物純化及定序 28
3.2.3 昆蟲細胞與桿狀病毒 28
3.2.4 重組桿狀病毒之製備 29
3.2.4.1 Bacmid-env之轉染作用 29
3.2.4.2 轉染作用之確認 29
3.2.4.3重組病毒Baculovirus-env增殖 30
3.2.4.4 病毒力價測定 30
3.2.5 重組蛋白質表現及純化 30
3.2.5.1 重組蛋白質表現及最佳化 30
3.2.5.2 重組蛋白質純化 31
3.2.5.3 蛋白質濃縮 32
3.2.5.4 SDS-PAGE與Western blotting分析 32
3.2.5.5 蛋白質定量 33
3.2.6 以原核系統表現重組封套蛋白質 34
3.2.6.1 病毒來源 34
3.2.6.2重組封套基因pET-32b-env之建構 34
3.2.6.3 重組封套蛋白質表現 34
3.2.6.4 透析 35
第三節 單株抗體 35
3.3.1 單株抗體之製備 35
3.3.2 純化單株抗體 36
3.3.2.1 以Protein A Sepharose純化抗體 36
3.3.2.2 單株抗體定量 36
第四節 酵素連結免疫吸附法之應用 36
3.4.1 桿狀病毒表現之重組封套蛋白的間接型ELISA 36
3.4.1.1 重組封套蛋白質之辨識能力 36
3.4.1.2 間接型ELISA之條件最佳化 37
3.4.1.3 間接型ELISA製備 37
3.4.1.4 計算間接型ELISA之cut-off值 38
3.4.1.5 敏感性與特異性分析 38
3.4.2 原核表現之重組封套蛋白質的阻斷型ELISA 39
3.4.2.1 單株抗體標示過氧化氫(horseradish peroxidase, HRP) 39
3.4.2.2 阻斷型ELISA之條件最佳化 39
3.4.2.2.1 阻斷型ELISA之條件 39
3.4.2.2.2阻斷型ELISA最佳化 39
3.4.2.3 阻斷型ELISA製備 40
3.4.2.4 計算阻斷型ELISA之cut-off值 40
3.4.2.5 敏感性與特異性分析 40
第四章 結果 41
第一節 REV病毒增殖及力價測定 41
4.1.1 病毒增殖 41
4.1.2 病毒力價測定 41
4.1.3 使用田間樣本進行血清中和試驗 41
第二節 重組病毒蛋白質之建構與表現 41
4.2.1 重組bacmid-env質體確認 41
4.2.2 重組桿狀病毒(baculovirus-env)製備 42
4.2.3 重組封套蛋白質表現及確認 42
4.2.4重組蛋白質表現條件最佳化 42
4.2.5 重組蛋白質純化 43
4.2.6 以原核系統表現重組封套蛋白質 43
4.2.6.1重組封套蛋白質表現與確認 43
4.2.6.2 單株抗體定量 44
第三節 酵素連結免疫吸附法之應用 44
4.3.1 桿狀病毒表現之重組封套蛋白的間接型ELISA 44
4.3.1.1 間接型ELISA之塗鍍抗原濃度及樣本稀釋倍數 44
4.3.1.2 以Receiver operator characteristic curve (ROC curve)畫出間接型ELISA最佳敏感性及特異性 44
4.3.1.3 間接型ELISA之敏感性與特異性分析 45
4.3.2 原核表現之重組封套蛋白質的阻斷型ELISA 45
4.3.2.1 阻斷型ELISA之塗鍍抗原濃度及tracer稀釋倍數 45
4.3.2.2 阻斷型ELISA條件最佳化 46
4.3.2.3 以Receiver operator characteristic curve (ROC curve)畫出阻斷型ELISA最佳敏感性及特異性 46
4.3.2.4 阻斷型ELISA之敏感性與特異性分析 46
4.3.3 間接型ELISA與阻斷型ELISA之敏感性與特異性分析 47
4.3.4 三種檢測方式比較 47
第五章 討論 48
第六章 參考文獻 54
表一、本實驗所使用之特異性引子 70
表二、本實驗所使用之樣本資料來源 71
表三、以血清樣本畫出ROC curve並計算間接型ELISA之cut-off值 73
表四、間接型Env-ELISA與血清中和試驗之結果比較 75
表五、以血清樣本畫出ROC curve並計算阻斷型ELISA之cut-off值 76
表六、阻斷型Env-ELISA與血清中和試驗之結果比較 79
表七、間接型Env-ELISA與阻斷型Env-ELISA之結果比較 80
表八、血清中和試驗、間接型ELISA及阻斷型ELISA之檢測結果比較 81
圖一、反轉錄病毒顆粒之結構及帶有蛋白質之示意圖 82
圖二、ELISA方式之示意圖 83
圖三、REV(goose/3410/06)病毒增殖 84
圖四、測定病毒之力價(TCID50) 85
圖五、本實驗所使用之載體 86
圖六、確認重組之bacmid-env 87
圖七、確認重組桿狀病毒之形成及複製 88
圖八、病毒斑試驗(virus plaque assay) 89
圖九、昆蟲細胞表現重組封套蛋白質之情形 90
圖十、重組蛋白質表現條件最佳化 91
圖十一、重組封套蛋白質之純化 92
圖十二、E. coli表現重組封套蛋白質之情形 93
圖十三、以棋盤格方式找出最適之間接型ELISA的塗鍍抗原濃度及樣本稀釋倍數 94
圖十四、以Receiver operator characteristic curve(ROC curve)分析間接型ELISA之最佳敏感性及特異性 95
圖十五、間接型ELISA之敏感度分析 96
圖十六、間接型ELISA之特異性分析 97
圖十七、以棋盤格方式找出最適之阻斷型ELISA的塗鍍抗原濃度及tracer稀釋倍數 98
圖十八、以Receiver operator characteristic curve(ROC curve)分析阻斷型ELISA之最佳敏感性及特異性 99
圖十九、阻斷型ELISA之敏感度分析 100
圖二十、阻斷型ELISA之特異性分析 101
dc.language.isozh-TW
dc.subject桿狀病毒/昆蟲細胞表現zh_TW
dc.subject家禽網狀內皮增生症病毒zh_TW
dc.subject間接型酵素連結免疫吸附法 (ELISA)zh_TW
dc.subject重組封套蛋白質zh_TW
dc.subjectbaculovirus/insect cells expressionen
dc.subjectreticuloendotheliosis virusen
dc.subjectindirect enzyme linked immunosorbent assay (ELISA)en
dc.subjectrecombinant envelope proteinen
dc.title桿狀病毒表現家禽網狀內皮增生症病毒封套蛋白質之酵素連結免疫吸附法檢測抗體開發zh_TW
dc.titleDetection of reticuloendotheliosis antibody by enzyme-linked immunosorbent assay with baculovirus expressed envelope proteinen
dc.typeThesis
dc.date.schoolyear102-2
dc.description.degree碩士
dc.contributor.oralexamcommittee謝快樂(Happy K. Shieh),沈瑞鴻(Jui-Hung Shien),張伯俊(Poa-Chun Chang),陳慧文(Hui-Wen Chen)
dc.subject.keyword家禽網狀內皮增生症病毒,桿狀病毒/昆蟲細胞表現,重組封套蛋白質,間接型酵素連結免疫吸附法 (ELISA),zh_TW
dc.subject.keywordreticuloendotheliosis virus,baculovirus/insect cells expression,recombinant envelope protein,indirect enzyme linked immunosorbent assay (ELISA),en
dc.relation.page101
dc.rights.note有償授權
dc.date.accepted2014-07-31
dc.contributor.author-college獸醫專業學院zh_TW
dc.contributor.author-dept獸醫學研究所zh_TW
Appears in Collections:獸醫學系

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