| Tuberculosis (TB) is a major threat to global health and it is estimated that up to one-third of the world's population, or 2 billion individuals, has been infected with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB). The remarkable success of M. tuberculosis is attributed in part to the capacity of this organism to persist for months or years in the host issues even with a normal host immune response. Because the majority of these infected people are considered latent carriers with no signs of symptom, and these latently infected persons may represent a significantly important reservoir for disease reactivation and embody a major obstacle in achieving worldwide control of TB. In latently infected but otherwise healthy individuals, the risk of reactivation is estimated to be 2 to 23% over their lifetimes; however, in infected persons with immunodeficiency like AIDS, the risk is a considerably higher 10% per year. So it is crucial to address the reservoir of latent infection in order to control the TB epidemic. In vitro model developed by Zhang Ying, cells of MTB grow to and maintain in"dormant"or"non-culturable"phase in batch culture. These"non-culturable"organisms have extremely low metabolic activity and can not restart after the reintroduction of oxygen. They must require specialized treatment such as Rpf proteins to promote their resuscitation. In many latently infected people, the cultivation of M. tuberculosis cells in sputum culture may not be positive and it is not sufficient to determine who are latently infected only by tuberculin skin tests (TST), so we need to find new methods to diagnose latent infection.The surface-located or secreted protein, Resuscitation promoting factor (Rpf), was first discovered in Micrococcus luteus, which is essentially required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. M. luteus seems to contain only one rpf gene, but most organisms contain several rpf-like genes, whose products are functionally redundant. Rv0867c, Rv1884c and Rv2389c proteins are three members of the five Rpf-like proteins of M. tuberculosis, which all are secreted proteins and share the so-called Rpf domain, a highly conserved protein domain of approximately 70 amino acids. They all have also been demonstrated that they can not only stimulate the resuscitation of stationary-phase mycobacterial, including M. bovis BCG and M. tuberculosis, but also be a target antigen of immune system of host. In this study, we expressed and purified Rv0867c, Rv1884c and Rv2389c fusion proteins in E. coli or M. vaccae and studied the immunological and biological characteristics of them, in order to evaluate the possibility of using it to rapidly diagnose the infected person as a candidate component of culture.Aim: To high efficiently express the MTB Rpf-like proteins (Rv0867c, Rv1884c, and Rv2389c) in E. coli and M. vaccae, and study the biological activity and immunological properties of these proteins.Methods and Results: All 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 and Rv2389c) were amplified by PCR from genome of MTB H37Rv strain, and then inserted into cloning vector pGEM-T-easy respectively for sequencing purpose. The pGEM-T-easy- Rv0867c plasmid gifted by Mike Young was also sequenced. Genetic of Rv0867c, Rv1884 and Rv2389c were identical with that of GenBank reported. Then genes of Rv1884c and Rv2389c were digested by restriction endonuclease and cloned into expression vector pPRO-EXHT-B or pDE-22, and recombinated into E.coli DH5αor M. vaccae respectively for induction; genes of Rv0867c were cloned into expression vector pET19b and recombinated into E.coli DE3 for induction. Finally, we have got five kinds of recombinant fusion proteins: Rv0867c (E. coli), Rv1884c (E. coli), Rv1884c (M. vaccae), Rv2389c (E. coli) and Rv2389c (M. vaccae). Expressed fusion Rpf proteins were confirmed by Western-blot analysis with mouse-specific monoclonal antibody against 6×His. Fused expression proteins were purified by Ni2+-NTA purification system.2. Biological Activity and Immunological Properties of MTB H37Rv Rpf-like proteins2.1 Biological Activity of Rpf-like proteinsEstablished the in vitrol model of dormant MTB H37Ra according to the method of Zhang Ying. Then the dormant MTB H37Ra strain was diluted at 1:1000 using 7H9 medium (supplemented with 10% ADC and 0.05%Tween-80), and then added to 10-ml Pyrex screw-capped tubes. Purified fusion Rpf-like proteins were added at different concentrations, the tubes were incubated at 37℃without shaking for 60 d. Taking 100μl from each tubes every 5 days to detect the absorbance at OD600; After we knew their best concentrations of activity, the dormant MTB H37Ra strain was divided into three groups: Rv0867c (E. coli), Rv2389c (E. coli) and Rv2389c (M. vaccae) were added at their best concentrations respectively, with rabbit-specific antiserum against Rv0867c (E. coli) or Rv2389c (E. coli). The tubes were incubated at 37℃without shaking for 60 days and we took 100μl from each tube every 5 days to detect the absorbance at OD600. The results showed that Rv0867c (E. coli), Rv2389c (E. coli) and Rv2389c (M. vaccae) could significantly stimulate the resuscitation of MTB H37Ra, and the two kinds of antiserum could completely inhibit the resuscitation promoting activity of them2.2 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 in their groin with Rv0867c fusion proteins, Rv1884c fusion proteins derived from E. coli and M. vaccae, Rv2389c fusion proteins derived from E. coli and M. vaccae, which were transferred to NC membranes beforehand. The titers of specific antibody in C57BL/6 mice sera immunized with Rv0867c (E. coli) was 1:1600;That of Rv1884c (M. vaccae) was 1:400 and that of Rv1884c (E. coli) was 1:200;That of Rv2389c (M. vaccae) was 1:200 and that of Rv2389c (E. coli) was 1:800。To evaluate cell-mediated immune response, stimulating index (SI) of the spleen lymphocytes in immunized mice was measured by MTT method. The SI of Rv0867c (E. coli) was 3.57, significantly higher than that of negative control group (P<0.05), but still lower than that of BCG immunized group (P<0.05); The second was Rv2389c (E. coli), which SI was 2.64 and the third was Rv2389c (M. vaccae), which SI was 2.08, both of them were higher than that of negative control group (P<0.05), but still lower than that of Rv0867c immunized group (P<0.05); The SI of Rv1884c (M. vaccae) was 1.14 and the SI of Rv1884c (E. coli) was 1.39, both of which were not higher than that of negative control group (P>0.05).The levels of IFN-γsecretion stimulated by antigen-specific were detected by indirect ELISA. IFN-γconcentration in cultured supernatant of spleen lymphocytes from mice immunized with Rpf-like fusion protein Rv0867c (E. coli) was 613.55±73.71ng/L, significantly higher than that of negative control group (P<0.05), but still lower than that of BCG immunized group (P<0.05); Rv2389c (E. coli) was 401.01±43.46ng/L and Rv2389 (M. vaccae) was 388.58±30.92ng/L, both of them were higher than that of negative control group (P<0.05), but still lower than that of Rv0867c immunized group (P<0.05); Rv1884c (E. coli) was 133.76±22.53ng/L and Rv1884 (M. vaccae) was 149.61±20.06ng/L, both of which were not higher than that of negative control group (P>0.05).The levels of IL-2 secretion stimulated by antigen-specific were also detected by indirect ELISA. IL-2 concentration in cultured supernatant of spleen lymphocytes from mice immunized with Rpf-like fusion protein Rv0867c (E. coli) was 477.62±45.92ng/L, significantly higher than that of negative control group (P<0.05), but still lower than that of BCG immunized group (P<0.05); Rv2389c (E. coli) was 275.49±25.53ng/L and Rv2389 (M. vaccae) was 291.29±15.41ng/L, both of them were higher than that of negative control group (P<0.05), but still lower than that of Rv0867c immunized group (P<0.05); Rv1884c (E. coli) was 120.61±4.65ng/L and Rv1884 (M. vaccae) was 114.93±20.58 ng/L, both of which were not higher than that of negative control group (P>0.05).In order to test Rpf-like proteins efficacies against MTB, two weeks after the final immunization, C57BL/6 mice were intravenously infected with 106 CFU MTB H37Rv. Four weeks later, the numbers of MTB CFU in spleens were determined. Bacteria loads were reduced in C57BL/6 immunized with fusion proteins Rv0867c (1.27Log10, P<0.05), Rv2389c (E.coli) (0.76Log10, P<0.05) and Rv2389c (M. vaccae) (0.61Log10, P<0.05). But the protection efficacies of mice immunized with fusion proteins were lower than that of BCG vaccination group. The bacteria loads of both the two Rv1884c fusion proteins were similar to negative control (P>0.05).Conclusion: Rpf-like fused expression proteins Rv0867c (E. coli), Rv2389c (E. coli) and Rv2389c (M. vaccae) of MTB H37Rv could stimulate the resuscitation of dormant MTB H37Ra cells. Rv0867c fusion protein also could induce high level cell-mediated immune response and confer effective protection against TB, so they may be an appropriate candidate for new diagnostic reagents and subunit vaccine to TB control and prophylaxis.
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