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| 研究生： |
莊 勤讓 Chuang, Chin-Jang |
|---|---|
| 論文名稱： |
增進豬流行性下痢病毒(PEDV)疫苗效力之研究 Study on Improving the Efficacy of Porcine Epidemic Diarrhea Virus (PEDV) Vaccine |
| 指導教授： |
鍾曜吉
Chung, Yao-Chi |
| 學位類別： |
碩士 Master |
| 系所名稱： |
獸醫學院 - 動物疫苗科技研究所 Graduate Institute of Animal Vaccine Technology |
| 畢業學年度： | 110 |
| 語文別： | 中文 |
| 論文頁數： | 73 |
| 中文關鍵詞： | PEDV 、不活化疫苗 、油質佐劑 、維他命A |
| 外文關鍵詞： | PEDV, inactivated vaccine, Oil adjuvant, Vitamin A |
| DOI URL： | http://doi.org/10.6346/NPUST202200024 |
| 相關次數： | 點閱：100 下載：24 |
| 分享至: |
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豬流行性下痢 (PED)乃是全球養豬業界危害非常嚴重的消化道傳染疾病，尤其是造成哺乳仔豬極為嚴峻的損失。台灣在2014年爆發PED，至今仍影響台灣的養豬產業。傳統的疫苗免疫方式無法讓初生乳豬產生足夠免疫力，因此本研究將針對誘發黏膜免疫反應的口服疫苗進行開發，利用多種油質測試作為口服疫苗之佐劑。而最終研究採月見草油及角鯊烯油作為口服疫苗之油質佐劑；另則以SPAN85與Tween80當介面活性劑並在適當的HLB (Hydrophilic and lipophilic balance)值以及合適的SOR (Surfactant oil ratio)比例下配製70%的油相佐劑加30%的水相病毒抗原之口服疫苗。利用滴水測試，離心動作，顯微鏡鏡檢、之後放置於4度C，室溫，37度C三種狀態，進行穩定觀察4周以上，最終再以人工胃液來作測試進行分析。所得配方混和維他命A及不活化病毒進行小鼠動物實驗。實驗結果中，我們利用總IgG、IgA、細胞增生實驗及中和抗體分析檢視佐劑配方是否能夠達到預期的保護效果，並且製作一支有效之口服油質佐劑不活化疫苗。
Porcine epidemic diarrhea (PED) is a very serious infectious disease in the global pig industry. This disease has caused high mortality to suckling piglets. The outbreak of PED in Taiwan in 2014 still affected Taiwan's pig industry. Traditional immunization methods cannot produce sufficient immunity for newborn suckling pigs. Therefore, this study will aim to develop oral vaccines that induce mucosal immune responses against PED. We have tested various oil as adjuvants for developing oral vaccines. In the final study, evening primrose oil and squalene oil were used as the oil-based adjuvants for oral vaccines; SPAN85 and Tween80 were used as surfactants, and 70% oil-phase adjuvants were formulated under appropriate HLB (Hydrophilic and lipophilic balance) values and appropriate SOR (Surfactant oil ratio) ratios. An oral vaccine containing 30% aqueous viral antigen. After the formulation, the oil adjuvants were analyzed by dripping test, centrifugation, microscopic examination. Then the oil adjuvants were storage at 4°C, room temperature or 37°C observation for more than 3 months. After the storage time, the adjuvant was tested in artificial gastric juice. The obtained formulations were mixed with vitamin A and inactivated virus for oral vaccine in mouse animal experiments. In the experimental results, we use total IgG, IgA, cell proliferation assay and neutralizing antibody analysis to check whether the adjuvant formula can achieve the expected protective effect, and make an effective oral oil-based adjuvant inactivated vaccine.
目錄
中文摘要 i
Abstract iii
謝 書 v
目錄 ix
圖表目錄 xiv
第一章 緒言 1
第二章 文獻回顧 4
2.1 豬的流行性下痢 (Porcine Epidemic Diarrhea) 4
2.1.1 豬的流行性下痢之傳統歷史背景 4
2.1.2 近代之變異 4
2.1.3 豬流行性下痢 (PED) 的臨床症狀 5
2.1.4 豬流行性下痢 (PED)之傳播途徑 6
2.1.5 豬流行性下痢 (PED)之致病過程 6
2.1.6 鏡檢及肉眼組織病理學變化 7
2.1.7 病原體特性 7
2.1.8 病原體結構 8
2.1.9 病毒的培養 8
2.1.10 病毒的流行病學 9
2.2 反轉錄聚合酶鍊所反應 (reverse transcription-polymerase chain reaction， RT-PCR) 9
2.3 酵素聯結免疫吸附分析法 (enzyme-linked immunosorbent assay，ELISA) 10
2.4 中和抗體試驗檢測 (Neutralizing Antibody Detection) 10
2.5 病毒的生物防治 11
2.6 乳源性免疫和豬流行性下痢病毒疫苗 11
2.6.1豬流行性下痢 (PED) 之疫苗防治 12
2.7 口服型疫苗 ( Oral vaccine) 13
2.7.1 生物製藥（例如疫苗）口服給藥系統的挑戰與進展 13
2.8 不活化疫苗 ( Inactivated vaccine ) 14
2.8.1 滅活病毒的方法乃多元性 (Methods of inactivating viruses are diverse) 14
2.9 口服維他命A補充劑在PEDV感染孕母豬後之IgA和泌乳免疫保護乳仔豬 15
2.9.1 維他命 A 需求、代謝、輸送，功能 16
2.10機體抵抗感染的第一道防線是黏膜免疫系統（Mucosal immune system, MIS） 17
2.10.1常見黏膜免疫系統 17
2.10.2黏膜耐受性 (MUCOSAL TOLERANCE) 18
2.10.3增強黏膜免疫的策略(STRATEGIES TO ENHANCE MUCOSAL IMMUNITY) 18
2.10.4 IgA與sIgA 18
2.10.5膜黏佐劑 (Mucosal Adjuvants): 18
2.11關於油質佐劑 19
2.12 佐劑在疫苗中所扮演的角色 20
2.13 疫苗佐劑在自體免疫的安全性 22
2.14油包水乳化體系在疫苗中的使用優點 22
油包水乳化體系在疫苗實務上的明顯的缺點 22
第三章 材料與方法 24
3.1 實驗的方法與簡單架構 24
3.2 實驗的主要器材 24
3.3 實驗的主要原料 24
3.4 不活化病毒的準備方法 25
3.5 人工胃液製備方法 25
3.6含維他命A之口服佐劑製備方法 26
3.6.1 補充劑製備方法 26
3.6.2 疫苗劑製備方法 27
3.7 在確定的HBL值情況下，如何計算兩種乳化劑各占多少百分比的方法 27
3.8油包水疫苗製備方法與程序 28
3.9實驗動物小白鼠的準備 30
3.10使用Indirect ELISA方法測定血清中total IgG Antibody 30
3.11 ELISA分析測定小腸黏液中的IgA antibody level 31
3.11 細胞增殖試驗 (Cell proliferation assay) 31
3.11.1 小鼠脾臟製備 31
3.11.2. CCK-8測定 33
3.12血清中和抗體力價測定方法 33
第四章 結 果 35
4.1 細胞感染病毒的結果 (Figure 1.) 35
4.2 通過逆轉錄-PCR 確認病毒 ( Reverse transcription-PCR) (Figure 2.) 35
4.3 病毒通過 Mycoplasma 檢測測試確認病毒 (Figure 3.) 35
4.4概述油質佐劑的篩選過程及標準 36
4.5 油質佐劑的滴水試驗 (Figure 4.) 36
4.8動物實驗分組 (Table 2.) 38
4.8.1 小白鼠之動物實驗分組結果 38
4.9 免疫後小白鼠血液中之total IgG檢測結果 (Figure 8.) 39
4.10免疫後小白鼠腸道中之total IgA檢測結果 (Figure 9.) 39
4.11免疫後小白鼠之脾臟細胞增生(Cell proliferation)分析 (Figure 10.) 40
4.12 免疫後小白鼠之血清中和抗體力價(neutralizing antibody titer)分析 (Figure 11.) 40
第五章 討論 54
第 六 章 心得與感想 58
6.1 筆者個人對於豬流行性下痢 (PED)之看法 58
6.2 關於油包水乳化體系穩定因素之看法 58
6.3 油質佐劑之選擇與測試心得 59
6.3.1 油質佐劑乳化剪切及攪拌 62
6.4 關於動物實驗操作之感想 62
第 七 章 參考文獻 63
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