Prokaryotic Expression,Protein Purification And Antibody Preparation Of IFT139 In Cilia Of Chlamydomonas Reinhardtii

IFT139 is a key component of the intraflagellar transport(IFT)complex A of Chlamydomonas reinhardtii cilia.The expression of IFT139 in E.coli,protein purification and preparation of polyclonal antibody lay the theoretical foundation for future research of IFT139 and the relationship between IFT139 and other transport protein in the cilia,but also provide a favorable basis for the research progress of cilia-related diseases.The research:(1)pBlu-ift139 plasmid as a template to obtain ift139 gene with the corresponding restriction sites by PCR method,it can be successfully complete the construction of the prokaryotic expression vector pGEX-2T-ift139,pMAL-c2-ift139 and pET-28a(+)-ift139(2)The recombinant plasmids were transformed into BL21,IPTG(concentration of 0.2 mmol/L)induced expression,GST-139,MBP-139 and HIS-139 three fusion proteins were all expressed after IPTG induction.The MBP tagged protein expression mostly distributed in the supernatant,GST-tagged fusion protein expressed in the supernatant and the precipitate,but expression level was low.HIS tagged fusion proteins are precipitated in the pellet.Finally,select the purified MBP-IFT139 fusion protein as antigen for immunization and HIS-IFT139 fusion protein as antibody purification.(3)The MBP-IFT139 fusion protein as antigen was purified by affinity chromatography,and then immunized rabbits,prepared antiserum,measured antisera titers by indirect ELISA method and the titer was 1:256,000.Followed by use of Protein A purification and NC membrane purified antibodies obtained antibody,and specifically identified by Western blotting.it was observed in the protein size 139 kDa have a clear target band,indicating IFT139 specific polyclonal antibody was successfully prepared.