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研究生:陳亮丞
論文名稱:家禽流行性感冒病毒核蛋白之核酸序列分析與酵素連結免疫吸附分析(ELISA)套組之開發
論文名稱(外文):Analysis of nucleotide sequences and development of ELISA kit for avian influenza virus nucleoprotein
指導教授:沈瑞鴻
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:家禽流行性感冒病毒核蛋白
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因為核蛋白 (NP) 在A型流行性感冒病毒具有良好保留性且變異性遠比流行性感冒病毒表面醣蛋白來得慢,所以常使用於血清學診斷方面。因此本研究分析禽類15種H血清亞型NP基因開讀框之胺基酸序列,得知禽類15種H血清亞型轉譯出的NP胺基酸序列相似度大於95%,顯示NP蛋白在禽類15種H血清亞型間具有保留性。將A / Chicken / Taiwan / NCHU-0507 / 99 (H6N1) 毒株其NP基因開讀框使用E. coli表現系統表現,並使用於血清學診斷方面。E. coli表現之重組核蛋白 (rNP) 使用SDS-PAGE及西方轉漬法分析 (Western blot) 分析其表現狀況並以親和性樹脂管柱加以純化,以得到大量之純化rNP。所得之純化rNP使用於Western blot可檢測出15種H血清亞型之抗體,而將其使用於酵素連結免疫吸附法 (ELISA),也可檢測出15種H血清亞型之抗體。進一步檢測469隻田間雞隻血清及165隻實驗感染及非感染雞隻血清,以評估純化rNP使用於ELISA的表現性。並以血球凝集抑制試驗 (HI) 確認純化rNP使用於ELISA檢測結果之特異性。相對於膠體免疫擴散試驗 (AGID),純化rNP使用於ELISA之敏感性為100%及特異性為80.4%。而純化rNP使用於ELISA相對於HI之檢測結果一致性為94.8%。這些結果顯示純化rNP使用於ELISA與AGID具有高度之相關性且比AGID更具敏感性。相對於商品化ELISA套組,純化rNP使用於ELISA之敏感性為99.6%及特異性為90.4%。結果顯示純化rNP使用於ELISA與商品化ELISA套組一樣具有良好之表現性。所以使用純化rNP並使用於ELISA可發展建立為高敏感性及特異性且達到快速檢測大量雞隻家禽流行性感冒抗體樣品的血清學診斷技術。
目次
中文摘要...................................................... I
英文摘要...................................................... II
目次.......................................................... III
表次.......................................................... V
圖次.......................................................... VI
第一章 緒言................................................... 1
第二章 文獻探討............................................... 2
第一節 家禽流行性感冒病毒歷史背景....................... 2
第二節 家禽流行性感冒病毒性狀........................... 4
2-2.1 病毒特性........................................ 4
2-2.2 病毒結構及基因體................................ 5
2-2.3 病毒的複製...................................... 8
2-2.4 病毒物理化學特性................................ 9
2-2.5 病毒抗原性改變特性.............................. 10
第三節 宿主範圍......................................... 10
第四節 病毒實驗室診斷................................... 12
2-4.1 病原直接分離與同定.............................. 12
2-4.2 直接證實病毒之存在.............................. 13
2-4.3 免疫螢光試驗病毒蛋白............................ 13
2-4.4 證實病毒核酸之存在.............................. 13
2-4.5 由血清抗體證實感染.............................. 14
第五節 酵素連結免疫吸附測定之應用原理................... 15
第三章 材料與方法............................................ 17
第一節 病毒增殖、純化及其力價測定........................ 17
3-1.1 病毒來源........................................ 17
3-1.2 實驗動物........................................ 17
3-1.3 病毒增殖........................................ 17
3-1.4 病毒純化........................................ 17
3-1.5 病毒力價測定.................................... 18
第二節 病毒之核酸序列分析............................... 18
3-2.1 病毒核酸的萃取.................................. 18
3-2.2 反轉錄聚合酶鏈反應人工引子與反應條件............ 19
3-2.3 反轉錄聚合酶鏈反應產物的確認.................... 20
3-2.4 反轉錄聚合酶鏈反應產物的純化.................... 20
3-2.5 反轉錄聚合酶鏈反應產物選殖...................... 21
3-2.6 反轉錄聚合酶鏈反應產物之定序反應................ 21
3-2.7 定序結果之序列分析.............................. 22
第三節 病毒重組蛋白之製備............................... 22
3-3.1 基因之增幅...................................... 22
3-3.2 基因之選殖...................................... 23
3-3.3重組質體之表現、純化及確認........................ 25
第四節 血清............................................. 28
3-4.1 實驗血清........................................ 28
3-4.2 田間血清........................................ 29
第五節 血清抗體檢測..................................... 29
3-5.1 膠體免疫擴散試驗................................ 29
3-5.2 血球凝集抑制試驗................................ 30
3-5.3 AIV Ab Test Kit (IDEXX)............................ 30
3-5.4 Indirect NP-ELISA................................. 31
第四章 結果................................................... 33
第一節 病毒增殖、純化及其力價測定........................ 33
4-1.1 病毒增殖........................................ 33
4-1.2 病毒純化........................................ 33
4-1.3 病毒力價測定.................................... 33
第二節 病毒之核酸序列分析............................... 33
4-2.1 RT-PCR產物之確認............................... 33
4-2.2 NP基因序列分析與親緣樹圖.................... 34
4-2.3 NP基因開讀框胺基酸序列分析與親緣樹圖............ 34
第三節 病毒重組蛋白之製備............................... 34
4-3.1 NP基因之選殖.................................... 34
4-3.2 重組質體之表現、純化............................. 35
4-3.3 重組蛋白特異性分析與確認........................ 35
第四節 Indirect NP-ELISA之建立與應用..................... 35
4-4.1 建立Indirect NP-ELISA之檢測方法.................. 35
4-4.2 評估indirect NP-ELISA應用於檢測實驗血清.......... 36
4-4.3 評估indirect NP-ELISA應用於檢測田間血清.......... 37
第五章 討論................................................... 59
參考文獻...................................................... 64
附錄一. 15種H血清亞型之家禽流行性感冒病毒NP基因開讀框
的胺基酸序列............................................
74
表次
表1. 本研究所使用之家禽流行性感冒病毒病毒株.................... 38
表2. 以AGID、HI、AIV Ab Test Kit (IDEXX) 及indirect NP-ELISA 檢測SPF雞接種A / Chicken / Taiwan / NCHU-0507 / 99 (H6N1) 血清中之抗體反應..................................................
39
表3. Indirect NP-ELISA與AGID檢測田間血清之比較................. 40
表4. Indirect NP-ELISA與AIV Ab Test Kit (IDEXX) 檢測田間血清之比較 41
表5. Indirect NP-ELISA與HI檢測田間血清之一致性的比較........... 42
圖次
圖1. 家禽流行性感冒病毒結構圖................................. 43
圖2. Ribonucleoprotein(RNP)核醣核蛋白之示意圖................... 43
圖3. 以蔗糖梯度離心純化病毒................................... 44
圖4. RT-PCR及Colony PCR產物之Agarose gel電泳圖................ 45
圖5. 使用DNASTAR®的MegAlign軟體,以Clustal的方法分析後,15
種H血清亞型之家禽流行性感冒病毒NP基因核苷酸序列相似性與
差異性之比較。............................................
46
圖6. 15種H血清亞型之家禽流行性感冒病毒NP基因核苷酸序列之親
源樹圖...................................................
47
圖7. 使用DNASTAR®的MegAlign軟體,以Clustal的方法分析後,15
種H血清亞型之家禽流行性感冒病毒NP基因開讀框轉譯的胺基酸
序列相似性與差異性之比較.................................
48
圖8. 15種H血清亞型之家禽流行性感冒病毒NP基因開讀框的胺基酸
序列之親源樹圖...........................................
49
圖9. 重組質體pET32a-NP於E. coli BL21(DE3)中表現結果之SDS-PAGE
圖.......................................................
50
圖10. 重組質體pET32a-NP於E. coli BL21(DE3)中表現結果之Western
blot圖..................................................
51
圖11. 以SDS-PAGE (A) 及Western blot (B) 分析純化之重組NP蛋白.. 52
圖12. 以Western blot分析純化之重組NP蛋白...................... 53
圖13. 重組NP蛋白應用於ELISA之棋盤式力價測定................. 54
圖14. 以E. coli BL21(DE3)經超音波擊碎菌體後取其上清液作血清前
處理之結果..............................................
55
圖15. 以TG-ROC (two-graph receiver operating characteristics) 之方法分析Indirect NP-ELISA檢測各50隻陽性雞隻血清及陰性雞隻血清之結果..................................................
56
圖16. 以indirect NP-ELISA檢測陰性血清及陽性血清................ 57
圖17. 以indirect NP-ELISA檢測H1-H15血清亞型之雞隻高免血清及其
它禽類病毒NDV,ARV,IBV,IBDV之雞隻高免血清...........
58
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