人SUMO1蛋白表达纯化及单克隆抗体的制备、鉴定

目的 表达重组人SUMO1(small ubiquitin-related modifier 1)蛋白并制备单克隆抗体.方法 构建含人SUMO1基因的重组表达质粒pET32a-HIS-SUMO1,在大肠杆菌中表达重组蛋白HIS-SUMO1;以纯化后的HIS-SUMO1蛋白为抗原免疫BALB/c小鼠,利用杂交瘤技术,通过ELISA和Western blot方法筛选稳定分泌抗体的杂交瘤细胞株,用免疫双向扩散法鉴定抗体Ig的类型及亚类;取一株筛选细胞按照常规方法制备腹水,利用Millipore抗体纯化试剂盒进行抗体纯化,Western blot方法检测抗体效价.结果 表达纯化了重组人HIS-SUMO1蛋白;3株稳定分泌特异性抗人SUMO1的单克隆抗体杂交瘤细胞株被筛选出,其免疫球蛋白类型均为IgG1类;通过腹水制备和纯化获得效价较高的鼠抗人SUMO1单克隆抗体.结论 通过表达纯化人SUMO1重组蛋白,制备出高效价鼠抗人SUMO1的单克隆抗体,该抗体可用于蛋白质SUMO化的研究.

Preparation and identification of monoclonal antibody against human SUMO1

Objective To express the human recombinant SUMO1 protein and prepare monoclonal antibody(mAb) against it.Methods The recombinant expression plasmid pET32a-HIS-SUMO1 was made and transformed into E.coli(BL21),then the recombinant fusion protein HIS-SUMO1 was expressed and purified.The BALB/c mice were immuned with pure protein HIS-SUMO1 as antigen.Monoclonal antibody against SUMO1 was prepared with standard hybridoma technology.The hybridoma cell lines were obtained by ELISA and Western blot screening procedure,the isotype of the mAbs were further identified by immune-double diffusion.Ascites were collected from one propagated hybridoma cell line and mAbs were purified by using the Kit of Millipore.The valence of mAb was detected by Western Blot.Results The recombinant protein HIS-SUMO1 is expressed and purified.Three hybfidmas producing antibodies against SUMO1 were obtained,the isotypes of three mAbs are IgG1,Western blot showed that the antibodies were specific for SUMO1.The antibody purified from the ascites has better specificity.Conclusion The SUMO1 mAb prepared by using recombinant SUMO1 protein as antigen can be used for detectingthe protein sumoylation.