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研究生:溫芷瑀
研究生(外文):Chih-Yu Wen
論文名稱:以牛型結核菌素重組純化蛋白衍生物作為ELISA檢測抗原之評估
論文名稱(外文):Evaluation of recombinant purified protein derivatives of bovine tuberculin as diagnostic antigens in ELISA
指導教授:郭致榮
口試委員:劉正哲賈敏原
口試日期:2020-07-20
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
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:2020
畢業學年度:108
語文別:中文
論文頁數:94
中文關鍵詞:牛型結核病牛型結核菌素純化蛋白衍生物酵素連結免疫吸附分析抗原性
外文關鍵詞:Bovine tuberculosisbovine purified protein derivativeenzyme-linked immunosorbent assayantigenicity
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牛型結核病 bovine tuberculosis簡稱bTB,是由牛型分枝桿菌(Mycobacterium bovis , M.bovis)所引起的疾病,主要感染草食性動物,亦是重要的人畜共通傳染病。此疾病不僅對農業經濟帶來衝擊,也造成公共衛生的問題。目前牛型結核病的標準檢驗方式為利用牛型純化蛋白衍生物 (bovine purified protein derivative, bPPD) 進行皮內結合核菌素測試 (intradermal tuberculin test, ITT) 或是以鳥型純化蛋白衍生物 (avian purified protein derivative, aPPD) 進行駢比皮內結核菌素測試 (comparative intradermal tuberculin test, CITT)。而當ITT及CITT可能在感染後期出現偽陰性的結果,因此為了提高感染後期的檢驗準確率,血清學診斷 (serological test)如酵素連結免疫吸附分析 (enzyme-linked immunosorbent assay, ELISA) 成了不可或缺的輔助診斷方法。本篇研究從牛型純化蛋白衍生物中選殖15個特異性基因,構築成表達質體後經轉型作用送入大腸桿菌 (Escherichia coli) 進行大量表現,最後以親和性層析管柱 (affinity chromatography column) 進行蛋白純化。再透過ELISA的方式評估15個重組蛋白對於牛型結核病陽性 (n=62)及陰性 (n=82)血清之抗原性。另一方面,利用本實驗室先前篩選出的抗原嵌合體(antigen cocktails)分析2019年1月到2020年4月所收集之血清,共含乳牛257頭與鹿313頭,其檢測陽性率分別為8/257 (3.1%)與94/313 (30.0%)。若將上述血清以台灣現行常規使用之牛型結核病ELISA檢驗套組 (BTB Ab ELISA 2.0),其檢測陽性率分別為0/257 (0%)與63/313 (20.1%)。由此可證,使用抗原嵌合體作為抗原可提升牛型結核病ELISA血清抗體檢測之檢測率。
Bovine tuberculosis (bTB), a serious zoonotic infectious disease in herbivore, mainly caused by Mycobacterium bovis (M. bovis) can be transmitted to humans. This disease has a significant impact on agricultural economy and public health issues. Nowadays, official diagnoses of animal TB are based on the cell-mediated immunity assays, such as intradermal tuberculin test (ITT) and comparative intradermal tuberculin test (CITT) by using bovine purified protein derivative (bPPD) and avian purified protein derivative (aPPD), respectively. However, false negative results may occur in the late stage of infection. Therefore, serological test has become an auxiliary diagnostic tool to improve the diagnostic accuracy. In this study, fifteen protein candidates selected from bPPD were overexpressed by E. coli and purified by the affinity chromatography columns. Antigenicities of 15 recombinant proteins were evaluated by ELISA using bTB positive (n=62) and negative (n=82) serum samples. On the other hand, bovine (n=257) and deer (n=313) serum samples collected from January 2019 to April 2020 were tested using antigen cocktails (containing 8 antigens) we reported previously. The results showed that the positive rates of bovine and deer samples were estimated as 8/257 (3.1%) and 94/313 (30.0%), respectively. By using the commercial ELISA kit that has been currently used for routine bTB diagnosis in Taiwan, the positive rates were respectively measured as 0/257 (0%) and 63/313 (20.1%), for bovine and deer samples. As a result, the antigen cocktails were demonstrated to possess excellent diagnostic capabilities in bTB serum antibody detection by an ELISA
誌謝辭 i
中文摘要 ii
Abstract iii
表次 vi
圖次 vii
第一章 前言 1
第二章 文獻探討 2
第一節 牛型分枝桿菌 2
一、 病原特性 2
二、 染色特點 2
三、 傳播途徑 2
四、 致病機制 3
第二節 牛結核病之檢測方法 5
一、 生前檢驗法 5
二、 死後檢驗法 9
第三節 牛型結核菌素純化蛋白衍生物 (Bovine tuberculin purified protein derivative, bPPD) 10
一、 常見之製造方法 10
二、 牛型結核菌素純化蛋白衍生物之研究概況 10
第三章 材料與方法 11
第一節 實驗設計 11
第二節 實驗試驗藥品及配方 11
第三節 重組結核菌素蛋白衍生物之基因選殖 12
一、 試驗藥劑及商業套組 12
二、 目標PPD轉殖基因之製備 12
三、 表現載體 (vector) 之製備 13
四、 接合反應 (Ligation) 14
五、 轉型反應 (Transformation) 14
六、 重組質體之篩選 (Colony PCR) 14
七、 定序及重組質體之保存 14
第四節 重組蛋白之表現、純化及分析 15
一、 試驗藥劑及商業套組 15
二、 重組蛋白之大量表現 (Overexpression) 及誘導 (Induction) 15
三、 重組蛋白質之純化 15
四、 重組蛋白之分析及保存 16
第五節 酵素連結免疫吸附分析 (ELISA) 18
一、 試驗藥劑及商業套組 18
二、 乳牛與鹿血清樣本收集 18
三、 重組PPD蛋白樣本 18
四、 間接型ELISA之方法建立 18
第四章 結果 20
第一節 重組結核菌素蛋白衍生物之基因選殖 20
第二節 重組蛋白之表現、純化及分析 20
第三節 酵素連結免疫吸附分析 (ELISA) 20
第五章 討論 23
第一節 重組蛋白之表現 23
第二節 牛型結核菌表面蛋白之功能探討 23
第三節 組合式抗原應用之探討 24
第六章 參考文獻 26
附表 34
附圖 40
附錄一 69
附錄二 77
附錄三 78
附錄四 93
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