Prokaryotic Expression And Purification Of A Small Gtpase BBS3b In Escherichia Coli And Preparation Of Polyclonal Antibody

Chlamydomonas reinhardtii is a single-celled model organism,with two equal length cilia.It is suitable for genetic analysis because it has many advantages including short of cell cycle,have a high survival rate and a fast growing speed and so on.Cilium is a very conservative organells,which is widely distributed in the surface of eukaryotic cells.When the structure or function of cilia is dysfunctional,people will suffer diseases called"ciliopathy",including obesity,polycystic kidney disease,retinal degeneration,and Bardet-Biedl syndrome.The intraflagellar transport(IFT),an active transport process within cilia mediated by a bidirectional movement of multiprotein complexes,is important to assembly,maintenance and signal transduction of cilia.BBS3b is one of BBS proteins,it interacts with other IFT proteins.The mutation will cause BBSome syndrome but its specific function is unclear.There is now no effective way to prevent and treat these"ciliopathy",which is due to people have not been a clear largely understanding to the formation,depolymerization of cilium and the pathogenesis of the"ciliopathy"at the molecular level.Making the polyclonal antibody is one of the most important contents of more extensive and in-depth research.In this issue,the polyclonal antibody of model organism Chlamydomonas reinhardtii small G protein BBS3b was maded.A GST-tagged and a 6×His-tagged prokaryotic expression vector of bbs3b gene were constructed,pGEX-2T-BBS3b and pET-28a(+)-BBS3b.The fusion protein was expressed in Escherichia coli and identified with 12%SDS-PAGE,the molecular weight of the fusion protein GST-BBS3b and 6xHis-BBS3b were 46 kDa and 20 kDa.After purified by affinity chromatography,the fusion protein GST-BBS3b was used as antigen to immune rabbits to prepare polyclonal antibody.The 4th immune blood was collected and separated the serum,the titer was determined by ELISA,the result of ELISA showed that the antibody titer was about 512000.The antiserum was purified by the affinity purification of Protein A and nitrocellulose membrane,the specificity of polyclonal antibodies was evaluated by Western Blotting.The results of Western blotting showed that the polyclonal antibody was recognition to the BBS3b protein in Chlamydomonas reinhardtii.The antibody possess great sensitivity and selectivity.It laid the foundation to continue to study the functional mechanism of BBS3b in cilia.