臺灣博碩士論文加值系統

溶血性曼哈米亞桿菌 (Mannheimia haemolytica) 屬於巴氏桿菌科，當牛羊遭受到環境上的緊迫，進而使 M. haemolytica引起巴氏桿菌症，屬於急性纖維素性肺炎，死亡率高達30%，急須開發新一代疫苗來降低經濟上的損失。由前人研究發現 M. haemolytica表面的外膜蛋白PlpE (Pasteurella haemolytica lipoprotein E) 是一個重要的抗原，而白血球毒素則是增加肺部病變的元兇。本研究選殖自不同菌株之plpe以及lkt基因，利用原核系統表現重組蛋白rPlpE和rLkt’C並分析其親源性。結果証實重組蛋白可被羊免疫血清所辨識，添加水包油包水佐劑免疫動物，於小鼠抗體力價試驗中，免疫組與對照組相較下，均有顯著性之差異，細胞增生試驗中，免疫組可剌激細胞的增生(p<0.05)，小鼠攻毒試驗中，存活率達80%以上。羊隻抗體試驗中，死菌苗組及rLkt’C組分別與控制組比較，於免疫後第四週具有顯著性差異 (p<0.05)。綜合上述結果，証實重組蛋白之抗原性佳，具有發展為溶血性曼哈米亞桿菌重組蛋白疫苗之潛力。

Mannheimia haemolytica is a member of the Pasteurellaceae family, which is the major cause of acute fibrinous pneumonia when ruminant face the situation of stress. The mortality rate of pneumonic pasteurellosis is 30% approximately. It is urgency to develop new vaccine to prevent M. haemolytica infection and reduce the economical loss. The previous studies indicated that the outer membrane protein PlpE (Pasteurella haemolytica lipoprotein E) is the most important antigen and the leukotoxin (Lkt) play a major role in lung injury. In this study, we cloned the gene of lkt and plpe from different strains to express recombinant proteins (rLkt’C, rPlpE) by the prokaryotic system and sequences pair-wise alignment. The rLkt’C and rPlpE were recognized by sera from immunized goat. The vaccines made of inactivated M. haemolytica and recombinant protein (rPlpE, rLkt’C) with w/o/w adjuvant. In mice study, the antibody titer and T cell proliferation index of immunized group was significantly higher than control group
(p<0.05), and the survival rate of protection efficacy was 80%. In goat study, both inactivated M. haemolytica and rLkt’C group show significant higher ELISA antibody titers than control group at the four weeks post-vaccination (p<0.05). In summary, these expressed proteins may be used as recombinant protein vaccine candidate against M. haemolytica.

中文摘要....................................................................................................... Ⅰ
Abstract......................................................................................................... Ⅱ
謝誌……………………………………………………………………….. Ⅳ
目錄............................................................................................................... Ⅴ
圖表目錄...................................................................................................... Ⅸ
第1章 前言................................................................................................. 1
第2章 文獻回顧......................................................................................... 5
2. 1 巴氏桿菌症之簡介............................................................................ 5
2. 1. 1 巴氏桿菌症之流行病學…………….....…………………….. 5
2. 1. 2 巴氏桿菌症之臨床症狀和病變.……………………….......... 5
2. 1. 3 巴氏桿菌症之經濟損失及重要性...……………………….... 6
2. 2. 溶血性曼哈米亞桿菌 (Mannheimia haemolytica)之性狀……….. 7
2. 2. 1 溶血性曼哈米亞桿菌之型態與生化特性……………..…... 7
2. 2. 2 溶血性曼哈米亞桿菌之生物分類.………………………… 7
2. 2. 3 溶血性曼哈米亞桿菌之致病機轉….……………………… 9
2. 2. 4 溶血性曼哈米亞桿菌之毒力因子.…………………………
10
2. 2. 4. 1 莢膜 (Capsular)……………………….……............. 10
2. 2. 4. 2 外膜脂蛋白 (outer membrane lipoprotein)................ 11
2. 2. 4. 3 白血球毒素 (lukotoxin)………................................. 12
2. 3 巴氏桿菌肺炎症之治療與預防……………………………......... 14
2. 4 巴氏桿菌肺炎症之診斷……………………………..................... 15
2. 5 巴氏桿菌疫苗開發現況…………………………………………. 15
第3章 材料與方法……………………………………………………... 17
3. 1 本試驗所用之菌株與載體……………..…………………………. 17
3. 1. 1 Mannheimia haemolytica….…………………………………. 17
3. 1. 2 Escherichia coli DH5α.….………………………………….... 17
3. 1. 3 Escherichia coli BL21 (DE3)……………………………….... 17
3. 1. 4 pET-32a (+)……………………………….................................. 17

3. 2 M. haemolytica之培養…………………….………......................... 17
3. 3 M. haemolytica plpe 與lkt之選殖與定序..………......................... 18
3. 3. 1 M. haemolytica genomic DNA之萃取…...…………………... 18
3. 3. 2 聚合酶連鎖反應 (polymerase chain reaction; PCR)……........ 18
3. 3. 2. 1 M. haemolytica plpe之選殖...........................…………. 19
3. 3. 2. 2 M. haemolytica lkt’C之選殖…………………………... 19
3. 3. 3 PCR產物之膠體電泳法……………………………………. 19
3. 3. 4 PCR產物之回收……………………………...…………….. 19
3. 4 以原核表現系統表現plpe及lkt’C之重組蛋白質.……………… 20
3. 4. 1 plpe及lkt’C基因之製備……………………………………. 20
3. 4. 2 表現載體之製備…..…………………………………………. 20
3. 4. 3 plpe及lkt’C 與表現載體之接合反應 (ligation)………….. 20
3. 4. 4 勝任細胞 (competent cell)之製備…………..…...………….. 20
3. 4. 5 轉形作用至E. coli BL21 (DE3) (Transformation)………….. 21
3. 4. 6 小量質體DNA之萃取…..……………………...………….. 21
3. 5 重組蛋白質rPlpE及rLkt’C之表現與分析………...................... 21
3. 5. 1 重組蛋白rPlpE及rLkt’C之表現………………………….. 22
3. 5. 2 蛋白質電泳(SDS-PAGE)……………………………………... 22
3. 5. 2. 1 聚丙烯醯胺膠體之製備…............................................. 22
3. 5. 2. 2 重組蛋白質 rPlpE及rLkt’C之製備……………........ 22
3. 5. 2. 3 重組蛋白質之定量………………................................. 22
3. 5. 3 重組蛋白質之抗原性分析-西方墨點法 (western blotting).. 23
3. 6 疫苗之製備……………………..………....................................... 23
3. 6. 1 M. haemolytica 之定量與不活化………………………….. 23
3. 6. 2 重組蛋白之培養與不活化…………………………………. 24
3. 6. 3 疫苗之調配…………………………………………………. 24
3. 7 動物試驗………..………………………………………………..... 24
3. 7. 1 小鼠安全效力試驗-rPlpE.....………………………………. 24
3. 7. 2 小鼠安全效力試驗-rLkt’C.....……………………………… 25
3. 7. 3 仔羊安全及效力試驗.….…................................................... 25
3. 7. 4 羊隻攻毒模式之建立………………………………………. 26
3. 8 免疫反應之分析……..…………………………………............... 26
3. 8. 1 酵素連結免疫吸附法(ELISA)…..……………...………….. 26
3. 8. 2 免疫細胞增生試驗…………………………………………. 26
3. 8. 2. 1 小鼠免疫……………………………………………….. 26
3. 8. 2. 2 脾臟細胞之分離………………………………………. 27
3. 8. 2. 3 周邊血液單核球細胞 (PBMC)之抽取.…….………… 27
3. 8. 2. 4 細胞計數………………………………………………. 27
3. 8. 2. 5 抗原刺激………………………………………………. 27
3. 8. 2. 6 MTT assay……………………………………………… 28
3. 8. 2. 7 細胞增生指數 SI值計算……………………………... 28
3. 9 分泌型白血球毒素 (sLkt) 之製備.…………………………….... 28
3. 9. 1 分泌型白血球毒素之抽取….…..……………...…………… 28
3. 9. 2 白血球毒素之抗原性分析….…..……………...…………… 29
3. 10 統計分析………………………………………………………… 29
第4章 結果…………………………………………………….………... 30
4. 1 M. haemolytica plpe 之選殖、定序與胺基酸比對....................... 30
4. 2 M. haemolytica lkt’C 之選殖、定序與胺基酸比對…………….. 30
4. 3 重組蛋白質rPlpE及rLkt’C 之表現與分析……….………........ 30
4. 4 白血球毒素之製備與抗原性分析………………………………. 31
4. 5 動物試驗………………………………………………………….. 31
4. 5. 1 小鼠效力試驗 – rPlpE………………………………………. 31
4. 5. 2 小鼠細胞性免疫反應試驗…………………………………. 31
4. 5. 3 小鼠效力試驗 –rLkt’C…………………………………….. 32
4. 5. 4 小鼠攻毒試驗………………………………………………. 32
4. 5. 5 仔羊效力試驗………………………………………………. 32
4. 5. 6 羊隻攻毒模式之建立………………………………………. 33
第5章 討論…………………………………………………..……….... 58
參考文獻…………………………………………………………………. 62
附錄………………………………………………………………………. 71
Appendix 1. The positive percentage of bovine diseases in Taiwan…… 71
Appendix 2. The positive percentage of bovine diseases cross infection
in Taiwan…………………………………………………. 72
作者簡介……….…………………………………………………………. 73

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