Design,Biological Characteristics And Antibacterial Mechanism Of ?-Helix Peptide With PRRP As Central Axis

Antimicrobial peptides are an important part of animal nonspecific immunity and the first line of defense against foreign invasive microorganisms,which are widely distributed in nature.Antimicrobial peptides are sterilized by non-receptor membrane dissolution mechanism.This sterilization mechanism,which is completely different from antibiotics,makes it difficult for bacteria to produce drug resistance.Therefore,antimicrobial peptides are potential substitutes for antibiotics.Because some natural antibacterial peptides have the disadvantages of poor activity,high toxicity and instability,their clinical application is hindered.Therefore,referring to the structural parameters of antibacterial peptides,a brand-new design and transformation can solve the problems above to a certain extent.First of all,proline(Pro,P)can reduce the spatial structure of protein and reduce the toxicity of peptide to host cells.Therefore,in this experiment,PRRP was inserted into two mirror symmetrical repeated heptapeptide sequences as the central axis in order to obtain antibacterial peptides with low cytotoxicity and high stability.The template sequence of the designed peptide is XRRXXRXPRRPXRXXRRX-NH₂,where X represents Phe,Leu,Val,Ile.The biological activities(antibacterial activity,hemolytic activity,cytotoxicity,etc.)of the synthetic peptide were determined.The results showed that the newly synthesized peptide LR18has broad-spectrum antibacterial activity,and its hemolytic activity and cytotoxicity are significantly lower than the natural antibacterial peptide melittin.Among them,the average minimum inhibitory concentration GM_MIC=3?M and the therapeutic index TI=42.7.Secondly,the stability of antimicrobial peptides is also one of the important factors affecting its clinical application.In this study,the salt ion stability,temperature and PH value stability and protease stability of the designed peptide were detected and analyzed.The results showed that the antibacterial peptide LR18 had higher salt ion,temperature and PH value stability.After incubation at 37?for 30 min in all tested physiological salt solutions,the antimicrobial activity hardly changed;The antibacterial activity of LR18 was detected after incubation at 0?and 37?for 30 min,respectively,and the result showed its MIC value hardly changed.After LR18 was incubated in p H 6 and p H 8 solutions at 37?for 30 min,its antibacterial activity hardly changed.Finally,in order to study the antibacterial mechanism of LR18,the effect of LR18 on bacterial cell membrane was detected by fluorescent dye and electron microscopy,and the binding effect of LR18 on genomic DNA was detected by gel retardation test.The results showed that antibacterial peptide LR18 affected the integrity of bacterial cell membrane,and could rapidly increase the permeability of intracellular and outer membranes,causing the change of membrane potential energy,membrane rupture,leading to the outflow of contents,and finally causing bacterial death.The results of gel retardation test showed that the antibacterial peptide LR18 could bind to genomic DNA at the concentration of 32×MIC,and block its migration to the positive electrode.The results indicated that antibacterial peptide LR18 mainly caused the outflow of bacterial contents and lead to bacterial death by destroying bacterial cell membrane.