Ten antibacterial peptides and application thereof in preparation of antibacterial infection drugs

Abstract

The invention relates to application of ten antibacterial peptides in the antimicrobial field. The ten antibacterial peptides have strong antibacterial activity on multi-drug-resistant bacteria, including but not limited to gram-negative bacteria such as escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and the like, and gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis and the like. Activity research proves that any one of the ten antibacterial peptides has a remarkable effect of inhibiting, killing or preventing microbial infection. Due to the strong antibacterial property of any antibacterial peptide, the antibacterial peptide has wide application potential in the aspects of preparing antibacterial drugs, preservatives, antibacterial coatings, antibacterial fibers, antibacterial packaging materials and the like. Due to the unique property of the antibacterial peptide in the aspect of antibacterial performance, a new direction is provided for the development of efficient and reliable antibacterial agents and antibacterial technologies and products in multiple fields.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to ten antimicrobial peptides with high antimicrobial activity and applications thereof. Background Art

[0002] The emergence of drug-resistant bacteria, especially multidrug-resistant bacteria, poses an increasing threat to human health. "ESKAPE" strains (including Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter) are particularly dangerous. These strains have become the main pathogens of hospital infections due to their resistance to multiple antibiotics, and treatment options are very limited. In this context, antimicrobial peptides (AMPs), as a type of polypeptide substance with a significantly different mechanism of action from traditional antibiotics, have the potential to quickly kill bacteria, fungi, viruses and parasites, and have become a new type of drug for the treatment of infectious diseases.

[0003] Antimicrobial peptides mainly act on bacterial cell membranes, interact with them and lyse the cell membranes, leading to bacterial death. Since the basic components of bacterial cell membranes are relatively stable and not prone to drug resistance, antimicrobial peptides show significant advantages in combating bacterial infections. However, antimicrobial peptides may also produce cytotoxicity to eukaryotic cells, which limits their widespread clinical application.

[0004] In the study of antimicrobial peptides, how to improve selective toxicity is a key goal. Selective toxicity means that antimicrobial peptides can kill bacteria more specifically without damaging human cells. Factors affecting selective toxicity include environmental conditions and the properties of the antimicrobial peptides themselves. Environmental conditions such as ionic strength, pH, and serum composition have a significant effect on the activity of antimicrobial peptides, so these factors need to be considered when designing antimicrobial peptides. In addition, the properties of antimicrobial peptides such as net charge, helicity, hydrophobicity, and amphipathicity are also closely related to their interactions on bacterial and eukaryotic cell membranes.

[0005] Adjusting the net charge of antimicrobial peptides so that they interact more selectively with bacterial cell membranes is one of the key strategies to improve selective toxicity. In addition, through synthetic modification and structural optimization, the hydrophobicity and amphiphilicity of antimicrobial peptides can be adjusted to enhance their selectivity for bacteria. The application of nanotechnology also provides a way to reduce the impact on eukaryotic cells by carrierizing antimicrobial peptides to improve their localization on bacteria. Through these multi-faceted optimizations, researchers are committed to finding a balance to improve the selective toxicity of antimicrobial peptides and provide new directions for the development of future anti-infection treatments.

[0006] The history of antibiotics can be traced back to the discovery of penicillin, a milestone that has gradually expanded the library of drugs for treating microbial infections. However, with the widespread use and abuse of antibiotics, bacteria have gradually evolved resistance to these drugs, causing many bacteria to become multidrug-resistant strains, posing a huge threat to human health. The cross-regional spread of drug-resistant microorganisms and drug-resistant genes around the world has made antibiotic resistance an urgent global issue. Against this challenging background, the search for new antimicrobial drugs is particularly urgent.

[0007] Antimicrobial peptides have become potential alternatives to antibiotics due to their unique mechanism of action and broad antimicrobial activity. Compared with traditional antibiotics, antimicrobial peptides have highly effective bactericidal effects on both Gram-negative and Gram-positive bacteria, and their bactericidal rate is relatively fast. At the same time, antimicrobial peptides are less likely to cause bacterial resistance, which makes them a focus of attention in the field of anti-infection treatment. However, the research on antimicrobial peptides also faces some challenges, such as their stability, bioavailability and toxicity.

[0008] At present, the hot topic of antimicrobial peptide research is how to improve its selective toxicity and reduce its toxicity to eukaryotic cells. The current research focus is to carefully design the antimicrobial peptide sequence, optimize its structure and properties, and improve its stability and bioavailability. This will help develop safer and more effective anti-infection treatments and provide a new solution to overcome antibiotic resistance. Summary of the invention

[0009] The present invention aims to solve the deficiencies of the prior art and provides ten antimicrobial peptides with high antimicrobial activity, which can effectively inhibit bacterial activity and achieve the effect of killing bacteria. These antimicrobial peptides and their derivatives can be used to prepare antimicrobial drugs.

[0010] Cecropin B is a naturally occurring antimicrobial peptide with broad-spectrum antimicrobial activity, showing significant antibacterial effects on a variety of Gram-negative and Gram-positive bacteria. The amino acid sequence of Cecropin B is KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL, which mainly destroys the integrity of the bacterial membrane by interacting with the bacterial cell membrane, thereby achieving an antibacterial effect. In order to further improve the antibacterial activity, stability and selectivity of Cecropin B, the present invention designed and synthesized a series of antimicrobial peptide derivatives based on the sequence of Cecropin B, and finally obtained 10 optimized antimicrobial peptides through systematic screening, which were named AMP1 to AMP10.

[0011] Design and optimization ideas.

[0012] 1. Optimization of amino acid sequence: Based on the amino acid sequence of Cecropin B, its key amino acid residues were first screened and replaced. By introducing or replacing specific amino acids, such as introducing more positively charged amino acids (lysine K, arginine R), the interaction between the peptide chain and the bacterial membrane is increased, and the antibacterial effect is enhanced. At the same time, by introducing hydrophobic amino acids (such as leucine-L, isoleucine-I, phenylalanine-F), the ability of the peptide chain to insert into the membrane is improved.

[0013] 2. Diversification of sequence length and structure: In order to optimize the biological activity of antimicrobial peptides, derivatives of different lengths were designed, ranging from 25 to 35 amino acids. The structural stability and membrane binding ability of antimicrobial peptides were improved by modifying the ends of the peptide chains, such as cyclization or adding a hydrophobic tail. In addition, the middle segment of the peptide chain was structurally adjusted to optimize the secondary structure of the peptide chain and enhance its interaction with the bacterial membrane.

[0014] 3. Introduction of D-amino acids: In order to improve the stability of antimicrobial peptides in the body and reduce the possibility of degradation by enzymes in the body, D-amino acids are introduced into some derivatives to replace natural L-amino acids, especially at sites where the peptide chain is easily cleaved by enzymes, to improve the biological stability of the peptides.

[0015] 4. Adjustment of isoelectric point and instability index: By adjusting the isoelectric point and instability index of the peptide chain, the solubility and half-life of the antimicrobial peptide in vivo are optimized. The composition of amino acids is selectively adjusted so that the isoelectric point of some derivatives is biased towards the physiological pH value of bacteria to enhance their activity in the pathogen environment. At the same time, by reducing the amino acid residues that are prone to instability, the overall stability of the peptide chain is improved.

[0016] Through the above design and optimization, the present invention synthesized hundreds of antimicrobial peptide derivatives. Through in vitro antimicrobial activity tests, cytotoxicity evaluations, and in vivo model experiments, 10 antimicrobial peptides with excellent antimicrobial activity and good biosafety were screened out, named AMP1 to AMP10. These derivatives all showed stronger antimicrobial activity and better biostability than Cecropin B, and had lower toxicity to mammalian cells.

[0017] Antimicrobial peptide sequences: The present invention relates to ten antimicrobial peptides, namely AMP1-10. Their amino acid sequences are: AMP1:KWKKWKDIIKGRNRVKAGPAIAVLGEAKALD AMP2:KWKRFKKLIKMGNRNGIVKAGPAIAVLG AMP3:KWKVWKKIEKMGNIRNGIVKAGPAIAVLGA AMP4:KWKVFKKIEMGRNIRNGIVKKKAGPAIALG AMP5:KWKKFKKIEMGNIRNGIVKAGPAIAVLLGE AMP6:KWKVFGRIEMGRNIRNGIVKAGPAIAVLLG AMP7:KWKVFKKIEMGRNIRNGLVKAGPAIAVKGA AMP8:KWKKFKKIEMGNIRNGIVKAGPAIAVLGAK AMP9:KWKVFKKIEMGRNIRNGIVKAGPAIAVLGD AMP10:KWKKWKDFFKGRNRVKAGFAIAVLGEAKD

[0018] Application fields: The antimicrobial peptide of the present invention can be widely used in the preparation of antimicrobial drugs, especially for the preparation of antimicrobial drugs against Gram-negative bacteria.

[0019] Targeting Gram-negative bacteria: The present invention is preferably used to treat Gram-negative bacterial infections, including Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli.

[0020] Multi-field application: The antimicrobial peptide of the present invention can not only be used to prepare medical imaging reagents, preservatives, feed additives, and daily chemical cleaning products, but can also be applied to multiple fields such as medical devices with antibacterial effects.

[0021] Compositions and dosage forms: The invention includes a medical imaging agent composition, which includes an antimicrobial peptide and an imaging agent. In addition, it also involves biological antimicrobial agents, preservatives, animal feed, daily chemical cleaning products, and medical dressings containing antimicrobial peptides and other pharmaceutical excipients.

[0022] Advantages and safety: Antimicrobial peptides perform well in inhibiting and killing bacteria. They do not show toxicity to cells in in vitro cytotoxicity experiments and are extremely safe.

[0023] Preparation process: The preparation process of antimicrobial peptides is mature and easy to obtain, providing a feasible solution to the needs in the research and development of antimicrobial drugs.

[0024] In general, the antimicrobial peptides of the present invention have broad application prospects in the treatment of bacterial infections and many other fields, have significant antibacterial effects and high safety, and have mature preparation processes and are easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1These are the bactericidal curves of ten antimicrobial peptides in Example 3.

[0026] Figure 2 This is the effect of ten antimicrobial peptides and the control group on the bacterial NPN test in Example 4.

[0027] Figure 3 This is the effect of ten antimicrobial peptides and the control group on the bacterial PI test in Example 5. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments.

[0029] Example 1 Preparation of antimicrobial peptides

[0030] The present invention adopts a solid phase synthesis method to prepare the polypeptide, and the specific steps are as follows:

[0031] (1) Weigh an appropriate amount of Fmoc-Gly Wang Resin with a degree of substitution of 0.35 mmol / g and place it in a medium-sized reaction column, then soak it in DMF for 120 minutes.

[0032] (2) After draining DMF, add 20% Pip / DMF in an amount 3 times the volume of the resin and blow nitrogen for 30 minutes to remove Fmoc. Then wash with DMF 5 times and use ninhydrin to detect the dark blue color.

[0033] (3) Add the raw materials in proportion, add an appropriate amount of DMF, and react by blowing nitrogen gas until the ninhydrin test becomes transparent.

[0034] (4) Repeat steps 2-3 to complete the synthesis of the entire polypeptide sequence.

[0035] (5) Drain DMF, add 20% Pip / DMF (3 times the volume of the resin), and blow nitrogen for 30 minutes to remove Fmoc. Then wash with DMF2, MeOH2, DCM2, and MeOH2.

[0036] (6) Drain the resin in the reactor and transfer it to a cut tube. Add 40 mL of liquid F and control the temperature on a shaker for 2.5 hours.

[0037] (7) Filter by suction, collect the cutting filtrate into a centrifuge tube, add 6 times the volume of ice ether, and precipitate by low-speed centrifuge.

[0038] (8) The precipitate was washed with ether three times to obtain a crude product.

[0039] (9) The crude product is placed in a drying pot and vacuum dried overnight, and then sent to the purification department for purification to obtain the polypeptide of the target purity.

[0040] (10) Finally, high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to identify the molecular weight and purity of the peptide.

[0041] In order to evaluate the antibacterial activity of the polypeptides of the present invention, the minimum inhibitory concentration of antimicrobial peptides was determined.

[0042] Example 2 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0043] Test strains: Reference strains, such as Escherichia coli ( Escherichia coli ATCC25922, Pseudomonas aeruginosa ( Pseudomonas aeruginosa PAO1), Acinetobacter baumannii ( Acinetobacter baumannii ATCC49096), Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC 43816) and other Gram-negative bacteria as well as Staphylococcus aureus ( Staphylococcus aureus ATCC 25923), Enterococcus faecalis ( Enterococcus faecalis ATCC 29212), as well as clinical multidrug-resistant strains such as Escherichia coli E11 and E19, Pseudomonas aeruginosa PA16 and PA18, Acinetobacter baumannii A8 and A10, Klebsiella pneumoniae K47 and K56, Staphylococcus aureus SA72 and SA35, and Enterococcus faecalis EF6 and EF9.

[0044] The specific steps for determining the minimum inhibitory concentration are as follows:

[0045] (1) Cultivate bacteria: All the strains to be tested were streaked onto agar plates and cultured overnight at 37°C in a microbial incubator. Monoclonal colonies were selected and cultured in ordinary MHB liquid culture medium at 37°C and 200× rpm until the logarithmic growth phase. Dilute the bacterial solution in LB liquid culture medium to 10 6 CFU / ml.

[0046] (2) Preparation of antimicrobial peptide solution: The antimicrobial peptide was dissolved in MHB medium and solutions of different concentrations were obtained using the 2-fold microdilution method.

[0047] (3) Determination of bactericidal activity: 100 μl of antimicrobial peptide solution of different concentrations was mixed with 100 μl of diluted bacterial solution and incubated in a sterile 96-well plate for 18 hours. Subsequently, the absorbance of each well of the 96-well plate was detected at 600 nm using a multifunctional microplate reader. The positive control and negative control were set as no antimicrobial peptide, only 100 μl of MHB culture medium and 100 μl of diluted bacterial solution, or no antimicrobial peptide or antimicrobial peptide derivative, and only 200 μl of MHB culture medium.

[0048] (4) Detect the OD of each well 600The values ​​were compared with those of negative and positive controls to obtain the minimum inhibitory concentration (MIC) of antimicrobial peptides and antimicrobial peptide derivatives.

[0049] The results of the minimum inhibitory concentration of antimicrobial peptides against the test strains are shown in Table 1. As can be seen from the table, the minimum inhibitory concentrations of all antimicrobial peptides against the four tested Gram-negative bacteria were ≤2g / mL, showing a strong antibacterial effect. In addition, the minimum inhibitory concentrations of all antimicrobial peptides against the two tested Gram-positive bacteria were ≤8g / mL. A comprehensive comparison found that compared with the overall antibacterial effects of the 10 antimicrobial peptides on all Gram-negative bacteria and Gram-positive bacteria, the antimicrobial peptides were more sensitive to Gram-negative bacteria, which is consistent with the existing research results on Cecropin B antimicrobial peptides.

[0050] In order to study the antibacterial mechanism of the invented antimicrobial peptides, we designed NPN and PI staining experiments. Based on the results, we speculated that these antimicrobial peptides may exert their antibacterial and bactericidal activities by destroying the integrity of bacterial cell membranes.

[0051] Table 1 Minimum inhibitory concentration results of antimicrobial peptide AMP1-10

[0052]

[0053] Example 3 Activity test of antimicrobial peptides against planktonic bacteria

[0054] The specific steps are as follows:

[0055] (1) Cultivate bacteria: Take a single colony from the purified test bacteria plate and inoculate it into 10 mL of MHB medium. Cultivate at 37°C for 5-6 hours to allow the test bacteria to reach the logarithmic growth phase.

[0056] (2) Preparation of antimicrobial peptide solution: Dissolve the antimicrobial peptide in MHB medium with final concentrations ranging from 0.01 to 100 times the MIC.

[0057] (3) Incubation of bacteria and antimicrobial peptides: Inoculate bacteria into the culture medium containing antimicrobial peptides, with the final concentration of bacteria being 10 6 CFU / ml.

[0058] (4) Incubation time: Place the incubation dish in an incubator at 37°C and incubate for 24 hours.

[0059] (5) Determination of bacterial growth: After the incubation period, plate counts were performed on all test samples.

[0060] (6) Record results: Record the growth of bacteria in each culture dish and determine the activity of antimicrobial peptides against planktonic bacteria.

[0061] (7) Data analysis: Conduct statistical analysis on the results and draw a concentration-sterilization curve.

[0062] The results showed that all antimicrobial peptides (AMP1-AMP10) and the control group CecropinB could significantly reduce the colony count (CFU) to an extremely low level in a short period of time (about 1 hour), and maintained the antibacterial effect in the subsequent period of time. This shows that all antimicrobial peptides have significant and rapid bactericidal activity, which can effectively reduce the number of colonies and maintain a low level.

[0063] Example 4 NPN accumulation experiment

[0064] N-Phenyl-1-naphthylamine (NPN) is a fluorescent dye used to detect bacterial membrane permeability. In this experiment, Cecropin-B (H.cecropia) was used as a positive control, and NPN was used to evaluate the effects of ten antimicrobial peptides on the membrane permeability of Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923. The following are the specific steps:

[0065] (1) Cultivate bacteria: Take a single colony from the purified test bacteria plate and inoculate it into 10 mL of MHB medium. Cultivate at 37°C for 5-6 hours to allow the test bacteria to reach the logarithmic growth phase.

[0066] (2) Centrifuge the bacterial solution in the logarithmic growth phase at 10,000 rpm / min for 2 minutes to collect the bacteria.

[0067] (3) Wash the cells with PBS buffer solution twice, then resuspend the cells in MHB medium to a concentration of 1×10 8 CFU / mL.

[0068] (4) Add antimicrobial peptides to the resuspended bacterial solution to make the final concentration of each antimicrobial peptide 1 times the MIC, and gently shake to mix. For the blank control group, add an equal volume of PBS buffer solution to the resuspended bacterial solution and incubate at 37°C for 1 hour.

[0069] (5) After incubation, centrifuge at 10,000 rpm / min for 2 minutes, discard the supernatant, and resuspend the bacterial pellet in 200 μL of cell Tris buffer.

[0070] (6) Add 5 μL of NPN dye solution to the bacterial resuspension to make the final concentration of 20 μM, place in a 96-well plate, mix well, and incubate at 37°C for 30 min.

[0071] (7) After the incubation, set the microplate reader to emit light at 340 nm and absorb light at 410 nm, and place the 96-well plate in the microplate reader to detect the fluorescence value of the sample to be tested.

[0072] Through the results of the NPN accumulation experiment, the fluorescence intensity of the antimicrobial peptide treatment group can be observed to evaluate the effect of the antimicrobial peptide on the permeability of the bacterial outer membrane. Compared with the blank control group, the increased fluorescence signal in the antimicrobial peptide treatment group indicates that the bacterial membrane permeability has increased. This result will help to gain a deeper understanding of the mechanism of action of antimicrobial peptides on the bacterial outer membrane.

[0073] Example 5 PI staining test

[0074] PI (propidium iodide) is a fluorescent dye that can be used for DNA staining and is widely used in cell apoptosis detection. In this experiment, Cecropin-B (H.cecropia) was used as a positive control, and the PI staining method was used to evaluate the effects of ten antimicrobial peptides on the cell membrane damage of Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923, in order to preliminarily explore their possible antibacterial mechanisms. The following are the specific steps:

[0075] (1) Strain purification: Take a single colony from the purified test bacteria plate and inoculate it into 10 mL of MHB medium. Culture it at 37°C for 5-6 hours until the test bacteria reaches the logarithmic growth phase.

[0076] (2) Centrifuge the bacterial solution in the logarithmic growth phase at 10,000 rpm / min for 2 minutes to collect the bacteria.

[0077] (3) Wash the cells with PBS buffer solution twice, then resuspend the cells in MHB medium to a concentration of 1×10 8 CFU / mL.

[0078] (4) Add antimicrobial peptides to the resuspended bacterial solution to make the final concentration of each antimicrobial peptide 1 times the MIC, and gently shake to mix. For the blank control group, add an equal volume of PBS buffer solution to the resuspended bacterial solution and incubate at 37°C for 1 hour.

[0079] (5) After incubation, centrifuge at 10,000 rpm / min for 2 minutes, discard the supernatant, and resuspend the bacterial pellet in 200 μL of cell Tris buffer.

[0080] (6) Add 5 μL of PI dye solution to the bacterial resuspension to make the final concentration 5 μM, mix well, and incubate at 37°C for 60 min.

[0081] (7) After incubation, set the microplate reader to emit light at 493 nm and absorb light at 636 nm, and place the 96-well plate in the microplate reader to detect the fluorescence value of the sample to be tested. Through the PI staining results, it can be observed that after 1 hour of incubation, the fluorescence signal value of the antimicrobial peptide treatment group increased compared with the blank control group, indicating that the integrity of the bacterial membrane was destroyed. Therefore, it is speculated that these antimicrobial peptides may exert antibacterial and bactericidal activities by destroying the integrity of the cell membrane.

[0082] Example 6 Antimicrobial peptide erythrocyte hemolysis test

[0083] In this experiment, PBS buffer solution was used as negative control, 0.1% Trion X-100 was used as positive control, and the hemolytic activity of antimicrobial peptides at different concentrations on human red blood cells was detected by the cyanide methemoglobin method. The specific operation is as follows:

[0084] (1) Prepare antimicrobial peptide solutions with concentrations of 8 μg / mL, 32 μg / mL, 128 μg / mL, and 512 μg / mL.

[0085] (2) Place 200 μL of antimicrobial peptide and 200 μL of diluted red blood cell suspension in a 1 mL EP tube, gently shake to mix, and incubate at 37°C for 1 hour.

[0086] (3) After incubation, centrifuge the mixed solution at 800 rpm / min for 10 min. After centrifugation, use a pipette to pipette 200 μL of supernatant into a 96-well cell culture plate.

[0087] (4) Use a multifunctional microplate to measure the absorbance of each sample well at a wavelength of 540 nm.

[0088] (5) According to the formula: Hemolysis rate = (AAMP - APBS) / (A0.1% Trion X-100 - APBS) x100%

[0089] The results showed that the hemolysis rate of all antimicrobial peptides was less than 5% at the tested concentrations, and the detailed data are shown in Table 2. Through this experiment, it can be judged that at the tested concentrations, all antimicrobial peptides have no hemolytic effect on sheep erythrocytes, indicating that these antimicrobial peptides may be relatively safe in vivo.

[0090] Table 2 Results of the hemolysis experiment of ten antimicrobial peptides on red blood cells

[0091]

[0092] Example 7 Cytotoxicity of antimicrobial peptide WY-21

[0093] In this experiment, PBS buffer solution was used as negative control, 1% Trion X-100 was used as positive control, and the cytotoxicity of antimicrobial peptides at different concentrations on HEK293 cells was detected by detecting the release rate of LDH. The specific operation is as follows:

[0094] (1) Prepare cell suspension: HEK293 cells were isolated and sealed from porcine peripheral blood, and then resuspended in RMPI1640 medium to a concentration of 1 × 10 6 Pieces / mL.

[0095] (2) Treat the experimental plate: Add 90 μL of cell suspension to each well of the 96-well plate. Set up background control wells (add 100 μL of culture medium), negative control wells (add 90 μL of cell suspension and 10 μL of PBS), and positive control wells (add 90 μL of cell suspension and 10 μL of 10% TritonX-100).

[0096] (3) Adding antimicrobial peptide WY-21: Add 10 μL of different concentrations of antimicrobial peptide WY-21 to each well, so that the final concentrations are 512 μg / mL, 128 μg / mL, 32 μg / mL, and 8 μg / mL, respectively. Each concentration is treated with 6 replicates.

[0097] (4) Cell culture: Place the 96-well plate in an incubator at 37°C, 5% CO2 for 24 hours.

[0098] (5) Sample treatment: After 24 hours of culture, add 10 μL of 10% TritonX-100 to the control wells and mix well with a pipette, and continue to culture for 15 minutes. After the culture is completed, centrifuge each well and carefully transfer 60 μL of cell supernatant to the corresponding well of a 96-well culture plate with a transparent bottom.

[0099] (6) LDH assay: Add 30 μL of LDH assay reagent to each well, incubate in the dark for 30 minutes with shaking, and then use an ELISA reader to measure OD490nm.

[0100] (7) Calculate the LDH release rate: According to the formula, LDH release rate (%) = 100% * (OD test - OD negative control) / (OD positive control - OD negative control).

[0101] Result analysis: Based on the LDH release rate results, the cytotoxicity of antimicrobial peptides to HEK293 cells was evaluated. The results showed that all antimicrobial peptides showed no cytotoxicity at the highest tested concentration of 512 μg / mL. Detailed data are shown in Table 3.

[0102] Table 3 Cytotoxicity test results of ten antimicrobial peptides on HEK293 cells

[0103]

Claims

1. Antimicrobial peptide, characterized in that Selected from the following polypeptides represented by the amino acid sequence AMP1:KWKKWKDIIKGRNRVKAGPAIAVLGEAKALD AMP2:KWKRFKKLIKMGNRNGIVKAGPAIAVLG AMP3:KWKVWKKIEKMGNIRNGIVKAGPAIAVLGA AMP4:KWKVFKKIEMGRNIRNGIVKKKAGPAIALG AMP5:KWKKFKKIEMGNIRNGIVKAGPAIAVLLGE AMP6:KWKVFGRIEMGRNIRNGIVKAGPAIAVLLG AMP7:KWKVFKKIEMGRNIRNGLVKAGPAIAVKGA AMP8:KWKKFKKIEMGNIRNGIVKAGPAIAVLGAK AMP9:KWKVFKKIEMGRNIRNGIVKAGPAIAVLGD AMP10:KWKKWKDFFKGRNRVKAGFAIAVLGEAKD.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs, characterized in that It is preferably used to prepare antibacterial drugs against Gram-negative and Gram-positive bacteria.

3. The use according to claim 2, characterized in that The Gram-negative bacteria include, but are not limited to, one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa; the Gram-positive bacteria include, but are not limited to, one or more of Staphylococcus aureus and Enterococcus faecalis.

4. Use of the antimicrobial peptide according to claim 1 in the preparation of broad-spectrum antimicrobial drugs, disinfectants, detergents, preservatives, feed additives, daily chemical cleaning products or packaging materials.

5. A broad-spectrum antibacterial drug, characterized in that The invention comprises any one peptide or a combination of multiple peptides as described in claim 1 and pharmaceutically acceptable excipients.

6. A disinfectant and cleaner, characterized in that The invention comprises any one peptide or a combination of multiple peptides as described in claim 1 and an emulsifier allowed to be added in the chemical industry.

7. A preservative, characterized in that Comprising any one peptide or a combination of multiple peptides as described in claim 1.

8. An animal feed additive, characterized in that Comprising any one peptide or a combination of multiple peptides as described in claim 1 and a basic diet.

9. A daily cleaning product composition, characterized in that The method comprises any one of the peptides or a combination of multiple peptides as described in claim 1 and one or more surfactants.

10. A medical dressing, characterized in that The method comprises any one of the peptides or a combination of multiple peptides as described in claim 1 and a matrix.

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