【摘要】 目的制备特异的能区分人跨膜型肿瘤坏死因子(TM TNFα)和分泌型肿瘤坏死因子(sTNFα)的多克隆抗体。方法借助抗原肽预测软件对TM TNFα的抗原表位进行预测,合成TM TNFα特异的20个氨基酸的多肽,将之与匙孔碱血蓝蛋白(KLH)耦联,BALB/c小鼠皮背部多点注射,收集分离血清,采用酶联免疫吸附试验(ELISA)和流式细胞术鉴定其特异性。结果成功免疫BALB/c小鼠,ELISA证实该抗血清与免疫多肽有良好的结合,而与sTNFα、白细胞介素2(IL2)和γ干扰素(IFNγ)无交叉反应,流式细胞术显示该抗血清可与细胞系U937表达的TM TNFα发生特异性结合。结论成功制备了TM TNFα特异性多克隆抗体,为制备TM TNFα特异性单克隆抗体奠定了基础,为区分TM TNFα和sTNFα的生物学作用提供了有力的工具。更多还原

【Abstract】 Objective To prepare a kind of polyclonal antibody that can differentiate human transmembrane tumor necrosis factor-α (TNF-α) from secreted TNF-α.Methods Based on the results of antigen anticipating of human transmembrane TNF-α by using a software, the human transmembrane TNF-α specific peptide of 20 amino acids was synthesized and conjugated with key hole limpet hemocyanin (KLH) to immunize BALB/c mice by intradermal injection of several points on back. Enzyme linked immunosorbent assay (ELISA) and flow cytometer technique were used to test the specificity of serum collected from the immunized mice.Results The BALB/c mice were successfully immunized. The ELISA revealed this antiserum could combine the immunogen peptide, while could not crossed with soluble TNF-α, interleukin-2 (IL-2) and interferon-γ (IFN-γ). Flow cytometer technique showed that this antiserum could specifically combine with transmembrane TNF-α expressed on U937 cell line.Conclusion Human transmembrane TNF-α specific polyclonal antibody was successfully prepared. This throws a light on the preparation for monoclonal antibody of transmembrane TNF-α and provides a useful tool to investigate the biological functions of transmembrane TNF-α and secreted TNF-α.更多还原