Prokaryotic Expression and Purification of Chlamydomonas reinhardtii IFT25 in Escherichia coli and Preparation of Polyclonal Antibody

Abstract:

IFT25 is one of the important components of IFT-B complexes，which are necessary to maintain cilia movement and perception. To explore IFT25 mechanism especially in the model organism Chlamydomonas reinhardtii plays an important role. This experiment adopts economic and simple method to prepare the rabbit polyclonal antibody of the Chlamydomonas reinhardtii IFT25.The target gene ift25 was cloned into vectors of pMAL-c2X- and pET-28a（+）-，respectively. Then the plasmids were transformed into E. coli BL21（DE3） to induce highly expressedby IPTG. After purified by affinity adsorption purification，the purity of fusion protein is both higher than 90%.Through the back of the neck skin repeatedly immune eight weeks of the New Zealand white rabbit to prepare polyclonal antibody. After the fifth immune antiserum titer was determined by ELISA，which titer more than 12 800. Polyclonal antibodies were then successively prepared by Protein A affinity adsorption purification and nitrocellulose membrane purification. And the specificity of polyclonal antibodies was detected by Western blotting. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT25 in Chlamydomonas reinhardtii，which could provide important theoretical and methodological supports to clarify mechanisms of the action of IFT25 and diagnose diseases associated with cilia.