雞傳染性華氏囊病 (Infectious bursal disease，IBD)與禽流感 (Avian influenza，AI)為具高度傳染性之禽類病毒性疾病，IBD由傳染性華氏囊病毒 (Infectious bursal disease virus，IBDV)所引起。禽流感由禽流感病毒 (Avian influenza virus，AIV)所感染，屬於A型流感病毒，此兩種疾病皆使家禽產業遭受嚴重的經濟損失。目前研究指出傳染性華氏囊病毒之類病毒顆粒 (Virus-like particle，VLP) 可作為一種安全並且具有良好效力的IBD疫苗，除此之外，IBDV的類病毒顆粒還能作為載體攜帶外源基因，具有發展為雙價疫苗的潛力，因為DNA疫苗具有能引發體液性免疫反應，也能引發細胞性免疫反應之特點，因此，本篇論文的目的是以DNA疫苗的架構發展對抗傳染性華氏囊病與禽流感的雙價疫苗。在質體的構築上，選用H6N1亞型的AIV之HA1基因取代部分VP3基因，製備成重組質體pTri/VP243-HA1，另外也將IBDV之VP243基因與H6N1之HA1基因分別構築於pTriEx-3載體，作為對照組質體。藉由免疫螢光染色分析 (Immunofluorescence assay)與西方墨點法 (Western blotting)確認重組質體均能成功表現目標蛋白。第一次動物實驗以純化之質體免疫一週齡雞隻，共免疫三次，採集血液偵測抗體反應，並利用IBDV高病原株 (TWN strain)攻毒試驗評估疫苗保護力，結果顯示免疫之雞隻無法產生對IBD與AI之保護力。因此第二次動物實驗，增加DNA疫苗免疫次數與使用次單位疫苗作為補強免疫。次單位疫苗之重組抗原蛋白由大腸桿菌 (Escherichia coli，E.coli)與人類胚胎腎細胞 (HEK-293T cells)所表現。本論文結果顯示無論是單純免疫pTri/VP243-HA1的DNA疫苗或以VP243-HA1次單位疫苗補強免疫的組別，皆可誘發雞隻產生保護力對抗IBDV之攻毒試驗；而利用自製的間接型酵素連結免疫吸附試驗則觀察到抗HA1抗體的揚升。

Infectious bursal disease (IBD) and avian influenza (AI) are highly contagious avian viral diseases which cause severe economic loss in poultry industry. Current researches showed that virus-like particles (VLP) of infectious bursal disease (IBDV) has been proved as a safe and efficacious vaccine against IBD. Besides, the VLP of IBDV can also play a role as a vector to carry a foreign protein to form a bivalent vaccine. The purpose of this study is to develop a bivalent vaccine against infection of IBDV and AIV. DNA vaccines are a powerful means of inducing humoral and cellular immune responses and chosen for this study to form IBDV VLP carrying HA protein of AIV. Partial VP3 gene of IBDV was substituted with HA1 gene of AIV (H6N1) and the VP243-HA1 gene was cloned into pTriEx-3 vector, named as pTri/VP243-HA1. The plasmid pTri/VP243 carrying VP243 gene and pTri/HA1 carrying HA1 gene were also constructed as control plasmids. Those plasmids were verified by immunofluorescence assay and western blotting. Purified plasmids and proteins expressed by Escherichia coli expression system and HEK-293T cells were prepared for animal experiment. The result indicated that chickens vaccinated with DNA vaccines and boosted with DNA vaccines or subunit vaccines showed protection against IBDV challenge and positive antibodies against HA1 by ELISA.

誌謝 i
摘要 ii
Abstract iii
目次 iv
表次 viii
圖次 ix
第壹章、 緒言 1
第貳章、 文獻探討 3
第一節 雞傳染性華氏囊病 3
(一) 雞傳染性華氏囊病之歷史背景 3
(二) 傳染性華氏囊病之臨床症狀 3
(三) 傳染性華氏囊病之病變 4
(四) 傳播途徑 5
(五) 診斷方法 5
(六) 預防與控制 7
第二節 雞傳染性華氏囊病病毒 8
(一) 病毒特性 8
(二) 病毒致病機制 9
(三) 病毒型態、基因體與蛋白 10
(四) 病毒顆粒 11
(五) 類病毒顆粒 13
第三節 禽流感及禽流感病毒 14
(一) 禽流感之歷史背景 14
(二) 禽流感病毒 14
(三) 禽流感之亞型 21
(四) 禽流感之臨床症狀 21
(五) 傳播途徑與自然宿主 22
(六) 診斷 22
(七) 預防 24
第四節 DNA疫苗與次單位疫苗 24
(一) DNA疫苗 24
(二) 次單位疫苗 27
第五節 傳染性華氏囊病與禽流感雙價疫苗之開發 29
第參章、 材料與方法 31
第一節 重組表現質體之構築 31
(一) 病毒株來源及pTri/VP243質體 31
(二) 原核與真核表現載體pTriEx-3 31
(三) 禽流感病毒紅血球凝集基因(AIV-HA)基因片段之選殖 32
(四) 真核與原核重組表現質體之構築 34
(五) 轉形作用 (Transformation) 35
(六) 質體DNA之小量萃取與確認 36
(七) 菌種保存 37
第二節 重組質體之短暫表現 37
(一) 人類胚胎腎臟細胞株 (HEK-293T)之培養 37
(二) 轉染作用 38
(三) 間接免疫螢光分析 38
第三節 原核細胞重組蛋白之表現 39
(一) 轉形作用 39
(二) 原核細胞重組蛋白之小量誘導表現 40
第四節 真核細胞(HEK-293T)之重組蛋白表現 41
(一) 真核HEK-293T細胞中重組蛋白表現 41
第五節 重組蛋白之分析 42
(一) 蛋白質膠體電泳分析 42
(二) 西方轉漬法 43
第六節 DNA疫苗之製備 44
(一) 質體DNA之大量萃取 44
(二) 質體DNA之濃度及純度分析 45
(三) DNA疫苗之確認 45
(四) 質體DNA之短暫表現 45
第七節 次單位疫苗之製備 46
(一) 重組蛋白之大量誘導表現 46
(二) 重組蛋白之純化 46
(三) 重組蛋白於HEK-293T細胞中表現 47
(四) 重組蛋白之電泳分析 47
(五) 重組蛋白之定量 47
(六) 次單位疫苗之配製 50
第八節 DNA疫苗與重組次單位疫苗之保護效力評估 50
(一) 第一次動物試驗之免疫計畫 50
(二) 第二次動物試驗之免疫計畫 51
(三) 血清抗體檢測 52
(四) 臨床症狀與組織病理學檢查 55
(五) 華氏囊中IBDV之偵測 56
第九節 統計分析 57
第肆章、 結果 58
第一節 重組表現質體之構築 58
第二節 重組表現質體於真核細胞進行短暫表現之確認 58
第三節 原核重組蛋白之表現與確認 59
第四節 重組蛋白之純化 59
第五節 真核重組蛋白之表現與確認 60
第六節 DNA疫苗之製備 60
第七節 次單位疫苗之製備 61
(一) 重組蛋白定量 61
(二) 次單位疫苗之配置 63
第八節 DNA疫苗與重組次單位疫苗之保護效力評估 63
(一) 第一次動物試驗 63
(二) 第二次動物試驗 65
第伍章、 討論 100
參考文獻 106
附錄 120
附錄一、實驗材料之配置 120
附錄二、AIV H6N1之HA基因全長序列 126

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