Isolation And Purification Of The Antimicrobial Peptide AMP Of Cycloplta Parva Distant And Analysis Of Properties

Antibiotics are the most pervasive chemotherapeutic agents in the clinical application,but in the last ten years.emergence of the multi-resistant bacterial strains have led to the serious difficulty of disease therapy,so it is importune to search for the novel and potent antimicrobial agent.Peptide antibiotics are produced by all species of the natural host defense molecules of these species and are major players in the insect'innate immune response.Insects have been remarkably successful in evolution and account for 90% of all extent animal species.When infection occurs there is the rapid and transient synthesis of a battery of antimicrobial peptides by fat body of the insect and secreted into the hemolymph,so the natural peptides are inducible,small-sized and exihibting a broad antimcrobial activity spectrum directly against bacteria and/or fungi.lt is now fully appreciated that the antimicrobial peptides are primordial elements of the innate immune defense against infections.Since the original discovery of cecropins in the diapausing paupae of the lepidopteran Hyalophora cecropiajn the early 1980s,an unexpectedly high number of the insect antimicrobial peptides have been isolated and purified from a varity of insects,such as Lepidoptera, Diptera, Coleoptera,Hymenoptera et al. in exopterygote insects and Hemiptera, Orthoptera,Dictyoptera et al. in endoptergote insects.The analysis to their structures reveals a fairly large diversity.This paper elucidated the extraction,isolation and purification of the antimicrobial peptide AMP of the bug C.parva Distant adults.and analyze its properties as following.The bug C.parva Distant adults obtained outdoors were pricked with a fine needle dipped into a combined bacterial pellet of S.aureus and E.coli,so the antimicrobial defense response was induced and extracted with the insect physiological saline.The extract are acidified to 0.1%trifluoroacetic acid concentration with TFA.The acidic extraction was performed for 30 min and ultracentrifugation,then the supernatant was ultrafiltered by lOkDa membrane in the Millipore ultrafilteration system.The antimicrobial activity appeared in the filtered liquid.In order to isolate and purify the molecules responsible for antimicrobial activity, the filtered liquid was directly applied to a Sep-Pak C18 Cartridge for solid-phase extraction.The active fractions were pooled and loaded onto Sep-Pak C18 Cartridges(Waters) and fractionated.Elutions wereperformed with 2,40 and 80%acetonitrile in acidified water.The 40%Sep-Pak fraction contains the potent antimicrobial peptide which was purified by reverse-phase HPLC on an Aquapore OD300 C18 column(250 X 4.6mm, Brownlee) developed with the gradient elution system composed of ACN-water-TFA.Then the 27%ACN eluted fraction with the antibacterial activity was further applied on the Superdex Peptide column developed with the 0.05%TFA containing the 30%ACN organic solvent over 50min at a flow rate of 0.5ml/min.The potent antimicrobial fraction isolated by GP-HPLC was further applied on a narrow bore reversed-phase column(Symmetry 300 C18 Column(4.6 X 250mm))and the active compound was a single peak.The peptide purity was ascertained by the another reversed-phase column(PE PepMap C18 Column(4.6 X 250mm ; 5m,300A)).The pure antimicrobial compound obtained was named AMP.The three steps of HPLC purification were performed with a Beckman Gold HPLC system equipped with a Beckman 166 UV absorbance detector.During the purification procedure,the column effluent was monitored by absorbance at 225nm.The peaks of antimicrobial activity were hand-collected in order to have one individual peak per fraction.The active fractions were monitored by the plate-inhibition assay.AMP had a broad antimicrobial activity spectrum and inhibit growth of the most gram-positive and gram-negative bacterial.AMP has a low MIC values against the representative strains in the lab.Biochememical assay showed that AMP can react with ninhydrin dye staining and purple can be visualized.AMP have UV absorbance at 280nm,as ind...