抗新型锌指蛋白ZBTB45单克隆抗体的制备与应用

Preparation and application of monoclonal antibodies against novel zinc finger protein ZBTB45

目的制备新型锌指蛋白ZBTB45的单克隆抗体,建立该分子的体内外免疫学检测方法,为研究其生物学功能提供技术手段。方法制备小鼠ZBTB45的多表位抗原融合蛋白,免疫小鼠,取其脾细胞与骨髓瘤细胞融合,经ELISA检测、多次有限稀释亚克隆建立分泌ZBTB45单克隆抗体的杂交瘤细胞株,从荷瘤小鼠的腹水中通过蛋白A柱纯化单克隆抗体,鉴定分析所制备抗体在免疫印迹、免疫沉淀和免疫组织化学中的应用效果。结果成功制备了6株抗ZBTB45的特异性单克隆抗体,其中3E5、2G4、4D2和1D4可成功用于免疫印迹和免疫沉淀分析,3E5、6D8和8E3可用于免疫组织化学检测内源性ZBTB45,具有较好的敏感性、特异性。结论首次成功制备了抗ZBTB45单克隆抗体,可应用于体外和体内的免疫学检测,为深入研究ZBTB45的生物学功能提供了技术支持。

Objective To generate monoclonal antibodies against novel zinc finger protein ZBTB45 of mouse for in vitro and in vivo immunoassay of the protein,so as to contribute to the study on its biological functions.Methods Multi-epitope antigen of ZBTB45 was prepared by prokaryotic fusion expression and was used to immunize BALB/c mice,and then the splenocytes of mice were fused with the myeloma cells.The resultant fused cells were subjected to screening culture,ELISA assay and subcloning by limited dilution to establish monoclonal hybridomas secreting anti-ZBTB45 antibody.The antibody was purified by affinity chromatography with protein A column from the ascites of hybridoma-bearing mice,and was tested by Western blotting analysis,immunoprecipitation,and immunohistochemistry.Results We successfully established 6 cell lines of monoclonal hybridoma secreting anti-ZBTB45 antibody,of which monoclonal antibodies 3E5,2G4,4D2,and 1D4 were applicable for Western blotting analysis and immunoprecipitation,and 3E5,6D8,and 8E3 were applicable for detecting endogenous ZBTB45 by immunohistochemistry,with a satisfying sensitivity and specificity.Conclusion We have for the first time successfully generated monoclonal antibodies against mouse ZBTB45,which are applicable in various immunoassays for ZBTB45 both in vitro and in vivo.