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研究生:江翰鍇
研究生(外文):Han-KaiJiang
論文名稱:石斑魚神經壞死病毒及虹彩病毒多價重組次單位疫苗之開發
論文名稱(外文):Development of the multivalent recombinant subunit vaccine of nervous necrosis virus and iridovirus for grouper
指導教授:陳宗嶽
指導教授(外文):Tzong-Yueh Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:118
中文關鍵詞:神經壞死病毒虹彩病毒多價重組次單位疫苗
外文關鍵詞:NNVIridovirusTR-PCRrecombinant subunit vaccine
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神經壞死病毒 (nervous necrosis virus, NNV) 與虹彩病毒 (Iridovirus) 對台灣石斑養殖產業造成嚴重損失,本研究主旨為開發能同時對抗兩種病毒之多價重組次單位疫苗。本研究以龍膽石斑為試驗對象,首先建立病毒之攻毒平台,所測定 NNV 之半致死劑量 (median lethal dose, LD50) 為 105.2 TCID50/mL;Iridovirus 之 LD50 為 2.76 × 107.5 copies/mL。在疫苗開發部份,篩選可能的 NNV 鞘蛋白 (NNV coat protein, NNVCP) 與 Iridovirus 主要外鞘蛋白 (major capsid protein, MCP) 作為抗原決定域 (epitope),以 TR-PCR 技術製備重組蛋白作為候選次單位疫苗,並與神經壞死病毒之類病毒顆粒 (NNV-VLPs) 疫苗做比較測試。以 real-time PCR 分析重組次單位疫苗所誘發之免疫相關基因表現,實驗結果發現較偏向Th2 免疫路徑,產生抗體免疫反應。在安全性測試部分,實驗結果顯示重組次單位疫苗對魚體具備高度安全性;在多價次單位疫苗效力測試部分,以 NNV 攻毒後,NNVCP-S5E 混合 IMCP400-8E 組之相對存活率 (relative percent survival, RPS) 為 69%;以 Iridovirus 攻毒後之 RPS 為50%。實驗結果顯示多價重組次單位疫苗對魚體具保護作用,可以延緩發病,具較低之得病死亡率。
In recent years, nervous necrosis virus (NNV) and Iridovirus infection caused high mortality and serious losses in Taiwan grouper industry. Therefore, the development of the multivalent recombinant subunit vaccines would be an ideal strategy for disease control and prevention. Firstly, to evaluate the efficacy of vaccine, we determine the half lethal dose of NNV and Iridovirus on giant grouper (Epinephelus lanceolatus). The results showed that the LD50 in NNV was 105.2 TCID50/mL, and the LD50 in Iridovirus was 2.76 × 107.5 copies/mL on giant grouper. On the other hand, nervous necrosis virus-like particle (NNV-VLPs) vaccine was used in this study. The predictable epitopes were chosen and amplified by template-repeat polymerase chain reaction (TR-PCR) from NNV coat protein (NNVCP) and major capsid protein (MCP) of Iridovirus as candidates for vaccine development. The immune-related gene expression at various times in spleen and head kidney were analyzed. Our results showed that Th2 immune pathway might be involved and leaded to antibody production. The results of safty trials showed that these vaccines were highly safety for grouper. And the results of efficacy trials demonstrated the vaccines were effective in preventing virus infection. After challenge with NNV and Iridovirus respectively, the relative percent survival (RPS) values in vaccine NNVCP-S5E mixed with IMCP400-8E were 69% and 50%. The results showed that the mortality of vaccinated groups were much lower than unvaccinated groups.
目錄
中文摘要 ..............................................I
英文摘要 ............................................... II
誌謝 ...............................................VI
目錄 ..............................................VII
表目錄 ............................................. XII
圖目錄 .......................................... XIII
縮寫表 ......................................... XV
一、研究背景 ...................................... 1
1-1 石斑魚簡介 ................................. 1
1-2 神經壞死病毒 ................................... 2
1-3 虹彩病毒 ....................................... 3
1-4 病毒共感染現象 ................................. 5
1-5 魚用疫苗簡介 ................................... 7
1-6 魚病防治方法 ................................. 7
1-7 魚用疫苗研究歷程 ........................................ 8
1-8 去活化疫苗 ...................................... 11
1-9 不活化疫苗 ................................. 12
1-10 重組次單位疫苗 ..................................... 13
1-11 核酸疫苗 ................................ 16
1-12 單價疫苗與多價疫苗之比較
1-13 利用 TR-PCR 建立重組次單位疫苗生產平台 ................. 18
1-14 利用 NNV-VLPs 建立重組次單位疫苗生產平台 ......... 20
1-15 研究目的 ........................................ 21
二、實驗材料 ............................... 22
2-1 生物材料 ................................. 22
2-2 質體 ...................................... 22
2-3 反應試劑組 .............................. 22
2-4 其他藥品與試劑 ...................................... 22
三、實驗方法 ...................................... 24
3-1 組織 RNA 萃取 ....................................... 24
3-2 反轉錄聚合酶鏈鎖反應 ................................ 24
3-3 即時螢光定量 PCR .................................. 25
3-4 細胞培養 ................................ 25
3-5 病毒之分離 ....................................... 26
3-6 病毒之純化 ......................................... 26
3-7 病毒力價測定 ...................................... 27
3-8 中和抗體試驗 ......................................... 28
3-9 負染色法 ............................................. 28
3-10 重複模版聚合酶鏈鎖反應 .............................. 29
3-11 轉接短鏈聚合酶鏈鎖反應 ............................... 30
3-12 核酸接合反應 .................................... 30
3-13 勝任細胞製備方法 .................................. 30
3-14 細胞轉型 ........................................ 31
3-15 菌種之保存 ...................................... 31
3-16 質體小量製備 ................................ 32
3-17 核酸瓊脂洋菜膠體電泳分析 .............................. 32
3-18 膠體回收 ...............................33
3-19 核酸限制酵素處理 ..................................... 34
3-20 蛋白質誘導 ......................................... 34
3-21 聚丙烯醯胺膠體製備 .......................... 35
3-22 聚丙烯醯胺膠體電泳 ................................... 35
3-23 重組蛋白之純化 .................................... 36
3-24 利用 BCA 法偵測蛋白質濃度 .......................... 37
3-25 西方墨點法 .......................................... 37
3-26 神經壞死病毒半致死劑量測定 ..................... 38
3-27 虹彩病毒半致死劑量測定 ................................ 39
3-28 免疫石斑魚 ............................. 39
3-29 疫苗效力測試 ..................................... 40
3-30 疫苗安全性測試 .................................. 40
3-31 酵素免疫吸附實驗 .............................. 40
四、實驗結果 ................................... 42
4-1 病材收集與 NNV 檢測 ................................. 42
4-2 人工接種不同病毒力價之 NNV 與 LD50 測定結果 ............... 42
4-3 病材收集與 Iridovirus 檢測 ........................ 43
4-4 人工接種不同病毒力價之 Iridovirus 與 LD50 測定結果 ........ 43
4-5 建立抗 NNV 重組次單位疫苗生產平台 ....................... 44
4-6 分析 NNV-VLPs 與 NNVCP-S5E 免疫後抗體變化情形 ...... 45
4-7 抗 NNV 重組次單位疫苗之效力測試結果 ...................... 47
4-8 抗 NNV重組次單位疫苗之安全性測試結果 .................... 49
4-9 建立抗 Iridovirus 重組次單位疫苗生產平台 ................. 50
4-10 分析 IMCP200-4E 與 IMCP400-8E 免疫後抗體變化情形 ... 51
4-11 抗 Iridovirus重組次單位疫苗之效力測試結果 ............... 52
4-12 抗 Iridovirus 重組次單位疫苗之安全性測試結果 ............ 53
4-13 以 IMCP200-4E 與 IMCP400-8E 混合免疫測試結果 .......... 54
4-14 以 NNVCP-S5E 與 IMCP400-8E 混合免疫效力測試結果 ... 54
4-15 分析 NNV-VLPs 與 NNVCP-S5E誘發免疫相關基因表現.. 55
五、討論 ..................................... 57
5-1 建立 NNV攻毒模式 ..................................... 57
5-2 建立 Iridovirus 攻毒模式 .............................. 58
5-3 抗 NNV 重組次單位疫苗製備與測試 ................... 59
5-4 抗 Iridovirus 重組次單位疫苗製備與測試 ........... 62
5-5 疫苗混用效力測試與多價疫苗效力測試之探討 .......... 63
5-6 重組次單位疫苗誘發龍膽石斑走向 Th2 免疫途徑 ................ 64
5-7 疫苗效力改善與總結 ............................... 67
六、參考文獻 ........................................ 69

表目錄
表一、NNV 檢體收集相關資訊歸納 .......................... 84
表二、龍膽石斑NNV半致死劑量測定結果 ........................ 85
表三、NNVCP-S5E 免疫血清中和抗體測試結果 ..................... 86
表四、NNV-VLPs 免疫血清中和抗體測試結果 ..................... 87
表五、Iridovirus 檢體收集相關資訊歸納 ....................... 88
表六、龍膽石斑 Iridovirus 半致死劑量測定結果 ............... 89
表七、重組次單位疫苗安全性測試結果 ........................ 90
表八、單價重組次單位疫苗效力測試結果....................... 91
表九、多價重組次單位疫苗效力測試結果...................... 92
表十、實驗中所使用之引子序列總表 .......................... 93

圖目錄
圖一、表現載體 pNNVCP-S5E 上之標的蛋白表現及純化結果 ........... 94
圖二、表現載體 pSUMO-NNVCP 上之標的蛋白表現及純化結果 ...... 95
圖三、分析 SUMO-NNVCP 經 SUMO-protease 處理結果 ......... 96
圖四、利用 SDS-PAGE 與 western blot分析 NNV-VLPs 組裝情形 .. 97
圖五、利用穿透式電子顯微鏡確認 NNV 與 NNV-VLPs 相似性 ....... 98
圖六、分析龍膽石斑免疫 NNVCP-S5E 後血清抗體效價變化 .......... 99
圖七、分析龍膽石斑免疫 NNV-VLPs 後血清抗體效價變化 ........ 100
圖八、疫苗安全性試驗與效力試驗之實驗流程 ...................... 101
圖九、以 1.1 × 107 TCID50/mL 劑量 NNV 攻毒致死率分析..... 102
圖十、以 1.1 × 106 TCID50/mL 劑量 NNV 攻毒致死率分析 ...... 103
圖十一、以 1.1 × 105 TCID50/mL 劑量 NNV 攻毒致死率分析 ...... 104
圖十二、表現載體 pIMCP200-4E 上之標的蛋白表現及純化結果 ..... 105
圖十三、表現載體 pIMCP400-8E 上之標的蛋白表現及純化結果 ..... 106
圖十四、分析龍膽石斑免疫 IMCP200-4E 後血清抗體效價變化 ....... 107
圖十五、分析龍膽石斑免疫 IMCP400-8E 後血清抗體效價變化 ....... 108
圖十六、以 2.76 × 109 copies/mL 劑量 Iridovirus 攻毒致死率分析.... 109
圖十七、以 2.76 × 108 copies/mL 劑量 Iridovirus 攻毒致死率分析.... 110
圖十八、以 2.76 × 107 copies/mL 劑量 Iridovirus 攻毒致死率分析.... 111
圖十九、混合免疫後以 Iridovirus 攻毒致死率分析.............. 112
圖二十、多價疫苗免疫後以 NNV 攻毒致死率分析 ................. 113
圖二十一、多價疫苗免疫後以 Iridovirus攻毒致死率分析 .......... 114
圖二十二、分析龍膽石斑免疫後脾臟中免疫相關基因表現 .............. 115
圖二十三、分析龍膽石斑免疫後頭腎中免疫相關基因表現 .............. 117

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