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

To prepare rabbit polyclonal antibody (pAb) against Ras-related C3 botulinum toxin substrate 1 (Rac1) and identify the functions of the antibody.The Rac1 fragment was amplified from cDNA of human cervical cancer HeLa cells, and then inserted into the vector pET-32a to construct the recombinant plasmid pET-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.The fusion protein His-Rac1 was successfully expressed in E.coli. The pAb could specifically detect Rac1 in human HeLa cells and MCF-7 cells in Western blotting. Moreover, the pAb could specifically recognized Rac1 protein in human kidney tissues.The pAb of human Rac1 protein was successfully prepared, which could specifically recognize Rac1 protein in several natural samples.