Preparation And Application Of Monoclonal Antibody Against Equine Macrophage Polarization Markers CD80 And CD163

Infection causes changes in the immune system,and inflammation is a common early symptom.The degree of inflammatory response caused by different pathogens is different.Mild inflammation can promote the body to remove pathogens,but does not cause inflammatory damage to organs,while severe inflammation can cause excessive stress reaction of immune cells.When a pathogen invades a host,macrophages not only play a key role in host immune response,but also regulate the balance of inflammatory response.Macrophages are generally divided into two types of inflammatory polarization phenotypes: classical activation(M1)and alternative activation(M2).Different phenotypes have different surface markers and secrete different cytokines,chemokines and enzymes to better respond to changes in the physiological environment.Therefore,it is important to identify the polarization phenotype of macrophages when pathogens infect the host for the mechanism research and treatment strategies of the disease.Currently,the corresponding monoclonal antibody for the polarization markers of horse macrophages is still lacking,so the method of identifying the phenotype is still not established.The purpose of this study was to prepare monoclonal antibodies of the polarization markers CD80 and CD163 of horse macrophage and to use them to establish a method to identify the polarization phenotype of horse macrophage.This method can be used to detect the polarization phenotypes of Salmonella abortus equi(S.Abortus equi),Equine herpesvirus(EHV-1),Equine arteritis virus(EAV)and Equine infectious anemia virus(EIAV)infected horse macrophages.Firstly,the nucleotide sequences of horse source CD80 and CD163 were obtained by reverse transcription amplification using horse peripheral blood macrophage RNA as template.Two proteins were obtained by synthetic peptide and recombinant expression and immunized mice with them as immunogens.Western blotting showed that these antibodies could specifically identify eukaryotic proteins and bind to endogenous levels of horse proteins,with good specificity and sensitivity.Polarization phenotypic markers to determine Ma Ju macrophages,use of M1 type activator lipopolysaccharide(LPS)and interferon(IFN)-? and M2 type activator interleukin(IL)-4 or IL-10 respectively stimulate Equine macrophages,respectively by fluorescence quantitative PCR technique to detect the M1 type marker genes(TNF-?,IL-1?,IL-12 and CD80)and M2 type marker genes(TGF-?,IL-10,CD206 and CD163)mRNA level,ultimately determine the eight kinds of genes as Equine macrophages polarization phenotypic markers.On this basis,CD163 monoclonal antibody and fluorescence quantitative PCR were used to identify the polarization typing of S.Abortus equi,EHV-1,EAV and EIAV infected horse macrophages.S.Abortus equi,EHV-1,and EAV induce macrophages to become M1-type and release a large number of pro-inflammatory factors in the early stages of infection.EIAV induces macrophages to polarize into the M2 type and secrete different anti-inflammatory factors.In summary,in this study,we successfully prepared matogenic CD80 and CD163 monoclonal antibodies,which can be used for the identification of horse macrophage polarization after S.Abortus equi,EHV-1,EAV and EIAV stimulation.By referring to other specie-specific markers,8 markers of M1-type and M2-type of horse were identified through verification,which can help to identify the horse macrophage phenotype,so as to better understand their respective roles in the development of disease,and provide a biological tool for further research on the mechanism of disease caused by pathogen infection of macrophages.