【摘要】 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。更多还原

【Abstract】 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.更多还原