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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 (m Ab) of bovine phosphoenolpyruvate carboxykinase (PEPCK) and to identify its specificity. Methods The recombinant plasmid containing PEPCK gene was constructed and transformed into E.coli. The expression product PEPCK recombinant protein was purified. PEPCK recombinant protein was used as antigen to immunize BALB / c mice. Spleen cells and mouse bone marrow Hybridoma cells were obtained by fusion of tumor cells. Positive clones were screened by indirect ELISA, and cell lines stably secreting anti-PEPCK m Ab were obtained. The cell lines were identified by Western blot, immunohistochemistry and immunoprecipitation. Results The hybridoma cell line stably secreting anti-PEPCK m Ab was successfully obtained. Western blot, immunohistochemistry and immunoprecipitation results showed that the antibody could specifically bind to bovine PEPCK protein. Conclusion Anti-bovine PEPCK m Ab was successfully obtained.