行政院國家科學委員會專題研究計畫期中進度報告:蝦類過敏原之生化及免疫特性研究(2/3)

食物過敏是因為某些特定的食物所引起的過度免疫反應。通常在食入或吸 入特定食物後，很快地就可見IgE 所媒介的第一型過敏反應的發生。據統計， 將近 8 % 的小孩與約2 % 的成人會有食物過敏現象。 由於蝦類已知是常見的海鮮過敏原之一，故本實驗選擇國人經常食用且廣 布在東南亞地區的草蝦作為研究蝦子過敏的材料。在第一年的實驗中我們利用蝦 子過敏病人血清結合蛋白質體學的技術（ 雙向電泳、膠內酵素切割與 MALDI-TOF 質譜儀），鑑定到除了已知的海鮮過敏原:原肌球蛋白 (tropomyosin) 外，還鑑定到一個分子量約為 40 kDa，等電點為6.0，新的蝦子過敏原，並將之 命名為 Pen m 2。接著利用離子交換樹脂成功地純化出具有激 活P性en m 2 的 分子，並證實其具IgE 結合能力， 因此認為精胺酸激 是一種新發現的蝦類過敏 原。在第二年的研究中，我們利用蛋白質定序所得N 端序列與經質譜分析所得 之蛋白質內部高度保留區域之序列來設計引子，進而進行Pen m 2 之選殖與重組 蛋白之表現。再利用該重組蛋白純化製備出多株抗體，並進行重組蛋白質之免疫 特性研究，證實該重組蛋白仍具有IgE 結合能力。 本實驗係第一次利用蛋白質體的方法學來鑑定海鮮過敏原。該種快速且正 確的蛋白質鑑定系統將加速我們對海鮮過敏研究的腳步，並且提供海鮮過敏相關 資訊於臨床診斷與治療上的新應用。Food allergy is defined as the exaggerated response of the immune system after the ingestion of a food or food additive. IgE-mediated type I hypersensitivity reactions to food occur rapidly following ingestion or inhalation of a food antigen to which an individual is sensitive. Up to 8 % of children less than 3 years of age and approximately 2 % of the adult population experience food-induced allergic disorders. Shrimp is one of the important reported seafood allergen. In this study, We chose the black tiger shrimp, P. monodon, one of the most common shrimp species in Southeast Asia as material and demonstrated the identification and characterization of the shrimp allergen. In the first year, we used proteomics approach to identify not only the well known allergen, tropomyosin, but also a novel allergen, designated Pen m 2. Comparison of the Pen m 2 sequence with known protein sequences revealed extensive similarity with those of arginine kinase (EC 2.7.3.3) from crustaceans. A purified Pen m 2 was obtained by anion exchange chromatography and remained its arginine kinase and IgE -binding activity. In the second year, using sequence information obtained by mass spectrometry and N-terminal sequencing , we cloned and expressed recombinant Pen m 2。Followed by purification of recombinant Pen m 2, we generated polyclonal antibody of Pen m 2。 By means of immunological analysis, confirming IgE-binding ability of recombinant Pen m 2。 This is the first time to identify seafood allergen by proteomics approaches. This strategy provides a powerful tool for the identification of allergenic proteins from heterogeneous food sources and should greatly improve the further studies in food allergy sensitization. In addition, it could provide important information on the development of a new class of biopharmaceuticals and future application of diagnosis for food allergic disease