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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。
Objective To prepare anti-TOP2A (DNA topoisomerase type Ⅱ α) monoclonal antibody and to identify its biological characteristics. Methods The prokaryotic expression vector containing human TOP2A coding sequence was constructed by molecular biology technique, and the human TOP2A protein was expressed and purified. The BALB / c mice were immunized with TOP2A protein subcutaneously and intraperitoneally. The spleen was fused with mouse myeloma SP2 / 0 cells. The hybridoma cells were screened by indirect ELISA and subcloned by limiting dilution. The antibodies were specifically identified by immunohistochemical staining and flow cytometry. Results Three hybridoma cells that could secrete TOP2A monoclonal antibody were obtained and named as 11G5, 3B10 and 10A3, respectively. All of them were positive by immunohistochemistry and flow cytometry. Conclusion The TOP2A monoclonal antibody was successfully prepared, which laid the foundation for further research on the relationship between the expression of TOP2A protein and tumor.