苦荞过敏原TBa的表位突变及免疫活性鉴定

Immunological Characterization and Mutational Analysis of Allergen TBa in Tartary Buckwheat

前期研究曾分离获得一种纯度均一的天然苦荞过敏蛋白(tartary buckw heatallergena,TBa),免疫检测发现其为苦荞中的主要过敏原。本实验采用定点突变的方法对得分较高的表位E1的基因进行分子改造,构建5个突变体L39R、L42R、L47R、V52R、L54R。在大肠杆菌BL21中分别进行表达,经Ni2+-NTA亲和纯化后得到纯度较高的重组突变体蛋白。ELISA和点杂交检测发现,与突变前的E1相比,突变体L42R、L47R、L54R的免疫活性明显降低,这表明Leu42、Leu47、Leu54在TBa的过敏性中起着至关重要的作用。

Buckwheat not only contains abundant nutriments such as flavoniods, but also has allergenic proteins that cause hypersensitive reactions. A natural allergenic protein with molecular weight of 24 kD named TBa （tartary buckwheat allergen A） has been obtained after separation and purification in our previous studies. According to its biological information, 5 mutants including L39R, L42R, L47R, V52R and L54R were constructed through site-directed mutagenesis and expressed in Escherichia coli BL21. The expressed products were purified by Ni2＋ affinity chromatography. Purified recombinant proteins were examined using ELISA and dot blot and mutants L42R, L47R and L54R presented a weaker ability to bind tartary buckwheat allergic patients serum IgE than original E1, which suggests that Leu42, Leu47 and Leu54 might play a significant role in TBa allergenity.