【摘要】 目的对重组的苦荞过敏蛋白TBb进行免疫学活性鉴定,并预测其B细胞表位。方法根据已获得的苦荞过敏蛋白N端结构域TBb的基因序列,构建原核表达载体pET-32m-TBb,然后转入E.coliBL21(DE3)中表达,表达产物用Ni2+-NTA琼脂糖柱亲和纯化,并用ELISA分析其免疫学活性,综合分析TBb的二级结构、亲水性、可及性、可塑性、抗原性指数,并预测其B细胞表位的分布。结果获得了纯度95%以上的重组过敏蛋白TBb,获得的重组蛋白能与荞麦过敏病人血清中的IgE抗体特异性结合,具有较高的免疫学活性,在TBb蛋白的320个氨基酸残基中,预测到的B细胞表位位于6-17,31-45,50-57,88-94,103-134,138-146,156-163,178-185,192-220,240-260,267-299区段。结论TBb蛋白的活性分析及B细胞表位的预测为进一步研究该蛋白的分子特征及应用奠定了基础。更多还原

【Abstract】 Objective To identify the immunological activity of allergic protein TBb in tartary buckwheat and to predict its B cell epitope.Methods The TBb gene was cloned into the expression vector pET-32m,and then expressed in E.coli BL21(DE3)host cell.The expressed product was purified by Ni2+-NTA agarose affinity chromatography column.The immunological activity of the protein was analyzed by ELISA.The secondary structure,hydrophilicity,and antigenic index of the protein were analyzed using DNA star software,and B cell epitopes was predicted.Results The purity of the target protein reached over 95% and the result of ELISA indicated that the recombinant TBb,implied high immunological activity,had a specific binding activity with IgE antibody from sera of buckwheat-allergic patients.The B cell epitopes were located at or adjacent to these regions:6-17,31-45,50-57,88-94,103-134,138-146,156-163,178-185,192-220,240-260,and 267-299.Conclusion The analysis of activity and prediction of B cell epitopes lay a foundation for the further research of molecular character and application of allergic protein in tartary buckwheat.更多还原