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研究生:周和源
研究生(外文):CHOU HO YUAN
論文名稱:家禽傳染性支氣管炎病毒之序列分析、抗體力價評估技術及病毒定量方法之研究
論文名稱(外文):Study on the nucleotide sequence, evaluation of antibody titer, and virus quantitation of avian infectious bronchitis virus
指導教授:張伯俊
指導教授(外文):CHANG POA CHUN
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:傳染性支氣管炎病毒序列分析ELISA血球凝集抑制試驗病毒定量方法定量PCR
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家禽傳染性支氣管炎病毒(Avian infectious bronchitis virus,IBV),所引起的家禽傳染性支氣管炎(Avian infectious bronchitis,IB),是造成養雞事業經濟損失的主要原因之一。IBV之特性即為抗原性的經常改變,因此常有免疫效果不佳,或是抗體力價測定結果解釋不易的問題,而影響IB的防治。為了瞭解台灣IBV與世界上其它區域IBV之抗原性差異,我們決定了台灣IBV其基因體中可決定病毒血清型別特異性,並可引起中和抗體抗原決定位的棘突蛋白(spike protein)基因,以及包含有可引起細胞免疫抗原決定位的核蛋白衣蛋白(nucleocapsid protein)基因的序列,再與世界上已發表的其它IBV進行親緣樹分析比較。結果發現其相似性分別介於39.1-99.1%與55.0-99.3%,且台灣IBV分離株之S1序列與普遍使用作為疫苗的麻州型H120病毒株的相似性介於81.0~82.1%。另外,為了瞭解不同的IB抗體力價評估技術,即ELISA與血球凝集抑制試驗對於不同血清型抗體測定結果的關連性,我們將上述台灣IBV及疫苗株H120,分別免疫雞隻以製備抗血清。接著將抗血清進行ELISA與HI 試驗,利用這些測定結果進行比較研究。結果發現,商品化的ELISA套組並無法準確的反映出以台灣IBV分離株免疫所得之血清抗體力價,且以疫苗株H120免疫所得之血清抗體以台灣I型IBV製成的HA抗原所測定的HI力價也很低。最後,有鑑於傳統的病毒力價測定方式耗力費時,因此我們發展了一個運用TaqMan技術的定量RT-PCR之即時核酸偵測方法,以建立病毒力價(EID50與HA unit)與病毒基因體數量之間的關係。結果發現,此一技術之敏感度是一般RT-PCR的100倍;且1個EID50/0.2 mL的病毒基因體數量約為452到1000個;而1個HA單位/50 L的病毒基因體數量約為4.7 ×105到9.8 ×106個,依所使用的病毒株不同而有不同的結果。此一具有再現性、快速而簡便的技術可以彌補傳統病毒力價測定方式耗力費時的缺點。

摘要 …………......………………………………...... I
Abstract ...……………………..……………………………… II
目次 …..……………………..…………………………… III
表次 ……..…………………..…………………………… V
圖次 ………..………………..…………………………… VI
第一章 緒言………………………..………………………… 1
第二章 文獻探討………………………………………..…… 2
2-1 雞傳染性支氣管炎之背景…………………...……… 2
2-2 雞傳染性支氣管炎之特性…………………………… 3
2-3 雞傳染性支氣管炎病毒之特性……………………… 4
2-4 雞傳染性支氣管炎之診斷方法…...………………… 9
2-5 病毒株分型…………………………………………… 10
2-6 雞群抗體測定的重要性……………………………… 13
2-7 定量PCR的應用原理…………………………………… 15
第三章 材料與方法…………………………………………… 18
3-1 病毒選擇、純化、增殖及力價之測定……………… 18
3-2 病毒之序列分析……………………………………… 19
3-2.1 病毒核酸之萃取及確認……………………………… 19
3-2.2 定序引子的設計……………………………………… 20
3-2.3 反轉錄聚合酵素連鎖反應…………………………… 20
3-2.4 定序用重組質體之構築……………………………… 22
3-2.5 RT-PCR產物或質體的定序…………………………… 24
3-2.6 核酸序列整理與分析………………………………… 24
3-3 抗體力價測定技術之評估…………………………… 24
3-3.1 抗血清之製備………………………………………… 24
3-3.2 血清之抗體力價測定………………………………… 25
3-4 IBV之定量技術研究…………………………………… 27
3-4.1 病毒核酸之萃取……………………………………… 27
3-4.2 定量RT-PCR之引子與探針的設計…………………… 27
3-4.3 標準RNA的製備……………………………………… 28
3-4.4 定量RT-PCR…………………………………………… 30
3-4.5 定量RT-PCR標準曲線之建立………………………… 31
3-4.6 敏感度試驗…………………………………………… 31
3-4.7 精確性試驗……............................... 32
3-4.8 病毒力價與病毒基因體數量的比較………………… 32
第四章 結果……………………………………………...…… 33
4-1 IBV序列分析………….......................... 33
4-1.1 萃取核酸的確認……………………………………… 33
4-1.2 基因全長的增幅……………………………………… 33
4-1.3 RT-PCR產物的選殖與確認…………………………… 33
4-1.4 S1與N基因之全長定序與親緣分析……..…………… 34
4-2 抗體力價測定技術之評估…………………………… 35
4-3 IBV之定量技術研究…………………………………… 36
4-3.1 定量RT-PCR之引子確認……………………………… 36
4-3.2 定量RT-PCR中標準RNA的製備與標準曲線之建立…… 36
4-3.3 敏感度試驗……………………………………………..37
4-3.4 精確性試驗………………………………………………37
4-3.5 病毒力價與病毒基因體數量的比較………………… 38
第五章 討論……...…………………………………………… 58
參考文獻 ………………………………………………………… 65
表次
表1. S1醣蛋白基因胺基酸序列相似性與相異性百分比… 39
表2. N蛋白基因胺基酸序列相似性與相異性百分比……… 39
表3. 定量RT-PCR的精確性試驗…………………………… 40
表4. 病毒力價與病毒基因體數量的比較………………… 41
圖次
圖1. IBV之基因體結構簡圖………………………………… 42
圖2. IBV的S1及N基因,全長增幅及定序用引子的序列及其與基因的相關位置………………………….......................... 43
圖3. 台灣IB分離株與日本分離株萃取核酸的確認……… 44
圖4. IBV的S1醣蛋白及N蛋白基因全長之增幅結果……… 45
圖5. 定序用重組質體的電泳分析圖……………………… 46
圖6. 台灣IB分離株與日本分離株等81株IBV的S1醣蛋白基因胺基酸序列的親緣樹狀圖…………………………………………….....47
圖7. 台灣IB分離株與日本分離株等29株IBV的N蛋白基因胺基酸序列的親緣樹狀圖………………………………………………….....48
圖8. IBV之S1醣蛋白胺基酸相似與相異區域之分析……… 49
圖9. H120抗血清抗體力價變化之情形…………………… 50
圖10. 台灣I型IBV抗血清抗體力價變化之情形…………… 51
圖11. 台灣II型IBV抗血清抗體力價變化之情形…………… 52
圖12. 定量RT-PCR引子之測試……………………………… 53
圖13. 標準RNA的製備………………………………………… 54
圖14. 標準RNA的確認………………………………………… 55
圖15. 定量RT-PCR標準曲線之建立………………………… 56
圖16. 定量RT-PCR與一般RT-PCR敏感度之比較…………… 57

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