粉尘螨变应原第7组分(Der f 7)重组蛋白单克隆抗体的制备

Preparation of monoclonal antibodies against recombinant Der f 7 allergen

目的:克隆表达粉尘螨变应原第7组分(简称Der f 7)重组蛋白,制备抗Der f 7单克隆抗体,并进行鉴定。方法:用p ET28a(+)-Der f 7原核表达体系表达Der f 7重组蛋白,以此免疫BALB/c小鼠,将骨髓瘤细胞和脾细胞融合,经ELISA法筛选杂交瘤细胞。用杂交瘤细胞注射入小鼠腹腔,获得腹水,经蛋白A琼脂糖介质纯化,后鉴定抗体的效价、纯度,采用免疫印迹法鉴定抗体的特异性。结果:获得三株稳定分泌重组Der f 7单克隆抗体的杂交瘤细胞,产生的抗体纯度高,效价高于1∶243 000。Western blot显示能很好地识别Der f 7重组蛋白。为Der f 7变应原的定量、定位及相关过敏性疾病的诊疗奠定基础。结论:成功获得粉尘螨变应原第7组分(Der f7)重组蛋白单克隆抗体,且抗体效价纯度及特异性较好。

Objective：To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody. Methods： The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a（ ＋ ） -Der f 7. BALB/c mice were immunized with the recombinant protein. Myeloma cells and spleen cells were fused, and hybridoma cells were screened by ELISA. Hybridoma cells were injected into the mice abdominal cavity to obtain aseites. It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody. The specificity of the antibody was identified by Western blot. Results： Three hybridoma ceils which stably secret recombinant Der f 7 monoclonal antibody were obtained. The monoclonal antibody had high purity, the titer was higher than 1：243 000. Western blot showed that Der f 7 recombinant protein could be recognized well. Conclusion： We successfully obtained Der f 7 monoclonal antibody, which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.