Micromolecular antibacterial polypeptide, and preparation method therefor and use thereof

Abstract

The present disclosure relates to the technical field of antibacterial drugs, and specifically relates to a micromolecular antibacterial polypeptide, and a preparation method therefor and the use thereof. The antibacterial peptide provided in the present disclosure has a stronger antibacterial activity compared with most other derived polypeptides and the antibacterial polypeptides disclosed in the existing reports. Bactericidal activity evaluation is carried out by using a plurality of strains known to have drug resistance, and the results show that the antibacterial peptide provided in the present disclosure has broad-spectrum antibacterial activity against drug-resistant strains and more comprehensive antibacterial effects, and allows for wider indications.

Description

Small molecule antibacterial polypeptide, preparation method and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2023108801562 filed on July 18, 2023, and this application cites the full text of the above-mentioned Chinese patent application. Technical Field

[0003] The present disclosure relates to the technical field of antibacterial drugs, and in particular to small molecule antibacterial polypeptides, preparation methods and uses thereof. Background Art

[0004] In recent decades, the frequent and irregular use of antibiotics has led to bacterial resistance, a public health crisis. According to the latest research from the U.S. Centers for Disease Control and Prevention (CDC), antibiotic resistance causes millions of illnesses worldwide each year, and the death toll from antibiotic resistance is projected to reach tens of millions by 2050. Therefore, the development of new antimicrobial drugs has become a top priority for anti-infection treatment.

[0005] Antimicrobial peptides (AMPs) have demonstrated unprecedented advantages as promising antimicrobial agents against multidrug-resistant bacteria. Unlike antibiotics, which interfere with pathogenic microorganisms' metabolic processes, AMPs typically exert their antimicrobial effects by physically disrupting microbial cell membrane lipids and inducing leakage of cellular contents, resulting in minimal impact on the evolution of bacterial resistance. Due to their potent antimicrobial potential and unique mechanism of action, AMPs are currently considered ideal candidates to replace antibiotics.

[0006] Cathelicidin peptides from the Bungarus bungeus family are multifunctional antimicrobial peptides with broad-spectrum antimicrobial activity. They possess strong bactericidal activity against Gram-positive and Gram-negative bacteria, certain fungi, and viruses, and are also active against many clinically drug-resistant bacteria. BF30, an active peptide isolated from Bungarus bungeus venom by Lai Ren et al. at the Kunming Institute of Zoology, Chinese Academy of Sciences in 2008, is a 30-amino acid single peptide drug synthesized artificially. It is encoded by the Bungarus cathelicidin gene and is a linear peptide with an N-terminal α-helix, containing 30 amino acid residues and a molecular weight of 3636.24 Da.

[0007] Given that the antimicrobial peptide from Bungarus serratus has high activity, is not prone to drug resistance, and has a broad spectrum of effects, it has become a very good lead molecule for screening new anti-infective drugs. However, the sequence of the antimicrobial peptide from Bungarus serratus BF30 is relatively complex, the synthesis cost is high, and its antimicrobial activity still needs to be improved.

[0008] Summary of the Invention

[0009] The present invention aims to improve the activity of Bungarus fasciatus antimicrobial peptides and reduce the synthesis cost of Bungarus fasciatus antimicrobial peptides. The amino acid sequence and structure of Bungarus fasciatus antimicrobial peptides are modified and optimized to obtain mutant antimicrobial peptides with broad spectrum and high activity. The preparation method and use of the mutant antimicrobial peptides are also provided.

[0010] In a first aspect, the present disclosure provides an antimicrobial peptide having an amino acid sequence of X1KRFKKFX2X3KLKKWV, wherein X1 is selected from non-polar side chain amino acids, X2 is selected from amino acids with aromatic side chains, and X3 is selected from amino acids with aromatic side chains or amino acids with basic side chains.

[0011] In an optional embodiment, the X1 is selected from valine or isoleucine, the X2 is selected from phenylalanine or tryptophan, and the X3 is selected from phenylalanine, tryptophan or arginine.

[0012] In an optional embodiment, the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NOs. 1 to 4.

[0013] In an optional embodiment, the N-terminus of the antimicrobial peptide contains a modified functional group, and the modified functional group is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanoyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.

[0014] In an optional embodiment, the modified antimicrobial peptide contains at least one D-type or β-type amino acid.

[0015] In a second aspect, the present disclosure provides a method for preparing the antimicrobial peptide described in any of the aforementioned embodiments, wherein amino acids with side chain protecting groups are coupled sequentially according to the amino acid sequence using a solid-phase synthesis or liquid-phase synthesis method, and then the side chain protecting groups are removed, extracted, and purified to obtain the antimicrobial peptide.

[0016] In an optional embodiment, the method for preparing the antimicrobial peptide further comprises the step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptide, and the functional group used for the N-terminal modification is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanoyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, p-toluenesulfonyl or 2-methylthiazole-5-carbonyl.

[0017] In an alternative embodiment, at least one of the amino acids used in the solid phase synthesis is a D- or β-amino acid.

[0018] In a third aspect, the present disclosure provides the use of the antimicrobial peptide described in any of the preceding embodiments or the antimicrobial peptide obtained by the preparation method described in any of the preceding embodiments in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, preservatives, animal feed or cosmetic precursors.

[0019] In a fourth aspect, the present disclosure provides an antimicrobial pharmaceutical preparation, comprising the antimicrobial peptide according to any one of the aforementioned embodiments or the antimicrobial peptide prepared by the preparation method according to any one of the aforementioned embodiments.

[0020] In an optional embodiment, the effective dose of the antimicrobial peptide is 0.01 to 512 μg / ml, preferably 0.125 to 256 μg / ml.

[0021] In an optional embodiment, the pathogenic microorganisms having the antibacterial effect include bacteria and / or fungi.

[0022] Preferably, the bacteria include Gram-positive bacteria, such as Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.

[0023] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0024] In an optional embodiment, the dosage form of the antibacterial drug preparation includes an oral preparation, an external preparation or an injection; the oral preparation includes granules, tablets, pastes or oral solutions; the external preparation includes ointments, gels, suppositories, medicated baths, hoof baths or sprays.

[0025] In a fifth aspect, the present disclosure provides a method for treating a condition associated with infection by pathogenic microorganisms, the method comprising administering the aforementioned antimicrobial peptide or antimicrobial drug preparation to a subject;

[0026] In an alternative embodiment, the pathogenic microorganisms include bacteria and / or fungi.

[0027] In alternative embodiments, the bacteria include Gram-positive bacteria including Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0028] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0029] In a sixth aspect, the present disclosure provides an antimicrobial peptide or an antimicrobial drug preparation for treating diseases associated with infection by pathogenic microorganisms. The antimicrobial peptide is as described in the aforementioned aspects or obtained by the aforementioned preparation method, and the antimicrobial drug preparation is as described in the aforementioned aspects.

[0030] In an alternative embodiment, the pathogenic microorganisms include bacteria and / or fungi.

[0031] In alternative embodiments, the bacteria include Gram-positive bacteria including Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0032] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0033] The beneficial effects achieved by the present disclosure include:

[0034] The antimicrobial peptides provided in this disclosure exhibit stronger antibacterial activity than most other derivative peptides and those reported in existing studies. Bactericidal activity was evaluated against a variety of strains known to be drug-resistant. The results showed that the antimicrobial peptides provided in this disclosure exhibit broad-spectrum antibacterial activity against drug-resistant strains, demonstrating a more comprehensive antibacterial effect and a wider range of applications.

[0035] Furthermore, the safety of the antimicrobial peptides provided by the present disclosure was evaluated using hemolytic activity. It was determined that the preferred antimicrobial peptides of the present disclosure showed no hemolytic toxicity at the highest concentration of 256 μg / ml, indicating high safety.

[0036] Therefore, the present disclosure provides an antimicrobial peptide with stronger antimicrobial activity, wider scope of application, and greater potential development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG1 is a high performance liquid chromatogram of the antimicrobial peptide WZ-2 synthesized in Example 1;

[0038] FIG2 is a mass spectrum of the antimicrobial peptide WZ-2 synthesized in Example 1;

[0039] FIG3 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2;

[0040] FIG4 is a mass spectrum of the antimicrobial peptide Ac-WZ-2 synthesized in Example 2;

[0041] FIG5 is a high performance liquid chromatogram of the antimicrobial peptide WZ-4 synthesized in Example 3;

[0042] FIG6 is a mass spectrum of the antimicrobial peptide WZ-4 synthesized in Example 3;

[0043] FIG7 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3;

[0044] FIG8 is a mass spectrum of the antimicrobial peptide Ac-WZ-4 synthesized in Example 3;

[0045] Figure 9 is a high performance liquid chromatogram of the antimicrobial peptide WZ-16 synthesized in Example 4;

[0046] Figure 10 is a mass spectrum of the antimicrobial peptide WZ-16 synthesized in Example 4;

[0047] Figure 11 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4;

[0048] Figure 12 is a mass spectrum of the antimicrobial peptide Ac-WZ-16 synthesized in Example 4;

[0049] Figure 13 is a high performance liquid chromatogram of the antimicrobial peptide WZ-21 synthesized in Example 5;

[0050] Figure 14 is a mass spectrum of the antimicrobial peptide WZ-21 synthesized in Example 5;

[0051] Figure 15 is a high performance liquid chromatogram of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5;

[0052] Figure 16 is a mass spectrum of the antimicrobial peptide Ac-WZ-21 synthesized in Example 5;

[0053] FIG17 is a graph showing the hemolytic activity experimental results of different antimicrobial peptides provided by the present disclosure;

[0054] FIG18 is a graph showing the hemolytic activity experimental results of different antimicrobial peptides modified with acetyl groups provided by the present disclosure. DETAILED DESCRIPTION

[0055] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only one embodiment of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0056] Throughout this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of this application, definitions and explanations of relevant terms are provided below.

[0057] It will also be understood that in certain methods described herein that include more than one step or action, the order of the steps or actions of the method are not necessarily limited to the order in which the steps or actions of the method are listed unless the context dictates otherwise.

[0058] definition

[0059] As used herein, the terms "a," "an," "the," and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0060] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0061] As used herein, the conjunction term "and / or" between multiple described elements is understood to include both individual options and combined options. For example, when two elements are linked together by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The concurrent applicability of multiple options is also understood to be within the meaning of the term and therefore meets the requirements of the term "and / or."

[0062] As used herein, the term "antimicrobial peptide," which may also be referred to as "antimicrobial polypeptide," is synonymous with "antimicrobial protein" and "antimicrobial protein" and refers to a polymer of amino acid residues that possesses antimicrobial, bacteriostatic, or bactericidal function. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0063] As used herein, the term "drug resistance", also known as drug resistance, refers to the tolerance of microorganisms, parasites and tumor cells to the effects of chemotherapy drugs. Once drug resistance occurs, the chemotherapy effect of the drug is significantly reduced. Drug resistance can be divided into acquired resistance and natural resistance according to the cause of its occurrence. Pathogens in nature, such as a certain strain of bacteria, can also have natural drug resistance. When antibiotics are used for a long time, the majority of sensitive strains are continuously killed, and the resistant strains multiply in large numbers, replacing the sensitive strains, and the resistance rate of bacteria to this drug continues to increase. It is currently believed that the latter method is the main reason for the generation of drug-resistant bacteria. In order to maintain the effectiveness of antibiotics, their rational use should be emphasized.

[0064] As used herein, the term "MRSA" refers to methicillin-resistant Staphylococcus aureus, a generally resistant strain. It exhibits broad-spectrum resistance to β-lactams and cephalosporins, and to varying degrees of resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, and rifampicin (WHO's list of priority pathogens for new antibiotic development: highly important).

[0065] As used herein, the term "MSSA" is methicillin-sensitive Staphylococcus aureus.

[0066] As used herein, the term "D-amino acid" refers to two isomers of the same amino acid with different optical rotations, as opposed to L-amino acids. According to the Fischer projection, L-amino acids have the amino group on the left, while D-amino acids have the amino group on the right. Natural amino acids are generally L-amino acids, while D-amino acids must be synthesized.

[0067] As used herein, the term "β-amino acid" refers to an amino acid in which the amino group is attached to the β-carbon atom. The only common naturally occurring β-amino acid is β-alanine. While β-alanine is often a component of biologically active macromolecules, β-peptides do not generally occur in nature. For this reason, β-peptide antibiotics are being used to address the problem of antibiotic resistance.

[0068] As used herein, the term "conservative substitutions" or "conservative sequence modifications" of a sequence refers to nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-MASP-2 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abrogate antigen binding are well known in the art.

[0069] Exemplary amino acids that can be conservatively substituted are shown in Table 1 below:

[0070] Table 1 Examples of conservative amino acid substitutions

[0071] As used herein, the term "effective dose" is synonymous with "effective amount" and refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0072] Detailed description of specific implementation plan

[0073] In a first aspect, the present disclosure provides an antimicrobial peptide having an amino acid sequence X1KRFKKFX2X3KLKKWV, wherein X1 is selected from non-polar side chain amino acids, X2 is selected from amino acids with aromatic side chains, and X3 is selected from amino acids with aromatic side chains or amino acids with basic side chains.

[0074] In an optional embodiment, the X1 is selected from valine or isoleucine, the X2 is selected from phenylalanine or tryptophan, and the X3 is selected from phenylalanine, tryptophan or arginine.

[0075] In an optional embodiment, the antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NOs. 1 to 4.

[0076] SEQ ID NO.1: VKRFKKFFWKLKKWV;

[0077] SEQ ID NO.2: VKRFKKFWFKLKKWV;

[0078] SEQ ID NO.3: IKRFKKFFRKLKKWV;

[0079] SEQ ID NO.4: IKRFKKFFWKLKKWV.

[0080] SEQ ID NO. 2 is obtained by conservative substitutions at positions 8 and 9 of SEQ ID NO. 1, SEQ ID NO. 4 is obtained by conservative substitutions at position 1 of SEQ ID NO. 1, and SEQ ID NO. 3 is obtained by non-conservative substitutions at position 9 of SEQ ID NO. 4. The core amino acid sequence of the antimicrobial peptide provided herein comprises 15 amino acid residues. With 5 amino acid residues as a fragment, the core fragment of the antimicrobial peptide provided herein is divided equally into three fragments, namely, front, middle, and back, from the N-terminus to the C-terminus. It can be seen that the front fragment is a tryptophan-free polypeptide fragment.

[0081] In an optional embodiment, the N-terminus of the antimicrobial peptide contains a modified functional group, and the modified functional group is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanoyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.

[0082] The above-mentioned modified functional group is a modified functional group additionally added to the N-terminus of the antimicrobial peptide for the purpose of improving the antibacterial activity of the antimicrobial peptide. It is understandable that other conventional modified groups that can be used to improve the activity of the antimicrobial peptide should also be understood to be within the scope of protection of the present disclosure.

[0083] In an optional embodiment, the modified antimicrobial peptide contains at least one D-amino acid or β-amino acid.

[0084] In a second aspect, the present disclosure provides a method for preparing the antimicrobial peptide described in any of the aforementioned embodiments, wherein amino acids with side chain protecting groups are coupled sequentially according to the amino acid sequence using a solid-phase synthesis or liquid-phase synthesis method, and then the side chain protecting groups are removed, extracted, and purified to obtain the antimicrobial peptide.

[0085] It is understandable that peptide synthesis is a process of repeated addition of amino acids. The order of solid-phase synthesis is generally from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus), which can greatly reduce the difficulty of product purification at each step. In order to prevent the occurrence of side reactions, the side chains of the amino acids participating in the reaction are protected, the carboxyl terminus is free, and must be activated before the reaction. There are two solid-phase synthesis methods, namely Fmoc and tBoc. Liquid-phase synthesis mainly has two strategies: stepwise synthesis and fragment combination. Stepwise synthesis is simple and rapid, and is used to synthesize various biologically active peptide fragments; the fragment combination method provides the most promising route for the synthesis of peptides containing more than 100 amino acids, and has successfully synthesized a variety of biologically active peptides. Its biggest feature is that it is easy to purify.

[0086] In an optional embodiment, the method for preparing the antimicrobial peptide further comprises the step of modifying the N-terminus of the solid-phase synthesized antimicrobial peptide, wherein the functional group used for the N-terminal modification is selected from 5-isoxazole-5-carbonyl (Ez), acetyl (Ac), 2-(1-imidazolyl)acetyl (Mz), benzoyl (Bz), decacarbonyl (Dec), 2-morpholinoacetyl (Ml), pyrazinecarbonyl (Pz), dodecanoyl (Dodec), toluenesulfonyl (Ts) or 2-methylthiazole-5-carbonyl (Sz), and the structural formula of each functional group is as follows:

[0087] In an alternative embodiment, at least one of the amino acids used in the solid phase synthesis is a D-amino acid or a β-amino acid.

[0088] In a third aspect, the present disclosure provides the use of the antimicrobial peptide described in any of the preceding embodiments or the antimicrobial peptide obtained by the preparation method described in any of the preceding embodiments in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, preservatives, animal feed or cosmetic precursors.

[0089] In a fourth aspect, the present disclosure provides an antimicrobial pharmaceutical preparation, comprising the antimicrobial peptide according to any one of the aforementioned embodiments or the antimicrobial peptide prepared by the preparation method according to any one of the aforementioned embodiments.

[0090] In an optional embodiment, the effective dose of the antimicrobial peptide is 0.01 to 512 μg / ml, preferably 0.125 to 256 μg / ml.

[0091] In an optional embodiment, the pathogenic microorganisms having the antibacterial effect include bacteria and / or fungi.

[0092] In alternative embodiments, the bacteria include Gram-positive bacteria, such as Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0093] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0094] In an optional embodiment, the dosage form of the antibacterial drug preparation includes an oral preparation, an external preparation or an injection; the oral preparation includes granules, tablets, pastes or oral solutions; the external preparation includes ointments, gels, suppositories, medicated baths, hoof baths or sprays.

[0095] In a fifth aspect, the present disclosure provides a method for treating a condition associated with infection by pathogenic microorganisms, the method comprising administering the aforementioned antimicrobial peptide or antimicrobial drug preparation to a subject;

[0096] In an alternative embodiment, the pathogenic microorganisms include bacteria and / or fungi.

[0097] In alternative embodiments, the bacteria include Gram-positive bacteria including Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0098] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0099] In a sixth aspect, the present disclosure provides an antimicrobial peptide or an antimicrobial drug preparation for treating diseases associated with infection by pathogenic microorganisms. The antimicrobial peptide is as described in the aforementioned aspects or obtained by the aforementioned preparation method, and the antimicrobial drug preparation is as described in the aforementioned aspects.

[0100] In an alternative embodiment, the pathogenic microorganisms include bacteria and / or fungi.

[0101] In alternative embodiments, the bacteria include Gram-positive bacteria including Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii.

[0102] In an optional embodiment, the pathogenic microorganism is drug-resistant, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, or rifampicin.

[0103] The embodiments of the present disclosure will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0104] In the present disclosure, the detection method can be a conventional method for detecting antimicrobial polypeptides and antimicrobial proteins in the art, including but not limited to MIC determination, MBC determination, yield detection, purity detection and hemolytic activity evaluation.

[0105] Exemplary:

[0106] The MIC determination method is as follows:

[0107] (1) Preparation of CAMHB culture medium: Weigh 22.5 g of CAMHB culture medium powder (Shenzhen Haibo Biotechnology Co., Ltd., product number: HB6231-1), add 1000 mL of distilled water, and sterilize for use; CAMHB culture medium contains beef extract powder and acid hydrolyzed casein, which can provide the nitrogen source, vitamins and other growth factors required for bacterial growth; the soluble starch contained in it provides a carbon source and energy for bacteria; the calcium chloride contained in it can adjust the pH and is also an activator of certain enzymes. It is often used for rapid bacterial growth and cultivation of aerobic microorganisms in clinical samples or other samples. The present invention is used for antimicrobial peptide MIC determination. As an alternative culture medium, MHB culture medium or LB culture medium can also be used.

[0108] (2) Sterilization of instruments: Place the prepared CAMHB medium, 0.9% (w / v) NaCl, pipette tips, and centrifuge tubes in an autoclave at 121°C for 30 minutes. Place the pipette tips, centrifuge tubes, and test tubes in a drying oven and dry them for 24 hours. The 96-well plate is sterilized by UV light on a clean bench for 30 minutes.

[0109] (3) Preparation of antibacterial peptide stock solution: Peptide samples were prepared into 10 mg / ml stock solution with sterilized 0.9% (w / v) NaCl. A maximum concentration of 512 μg / ml was prepared using CAMHB medium, and then diluted to 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml according to the system.

[0110] (4) Preparation of positive control drugs: Daptomycin (Shanghai Yuanye Biotechnology Co., Ltd., B27420), BF-30 (Gill Biochemical (Shanghai) Co., Ltd., purity>99%), and ZY13 (Gill Biochemical (Shanghai) Co., Ltd., purity>99%) were prepared as stock solutions with sterilized 0.9% (w / v) NaCl, and prepared to a maximum concentration of 512 μg / ml using CAMHB medium, and diluted to the corresponding drug concentrations of 256 μg / ml, 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml.

[0111] (5) Preparation of bacterial suspension: Gram-negative bacteria were shaken in advance with LB (Thermo Fisher Scientific, Hangzhou, 1278005) medium (lysate broth medium), and Gram-positive bacteria were shaken with TSB (Thermo Fisher Scientific, Hangzhou, CM0129) medium (tryptone soy broth medium) at 220 rpm and 37°C overnight. The suspension was diluted 1:1000 before the experiment (the final concentration of the system was 1:2000).

[0112] (6) Specific operation steps

[0113] Step 1: Preparation and reagent preparation

[0114] Wipe the work surface with alcohol and prepare pre-sterilized pipette tips, pipettes, EP tubing, culture dishes, racks, 96-well plates with lids, drugs, CAMHB medium, 0.9% (w / v) NaCl, and bacterial suspension. (For example, 8 mg of drug should be dissolved in 800 μl of 0.9% (w / v) NaCl to prepare a 10 mg / ml stock solution. Add 25.6 μl of the stock solution to 474.4 μl of CAMHB medium for a maximum treatment concentration of 512 μg / ml.)

[0115] Step 2: Two-fold dilution method

[0116] Taking A1 to 12 of a 96-well plate as an example, add 200 μl of 512 μg / ml test sample (control antibiotic drug, different antimicrobial peptide samples provided by the present invention, etc.) to A12, and add 100 μl of CAMHB culture medium to A1 to 11 respectively. Take 100 μl of A12 solution, add A11 and pipette to mix, aspirate 100 μl and add A10 and pipette to mix, and continue this operation until A2. After A2 is pipetted and mixed, aspirate 100 μl and discard. Ensure that A1 is a blank control and no drugs are added. Then add 100 μl of bacterial solution with a gun. Place in a bacterial incubator at 37°C and 62% humidity and culture for 24 hours.

[0117] Step 3: Observation and judgment

[0118] After 24 hours, remove the 96-well plate. Under a light source, hold the plate steady above your head and observe the bacterial growth in each well with the naked eye. The test is meaningful only when there is obvious bacterial growth in the negative control well (i.e., without the test sample). The clear CAMHB state with no colonies is the MIC for that group (Note: OD value can also be used for judgment). When a single jump well occurs in the microculture dilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump wells occur, the results should not be reported and the test should be repeated.

[0119] The MBC determination method is as follows:

[0120] (1) Preparation of TSB agar medium: Weigh 30 g of TSB medium powder and 15 g of agar, mix them, add 1000 mL of distilled water, and sterilize them for use.

[0121] (2) Preparation of TSB agar plates: Cool the sterilized TSB medium to about 45°C (about 15-20 mL) and pour it into a culture dish. Cover the dish with a lid and shake it gently. After the plate cools and solidifies (about 5-10 minutes), turn the plate upside down with the lid at the bottom and the bottom at the top.

[0122] (3) Pipette 100 μl of the bacterial solution at the MIC and then at 2 times, 4 times, and up to the maximum concentration onto a pre-marked TSB agar plate. Cover the plate and gently shake the plate back and forth until the bacterial solution covers the entire plate. Place the plate in a bacterial incubator for overnight culture. The next day, observe the number of colonies growing on each plate. The lowest drug concentration in the corresponding culture medium tube is the minimum bactericidal concentration (MBC) of the drug if the number of colonies growing on the plate is less than 0.1% of the inoculum volume.

[0123] The hemolytic activity evaluation method is as follows:

[0124] Blood from healthy rabbits (cynomolgus monkeys or humans may also be used) is collected and placed in an Erlenmeyer flask containing glass beads and shaken for 10 minutes, or stirred with a glass rod to remove fibrinogen and render the blood defibrillated. Approximately 10 times the volume of 0.9% (w / v) sodium chloride solution is added, shaken thoroughly, and centrifuged at 1000-1500 rpm for 15 minutes. The supernatant is removed, and the precipitated red blood cells are washed two to three times with 0.9% (w / v) sodium chloride solution as described above, until the supernatant no longer appears red. A 2% (w / v) suspension of the resulting red blood cells is prepared in 0.9% (w / v) sodium chloride solution for testing. 100 μL of the suspension is added to a 96-well round-bottom polystyrene microplate. 100 μL of the sample to be tested is added to each well at a starting concentration of 256 μg / mL and serially diluted two-fold. 1% (w / v) triton is used as a positive control, and DMSO is used as a negative control. The mixture was then incubated at 37°C on a shaker at 60 rpm for 1 hour. After incubation, the mixture was centrifuged at 1000 × g for 3 minutes, and 100 μL of the supernatant per well was transferred to a new 96-well plate. If the solution in the tube is clear red with no cells or a small amount of red blood cells remaining at the bottom of the tube, hemolysis has occurred. If all red blood cells sink and the supernatant is colorless and clear, or if the supernatant is clear and has color, hemolysis has not occurred. Alternatively, absorbance at 450 nm can be measured for hemolysis.

[0125] The strains used in this disclosure are as follows:

[0126] Conventional strains: Escherichia coli (E. coli) ATCC25922, Enterococcus faecalis (E. faecalis) ATCC29212 standard strains are purchased from ATCC official. Staphylococcus aureus (S. aureus) SA113, Staphylococcus aureus (S. aureus) CHS101, Staphylococcus aureus (S. aureus) YUSA139, Staphylococcus aureus (S. aureus) YUSA145, Enterococcus faecalis (E. faecalis) 16C166, Pseudomonas aeruginosa (P. aeruginosa) PA2237, Pseudomonas aeruginosa (P. aeruginosa) ATCC27 853, Escherichia coli (E.coli) Eco2242, Escherichia coli (E.coli) ATCC25922, Klebsiella pneumoniae (K.pneumoniae) K2044, Acinetobacter baumannii (A.baumannii) Ab2201, and Acinetobacter baumannii (A.baumannii) Ab2202 are all preserved in the Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, with the preservation address being No. 89, Taoyuan Road, Nanshan District, Shenzhen. The preservation number is the specific strain number.

[0127] Drug-resistant strains: Staphylococcus aureus (S.aureus) YUSA132 and Staphylococcus aureus (S.aureus) YUSA139 are both preserved in the Department of Infectious Diseases, Shenzhen Nanshan People's Hospital, with the preservation address being No. 89, Taoyuan Road, Nanshan District, Shenzhen. The preservation number is the specific strain number.

[0128] Example 1: Synthesis of antimicrobial peptide VKRFKKFFWKLKKWV (hereinafter referred to as WZ-2 SEQ ID NO. 1)

[0129] (1) Peptide synthesis

[0130] On Rink Amide MBHA resin (Gill Biochemical (Shanghai) Co., Ltd., 49006), according to the core peptide sequence of antimicrobial peptide WZ-2, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, FmocArg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Val-OH were coupled in sequence. After the synthesis was completed, the mixture was washed with DMF, DCM, and methanol in sequence and dried to obtain the peptide resin.

[0131] (2) Lysis and peptide purification

[0132] The peptide resin obtained in step (1) was cleaved using a cleavage solution containing TFA (trifluoroacetic acid, CAS#: 76-05-1, supplier: Shanghai MacLean Biochemical Technology Co., Ltd.), TIS (triisopropylsilane, CAS#: 6485-79-6, supplier: Shanghai MacLean Biochemical Technology Co., Ltd.), EDT (2,2′-(1,2-ethylenedioxy)bis(ethanediol, CAS#: 14970-87-7, supplier: Shanghai MacLean Biochemical Technology Co., Ltd.) and H2O in a volume ratio of 91:3:3:3. The resin was then filtered off, the resin was washed with a small amount of TFA, the filtrate was combined, and added to anhydrous ether to precipitate a white solid. The solid was centrifuged, washed with anhydrous ether, and vacuum dried to obtain a crude WZ-2 peptide. The crude peptide was purified by HPLC (purification conditions are as follows) to obtain the refined WZ-2 peptide. The MS detection conditions are as follows. The detection results are shown in Figure 2. The antimicrobial peptide WZ-2 was successfully synthesized.

[0133] HPLC conditions:

[0134] MS conditions:

[0135] Example 2: Synthesis of N-terminal acetyl-modified antimicrobial peptide VKRFKKFFWKLKKWV (hereinafter referred to as Ac-WZ-2)

[0136] On Rink Amide MBHA resin (Gill Biochemical (Shanghai) Co., Ltd., 49006), according to the core peptide sequence of antimicrobial peptide WZ-2, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, After the synthesis of Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Val-OH, acetic anhydride and pyridine were added for acetylation modification, followed by washing with DMF, DCM, and methanol in sequence and drying to obtain a peptide resin. The purification and detection steps were the same as in Example 1. The HPLC and MS detection results are shown in Figures 3 and 4, respectively. The acetyl-modified antimicrobial peptide Ac-WZ-2 was successfully synthesized with a purity of 98.3%.

[0137] Example 3: Synthesis of antimicrobial peptides WZ-4 and Ac-WZ-4 (SEQ ID NO. 2)

[0138] With reference to Examples 1 and 2, respectively, an antimicrobial peptide VKRFKKFWFKLKKWV was synthesized, designated WZ-4, and an N-terminally acetyl-modified antimicrobial peptide VKRFKKFWFKLKKWV, designated Ac-WZ-4. The HPLC and MS analysis results for WZ-4 are shown in Figures 5 and 6, respectively. The HPLC and MS analysis results for Ac-WZ-4 are shown in Figures 7 and 8, respectively. The purity of the prepared WZ-4 was 98.4%, and the purity of Ac-WZ-4 was 98.3%.

[0139] Example 4: Synthesis of antimicrobial peptides WZ-16 and Ac-WZ-16 (SEQ ID NO. 3)

[0140] With reference to Examples 1 and 2, respectively, an antimicrobial peptide IKRFKKFFRKLKKWV was synthesized, designated WZ-16, and an N-terminally acetyl-modified antimicrobial peptide IKRFKKFFRKLKKWV, designated Ac-WZ-16. The HPLC and MS analysis results for WZ-16 are shown in Figures 9 and 10, respectively. The HPLC and MS analysis results for Ac-WZ-16 are shown in Figures 11 and 12, respectively. The purity of the prepared WZ-16 was 98.7%, and the purity of Ac-WZ-16 was 98.4%.

[0141] Example 5: Synthesis of antimicrobial peptides WZ-21 and Ac-WZ-21 (SEQ ID NO.4)

[0142] With reference to Examples 1 and 2, respectively, an antimicrobial peptide IKRFKKFFWKLKKWV was synthesized, designated WZ-21, and an N-terminally acetyl-modified antimicrobial peptide IKRFKKFFWKLKKWV, designated Ac-WZ-21. The HPLC and MS analysis results for WZ-21 are shown in Figures 13 and 14, respectively. The HPLC and MS analysis results for Ac-WZ-21 are shown in Figures 15 and 16, respectively. The purity of the prepared WZ-21 was 98.7%, and the purity of Ac-WZ-21 was 98.8%.

[0143] Example 6: Comparison of antibacterial activity of different antimicrobial peptides

[0144] This example compares the antimicrobial properties of the antimicrobial peptide WZ-2 and the antimicrobial peptide ZY-13 disclosed in patent CN103275190A, demonstrating that the antimicrobial peptide provided in Example 1 has superior antimicrobial activity.

[0145] Table 2 Comparison of antibacterial properties of WZ-2 and ZY13 against different pathogens

[0146] Table 3 Comparison of the antibacterial effects of WZ-2 and ZY-13 on methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus

[0147] Table 4 Comparison of the antibacterial effects of WZ-2 and ZY-13 on methicillin-resistant Acinetobacter baumannii and methicillin-sensitive Acinetobacter baumannii

[0148] Table 5 Comparison of the antibacterial effects of WZ-2 and ZY-13 on methicillin-resistant Klebsiella pneumoniae and methicillin-sensitive Klebsiella pneumoniae

[0149] Table 6 Comparison of the antibacterial effects of WZ-2 and ZY-13 on methicillin-resistant Escherichia coli and methicillin-sensitive Escherichia coli

[0150] Table 7 Comparison of the antibacterial effects of WZ-2 and ZY-13 on methicillin-resistant Pseudomonas aeruginosa and methicillin-sensitive Pseudomonas aeruginosa

[0151] As can be seen from Tables 2 to 7 above, WZ2's antibacterial and bactericidal activities against a variety of standard pathogenic bacteria are comparable to or superior to those of ZY13. Furthermore, based on the systematic antibacterial activity data against Staphylococcus aureus, WZ2 outperformed the positive drug ZY13 (Table 3), both against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus. Regarding the activity against a variety of other clinically derived strains, the antimicrobial peptide WZ2 exhibited overall superior antibacterial activity to ZY13 against Acinetobacter baumannii (Table 4), Klebsiella pneumoniae (Table 5), Escherichia coli (Table 6), and Pseudomonas aeruginosa (Table 7).

[0152] Example 7

[0153] In this example, with reference to the synthesis method of Example 1, a variety of antimicrobial peptides with different amino acid sequences were synthesized, and the physicochemical parameters and antibacterial activity of these antimicrobial peptides were compared with those of the antimicrobial peptides obtained in Examples 1, 3 to 5. The antimicrobial properties were compared with those of the antimicrobial peptide ZY-13 disclosed in CN103275190A, the antimicrobial peptide ZY-4 disclosed in patent WO2016201972A1, BF-30 (IND obtained in 2018 for the treatment of bacterial vaginosis, effervescent tablets), Ampicillin, Vancomycin and Gentamicin, demonstrating that the antimicrobial peptides provided in Examples 1, 3 to 5 have superior antibacterial activity.

[0154] Table 8 Comparison of amino acid sequences and physicochemical parameters of different antimicrobial peptides

[0155] Table 9 Comparison of antibacterial activity of different antimicrobial peptides

[0156] It can be seen from Tables 8 and 9 above that the antimicrobial peptides provided in Examples 1 and 3 to 5 have superior antimicrobial activity compared to other antimicrobial peptides with similar amino acid sequences, the three reported antimicrobial peptides and the three antibiotics.

[0157] Example 8

[0158] In this example, the amino acid sequences of various antimicrobial peptides synthesized in Example 7 were referenced. In combination with the synthesis method of Example 2, acetyl groups were introduced into the N-termini of these antimicrobial peptides for modification. The bactericidal activity of these peptides was then compared with that of two clinical candidate drugs, PL-5 (CAS#: 850761-47-6, currently in clinical phase II-III, for diabetic foot ulcers, spray) and PL-18 (patent CN102219831B, for the treatment of bacterial / fungal vaginitis, suppository). This demonstrated that the N-terminally modified antimicrobial peptides provided by the present disclosure have superior antimicrobial activity (as shown in Table 10).

[0159] Table 10 Comparison of antibacterial activity of different antimicrobial peptides modified with acetyl groups

[0160] In addition, in this example, the N-terminal modified acetyl group was replaced with other modified functional groups, or all D-amino acids were used to synthesize antimicrobial peptides. The resulting antimicrobial peptides all had antibacterial activities comparable to or better than those with acetyl group (as shown in Table 11).

[0161] Table 11 Comparison of antibacterial activity of antimicrobial peptides modified with different functional groups

[0162] Example 9: Hemolytic activity evaluation

[0163] According to the above-mentioned hemolytic activity evaluation method, the antimicrobial peptides WZ-2, WZ-4, WZ-16, WZ-21, Ac-WZ-2, AC-WZ-4, Ac-WZ-16, Ac-WZ-21, ZY-13 and BF-30 obtained in the above examples were subjected to hemolytic activity experiments. The results are shown in Figures 17 and 18. It can be seen that the antimicrobial peptides WZ-2, WZ-4, WZ-16, WZ-21, Ac-WZ-2, AC-WZ-4, Ac-WZ-16 and Ac-WZ-21 provided by the present disclosure showed no hemolytic toxicity at the highest concentration of 256 μg / ml, demonstrating that the antimicrobial peptides provided in the above examples are safe and effective.

[0164] Although the specific embodiments of the present disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present disclosure.

Claims

1. An antimicrobial peptide, characterized in that The antimicrobial peptide has an amino acid sequence of X1KRFKKFX2X3KLKKWV, wherein X1 is selected from non-polar side chain amino acids, X2 is selected from amino acids with aromatic side chains, and X3 is selected from amino acids with aromatic side chains or amino acids with basic side chains.

2. The antimicrobial peptide according to claim 1, characterized in that The X1 is selected from valine or isoleucine, the X2 is selected from phenylalanine or tryptophan, and the X3 is selected from phenylalanine, tryptophan or arginine.

3. The antimicrobial peptide according to claim 1 or 2, characterized in that The antimicrobial peptide has an amino acid sequence shown in any one of SEQ ID NOs. 1 to 4.

4. The antimicrobial peptide according to claim 3, characterized in that The N-terminus of the antimicrobial peptide contains a modified functional group, which is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.

5. The antimicrobial peptide according to claim 4, characterized in that The modified antimicrobial peptide contains at least one D-type or β-type amino acid.

6. The method for preparing the antimicrobial peptide according to any one of claims 1 to 3, characterized in that: The amino acids with side chain protecting groups are coupled in sequence according to the amino acid sequence by solid phase synthesis or liquid phase synthesis, and then the side chain protecting groups are removed, extracted and purified in sequence to obtain the antimicrobial peptide.

7. The preparation method according to claim 6, characterized in that: The method also includes the step of modifying the N-terminus of the solid phase synthesized antimicrobial peptide, wherein the functional group used for the N-terminal modification is selected from 5-isoxazole-5-carbonyl, acetyl, 2-(1-imidazolyl)acetyl, benzoyl, decanyl, 2-morpholinoacetyl, pyrazinecarbonyl, dodecanoyl, toluenesulfonyl or 2-methylthiazole-5-carbonyl.

8. The preparation method according to claim 6 or 7, characterized in that: At least one of the amino acids used in the solid phase synthesis is a D-type or β-type amino acid.

9. Use of the antimicrobial peptide according to any one of claims 1 to 5 or the antimicrobial peptide obtained by the preparation method according to any one of claims 6 to 8 in the preparation of antimicrobial drugs, anti-infection drugs, wound repair products, acne treatment drugs, radiation dermatitis prevention and treatment drugs, preservatives, animal feed or cosmetic precursors.

10. An antibacterial drug preparation, characterized in that: The antimicrobial pharmaceutical preparation contains the antimicrobial peptide according to any one of claims 1 to 5 or the antimicrobial peptide prepared by the preparation method according to any one of claims 6 to 8.

11. The antibacterial drug preparation according to claim 10, characterized in that: The effective dose of the antimicrobial peptide is 0.01 to 512 μg / ml, preferably 0.125 to 256 μg / ml.

12. The antibacterial drug preparation according to claim 10 or 11, characterized in that: The pathogenic microorganisms of the antibacterial effect include bacteria and / or fungi; Preferably, the bacteria include Gram-positive bacteria including Staphylococcus aureus or Enterococcus faecalis; and / or Gram-negative bacteria including Pseudomonas aerμginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.

13. The antibacterial drug preparation according to claim 12, characterized in that: The pathogenic microorganism is resistant to antibiotics, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.

14. The antibacterial drug preparation according to claim 13, characterized in that: The dosage form of the antibacterial drug preparation includes oral preparation, external preparation or injection; The oral preparation includes granules, tablets, pastes or oral solutions; The topical preparations include ointments, gels, suppositories, medicated baths, hoof baths or sprays.

15. A method for treating a disease associated with infection by pathogenic microorganisms, characterized in that: The method comprises administering to a subject the antimicrobial peptide according to any one of claims 1 to 5, the antimicrobial peptide obtained by the preparation method according to any one of claims 6 to 8, or the antimicrobial pharmaceutical preparation according to any one of claims 10 to 14; Preferably, the pathogenic microorganisms include bacteria and / or fungi; More preferably, the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and / or, Gram-negative bacteria, including Pseudomonas aerμginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii; Preferably, the pathogenic microorganism is resistant to antibiotics, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.

16. An antimicrobial peptide or antimicrobial drug preparation for treating a disease associated with infection by pathogenic microorganisms, wherein the antimicrobial peptide is as claimed in any one of claims 1 to 5 or is obtained by the preparation method as claimed in any one of claims 6 to 8, and the antimicrobial drug preparation is as claimed in any one of claims 10 to 14; Preferably, the pathogenic microorganisms include bacteria and / or fungi; More preferably, the bacteria include Gram-positive bacteria, including Staphylococcus aureus or Enterococcus faecalis; and / or, Gram-negative bacteria, including Pseudomonas aerμginosa, Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii; Preferably, the pathogenic microorganism is resistant to antibiotics, and the types of antibiotics it tolerates include at least one of β-lactams, cephalosporins, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides or rifampicin.

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