過敏原蛋白在大腸桿菌系統下之表現與純化

Abstract

過敏是一種現代人常見的疾病，難以預防以及治療，因為環境中過敏原的增加而造成現今過敏人口持續上升中，為了預防過敏的發生，可使用過敏原檢測及相關檢敏療法，但是目前醫療上可檢驗的過敏原種類太少。因此我們的目標是透過大腸桿菌系統大量表現並純化過敏原蛋白，以了解免疫反應引發的過敏分子機制，並應用在生物醫學檢測上。我們總共挑選18個過敏原蛋白作為研究目標，依序可分為五類，包括食物類 (Ara h 1、Ara h 2、Gly m 5和Pen a 1)，花粉類 (Phl p 1、Phl p 5、Amb a 1、Art v 1、Par j 2、Cry j 1、Cry j 2、Cup s 1、Jun a 2和Pla a 1)，橡膠類 (Hev b 5和Hev b 6)，毒液類 (Ves v 1) 以及真菌類 (Asp f 1)。將所有的過敏原基因我們轉殖到pET22b或是pET28a的質體上，利用大腸桿菌系統進行蛋白質表現，再透過管柱層析法萃取蛋白質，並將過敏原蛋白合成抗體，以供日後作為生物醫學檢測上的應用。此外，我們希望透過結構生物學解析過敏原蛋白之晶體結構，更深入探討過敏原所造成的免疫反應之分子機制。

Allergy is a common disease for modern people, and is difficult to prevent and treat. Due to the increase of allergens in the modern environment, people suffering allergy become more and more. Hypersensitivity to allergy can be prevented by combining allergen specific tests and immunotherapy, but the types of allergens that have been tested are very few. Therefore, we aim to purify allergenic proteins in large scale through the E.coli expression system in order to understand the mechanism of allergen induced by immune responses and for applyication in biomedical detection. We selected 18 allergenic proteins, which are divided into five types: the food type (Ara h 1, Ara h 2, Gly m 5 and Pen a 1), the pollen type (Phl p 1, Phl p 5, Amb a 1, Art v 1, Par j 2, Cry j 1, Cry j 2, Cup s 1, Jun a 2 and Pla a 1), the rubber type (Hev b 5 and Hev b 6), the venom type (Ves v 1) and the microorganism type (Asp f 1). The allergenic genes were cloned into pET22b or pET28a plasmids and then expressed through the E. coli expression system. These proteins were further purified in a homogenous state by column chromatography and used on antibody synthesizion for application in biomedical detection. In addition, we will try to determine the crystal structures of these allergenic proteins to understand the allergen induced immune responses.