| Immuno-costimulating molecules CD80 and CD86 are Class I transmembrane glycoproteins,both belonging to the immunoglobulin superfamily and typically expressed in macrophages(MΦ)、Dendritic cells(DC)and activated B cells.When T cells undergo TCRpeptide-MHC activation,CD80 and CD86 form heterodimers that can bind to CD28 or CTLA4 receptors on T cells,providing a second co-stimulatory signal for T cell activation or inhibition;This signal axis plays a crucial role in initiating,maintaining and regulating T cell activation,proliferation or inhibition.At present,human or mouse CD80 and CD86 have been extensively studied,and by using of anti-human or mouse CD80 and CD86 antibodies,we can delve deeper into the research on the phenotype and function of antigen presenting cell,as well as the mechanism of T cell activation.However,so far,the research on poultry,especially chicken CD80 and CD86(chCD80、chCD86)are still very scarce.Due to the lack of functional anti-chCD80 and chCD86 antibodies,it is not possible to define and study the phenotype and function of chicken antigen presenting cells.Therefore,this study prepared recombinant extracellular domain protein of chCD86 through prokaryotic expression;Monoclonal antibodies capable of recognizing natural chCD86 were prepared using hybridoma technology and used for flow cytometry detection of cells expressing chCD86.At the same time,we constructed an eukaryotic vector of chCD80 and used it as an immunogen to prepare a multi antibody serum that can recognize both eukaryotic and natural chCD80 through spleen immunization.This lays the foundation for defining and in-depth studying the phenotype and function of chicken macrophages and dendritic cells.The specific research content is as follows:1.Recombinant expression and protein purification of chCD86Based on the chCD86 gene sequence included in GenBank,we designed specific primers and extract the total RNA of chicken spleen mononuclear cells.The full length of chCD86 and the extracellular domain sequence of chCD86(ed-chCD86)were amplified by RT-PCR.Through phylogenetic tree analysis,Through phylogenetic tree analysis,it was found that chicken CD86 has a distant phylogenetic relationship with mammals,with less than 30%homology at the amino acid level.The sequence of the extracellular domain of chCD86 and the full-length gene were cloned into the prokaryotic expression vector pET-2 8a(+)and the eukaryotic expression vector pCAGGS,respectively,and the recombinant vectors pet28a-edchCD86 and pCAGGS-chCD86 were verified by sequencing.Transforming the prokaryotic recombinant plasmid into E.coli BL21(DE3),the recombinant extracellular domain fusion protein(His-chCD86)was successfully expressed after IPTG induction,SDS-PAGE,and Western blotting identification.The target protein was expressed as an inclusion body,with a size of 28 kDa and an expression level of 19.5mg/L of bacterial fluid.pCAGGS-chCD86 was transfected into DF-1 cells,and after indirect immunofluorescence(IFA)identification,the eukaryotic protein chCD86 was expressed,with a size of 65 kDa,indicating the possibility of glycosylation modification.2.Preparation and identification of monoclonal antibodies against chCD86The purified recombinant His-chCD86 was used to immunize mice.Through hybridoma technique,nine monoclonal antibodies(mAbs)against chCD86 were obtained,namely 1C2,1D3,2G10,3C3,3D4,3F6,4A2,4B2 and 5A1,respectively.Through Ig subclass identification,the heavy chains of 1D3、3C3、3F6、4B2 and 5A1 are IgG1,2G10 is IgG2a,1C2、3D4 and 4A2 are IgG2b.Antibody light chains are all κ chain;The titer of ascites determined by indirect ELISA is greater than 1:409600.Through IFA and Western blotting identification,it was found that all monoclonal antibodies specifically recognize the prokaryotic and eukaryotic expression of chCD86 protein;Flow cytometry surface staining showed that three monoclonal antibodies(1C2,1D3,3C3)could specifically detect chCD86 positive cells in peripheral blood mononuclear cell(PBMC)of chicken,with a detection frequency of 1.4%.The results indicate that the prepared anti-chCD86 monoclonal antibody has the ability to recognize the natural conformation of chCD86 and can be preliminarily applied to flow cytometry surface staining.3.Eukaryotic expression of chCD80 and preparation of polyclonal antibodiesAccording to the chCD80 gene sequence included in GenBank,specific primers were set to amplify the chCD80 without signal peptide(sd-chCD80)gene and the full length of the chCD80 gene by RT-PCR,the sd-chCD80 sequence and the full length sequence were respectively constructed on the prokaryotic expression vector pET-28a(+)and the eukaryotic expression vector pCAGGS,and the recombinant vectors pET28a-sd-chCD80 and pCAGGSchCD80 were obtained by sequencing and identification.The prokaryotic recombinant plasmid pET28a-sd-chCD80 was transformed into Rosetta(DE3)and induced by IPTG.The recombinant chCD80 fusion protein(His-chCD80)was expressed,with a size of 35 kDa and an expression level of up to 2.25mg/L of bacterial fluid.PCAGGS chCD80 was transfected into CHO cells,and after indirect immunofluorescence(IFA)identification,recombinant chCD80 was obtained for eukaryotic expression.This plasmid was immunized twice through the mouse intrasplenic immunization to prepare a mouse polyclonal antibody against chCD80.Verified by IFA and WB,the mouse polyclonal antibody against chCD80 can recognize eukaryotic expression and natural chCD80 protein.In summary,this study used the prepared recombinant extracellular domain protein of chCD86 as an immunogen and prepared a monoclonal antibody that can specifically recognize natural chCD86 through hybridoma technology,which can be preliminarily applied to flow cytometry surface staining.The chCD80 polyclonal antiserum was generated by intrasplenic immunization of the chCD80 eukaryotic plasmid.These results provide a foundation for studying the function of chicken antigen-presenting cells... |
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