臺灣博碩士論文加值系統

豬胸膜肺炎放線桿菌（Actinobacillus pleuropneumoniae, A.p.）是屬於溶血性革蘭氏陰性菌，共有15種血清型，會引起豬隻出血性、纖維性和壞死性胸膜肺炎，造成豬場嚴重之經濟損失。A.p.致病因子包括莢膜多醣、脂多醣、外膜蛋白(outer membrane lipoprotein , OMP )及外毒素，其中又以屬於RTX (repeat in the structural toxins)家族之Apx （Actinobacillus pleuropneumoniae -toxin）外毒素為重要毒力因子及具免疫原特性。因不同的血清型間無足夠的交叉免疫保護性，保護效力上仍有限制性，須發展新型疫苗以提高保護力。本研究結果顯示選殖的Apx I、Apx II、Apx IV和OMP基因已成功表現重組蛋白，利用西方墨點法分析抗原性可被接種過豬隻的血清所辨識。將重組蛋白添加佐劑，進行小鼠效力試驗以ELISA 檢測抗體，均可以提昇較高力價，以A.p 血清型第一、五型(1×108 CFU/mL)攻毒後，免疫組的存活率為66.6％，對照組及市售疫苗組則全部死亡。以豬隻免疫後攻毒，對照組豬隻持續有發燒症狀，並出現厭食、活力減低與呼吸困難等臨床症狀，於解剖病變顯示，試驗組比對照組可減輕病變程度達63%，證實rApx 對於豬胸膜肺炎放線桿菌具保護作用。以流式細胞儀分析CD4+及CD8+免疫細胞種類比例，CD8+ 細胞所佔之比例，以免疫組為35.6% ，對照組為19% 。綜合以上結果顯示豬胸膜肺炎放線桿菌重組毒素抗原具有發展為次單位疫苗的潛力。

Actinobacillus pleuropeumoninae (A.p.) is a type of hemolytic Gram-negative bacteria, which includes a total of 15 serotypes, and can cause fibrinonecrotic and hemorrhagic pleuropneumonia, resulting in serious economic loss in farms. The pathogenic factors of A.p. are capsular polysaccharides, lipopolysaccharides, outer membrane proteins (OMP), adsorption factors, and exotoxins. Actinobacillus pleuropneumoniae toxins (Apxs) belong to RTX (repeat in the structural toxin) family, are exotoxins and important virulence factors with immunogenicity, is more efficient than others to induce immune response. Due to weak cross reactivity among different serotypes, the protective ability of traditional vaccines is still limited. Therefore, it is important to develop a new effective vaccine. The results in this study showed successfully expressed these recombinant (Apx I, Apx II, Apx IV and OMP) proteins and can be recognized by antiserum form infected pigs. The ELISA antibodies of immunized mice with these recombinant proteins combined with adjuvant higher than those from control mice. When challenged with A.p. virulent serotype 1 and 5 (1×108 CFU/mL), the survival rate of immunized mice was 66.6% compared to 0% of control and commercial vaccine group. After immunization the pigs challenged with A.p. virulent serotype 1 (8×107 CFU/mL), control group showed the clinical signs including fever, anorexia, and dyspnea . In anatomy result showed the immunized pig was reduce the pathological levels 63% than control. It is demonstrated the rApx could protect pigs suffered from A.p invade. Futhermore, the flow cytometry results in vaccine group CD8+ percentage is 35.6% higher than control group (19%) . The results indicated rApx may have potential as the subunit vaccine candidate.

目 錄
中文摘要....................................................................................................... Ⅰ
Abstract......................................................................................................... Ⅲ
謝誌............................................................................................................... Ⅴ
目錄............................................................................................................... Ⅵ
圖表目錄....................................................................................................... Ⅹ
第1章 緒言................................................................................................. 1
第2章 文獻回顧......................................................................................... 3
2. 1 名稱之演變…….............................................................................. 3
2. 2. Actinobacillus pleuropneumoniae 血清型分類及流行病學…… 3
2. 2. 1 生化特性及培養...…………………………………………… 3
2. 2. 2. 血清型分類………………………………………………… 4
2. 2. 3. 流行病學……….....……...………………………………… 6
2. 3. 豬感染Actinobacillus pleuropneumoniae之病理變化………… 7
2. 3. 1. 臨床病徵及肉眼病變……………………………………… 7
2. 3. 2. 肉眼病變……………………………………………………. 7
2. 3. 3. 顯微鏡下主要病變……......................................................... 8
2. 4. RTX (repeats in toxin)毒素家族…….............................................. 8
2. 5. Actinobacillus pleuropneumoniae 之毒力因子…………………. 8
2. 5. 1. Apx toxins 介紹………………………………………………. 9
2. 5 1. 1. Apx I………………………………………………………… 9
2. 5. 1. 2. Apx II……………………………………………………….. 10
2. 5. 1. 3. Apx III………………………………………………………. 10
2. 5. 1. 4 Apx IV………………………………………………………. 11
2. 5. 2. 內毒素………………………………………………………….. 11
2. 5. 3. 線毛……………………………………………………………. 11
2. 5. 4. 莢膜……………………………………………………………. 13
2. 5. 5. 運鐵蛋白結合蛋白……………………………………………. 13
2. 5. 6. 外膜蛋白………………………………………………………. 14
第3章 材料與方法…………………………………………………….. 15
3. 1 .1 A. pleuropneumoniae之來源……..…………………………. 15
3. 1. 2 A. pleuropneumoniae serotype 1 outer membrane protein(OMP) 15
3. 2 A. pleuropneumoniae 之培養…………………………………… 15
3. 3 A. pleuropneumoniae 之培養與ApxI﹑II和ApxIV 之選殖與定序……….......................................................................................... 15
3. 3. 1 A. pleuropneumoniae DNA之萃取....…………………......... 15
3. 3. 2 聚合酶連鎖反應…….….…………………………………... 16
3. 3. 2. 1 A. pleuropneumoniae ApxI之選殖..........………….. 16
3. 3. 2. 2 A.pleuropneumoniae ApxII之選殖………………… 16
3. 3. 2. 3. A.pleuropneumoniae ApxIV之選殖…………………. 17
3. 3. 3 DNA之膠體電泳法……………………………………….. 17
3. 3. 4 PCR反應產物之純化………………………...…………….. 17
3. 3. 5 PCR反應產物之定序………………………………………. 17
3. 4 以原核表現系統表現ApxI﹑II和ApxIV之重組蛋白質…….. 18
3. 4. 1 ApxI﹑II和ApxIV基因之製備…………………………… 18
3. 4. 2 表現載體之製備……………………………………………. 18
3. 4. 3 ApxI﹑II和ApxIV與表現載體接合反應………………… 19
3. 4. 4 勝任細胞之製備………………………………...………….. 19
3. 4. 5 轉形作用……………………………………...…………….. 19
3. 4. 6 小量質體DNA之萃取…..……………………...…………. 19
3. 5 重組蛋白質ApxI﹑II和ApxIV之表現與分析…………………. 20
3. 5. 1 重組蛋白ApxI﹑II和ApxIV之表現……………………… 20
3. 5. 2 蛋白質電泳……………………………...………………….. 20
3. 5. 2. 1 蛋白質之製備……………………………..................... 20
3. 5. 2. 2 聚丙烯醯胺膠體之製備…………................................. 21
3. 5. 2. 3 重組蛋白質之定量…………………........................... 21
3. 5. 3 重組蛋白質之抗原性分析-西方墨點法…………………… 21
3. 6 疫苗之製備……………………..………....................................... 22
3. 6. 1 A. pleuropneumoniae 之定量與不活化…………………… 22
3. 6. 2 重組蛋白之培養與不活化………………………………….. 22
3. 6. 3 疫苗之調配………………………………………………….. 23
3. 7 動物試驗………..………………………………………………... 23
3. 7. 1 小鼠半致死劑量試驗……………………………………… 23
3. 7. 2 小鼠安全效力試驗.………...……………………………… 23
3. 7. 3 仔豬安全及效力試驗.…….…............................................... 24
3. 8 免疫反應之分析……..…………………………………................ 24
3. 8. 1 酵素連結免疫吸附法……....…………………...………….. 24
3. 8. 2 周邊血液淋巴球細胞 (peripheral blood mononuclear cell; PBMC) 之抽取……………………………………………… 25
3. 8. 3 以流氏細胞儀(FACS FLOW cytometry )分析CD4＋及CD8＋免疫細胞…………………………………………………… 25
3. 8. 4. 統計分析…………………………………………………….. 26
第4章 結果…………………………………………………..………..... 27
4. 1 A. pleuropneumoniae之菌體培養……......................................... 27
4. 2 構築A. pleuropneumoniae之ApxI﹑II和ApxIV選殖………… 27
4. 3 重組蛋白質rApx 之表現與西方墨點法………………………. 27
4. 4 動物試驗………….......................................................................... 27
4. 4. 1 小鼠半致死劑量 (LD50) ........................................................ 27
4. 4. 2 小鼠安全效力試驗.................................................................. 28
4. 4. 2. 1 小鼠安全試驗................................................................ 28
4. 4. 2. 2 小鼠效力試驗................................................................. 28
4. 4. 2. 3 小鼠攻毒試驗……………………….. ………………... 28
4. 4. 2. 4. 分別以血清型第一型和第五型小鼠攻毒試驗………… 29
4. 4. 3. 仔豬安全效力試驗…………………………………………… 29
4. 4. 3. 1. 豬的ELISA 抗體力價…………………………………. 30
4. 4. 3. 2. 攻毒後體溫之差異……………………………………... 30
4. 4. 3. 3. 攻毒後解剖病變差異…………………………………... 30
4. 4. 4. 免疫細胞增生試驗…………………………………………… 30
4. 4. 4. 1. 以流式細胞儀分析CD4+/CD8+免疫細胞種類比例…… 30
第5章 討論…………………………………………………..………... 56
參考文獻………………………………………………………………….. 60
附錄……………………………………………………………………….. 67
Appendix 1. Apx toxin 基因操縱子(operon)………………………………. 67
Appendix 2. 豬胸膜肺炎放線桿菌接收鐵離子之路徑…………………… 68
Appendix 3. pET-32a vector map………………………………………………... 69

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