Regulation Of Mycobacterium Bovis PtpA On NF-?B Signaling Pathway

Bovine tuberculosis is a zoonotic infectious disease caused mainly by Mycobacterium bovis(M.bovis).M.bovis is a major pathogenic bacterium that can infect humans and cattle at the same time.Although it has a genetic similarity of more than 99.9% with Mycobacterium tuberculosis(M.tuberculosis),there is a huge difference in the pathogenic role played by the infected host.M.bovis has a wider range of host infection than human M.tuberculosis.Controlling the spread of bovine tuberculosis is not only beneficial to the healthy development of animal husbandry,but also has great significance for ensuring human health and safety.Protein tyrosine phosphatase A(PtpA)is an important gene in the mycobacterial genome that plays an important role in mycobacteria infestation.Mycobacterial PtpA can make mycobacteria evade the body's immune clearance after entering the body through phosphorylation,PtpA phosphorylation play a huge influence especially in the antiphagocytosis of macrophages.The specific mechanism of how the M.bovis PtpA gene contributes to immune scavenging by M.bovis through the regulation of cell signaling pathways,it is unclear whether it is similar to the PtpA gene in M.tuberculosis.A signaling mechanism can provide a new direction for the prevention and treatment of bovine tuberculosis and vaccine research.In this study,M.bovis DNA was used as template to obtain PtpA clones by PCR.After cloning,the PtpA fragment was attached to the plasmid pMD18-T Vector.After sequencing,the target gene was subcloned into the expression vector.In pET-32a(+)and eukaryotic expression vectors p3×FLAG-CMV-10,recombinant pET-PtpA and FLAG-PtpA plasmids were established,and pET-PtpA was transformed into E.coli BL21(DE3),followed by IPTG.Induced expression,the size of PtpA recombinant protein was about 35 kDa(containing 6× histidine tag)analyzed by SDS-PAGE electrophoresis.The best induction conditions of the recombinant protein were as follows: 26?,IPTG final concentration 1 mmol/L,and 170 rpm concussion culture for 5 hours.By Western-Blot analysis,the recombinant protein can be combined with the specific antibodies in the positive serum of bovine tuberculosis that identified by commercial kits,It is suggested that the protein can be used as an immune antigen as an effective material for subsequent detection of bovine tuberculosis.The expression of the recombinant protein of PtpA was a soluble protein that analyzed by SDSPAGE electrophoresis.The results showed that the recombinant protein of PtpA was mainly in the broken supernatant of the expression strain.Then the recombinant protein PtpA was purified by nickel column affinity chromatography,and a high purity PtpA recombinant protein could be obtained.The antiserum containing anti PtpA polyclonal antibody was isolated from PtpA recombinant protein as antigen and mixed with Freund's adjuvant after multiple immunization after complete emulsification.The titer of anti PtpA polyclonal antibody was more than 1:1 000 000 that detected by indirect ELISA,with the final concentration of PtpA protein at 2 ug per pore,and the specificity of PtpA polyclonal antibody was analyzed by Western-blot experiment.The experimental results showed that the prepared PtpA polyclonal antibody could be effectively combined with the eukaryotic expression of PtpA protein.The results of the titer test of the synthetic antibody were analyzed in the specific experiment.The anti PtpA polyclonal antibody prepared by the recombinant protein PtpA after immunization had good sensitivity and specificity,and could be used as an experimental material for the subsequent screening of PtpA interaction protein.The recombinant plasmid FLAG-PtpA and the gene reporter plasmid pGL4.32[luc2P/NF-?B-RE/Hygro] were co-transfected into HEK293 T cells.The transfected cells were stimulated by the TNF-? with stimulation of 10 ng/mL.The regulation of PtpA protein on the NF-?B signaling pathway was analyzed by detecting the relative fluorescence intensity at different time points.The results showed that the overexpression of PtpA protein significantly inhibited the activation of NF-?B signaling pathway.The effects of PtpA protein on the expression of NF-?B signal tr-ansduction related cytokines(IL-6,GM-CSF,BIRC-2,BIRC-3)mRNA were analyzed by fluorescence quantitative PCR(qRT-PCR)at different time points.The experimental results showed that PtpA protein had a significant inhibitory effect on the expression of NF-?B signaling pathway related cytokines(IL-6,GM-CSF,BIRC-2,BIRC-3)at the early stage of external stimulation.