粉尘螨过敏原 Der f15的表达、纯化和生物信息学分析

摘要

Objective To obtain purified recombinant dust mite allergen Der f15,and to predict its three-dimensional structure and B-cell epitopes.Methods The recombinant plasmids encoding Der f15 were introduced into Escherichia coli Rosetta,and the expression of recombinant Der f15 was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).Der f15 protein was purified by affin-ity chromatography,and the allergenic activity was detected by Western blot.The three-dimen-sional structure and B-cell epitopes were predicted by bioinformatics analysis.Results After in-duction with IPTG,Der f15 protein was highly expressed in Escherichia coli.SDS-PAGE analysis showed that the molecular weight of the recombinant Der f15 protein was 107 ku.After purifica-tion by affinity chromatography,Western blot was performed with serum from dust mite-allergic patients as the primary antibody.The recombinant Der f15 protein specifically bound serum IgE, and no reactive bands appeared in negative controls.Western blot analysis showed that the recom-binant Der f15 protein had good antigenicity.Bioinformatics analysis showed that regions 20-35, 121-131,340-359 and 485-500 were potential B-cell epitopes.Conclusion The expression,purifica-tion and bioinformatics analysis of dust mite allergen Der f15 help to understand the molecular and biological characteristics of Der f15 protein and lay a theoretical foundation for the specific di-agnosis and vaccine treatment of dust mite allergy disease.%目的：表达纯化出粉尘螨过敏原 Der f15蛋白，并预测其三维结构及 B 细胞抗原表位。方法利用实验室已有的 Der f15质粒转化到大肠杆菌 Rosetta，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，通过亲和层析纯化蛋白，经免疫印迹法（Western-blot）分析 Der f15免疫原性，用生物信息学方法分析 Der f15的三维结构及 B 细胞抗原表位。结果高效表达出 Der f15蛋白。SDS-PAGE 结果显示：表达的产物分子量约107 ku。经亲和层析成功纯化蛋白，纯化蛋白以尘螨过敏患者血清为一抗进行 Western-blot，结果显示：Der f15重组蛋白能与患者血清 IgE 发生特异性结合，而阴性对照没有反应条带出现，表明 Der f15具有良好的抗原性。对 B 细胞抗原表位的预测表明， Der f15蛋白的第20～35、121～131、340～359及485～500是潜在 B 细胞抗原表位。结论粉尘螨 Der f15表达、纯化和生物信息学分析研究有助于了解 Der f15蛋白的分子生物学特性，为粉尘螨过敏反应性疾病的特异性诊断和疫苗治疗奠定理论基础。