Preparation Of Polyclonal Antibody Of Intraflagellar Transport Protein 57(IFT57) In The Cilia Of Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii is single-celled eukaryotic algae with two equal length cilia.It has the advantages of short growth cycle and simple experimental operation.Also is a more comprehensive model organism in genetics research.Cilia is a kind of subcellular structure of the cell surface,which composed of cell microtubules.The assembly and maintenance of cilia are known to be dependent on intraflagellar transport(IFT).IFT is an active transport process within cilia mediated by a bi-directional movement of multiprotein complexes.When the IFT complexes mutation,the cilia structure or function become defects,will cause multiple human diseases,such as obesity,diabetes,congential heart disease,polycystic kidney disease,retinitis pigmentosa,refinement and respiratory infection,infertility,mental retardation and excessive Numbers of fingers.Therefore,research on IFT protein particle composites correlation function is particularly important for cilia disease pathogenesis and prevention.Recently,the biology study found IFT complexes B are composed of complex IFT-B1 and IFT-B2,IFT57 in the between of IFT-B1 and IFT-B2.IFT57 become a movement protein,it plays an important role in cilium assembly.The lack of IFT57 will lead to the defect of the cilia,affect cells swimming.The IFT57 antibody preparation,it laid the foundation to continue to study the functional mechanism of IFT57 in cilium assembly.To express ift57 gene,select the n-terminal 319?469 amino acids(aa)hydrophilic sequence,DNA fragments were amplified by PCR and cloned into two in-house modified version of the pET-28a and pMAL-c2X vector.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 were 18 KDa and 57.2 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 256000.After the affinity purification of Protein A,we got a high specificity and sensitivity polyclonal antibody.The results of Western blotting showed that thepolyclonal 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.