| Objective:Mycobacterium avium(MAV)is one of the most common and common pathogenic Nontuberculous mycobacteria(NTM).To search for the pathogenic factors of MAV and study its related pathogenic mechanism.It is of great significance for the prevention and treatment of MAV and the research of new vaccines.The PE/PPE family proteins are mainly distributed in pathogenic mycobacteria,which can regulate the innate immune response of the host,and are related to the pathogenesis of pathogenic mycobacteria,and can be used as potential vaccine components.In this study,bioinformatics method was used to analyze the structure and function of PPE25-MAV protein,induce the expression and purify the purified PPE25-MAV protein,and preliminarily explore the apoptosis and immunological mechanism of mouse immune cells induced by PPE25-MAV protein in vivo and in vitro.Methods:1.The amino acid sequence information of the protein encoded by MAV＿2928 gene was obtained from NCBI database;The physicochemical properties,hydrophilicity,signal peptide,subcellular localization,transmembrane region,phosphorylation site,structure and epitope of PPE25-MAV protein were predicted and analyzed using online database or software.2.Optimal expression conditions were explored to induce the recombinant plasmid to express PPE25-MAV protein in large quantities,and purified by nickel agarose gel chromatography column.3.PPE25-MAV protein is used in the function in mice spleen lymphocytes in vitro,and to the BALB/c mice subcutaneously and administration of the purified PPE25-after the MAV protein extraction of spleen lymphocytes in mice and mice abdominal cavity macrophage,flow cytometry to detect cell apoptosis,and Western Blot(WB)method to detect cell NLRP3,ASC,caspase1-p20 and apoptosis related proteins expression of caspase3,8,9.4.The secretion of inflammatory cytokines IL-6,IL-12 and TNF-α in spleen lymphocytes and peritoneal macrophages of mice was determined by ELISA.Results:1.Bioinformatics analysis showed that the full length of MAV＿2928 gene was1266 bp,encoding PPE25-MAV protein containing 421 amino acids,which was an unstable hydrophobic protein with a fat coefficient of 72.66,no signal peptide and transmembrane region,and most likely located on the bacterial cell membrane.There are52 phosphoric acid sites and 1 conserved domain structure.The secondary structure is a monomer protein with a relatively loose structure.PPE25-MAV was predicted to contain7 dominant epitopes of B cells and 24 epitopes of Th cells.2.was successfully expressed and purified to the target protein and identified as PPE25-MAV protein.3.PPE25-MAV protein can induce apoptosis of spleen lymphocytes and peritoneal macrophages in mice after subcutaneous injection and intraperitoneal injection,respectively.In vitro experiments,the apoptosis of spleen lymphocytes in mice was mainly induced by late apoptosis.4.PPE25-MAV protein can activate the NLRP3 inflammasome in spleen lymphocytes and peritoneal macrophages of mice,increase the expression of related molecules NLRP3,ASC and caspase1-P20,increase the secretion of inflammatory cytokines IL-6,IL-12 and TNF-α,increase the activation fragments of caspase8 and caspase3 and 9 associated with apoptosis pathway.Conclusion:1.The PPE25-MAV protein has multiple phosphorylation sites and plays an important role in cell signal transduction.It contains seven B cell dominant epitopes and multiple Th and CTL cell epitopes,which provides a theoretical basis for the protein to be used as a candidate vaccine antigen.2.PPE25-MAV protein successfully expressed and purified can induce apoptosis of mouse immune cells,increase the expression of inflammatory cytokines,and activate NLRP3 inflammasome,causing inflammatory response.3.The immunological mechanism of PPE25-MAV protein inducing apoptosis of mouse peritoneal macrophages and splenic lymphocytes is mainly through activating caspase-9 and cleaving downstream caspase-3 to form the survival substrate necessary for cleavd-caspase-3 degrading cells,which leads to cell apoptosis. |
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