杨絮热休克蛋白70(HSP70)结构域蛋白的生物信息学分析、原核表达及生物学活性鉴定

Bioinformatics analysis,prokaryotic expression and biological activity of HSP70 domain-containing proteins derived from pollen of Populus deltoides

目的揭示杨絮热休克蛋白70(HSP70)结构域蛋白生物信息学特征,并探索该类蛋白外源表达方法及生物学活性。方法通过生物信息学软件对已获得的3个含HSP70结构域蛋白的理化性质进行分析,利用免疫表位数据库和分析资源(IEDB)对该类蛋白的T、B细胞表位进行预测;根据Uniprot数据库提供的氨基酸序列合成相关基因并克隆入pET28a(+)质粒进行原核表达,采用SDS-PAGE检测蛋白表达,表达产物经镍柱纯化后行Western blot鉴定目的蛋白;蛋白浓度定量试剂盒检测蛋白浓度后,分别以鉴定出的目的蛋白为抗原制备BALB/c小鼠哮喘模型,ELISA检测小鼠血清中IgE抗体浓度。结果经生物信息学分析B9N9W6、B9GX02及A0A2K2AYN8蛋白相对分子质量(Mr)分别为71 900、94 600和75 200,均为亲水性蛋白且稳定性较好;经在线预测,共鉴定出3种蛋白的高亲和力T细胞表位13个,B细胞表位14个;SDS-PAGE显示,B9N9W6蛋白、B9GX02蛋白及A0A2K2AYN8蛋白分别在约72 000、95 000和75 000处出现特异性条带,Western blot法也在相应位置出现特异性条带;ELISA检测显示,提取液组和A0A2K2AYN8组IgE表达水平高于PBS组;同A0A2K2AYN8组比较,B9N9W6组及B9GX02组IgE浓度显著增高。结论杨絮HSP70结构域蛋白A0A2K2AYN8、B9GX02及B9N9W6均可原核表达,为可溶性蛋白,且具有生成IgE的生物活性。

Objective To study the bioinformatics characteristics of HSP70 domain proteins derived from pollen of Populus deltoides(P. deltoides), optimize the prokaryotic expression methods, and identify the biological activity of these proteins. Methods Physicochemical characteristics of three kinds of HSP70 domain-containing proteins were analyzed by bioinformatics software. The T/B cell epitopes of these proteins were predicted by Immune Epitope Database and Analysis Resource(IEDB). According to the amino acid sequence provided by Uniprot database, their nucleotide sequences were synthesized and cloned into pET28a(+) plasmid for prokaryotic expression. Protein expression was detected by SDS-PAGE, then the expressed products were purified by nickel column and identified by Western blotting. The protein concentration was measured by protein quantitative kit. Then the three proteins were used as antigens to prepare mouse asthma models, and the concentration of serum total IgE antibody was determined by ELISA. Results The bioinformatics analysis showed that the relative molecular mass(Mr) of B9N9W6, B9GX02 and A0A2K2AYN8 were 71 900, 94 600 and 75 200, respectively. The 13 T-cell epitopes and 14 B-cell epitopes were identified in the three proteins which had high hydrophilia and stability. SDS-PAGE analysis revealed that the genes encoding the three proteins were expressed with three specific bands of approximately Mr 72 000, 95 000 and 75 000, respectively. Western blotting showed the specific bands at the corresponding sites. ELISA showed that the IgE level in the extract group and the A0A2K2AYN8 group were higher than that in the PBS group. Compared with the A0A2K2AYN8 group, the IgE concentration in the B9N9W6 group and B9GX02 group increased significantly. Conclusion The soluble HSP70 domain-containing proteins A0A2K2AYN8, B9GX02 and B9N9W6 derived from pollen of P. deltoides can be expressed as well as purified, and have the biological activity of producing IgE antibodies.