Smurf2单克隆抗体的制备与鉴定

Preparation and Characterization of Anti-Smurf2Monoclonal Antibody

Smad泛素调节因子家族的两个E3泛素连接酶成员Smurf1和Smurf2(Smad ubiquitin regulatory factor 1and 2)具有至少80%的同源性.为了更好地研究它们的功能,需要获得一个能区分Smurf1和Smurf2的单克隆抗体.选取Smurf2与Smurf1蛋白序列中仅有的非同源区域序列作为抗原免疫Bal b/C小鼠,成功制备若干株能稳定分泌抗Smurf2抗体的杂交瘤细胞株.对所获得的单克隆抗体的效价和特异性等进行一系列检测,结果表明该抗体能特异性地识别过表达及细胞内源Smurf2蛋白.此外该抗体能被应用于Western blot和免疫荧光实验,从而为深入研究Smurf2的细胞生物学功能提供了良好的实验基础.

Two members of Smad ubiquitin regulatory factor family,Smurf1 and Smurf2,share at least 80% homologous in their protein sequences.A specific Smurf2 monoclonal antibody was required to distinguish Smurf2 from Smurf1,therefore a protein sequence of Smurf2 which has least homolog with Smurf1 was selected as an antigen to immunize mice.After making hybridomas and several rounds of screening,9 positive hybridoma cell lines were obtained.With monoclonal antibodies produced and purified from mouse ascites,specificity and application of Smurf2 monoclonal antibody were tested by ELISA,Western blot and immunostaining.Primary data indicated that this Smurf2 monoclonal antibody has good specificity,and it can recognize both over-expressed and endogernous Smurf2 protein,therefore this antibody would be very useful for studying Smurf2 function in future.