Investigation The Immunocompetence Of Rv0220, Rv2958c, Rv2994 And Rv3347c Of Mycobacterium Tuberculosis In Mice And Their Protential Application In Serodiagnosis Of Tuberculosis

[Objective] The combinant proteins Rv0220, Rv2958c, Rv2994 and Rv3347c of Mycobacterium tuberculosis were expressed and purified, and the immunocompetence of recombinant proteins were further analyzed to evaluate their potential value for the serodiagnosis of tuberculosis.[Methods](1) The four genes coding Rv0220, Rv2958c, Rv2994 and Rv3347c were amplified by Polymerase Chain Reaction(PCR) from M. tuberculosis H37 Rv genomic DNA, and then respectively transformed into the prokaryotic expression vector p ET-28 a, and then transformed into E. coli BL21. The recombinant proteins rRv0220, rRv2958c, rRv2994 and rRv3347c were expressed in E.coli BL21 and purified by affinity chromatography technique, and identified by SDS-PAGE and Western blotting assay. The concentrations of recombinant proteins were determined by the BCA protein assay kit.(2) BALB/c female mice were separately immunized with the recombinant proteins rRv0220, rRv2958c, rRv2994 and rRv3347c. The level of Ig G, Ig G1 and Ig G2 a antibodies in the sera of immunized mice were detected by ELISA. The levels of IL-2, IL-4, IL-10 and IFN-? in the mice splenocytes culture supernatants were detected by flowcytometry(FCM). The stimulation index(SI) of splenic lymphocyte were determined by CCK8.(3) The potential serodiagnosis values of rRv0220, rRv2958c, rRv2994 and rRv3347c for M. tuberculosis were further evaluated by indirect ELISA assay to detect the specific antibodies in sera from patients with tuberculosis, and the cross-reaction between recombinant proteins and sera from Mycoplasma pneumoniae infected patients were also detected by indirect ELISA.[Results](1) The four coding genes of Rv0220, Rv2958c, Rv2994 and Rv3347c were successfully amplified, and transformed into the prokaryotic expression vector p ET-28 a. The prokaryotic recombinant vetors p ET-28a/ Rv0220, p ET-28a/Rv2958c, p ET-28a/Rv2994 and p ET-28a/Rv3347c were successfully constructed and the fusion protein rRv0220(46k Da), rRv2958c(50k Da), rRv2994(38k Da) and rRv3347c(38k Da) were attained after expression and purification.(2) BALB/c mice were respectively immunized subcutaneouly with fusion proteins rRv0220, rRv2958c, rRv2994 and rRv3347c. The specific antibodies including Ig G, Ig G1, Ig G2 a were effectively induced by the fusion protein, and the levels of sera antibodies in the immunized groups were significantly higher than that in controls(P<0.01). At the sixth week, the titers of the Ig G, Ig G1 and Ig G2 a antibodies in the groups of immunized mice more than 1:6400 after 3 vaccinations, respectively. At the second, fourth and sixth week, the sera Ig G2a/Ig G1 ratios of the immunized groups were 2.704, 2.386 and 1.828; 2.277, 1.635 and 1.767; 1.772, 2.065 and 2.429; 1.882, 2.595 and 2.030, respectively. All ratios were more than 1. At the sixth week, the stimulation index of the splenic lymphocyte in the immunized groups were significantly higher than PBS group(P<0.01). The four purified fusion proteins exhibited good reactivity with sera from immunized mice.(3) The levels of IL-2, IL-4, IL-10 and IFN-? in immunized groups were significantly higher than control group(P<0.01).(4)Western blot proved that rRv0220, rRv2958c, rRv2994 and rRv3347c can specifically react with M. tuberculosis Ig G-positive sera, respectively. Indirect ELISA was developed to detect the antibody to M. tuberculosis in human clinical sera, when compared with TB colloidal gold method, the sensitivities and specificities of four proteins by Indirect ELISA were 98.6% and 98.3%; 100.0% and 96.5%; 100.0% and 96.5%; 98.6% and 94.7%, respectively. The concordance of results between the ELISA test and the TB colloidal gold method were 98.4%, 98.4%, 98.4% and 96.9%. [Conclusion](1)The coding genes Rv0220, Rv2958c, Rv2994 and Rv3347c were successfully cloned into expression vector p ET28 a and expressed as 46 k Da, 50 k Da, 38 k Da and 38 k Da recombinant proteins, respectively.(2) Specific humoral immunity and cellular immune response, especially for Th1 cellular immunological response, could be induced by rRv0220, rRv2958c, rRv2994 and rRv3347c in mice.(3) Four recombinant proteins rRv0220, rRv2958c, rRv2994 and rRv3347c showed high sensitivity and specificities for detection of antibodies of M.tuberculosis in human sera, indicating that four proteins are fine candidate antigens for serological diagnosis in tuberculosis.