Cloning,expression And Function Of Two Secreted Proteins PPE25 And ESAT-6 Of Mycobacterium Avium

Objective:The global incidence of non-tuberculous mycobacterial diseases has risen rapidly and has become an important public health problem threatening human health.However,there are few basic studies on Non-tuberculous mycobacteria(NTM)infection.Mycobacterium avium(MAY)is a pathogen in NTM,and AIDS patients often develop M.avium infection.In order to reveal the pathogenic mechanism of Mycobacterium avium,this paper explored the apoptosis of host macrophages by studying the secretion of PPE25 and ESAT-6 from Mycobacterium avium to understand the changes of immune system caused by Mycobacterium avium infection.Its interaction mechanism with the host provides a theoretical basis for the prevention of mycobacterial diseases and the development of targeted drugs.Methods:(1)Primers of PPE25(MAV-2928)and ESAT-6(MAV-2921)genes were designed,and the PPE25 and ESAT-6 gene sequences were amplified by PCR using M.avium DNA as a template.(2)The PPE25 gene and the ESAT-6 gene were ligated to the pET-his and pGEX-4T-3 expression vectors,respectively,to construct recombinant vectors pET-PPE25 and pGEX-ESAT-6.(3)The recombinant expression vector was transformed into E.coli BL21(DE3)competent cells,and the fusion protein was induced to express under the action of IPTG.(4)PPE25 and ESAT-6 proteins were separated and purified by Ni-NTA nickel column and glutathione agarose gel beads,respectively.(5)The purified PPE25 and ESAT-6 proteins were applied to macrophage U937 at different time points,and the apoptosis rate of the cells was detected by flow cytometry.The change of the expression level of cytokine TNF-? and IL-12 gene in the cell supernatant was detected by ELISA.Results:PPE25 gene(1266 bp)and ESAT-6 gene(285 bp)were successfully cloned from Mycobacterium avium by PCR;PPE25(42 kDa)and ESAT-6 fusion protein(31.5 kDa)was successfully expressed by E.coli system.The PPE25 protein and ESAT-6 protein were successfully isolated from Escherichia coli by Ni-NTA nickel column and glutathione sepharose beads.Flow cytometry analysis showed that PPE25 and ESAT-6 protein infected macrophages U937,and the apoptosis rate increased after 0,6,12,24 and 48 hours,and the apoptosis rate increased with time.ELISA analysis showed that PPE25 and ESAT-6 protein could increase the expression of cytokine TNF-? and increase with the prolongation of action time.Within 24h,the expression of IL-12 gene showed a gradual upward trend.After 48h,the expression of IL-12 gene showed a downward trend,indicating that PPE25 and ESAT-6 proteins may interfere with the expression of IL-12 gene.The infected host cells phagocytose the fusion and maturation of lysosomes,thereby escaping immune killing mechanism of host cells.Conclusion:PPE25 and ESAT-6 proteins can not only induce macrophage apoptosis,but also stimulate macrophage-mediated inflammation and play an important role in immune regulation.