The Screening And Related Research Of Antibacterial Peptide Secreted By Skin Of Xenopus Laevis

Antibacterial peptide is a kind of polypeptides that generate by biological immune system to resist to infection caused by heterogeneous pathogens they ubiquitously exist in insects,plants and human bodies. Besides nonspecific resistance to bacterias,fungi and viruses, they still can function anti-tumor-cell.Especially their lethal effect to mutidrug resistant bacteria and tumor cells without destroy the normal cells of human, that is a new pattern of antibacterial with tremendous development potential,and is a hotspot of pharmaprojects that function resistant to pathogenic microorganism. Amphibia animals'skins are smooth and glossy, naked, and no scalinesses, shells or hairs,but they can secrete skin mucilage possessing protective effect. The skin mucilage contain mount of antimicrobial active substances that have special molecular structure and complicated and diverse functions,and antibacterial peptide is one of most important bioactive substances that consist of important parts of connatural defense system of Amphibian and extreme abundance in their skins.Using Xenopus laevis daudin,Rana temporaria chensinensis,Rana dybowskii Gurnther as experiment materials,the research extracted skin secretion four species of Amphibians through two induction methods,and measured a series of bioactivities.Among measured bioactivities, skin secretion of differerent species of Amphibians have different bioactivities,while the same Amphibians skin secretion extracted by different induction methods also have different bioactivities. Results of bacteriostatic activity test in vitro showed that skin secretion of four species of amphibians have didderent bacteriostatic effect to different bacteria strains while Xenopus laevis daudin possesses the strongest effect; Addition to this, skin secretion of Xenopus laevis daudin can also resrain, wound and kill tumor cells;they still have trypsogen inhibitors activity and extremely feeble haemolysis activity.Therefore,we made Xenopus laevis daudin as experiment materials,screened antibacterial peptides from their skin secretion and studied their structures and functions.Using four steps separation and purification:SephadexG-25 gel filtration chromatography, preparation mode of reverse phase,ion exchange chromatography,analysis mode of reversed-phase high-performance liquid chromatography,we separated and purified two antibacterial peptides:AMP-1 and AMP-2, combining amino acid sequencing and MALDI-TOF/TOF-MS methods identitied primary structures of antibacterial peptides AMP-1 and AMP-2.After comparing with sequences that searched database on Internet, it can be identitied that those two antibacterial peptides are newfound antibacterial peptides.Then conducting those two newfound antibacterial peptides with minimal inhibitory concentraton test(MIC); trypsinase inhibitory activity test; haemolysis activity test; anti-tumor activity test.Results displayed:the two antibacterial peptides both showed antibacterial activity to both Gram-Positive Bacteria Gram-Negtive Bacteria, AMP-2 showed faint antibacterial activity to seudomonas Aeruginosa, AMP-1 and AMP-2 manifested different degree of antibacterial activity to drug resistant strains.Because the origin of Microorganisms that used to test antibacterial activity cannot be identical with Microorganisms pedigree that existed in amphibian habitat, therefore, the possible differences of Microorganisms between we've tested and specific antibacterial peptides targeted may cuased that we hadn't tested antibacterial peptides'antibacterial activity to individual strains, in may also be the cause that antibacterial peptides showed low hyposensitivity to individual strains.Besides antibacterial activity, AMP-1 and AMP-2 both showed comparatively strong trypsinase inhibitory activity,meanwhile both showed extemly feeble haemolysis activity.Anti-tumor results showed that:AMP-1 had lethal effect on tumor cells while AMP-2 showed well anti-tumor effect,when sample concentration reached 146.6μg/ml,it can killed breast cancer cells MCF-7 up to 81.1%.In order to futher study structures and functions of the two antibacterial peptides, this study predicted their secondary structures and spatial structures through secondary structure prediction software Jpred and tertiary structure prediction software CPHmodels, reformed AMP-1by replacing and deleting its lysines,and thus gained nine new antibacterial peptides,after searching sequence-similar antibacterial peptides in NCBI, using amine acid solid-phase synthesis methods synthesized fifteen antibacterial peptides,after purifying with high efficiency liquid chromatography,obtaining five antibacterial peptides that purity reached above 99%,and nine above 95% Using Staphylococcus aureus as indicator to test antibacterial activity of fourteen antibacterial peptides, and combined with prediction results to predict the functions of every lysine to structure and antibacterial activity of AMP-1.Because antibacterial peptides generally containsα-helices, hydrophobicity and charge number affect their bioactivity extraordinarily. as far as antibacterial peptide is concerned,the mutation of a single of amine acid is possible to a great extent to change its spatial structure and bioactivity. In the process of reforming antibacterial peptide,we again obtain another four new antibacterial peptides that possessing antibacterial activity.In order to further explain the antibacterial activity mechanism of antibacterial peptides originated from Xenopus laevis, After co-incubating E.coli ATCC25922 and S.aureus ATCC25923 with AMP-land AMP-2 respectively, using transmission electron microscope to observe the effect that antibacterial peptides of Xenopus laevis imposed on bacterial morphous andstructure.Before the treatment, E.coli ATCC25922 had intact cell wall and cell membrance,and moreover had complete cellularity,the surface was smooth and glossy,and can be observed intact pili,the cell surface had no injure. In addition, intracellular proteins can combine the heavy metalions that exist in staining solution,and thus make bacteria as Pitch black. E.coli ATCC25922 treated with AMP-land AMP-2 leaked parts of cell entocyte,and cells would be damaged or swelled, thalli would fracture partly or completely acccompanied with cytoplasm being diluent.As a result of permeate of cell entocyte, cytoplasm fractured and formed hollow cave,therefore made the color of stained cells faded.All above indicated that: AMP-land AMP-2 could make cell wall of E.coli ATCC25922 breakage or form the hole,caused cell entocyte leakage and finally led to the death of bacteria.Conclusions:the study separated and purified two new antibacterial peptides AMP-land AMP-2,both of them had broad spectrum of antibacterial activity, trypsinase inhibitory activity and feeable haemolysis activity, AMP-2 also possessed well anti-tumor activity.After structure perdiction,reformed the structure of antibacterial peptide AMP-1,and obtained four new antibacterial peptides with antibacterial activity:AMP-1-2,AMP-1-3,AMP-1-4 and AMP-1-5. Observed AMP-land AMP-2could to different extent destroy the cell wall.