臺灣博碩士論文加值系統

牛疱疹病毒1型 (Bovine Herpes Virus type 1；BHV-1) 主要引起以鼻氣管炎為主徵之急性熱性呼吸器疾病，常被稱為牛傳染性鼻氣管炎(Infectious Bovine Rhinotracheitis；IBR)。流行熱病毒 (Bovine ephemeral fever virus；BEFV) 在牛主要引起以肺氣腫或肺水腫為主徵之急性熱呼吸器疾病，這些疾病時而侵襲動物，造成酪農戶重大的經濟損失。本研究分別以MDBK、BHK 細胞增殖BHV-1及BEF病毒後，於最適條件下添加不活化劑BEI予以不活化完全，並與w/o/w佐劑混合成試製疫苗。分別免疫小鼠及牛隻，均無任何不良反應產生，牛隻第一次免疫4週後補強注射一劑量，於8週後之ELISA及中和抗體結果顯示較控制組具有顯著性差異 (p < 0.01) ，並可持續10個月之久。此外，利用細胞增生與分析CD4+及CD8+之免疫細胞含量試驗，評估混合疫苗之效力。

Bovine herpesvirus type I (BHV-1) may cause acute febrile respiratory diseases, called infectious bovine rhinotracheitis (IBR). Bovine ephemeral fever virus (BEFV) causes acute febrile respiratory disease, the main symptoms are pulmonary emphysema and pulmonary edema. These disease invade domesticated herds lead to enormous economic losses. In this study, we try to culture MDBK, BHK cell to amplify BHV-1 and BEFV virus and add binary ethylenimine (BEI) to inactivate virus at appropriate conditions. We combined BHV-1 and BEFV with water-in-oil-in-water adjuvant to immunize mice and cattle and no adverse reactions were observed. The immunized cattle showed significant higher ELISA and neutralizing antibody titers than the control post vaccination eight weeks (p < 0.01), and lasting for
10 months. Furthermore, evaluate the vaccine efficacy with cell proliferation, and the percentage of CD4+ or CD8+ T cells with flow cytometry.

中文摘要……………………………………………………………………...I
Abstract Ⅱ
謝誌 Ⅳ
目錄…………………………………………………………………………..V
圖表目錄 Ⅸ
第1章 緒言 1
第2章 文獻回顧 3
2.1 牛疱疹病毒之簡介 3
2.1.1 牛疱疹病毒之背景 3
2.1.2 BHV-1感染牛隻之臨床症狀 3
2.1.3 BHV-1 流行病學 3
2.1.4 BHV-1之分類與結構 4
2.1.5 BHV-1之基因體 5
2.1.6 BHV-1之複製週期 5
2.1.7 BHV-1之物理與化學特性 6
2.1.8疫苗 6
2.1.9佐劑 7
2.2牛流行熱病毒之簡介 8
2.2.1牛流行熱病毒之背景 8
2.2.2 BEF感染牛隻之臨床症狀 8
2.2.3 BEF 流行病學 8
2.2.4 BEFV之分類與結構 9
2.2.5 BEFV之基因體 9
2.2.6 BEFV之複製週期 10
2.2.7 BEFV之物理與化學特性 11
2.2.8預防與控制 11
第3章 材料與方法 12
3.1 病毒分離及分子診斷 12
3.1.1 BHV-1分離及純化 12
3.1.1.1細胞培養 12

3.1.1.2病毒增殖與純化 12
3.1 2 BEFV病毒增殖 12
3.2聚合酶鏈鎖反應 12
3.2.1鑑定牛疱疹病毒I型 12
3.2.1.1萃取BHV-1病毒DNA 12
3.2.1.2引子序列 13
3.2.1.3 PCR反應條件 13
3.2.1.4 DNA電泳分析 13
3.2.2鑑定牛流行熱病毒 13
3.2.2.1萃取BEFV病毒RNA 13
3.2.2.2 RT-PCR 14
3.2.2.3引子序列 14
3.2.2.4 PCR反應條件 14
3.2.2.5 DNA電泳分析 15
3.3 BHV-1之製備 15
3.3.1培養抗原之條件 15
3.3.2培養抗原之生長曲線 15
3.3.3不活化抗原之動力曲線 15
3.4蛋白質之分析 15
3.4.1病毒離心 15
3.4.2蛋白質電泳 ( SDS-PAGE ) 16
3.4.3西方墨點法 ( Western blotting ) 16
3.5病毒力價測定 (50% tissue culture infective dose, TCID50) 17
3.6試製疫苗之調配 17
3.7動物試驗 17
3.7.1安全試驗-小白鼠 17
3.7.2安全試驗-仔牛 17
3.7.3效力試驗-小白鼠 18
3.7.4效力試驗-仔牛 18
3.8免疫分析方法 18
3.8.1酵素連結免疫吸附法 ( enzyme-linked immunosorbent assay；　
　　 ELISA) 18
3.8.1.1 抗原定量 18
3.8.1.2 ELISA抗體-小白鼠 18
3.8.1.3 IBR ELISA Kit抗體-仔牛 19
3.8.1.4 IBR ELISA IgG2抗體- 仔牛 19
3.8.1.5統計分析 19
3.8.2中和抗體 ( neutralizing antibody ) 20
3.8.2.1血清非働化 20
3.8.2.2中和抗體-仔牛 20
3.9細胞增生免疫反應試驗 20
3.9.1免疫小白鼠 20
3.9.2小白鼠脾臟細胞培養 20
3.9.3免疫仔牛 21
3.9.4抽取PBMC ( peripheral blood mononuclear cell ) 21
3.9.5細胞計數 21
3.9.6抗原刺激 21
3.9.7測定細胞增生數 21
3.10以流式細胞儀 ( FACS Flow cytometry )分析CD4+及CD8+免疫細
胞 21
第4章 結果…………………………………………………………………23
4.1病毒株鑑定 23
4.1.1 BHV-1之鑑定 23
4.1.2 BEFV之鑑定 23
4.2 BHV-1製備之結果 23
4.2.1 BHV-1病毒生長曲線 23
4.2.2 BHV-1病毒不活化動力曲線 23
4.3蛋白質之表現與分析 23
4.3.1 BHV-1之表現與分析 23
3.2 BEFV之表現與分析 24
4.4 動物安全試驗 24
4.4.1 小白鼠之安全試驗 24
4.4.2 仔牛之安全試驗 24
4.5 ELISA抗體 24
4.5.1小白鼠之BEFV抗體 24
4.5.2仔牛之BHV-1抗體 24
4.5.3 仔牛之IgG2抗 25
4.6中和抗體 25
4.6.1仔牛之BHV-1抗體 25
4.6.2仔牛之BEFV抗體 25
4.7細胞增生試驗 25
4.7.1小鼠脾臟細胞增生試驗 26
4.7.2仔牛免疫細胞增生試驗 26
4.7.3以流式細胞儀分析CD4+細胞含量 26
4.7.4以流式細胞儀分析CD8+細胞含量 26
第5章 討論 52
參考文獻 55
作者簡介 61

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