粉尘螨过敏原Der f15的表达、纯化和生物信息学分析  

Expression,Purification and Bioinformatics Analysis of Dust Mite Allergen Der f15

作　　者：胡维[1] 詹政科[1] 刘玉琳[2] 喻海琼[1] 杨小猛[1] 刘晓宇[1] 

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,广东深圳518060 [2]南昌大学基础医学院免疫学教研室,南昌330006

出　　处：《南昌大学学报（医学版）》2015年第4期1-4,共4页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(31328014;31400786);广东省高等学校国际暨港澳台科技合作创新平台项目(2012gjhz0009);深圳市科技计划国际科技合作项目(GJHZ20130408174112021);深圳市科技计划项目(JCYJ20130329110735981;JCYJ20120613173233810);深圳市南山区研发项目(KC2012JSYB0003A)

摘　　要：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107ku。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。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 affinity chromatography,and the allergenic activity was detected by Western blot.The three-dimensional structure and B-cell epitopes were predicted by bioinformatics analysis.Results After induction 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 purification 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 recombinant 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,purification 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 diagnosis and vaccine treatment of dust mite allergy disease.

关 键 词：粉尘螨 Der f15 表达 免疫印迹法 

分 类 号：R392.8[医药卫生—免疫学]