Biological Activity And Immunological Properties Of Mycobacterium Tuberculosis Rpf-like Proteins

Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB) . TB remains an important global public health problem, because about one-third of the world's populations are infected with the MTB and there are 10 million new cases and 3 million deaths annually. In recent years, such factors as increasing frequency of multidrug-resistance(MDR) MTB isolates and incidence of HIV-associated tuberculosis and increasement of population movement, have further aggravated the opportunity of people worldwide to face the threat of TB. In China, the number of TB patients was the second in the world,approximately 400 million people infected, over 5 million got sick, and 0.25 million died of TB annually. So it is a urgent need for new diagnosis methods and new vaccines to prevent TB.Viable cells of Micrococcus luteus secreted a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitation-promoting factor(Rpf) is a protein which can increase the viable cell count of dormant M. luteus cultures at least 100-fold and can alsostimulate the growth of viable cells. Similar genes are widely distributed among high G + C Gram-positive bacteria, genome sequencing has uncovered examples in Mycobacterium leprae, Mb.tuberculosis, M.bovis, Streptomyces spp and Corynebacterium glutamicum. Both Mb.tuberculosis and M.bovis contain five rpf-like genes that share a conserved segment which encodes an Rpf-like domain70 residues long, the presence of these genes in mycobacteria suggests that one or more corresponding products may be involved in mycobacterial reactivation. Sequence analysis suggests that all five proteins probably have extracytoplasmic functions, making them potential targets for recognition by the host immune system at the stage of reactivated disease. Aim:To high efficiently express the MTB Rpf-like proteins(Rv 1884c, Rv2450, Rv2389c and Rvl009)in E.coli and study the biological activity and immunological properties of these proteins.Methods And ResultsrAll the studies can be divided into two parts according to experimental aims. 1. Purification of Rpf-like proteins of MTBThe genes of MTB rpf (Rv1884c, Rv2450, Rv2389c and Rvl009) were amplified by PCR from genome of MTB H37Rv strain, and then inserted into cloning vector pGEM-T-easy respectively for sequencing purpose.Genetic of Rv1884c, Rv2450, Rv2389c and Rv1009 were identical with that of GenBank reported. Then genes of Rv1884c and Rv2450 were digested by restriction endonuculease and cloned into expression vector pPRO EX HT, genes of Rv2389c and Rvl009 were cloned into expression vector pET30. The recombinant plasmids were transformed into E.coli DH5a,induced with IPTG. Expressed fusion Rpf proteins were confirmed by Western-blot analysis with mouse-specific monoclonal antibody against 6× His. Fusedexpression proteins were purified by Ni-NTA purification systerm.2. Biological Activity And Immunological Properties Of Mycobacteriumtuberculosis Rpf proteins2.1 Biological Activity of Rpf-like proteinsH37Rv strains was at a 1:1000 dilution use 7H9 medium(supplemented with 10% ADC and 0.05%Tween80), and then added to 5 replicate/each group 10-ml Pyrex screw-capped tubes. Purified fusion Rpf-like proteins added to a concentration of between 5μg and 30μg, the tubes were incubated at 37 ℃ without shaking for 2-3 weeks. Tubes without Rpf-like proteins were counted as negative control.Taken 200μl from each tubes at different time to detected the OD₆₀₀. The results showed that Rv2450, Rv1009 could significately stimulate the growth of MTB H37Rv.2.2 Immunological Properties of Rpf-like proteinsTo test immunological properties of the fusion proteins, C57BL/6 mice were immunized three times at 2-week interval subcutaneously on their backs with fusion proteins Rvl884c, Rv2389c, Rv2450 and Rvl009 respectively, which were transferred to NC membranes beforehand. The titers of specific antibody in C57BL/6 mice sera immunized with Rvl884c is 1:1600; and that of Rv2389c is 1:800; that of Rv2450 is 1:3200 and that of Rv1009 is l:1600oTo evaluate cell-mediated immune response, stimulating index(SI) of the spleen lymphocytes in immunized mice were measured by MTT method, the SI of Rv2389c, Rv2450 and Rvl009 groups were significantly higher(which were 2.46 for Rv2389c, 3.76 for Rv2450, 2.65 for Rvl009)than that of normal saline controls, only Rvl 884c groups were similar to negative control.The levels of IFN-γ secretion stimulated by antigen-specific were detected by indirect ELISA. IFN-γ concentration in cultured supernatant of spleen lymphocytes from mice immunized with Rpf-like fusion protein...