Studies On Isolation, Purification And Biological Activities Of Antiviral Protein From The Larvae Of Housefly, Musca Domestica

In this study,protein-enriched fraction and chitosan（byproduct） were extracted from the maggot of housefly,Musca domestica.Antiviral protein（HAP） was purificated from the protein-enrich fractions.This paper studied and discussed the preparation,purification and structure characterization as well as biological activities of HAP by using modem separation technology and modem chemical analysis method and medicinal experiments. Moreovr,antioxidant and immunomodulatory activities of protein-enrich fractions and biological activities of chitosan were analyzed.The main results were shown as follows:1.Antiviral and antioxidant activities of protein-enriched fraction（PEF） from the larvae of houseflyThe protein-enriched fraction（PEF） was isolated and purified from the larvae of housefly.This study was designed to investigate antiviral activities and antioxidant effects of PEF.The effects of PEF treatment were studied on aged mice liver lipid peroxidation and antioxidant enzyme activities which included superoxide dismutase （SOD） and glutathione peroxidase（GSH-Px）.Results:PEF could inhibit proliferation of two insect viruses（AcMNPV and BmNPV） and possess excellent antiviral activities. Moreover,PEF not only inhibited H₂O₂ stimulated oxidative hemolysis of erythrocytes of mice,but also depressed MDA production in mice liver homogenate by auto-oxidation and hepatic mitochondria expanded induced by Fe²⁺-ascorbic acid system.Compared to control group,treatments of PEF significantly increase SOD and GSH-Px activity of serum and liver homogenate in aged mice.MDA level of serum and liver homogenate decreased significantly in aged mice.In conclusion,this study demonstrates that PEF possesses antioxidative activity and might be valuable sources of natural antioxidative agents.2.Immunomodulatory effects of protein-enriched fraction（PEF） from the larvae of houseflyIn this study,immunomodulatory effects of protein extracts obtained from the larvae of housefly were investigated.The results showed that PEF possessed immunomodulatory activity.Phagocytic index of the treated groups was increased significantly comparing with the control group and the low-dose,middle-dose and high-dose group significantly augment the ear swelling.Comparing with the control group,PEF at three doses significantly enhanced the abilities of the proliferation of spleen cells comparing with the control group,whether the mitogens were added or not,and significant increments were also observed in the hemolytic activity of PEF treated groups.The NK cell activities of low-dose,middle-dose and high-dose group were significantly higher than that of the control group.All these results suggest that PEF can modulate immune function in mice.3.Isolation and purification of antiviral protein（HAP） from the larvae of houseflyDEAE-52 ion-exchange chromatography and Sephadex Gel filtration chromatography were used to separate and purify antiviral protein（HAP） from the larvae of housefly. Some methods such as spectrophotometer and chromatography and electrophoresis and RP-HPLC were used to identify the purity of HAP.It was identified to be electrophoresis and chromatography grade pure.Moreover,antiviral activities and antioxidant effects were analyzed.And PEF could significantly enhance the abilities of the proliferation of spleen cells.4.Structural characteristics and biological activities of antiviral protein（HAP） from the larvae of houseflySome methods such as UV spectra,FTIR spectra,Fluorescence spectra and CD spectra were used to identify the structure characteristics of HAP.As the result of CD spectrum and FTIR spectrum shown,the secondary structure of HAP was mainlyβ-sheet or coil and the content ofα-helix was very small.The content ofβ-sheet,coil and the content ofα-helix from HAP were respectively 79.1%,17.3%,3.6%.The largest emission wavelength and most excellent excitation wavelength were respectively 332nm,283nm.5.Biological activities of chitosan（byproduct） from the larvae of housefly Chitosan（byproduct） was isolated from the larvae of housefly.The antitumor, antioxidant and antifungal activities of chitosan from the larvae of Musca domestica were tested.The results showed that the chitosan from the larvae of housefly had excellent reducing power,metal ion chelating ability and strong scavenging activity on radical.The chitosan from the larvae of housefly had excellent antifungal activity against seven plant deleterious fungi（Rhizoctonia solani,Curvularia lunata,Sclerotium rolfsii,Fulvia fulva, Thanatephorus cucumeris,Sclerotina sclerotiorum and Rhizopus stolonifer）.And it could significantly inhibit the growth of R.stolonifer in the low concentration,the half inhibitory concentration is 0.20 mg/mL Moreover,chitosan from housefly larvae also exhibited significant antitumor activity against HeLa and S-180 tumor cell lines in vitro.