Inhibitory Effect Of Myeloid Differentiation Protein-2 Mimetic Peptide On The Phagocytosis Of Gram-negative Bacteria By Macrophage

Objective To investigate the effect of myeloid differentiation protein-2 mimetic peptide(MDMP) on phagocytosis of gram-negative bacteria by macrophage and the inhibition of TNF-a and IL-6 releasing in this process.Methods Enhanced green fluorescent protein(EGFP) gene fragment was amplified from PEGFP-C3 plasmid by PCR technology. The prokaryotic expression vector pET-32a(+)-EGFP was constructed by gene recombination method. After sequencing, the vector was transformed into E.coli BL21(DE3) PLysS to screen the stable expression recombinant-bacteria pET-32a(+)-EGFP-BL21(DE3) PLysS(EGFP-BL21). The expression of recombinant plasmid was induced by IPTG, and the phagocytic ability of RAW264.7 cells to EGFP-BL21 was detected by immunofluorescence technique and flow cytometry to verify the construction of EGFP-BL21. And then, the effect of MDMP(1, 10 and 100ug/ml) on the phagocytosis of EGFP-BL21 in Raw264.7 cells or the murine bone marrow-derived macrophages was analyzed by flow cytometry and the secretion level of IL-6 and TNF- ? were measured by Elisa assay.Results The recombinant E.coli EGFP-BL21 with stable expression EGFP was obtained. After pretreatment with MDMP, the phagocytosis of EGFP-BL21 was significantly inhibited in Raw264.7 cells and the murine bone marrow-derived macrophages, the phagocytic index(PI) of the pretreatment group(10, 100ug/ml) was significantly lower than the positive control group(P<0.01). Furthermore, MDMP(10, 100ug/ml) significantly decreased the secretion level of TNF-? and IL-6 in EGFP-BL21-stimulated macrophages.Conclusion The recombinant E.coli EGFP-BL21 with stable expression EGFP could be used to study the phagocytosis of macrophages on Gram negative bacteria. The intervention experimental results showed that MDMP pretreatment could inhibit the phagocytosis of gram-negative bacteria by macrophages, and reduce the secretion of inflammatory factors such as TNF-? and IL-6.