臺灣博碩士論文加值系統

新城病（Newcastle disease；ND）是對所有禽類具有高度傳染力及致病力的急性接觸性傳染病，由新城病病毒 (Newcastle disease virus；NDV)引起，造成禽類快速死亡。本病的預防策略主要以施打疫苗來達成，但2000年後第七基因型病毒仍爆發於世界各地，造成家禽產業嚴重的經濟損失。目前疫苗研發策略除了開發基因改造病毒及核酸疫苗外，具安全性及低風險的次單位蛋白質疫苗也是策略之一。研究指出NDV的F及HN蛋白可產生具保護力的中和抗體反應，但F是主要的免疫原性蛋白。因此，本篇論文研發策略是使用桿狀病毒和昆蟲細胞表現系統來表現第七基因型病毒的F蛋白，並混合佐劑ISA 71 VG，進行動物試驗並評估其效力、血清學HI、ELISA及VN檢驗方法。使用pTriEx-3質體將增幅之F基因構築成pTri/ND-F質體，與昆蟲桿狀病毒DNA共轉染至Sf9細胞重組成帶有F基因昆蟲桿狀病毒後，在持續增殖病毒的昆蟲細胞中使用免疫螢光染色分析與西方墨點法，均能確認F蛋白的表現情形。第一次動物試驗結果顯示，免疫一次3.5 μg或10 μg的F蛋白次單位疫苗雞隻，免疫2週後使用Sato強毒株攻毒後均死亡，NDV shedding為100％；HI、ELISA及VN結果均呈現無力價或極低力價。第二次動物試驗結果顯示，免疫三次（間隔2週）10 μg的F蛋白次單位疫苗雞隻，攻毒後均存活，NDV shedding分別為16.67％（共泄腔）及0％（喉頭）。HI結果無法呈現血清的抗體力價，但ELISA及VN結果隨著免疫次數增加，平均抗體力價有上升趨勢。本論文結果指出免疫三次10 μg的F蛋白次單位疫苗可以保護雞隻免於攻毒後死亡，並減少NDV shedding；血清學ELISA及VN結果均能呈現抗體力價，2者間具非常顯著高度相關性。結果顯示桿狀病毒和昆蟲細胞系統表現之NDV F蛋白具有成為對抗NDV病毒感染之次單位疫苗的潛力。

Newcastle disease (ND), an acute contagious disease, is highly infectious and pathogenic to all poultry. Currently, ND is controlled by vaccination with attenuated and/or killed vaccines. The objective of this study was to develop a subunit vaccine containing genotype VII NDV fusion (F) protein expressed by baculovirus-inset cell expression system against ND virus infection in chickens. The subunit vaccine was prepared by mixing F protein with the adjuvant ISA 71 VG. SPF chickens were injected with subunit vaccine (10 μg of F protein) or commercial killed vaccine (as control) three times at two-week intervals from four weeks of age, challenged with Sato strain at ten weeks of age and observed for one week. The immune responses were evaluated by serological HI, ELISA and VN titers and the efficacy was determined by the detection of shedding NDV and observations of clinical signs and mortality. Chickens received subunit vaccine had 16.67 % (anal) and 0 % (throat) of NDV shedding and no chicken showed clinical signs and mortality. As expected, F protein did not induce any HI antibody titer. The antibody titer of ELISA and VN showed an upward trend with the increase of the numbers of vaccinations. There was a significant correlation (r = 0.711, P < 0.01) between the ELISA and VN antibody titers. Chickens received subunit vaccine had significant higher ELISA and VN titers than those in chickens without vaccination and challenge, but significant lower titers than those in chickens vaccinated with commercial killed vaccine. The results of this study indicated that the ND subunit vaccine was able to induce protective immune responses, reduced virus shedding and prevent chickens from death and showing clinical signs.

摘要 i
Abstract ii
目次 iii
表次 viii
圖次 ix
第壹章、緒言 1
第貳章、文獻探討 3
第一節 新城病 3
(一) 新城病流行概況 3
(二) 新城病之臨床症狀 4
(三) 新城病之病理變化 5
(四) 傳播途徑 5
(五) 診斷方法 6
(六) 預防與控制 7
第二節 新城病病毒 8
(一) 病毒分類 8
(二) 病毒顆粒與基因體 8
(三) 病毒蛋白 9
(四) 病毒複製 12
(五) 病毒致病機制 13
(六) 病毒基因型 14
(七) 病毒血清型 14
第三節 新城病疫苗研發概況 15
(一) 基因改造病毒疫苗 15
(二) 次單位蛋白質疫苗 16
(三) 病毒載體疫苗 16
(四) 核酸疫苗 16
(五) 馴化或不活化疫苗 17
第四節 開發新城病次單位疫苗與檢驗方法介紹 17
(一) 疫苗研發種類之評估 17
(二) 次單位疫苗簡介 18
(三) 桿狀病毒於昆蟲細胞株表現系統 19
(四) 佐劑 21
(五) 新城病疫苗檢驗方法 21
(六) 總結 22
第參章、材料與方法 24
第一節 F基因質體之構築 24
(一) 病毒株來源 24
(二) pTriEx-3質體 24
(三) F基因片段之選殖 25
(四) pTri/ND-F之構築 26
(五) 轉形作用 (Transformation) 27
(六) 質體DNA之小量萃取與確認 27
(七) 菌種保存 28
第二節 pTri/ND-F質體之短暫表現 29
(一) 雞纖維母細胞 (Chicken Fibroblast Cell；DF-1)之培養 29
(二) 轉染作用 (Transfection) 30
(三) 間接免疫螢光分析 30
第三節 F基因重組桿狀病毒製備與增殖 31
(一) F基因重組桿狀病毒製備 31
(二) F基因重組桿狀病毒力價測定 32
(三) 重組桿狀病毒內F基因偵測 33
(四) F基因重組桿狀病毒增殖 34
(五) F基因重組桿狀病毒間接免疫螢光分析 34
第四節 重組桿狀病毒之F蛋白分析 36
(一) 蛋白質溶液 36
(二) 蛋白質膠體電泳分析 36
(三) 西方轉漬法 37
第五節 重組桿狀病毒之F蛋白表現 38
(一) 昆蟲細胞 (Trichoplusia ni cell line, Tni)製備 38
(二) 間接免疫螢光分析 39
(三) F蛋白表現最佳條件測試（Optimization） 40
(四) F蛋白分析 40
第六節 次單位疫苗之製備 41
(一) F蛋白大量表現 41
(二) F蛋白之純化 41
(三) 純化過程之蛋白質溶液濃度測定 42
(四) 純化過程之蛋白質溶液分析 43
(五) 純化之F蛋白濃度 43
(六) 次單位疫苗之配製 44
第七節 動物試驗計畫 45
(一) 第一次動物試驗 45
(二) 第二次動物試驗 46
第八節 重組次單位疫苗之攻毒效力評估 46
(一) 臨床症狀評估 46
(二) 攻毒後NDV之偵測 47
第九節 重組次單位疫苗之血清學檢測方法評估 48
(一) 雞隻血液採集與血清分離 48
(二) 血清學檢測方法 48
第十節 統計分析 51
第肆章、結果 52
第一節 F基因質體之構築 52
第二節 pTri/ND-F質體之短暫表現 52
第三節 F基因重組桿狀病毒製備與增殖 53
第四節 重組桿狀病毒之F蛋白分析 54
第五節 重組桿狀病毒之F蛋白表現 55
第六節 次單位疫苗之製備 56
第七節 動物試驗計畫 57
第八節 重組次單位疫苗之攻毒效力評估 58
第九節 重組次單位疫苗之血清學檢測方法評估 59
第伍章、討論 89
參考文獻 94
附錄 103
附錄一、實驗材料之配置 103
附錄二、新城病第Ⅶ基因型病毒之增幅F基因全長序列 (1662 bp) 107

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