摘要
目的制备牛磷酸烯醇式丙酮酸羧激酶(PEPCK)单克隆抗体(m Ab),并对其特异性进行初步鉴定。方法构建含PEPCK基因的重组质粒,将其转化大肠杆菌,并进行诱导表达,对表达产物PEPCK重组蛋白进行纯化,以PEPCK重组蛋白为抗原,免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞融合获得杂交瘤细胞,用间接ELISA筛选阳性克隆,获得稳定分泌抗牛PEPCK m Ab的细胞株,并用Western blot法,免疫组织化学染色和免疫沉淀鉴定其特异性。结果成功获得稳定分泌抗牛PEPCK m Ab的杂交瘤细胞株。Western blot法、免疫组织化学和免疫沉淀结果表明,该抗体能与牛PEPCK蛋白特异性结合。结论成功获得抗牛PEPCK m Ab。
Objective To prepare the monoclonal antibody against bovine phosphoenolpyruvate carboxykinase （PEPCK） and characterize its biological functions. Methods The recombinant plasmid containing PEPCK gene was constructed and used to transfect Escherichia coli. After expression induction in E. coli, the recombinant protein PEPCK was purified and used to immunize BALB/c mice. After the spleen B cells in the immunized mice were fused with murine myeloma cells, the positive clones were identified and selected by indirect ELISA for titer determination. PEPCK mAb produced by the positive hybridoma cells was enriched and its biological functions were examined by Western blotting, immunohistochemistry and immunoprecipitation. Results One hybridoma cell line steadily secreting PEPCK mAb was successfully generated, namely 3D36H. Western blotting, immunohistochemistry and immunoprecipitation showed that the PEPCK mAb was able to specifically bind to bovine PEPCK protein. Conclusion The bovine PEPCK mAb was prepared successfully and had a good ability and specificity.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第6期829-832,837,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
"973"计划项目(2009CB918904)
国家自然科学基金(81070671)
