| Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis complex (MTC) and exhibits as respiratory infections in most cases, infecting one-third of world’s population. The Calmette-Guerin (BCG) vaccine can provide an effective protection to pulmonary TB of children, but the efficiency to adults is limited. The multigenic PE/PPE proteins comprise about 10% of the coding potential of the Mycobacterium tuberculosis (MTB) genome. These proteins are thought to be virulence factors that participate in impounding the host immune responses. Members of this family are present only in pathogenic mycobacteria and indicate their importance in disease pathogenesis, but the functions of most PE/PPE proteins are remained to be explored. In this study, Mycobacterium smegmatis was used to express ppe25, ppe27gene, and the immunobiological characteristics of recombinant bacteria were evaluated. It laid the foundation to develop novel vaccines against TB.1. Construction and identification of recombinant bacteria M.S. (pMV261-ppe25-flag) and M.S. (pMV261-ppe21-flag)ppe25 and ppe27 gene were PCR amplified from MTB H37Rv genomic DNA, and FLAG tag was added in the reverse primers. After sequencing, the positive recombinant plasmids pMV261-ppe25 and pMV261-ppe27 were transformed into Mycobacterium smegmati by electroporation, respectively. The recombinant strains M.S. (pMV261-ppe25-flag) and M.S. (pMV261-ppe27-flag) were induced at 45℃ for 30min. Their induced fusion proteins PPE25, PPE27 were detected by Western blotting assay, and showed that anti-FLAG monoclonal antibody could specifically recognize these induced products at the desired size of 39.51 kD and 37.9 kD, respectively.2. Biochemistry characteristics of recombinant strains M.S.(pMV261-ppe25-flag) andM.S.(pMV261-ppe27-flag)The recombinant strains M.S. (pMV261-ppe25-flag) and M.S. (pMV261-ppe27-flag) were cultured in 7H9. Compared to the parent strain Mycobacterium smegmatis, the growth rate of M.S. (pMV261-ppe25) and M.S. (pMV261-ppe27) had no obvious change as shown in their growth curves. In order to evaluate whether PPE25 and PPE27 proteins were related to cope with a variety of stress environments, the survival of recombinant bacteria under SDS surface pressure, starvation and acid stress were further detected. It was shown that PPE25 and PPE27 fusion proteins are located on the cell surface of the recombinant strains. The starvation capacity and the surface pressure tolerance of these two strains were increased, but decreased their acid tolerance, compared to the parent strain. It demonstrated that PPE25 and PPE27 proteins significantly enhanced the survival of recombinant stairns in macrophages, and induced macrophage apoptosis and necrosis (p<0.05)3. Immunological properties of recombinant strains M.S. (pMV261-ppe25-flag) and M.S. (pMV261-ppe27-flag)BALB/c mice were subcutaneously immunized twice at the internal of 2 weeks at the base of tail with 5 X 107 CFU/mouse of recombinant strains M.S.(pMV261-ppe25-flag) and M.S. (pMV261-ppe27-flag). Four weeks later, all the mice were bleeded and sacrified. The immunized mice significantly produced specific antibodies against PPE25, PPE27 proteins and PPE25/27:56-71 peptide, respectively. And the subtype of these antibodies was IgG1. Splenocytes from M.S. (pMV261-ppe25-flag) or M.S. (pMV261-ppe27-flag) immunized mice could proliferate with PPE25/PPE27 protein or PPE25/27:44-52 peptide stimulation compared with M.S. (pMV261) control group (p<0.05). Mice immunized with M.S. (pMV261-ppe27-flag) significantly produced IL-4 and IL-6 (p<0.05) compared to the control, which indicate Th2-biased immune responses induced by PPE27 protein. This study laid a foundation for exploring the potential of PPE25 and PPE27 protein in the development of novel TB vaccines. |
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