家蝇幼虫血淋巴抗真菌肽的特性和分子生物学研究

The fungi diseases of human being and animals is becoming a very important health problem. For us there are works to discover a new kind of antibiotics which is high curative effect, less side-effect and hard to get resistant by the pathogen. It is to prove that Insects have an excellent immunes defense system. When being challenged by a pathogen they can quickly produce nonspecific peptides antibiotics and other material against bacteria. Some peptides from Sarcophaga and Drasophila have ability of inhibiting reproduction of fungi. According to our studies the secretions of larvae of the housefly (Musca demostic ) can inhibit reproduction of fungi, and furthermore there is an anti-fungus activity of their hemolymph. There are not any other reports of studies on anti-fungus activity of peptides of hemolymph of housefly, therefore we started, granted by national science fund, to research the anti-fungus activity and characteristics of molecules biology of peptides from housefly hemolymph.1.2 Aim： To isolate,purify and characterize new antifungal peptides from the hemolymph of housefly.1.3 The profiles of the studies 1.3.1 A study of the antifungal activity of peptides of the hemolymph from the non-induced .larvae of housefly：1.3.2 A study of the antifungal activity of peptides of the hemolymph from the immune-induced larvae of housefly;1.3.3 A preliminary study on antiviral capability of the hemolymph from larvae of housefly; 1.3.4 A study on the effect of housefly larval secretions on development of Ascaris suum eggs .1.4 The key results and its essential data.1.4.1 Isolating and purifying of the antifungal peptides 280nmOD values of Hemolymph of third instar larvae of housefly were 2.311±0.124 for immune-induced hemolymph and 1.729±0.120 for nonimmune-induced one respectively after being diluted by 500 times with Millipore water( P<0.05). The concentration of protein of the immune-induced hemolymph was higher than the nonimmune-induced one. Anti-fungal fractions were taken by precipitation with saturation solution of ammonium sulfate and then prepurifed by solid-phase extraction on reversed-phase Sep-Pak C18 cartridges(Waters). The results showed that three fractions eluted with 30%,60%,70% acetonitrile can inhibited reproduction of Candida albicans(ATCC76615). Their electrophoretic bands were between 3.1-14.4 KD. From the immune-induced and unimmune-induced hemolymph, the Sep-Pak fraction eluted with 30% acetonitrile revealed antifungal activity against the medium sensitive strain(08) and resistant strain(19) to fluconazole, and from the immune-induced hemolymph the Sep-Pak fractions eluted with 60% and 70% acetonitrile revealed antifungal activity against the sensitive(09) and resistant strains. The antifungal capability of the fraction against the medium sensitive strain was lower than against the sensitive and resistant strains. After being purified by reversed-phase HPLC, the Sep-Pak fraction eluted with 60% acetonitrile remained antifungal activity, its electrophoretic band and antifungal activity disappeared after the fraction was digested with protease K. There was no antifungal activity from the nonimmune-induced control. 1.4.2 The concentration and activity of the antifungal peptides .The peptide concentration of Sep-Pak fraction eluted with 30% acetonitrile was 0.073 mg/ml, 60% was 0.101 mg/ml，70% was 0.107 mg/ml。The activity of Sep-Pak fraction eluted with 60% acetonitrile was 0.552±0.0147 for the sensitive strain(09), 0.408±0.0081 for the medium sensitive strain and 0.479±0.0105 for the resistant strain(u＝ ). The peptide concentration of the purified material of the 60% Sep-Pak eluted fraction by reversed-phase HPLC was 0.043 mg/ml, its antifungal activity was 0.566±0.0374，0.307±0.0057 and 0.480±0.0116 respectively, otherwise the concentration of Ketoconazole was 11 mg/ml and its antifungal activity was 0.539±0.0496、0.897±0.0674 and 0.488±0.0058 respectively。1.4.3 The determination of biologic activity of extractions from housefly larvae sensitive fung

分离提纯家蝇幼虫血淋巴抗真菌肽，设定空白河抗真菌药物阳性对照：测定体外抗人病原真菌活性、抗真菌谱及理化性质；比较驯化和野生家蝇抗真菌肽活性差异；观察诱导原刺激幼虫对其抗真菌肽活性的影响；动物实验观察抗真菌肽氨基酸及核苷酸序列，确定其组织来源。为寻找抗人体真菌感染新药来源提供实验和理论依据。