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研究生:楊莉嘉
研究生(外文):li chia yang
論文名稱:豬隻口蹄疫病毒非結構蛋白ELISA診斷試劑之開發與應用
論文名稱(外文):Development and application of ELISA Kit for the detection of antibodies to the non-structural proteins of foot-and-mouth disease(FMD)virus in pigs
指導教授:鍾文彬鍾文彬引用關係
指導教授(外文):wen bin chung
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:94
中文關鍵詞:口蹄疫非結構性蛋白酵素結合免疫吸附試驗
外文關鍵詞:foot-and -mouth diseasenon-structural proteinenzyme -linked immunosorbent assays
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口蹄疫為高度傳染性之病毒性疾病,造成偶蹄類動物之水疱性疾病。該病毒在複製過程中會產生非結構蛋白(non-structureal protein;NSP),但市售不活化疫苗為純化的病毒,不含或只含極微量的某些NSP,因此,自然感染之動物體內會產生這些NSP抗體,但施打疫苗者則無,本試驗應用此原理,發展鑑別診斷口蹄疫自然感染及疫苗免疫抗體之試劑。首先,將O/TWN/97病毒株之3A及3AB基因應用pRSET、pET43及pBlueBacHis2等載體系統進行選殖,再分別應用大腸桿菌及桿狀病毒進行蛋白質之表現,應用純化後之NSP作為抗原進行酵素結合免疫吸附試驗(ELISA),結果發現,以桿狀病毒所表現之3AB蛋白作為抗原進行間接型ELISA,其敏感性及特異性分別只有62.4﹪及86.8-91.6﹪,效果不佳有待改善,但以大腸桿菌所表現之3AB作為抗原進行雙層三明治阻斷型ELISA,其敏感性及特異性分別為94.3﹪及97.9-100﹪,應用此試劑於現場血清樣本之測試,初步結果顯示可作為口蹄疫撲滅計畫中成效追蹤之用。
Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. The infection of the virus induces vesicular lesions in animals. Non-structural proteins (NSP) are produced during viral replication. The inactivated vaccines consist of purified viral particle without or with only minor contaminants of NSP and thus induce antibody mainly against structural proteins of virus. Differentiation of infection from vaccination can therefore be based on the detection of antibodies to NSP. In this study, the 3A and 3AB genes of O/TWN/97 were cloned using pREST, pET43 and pBlueBacHis2 plasmids. The 3A and 3AB proteins were expressed in the E. coli and baculovirus expression systems. Enzyme-linked immunosorbent assays (ELISA) were then established using the purified 3A and 3AB antigens. The sensitivity and specificity of the indirect ELISA using baculovirus expressed 3AB protein as the antigen were 62.4% and 86.8-91.7%, respectively. The result was not satisfied and requires further modification. The sensitivity and specificity of the double-sandwich blocking ELISA using E. coli expressed 3AB protein as the antigen were 94% and 98-100%, respectively. The application of this diagnostic kit in the field samples demonstrated that this detection system can be used in the monitoring of the progress of FMD eradication program.
目錄 Ⅲ
中文摘要 Ⅰ
英文摘要 Ⅱ
圖次 Ⅶ
表次 Ⅶ
第一章序言 1
第二章文獻探討
2.1口蹄疫之歷史背景 3
2.2症狀、病變及流行病學 4
2.3口蹄疫病毒
2.3.1病毒結構與分類 5
2.3.2病毒之理化特性 6
2.3.3病毒之生長特性 7
2.3.4病毒基因體 8
2.3.5病毒結構蛋白之功能 8
2.3.6病毒非結構蛋白之功能 9
2.3.7結構蛋白之免疫反應 10
2.3.8口蹄疫病毒自然感染或疫苗施打後之免疫反應 11
2.4 NSP抗體之檢測 13
2.5 NSP-ELISA之應用 17
2.6口蹄疫之標準診斷方法
2.6.1病毒分離 18
2.6.2補體結合試驗 19
2.6.3反轉錄聚核酶鏈反應 20
2.6.4病毒中和抗體試驗 21
2.6.5液相阻斷酵素聯合免疫吸附法 21
2.6.6酵素結合免疫吸附法 22
2.7實驗目的與展望 22
第三章材料與方法
3.1病毒來源 23
3.2病毒RNA的抽取與純化 23
3.3引子設計 24
3.4反轉錄聚合鏈反應 25
3.5非結構蛋白3AB 及3A基因片段之純化 26
3.6以大腸桿菌表現口蹄疫病毒3A及3AB蛋白
3.6.1載體製備 28
3.6.2非結構蛋白3A及3AB基因之選殖 28
3.6.3非結構蛋白3A及3AB基因之表現 33
3.7以昆蟲桿狀病毒表現口蹄疫病毒3AB蛋白
3.7.1非結構蛋白3AB基因之選殖 34
3.7.2基因重組桿狀病毒 36
3.7.3病毒純化
3.7.3.1病毒感染與瓊膠覆蓋 37
3.7.3.2基因重組桿狀病毒之培養與DNA萃取 38
3.7.3.3基因重組桿狀病毒DNA之PCR分析 39
3.7.4非結構蛋白3AB之表現 40
3.8 SDS-PAGE電泳分析 41
3.9免疫墨點法 41
3.10西方免疫轉漬法 42
3.11蛋白質純化
3.11.1管柱製備 43
3.11.2蛋白純化 43
3.11.3蛋白復性 44
3.11.4濃度測定 44
3.12間接型酵素結合免疫吸附試驗 44
3.13阻斷型酵素結合免疫吸附試驗 46
3.14丹麥3AB試劑及UBI 3B試劑之檢測方法 47
3.15口蹄疫NSP抗體標準陽性及陰性血清 49
3.16母豬血清、初乳及仔豬血清 49
3.17豬場口蹄疫爆發後之血清 49
3.18統計分析 50
第四章結果
4.1非結構蛋白3A及3AB表現基因之選殖 51
4.2非結構蛋白3A及3AB之表現 51
4.3非結構蛋白抗體檢測方法之初步測試 52
4.4 Eco-3AB阻斷型ELISA之陰陽性界定值 52
4.5 Bac-3AB間接型ELISA之陰陽性界定值 53
4.6 Eco-3AB阻斷型ELISA與其他檢測方法之比較 53
4.7母豬血清、初乳及其仔豬血清之NSP抗體檢測結果 54
4.8豬場口蹄疫爆發後之NSP抗體監測 54
第五章討論 62
第六章參考文獻 69
第七章附錄
7.1各項溶液配製 81
7.2大腸桿菌阻斷液之濃度試驗] 87
7.3抗原阻斷液之選擇比較 88
7.4抗原定量 89
7.5 Kappa值之計算 91
7.6 pET43-3AB質體之構築 92
7.7 pRSRT-3A質體之構築 93
7.8 pBlueBacHis2-3AB質體之構築 94
圖次
圖1. pET43-3AB、pRSET-3A及pBlueBacHis2-3AB表現質體以限制酶HindⅢ與BamHI切割後之電泳圖。……. 55
圖2. 純化後的非結構蛋白3A及3AB融合蛋白之SDS-PAGE電泳圖。………………………………………………. 56
圖3. 非結構蛋白3A及3AB之西方免疫轉漬圖。…………. 57
圖4. 以Eco-3AB阻斷型ELISA檢測豬隻血清中之NSP抗體,其S/N值之分佈圖。………………………………. 58
圖5. 以Bac-3AB間接型ELISA檢測豬隻血清中之NSP抗體,其S/P值之分佈圖。………………………………. 59
圖6. 台灣中部(A)及南部(B)豬場豬隻之血清於不同時期,口蹄疫爆發後之以Eco-3AB阻斷型ELISA進行NSP抗體監測之結果。……………………………… 60
表次
表1 Eco-3AB阻斷型ELISA檢測母豬血清、初乳及其仔豬血清不同檢體間之相關性。…………………………. 61
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