Purification And Bioactivity Identification Of Xenopus Laevis Skin Antibacterial Peptides

The discovery and applications of antibiotics provides an important protectionfor human health. With extensive and irrational use of antibiotics, the resistancebegan to appear, and seriously affect human health and the safety of livestockproducts. The study of new type antimicrobial drugs in order to replace antibioticsand reduce bacterial resistance has become a research focus at present.Antimicrobial peptides, also known as antimicrobial peptides, was a new typeof antibiotics and encoded by genes. The purpose of this study was to screen out newantimicrobial peptides and study their biological properties. And the same time, thestudy could provide a variety of candidate resources and basic research support tothe research and application of antimicrobial peptides.In this research, Xenopus laevis as experimental material, were stimulated byelectricity, and the skin secretion with antimicrobial activity was collected. Thepreparative and analytical high performance liquid chromatography was used toseparate the material collected, while S.aureus ATCC29213as an experimentalindicator bacteria was used to assay the activity. MALDI-TOF/TOF-MS and aminoacid sequencing techniques could be used to identify the molecular weight andamino acid sequence of the peptides. The homology comparison was executed byNational Center for Biotechnology Information and Peptide databases. The resultsshowed that seven antimicrobial peptides were successfully identified. And there wasone newly sequence of the Xenopus laevis skin antimicrobial peptides and namedCPF-Pan. The amino acid sequence was KAGLKIGTHFLGGAPQQ.The peptides CPF-Pan and CPF were manufactured by Peptide solid phasesynthesis method and the purification were executed by high performance liquidchromatography separation. The results of MIC showed that CPF-Pan took thestrong inhibitory effect against S.aureus, the control group CPF’s inhibitory effect was weaker. This results was in line with that S.aureus ATCC29213as theexperimental indicator bacteria. The hemolytic activity of antimicrobial peptides,reflected an important biochemical indicator of its side effects as drug candidates.Hemolysis test indicated that the antimicrobial peptides of CPF-Pan didn’t have asignificant role at16μg/mL, and MHC is greater than50μg/mL. The result of MTTshowed that the CPF-Pan possessed a certain degree of growth suppression of breastcancer cell MCF-7. But the effect of the control group CPF was weaker, and therewas no significant change while the concentration increased. In order to study themechanism of antimicrobial activity, the change of the bacterial membranes treatedby antimicrobial peptides was observed by transmission electron microscopy. Theresults showed that the cell membrane of E.coli form a pore, the contents escaped,and the bacteria shrank after the role of CPF-Pan. And part or all cell membrane ofS.aureus dissolved, the contents escaped, and the normal structure was not existed.Finally this experiment got the CPF-Pan, a newly antimicrobial peptides withbroad-spectrum antimicrobial activity, a very weak hemolytic activity, and growthinhibition to MCF-7cells. Observation of the bacterial under transmission electronmicroscopy showed that the cell membrane of gram-negative and gram-positivebacteria was damaged by CPF-Pan.