| Mycobacterium tuberculosis (Mtb) is one of the oldest pathogen which is still a great public threaten for human being. However, the Bacillus Calmette-Guerin (BCG), the most common vaccine, protects children, but failed to defend against adult pulmonary across the world. So it is important for us to understand the mycobacteria pathogenesis and evasion of the immune system. Mtb can survival within immunocytes such as dendritic cell, macrophage. The reported mechanisms involved in mycobacteria survival are arrested of phagosome-lysosome fusion, resistance against reactive nitrogen intermediate and reactive oxygen intermediate, interference with antigen presentation. Autophagy is not only a catabolic process for the degradation of cytosolic components to maintain the homeostasis, but also serves as an innate immune defense mechanism against infectious pathogen which was interacted with antigen presentation and cytokine modulation. Mtb impedes autophagosome-lysosome pathway, thus residues within macrophage. Otherwise, autophagy is also an effective defense mechanism inhibiting Mtb survival in macrophage while it was activated. And all of cytokines, Vitamin-D, ion homeostasis and ROS play a vital role on regulation of autophagy.TB10.4is a secretion protein which is an important candidate for vaccine development, but there is little knowledge about how TB10.4functions while Mtb infected parasitifer. Thus, we investigated whether TB10.4has an influence on autophagy by detecting LC3BⅡand Beclin-1, and whether TB10.4modulates cytokines secretion from macrophage. Besides, we constructed and characterized an eukaryotic vector expressed TB10.4for further investigating the function of TB10.4.1. TB10.4inhibited autophagy and modulated inflammatory cytokines in LP S-induced RAW264.7cellsThe LPS-induced RAW264.7cells were treated with TB10.4expressed from Escherichia coli. With detecting LC3BⅡ(molecular marker for autophagosome and autophagolysosome) and Beclin-1(core protein for initiating autophagy) by Western blotting assay, the results showed TB10.4rapidly inhibited autophagy within30min, and10μg/mL TB10.4was sufficient to control the synthesis of autophagosome. Thus it hypothesised that TB10.4might also affected cytokines secretion induced by LPS. The data showed TB10.4could decrease secretion of TNF-a and production of NO, increase secretion of IL-1β, but had no effect on secretion of IL-6. Furthermore, with TB10.4treatment, RAW264.7cells showed ERK/p70S6K activation. But AKT was first activated, and then reverted. TB10.4also protected BCG viability from Rapamycin treatment in RAW264.7cells. These results showed TB10.4might have an important role on mycobacteria infection under inflammation.2. Construction and characterization of an eukaryotic vector expressed TB10.4The TB10.4and GFP-TB10.4DNA fragments were cloned into pVAX1vector, respectively, then we identified the expression of pVAXl-TB10.4and pVAXl-GFP-TB10.4within mammal cells by RT-PCR and Indirect immune fluorescent assay. Furthermore the immune responses were analyzed in mice immunized with pVAXl-TB10.4and BCG. The results showed TB10.4and GFP-TB10.4were successfully expressed in cells. pVAXl-TB10.4could induce low level of specific antibodies against Bovine-PPD or recombinant TB10.4. And a significant release of IFN-γ and low level of IL-4release were detected from spleen cells of immunized mice. Among the different group(BCG, pVAXl-TB10.4, BCG/pVAX1-TB10.4, PBS), BCG/pVAX1-TB10.4group showed a higher production of IFN-y. Thus, we successfully acquired the recombinant plasmid expressed TB10.4in eukaryotic cells applied for further researches, which could induce immune response in immunized mice efficiently. |
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