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目的:制备抗人ANKIB1的小鼠单克隆抗体。方法:PCR扩增人ANKIB1(1-480)的编码序列,构建pGEX-5X-ANKIB1(1-480)表达载体。在大肠杆菌中诱导表达GST-ANKIB1(1-480),利用谷胱甘肽琼脂糖凝胶纯化重组蛋白。使用纯化蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,筛选杂交瘤细胞株,通过ELISA、Western blot和免疫沉淀实验鉴定抗体,并检测ANKIB1在哺乳动物细胞中的表达。结果:获得了一株杂交瘤细胞株(7A9),所分泌的抗体对人、大鼠和小鼠ANKIB1均具有很高的灵敏性和特异性。ANKIB1在哺乳动物细胞中高表达。结论:成功制备了抗ANKIB1的单克隆抗体,ANKIB1在人、大鼠和小鼠脑以及人胚肾细胞293ET中均有较高水平的表达。
Objective: To prepare a mouse monoclonal antibody against human ANKIB1. Methods: The coding sequence of human ANKIB1 (1-480) was amplified by PCR, and the pGEX-5X-ANKIB1 (1-480) expression vector was constructed. GST-ANKIB1 (1-480) was induced to express in E. coli and the recombinant protein was purified using glutathione sepharose. The purified protein was used to immunize BALB / c mice. The spleen cells were fused with SP2 / 0 myeloma cells, and the hybridoma cell lines were screened. The antibodies were identified by ELISA, Western blot and immunoprecipitation assays, and ANKIB1 was detected in mammalian cells expression. Results: A hybridoma cell line (7A9) was obtained. The secreted antibodies were highly sensitive and specific to human, rat and mouse ANKIB1. ANKIB1 is highly expressed in mammalian cells. Conclusion: Monoclonal antibodies against ANKIB1 were successfully prepared. ANKIB1 was highly expressed in human, rat and mouse brain as well as in human embryonic kidney 293ET cells.