Prokaryotic Expression and Purification of Chlamydomonas reinhardtii Intraflagellar Transport Protein 57（IFT57） Antigen and Preparation of Polyclonal Antibody

Abstract:

IFT57 is a subunit of the intraflagellar transport protein B complex and plays an important role in cilium assembly. To express ift57 gene，DNA fragments were amplified by PCR and cloned into an in-house modified version of the pET28a vector. The resulting proteins contained a 6×His tag at their N terminus. BL21（DE3） Escherichia coli cells harboring the expression plasmid were grown in lysogeny broth（LB） medium at 37 ℃ and then induced with isopropyl-b-D-thiogalactoside，SDS-PAGE（12%） results showed that the molecular weights of the fusion protein 6×His-IFT57 was 17.2 KDa. After purified by affinity chromategraphy，the fusion protein 6×His-IFT57 was used as antigen to immune rabbits to prepare polyclonal antibody. After 5 hours we collected the immune blood，then separated the serum and identified the antibody activity. The ELISA result showed that the antibody titer of serum was determined to be about 512 000. After the affinity purification of Protein A，we got a high specificity and sensitivity polyclonal antibody. The results of Western blotting showed that the polyclonal antibody was specificity recognition to the IFT57 protein in C. reinhardtii. It provided the certain reference value for expression and purification of difficult soluble protein，and antibody preparation of polyclonal antibody. It laid the foundation to continue to study the functional mechanism of IFT57 in cilium assembly.