Prokaryotic Expression,Purification And Polyclonal Antibody Preparation Of Chlamydomonas Reinhardtii BBSome Component Protein BBS4

Chlamydomonas reinhardtii is a unicellular eukaryotic alga,which has the advantages of simple culture,easy operation and mutants.It has two slender cilia on its cells,so it is widely used in the study of cilia.Cilia are tiny hair like organelles attached to the surface of cells.Various studies have shown that cilia play an important role in maintaining physiological functions of organisms and can participate in regulating cell movement.Cilia are tiny hair like organelles attached to the surface of cells.Various studies have shown that cilia play an important role in maintaining physiological functions of organisms and can participate in regulating cell movement.In the process of complex formation of cilia,coordination and cooperation of various transporters are needed.For example,in the process of transporting tubulin involved in assembly of cilia to the tip of cilia far away from the cell body,it is completed by an IFT system with two-way transport mode.The assembly protein can be effectively moved from the cell body to the corresponding assembly site through IFT,It can also complete reverse transportation,and IFT can maintain the length of cilia by constantly adjusting the balance between them.As one of the family members of bbsome protein complex,the absence of BBS4 will affect the assembly integrity of bbsome protein complex,and then affect its functional integrity.Bbsome exists as an octamer complex,mainly in the basal body of cilia,and participates in the assembly of IFT.Any mutation of BBS gene will cause budd-b syndrome(BBS).At present,there is still a lack of understanding about the mechanism of BBS4 protein in maintaining ciliary motor function.Therefore,the successful preparation of polyclonal antibody against Chlamydomonas reinhardtii BBS4 can effectively help us to further study cilia and cilia related diseases.In this study,we first obtained the BBS4 gene of Chlamydomonas reinhardtii by PCR,and then constructed it into prokaryotic expression vectors pET-28a(+)and pMAL-c2X.Then we induced the expression of the recombinant protein 6×His-BBS4 and MBP-BBS4,and the protein size of 45 kDa and 85 kDa,respectively.The 6×his-BBS4 recombinant protein was purified and then immunized to New Zealand white rabbits.After being immunized,the antiserum was separated.The obtained antiserum was further purified to obtain excellent BBS4 antibody.Western blotting showed that the prepared BBS4 polyclonal antibody could specifically bind to Chlamydomonas reinhardtii cc-125 at a molecular weight of about 45 kDa,and immunofluorescence assay could locate the position of BBS4 protein in cilia.It laid the foundation to continue to study the functional mechanism of BBS4 in cilia.