Peptide Sequence Optimization Of Antibacterial Peptide CC34 And Its Antibacterial Mechanism In Vitro

In this study,the antimicrobial peptide CC34 which was crossed by the antimicrobial peptide Cec Md of the housefly and the antimicrobial peptide Chensirin of the Chinese forest frog was used.To increase its bacteriostatic activity and reduce hemolytic activity,replacing from the beginning and replacing the two-two combination was adopted to construct an optimized peptide library of CC34.bioinformatics software Prediction of bioinformatics parameters of optimized antimicrobial peptides using bioinformatics software to optimize the bacteriostasis and reduce the hemolytic activity,and the method of substituting and replacing the two groups was adopted.The optimized peptide library of CC34 was constructed.The bioinformatics software was used to predict the bioinformatics parameters of the optimized antibacterial peptides,and four antibacterial peptides CC34P11,CC34K11,CC34 PL and CC34 PA were selected.The bioactivity test was carried out by using the drug-sensitive tablet method and the double dilution method,mainly including the antibacterial activity,the minimum inhibitory concentration,the bactericidal concentration,and the hemolytic assay;the in vitro antibacterial mechanism includes: the binding assay of the antimicrobial peptide to the simulated membrane The effect on the hydrophobicity of bacteria,the influence on the permeability of bacteria's inner and outer membranes,and the binding to DNA after incorporation and the effect on protein synthesis.The specific experimental results are as follows:(1)The four new peptides optimized and screened were CC34P11,CC34K11,CC34 PL and CC34 PA.The antibacterial activity was generally improved after optimization,and the hemolytic activity of CC34P11 and CC34K11 was lower.(2)Compared with the original peptide CC34,the inhibitory effect of the optimized peptide on Escherichia coli,Salmonella and Staphylococcus aureus was enhanced;the antibacterial activity of CC34P11,CC34K11 and CC34 PA on Pseudomonas aeruginosa was enhanced;CC34P11 and CC34 PL were stopped milk.The antibacterial effect of streptococcus was significantly improved;the antibacterial effect of CC34 PL on Streptococcus faecalis was improved.Compared with the original peptide,the four hemolysis peptides reduced the hemolysis rate of sheep red blood cells,and the lowest hemolysis rate was CC34K11;the hemolysis rate of chicken red blood cells decreased,and the lowest hemolysis rate was CC34 PA.(3)Five antibacterial peptides have effects on artificial biofilms.CC34 PL has the strongest binding ability to the simulated membranes,and is less likely to be quenched after binding with the simulated membranes.CC34 has the weakest binding ability to the simulated membranes.(4)Five antimicrobial peptides can reduce the hydrophobicity of the bacterial surface.CC34 PL had the greatest influence on the surface hydrophobicity of Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa.CC34 PA had the greatest influence on the hydrophobicity of Salmonella typhimurium,Streptococcus dysgalactiae,Streptococcus faecalis and Enterobacter aerogenes,while CC34 was stopped.In addition to the large surface hydrophobicity of Streptococcus mutans cells,the effect on the surface hydrophobicity of several other bacteria is not as good as optimizing peptides.(5)The results of bacterial outer membrane permeability test showed that the antibacterial peptides CC34P11,CC34K11,CC34 PL and CC34 PA had higher effects on the membrane permeability than the antimicrobial peptide CC34.(6)The effect of optimized peptide on Escherichia coli was greater than that of the original peptide CC34,but the permeability of CC34P11,CC34K11 and CC34 PL to other bacteria was not as large as that of the original peptide CC34.(7)All five peptides can destroy the membrane of Escherichia coli and Staphylococcus aureus,and the effect is different under the minimum inhibitory concentration of each peptide.The effect on the E.coli membrane from strong to weak is CC34 PA,CC34K11,CC34 PL,CC34P11,CC34;the effect on the Staphylococcus aureus membrane from strong to weak is CC34 PL,CC34P11,CC34 PA,CC34K11,CC34.(8)Binding effect of antibacterial peptide on bacterial DNA The test results show that high concentration of antibacterial peptide can bind DNA of Escherichia coli and Staphylococcus aureus,and lose the ability to bind to DNA at low concentration.Peptides bind DNA more strongly than CC34.(9)Effects of antimicrobial peptides on bacterial protein synthesis Experiments showed that the addition of different antimicrobial peptides reduced the bacterial protein content to varying degrees,and the effect of optimized peptides on protein synthesis was generally stronger than that of the original peptide CC34.