Cloning And Monoclonal Antibody Production Of Porcine B7-H4 Gene And Analysis Of Its Expression In Porcine Lung Tissues Upon Mycoplasma Pneumoniae Infection

The B7 molecules are important immune regulatory molecules in the immune system and belong to the immunoglobulin superfamily members.B7 molecules expressed on antigen-presenting cells bind to the corresponding receptors which provide the second signal for T cell activation and regulating the immune response of T cells.B7-H4,a newly discovered B7 family member,has 20-30%homology with other B7 family members at the level of amino acid,and is anchored on the cell membrane by the glycosylphosphatidylinositol,which belongs to type I transmembrane proteins.B7-H4 is widely distributed in the all tissues at mRNA level,but the expression of B7-H4 protein is limited only in activated B cells,APCs and mononuclear cells.The expression of B7-H4 in various malignant tumors is very high and is related to the immune escape of tumor in tumor microenvironment.At present,the study of the B7-H4 molecule mainly concentrates on the human and the mouse,but the research on the porcine B7-H4(pB7-H4)molecule has not been reported by now.In this study,the encoding region sequence of porcine b7-h4 gene was found by blast search using human b7-h4 gene sequence.The expression of porcine B7-H4 in various tissues was studied by RT-PCR and real-time PCR.The results showed that we successfully cloned the ORF region of pB7-H4 gene,the size of which was 849bp and encoded 282 amino acids.Bioinformatic analysis showed that the molecular weight of pB7-H4 protein was 30.7KDa,and the theoretical electric point was 5.47,which had signaling peptide and transmembrane region.The results showed that the pB7-H4 gene was expressed in healthy porcine lymph nodes,testis,small intestine,large intestine,gallbladder,kidney,spleen,parotid gland,liver,heart,muscle,brain,lung,bladder and stomach,among which the expression was relatively higher in the spleen and lower in the testis and muscle.In order to analyze the expression of pB7-H4 on immune cells,monoclonal antibodies against pB7-H4 were prepared.First,according to the pB7-H4 molecular genes and designing specific primers,we gain the extracellular region gene fragments of pB7-H4 by PCR.We gain recombinant pB7-H4 protein by prokaryotic expression system and use it to immune BALB/c mice.SP2/0 myeloma cells were fused with spleen cells from the BALB/c mice immunized with purified recombinant pB7-H4 expressed in E.coli.A hybridoma cell line of monoclonal antibody(MAb)against pB7-H4 protein(3C8)was screened with indirect ELISA.The mAb was identified to belong to IgGl subtype.It was labeled by fluorescence.The analysis of western blot and flow cytometry showed that the MAb could specifically bind to the pB7-H4 protein.The expression of pB7-H4 on T lymphocytes and macrophages of porcine peripheral blood,as well as alveolar macrophages,was analyzed by flow cytometry.The results showed that pB7-H4 molecules is highly expressed on alveolar macrophages and peripheral blood macrophage,but not detected in T lymphocytes.To further explore the expression of pB7-H4 in the porcine infected with mycoplasma pneumonia,we carry out the infection experiment with mycoplasma pneumonia and staining the porcine lung tissue sections with the monoclonal antibody against pB7-H4.Immunohistochemical results showed that pB7-H4 expressed in the lung tissue of porcine infectioned with Mycoplasma pneumoniae is significantly higher than healthy porcines,This result suggests that pB7-H4 may play a role in porcine Mycoplasma pneumoniae infection.In conclusion,the B7-H4 gene was cloned for the first time in the world,it is widely distributed at mRNA level in healthy lymph nodes,testis,small intestine,large intestine,gallbladder,kidney,spleen,parotid,liver,heart,muscle,brain,lung,bladder and stomach,in which the expression was relatively higher in the spleen and lower in the testis and muscle.In the FACS analysis,the pB7-H4 molecule was proved highly expressed on the peripheral blood macrophages and alveolar macrophages,but not expressed on T lymphocytes.the research preliminarily proved that the number of pB7-H4 positive cells is increased in the lung tissue infected with mycoplasma pneumoniae.it will provide a reference for further study of the immunological function of pB7-H4 molecule and the relationship between the pB7-H4 expression and disease occurrence.