Beta-folding structure broad-spectrum antibacterial peptide and application thereof

Abstract

The invention discloses a broad-spectrum antibacterial peptide with a beta-folding structure and application of the broad-spectrum antibacterial peptide, and relates to the technical field of biology. The amino acid sequence of the antibacterial peptide is as shown in any one of SEQ ID NO.1-7. According to the invention, seven antibacterial peptides are designed by constructing a typical sequence of a beta-sheet structure in which hydrophilic and hydrophobic amino acids are alternately sequenced and a pi-pi stacking structure based on diphenylalanine. Studies find that the seven antibacterial peptides have broad-spectrum antibacterial activity and excellent biocompatibility, still keep good antibacterial activity and stability in physiological salt, serum and protease environments, and have high application value. Technical support is provided for development of alternative drugs of traditional antibiotics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a beta-pleated structure broad-spectrum antimicrobial peptide and application thereof. Background Art

[0002] Antimicrobial peptides, as an important component of the innate immune system, exert their antimicrobial effects through physical mechanisms such as altering membrane permeability and disrupting cell membrane integrity. Due to their natural antimicrobial properties and resistance to developing drug resistance, antimicrobial peptides are considered promising alternatives to traditional antibiotics. Studies have shown that β-pleated antimicrobial peptides exhibit superior cell selectivity compared to α-helical peptides. However, due to the difficulty of short peptides in forming a stable β-pleated conformation, systematic research on β-pleated peptides is still insufficient.

[0003] This invention intends to develop new antimicrobial peptides by exploring the influence of the hydrophilic-hydrophobic alternating sequence β-pleated structure on the biological activity of peptide molecules, thereby providing technical support for the development of alternative drugs to traditional antibiotics. Summary of the Invention

[0004] The present invention aims to provide a broad-spectrum antimicrobial peptide with a β-pleated sheet structure and its application to address the aforementioned problems of the prior art. The antimicrobial peptide provided by the present invention has broad-spectrum antimicrobial activity and excellent biocompatibility, providing technical support for the development of alternatives to traditional antibiotics.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an antimicrobial peptide JK, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides an antimicrobial peptide JR, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides an antimicrobial peptide JH, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] The present invention also provides an antimicrobial peptide F-1, the amino acid sequence of which is shown in SEQ ID NO.4.

[0010] The present invention also provides an antimicrobial peptide FK, the amino acid sequence of which is shown in SEQ ID NO.5.

[0011] The present invention also provides an antimicrobial peptide FR, the amino acid sequence of which is shown in SEQ ID NO.6.

[0012] The present invention also provides an antimicrobial peptide FH, the amino acid sequence of which is shown in SEQ ID NO.7.

[0013] The present invention also provides the use of an antimicrobial peptide in the preparation of an antibacterial agent, wherein the amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NOs. 1-7.

[0014] Furthermore, the pathogens inhibited by the antibacterial agent include Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

[0015] The present invention also provides an antibacterial agent, the active ingredient of which includes an antibacterial peptide having an amino acid sequence as shown in any one of SEQ ID NOs. 1-7.

[0016] The present invention discloses the following technical effects:

[0017] This invention designed seven antimicrobial peptides by constructing a typical β-sheet structure with alternating hydrophilic and hydrophobic amino acids and a π-π stacking structure based on diphenylalanine. Research has shown that these seven antimicrobial peptides exhibit broad-spectrum antimicrobial activity and excellent biocompatibility. They maintain good antimicrobial activity and stability in physiological salt, serum, and protease environments, demonstrating high application value. This invention provides technical support for the development of alternatives to traditional antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is the mass spectrum of antimicrobial peptide JK;

[0020] Figure 2 is the mass spectrum of antimicrobial peptide JR;

[0021] Figure 3 is the mass spectrum of antimicrobial peptide JH;

[0022] Figure 4 is the mass spectrum of antimicrobial peptide F-1;

[0023] Figure 5 is the mass spectrum of antimicrobial peptide FK;

[0024] Figure 6 is the mass spectrum of antimicrobial peptide FR;

[0025] Figure 7 is the mass spectrum of antimicrobial peptide FH;

[0026] Figure 8 Circular dichroism spectra of antimicrobial peptide JK in three different environments;

[0027] Figure 9 Circular dichroism spectra of antimicrobial peptide JR in three different environments;

[0028] Figure 10 Circular dichroism spectra of antimicrobial peptide JH in three different environments;

[0029] Figure 11 Circular dichroism spectra of antimicrobial peptide F-1 in three different environments;

[0030] Figure 12 Circular dichroism spectra of antimicrobial peptide FK in three different environments;

[0031] Figure 13 Circular dichroism spectra of antimicrobial peptide FR in three different environments;

[0032] Figure 14 Circular dichroism spectra of antimicrobial peptide FH in three different environments;

[0033] Figure 15 The results of hemolytic activity test of antimicrobial peptides;

[0034] Figure 16 The activity statistics of RAW264.7 macrophages under the action of different antimicrobial peptides;

[0035] Figure 17 This is a laser confocal micrograph of S. aureus ATCC 29213 double-stained with Calcein-AM / PI live / dead cells;

[0036] Figure 18 is the SEM image of E. coli ATCC 25922 after treatment with antimicrobial peptides JR and FK;

[0037] Figure 19 is the SEM image of S. aureus ATCC 29213 after treatment with antimicrobial peptides JR and FK;

[0038] Figure 20 TEM images of E. coli ATCC 25922 after treatment with antimicrobial peptides JR and FK;

[0039] Figure 21 TEM images of S. aureus ATCC 29213 after treatment with antimicrobial peptides JR and FK. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1 Polypeptide sequence design and physicochemical parameter analysis

[0046] This study focused on β-sheet antimicrobial peptides, constructing a representative β-sheet sequence with alternating hydrophilic and hydrophobic amino acids. By constructing a π-π stacking structure of diphenylalanine and adjusting the number of positive charges in the sequence, the antimicrobial activity of the peptides was enhanced. Seven antimicrobial peptides were constructed, and their physicochemical parameters are shown in Table 1.

[0047] Table 1 Amino acid sequences and physicochemical parameters of antimicrobial peptides

[0048]

[0049] Note: aThe actual molecular mass is detected by mass spectrometer;

[0050] b Overall average hydrophilicity and c isoelectric point were calculated using an online tool (PepCalc.com—Peptidecalculator);

[0051] The total average hydropathicity value (GRAVY) is defined as the ratio of the sum of the hydropathicity values ​​of all amino acids in the sequence to the number of amino acids. A larger negative value indicates a stronger hydrophilicity, and a larger positive value indicates a stronger hydrophobicity.

[0052] Hydrophilic cationic residues and hydrophobic residues play important roles in electrostatic interactions with negatively charged bacterial membranes and in facilitating peptide penetration into hydrophobic domains of bacterial membranes, respectively. The positive charge counts of the antimicrobial peptides studied ranged from +3 to +4, providing appropriate electrostatic interactions for antimicrobial activity. The hydrophobicity of the antimicrobial peptides was assessed using the overall average hydrophilicity value, with higher positive values ​​indicating greater hydrophobicity. Among the antimicrobial peptides, F-1 exhibited the strongest hydrophobicity, followed by FK.

[0053] Example 2 Synthesis and Identification of Polypeptides

[0054] Antimicrobial peptides JK, JR, JH, F-1, FK, FR, and FH were synthesized by solid-phase synthesis. The specific method is detailed as follows:

[0055] 1. Synthesis of antimicrobial peptides

[0056] Weigh an appropriate amount of resin, add dichloromethane and soak for 5 minutes, wash with N,N-dimethylformamide, then remove the dichloromethane, and then use the prepared deprotecting agent to remove the Fmoc protecting group on the resin (decapping), and the decapping time is 20 minutes; weigh the second amino acid at the C-terminus, add a condensing agent, N,N-dimethylformamide, and place it in the reaction column for reaction (airing); use the ninhydrin method for detection, and the test result is that the solution is bright yellow, the resin is transparent, and there is no variegated color; use the deprotecting solution to remove the Fmoc protecting group on the second amino acid, wash with N,N-dimethylformamide 6 times, and repeat the operation for the remaining amino acids until the last amino acid; use the deprotecting solution to remove the last Fmoc protecting group, and then wash with N,N-dimethylformamide; after the reaction is completed, wash and shrink with dichloromethane and methanol respectively; use the cleavage solution to cut the polypeptide, and precipitate the liquid with ether; analyze with mass spectrometer (MS) and high performance liquid chromatography (HPLC) for purification.

[0057] 2. Purification of antimicrobial peptides

[0058] (1) Pre-analysis: Take an appropriate amount of sample and place it in a 0.5 mL tube. Dissolve it in ultrapure water. Filter the sample with a 0.45 μm membrane and analyze it by rapid gradient HPLC (10-100%).

[0059] (2) Sample preparation: Add 200 mg of sample to a 20 mL beaker, followed by 15 mL of H2O and 5 mL of methanol. Ultrasonicate the sample until it is completely dissolved, then filter the solution through a 0.45 μm membrane.

[0060] (3) High Performance Liquid Chromatography (HPLC): Samples were collected from 0 to 25 minutes. Pump A was 100% acetonitrile plus 0.1% trifluoroacetic acid, and pump B was 100% water plus 0.1% trifluoroacetic acid. The collected fractions were analyzed by HPLC to check purity.

[0061] The chromatogram showed that each antimicrobial peptide had a distinct absorption peak, and the purity of the purified peptides reached over 95%.

[0062] 3. Identification of antimicrobial peptides

[0063] The purified peptides were collected and the target peaks were identified by LC 6000 reverse phase preparative chromatography and Waters 2000 mass spectrometer, and the actual peptide molecular weight and purity were finally analyzed. Figure 1-Figure 7 shown.

[0064] Example 3 Determination of the secondary structure of antimicrobial peptides

[0065] 1. Determination of the secondary structure of antimicrobial peptides

[0066] The secondary structures of antimicrobial peptides JK, JR, JH, F-1, FK, FR and FH were determined by circular dichroism (CD).

[0067] The structures of the peptides were simulated in three different solution environments (aqueous, prokaryotic cell membrane, and hydrophobic biomembrane). The peptides were dissolved in 10 mM PBS buffer (pH 7.4), 30 mM sodium dodecyl sulfate (SDS), and 50% trifluoroethanol (TFE) to a final concentration of 150 μM. Circular dichroism spectrometer was used to scan the peptides at room temperature within the wavelength range of 190 nm to 250 nm at a scan rate of 1 nm / s.

[0068] The results are as follows Figures 8-14 As shown in Table 2.

[0069] Table 2 Secondary structure content of short peptides in different buffers

[0070]

[0071] In PBS buffer, all peptides except FH exhibited a random coil conformation, as evidenced by the appearance of a negative absorption peak at 198 nm. However, in SDS and TFE buffers, JK, JR, and FR exhibited a negative absorption peak at 195 nm and a positive absorption peak at 203 nm, representing a typical β-sheet conformation. This suggests that the alternating arrangement of hydrophilic and hydrophobic amino acids facilitates the formation of the peptide's β-sheet conformation in a simulated hydrophobic environment.

[0072] Example 4 Determination of antibacterial activity

[0073] Minimum Inhibitory Concentration (MIC) Determination: The antimicrobial activity of antimicrobial peptides JK, JR, JH, F-1, FK, FR, and FH was determined using the doubling microdilution method. Test strains were cultured to the logarithmic growth phase. Different concentrations of peptides and bacterial suspension were added to rows 1-11 of a 96-well plate and incubated at 37°C for 16 hours. Bacterial suspension and culture medium without peptides served as positive and negative controls, respectively. MIC values ​​for the peptides were expressed as the critical concentration at which no visible turbidity or precipitation occurred.

[0074] Determination of the minimum bactericidal concentration (MBC): After the MIC determination, 10 μL of sample was taken from the well where no turbidity was visible. The sample was plated with MHA medium and incubated at 37°C for 18 hours before colony count. The minimum peptide concentration that killed 99.9% of the bacteria was considered the minimum bactericidal concentration of the peptide. This experiment was repeated three times. The results are shown in Tables 3 and 4.

[0075] Table 3 MIC (MBC) values ​​of short peptides against Gram-negative bacteria (μM)

[0076]

[0077] Note: The data outside the brackets are MIC values, and the data inside the brackets are MBC values; the amino acid sequence of melittin is GIGAVLKVLTGLP ALISWIKRKRQQ (SEQ ID NO. 8).

[0078] Table 4 MIC (MBC) values ​​of short peptides against Gram-positive bacteria (μM)

[0079]

[0080] Note: The data outside the brackets are MIC values, and the data inside the brackets are MBC values.

[0081] All seven antimicrobial peptides exhibited broad-spectrum antibacterial activity. Their antibacterial activity against Escherichia coli ATCC 25922 was comparable to or equal to that of melittin, and they also exhibited significant antibacterial activity against Gram-positive bacteria (Staphylococcus albus CGMCC 1.3374 and Mycobacterium abscessus CGMCC 1.3384). This suggests that altering charge and hydrophobicity effectively enhances the antimicrobial activity of the antimicrobial peptides.

[0082] Example 5 Biocompatibility Analysis

[0083] 1. Determination of hemolytic activity

[0084] Healthy human blood was collected and centrifuged at 1000×g for 10 minutes to collect blood cells. The cells were washed three times with PBS and diluted to 1%. Equal volumes of blood cells were mixed with different concentrations of peptides in a 96-well plate, incubated at 37°C for 2 hours, and centrifuged (1000×g, 10 minutes). The supernatant was transferred to a new 96-well plate. The OD was measured using a microplate reader. 570nm The absorbance value under the following conditions was measured. Untreated blood cells were used as negative control, and blood cells treated with 0.1% Triton X-100 were used as positive control. The minimum peptide concentration that caused 10% hemolytic activity was defined as the minimum hemolytic concentration (MHC). Figure 15 It can be clearly seen that at the highest tested concentration (256 μM), the hemolytic activity of the seven antimicrobial peptides remained below 20%, while at 4 μM, the hemolytic rate of melittin reached 80%.

[0085] In order to accurately evaluate the antibacterial activity of the tested peptides, the geometric mean (GM) of the minimum inhibitory dose and the therapeutic index (TI) were calculated and shown in Table 5. Among them, the antimicrobial peptide FK showed the highest therapeutic index and had excellent cell selectivity.

[0086] Table 5 MHC, GM (μM) and TI values ​​of short peptides

[0087]

[0088] Note: a: The peptide concentration that causes 10% hemolysis is the minimum hemolytic concentration. When the hemolytic concentration is >128 μM, 256 μM is used for calculation; b: The geometric mean of the MIC value of the peptide. When it is >64 μM, 128 μM is used for calculation; c: TI (Therapeutic index) = MHC / GM. A larger therapeutic index (TI) indicates a stronger cell selectivity.

[0089] 2. Cytotoxicity

[0090] The cytotoxicity of antimicrobial peptides against mouse macrophages (RAW 264.7) was assessed using a CCK-8 assay. Cells were seeded into individual wells of a 96-well plate and exposed to varying concentrations of peptide solutions for 12 hours at 37°C in a 5% CO2 environment. After incubation, 10 μL of CCK-8 solution was added to each well. The cells were allowed to stand for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Positive controls included cells without peptide treatment, and negative controls included complete culture medium.

[0091] The results are as follows Figure 16 shown.

[0092] The experiments revealed that cell survival rates gradually decreased with increasing experimental concentrations. At a concentration of 4 μM, the cell survival rate of melittin was less than 20%. In contrast, the antimicrobial peptide designed in this invention achieved a cell survival rate of greater than 80% at the highest tested concentration of 64 μM. These results demonstrate that the antimicrobial peptide designed in this invention has good biocompatibility.

[0093] Example 6 Short Peptide Stability Analysis

[0094] The stability of antimicrobial peptides JK, JR, JH, F-1, FK, FR, and FH was evaluated using Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 as test strains. Untreated peptides were used as controls to determine changes in the MICs of the peptides in serum, salt solutions, and protease solutions. Final serum concentrations were 50%, 25%, and 12.5%, respectively. Salt concentrations were 150 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 6 mM NH4Cl, 8 mM ZnCl2, 4 mM FeCl3, and 2.5 mM CaCl2. The final concentration of trypsin, pepsin, papain, and chymotrypsin was 1 mg / mL.

[0095] The results are shown in Tables 6 to 11.

[0096] Table 6 MIC values ​​(μM) of antimicrobial peptides against E.coil ATCC 25922 at physiological salt concentrations

[0097]

[0098] Table 7 MIC values ​​(μM) of antimicrobial peptides against S. aureus ATCC 29213 at physiological salt concentrations

[0099]

[0100] Table 8 MIC values ​​(μM) of antimicrobial peptides against E.coil ATCC25922 in serum environment

[0101]

[0102] Note: GM is the geometric mean of MIC in different serum concentration environments.

[0103] Table 9 MIC values ​​(μM) of antimicrobial peptides against S. aureus ATCC 29213 in serum environment

[0104]

[0105]

[0106] Table 10 MIC values ​​(μM) of antimicrobial peptides against E.coil ATCC25922 in a protease environment

[0107]

[0108] Table 11 MIC values ​​(μM) of antimicrobial peptides against S. aureus ATCC 29213 in a protease environment

[0109]

[0110] Poor salt ion stability is one of the biggest obstacles to the clinical application of antimicrobial peptides. This study examined the changes in the minimum inhibitory activity of antimicrobial peptides against E. coli ATCC 25922 and S. aureus ATCC 29213 at different physiological salt concentrations and found that the antimicrobial peptides maintained substantial antibacterial activity against E. coli at physiological salt concentrations. Serum is a complex fluid containing anionic proteins and serum enzymes, and natural antimicrobial peptides exhibit poor stability in this environment, sometimes even losing their biological activity. The antimicrobial peptides designed in this study were found to maintain high biological activity, with either unaffected or reduced antimicrobial activity. Furthermore, the antimicrobial activity of the antimicrobial peptides decreased to varying degrees after treatment with different enzymes. With the exception of trypsin, the antimicrobial peptides maintained a certain degree of antimicrobial activity after treatment with papain and pepsin.

[0111] Example 7 Antibacterial Mechanism of Antimicrobial Peptides

[0112] 1. Live and dead cell staining analysis

[0113] Staphylococcus aureus was stained and analyzed using Calcein-AM / PI live / dead cell double staining reagent. Calcein-AM is a cell staining reagent that fluorescently labels live cells, emitting green fluorescence. Propidium iodide (PI) cannot cross the cell membrane of live cells, but can only pass through the disordered regions of the dead cell membrane to reach the cell nucleus, where it embeds into the cell's DNA double helix, producing red fluorescence. Since both Calcein and PI-DNA can be excited at 490 nm, live and dead cells can be observed simultaneously using a fluorescence microscope, allowing analysis at the live and dead cell levels.

[0114] The results are as follows Figure 17 After treatment with JR and FK, some Staphylococci were marked red by PI dye, while the E. coli in the control group all showed a green survival state, indicating that the antimicrobial peptides have a killing effect on Staphylococci, among which FK showed the strongest effect, with a higher number of bacterial deaths.

[0115] 2. Observation of cell membrane integrity

[0116] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the outer membrane morphology of the test strains E. coli ATCC25922 and S. aureus ATCC29213 to study the extent of damage to the bacterial cell outer membrane by the antimicrobial peptides. The experimental procedures are as follows:

[0117] (1) Bacterial preparation: The frozen bacteria were propagated to the second generation, cultured to the logarithmic growth phase, centrifuged to collect the bacterial sludge, rinsed three times with sterile PBS, and resuspended to OD 600nm =0.15.

[0118] (2) Antimicrobial peptide-bacteria reaction solution treatment: Antimicrobial peptides JK, JR, JH, F-1, FK, FR, and FH were added to the bacterial suspension to a final concentration of 1×MIC. The suspension was incubated on a shaker at 37°C for 1 h. The untreated bacterial suspension served as a blank control. After the reaction, the bacterial suspension was centrifuged and the supernatant discarded. The bacterial precipitate was collected and rinsed 3–4 times with PBS (pH 7.2).

[0119] (3) Fixation and dehydration: Add 2.5% glutaraldehyde (1 mL) to the bacterial pellet and fix it in a 4°C refrigerator for 12 h. After 12 h, collect the bacterial sludge and elute it with 50%, 70%, 90%, and 100% ethanol solutions in a gradient elution process of 10 min each. Mix equal volumes of 100% ethanol and tert-butanol solution, treat for 30 min, and then centrifuge (8000-10000 g, 5 min). Then treat with pure tert-butanol for 1 h and store at -20°C.

[0120] (4) Drying and coating: The sample is freeze-dried in a freeze dryer and then a layer of metal film is coated on the surface of the sample.

[0121] (5) Observation: Use a field-emission scanning electron microscope (TEM) to observe changes in the bacterial surface.

[0122] The results are as follows Figures 18-21 As shown in the figure. In the blank control group, Escherichia coli and Staphylococcus aureus were intact and plump, with smooth surfaces; the interiors of the cells were full and plump, with intact structures. When treated with the target peptide at 1×MIC, holes appeared on the surfaces of both negative and positive bacteria. As treatment time increased, the holes deepened, the wrinkles became uneven, E. coli showed signs of rupture, and Staphylococcus aureus spherically ruptured. Ultimately, both bacteria were no longer intact, with cells breaking and shattering. This suggests that the antimicrobial peptide penetrates the bacteria by rupturing the membrane, causing the contents to be destroyed and leak out of the bacteria, leading to their death.

[0123] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An antimicrobial peptide JK, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. An antimicrobial peptide JR, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. An antimicrobial peptide JH, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

4. An antimicrobial peptide F-1, characterized in that The amino acid sequence is shown in SEQ ID NO.

4.

5. An antimicrobial peptide FK, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

6. An antimicrobial peptide FR, characterized in that The amino acid sequence is shown in SEQ ID NO.

6.

7. An antimicrobial peptide FH, characterized in that: The amino acid sequence is shown in SEQ ID NO.

7.

8. Use of antimicrobial peptides in the preparation of antibacterial agents, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NOs. 1-7.

9. The use according to claim 8, characterized in that: The pathogens inhibited by the antibacterial agent include Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

10. An antibacterial agent, characterized in that The active ingredient comprises an antimicrobial peptide having an amino acid sequence as shown in any one of SEQ ID NO. 1-7.

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