小鼠Foxp3片段的表达和多克隆抗体的制备

Expression of Mouse Foxp3 Fragment and Preparation of Polyclonal Antibody Against Foxp3

克隆了小鼠Foxp3片段,采用基因重组方法,构建重组表达载体pGEX4T1Foxp3。应用在大肠杆菌BL21(DE3)中诱导产生的GSTFoxp3融合蛋白免疫家兔,制备兔抗Foxp3多克隆抗体。经过ELISA检测,抗血清的效价达1∶128000;采用Westernblot检测,转染Foxp3MIGR载体的Ψam细胞中表达Foxp3蛋白,而仅转染MIGR空载体的Ψam细胞中为阴性表达,表眀抗体具有特异性。

The pGEX-4T-1-Foxp3 expression vector has been constrncted with mouse Foxp3 fragment. The rabbit antibody against Foxp3 was obtained by immunizing a rabbit with GST-Foxp3 fusion protein expressed in E. coli. After the last immunization, the titer of antiserum against Foxp3 was about 1：128 000 determined by ELISA. To identify the specificity of antibody, the Ψam cells were transfected with either Foxp3 MIGR or MIGR vector. Foxp3 protein was only detected by Western blot analysis in Ψam cells transfected with Foxp3 MIGR vector, but not in cells transfected with the control vector. It was shown that the antibody was specific for Foxp3 protein.