Study On Immunoactive Compounds In Musca Domestic

Developing functional foods and new drugs to enhance the immunity is a main project in the field of food science and biotechnology. Nowadays immunity enhancers are mainly extracted from plant and blood of mammal. Their cost is high and their safety always calls problems. Musca domestic is an insect with high immune system which is easily to get. It is prosperous to be a new source of immune reagent.This paper systematically studied the purification and bioactivity of from Musca domestic pupae lectin(MPL). Isolation by glutaraldehydeized red blood cells (GRBC) adsorption and study of antibacterial activity and immunoactivity were first reported in this paper. To maximumly utilized the resource, another immunoactive compounds (chitin and chitosan) were isolated from Musca domestic.The purification procedure included glutaraldehydeized red blood cells (GRBC) adsorption and then affinity chromatography on Sepharose-4B. Diluted hemolymph was mixed with GRBC and incubated for 2.5h at RT under gentle agitation. Then, GRBC was precipitated to and the mixture of lectin-GRBC was obtained. Desorption of the proteins on GRBC was carried out using D-galactose solutions. The specific activity of crude extract was 533 times larger than the hemolymph. The specific activity of affinity-purified MPL was 1066 times larger than the hemolymph.The purified MPL was thought to be pure as it appeared as a single band in native PAGE. By SDS-PAGE with 2-mercaptoethanol, MPL yielded 3 bands corresponding to 16 KDa, 18 KDa and 50 KDa, respectively. By gel filtration, the affinity-purified MPL eluted as a single symmetrical peak. Compared with standard proteins its molecular mass was estimated to be 86 KDa .About 1.5 mg of MPL was obtained from 30 g of pupae. Carbohydrate estimation indicated that MPL contained about 12.3% neutral sugar.Hemagglutination and hemagglutination inhibition assay showed that MPL recognized D-galactose and did not recognize D-glucose, D-mannose, fructose, N-acetyl-D-glucosamin, lactose, maltose, sugar and D-ribose.Antibacterial activity of MPL has been observed against Escherichia coli, Bacillus subtilis and Salmonella typhi. The results of lymphocyte transformation test showed that MPL can increase cellular immunity and the result of plaque-forming cell assay showed that MPL can increase humoral immunity.Different conditions were studied to remove ash and protein from Musca domestic and to remove acetyl from chitin. The best condition is: 1g Musca Domestic powder was mixed with 20 ml 2mol/L HCL and incubated for 1h at RT under agitation. Then it was mixed with 2.5 mol/L NaOH and incubated for 8h in two sessions surrounded by boiling water. Musca domestic chitin was mixed with 40%NaOH and heated directly on the electric cooker for 8h in two sessions.