粉尘螨变应原Derf1基因的原核表达及多克隆抗体的制备

Prokaryotic expression and preparation of polyclonal antibody of recombinant group 1 allergen from Dermatophagoides farinae

目的:原核表达、纯化粉尘螨主要变应原Der f1重组蛋白,并制备其多克隆抗体。方法:大量诱导表达含pET-28a(+)-Der f1质粒的BL21菌株,SDS-PAGE电泳并割胶纯化目的蛋白,以此为抗原免疫新西兰大白兔,收集血清进行效价检测并用Western blot检测其特异性。结果:割胶回收可有效纯化Der f1重组蛋白,抗Der f1抗体效价为1∶32 000。结论:成功制备Der f1多克隆抗体,以期为尘螨过敏性疾病的诊断和免疫治疗提供参考。

Objective：To purify the recombinant protein of Dermatophagoides farinae（Der f1） through prokaryotic expression in Escherichia coli and prepare its polyclonal antibody.Methods： After recombinant plasmid Der f1/pET-28a（＋）Escherichia coli BL21 was successfully constructed,the bacterial pellet was resuspended and lysed by freezing-thawing treatment.The target protein was excised from the gel after SDS-PAGE electrophoresis and purified.Anti-Der f1 antibody was raised by immunization against Der f1 in New Zealand white rabbits through detection of specificity using Western blot method.Results： Def f1 was recovered by gel extraction,and the titer of anti-Der f1 antibody was 1∶32 000.Conclusion： Anti-Der f1 antibody was prepared successfully,which will provide a base for diagnosis and immunotherapy of allergic diseases.