蟑螂主要过敏原Blag7单克隆抗体的制备与鉴定

摘要

目的 制备蟑螂主要过敏原Bla g7的单克隆抗体(mAb).方法 用含编码Bla g7基因的大肠杆菌菌株Origami/pGS21a,诱导表达并纯化出Bla g7蛋白,以该蛋白为抗原免疫BALB/c小鼠,运用杂交瘤技术制备抗Bla g7 mAb;通过ELISA、Western blot技术鉴定mAb的特异性.结果 获得2株可稳定分泌抗Bla g7 mAb的杂交瘤细胞,分别命名为Bla g7-1和Bla g7-2;2株单克隆抗体的免疫球蛋白亚类均为IgG1；经间接ELISA法测定,杂交瘤细胞Bla g7培养上清中mAb的效价分别为1:16000和1:8000; Western blot结果表明Bla g7单抗对Bla g7蛋白具有高度特异性.结论 成功地诱导表达Bla g7蛋白,并制备出抗Bla g7 mAb,为进一步研究蟑螂过敏提供了有用的实验工具.%Objective Anti-cockroach allergen ( Bla g7) monoclonal antibodies (mAb) were produced and identified. Methods The Bla g7 was expressed in Origami/pGS2la E. Coli strain, and highly purified by affinity chromatography. BALB/c mice were immunized with Bla g7 protein and hybridoma cell lines were obtained by limited dilution procedure. The specificity of mAbs was characterized by enzyme-linked immunosorbent assay ( ELISA) and Western blot. Results Two hybridoma cell lines that secreted mAbs against Bla g7 were obtained, named Bla g7-l and Bla g7-2. The subtype of the two mAbs belongs to the IgGl. The titers of mAbs in the culture supernatant were 1: 16 000 and 1: 8 000 respectively, which were detected by indirect ELISA. Bla g7-l and Bla g7-2 mAbs can specifically bind with Bla g7, shown by Western blotting. Conclusion Bla g7 was successfully expressed and purified. Two mAbs against Bla g7 were obtained which will provide valuable tools for studies on cockroach related allergic diseases.