Effect Of PE22 Protein Of Mycobacterium Tuberculosis On Phenotype Of Mycobacterium Smegmatis And ANA-1 Function Of Macrophages

Objective:This study mainly focused on the role of PE22 protein in Mycobacterium tuberculosis（MTB）infection and its immunological function by taking gene encoding protein PE22 from important virulence gene PE/PPE family as the breakthrough point,to provide experimental evidence and theoretical basis for further analysis of the pathogenesis of MTB.Methods:1.Bioinformatics analysis and prokaryotic expression of PE22 protein:Such softwares as Expasy Prot Param,Prot Scale,TMHMM Server v.2.0,Signal P 5.0 Server,Net Phos-3.1,Cell-PLoc 2.0,SOPMA,SWISS MODEL,ABCpred,IEDB Analysis Resource,SYFPEITHI,Net MHC II pan 4.0 Server and Net CTL-1.2 were used for bioinformatics analysis of PE22 protein.Besides,prokaryotic expression and Western blot identification were carried out on PE22 protein.2.Effect of PE22 protein on the phenotype of Mycobacteria smegmatis:Adopting the serum obtained after immunizing BALB/c mice with purified PE22 protein as primary antibody,Western blot method was used to verify the Mycobacterium smegmatis（MS）which was transformed by electroporation and could express PE22protein heterologously.The colonial morphology and biofilm formation of the successfully constructed recombinant strains MS-vec and MS-PE22 were observed,and their growth curves were also determined.The biological characteristics of recombinant MS were preliminatively analyzed.The tolerance of MS-vec and MS-PE22 under different survival pressures such as malachite green,low p H,starvation,H₂O₂,lysozyme,Na NO₂and SDS was analyzed by drop plating method and colony-counting method（CFU）.3.Effect of PE22 protein on the ANA-1 function of macrophages:After infecting ANA-1 cells with recombinant strains MS-vec and MS-PE22,acid-fast staining was adopted for observing the infection situation,CCK8 method for detecting the proliferative activity of ANA-1 cells infected by recombinant strains,Griess method for detecting the NO content in cell culture supernatant and the involved signaling pathway,ANNEXIN V-FITC/PI double staining for detecting cell apoptosis,q RT-PCR method for detecting the RNA expression levels of i NOS,TNF-α,IL-6,IL-12p40,IL-10 and IL-1βas well as the signaling pathways involved in the secretion of TNF-αand IL-1β,and CFU method for detecting the intracellular Survival of recombinant strains.Results:1.Bioinformatics analysis and prokaryotic expression of PE22 protein:Bioinformatics analysis showed that the relative molecular mass of PE22 protein was10373.77,and the theoretical isoelectric point was 6.82.PE22 protein,composed of 98amino acids,was a stable hydrophobic protein without transmembrane domain or signal peptide,and it’s located in the cell wall and contained 9 phosphorylation sites,the secondary structure of which was mainly composed ofα-helices.Antigenic epitope prediction analysis showed that PE22 protein contained 10 linear B cell dominant epitopes,3 conformational B cell dominant epitopes,6 Th cell dominant epitopes and 7restricted CTL cell dominant epitopes.Colony PCR positive identification and Western blot identification showed that the recombinant plasmid p ET-32a-PE22 was successfully constructed,and the BL-21 strain containing the recombinant plasmid p ET-32a-PE22was induced by IPTG to obtain the PE22 protein expressed as inclusion body.2.Effect of PE22 protein on the phenotype of MS:The recombinant strain MS-PE22 expressing PE22 protein heterologously was successfully constructed.Comparison of the growth rate and colony morphology of MS-vec and MS-PE22indicated that the expression of PE22 protein had no significant effect on the colony morphology,growth rate and biofilm formation of recombinant strains（P>0.05）.Comparison of the tolerance of MS-vec and MS-PE22 under different survival pressures such as malachite green,low p H,starvation,H₂O₂,lysozyme,Na NO₂and SDS indicated that the expression of PE22 protein could enhance the tolerance of MS to H₂O₂（P<0.05）,but there was no significant difference in tolerance under other pressure conditions such as malachite green,low p H,starvation,lysozyme,Na NO₂and SDS（P>0.05）.3.Effect of PE22 protein on the ANA-1 function of macrophages:Observation of the infection situation by acid-fast staining showed that the recombinant strains MS-vec and MS-PE22 successfully infected ANA-1 cells.CCK8 method was used to detect the proliferative activity of ANA-1 cells infected by recombinant strains with different infection ratio,the results showed that a multiplicity of infection（MOI）of 10:1 did not damage cell viability.Compared with MS-vec infection group,Compared with MS-vec infection group,the apoptosis rate of ANA-1 cells in MS-PE22 infection group was significantly decreased（P<0.05）,the secretion of NO（P<0.001）,TNF-α（P<0.001）,IL-1β（P<0.05）and IL-12p40（P<0.05）was significantly increased,and the expression of i NOS was significantly increased（P<0.05）.The secretion of NO,TNF-αand IL-1βcould be significantly inhibited by NF-κB pathway inhibitor.Comparison of the intracellular survival rate of MS-vec and MS-PE22 indicated that the intracellular survival rate of MS-PE22 was higher than that of MS-vec（P<0.05）.Conclusion:1.PE22 protein is mainly expressed in the form of inclusion body,which contains several potential T and B cell epitopes and has certain immunogenicity.2.The heterologous expression of PE22 protein can change the phenotype of MS,enhance the intracellular viability of the strain,and regulate the immune response of macrophages by promoting the secretion of immune molecules such as NO,TNF-α,IL-12p40,IL-1βand i NOS by ANA-1 cells.