幽門螺旋桿菌高抗原性蛋白之鑑定及其診斷上之應用

Identification of immuno-dominant antigens in Helicobacter pylori and their application in the diagnosis of Helicobacter pylori infection

楊伊瑋(Yi-Wei Yang)

指導教授 ： 王錦堂

國立臺灣大學/醫學院/微生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01788

摘要

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。

關鍵字

幽門螺旋桿菌 ； 診斷 ； 高抗原性蛋白 ； 鑑定

並列摘要

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.

並列關鍵字

Helicobacter pylori ； immuno-dominant antigen ； diagnosis ； identfication

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