臺灣博碩士論文加值系統

傳染性華氏囊病 ( infectious bursal disease；IBD ) 中間偏強之活毒疫苗往往因使用時機不當而傷害到華氏囊或讓野外毒捷足先登，但無論何時給予疫苗，免疫空窗期還是難以避免。許多文獻指出蛋黃免疫球蛋白 ( immunoglobulin yolk；IgY ) 近年來已被廣泛地利用在被動免疫性治療或預防上，以此可應用於生產含高量抗IBD特異性IgY，以被動免疫的方式或許可以阻斷或延緩其吸附到標的細胞的機會。本實驗以IBDV為抗原，經由SPF雞隻增殖後，取脾臟及華氏囊研磨製成病毒乳劑，測出病毒含量為105.5 TCID50/mL，保存於-70℃。多次收集種雞及市售雞蛋蛋黃後，測出蛋黃IBDV-ELISA抗體反應力價之平均值為6083，冷凍乾燥使其成為蛋粉，其力價並未顯著增加或減少。確定抗原與抗體後，進行中和試驗，結果發現IgY於稀釋二倍時具有保護效力。接著並以IgY蛋粉及100 TCID50病毒液感作一小時後以灌食方式給予雞隻進行動物試驗，稱取體重和華氏囊重供計算華氏囊體重比 ( bursa-body weight ratio；BBR ) 並剖檢華氏囊肉眼病變 ( BGL ) 和華氏囊組織學病變 ( BHL ) 及反轉錄聚合酶鏈鎖反應 ( RT-PCR ) 判讀後，當IgY稀釋四倍時具有保護力。結果並不如預期，期望在提高IgY蛋粉的抗體力價，並找到最佳純化IgY之方法後，可獲得更佳的保護效果。

Due to the improper injecting time, bursal is often damaged by Infectious bursal disease (IBD) live vaccines or infected by wild virus in first. However, it is difficult to avoid the immunity risky period even giving the vaccine in any time period. Recently, the immunoglobulin yolk ( IgY ) has been widely used in the passive immunity treatment or the prevention. It has been suggested that can be applied to produce anti-IBD specificity IgY, when the virus enter the digestive tract which may block or delay it to target cell by the passive immunity. IBDV was used in this study and propagated in specific-pathogen-free (SPF) chicks; 105.5 TCID50/mL of virus was obtained, stored in -70℃. The 6083 titer of egg-yolk IBDV ELISA antibodies was collected. The titer was not distinct decreased after freeze-drying. According to neutralization test, the IgY neutralization titer was 2. In animal demonstration, chickens were fed with IgY powder (mixed with 100 TCID50 virus at 37℃ one hour). Then chickens and bursa were weighted to calculate BBR, BGL grading, BHL grading and RT-PCR. IgY was shown the protecting ability after the concentration was diluted to four times. Therefore, it may be suggested that the IBDV ELISA titer and purify anti-IBD specificity IgY can have the protective efficiency.

中文摘要....................................................................................................... Ⅰ
Abstract.......................................................................................................... Ⅲ
誌謝............................................................................................................... Ⅴ
目錄............................................................................................................... Ⅵ
圖表目錄....................................................................................................... Ⅸ
第1章 前言................................................................................................... 1
第2章 文獻回顧........................................................................................... 3
2. 1 傳染性華氏囊病之簡介....................................................................... 3
2. 1. 1 歷史背景...................................................................................... 3
2. 1. 2 病毒結構與特性.......................................................................... 3
2. 1. 3 IBDV之核苷酸及其蛋白質特性................................................ 4
2. 1. 4 IBD發生及流行病學................................................................... 6
2. 1. 5 IBDV的致病機轉與所造成的免疫缺陷.................................... 7
2. 1. 6 IBD的感染症狀及病變………………………………………... 8
2. 1. 7 IBDV診斷的分子應用工具…………………………………… 9
2. 1. 8 vvIBDV之預防與控制………………………………………... 11
2. 2 蛋黃免疫球蛋白簡介……………………………………………….. 12
2. 2. 1 蛋黃免疫球蛋白的優點............................................................. 12
2. 2. 2 蛋黃免疫球蛋白與哺乳動物免疫球蛋白之比較..................... 13
2. 2. 3 雞血清及雞蛋中之抗體............................................................. 13
2. 3 IgY分子的性質................................................................................... 14
2. 3. 1 IgY分子的構造.......................................................................... 14
2. 3. 2 IgY的抗體特性.......................................................................... 14
2. 3. 3 IgY分子的安定性...................................................................... 14
2. 3. 4 分離純化IgY 之方法................................................................. 17
2. 4 IgY之應用............................................................................................ 17
第3章 材料與方法...................................................................................... 20
3. 1 病毒來源............................................................................................... 20

3. 2 病毒增殖............................................................................................... 20
3. 2. 1 病毒乳劑配製.............................................................................. 20
3. 2. 2 病毒核酸萃取............................................................................ 20
3. 2. 3 PCR之引子對設計...................................................................... 21
3. 2. 4 反轉錄聚合酶鏈鎖反應 ( reverse transcription polymerase chain reaction; PCR )................................................ 21
3. 2. 5 RT-PCR產物鑑定及定序............................................................ 21
3. 2. 6 初代細胞培養.............................................................................. 22
3. 2. 7 病毒含量測定 ( 50％ tissue culture infective dose; TCID50 )....................................................................................... 22
3. 3 IgY製備................................................................................................ 22
3. 3. 1 蛋黃來源...................................................................................... 22
3. 3. 2 血清酵素連結免疫吸附試驗 ( enzyme-linked immunosorbent assay; ELISA ) 抗體檢測................................. 23
3. 3. 3 IgY抗體中和試驗....................................................................... 23
3. 3. 4 冷凍乾燥...................................................................................... 24
3. 4 IgY保護效力試驗................................................................................ 24
3. 4. 1 實驗動物....................................................................................... 24
3. 4. 2 實驗分組....................................................................................... 24
3. 4. 3 試驗觀察與判定........................................................................... 24
3. 4. 3. 1 華氏囊／體重比值 ( bursa／body-weight ratio; BBR ) 之測定...................................................................... 24
3. 4. 3. 2 華氏囊之肉眼及組織學病變分級...................................... 24
3. 4. 3. 3 病毒鑑定.............................................................................. 25
第4章 結果................................................................................................. 28
4. 1 以PCR診斷IBDV是否有增殖成功................................................... 28
4. 2 PCR產物序列之比對及分析............................................................... 29
4. 3 測定病毒的含量.................................................................................. 32
4. 4 IgY抗體力價....................................................................................... 33
4. 5 IgY抗體中和試驗............................................................................... 36

4. 6 IgY保護效力試驗................................................................................ 37
4. 6. 1 各組間BBR值測定...................................................................... 37
4. 6. 2 華氏囊肉眼及組織學病變分級................................................... 37
4. 6. 3 病毒鑑定....................................................................................... 39
第5章 討論................................................................................................. 46
參考文獻......................................................................................................... 50
作者簡介......................................................................................................... 68

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