王金和臺灣大學：獸醫學研究所陳奕成Chen, Yi-ChengYi-ChengChen2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/59909本研究目的以sandwich ELISA快速診斷H5亞型抗原，以blocking ELISA快速診斷H5亞型抗體。其方法為將3233/04之濃縮純化病毒與其HA重組蛋白免疫小鼠進行單源抗體的製備，並將製備好的單源抗體以免疫墨點法 (immunodot blot assay)、西方轉漬法 (western blot assay) 及血球凝集抑制試驗 (hemagglutination inhibition test) 分析其特性，接著進行單源抗體純化與過氧化氫酶標示後成為追蹤子 (tracer)，最後以blocking ELISA的架構進行野外H5亞型陽性血清檢測，以sandwich ELISA的架構進行H5亞型病毒檢測。在sandwich ELISA結果方面，只能偵測到3233/04 (H5N2) 病毒株，且病毒力價於106 EID50/0.1 mL以上時皆可被檢測到。在blocking ELISA結果方面，以228個H5陰性的血清樣本進行檢測，其特異性為97.36％ (222/228)；以234個H5陽性的血清樣本進行檢測，其敏感性為98.29％ (230/234)。 家禽流行性感冒病毒是否會經由母雞垂直傳染給小雞的證據目前仍缺乏，因此本實驗為將宜蘭雞場分離到的H6N1亞型病毒株2838K/00，一方面進行母雞攻毒試驗，視其病毒是否會經由母雞垂直傳染給小雞，而另一方面則進行無特定病原雞胚胎蛋攻毒試驗，視其小雞是否會將病毒直接帶毒出來。其方法為取108.65 EID50/0.1 mL的病毒量，以鼻內接種法攻毒於產蛋母雞，另將病毒液稀釋成1EID50/0.1 mL的劑量，直接將病毒液經由尿囊腔接種於無特定病原的雞胚胎蛋中，接著收取攻毒後母雞所生小雞與直接攻毒於SPF雞胚胎蛋出生小雞之氣管、腎臟與卵黃囊，以SPF雞胚胎蛋分離兩代後，抽取尿囊液以RT-PCR的方式進行AIV NP基因的檢測。其結果可從卵黃卵白中檢測到病毒核酸，但出生小雞檢體其病毒分離結果為陰性，依此結果推論家禽流行性感冒病毒不會有垂直傳播的情形發生。In this study, we used sandwich ELISA to detect H5 virus and blocking ELISA to detect H5 antibody. The concentrated purified virus (3233/04) and hemagglutinin (HA) recombinant proteins were used for the production of monoclonal antibodies. These monoclonal antibodies were confirmed to be specific to the H5 HA by immunodot blot assay, western blot assay and hemagglutination inhibition test. These monoclonal antibodies were purified and then labeled with horseradish peroxidase to become tracer. The tracer was used for the detection of H5 antibody in serum by blocking ELISA and for the H5 subtype virus by sandwich ELISA. The results showed that sandwich ELISA detected the H5 virus above the titer of 106 EID50/0.1 mL. The specificity and sensitivity for blocking ELISA were 97.36% (222/228) and 98.29% (230/234), respectively. Little information about AIV vertical transmission is available. This study aimed to prove the existence of vertical transmission of AIV from hens to embryos. We used a H6N1 low pathogenetic AIV Taiwanese isolate (Yilan, Taiwan), 2838K/00, in the vertical transmission test. After challenging the hens with 108.65 EID50/0.1 mL of 2838K/00, and inoculating the specific-pathogen-free embryonated eggs with 1EID50/0.1 mL of 2838K/00, the embryos were hatched. The tracheas, kidneys, and yolk sacs were collected from the hatched chicks for the virus isolation by the allantoic sac route. After two passages, we harvested the allantoic fluid and tested for the detection of NP gene of AIV by RT-PCR. The results showed that we detected the nucleic acid of AIV in the yolk and albumen mixture from unfertilized eggs. But we didn’t detect the AIV in the hatched chicks. According to above results, AIV seems to be no vertical transmission in chickens.目錄------------------------------------------------------------------------------------------------I 表次---------------------------------------------------------------------------------------------VII 圖次--------------------------------------------------------------------------------------------VIII 中文摘要-----------------------------------------------------------------------------------------X 英文摘要----------------------------------------------------------------------------------------XI 第一章 緒言-------------------------------------------------------------------------------------1 第二章 文獻回顧-------------------------------------------------------------------------------2 2-1 歷史背景------------------------------------------------------------------------------------2 2-2 病毒特徵------------------------------------------------------------------------------------5 2-3 病毒型態、結構與功能------------------------------------------------------------------6 2-3.1 血球凝集素-------------------------------------------------------------------------------6 2-3.2 神經胺酸酶-------------------------------------------------------------------------------7 2-3.3 核蛋白-------------------------------------------------------------------------------------8 2-3.4 基質蛋白----------------------------------------------------------------------------------8 2-3.5 非結構蛋白-------------------------------------------------------------------------------9 2-3.6 聚合酶蛋白-------------------------------------------------------------------------------9 2-4 病毒複製-----------------------------------------------------------------------------------10 2-5 病毒抗原變異性--------------------------------------------------------------------------11 2-6 病毒物理化學特性-----------------------------------------------------------------------11 2-7 宿主範圍-----------------------------------------------------------------------------------12 2-8 臨床症狀與病理變化--------------------------------------------------------------------13 2-9 病原性之評估-----------------------------------------------------------------------------13 2-10 病毒之命名------------------------------------------------------------------------------15 2-11 診斷方法----------------------------------------------------------------------------------15 2-11.1 偵測病毒抗原-------------------------------------------------------------------------15 2-11.2 偵測病毒抗體-------------------------------------------------------------------------17 2-12 酵素連結免疫吸附法之應用原理---------------------------------------------------19 2-13 單株抗體之歷史背景------------------------------------------------------------------20 2-13.1 細胞融合原理-------------------------------------------------------------------------20 2-13.2 細胞融合方式-------------------------------------------------------------------------20 2-13.3 HAT篩選原理-------------------------------------------------------------------------21 2-13.4 單株抗體之特性與應用-------------------------------------------------------------21 第三章 材料與方法---------------------------------------------------------------------------23 第一節 野鳥家禽流行性感冒帶毒之監測------------------------------------------------23 3-1.1 採樣時間與地點------------------------------------------------------------------------23 3-1.2 樣本數大小------------------------------------------------------------------------------23 3-1.3 病毒分離與鑑定------------------------------------------------------------------------23 3-1.3.1 樣本採集------------------------------------------------------------------------------23 3-1.3.2 樣本處理------------------------------------------------------------------------------24 3-1.3.3 病毒增殖------------------------------------------------------------------------------24 3-1.3.4 RNA之萃取---------------------------------------------------------------------------24 3-1.3.5 反轉錄聚合酶鏈反應---------------------------------------------------------------25 3-1.3.6 瓊膠醣凝膠電泳---------------------------------------------------------------------26 3-1.3.7 聚合酶鏈反應產物之純化---------------------------------------------------------27 3-1.3.8 聚合酶鏈反應產物之選殖---------------------------------------------------------27 3-1.3.9 Colony PCR---------------------------------------------------------------------------28 3-1.3.10 核酸序列之定序-------------------------------------------------------------------29 3-1.3.11 序列之分析-------------------------------------------------------------------------29 第二節 ELISA診斷試驗之開發-----------------------------------------------------------29 3-2.1 病毒來源--------------------------------------------------------------------------------29 3-2.2 屠宰場血清樣本採集-----------------------------------------------------------------30 3-2.3 血清樣本抗體檢測--------------------------------------------------------------------30 3-2.4 紅血球凝集試驗及紅血球凝集抑制試驗-----------------------------------------30 3-2.4.1 製備紅血球溶液---------------------------------------------------------------------30 3-2.4.2 紅血球凝集試驗---------------------------------------------------------------------31 3-2.4.3 紅血球凝集抑制試驗---------------------------------------------------------------31 3-2.5 病毒濃縮與純化-----------------------------------------------------------------------31 3-2.6 單株抗體--------------------------------------------------------------------------------32 3-2.6.1 單株抗體製備------------------------------------------------------------------------32 3-2.6.2 HA基因之選殖-----------------------------------------------------------------------32 3-2.6.3 建構表現載體------------------------------------------------------------------------33 3-2.6.4 免疫計畫------------------------------------------------------------------------------33 3-2.6.5 單株抗體篩選------------------------------------------------------------------------34 3-2.6.6 單株抗體純化------------------------------------------------------------------------34 3-2.6.7 蛋白質定量---------------------------------------------------------------------------34 3-2.6.8 單株抗體分析------------------------------------------------------------------------35 3-2.6.8.1 單株抗體亞型分析----------------------------------------------------------------35 3-2.6.8.2 免疫墨點法分析-------------------------------------------------------------------35 3-2.6.8.3 聚丙烯醯胺膠片電泳及西方墨點法分析-------------------------------------36 3-2.6.8.4 病毒中和試驗----------------------------------------------------------------------37 3-2.6.8.5 胜肽矩陣----------------------------------------------------------------------------38 3-2.6.9 以過氧化氫酶標示單株抗體------------------------------------------------------38 3-2.6.10 阻斷型ELISA之開發-------------------------------------------------------------38 3-2.6.10.1 阻斷型ELISA最佳化-----------------------------------------------------------38 3-2.6.10.2 單株抗體與15種禽流感病毒亞型高免疫血清之阻斷型ELISA試驗--39 3-2.6.10.3 阻斷型ELISA之敏感性與特異性分析--------------------------------------40 3-2.6.11 三明治型ELISA之開發-----------------------------------------------------------40 3-2.6.11.1 三明治型ELISA最佳化--------------------------------------------------------40 3-2.6.11.2 三明治型ELISA之敏感性與特異性分析-----------------------------------40 第三節 攻毒至SPF雞胚胎蛋探討孵出小雞之AIV帶毒情形-----------------------41 3-3.1 病毒株之挑選---------------------------------------------------------------------------41 3-3.2 病毒株之力價測定---------------------------------------------------------------------41 3-3.3 實驗設計---------------------------------------------------------------------------------41 3-3.3.1 SPF雞胚胎蛋攻毒試驗組-----------------------------------------------------------41 3-3.3.2 SPF雞胚胎蛋空白對照組-----------------------------------------------------------42 3-3.4 以SPF雞胚胎蛋繼代進行病毒增殖與鑑定---------------------------------------42 第四節 攻毒至產蛋母雞探討所生小雞之AIV帶毒情形------------------------------43 3-4.1 病毒株之挑選---------------------------------------------------------------------------43 3-4.2 病毒株之力價測定---------------------------------------------------------------------43 3-4.3 實驗動物---------------------------------------------------------------------------------43 3-4.4 飼料與飲水------------------------------------------------------------------------------43 3-4.5 飼養環境---------------------------------------------------------------------------------43 3-4.6 實驗設計---------------------------------------------------------------------------------44 3-4.7 攻毒母雞抗體監測---------------------------------------------------------------------45 第四章 結果------------------------------------------------------------------------------------46 第一節 野鳥家禽流行性感冒帶毒之監測------------------------------------------------46 4-1.1 家禽流行性感冒病毒之分離---------------------------------------------------------46 4-1.2 家禽流行性感冒病毒之鑑定---------------------------------------------------------46 4-1.3 家禽流行性感冒病毒之序列分析---------------------------------------------------46 第二節 ELISA診斷試劑之開發------------------------------------------------------------47 4-2.1 屠宰場血清樣本採集------------------------------------------------------------------47 4-2.2 血清樣本抗體檢測---------------------------------------------------------------------47 4-2.3 病毒濃縮與純化------------------------------------------------------------------------47 4-2.4 單株抗體---------------------------------------------------------------------------------47 4-2.4.1 單株抗體篩選------------------------------------------------------------------------47 4-2.4.2 蛋白質定量---------------------------------------------------------------------------47 4-2.4.3 單株抗體亞型分析------------------------------------------------------------------48 4-2.4.4 免疫墨點法---------------------------------------------------------------------------48 4-2.4.5 SDS-PAGE及Western blot assay--------------------------------------------------48 4-2.4.6 單株抗體HI分析--------------------------------------------------------------------49 4-2.4.7 病毒中和試驗------------------------------------------------------------------------49 4-2.4.8 胜肽矩陣------------------------------------------------------------------------------49 4-2.4.9 以過氧化氫酶標示單株抗體------------------------------------------------------49 4-2.4.10 阻斷型ELISA之開發--------------------------------------------------------------50 4-2.4.10.1 阻斷型ELISA最佳化-----------------------------------------------------------50 4-2.4.10.2 單株抗體與15種禽流感病毒亞型高免疫血清之阻斷型ELISA試驗--50 4-2.4.10.3 阻斷型ELISA之敏感性與特異性分析--------------------------------------50 4-2.4.11 三明治型ELISA之開發----------------------------------------------------------51 4-2.4.11.1 三明治型ELISA最佳化--------------------------------------------------------51 4-2.4.11.2 三明治型ELISA之特異性與敏感性分析-----------------------------------52 第三節 攻毒至SPF雞胚胎蛋探討孵出小雞之AIV帶毒情形------------------------52 4-3.1 病毒株之力價測定---------------------------------------------------------------------52 4-3.2 SPF雞胚胎蛋攻毒試驗組結果-------------------------------------------------------52 4-3.3 SPF雞胚胎蛋空白對照組-------------------------------------------------------------52 第四節 攻毒至產蛋母雞探討所生小雞之AIV帶毒情形------------------------------53 4-4.1 母雞產蛋之檢測結果------------------------------------------------------------------53 4-4.2 母雞抗體檢測之結果------------------------------------------------------------------53 第五章 討論與結論---------------------------------------------------------------------------54 第六章 參考文獻------------------------------------------------------------------------------612705755 bytesapplication/pdfen-US家禽流行性感冒病毒酵素連結免疫吸附法Avian influenza virusELISAthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59909/1/ntu-95-R93629002-1.pdf