In this study,the immunological characteristics of the PhoP protein were explored with a two-component system of Mycobacterium tuberculosis(Mtb).Bioinformatics was used to predict the B and T cell epitopes of the PhoP protein.A re-combinant expression plasmid was constructed by PCR analysis of the phoP sequence and cloning into the prokaryotic expres-sion vector pET-28a(+).Competent Escherichia coli BL21 cells were transformed with the recombinant plasmid and expres-sion was induced with IPTG.The recombinant PhoP protein was purified by affinity chromatography.Serum levels of PhoP-specific antibodies in Mtb-infected mice and tuberculosis(TB)patients were analyzed with an ELISA.BALB/c mice were im-munized with the PhoP recombinant protein by intramuscular injection.Sera of mice were collected and antibody titers were detected with an ELISA and specificity was assessed by West-ern blot analysis.Mouse splenocytes were isolated and the pro-portions of IFN-y-positive cells and cytokine levels were detec-ted with an ELISpot and ELISA,respectively.Bioinformatics i-dentified 24 B cell and 11 T cell epitopes of the PhoP protein.A prokaryotic recombinant vector of PhoP was successfully con-structed and the recombinant PhoP protein was obtained by purification.Specific antibody levels to PhoP in sera of Mtb in-fected mice and TB patients increased significantly,with preci-sion of 99.9％and 82.5％,and specificity of 100％,respectively.PhoP protein immunization successfully induced production of specific antibodies in mice.Stimulated by antigens in vitro,IL-2 and IFN-γ levels were significantly increased in the splenocytes of immunized mice.Immunization with the PhoP protein induce a humoral immune response and Thl-dominated cellular immu-nity,indicating that the PhoP protein was immunogenic with diagnostic efficacy for TB.These results lay a foundation to clari-fy the role of PhoP in Mtb infection and application for diagnosis and prevention of TB.

OBJECTIVE: To explore the relationship between tumor necrosis factor like weak inducer of apoptosis (TWEAK) gene and the pathogenesis of rheumatoid arthritis (RA) by detecting the DNA methylation level, mRNA expression level and serum protein concentration of TWEAK gene in peripheral blood. METHODS: The MassARRAY method was used to detect the DNA methylation level of the TWEAK gene in the peripheral blood of 112 RA patients and 86 matched healthy volunteers. The real-time quantitative polymerase chain reaction method was used to detect the mRNA expression level of the TWEAK gene in the peripheral blood of the subjects. The enzyme-linked immunosorbent assay method was used to detect the serum TWEAK protein concentration of the subjects. The TWEAK gene DNA methylation level, mRNA expression level and serum protein concentration between the RA group and the healthy control group were compared, and the relationship between it and the degree of disease activity analyzed. RESULTS: The overall DNA methylation level of TWEAK gene and the DNA methylation levels of CpG_11, CpG_17.18.19.20, CpG_40.41.42 site in the RA group were higher than those in the healthy control group (P=0.002, P=0.01, P=0.006, P=0.002, respectively). The DNA methylation level of CpG_55.56 site in the high disease activity group was higher than that in the medium and low disease activity group (P=0.041). The expression level of TWEAK gene mRNA in the peripheral blood of the RA group was lower than that of the healthy control group (P=0.023). The expression level of TWEAK gene mRNA in the high disease activity group was lower than that in the medium and low disease activity group (P=0.035). The serum TWEAK protein concentration of the RA group was not significantly different from that of the healthy control group (P=0.508), but it was positively correlated with the mRNA expression level (r=0.482, P < 0.001). CONCLUSION The TWEAK gene is closely related to the onset and progression of RA, and its hypermethylation state may be one of the epigenetic mechanisms regulating its low mRNA expression, and it can be used as one of the important indicators for clinical monitoring and evaluation of RA.
Arthritis, Rheumatoid/genetics* ; Cytokine TWEAK/genetics* ; DNA Methylation ; Humans ; Promoter Regions, Genetic