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目的:确定苦荞过敏原(TBa)的主要表位区段。方法:以TBacDNA为模板,利用PCR方法,构建TBa的4个表位区段(E12、E3、E6、E36)的重组原核表达载体,转入大肠杆菌BL21(DE3)中诱导表达。经Ni2+-NTA亲和纯化后,SDS-PAGE分析。采用间接和竞争ELISA以及点杂交技术进行免疫活性测定。结果:获得了纯度较高的TBa重组表位蛋白(E12、E3、E6、E36),与先前获得表位E1、E2的免疫活性比较表明,重组表位蛋白与荞麦过敏症患者血清均有IgE结合活性,其中E1的活性最强。结论:E1可能是苦荞过敏蛋白TBa中最重要的抗原结合区域之一。
Objective: To determine the major epitope segment of tartary buckwheat allergen (TBa). Methods: TBacDNA was used as a template to construct a recombinant prokaryotic expression vector of four epitopes (E12, E3, E6, E36) of TBa by PCR. The recombinant plasmid was transformed into E. coli BL21 (DE3) to induce expression. After Ni2 + -NTA affinity purification, SDS-PAGE analysis. Indirect and competitive ELISA and dot blot techniques were used to measure immunocompetence. Results: The higher purity of TBa recombinant epitope protein (E12, E3, E6, E36) was obtained. Compared with the previously obtained epitopes E1 and E2, the immunological activity of TBa showed that both recombinant epitope protein and serum from patients with buckwheat allergy had IgE The binding activity, of which the most active E1. Conclusion: E1 may be one of the most important antigen-binding regions in tartary buckwheat allergy protein TBa.