The Cloning,Prokaryotic Expression And Characterization Of The Cluster Of Differentiation 63 From Lamprey

Lamprey is a branch of jawless vertebrate,which is at the node between the jawless and jawed animal.They are unique from the perspective of evolution and immunology because of their possession of the innate and adaptive immune system,remarkably different from adaptive immunity of gnathostomes This is the first report that the cluster of differentiation 63gene(CD63)was cloned from lamprey and the characterization of CD63 was studied by bioinformatics analysis,real-time quantitive polymerase chain reaction(real time qPCR),recombinant functional fragment expression,Western blot assay and transfection into eukaryotic expression system.The results obtained in the present study will help us to understand the roles of CD63 in immune responses in lamprey.CD63 play multiple roles in a variety of cellular processes,like immunologic reaction,migration of cells and cancer metastasis and so on.The current study about CD63 focus much more on the mammalian amd cell lines in vitro.In this study,it is reported for the first time that a gene encoding cluster of differentiation 63 was cloned from lamprey,an ancient jawless animal,and its functional fragment,the extracellular loop 2(EC2)was expressed successfully.The open reading frame of LjCD63 has 738 base pairs(bp),and encodes 245 amino acids.Its predicted molecular weight is 26.48 kDa with the theoretical isoelectric point of6.79.LjCD63 which is a hydrophobic protein has 4 transmembrane domains consistent with CD63 from other species other than a signal peptide.LjCD63 may be modified by N-glycosylation and palmitoylation in vivo.There are two conservative motifs in the EC2 region containing “Q(X)4GCC(X)5DW”,“VPDSCC” and a “G(S/E)YE(Q)V(T)M(V)” motif in the C terminal included.They may play more important roles in the function of LjCD63.The result of real time qPCR shows that LjCD63 was upregulated in the liver of lamprey after LPS challenging,which demonstrated that LjCD63 may participate in the immunological reaction in the liver.It was much easier for expression of recombinant protein of LjCD63 EC2 region with the pET32a(+)(rLjCD63 EC2)than that of LjCD63 ORF recombinant protein(rLjCD63)in the prokaryotic expression system.And the best expression condition of the former was the concentration of IPTG at the 0.5mM and the temperature at20?.It was shown with the method of western blot that the rabbit polyclonal antibody specific to human CD63(hCD63)EC2 region could recognize the LjCD63 EC2 region.However,it is necessary to prepare antibody which is more specific to LjCD63 EC2 region oflamprey via immunohistochemistry assay.By transfection with LjCD63 open reading frame protein labeled with Green fluorescent protein(GFP),it was found that LjCD63 mainly located on the cellular membrane of human breast tumor cells and maybe some particular sub-cellular components or organelles,which was in accordance with CD63 from other species.This demonstrated that LjCD63 may interact with other molecules from the extracellular or internal environment to affect the function of the cells.However,the working mechanism of LjCD63 in the lamprey is further to investigate.