Ras相关C3肉毒毒素底物1(Rac1)多克隆抗体的制备及应用

Preparation and application of polyclonal antibody against Ras-related C3 botulinum toxin substrate 1

目的制备抗Ras相关C3肉毒毒素底物1(Rac1)兔多克隆抗体并进行初步应用。方法以人宫颈癌细胞系He La细胞c DNA为模板,构建重组表达质粒p ET-32a-Rac1。His-Rac1融合蛋白在大肠杆菌中成功表达,将纯化后的融合蛋白作为免疫原免疫家兔制备兔多克隆抗体。分别采用ELISA、Western blot法和免疫组织化学技术对抗Rac1抗血清的效价及特异性进行鉴定。结果 His-Rac1融合蛋白成功构建并高效表达。制备的抗Rac1兔多克隆抗体可以检测到He La细胞、MCF-7细胞的Rac1蛋白,并能识别人肾组织中的Rac1蛋白。结论成功制备了兔抗人Rac1多克隆抗体,该抗体可以识别多种天然样本中的Rac1蛋白。

Objective To prepare rabbit polyclonal antibody（p Ab） against Ras-related C3 botulinum toxin substrate 1（Rac1） and identify the functions of the antibody. Methods The Rac1 fragment was amplified from c DNA of human cervical cancer He La cells,and then inserted into the vector p ET-32 a to construct the recombinant plasmid p ET-32a-Rac1. The recombinant protein Rac1 was fused with His-tag and the fusion protein His-Rac1 was expressed in E. coli expression system. Thereafter,His-tagged Rac1 was purified to immunize rabbits as immunogen. The titer and specificity of rabbit polyclonal antibody against Rac1 were identified by ELISA,Western blotting and immunohistochemistry,respectively. Results The fusion protein His-Rac1 was successfully expressed in E. coli. The p Ab could specifically detect Rac1 in human He La cells and MCF-7 cells in Western blotting. Moreover,the p Ab could specifically recognized Rac1 protein in human kidney tissues. Conclusion The p Ab of human Rac1 protein was successfully prepared,which could specifically recognize Rac1 protein in several natural samples.