The Expression Of Drosomycin In Different Prokaryote Express System And It's Antifungal Activity Identification

In recent years,antibiotics has brought the drug resistance of bacteria,viruses and other problem,so it is anxious to find new alternatives to alleviate the situation.Antimicrobial peptides(AMPs)have many advantages,such as high efficiency,good stability,safety for humans and non-toxic for animals,and are expected to be new novel antibiotic drugs.But AMPs in animals is minimal,it is difficult to acquire from animals directly.Thus the construction of high efficient expression system for AMPs is very necessary.Drosomycin,which was extracted from the haemolymph of Drosophila Melanogaster,contains 44 amino acid residues with four disulfide bridges.It is the first antifungal protein from insects and the only active AMPs against some filamentous fungi.In this paper,Drosomycin was cloned and expressed in prokaryotic system.After the highest expression induced by IPTG was selected,the recombinant Drosomycin was purified,and its antibacterial activity was identified.The main results obtained are as follows:1.According to the codon preferences of E.coli,Drosomycin was remoulded and synthesized.Then three recombinant plasmids p ET-22b-Drs,pl188-TF-Drs and ppSUMO-Drs were successfully constructed,and transfered into E.coli respectively.2.The recombinant Drosomycin was expressed in E.coli induced by IPTG.The result of Tricine-SDS-PAGE demonstrated that the recombinant Drosomycin was secreted into periplasm when using signal peptide DsbA and pelB,and fusion proteins,Drosomycin-SUMO and TF-Drosomycin,were soluble expressed in the cytoplasm of E.coli.3.The fusion proteins,TF-Drs and SUMO-Drs,were purified using nickel column affinity chromatography,and digested by protease enzyme Factor Xa and SUMO respectively.Then Drosomycin(4.9 kDa)was acquired using nickel column affinity chromatography,and identified by mass spectrometry.4.The agar diffusion assay showed that Drosomycin inhibited the growth of Neurospora crassa,Fusarium oxysporum and Pyricularia oryzac Cavgra.And the result of the scanning electron microscopy displayed that the growth of N.crassa and F.oxysporum were destroyed after added Drosomycin.These results indicated that the recombinant Drosomycin had similar antimicrobial activity as native Drosomycin.5.Recombinant Drosomycin abtained from pilot fermentation using the engineering strain of ppSUMO-Drosomycin/BL21(DE3)showed strongest activity,which is beneficial to industrial production.6.Drosomycin didn't show affection to the mammalian cells of 293 T and 3T3-L1 by the CCK-8 assay.It implied that Drosomycin can be used as medical antifungal drugs or feed additives.All results in the thesis showed that Drosomycin expressed highly in E.coli exhibited antifungal activity and had no cytotoxic on the mammalian cells of 293 T and 3T3-L1,which were similar to the native protein.Taken together,Drosomycin can be a good application prospects for animal disease prevention and treatment,medical clinical pharmaceutical.