Antibacterial Mechanism And Application Of CF-14, A New Antimicrobial Peptide From Fish

In this paper,the antibacterial peptide"CF-14"?RIVELTLPRVSVRL-NH₂?isolated and purified from the surface mucus of catfish was taken as the research object.The basic properties of CF-14 amino acid sequence were predicted by bioinformatics,and the basic properties of CF-14 were characterized by the synthesis of fish-derived antimicrobial peptide CF-14.The bacteriostatic mechanism of CF-14 was further explored by using Shewanella putrefaciens and Escherichia coli as target bacteria.Finally,PLA/CF-14 nanofiber membrane was successfully prepared,and its properties and antibacterial effect were characterized and verified.The main results are as follows:1.The amino acid sequence of CF-14 from fish was analyzed by bioinformatics.It was found that the similarity of CF-14 in antimicrobial peptide database was only 40%,and it had cation,amphiphilic and thermal stability.Structural analysis showed that the middle and both ends of CF-14 presented irregular curl,the rest was divided into extension chain,and the extension chain and random coil accounted for 50%respectively.It is predicted that CF-14 will not form a transmembrane ion channel on the membrane,nor insert bacterial cell membrane to destroy it.It is predicted that CF-14 may not be a membrane active peptide;toxicity and hemolysis analysis show that CF-14 is not toxic and will not cause hemolysis of red blood cells.In addition,the antibacterial activity of CF-14 amino acid sequence was studied.It was found that the enhancement of charge and amidation had a great influence on the antibacterial activity of antimicrobial peptides.The proline in the middle of the peptide chain and the amino acids at both ends of the peptide chain were very important.2.CF-14 was synthesized according to its amino acid sequence,and the accuracy of the synthesized peptide was verified by mass spectrometry.The results of in vitro bacteriostatic test and related physical and chemical properties showed that CF-14 had a strong inhibitory effect on Gram-negative bacteria and Gram-positive bacteria,and the minimum inhibitory concentration of CF-14 was 62.5?g/mL and 31.3?g/mL for Shewanella putrefaciens and Escherichia coli;The hemolysis and toxicity experiments showed that CF-14 had no significant hemolysis on sheep red blood cells,and still had no effect on the survival rate of 293T cells at the concentration of 1mg/mL;by detecting the change of antibacterial activity of antibacterial peptide in different environments,it is concluded that CF-14 would be destroyed in high temperature and strong acid environment,and the sensitivity of CF-14 to the enzyme was strong.3.The bacteriostatic mechanism of CF-14 was studied by using S.putrefaciens and E.coli as target bacteria.In the detection of DNA leakage,CF-14 can make bacterial nucleic acid leak;scanning electron microscopy showed that there was slight perforation on the surface of the cell membrane at the beginning stage,but the whole cell remained intact.The cells treated with CF-14 were obviously broken and collapsed after 12 h;the detection of circular dichroism showed that when CF-14 was in the cell membrane environment,its?-helix structure was significantly increased;the observation of laser confocal microscopy confirmed that CF-14 could enter into the cell and accumulate in the cytoplasm,suggesting that there might be an intracellular target of the antibacterial peptide;nucleic acid electrophoresis experiment showed that high concentration of antimicrobial peptides could bind to bacterial DNA;molecular docking experiments showed that CF-14 competitively bound to the conventional substrate binding site of DnaK,thus inhibiting the immature peptide chain in bacterial cells to bind to DnaK,and ultimately affecting bacteria.4.PLA/CF-14 nanofiber membrane was successfully prepared by electrospinning.After adding CF-14,the diameter of PLA spinning fiber decreased and became more uniform,and the fiber direction randomly crossed into a network structure;infrared spectrum detection found that the characteristic peaks of CF-14 and PLA appeared in the co spun fiber at the same time,and both of them were physical combination;X-ray diffraction analysis found that the addition of CF-14 reduced the crystallinity of PLA fiber,the nucleation ability of molecular chain decreased,and finally led to the polymer becomes weak.The tensile test shows that the addition of CF-14can enhance the tensile resistance of the nanofiber film,decrease the elongation ability and decrease the toughness.In addition,PLA/CF-14 nanofiber membrane still has good antibacterial activity,which can obviously inhibit the growth of S.putrefaciens.