人TOP2A单克隆抗体的制备与初步应用

Preparation, identification and preliminary application of monoclonal antibodies against TOP2A

目的制备抗TOP2A(DNA拓扑异构酶Ⅱ型α)单克隆抗体并对其生物学特性进行鉴定。方法应用分子生物学技术构建含人TOP2A编码序列的原核表达载体,表达并纯化人TOP2A蛋白。TOP2A蛋白皮下多点和腹腔注射加强免疫Balb/c小鼠,取脾与小鼠骨髓瘤细胞SP2/0进行融合,利用间接ELISA方法筛选杂交瘤细胞,采用有限稀释法进行亚克隆,用免疫印迹法、免疫组化染色和流式细胞术对抗体进行特异性鉴定。结果获得了3株能够稳定分泌TOP2A单克隆抗体的杂交瘤细胞,分别命名为11G5、3B10和10A3,通过免疫印迹、免疫组化和流式结果验证均为阳性。结论成功制备TOP2A单克隆抗体,为进一步的TOP2A蛋白的表达与肿瘤的关系研究奠定了基础。

To prepare and identify the monoclonal antibody against TOP2 A, human TOP2 A was expressed and purified by molecular biology techniques, and then used to immunize Balb/c mice. Hybridism cell line secreting antibodies against human TOP2 A was determined by indirect ELISA（enzyme-linked immunosorbent assay） and limiting dilution assay; the specificity of monoclonal antibodies against human TOP2 A was evaluated with Western blot, immunohistochemical staining and flow cytometry, and the results indicated that all raised monoclonal antibodies could specifically react with human TOP2 A. The success in production of TOP2 A monoclonal antibody provides a foundation for evaluating the relationship between of TOP2 A and tumor.