| Tuberculosis caused by Mycobacterium tuberculosis complex is a major infectious disease of global concern. One third of the world’s population was infected tuberculosis and there are nearly nine million new cases annually. The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) multi-gene families code for approximately 10% of the Mycobacterium tuberculosis (MTB) genome. These proteins are thought to be virulence factors that participate in the host immune responses. While some members have been studied, the functions of most PE/PPE proteins are remained to be explored, including PPE25、PPE27 and PE19 proteins. In this study, PPE25, PPE27 and PE19 were successfully expressed in E.coli and purified, and their functions were initially analysed using a murine macrophage cell line RAW264.7. In order to further study their immuno-modulatory functions, recombinant Mycobacterium smegmatis harboring ppe25 gene with flag tag was constructed.1. Effects of PPE25, PPE27 and PE19 proteins from Mycobacterium tuberculosis on cytokine secretion and cell death of macrophage RAW264.7The purified recombinant PPE25, PPE27 and PE19 proteins were removed endotoxin and the concentrations were determinated by BCA method. RAW264.7 cells were stimulated by PPE25, PPE27 and PE19 with the concentration of 0,1.25,2.55.0 and 10.0 μg/mL respectively. The cell viabillities were detected at 24 h and the results demonstrated that all of the three proteins significantly inhibited the viability of RAW264.7 macrophage in a dose-dependent manner(p<0.05).Cell death types that the proteins induced were explored. The results showed that PPE25,PPE27 and PE19 could induce RAW264.7 nerosis after 24 and 48 h stimulation, which indicated that the recombinant protein PPE25, PPE27 and PE19 are toxic.Meanwhile in order to evaluate the effects of recombinant PPE25, PPE27 and PE19 on cytokine secretion, RAW264.7 cells were treated with various concentrations of targeting proteins. Culture supernatants were collected to measure the expression level of cytokines including IL-1β,IL-6, TNF-a and IL-12p40 by luminex200 method. After 24 and 48 h stimulation, recombinant PPE25 and PE19 significantly induced IL-6 secretion (p<0.05). High level of the PPE25-induced TNF-a and PE19-induced TNF-a were observed whereas there were no differences with control. Recombinant PPE27 significantly stimulated RAW264.7 cells to secrete low levels of IL-6 (p<0.05), as well as TNF-a. In addition, all the groups’IL-1β were less than 2.13 pg/ml, and accordance IL-12p40 were less than 2.09 pg/ml, which means they are under the detect limitation. These results demonstrated that PPE25, PPE27 and PE19 play a role in immune response on macrophage.2. Construction and identification of recombinant Mycobacterium smegmatis expressing the ppe25 gene of Mycobacterium tuberculosis.ppe25 gene was PCR amplified from MTB H37Rv genomic DNA and subcloned into pCR2.1 vector. FLAG tag was added in the reverse primer. After PCR amplificaton, restricted enzymes digestion and sequencing, verification, it was shown that ppe25 gene was suessfully ccinerted into the E.coli-mycobacterium shuttle vector pMV261. The recombinant vector pMV261-ppe25 was transformed into M.S by electroporation. After PCR comfirmaton, the recombinant MS was induced at 45℃ for 30min. Western blotting assay showed that both of the anti-PPE25 protein rabbit antiserum and anti-FLAG monoclonal antibody could specifically recognize the induced products at the desired size of 39.51kD. |
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