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研究生:紀又銘
研究生(外文):YU-MING CHI
論文名稱:口服豬沙氏桿菌抗體之研究
論文名稱(外文):The Study of Swine Salmonella Antibody by Oral Administration
指導教授:廖明輝廖明輝引用關係邱明堂
學位類別:碩士
校院名稱:國立屏東科技大學
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:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:蛋黃抗體沙氏桿菌
外文關鍵詞:SwineIGYSalmonella
相關次數:
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中文摘要內容:
近年來由於抗生素類藥物的濫用,加上其他因素如豬瘟或溶血性大腸桿菌及呼吸道疾病的併發,使沙氏桿菌更有機會呈現週期性發病的現象,且傳播迅速損失也高,讓管理人員防不勝防。為瞭解沙氏桿菌蛋黃抗體 ( Immunization Egg Yolk, IgY ) 對沙氏桿菌之防治效果,本研究針對市售沙氏桿菌蛋黃抗體 ( IgY ) 產品進行定性、安定性、特異性及田間活體功效試驗。結果顯示,蛋黃抗體經蛋白質電泳 ( SDS-PAGE ) 及西方免疫轉漬法 ( Western blotting assay ) 可確定其特異性蛋黃抗體存在。此特異性蛋黃抗體在酸-鹼 ( pH 2-11 ) 環境下作用二小時及高溫 ( 100℃,五分鐘 ) 處理後均極為穩定,而與沙氏桿菌 ( Salmonella choleraesuis及Salmonella typhimurium ) 作用呈現明顯之凝集反應。而以108 CFU/mL Salmonella choleraesuis及Salmonella typhimurium 10劑量口服攻毒於六週齡LYD三品系仔豬使其發病後,再以蛋黃抗體做治療處理,亦發現對於外觀症狀、死亡率及糞便中含菌量均有明顯之改善。
關鍵詞:豬、蛋黃抗體、沙氏桿菌
Abstract

The content of abstract in this thesis:
Due to the abusage of antibiotics in the field for these years, together with the explosion of classical swine fever and EHEC and the respiratory diseases. All these factors make Salmonella spp. reoccur constantly and periodically, and make the disease widespread speedily with a heavy financial loss to the breeders . This make the administrative staff get more difficult to conduct any prevention. This study is focused on the marketing product of Salmonella spp. IgY to conduct its quality, stability and potential activity of specific and the field test. At the tested result shows, the IgY antibody could be assured its certain specific antibody IgY existing through SDS-PAGE and Western blotting assay. This specific antibody IgY could be achieved an extremely stability after process under the environment at pH 2 ~ 11 for two hours and under a temperature of 100℃ for 5 minutes. And this specific antibody IgY shows a significant agglutinated response with Salmonella choleraesuis and Salmonella typhimurium. Attacked with ten doses of 108 CFU/ml of Salmonella choleraesuis and Salmonella typhimurium , via oral administration to 6-week-old three-interbreed piglets, after making these piglets to suffer from the illness , then treat them with the antibody IgY. And found there is a significant improve on the appearance symptom, mortality rate and contains bacterium quantity in feces.

Key words: Swine, IgY, Salmonella
目 錄
中文摘要....................................................................................................... Ⅰ
英文摘要...................................................................................................... Ⅱ
誌謝............................................................................................................. Ⅳ
目錄.............................................................................................................. Ⅴ
圖表目錄........................................................................................................ Ⅷ
第1章 前言................................................................................................... 1
第2章 文獻回顧........................................................................................... 3
2. 1 沙氏桿菌............................................................................................... 3
2. 2 抗體.. .................................................................................................... 13
2. 3 即時定量聚合酶鏈鎖反應(real-time PCR).................................... 21
第3章 材料與方法………………………………...................................... 23
3. 1 實驗材料…………….………………….……….…………………... 23
3. 1. 1 藥品..………..…………………………………………............. 23
3. 1. 2 培養基………………………………………………………….. 23
3. 1. 3 培養液…………………………………………………………. 24
3. 1. 4 套組……………………………………………………………. 24
3. 1. 5 抗體……………………………………………………………. 24
3. 1. 6 儀器……………………………………………………………. 25
3. 1. 7 器材……………………………………………………………. 26
3. 2 菌株採取………………………..………………………...…………. 27
3. 3. 沙氏桿菌的分離………….…………………………….…………… 27
3. 4 沙氏桿菌的生化鑑定……………………………………………...... 27
3. 4. 1 血清型鑑定……………………………………………..……... 28
3. 4. 2 O群別試驗………………………………..…………………... 28
3. 4. 3 H群別試驗…………………………………………………..... 29
3. 5 聚合酶連鎖反應 ( PCR ) 檢測………………...…………………... 29
3. 5. 1 核酸萃取………………………………………………..……... 29
3. 5. 2 聚合酶連鎖反應………………………..……………………... 29
3. 5. 3 電泳分析……………………..………………………………... 30
3. 5. 4 沙氏桿菌特異性 ( ST11ST15 ) 試驗………………………... 30
3. 5. 5 沙氏桿菌特異性 ( Is200-1 ) 試驗……………….…………... 31
3. 5. 6 Salmonella的保存….………..………………………………... 31
3. 6 IgY定性分析……..……………………………………..................... 31
3. 6. 1 IgY萃取……………………………………….………..……... 31
3. 6. 2 SDS-PAGE電泳分析……….…………………………..……... 31
3. 6. 3 西方免疫轉漬試驗……………………………………..……... 32
3. 6. 4 IgY抗體之安定性……………………………………..……... 32
3. 6. 5 蛋黃抗體之特異性……………………………………..……... 33
3. 7 即時定量聚合酶鏈鎖反應 ( Real-time PCR )………....................... 33
3. 7. 1 選殖用載體 ( Vector )…..…………………………………….. 33
3. 7. 2 載體之製備.……………………………………………………. 33
3. 7. 3 接合作用.………………………………………………………. 33
3. 7. 4 勝任細胞 ( TOP 10F’ ) 之製作.………………………………. 34
3. 7. 5 轉形作用.………………………………………………………. 34
3. 7. 6 菌落篩選.………………………………………………………. 34
3. 7. 7 限制酶切割確認重組成功與否.………………………………. 35
3. 7. 8 Real-time PCR標準曲線建立.…..……………………………. 35
3. 8 動物試驗……………………………………………...……………… 36
3. 8. 1 Salmonella菌數的定量….…………………….………………. 36
3. 8. 2 攻毒的方法……………………….…………………………… 36
3. 8. 3 田間活體功效試驗……………….…………………………… 36
第4章 結果………………………………….………………..................... 38
4. 1 沙氏桿菌的分離………………………..…………………………… 38
4. 2 沙氏桿菌之生化鑑定………………………………………………... 38
4. 2. 1 TSI生化鑑定…………………………………………………... 38
4. 2. 2 API 20E之生化鑑定…………………………………………... 38
4. 3 沙氏桿菌特異性 ( ST11ST15 ) 試驗結果………………………... 38
4. 4 沙氏桿菌特異性 ( Is200-1 ) 試驗結果……….……………….…... 39
4. 5 IgY抗體之定性結果…………………………………………….…... 39
4. 6 IgY抗體之安定性結果………………………………………….…... 39
4. 7 蛋黃抗體特異性結果……………………..…………………….…... 39
4. 8 Real-time PCR結果……………………………..…………………… 40
4. 9 田間活體功效試驗結果….………………..…………………….…... 40
4. 9. 1 對照組結果……..……………………………………………... 40
4. 9. 2 攻毒後立即灌服IgY抗體結果……..………………………... 40
4. 9. 3 攻毒兩天後,以IgY抗體治療結果……..……………………... 40
第5章 討論………………………………………………….…………...... 58
參考文獻………………………………………….……………………........ 62
附錄…………………………………………………………...…………...... 73
作者簡介...………………………………………………………………….. 76
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