TBa过敏原表位区段的融合表达及免疫活性分析

为了确定苦荞过敏原TBa(tartarybuckwheatallergen)的抗原表位及进一步了解荞麦过敏反应机制,本实验以苦荞过敏原TBacDNA序列为模板,设计引物,克隆苦荞过敏原TBa表位区段基因,分别构建TBa及两个表位区段原核表达载体,在大肠杆菌BL21(DE3)中进行表达并纯化,采用竞争ELISA对其免疫活性进行分析与比较。SDS-PAGE及WesternBlot鉴定和检测结果表明,目的蛋白在E.coliBL21(DE3)中可高效表达,其N端带有6个组氨酸标签。Ni2+-NTA琼脂糖柱亲和纯化得到了纯度较高的目的片段。ELISA实验结果显示,表达产物与荞麦食品过敏病人血清中的IgE具有特异的结合活性。本研究为揭示苦荞过敏蛋白结构与功能的关系奠定了基础。

Fusion Expression and Analysis of the Epitopes in Tartary Buckwheat Allergen

To map the epitopes for tartary buckwheat allergenic protein (TBa) and to reveal its reaction mechanism, the TBa and epitopes gene were cloned into the expression vector pET-32m, and expressed in E.coli BL21 (DE3) host cells. Furthermore, the expression products were purified by Ni2+-NTA agarose affinity chromatography column. Analysis of SDS-PAGE and western blot indicated that the recombinant fusion proteins can be largely expressed and the 6×His tag at N-terminus were confirmed. The results of ELISA indicated that the target protein had a specific binding activity with IgE antibody in the sera of the patient allergic to buckwheat food, which should lay a useful foundation for further study of the location of the epitope and reaction mechanism of tartary buckwheat allergen.