The Prokaryotic Expression And Purification Of Chlamydomonas Reinhardtii Intraflagellar Transport Protein 46 And Preparation Of Polyclonal Antibody

Chlamydomonas reinhardtii is single-celled eukaryotic algae with two equal length cilia which has many advantages.It is the model organism which can be used to study the assembly,depolymerization and function of cilia.Cilia is a kind of cell antenna,responsible for passing information from extracellular to intracellular.The assembly and maintenance of cilia are known to be dependent on Intraflagellar Transport(IFT),an active transport process within cilia mediated by a bi-directional movement of multiprotein complexes,known as IFT particles.The defects in cilia structure or function cause multiple human diseases,such as obesity,diabetes,congential heart disease and polycystic kidney disease.However,the molecular mechanism of how the transportation in cilia being regulated is still poorly understood.IFT46 was previously reported to be a complex B protein in C.reinhardtii.It is highly conserved across organisms that have cilia and plays an important role in IFT,the assembly and depolymerization of cilia.For further research on its function,protein level and molecular level research is necessary.So a high titer and good specific anti-IFT46 antibody is prepared.IFT46 was an Intraflagellar Transport complex B protein in C.reinhardtii.It played an important role in Intraflagellar Transport,the assembly and disaggregation of cilia.To study its functional mechanism,we constructed a GST-tagged and a MBP-tagged prokaryotic expression vector of ift46 gene of Chlamydomonas reinhardtii,pGEX-2T-IFT46 and pMAL-c2X-IFT46.The fusion protein was expressed in Escherichia coli and identified with 15%SDS-PAGE,the result showed that the molecular weight of the fusion protein was 70 kDa and 86 kDa.After purified by affinity chromatography,the fusion protein GST-IFT46(more than 95%purity)was used as antigen to immune rabbits to prepare polyclonal antibody.We collected the 5th immune blood and separated the serum and identified the antibody activity.The result of ELISA showed that the antibody titer of serum was determined to be about 256000.After the affinity purification of Protein A、MBP-IFT46 and nitrocellulose membrane,we got a high specificity and sensitivity polyclonal antibody.The results of Western blotting and immunofluorescence showed that the polyclonal antibody was very good recognition to the IFT46 protein in C.reinhardtii and we found that IFT46 was concentrated at basal body regions and was localized along the entire length of the flagellum as punctuated dots.It laid the foundation to continue to study the functional mechanism of IFT46 in cilia related diseases such as obesity,diabetes,polycystic kidney disease and so on.