粉尘螨变应原ProDer f 1编码基因重组pET28a-YARA-Ihc体系的构建及表达

Construction and expression of the recombinant plasmid pET28a-YARA-Ihc-ProDer f 1 of Dermatophagoides farina

目的构建粉尘螨变应原ProDerf1原核表达载体pET28a-YARA—IhC—ProDerf1,为评价其免疫治疗效果奠定基础。方法用分子克隆技术构建出表达载体pET28a—YARA—IhC—ProDerf1,在E.coli BL21(DE3)中表达融合蛋白YARA-IhC-ProDer f 1,并进行Ni^(2+)-NTA树脂柱亲和层析以纯化蛋白。结果经测序证实成功构建了表达载体pET28a-YARA-IhC-ProDer f 1,YARA-IhC-ProDer f 1融合蛋白在E.coli BL21(DE3)中得到表达,纯化后的蛋白浓度为278μg/ml。SDS-PAGE和Westem blot分析表明纯化蛋白为目的蛋白YARA-IhC-ProDer f 1。结论已成功制备出pET28a—YARA—IhC-ProD—er f 1原核表达载体,融合蛋白得到表达和纯化。

Objective To construct the prokaryotic expression vector pET28a-YARA-Ihc-ProDer f 1 and provide the foundation for exploring the fusion protein effect as vaccine for specific immunotherapy. Methods Two oligonucleotides encoding YARA were synthesized and annealed to generate YARA-encoding DNA. The fused genes, YARA-Ihc-ProDer f 1 was constructed and inserted into the prokaryotic expression vector pET28a（＋）. The fusion protein YARA-Ihc-ProDer f 1 induced with IPTG in E.coli BL21（DE3） was purified with Ni2＋-resin affinity chromatography and confirmed with SDS-PAGE and Western blot. Results Sequence analysis confirmed the construction of the expression vector pEt28a-YARA-Ihc-ProDer f 1 successfully, the fusion protein YARA-Ihc-ProDer f 1 was expressed and purified with the concentration of 278μg/ml. SDS-PAGE and Western blot demonstrated the fusion protein was YARA-Ihc-ProDer f 1. Conclusion The recombinant prokaryotic expression vectors, pET28a（＋）-YARA-Ihc-ProDer f 1 was successfully constructed, and the fusion protein was expressed and purified .