Study On The Mechanism Of Apoptosis Induced By Mycobacterium Avium MAV-5183 Protein In Mouse Ana-1 Macrophages

Objective:This study aims to analyze the protein structure,physicochemical properties and antigenic epitopes of MAV-5183 protein based on bioinformatics analysis;and to perform prokaryotic expression and purification of the target protein;finally,to establish an in vitro cell model to study and analyze the immunological mechanism of MAV-5183 protein on mouse immune cells,and to lay the theoretical foundation for the next clinical serological diagnosis and vaccine development.Methods:1.Expasy-Protparam,PHYRE2,IEDB,String were used to analyze the physicochemical properties,protein structure,T/B cell antigenic epitopes of MAV-5183protein;Signl P5.0 signal peptide prediction online site,TMHMM Server2.0,Port Scale,Net PHos3.1 Serve to analyze protein signal peptide,transmembrane domain,hydrophobicity,phosphorylation sites.2.p ET-21a-MAV-5183 recombinant plasmid was constructed,recombinant MAV-5183 protein was cloned,expressed and purified,and identified by anti-His tag antibody,in addition,antiserum was obtained by immunizing rabbits and antibody potency and immunoreactivity were detected by WB.3.Different concentrations of MAV-5183 protein were incubated with mouse Ana-1macrophages.Apoptosis was detected by flow cytometry based on ANNEXIN VFITC/PI double staining.Apoptosis was detected by fluorescence microscopy after fluorescent antibody staining.western Blot detected apoptosis-related antibodies Caspase-9/8/3,and inflammatory vesicles ASC,NLRP3,Cleaved-casp1.ELISA detected TNF-α,IL-6 and LDH,and ROS kit detected ROS secretion.q PCR detected Caspase-9/8/3,ASC,NLRP3,Caspase-1,IL-1β,Bax,Bcl-2,TNF-α,IL-6 m RNA expression.Results:1.MAV-5183 consists of 355 amino acids and is a stable exocrine protein.Its secondary structure contains 23% irregular convolutions,39% α-helix and 14% β-fold.The tertiary structure has a relatively stable and loose structure with multiple phosphorylation sites.In addition,MAV-5183 has 7 B-cell antigenic epitopes(score >0.5)and 11 CTL antigenic epitopes(score >20).2.The MAV-5183 protein was successfully cloned,induced and purified at a concentration of 0.2 mg/ml,and its good immunoreactivity was successfully verified by antiserum.3.The recombinant protein has good immunoreactivity,and the in vitro effect of MAV-5183 protein on Ana-1 macrophages significantly increased the expression of Caspase-9/8/3,ASC,NLRP3(P<0.01),and induced the secretion of ROS(P<0.05),thus promoting the secretion of inflammatory cytokines TNF-α and IL-6(P< 0.0001),however,LDH did not change significantly(P>0.05).Meanwhile,MAV-5183 induced apoptosis in Ana-1 macrophages(P<0.05).In addition,Caspase-9/8/3,ASC,NLRP3,TNF-α,IL-6,and pro-apoptotic factor Bax m RNA expression were elevated by q PCR(P<0.01),however,Caspase-1,apoptosis inhibitor Bcl-2 and L-1β m RNA expression were not altered(P>0.05).Conclusion:1.MAV-5183 protein has multiple dominant antigenic epitopes and phosphorylation sites,providing a theoretical basis for vaccine candidate protein and clinical serological diagnosis.2.We successfully induced the expression of MAV-5183 protein with good immunoreactivity.3.MAV-5183 protein can induce apoptosis of macrophages through endogenous and exogenous pathways and promote the secretion of inflammatory cytokines by secreting ROS.