Purification Of A Lectin From Musca Domestica Pupa And Its Effect On Immune Function Of Mouse Macrophages

The efficient defense mechanism of Musca domestica has already been one of the hotspots at home and abroad. Based on the previous research, a novel lectin form Musca domestica pupae was isolated and purified by Con A Sepharose4B affinity chromatography, DEAE weak anion exchange chromatography, ultrafiltration and freeze-drying method. The best component (MBL-1) was selected by hemagglutinating activity and the proliferation of mouse peritoneal macrophages. The investigations on SDS-PAGE electrophoresis and HPLC indicated that MBL-1was a single protein with the molecular weight of24kDa.The investigations of MBL-1indicated that the protein and oligosaccharide content was97.2%and2.55%respectively. Oligosaccharide chain was confirmed to be existed in MBL-1by periodic acid-schiff staining reaction. MBL-1was provided with agglutination activity on rabbit erythrocytes. The hemagglutinating activity of MBL-1was stable within the temperature range of0-40℃and pH range of6-9. D-mannose and D-galactose was the inhibitor of MBL-1hemagglutinating activity. Meanwhile, the results of β-elimination reaction, infrared spectroscopy, atomic force microscopy and protein sequencing instrument showed that MBL-1was the ellipsoidal-shaped monomer with60-100nm in diameter. Oligosaccharide chain and the N-terminal blocked peptide chain were linked by N-glycoside bond. Analysis of MALDI-TOF MS showed that sequence was similar to glutathione S-transferase1(gi|121696, from Musca domestica).MTT assay showed that the proliferation of macrophage could be promoted by adding MBL-1in a concentration-dependent manner. The best concentration and reaction time was40μg/mL and48h respectively. According to neutral red phagocytosis experiment, the phagocytic activity of macrophages was also enhanced by adding MBL-1. In addition, Expression of NO in macrophage was also increased in a dose-dependent manner. The analysis of scanning electron microscope analysis showed that the activation of macrophages could be also promoted by adding MBL-1. The secretion of cytokines IL-6, IL-12, TNF-α of macrophages were also increased in a concentration-dependent manner by adding MBL-1. The results of western blot showed that the ERK pathway of mouse peritoneal macrophages was activated by adding MBL-1.Moreover, ERK pathway also affected the secretion of IL-6, IL-12and TNF-α of macrophage. In sum, the lectin from Musca domestica pupae has a significant immunomodulation effect on mouse peritoneal macrophages. These results will lay foundation for future development of natural immunopotentiator.