Purification And Immunomudulatory Activity Of Lectin From Musca Domestica Pupae

Musca domestica is a kind of insect which lives in environments full of pathogens, so it is possible that Musca domestica possesses efficient defense mechanisms for its survival which has already been one of the focuseas at home and abroad studies. Recently, antibacterial and immunoactive substances in Musca domestica such as prophenoloxidase, antibacterial protein/peptide, lysozyme and some other secretion have been reported. However, the presence of lectin in Musca domestica pupae has not been reported before.A novel lectin (Musca domestica pupae lectin, MPL) with potent mitogenic activity was isolated from Musca domestica pupae using ultrafiltration and one single affinity chromatography on NCBr activated Sepharose 4B. D-galactose was detected to be the inhibitor of MPL hemagglutinating activity with the minimum inhibition concentration 15.6 mM. MPL agglutinated typsin treated rabbit blood cells strikingly (the optimum concentration was 20μg/mL). The activity of MPL was metal independent and relatively stable in the range of pH 7 - 9 and 4℃- 40℃. Detection of PAGE and HPLC showed that MPL was homogeneous with the molecular weight of 55 kDa. Oligosaccharide chain was confirmed to be existed in MPL by the method of gel staining using periodic acid-shiff s reaction staining, and then its structure was analyzed with the help of protein sequencing instrument, spectrophotometer color contrast,β- elimination reaction, infrared spectroscopy and atomic force microscopy. MPL was a global-shaped monomer with the diameter 75 nm or so and the protein and oligosaccharide content 97.36% and 2.1% respectively. Peptide chain and oligosaccharide chain was linked by O-glycoside bond with the N-terminal blocked. Analysis of peptide mass fingerprint showed that sequence was highly similar toβ-1 tublin (gi|158739, from drosophila). All above was the reliable theory for further analysis of its structure.The data from in vivo experiments of normal mice showed that injection of MPL at different doses could markedly increase the weight index of thymus and spleen, phagocytosis index, and half hemolysis value (P<0.05), augment phagocytotic functions of mononuclear macrophage. MPL concentration at 12 mg/kg group showed the most significant effect as compared to the control (P<0.01).To observe the influence of Musca domestica pupae hemolymph and MPL on mouse splenocytes proliferation. Different concentrations of Musca domestica hemolymph and MPL were cultured with splenocytes, T lymphocytes and B lymphocytes for 72 h, and the promotion proliferation effect was detected by the method of MTT test of optical density. Western blotting was used to examine the signal transduction pathway involved in B lymphocytes proliferation by MPL. The hemolyph and MPL with the molecular mass above 50 kDa significantly promoted the proliferation of mixed splenocytes and B lymphocytes than those of the control group, nevertheless, the proliferation effect of T lymphocytes was not significant. Results of western blotting showed that ERK signaling pathway in B lymphocytes proliferation was activated by MPL. These results showed that the Musca domestica pupae hemolymph and MPL have immunomodulation effect, which provide certain reference basis for the development of natural immunopotentiator.