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研究生:楊伊瑋
研究生(外文):Yi-Wei Yang
論文名稱:幽門螺旋桿菌高抗原性蛋白之鑑定及其診斷上之應用
論文名稱(外文):Identification of immuno-dominant antigens in Helicobacter pylori and their application in the diagnosis of Helicobacter pylori infection
指導教授:王錦堂王錦堂引用關係
指導教授(外文):Jin-Town Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:79
中文關鍵詞:幽門螺旋桿菌診斷高抗原性蛋白鑑定
外文關鍵詞:Helicobacter pyloriimmuno-dominant antigendiagnosisidentfication
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血清學的方法是目前廣泛使用於檢測是否有幽門螺旋桿菌( Helicobacter pylori ) 感染的方法之一,但目前使用的血清學檢測方法,在準確度、靈敏度上仍有一定的限制,並且需培養大量的幽門螺旋桿菌抽取蛋白質,因此進一步鑑定幽門螺旋桿菌中具有高抗原性的蛋白質,可能進一步提高血清學診斷的準確性並節省檢驗的成本。我們將幽門螺旋桿菌液態培養液中的蛋白質經二維電泳技術分離,樣本血清根據血清檢測試劑(HEL-pTEST®Ⅱ kit)以ELISA 方式檢驗及細菌培養確認幽門螺旋桿菌感染,將血清類分為真陽性及偽陰性,各取兩個血清進行西方墨點法篩選抗原。偵測結果發現有兩群共通的訊號出現,進一步以微量蛋白質N端定序及基質輔助雷射脫附游離/飛行時間質譜分析方法來鑑定其中數點,與資料庫比對分析結果,與幽門螺旋桿菌的蛋白質 catalase以及flagellar hook-associated protein 2(HAP2)有相當高的相似度。以分子生物學的技術表現catalase及HAP2的重組蛋白,接著再用27個真陽性,5個偽陰性、10個真陰性血清來分析其抗原性。其中catalase的靈敏度(sensitivity)和準確度(specificity)為25%和100%,而HAP2則為90.6%及60%,顯示HAP2具有較佳的抗原性,並對偽陰性血清樣本有很高的靈敏度,未來可與其他高抗原性蛋白組合應用於幽門螺旋桿菌感染的檢測。
Serological tests are commonly used methods used in the diagnosis of Helicobacter pylori infection. However, there are still some limitations in the sensitivity and specificity, and total crude lysate from H. pylori culture are necessary. Therefore, to identify immunodominant antigens of H. pylori may improve the accuracy of serological tests and save costs. We separated proteins recovered from broth culture medium by using two-dimensional gel electrophoresis and Western blotting were performed with two true positive and false negative sera proved by HEL-pTEST®Ⅱ ELISA test kit and bacteria culture. We found there were two common groups of signals. Using N-terminal sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques to identify these spots, we discovered that the proteins were homologus to H. pylori catalase and flagellar hook-associated protein 2 (HAP2). Recombinant proteins of catalase and HAP2 were expressed to test their antigenicity. We tested 27 true positive, 5 false negative and 10 true negative sera individually. The sensitivity and specificity of catalase was 25% and 100%, and of HAP2 was 90.6% and 60%. The results showed that the antigenicity of HAP2 is sensitive in detecting false negative sera group and may be applied to serodiagnosis in combination with other immunodominant antigens.
目錄 ................................................................................................................ 1
英文摘要 …………………………………………………………………………… 3
中文摘要 …………………………………………………………………………… 4
第一章 緒論 ……………………………………………………………………… 5
研究目的 ……………………………………………………………….. 14
第二章 材料 …………………………………………………………………….. 15
2.1 幽門螺旋桿菌(H. pylori)菌株 ………………………………………… 15
2.2 大腸桿菌(E. coli)菌株 ………………………………………………… 15
2.3 培養基 ……………………………………………………………………... 15
2.4 引子 ………………………………………………………………………... 16
2.5 血清 ………………………………………………………………………... 17
2.6 載體 ………………………………………………………………………... 17
2.7 抗生素 ……………………………………………………………………... 18
第三章 方法 …………………………………………………………………….. 19
3.1 幽門螺旋桿菌之液態培養與蛋白質回收 ……………………………...… 19
3.2 篩選具良好抗原性的幽門螺旋桿菌蛋白質 …………………………...… 20
3.3蛋白質鑑定 ………………………………………………………………… 23
3.4 重組幽門螺旋桿菌蛋白質表現質體的建構、表現及純化 ……………... 25
3.5 利用不同的血清樣本來測試重組蛋白的抗原性 ………………………... 36
3.6 以純化的蛋白質競爭抗體的結合 ………………………………………... 38
第四章 結果 …………………………………………………………………….. 39
4.1 幽門螺旋桿菌之培養與蛋白質之回收 …………………………………... 39
4.2 篩選具良好抗原性的幽門螺旋桿菌蛋白 ………………………………... 40
4.3 蛋白質鑑定 ………………………………………………………………... 41
4.4重組幽門螺旋桿菌蛋白質表現質體之建構、表現及純化 ……………….. 43
4.5利用不同的血清樣本來測試重組蛋白的抗原性 ………………………… 46
4.6以純化蛋白質競爭抗體的結合 ………………………………………… 46
第五章 討論 …………………………………………………………………….. 48
第六章 圖表 …………………………………………………………………….. 58
表一:血清測試結果 …………………………………………………………… 58
表二:根據血清測試結果加以分析重組蛋白之抗原性 ……………………... 58
圖一:pET-28a-c(+) 表現載體圖譜 ………………………………………….. 59
圖二:pGEX-4T-1表現載體圖譜 ……………………………………………... 60
圖三:將TCA沉澱回收之幽門螺旋桿菌液態培養基中的蛋白質,經二維電泳分離後以silver stain染色 ………………………………………. 61
圖四:以西方墨點法篩選具良好抗原性的幽門螺旋桿菌分泌性蛋白 ……… 62
圖五:MALDI-TOF MS分析圖譜 ……………………….…………………….. 63
圖六:catalase-His tag重組蛋白純化結果 …………….…………………….. 64
圖七:HAP2重組蛋白經GSTrap FF column純化結果 ……………………... 65
圖八:以西方墨點法及免疫偵測法分析HAP2的抗原性 ……………………. 66
圖九:以西方墨點法及免疫偵測法分析HAP2的抗原性 ……………………. 67
圖十:以西方墨點法及免疫偵測法分析catalase的抗原性 ………………… 68
圖十一:以西方墨點法及免疫偵測法分析catalase的抗原性 ………………. 69
第七章 參考文獻 ……………………………………………………………….. 70
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