苦荞过敏蛋白全长基因的克隆、表达及免疫学活性研究

Study on Cloning and Expression of A Full-length Gene of Allergenic Protein of Tartary Buckwheat and Its Immunological Activity

养麦属于一种常见的植物性过敏原。本实验在以往研究的基础上,以苦荞种子总RNA为模板,通过RT- PCR和5'-RACE等方法,扩增、克隆获得苦荞过敏蛋白全长cDNA序列。分析表明,该序列编码一个南515个氨基酸残基组成的功能蛋白,并与甜荞过敏性贮藏蛋白的氨基酸序列同源性达到90%以上。经原核表达及一步亲和纯化后得到纯度较高的重组苦荞过敏蛋白(rTBt)。采用竞争性ELISA对其免疫活性分析显示,目的蛋白与荞麦过敏病人血清中的IgE抗体可以特异结合。

Buckwheat is a common allergen in plants.In the present study,a full-length cDNA encoding 515 amino acid residues was amplified and cloned from the total RNA of tartary buckwheat by PCR and RACE methods.Its nucleotide sequence has high homology with main allergenic storage protein mRNA in common buckwheat.The recombinant allergenic protein of tartary buckwheat (rTBt) expressed in Escherichia.coli BL21 (DE3) was purified by using affinity chromatography and it can specifically bind to the IgE in patients ser...