Broad-spectrum antibacterial peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a broad-spectrum antibacterial peptide as well as a preparation method and application thereof. The antibacterial peptide or the salt thereof is disclosed for the first time, the antibacterial peptide has a good antibacterial effect on common pathogenic bacteria (such as staphylococcus aureus, escherichia coli, salmonella enteritidis and streptococcus suis) and oral pathogenic bacteria (such as fusobacterium nucleatum and porphyromonas gingivalis), and the antibacterial peptide shows slight cytotoxicity on mammalian cells only under high concentration; and the safety is relatively high.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a broad-spectrum antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] With the rapid development of the global economy and the acceleration of urbanization, pathogenic microorganisms are widely distributed in nature, and many strains are closely related to human health. For example, Staphylococcus aureus and Escherichia coli, as major pathogens, not only cause widespread gastrointestinal symptoms but may also lead to serious complications (vaginitis, urinary tract infections, etc.), especially in individuals with weakened immune systems. As a new generation of "antibiotics," most conventional antibiotics mainly act on intracellular targets, while antimicrobial peptides mainly achieve bactericidal effects by disrupting the bacterial cell membrane through electrostatic interactions or physical actions. At the same time, their unique bactericidal mechanism makes antimicrobial peptides less prone to developing drug resistance, effectively enhancing their bactericidal ability. Therefore, antimicrobial peptides have broad development prospects and significant research value.

[0003] Oral health is an important aspect of overall human health. The results of the Fourth National Oral Health Epidemiological Survey show that residents' oral health literacy is gradually improving. However, most Chinese people still suffer from a range of oral health problems, such as tooth decay, periodontitis, halitosis, and plaque, which frequently occur and seriously affect their health. Research indicates that oral problems such as tooth decay, periodontitis, halitosis, and plaque are inextricably linked to oral microorganisms. Fusobacterium nucleatum (… Fusobacterium nucleatum *Porphyromonas gingivalis* is a Gram-negative obligate anaerobic bacterium and the most abundant *Fusobacterium* found in the oral cavity. It can function as a symbiotic bacterium, promoting plaque biofilm formation, or it can induce the production of volatile sulfides, causing halitosis. Porphyromonas gingivalis (Syndromea rubrum) is a Gram-negative anaerobic bacterium that can disrupt the host cell's defense system and activate the host's immune response by secreting virulence factors. The nutrients produced by the damage to host cells promote the growth of other pathogenic bacteria, thereby disrupting the balance of the oral flora and triggering periodontitis.

[0004] Current treatments for pathogenic infections primarily rely on traditional antibiotics, which, with long-term use, can easily lead to bacterial resistance and dysbiosis. Therefore, there is an urgent need to develop novel drugs to avoid the risk of bacterial resistance. Antimicrobial peptides (AMPs) offer numerous advantages over traditional antibiotics: they are less likely to induce resistance in pathogens, thus mitigating antibiotic resistance and ensuring long-term effectiveness; they are also highly safe, have low residue risk, and minimal impact on the environment and food safety; and they can reduce environmental pollution. Summary of the Invention

[0005] The first aspect of the present invention is to provide an antimicrobial peptide or a salt thereof.

[0006] A second aspect of the present invention is to provide a conjugate.

[0007] A third aspect of the present invention is to provide a fusion protein.

[0008] The fourth aspect of this invention aims to provide biomaterials related to the antimicrobial peptide or its salt of the first aspect of this invention, the conjugate of the second aspect of this invention, and the fusion protein of the third aspect of this invention.

[0009] A fifth aspect of the present invention is to provide a composition.

[0010] The sixth aspect of this invention aims to provide the use of the antimicrobial peptide of the first aspect of this invention or its salt, the conjugate of the second aspect of this invention, the fusion protein of the third aspect of this invention, the biomaterial of the fourth aspect of this invention, or the composition of the fifth aspect of this invention.

[0011] The seventh aspect of this invention aims to provide a product.

[0012] The object of the eighth aspect of the present invention is to provide a method for inhibiting the growth of pathogenic microorganisms.

[0013] The object of the ninth aspect of the present invention is to provide a method for producing the antimicrobial peptide or a salt thereof of the first aspect of the present invention.

[0014] The tenth aspect of this invention aims to provide a method for preventing and / or treating diseases caused by pathogenic microorganism infection.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an antimicrobial peptide or a salt thereof, wherein the amino acid sequence of the antimicrobial peptide is as follows: a1) SEQ ID NO:1; or a2) A sequence of amino acids that has undergone substitution and / or deletion and / or addition of one or more amino acids in SEQ ID NO:1 and has the same function as SEQ ID NO:1; or a3) An amino acid sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, and 80% homology with SEQ ID NO:1 and has the same function as SEQ ID NO:1.

[0016] In some embodiments of the present invention, the salt of the antimicrobial peptide includes a metal salt of the antimicrobial peptide, or a salt formed by the antimicrobial peptide and an inorganic or organic acid.

[0017] In some embodiments of the present invention, the metal includes at least one of lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

[0018] In some embodiments of the present invention, the organic acid includes at least one selected from acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, or oxalic acid.

[0019] In some embodiments of the present invention, the inorganic acid includes at least one of hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, boric acid, carbonic acid, or nitric acid.

[0020] A second aspect of the present invention provides a conjugate comprising a modification portion and an antimicrobial peptide of the first aspect of the present invention.

[0021] The modification is an artificially induced drug, performed while preserving the peptide activity. The purposes of the modification include prolonging the half-life, increasing water solubility, and reducing or eliminating toxic side effects.

[0022] In some embodiments of the present invention, the modified portion is located at the N-terminus and / or C-terminus of the antimicrobial peptide.

[0023] In some embodiments of the present invention, the modified portion comprises at least one of chemical modification, targeting portion, fluorescent dye and protein tag.

[0024] In some embodiments of the present invention, the chemical modification includes at least one of amidation, acetylation, aminoation, methylation, phosphorylation, glycosylation, and ubiquitination.

[0025] In some embodiments of the present invention, the targeting portion includes at least one of a ligand, a receptor, and an antibody (such as a full-length antibody, Fab, scFv, F(ab')2, chimeric antibody, etc.).

[0026] In some embodiments of the present invention, the fluorescent dye comprises at least one of the following: Cy series (such as Cy3, Cy5.5, Cy7, etc.), Texas series (such as Texas Red-X succinimide ester, Texas Red Amine, etc.), Alexa series (such as Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, etc.), Rhodamine, Bodipy, Rox, FAM, FITC, MCA, TAMRA, and Dnp.

[0027] In some embodiments of the present invention, the protein tag includes at least one of His, Flag, GST, MBP, HA, Myc, GFP, and biotin.

[0028] A third aspect of the present invention provides a fusion protein comprising a first domain and a second domain; The first structural domain is the antimicrobial peptide of the first aspect of the present invention; The second domain has the effect of prolonging the in vivo half-life.

[0029] In some embodiments of the present invention, the first domain is located at the N-terminus and / or C-terminus of the antimicrobial peptide.

[0030] In some embodiments of the present invention, the second domain and the antimicrobial peptide are connected to the N-terminus and / or C-terminus of the antimicrobial peptide via a connector.

[0031] In some embodiments of the present invention, the connector includes a flexible linker, including but not limited to GSAS, (GGCAGCGCCAGC, SEQ ID NO:2). n , (GGCGGCGGCAGC, SEQ ID NO:3) n , (GGCGGCGGCGGCAGC, SEQID NO:4) n YAPVDV, (GGGS) n (GGSG) n (GGGGS) n (G) n , where 1≤n≤5, and n is an integer.

[0032] In some embodiments of the present invention, the second structural domain includes (but is not limited to): Immunoglobulin Fc region (such as human immunoglobulin Fc region); and / or Serum albumin (such as human HSA) or fragments thereof, domains that bind to serum albumin (such as anti-serum albumin antibodies, including nanobodies), polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof.

[0033] In some embodiments of the present invention, the immunoglobulin is one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0034] In some embodiments of the present invention, the IgG is selected from one or more combinations of IgG1, IgG2, IgG3 or IgG4 subtypes.

[0035] A fourth aspect of the present invention provides biomaterials relating to the antimicrobial peptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, said biomaterials comprising at least one of b1) to b8): b1) A nucleic acid molecule encoding the antimicrobial peptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention; b2) An expression cassette containing the nucleic acid molecule described in b1); b3) A carrier containing the nucleic acid molecule described in b1); b4) A carrier containing the expression box described in b2); b5) Transgenic cell lines containing the nucleic acid molecules described in b1); b6) Transgenic cell lines containing the expression cassette described in b2); b7) A transgenic cell line containing the vector described in b3); b8) A transgenic cell line containing the vector described in b4).

[0036] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0037] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0038] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0039] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0040] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0041] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0042] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0043] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0044] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0045] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0046] In some embodiments of the present invention, the cells include engineered cell lines such as CHO, CHO-K1, and CHO-GS, and lymphocyte lines such as T cells, NK cells, and CIK cells.

[0047] A fifth aspect of the present invention provides a composition comprising at least one of c1) to c3): c1) The antimicrobial peptide or its salt according to the first aspect of the present invention; c2) The conjugate of the second aspect of the present invention; c3) The fusion protein of the third aspect of the present invention.

[0048] In some embodiments of the present invention, the composition further comprises other active antibacterial ingredients.

[0049] In some embodiments of the present invention, the other active antibacterial components include at least one of the following: penicillin antibiotics (such as ampicillin, oxacillin, etc.), cephalosporin antibiotics (such as cefadroxil, cefuroxime, cefoperazone, etc.), carbapenem antibiotics (such as imipenem, meropenem, etc.), lipoprotein (glycopeptide) antibiotics (such as polymyxins and vancomycin, etc.), aminoglycoside antibiotics, tetracycline antibiotics (such as doxycycline, minocycline, oxytetracycline, etc.), lincosamide antibiotics (such as lincomycin, clindamycin, etc.), macrolide antibiotics (such as azithromycin, roxithromycin, erythromycin, etc.), fluoroquinolone antibiotics (such as ciprofloxacin, lomefloxacin, etc.), and fusidic acid.

[0050] In some embodiments of the present invention, the other active antibacterial components include at least one of the following natural antibacterial substances: cinnamaldehyde, eugenol, coptis chinensis, berberine, quercetin, baicalin, thymol, andrographolide, curcumin, kaempferol, apigenin, soy isoflavones, gallic acid, protocatechuic acid, chlorogenic acid, emodin, juglone, ellagic acid, condensed tannin, propolis, lysozyme, carvacrol, menthol, eucalyptol, cinnamaldehyde, linalool, artemisinin, berberine, sanguisorbin, quinine, allicin, allyl sulfide, sulforaphane, and lectin.

[0051] In some embodiments of the present invention, the bacteria include at least one of bacteria and fungi.

[0052] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis / Enterococcus urinaria, Bacillus anthracis, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Corynebacterium diphtheriae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhi, Salmonella non-typhi, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus suis, Salmonella enteritidis, enterotoxic Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Streptococcus Gordonii, Streptococcus mutans, and Porphyromonas gingivalis.

[0053] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Malassezia furfur, Trichophyton nodules, Trichophyton nigricans, and Pneumocystis jirovecii.

[0054] In some embodiments of the present invention, the bacteria are at least one of Escherichia coli, Staphylococcus aureus, Gardnerella vaginalis, Candida albicans, Fusobacterium nucleatum, Streptococcus Gordonii, Salmonella enteritidis, Streptococcus suis, and enterotoxic Escherichia coli.

[0055] A sixth aspect of the present invention provides the use of the antimicrobial peptide RK17 or a salt thereof from the first aspect of the present invention, the conjugate of the second aspect of the present invention, the fusion protein of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, and / or the composition of the fifth aspect of the present invention in any one of e1) to e3): e1) Antibacterial; e2) Prepare antibacterial products; e3) Prepare products for preventing and treating diseases caused by pathogenic microorganisms; In some embodiments of the present invention, the bacteria mentioned in e1) includes at least one of bacteria and fungi.

[0056] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis / Enterococcus urinaria, Bacillus anthracis, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Corynebacterium diphtheriae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhi, Salmonella non-typhi, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus suis, Salmonella enteritidis, enterotoxic Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Streptococcus Gordonii, Streptococcus mutans, and Porphyromonas gingivalis.

[0057] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Malassezia furfur, Trichophyton nodules, Trichophyton nigricans, and Pneumocystis jirovecii.

[0058] In some embodiments of the present invention, the bacteria are at least one of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis, Fusobacterium nucleatum, Salmonella enteritidis, Streptococcus suis, and enterotoxic Escherichia coli.

[0059] In some embodiments of the present invention, the product described in e2) comprises at least one of reagents, drugs, feed, feed additives, preservatives, daily chemical products, textiles, paper products, medical materials, and cell culture media.

[0060] In some embodiments of the present invention, the daily chemical products include at least one of hand sanitizer, shower gel, shampoo, mouthwash, toothpaste, soap, cosmetics (including but not limited to facial cleanser, face cream, serum, toner, face cream, etc.), feminine wash, laundry soap, laundry detergent, laundry powder, dish soap, disinfectant, and toilet cleaner.

[0061] In some embodiments of the present invention, the fabric includes at least one of clothing, bedding, disinfectant wipes, dressings, and bandages.

[0062] In some embodiments of the present invention, the paper product includes at least one of sanitary napkins, panty liners, diapers, diaper pads, and wet wipes.

[0063] In some embodiments of the present invention, the medical material comprises at least one of tissue regeneration material, tissue repair material, medical hydrogel material, and medical antibacterial material.

[0064] In some embodiments of the present invention, the dosage form of the drug includes at least one of injection, oral, and topical formulations.

[0065] In some embodiments of the present invention, the product described in e3) includes at least one of pharmaceuticals, daily chemical products, medical devices, and health products.

[0066] In some embodiments of the present invention, the diseases described in e3) include reproductive tract infections (such as urethritis, vaginitis, cervicitis, endometritis, salpingitis, pelvic peritonitis, etc.) and oral diseases (such as dental caries, gingivitis, chronic periodontitis, ulcerative gingivitis, thrush, angular cheilitis, etc.).

[0067] A seventh aspect of the present invention provides a product comprising at least one of d1) to d4): d1) The antimicrobial peptide or its salt according to the first aspect of the present invention; d2) The conjugate of the second aspect of the present invention; d3) The fusion protein of the third aspect of the present invention; d4) The composition of the fourth aspect of the present invention; The product is any one of f1) to f2): f1) Antibacterial products; f2) Products for preventing and treating diseases caused by pathogenic microorganisms.

[0068] In some embodiments of the present invention, the bacteria mentioned in f1) includes at least one of bacteria and fungi.

[0069] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis / Enterococcus urinaria, Bacillus anthracis, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Corynebacterium diphtheriae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhi, Salmonella non-typhi, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus suis, Salmonella enteritidis, enterotoxic Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Streptococcus Gordonii, Streptococcus mutans, and Porphyromonas gingivalis.

[0070] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Malassezia furfur, Trichophyton nodules, Trichophyton nigricans, and Pneumocystis jirovecii.

[0071] In some embodiments of the present invention, the bacteria are at least one of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis, Fusobacterium nucleatum, Salmonella enteritidis, Streptococcus suis, and enterotoxic Escherichia coli.

[0072] In some embodiments of the present invention, the product described in f1) comprises at least one of reagents, drugs, feed, feed additives, preservatives, daily chemical products, textiles, paper products, medical materials, and cell culture media.

[0073] In some embodiments of the present invention, the daily chemical products include at least one of hand sanitizer, shower gel, shampoo, mouthwash, toothpaste, soap, cosmetics (including but not limited to facial cleanser, face cream, serum, toner, face cream, etc.), feminine wash, laundry soap, laundry detergent, laundry powder, dish soap, disinfectant, and toilet cleaner.

[0074] In some embodiments of the present invention, the fabric includes at least one of clothing, bedding, disinfectant wipes, dressings, and bandages.

[0075] In some embodiments of the present invention, the paper product includes at least one of sanitary napkins, panty liners, diapers, diaper pads, and wet wipes.

[0076] In some embodiments of the present invention, the medical material comprises at least one of tissue regeneration material, tissue repair material, medical hydrogel material, and medical antibacterial material.

[0077] In some embodiments of the present invention, the dosage form of the drug includes at least one of injection, oral, and topical formulations.

[0078] In some embodiments of the present invention, the product described in f2) includes at least one of pharmaceuticals, daily chemical products, medical devices, and health products.

[0079] In some embodiments of the present invention, the diseases described in f2) include reproductive tract infections (such as urethritis, vaginitis, cervicitis, endometritis, salpingitis, pelvic peritonitis, etc.) and oral diseases (such as dental caries, gingivitis, chronic periodontitis, ulcerative gingivitis, thrush, angular cheilitis, etc.).

[0080] An eighth aspect of the present invention provides a method for inhibiting the growth of pathogenic microorganisms, comprising treating a sample to be treated with an antimicrobial peptide or a salt thereof of the first aspect of the present invention, a conjugate of the second aspect of the present invention, a fusion protein of the third aspect of the present invention, a biomaterial of the fourth aspect of the present invention, a composition of the fifth aspect of the present invention, or a product of the seventh aspect of the present invention; wherein the sample to be treated comprises pathogenic microorganisms or a sample containing pathogenic microorganisms.

[0081] In some embodiments of the present invention, the pathogenic microorganism comprises at least one of bacteria and fungi.

[0082] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis / Enterococcus urinaria, Bacillus anthracis, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Corynebacterium diphtheriae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Escherichia coli, Salmonella typhi, Salmonella non-typhi, Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus suis, Salmonella enteritidis, enterotoxic Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Streptococcus Gordonii, Streptococcus mutans, and Porphyromonas gingivalis.

[0083] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Malassezia furfur, Trichophyton nodules, Trichophyton nigricans, and Pneumocystis jirovecii.

[0084] In some embodiments of the present invention, the pathogenic microorganism is at least one of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis, Fusobacterium nucleatum, Salmonella enteritidis, Streptococcus suis, and enterotoxic Escherichia coli.

[0085] A ninth aspect of the present invention provides a method for producing the antimicrobial peptide or a salt thereof of the first aspect of the present invention, the method comprising chemical synthesis or by culturing the transgenic cell line of the fourth aspect of the present invention.

[0086] In some embodiments of the present invention, the chemical synthesis includes polypeptide solid-phase synthesis.

[0087] A tenth aspect of the present invention provides a method for preventing and / or treating diseases caused by pathogenic microorganism infection, comprising the steps of administering to a subject an effective dose of an antimicrobial peptide of the first aspect of the present invention or a salt thereof, a conjugate of the second aspect of the present invention, a fusion protein of the third aspect of the present invention, a biomaterial of the fourth aspect of the present invention, a composition of the fifth aspect of the present invention, or a product of the seventh aspect of the present invention.

[0088] In some embodiments of the present invention, the pathogenic microorganism includes at least one of bacteria and fungi.

[0089] In some embodiments of the present invention, the pathogenic microorganism is at least one of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis, Fusobacterium nucleatum, Salmonella enteritidis, Streptococcus suis, and enterotoxic Escherichia coli.

[0090] In some embodiments of the present invention, the antimicrobial peptide or its salts, conjugates, fusion proteins, compositions and / or products are administered at least once daily.

[0091] In some embodiments of the present invention, when the number of administrations is more than twice a day, the same dose is administered each time.

[0092] In some embodiments of the present invention, when the administration frequency is more than twice a day, a different dose is administered each time.

[0093] The term "effective dose" refers to the amount that is effective for treatment or prevention in the subject being treated. The precise amount of a therapeutic composition also depends on the practitioner's judgment and may be unique to each individual. Appropriate regimens for initial dosing and synergists are also variable, but are represented by initial and subsequent dosing. Factors affecting dosage include the patient's physical and clinical condition, route of administration, intended therapeutic goal (symptom relief or cure), and the efficacy, stability, and toxicity of the specific therapeutic substance or the efficacy of other treatments the subject may be receiving.

[0094] The term "subject" refers to any mammal. A subject described as "in need" refers to a mammal requiring treatment (or prevention) for a disease. This includes, for example, any one or more animals within the class Mammalia, such as Monotremes, Marsupials, Insectivora, Flying Lepidoptera, Climbing Therapsids, Dermoptera, Chiroptera, Primates, Phenotypica, Pholidota, Lagomorpha, Rodentia, Carnivora, Sirenia, Hyracodontia, Tubularodontia, Perissodactylus, Artiodactylus, Cetacea, etc. Common mammals include Rodentia (e.g., mice, rats, hamsters, guinea pigs), Lagomorpha (e.g., rabbits), Perissodactylus (e.g., horses, donkeys), Artiodactylus (e.g., sheep, goats, camels, cattle, pigs), Primates (e.g., monkeys, gorillas, chimpanzees, humans), and Carnivora (e.g., dogs, cats). It is understood that a subject may be healthy or may have a disease at any stage of development.

[0095] The beneficial effects of this invention are: This invention discloses for the first time an antimicrobial peptide (SKRIFRKRWRKPRRLRRRTRKKPAAHTRF, SEQ ID NO:1) or its salt, which has good antibacterial effects against common pathogenic bacteria (such as Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Streptococcus suis), antibacterial effects against oral pathogenic bacteria (such as Fusobacterium nucleatum and Porphyromonas gingivalis), and exhibits only slight cytotoxicity to mammalian cells at high concentrations.

[0096] Specifically, the antimicrobial peptide provided by this invention has the following characteristics compared to existing antimicrobial drugs: 1. Reduced Drug Resistance: Antimicrobial peptides interfere with the survival environment of pathogens through multiple mechanisms of action, reducing the development of drug resistance. Compared to traditional antibiotics, antimicrobial peptides effectively avoid drug resistance problems and ensure efficacy during long-term use.

[0097] 2. Regulating the balance of the microecology: Antimicrobial peptides can selectively inhibit the growth of harmful bacteria, protect and promote the proliferation of probiotics, thereby helping to maintain the stability of the microecology and reduce the risk of recurrence and reinfection.

[0098] 3. High safety and no significant side effects: Antimicrobial peptides are widely available and naturally exist in living organisms. They have no obvious toxic side effects on the human body and do not damage liver and kidney function during use, thus avoiding the safety risks associated with antibiotics.

[0099] 4. Precision treatment: Antimicrobial peptides have strong selectivity and can act on specific pathogens, thereby achieving the purpose of precision treatment without widely affecting the growth of other beneficial microorganisms.

[0100] 5. Potential to replace antibiotics: With the increasing severity of antibiotic abuse, antimicrobial peptides, as a natural and green alternative, meet the needs of modern health, safety and environmental protection, and have broad application prospects. Attached Figure Description

[0101] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The tertiary structure of the antimicrobial peptide (SKRIFRKRWRKPRRLRRRTRKKPAAHTRF, SEQ ID NO:1) of this invention is the result predicted by the AlphaFold3 protein prediction tool.

[0102] Figure 2 The present invention describes the antimicrobial peptide's inhibitory effects at a level of 10 μmol / L against common pathogens (Staphylococcus aureus ATCC6538), oral pathogens (Fusobacterium nucleatum ATCC25586, Porphyromonas gingivalis ATCC33277), and at a level of 8 μmol / L against other pathogens (Escherichia coli ATCC25922, Salmonella enteritidis CICC 20082847, Streptococcus suis CVCC4268, and enterotoxic Escherichia coli K88).

[0103] Figure 3 The results show the minimum inhibitory concentration (MIC) test results of the antimicrobial peptides of this invention against Escherichia coli ATCC25922 and Staphylococcus aureus ATCC6538.

[0104] Figure 4 The results show the minimum inhibitory concentration (MIC) test results of the antimicrobial peptides of this invention against enterotoxic Escherichia coli K88, Salmonella enteritidis CICC 20082847, and Streptococcus suis CVCC 4268.

[0105] Figure 5 The results show the hemolytic effect of the antimicrobial peptides of this invention (using porcine erythrocytes as test cells).

[0106] Figure 6 This is the result of the cytotoxicity test (using HT-29 cells as test cells) of the antimicrobial peptide of the present invention. Detailed Implementation

[0107] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0108] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0109] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0110] Example 1: Discovery and Screening of Antimicrobial Peptides By mining and screening potential antimicrobial peptide genes in the pig gut microbiome, an antimicrobial peptide was obtained. The amino acid sequence of this antimicrobial peptide is SKRIFRKRWRKPRRLRRRTRKKPAAHTRF (SEQ ID NO:1).

[0111] Example 2 Physicochemical Properties and Structural Biological Analysis of Antimicrobial Peptides Based on the amino acid sequence of the antimicrobial peptide in Example 1, the physicochemical properties of the antimicrobial peptide were analyzed and statistically analyzed in this example. At the same time, the three-dimensional structure of this antimicrobial peptide was predicted using the Alphafold protein three-dimensional structure prediction tool.

[0112] The physicochemical properties of the antimicrobial peptides are shown in Table 1, and their three-dimensional structures are shown in Table 1. Figure 1 As shown, the antimicrobial peptide exhibits a distinct α-helix structure.

[0113] Table 1 Physicochemical properties of antimicrobial peptides

[0114] The above-mentioned antimicrobial peptides were further synthesized using solid-phase peptide synthesis (SPPS), and the general process is as follows: (1) Calculate the weight of each raw material (with protection) based on the weight of the target polypeptide; (2) Place the resin in a 150 ml reactor and add 50 ml of dichloromethane to soak for 2 hours; (3) Wash the resin with dichloromethane and then dry it. Repeat this process four times to dry the resin. (4) Weigh the first amino acid (with protection) at the C-terminus of the target polypeptide into the reactor, add dichloromethane and N-diisopropylethylamine, and then place the reactor in a shaker at 30°C for 2 hours. (5) Seal with methanol solution (methanol: N-diisopropylethylamine: dichloromethane = 1:1:2) for half an hour, then wash with dichloromethane four times and dry; (6) Add a 20% piperidine solution to the reactor to remove the 9-fluorenylmethoxycarbonyl protecting group. After removing the protecting group, wash four times with dichloromethane and then dry. (7) Take a small amount of resin and test it using the ninhydrin method. If the resin is colored, it indicates that the protecting group has been successfully removed. (8) Weigh the second amino acid (with protection) at the C-terminus and add it to the reactor. Then add 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide. Then place the reactor in a shaker at 30°C and react for 1 hour. (9) Take a small amount of resin for testing. Use the ninhydrin method to test. If the resin is colored, it indicates that the condensation is incomplete and the reaction should continue. If the resin is colorless, it indicates that the reaction is complete. After the reaction is complete, wash the resin four times with dichloromethane and then dry it. (10) Add a certain amount of 20% piperidine (piperidine / dichloromethane = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the 9-fluorenylmethoxycarbonyl protecting group on the resin. After removing the protecting group, wash it four times with dichloromethane, and then dry it to check whether the protection has been removed; (11) Take a small amount of resin and test it using the ninhydrin method. If the resin is colored, it indicates that the protecting group has been successfully removed. (12) Connect the amino acids sequentially according to steps (8) to (11); (13) Add Boc anhydride (Di-tert-butyl dicarbonate) to protect the N-terminal amino group; (14) Take a small amount of resin for testing. Use the ninhydrin method to test. If the resin is colored, it indicates that the condensation is incomplete and the reaction should continue. If the resin is colorless, it indicates that the reaction is complete. After the reaction is complete, wash the resin four times with dichloromethane and then dry it. (15) Remove the special protecting group from the lysine side chain and connect the small molecules with the side chain sequentially according to steps (8) to (11); (16) Use a cleavage reagent to remove all the protecting groups of the polypeptide and cut the polypeptide off the resin for further purification.

[0115] Example 3: Verification of the antibacterial effect of antimicrobial peptides This embodiment is used to verify the antibacterial effect of the antimicrobial peptide in Example 1, as detailed below: 1. Preliminary confirmation of the antibacterial activity of antimicrobial peptides Seven common pathogens causing genital tract, oral cavity and other infections were used in this test (Table 2).

[0116] Table 2 Pathogenic bacteria used in antibacterial tests

[0117] The antimicrobial effect of the antimicrobial peptide from Example 1 at fixed concentrations (8 μmol / L or 10 μmol / L) was preliminarily confirmed as follows: For bacteria, the pathogens were inoculated into Mueller Hinton broth (MHB) and cultured overnight at 37°C with shaking at 220 rpm. For anaerobic bacteria, the pathogens were inoculated into BHIS liquid medium and cultured overnight in an anaerobic incubator at 37°C. The culture was then diluted to the initial concentration (OD). 600 =0.1, approximately equivalent to 1×10 8 CFU / mL), then diluted 100-fold to obtain the bacterial suspension. Antimicrobial peptides (final concentration 8 μmol / L or 10 μmol / L) were added to the bacterial suspension. 200 μL of the bacterial suspension containing the antimicrobial peptides (final concentration 8 μmol / L or 10 μmol / L) was added to a 96-well plate. A blank control group (200 μL MHB solution) and a negative control group (100 μL of peptide-free bacterial suspension + 100 μL MHB / BHIS solution; the culture medium was selected according to the pathogens mentioned above) were also set up. After incubation at 37℃ for 24 hours, the OD was measured. 600 Value. The effective value of antimicrobial peptides is defined as OD. 600 The mean value was less than 70% of that of the negative control group, and the difference between the two groups was statistically significant. P Value < 0.05). All experiments were performed in triplicate.

[0118] The results are as follows Figure 2 As shown, at selected concentrations (8 μmol / L or 10 μmol / L), the antimicrobial peptides exhibit significant antimicrobial effects against common pathogens and have an inhibitory effect on oral pathogens (Fusobacterium nucleatum and Porphyromonas gingivalis).

[0119] 2. Minimum inhibitory concentration (MIC) test of antimicrobial peptides Five pathogenic bacteria were used in this test, namely Escherichia coli (E. coli) Escherichia coli ATCC 25922), Staphylococcus aureus ( Staphylococcus aureus ATCC 6538), enterotoxic Escherichia coli (ETEC K88), Salmonella enteritidis (ATCC 6538), enterotoxic Escherichia coli (ETEC K88), enterotoxic Salmonella ... Salmonella enteritidis CICC 20082847), Streptococcus suis ( Streptococcus suits (CVCC4268) The inhibitory effects of a series of antimicrobial peptide solutions at various concentrations on these bacteria were tested. The specific procedure was as follows: The pathogen was inoculated into Mueller Hinton broth (MHB) medium and cultured overnight at 37°C with shaking at 220 rpm. The culture was then diluted to the initial concentration (OD). 600 =0.1, approximately equivalent to 1×10 8CFU / mL), then diluted 100-fold. Antimicrobial peptides (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, final concentrations of 0.1, 0.3, 1, 3, 10 μmol / L; enterotoxic Escherichia coli K88, Salmonella enteritidis CICC 20082847, Streptococcus suis CVCC 4268, final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8 μmol / L) were added to the bacterial suspension. 200 μL of bacterial suspension containing different concentrations of antimicrobial peptides was added to a 96-well plate. A blank control group (200 μL MHB solution) and a negative control group (100 μL of sterile peptide-free bacterial suspension + 100 μL MHB solution) were also established. OD was measured after incubation at 37℃ for 24 hours. 600 The MIC (Minimum Intake) was defined as the lowest concentration of antimicrobial peptide that differed from the mean of the negative control group by three standard deviations. All experiments were performed in triplicate.

[0120] The results are as follows Figures 3-4 As can be seen, the antibacterial ability of the antimicrobial peptide solution gradually increases with increasing concentration, and the OD... 600 The values ​​gradually decreased. The MIC value of the antimicrobial peptide against Escherichia coli ATCC25922 was 10 μmol / L, the MIC value against enterotoxic Escherichia coli K88, Salmonella enteritidis CICC 20082847 and Streptococcus suis CVCC 4268 was 8 μmol / L, and the MIC value against Staphylococcus aureus ATCC6538 was 4 μmol / L (Table 3).

[0121] Table 3. Minimum Inhibitory Concentration (μmol / L)

[0122] Example 4 Biosafety Testing of Antimicrobial Peptides 1. Hemolysis test Using porcine erythrocytes as test cells, the hemolytic effect of the antimicrobial peptides of Example 1 at different concentration gradients (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μmol / L) was detected to investigate the toxic effects of the antimicrobial peptides on erythrocytes. The specific experimental procedures were as follows: A 4% porcine erythrocyte suspension (Solarbio) was centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The erythrocytes were then resuspended in PBS buffer containing gradient concentrations (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 µmol / L) of antimicrobial peptides and incubated at 37°C for 1 hour. Triton X-100 was used as a positive control (highest concentration 1%, subsequent concentrations 2-fold dilutions). After the reaction, the cells were centrifuged at 3000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 570 nm. All experiments were independently repeated three times.

[0123] The results are as follows Figure 5 As shown, at the highest concentration of 128 μmol / L, the antimicrobial peptide only exhibited a slight hemolytic reaction, indicating that the antimicrobial peptide of Example 1 has low toxicity to mammalian erythrocytes.

[0124] 2. Toxicity test on mammalian cells The toxic effects of the antimicrobial peptide of Example 1 on HT-29 cells were tested using different concentration gradients (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μmol / L) of the HT-29 cell line (human colorectal cell line). The specific experimental procedures were as follows: HT-29 cells in logarithmic growth phase were seeded in 96-well plates and cultured in DMEM medium (Gibco) containing 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) penicillin-streptomycin antibiotic (Solarbio); then incubated at 37°C in a 5% CO2 incubator for 12 hours. After discarding the original medium, the medium was replaced with DMEM medium containing 10% (v / v) FBS and gradient concentrations (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 µmol / L) of antimicrobial peptides and cultured for another 24 hours; Triton X-100 was used as a positive control (highest concentration 1%, followed by 2-fold dilutions); finally, cell viability was assessed using the MTT assay kit (Beyotime), with absorbance measured at 570 nm. Each antimicrobial peptide concentration was tested in four independent replicates.

[0125] The results are as follows Figure 6As shown, the antimicrobial peptide exhibited certain toxicity to HT-29 cells with increasing concentration. At a concentration 4 times the MIC (16 μmol / L), the survival rate of mammalian cells remained at 100%, indicating that the antimicrobial peptide had low cytotoxicity to mammalian cells at its effective concentration.

[0126] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An antibacterial peptide or a salt thereof, the amino acid sequence of the antibacterial peptide being: a1) SEQ ID NO: 1; or a2) an amino acid sequence obtained by substitution and / or deletion and / or addition of one or several amino acids of SEQ ID NO: 1 and having the same function as SEQ ID NO: 1; or a3) an amino acid sequence having at least 80% homology with SEQ ID NO: 1 and having the same function as SEQ ID NO:

1. 2.A conjugate comprising a modification moiety and the antibacterial peptide of claim 1. Preferably, the modification moiety is located at the N-terminal and / or C-terminal of the antibacterial peptide. Preferably, the modification moiety comprises at least one of a chemical modification, a targeting moiety, a fluorescent dye and a protein tag. Preferably, the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination. Preferably, the targeting moiety comprises at least one of a ligand, a receptor, an antibody. Preferably, the protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP, biotin. 3.A fusion protein comprising a first domain and a second domain. The first domain is the antibacterial peptide of claim 1. The second domain has the effect of prolonging the half-life in vivo. Preferably, the first domain is located at the N-terminal and / or C-terminal of the antibacterial peptide. Preferably, the second domain and the antibacterial peptide are connected to the N-terminal and / or C-terminal of the antibacterial peptide through a linker. 4.A biological material related to the antibacterial peptide of claim 1, the conjugate of claim 2 or the fusion protein of claim 3, the biological material comprising any one of b1) to b8): b1) a nucleic acid molecule encoding the antibacterial peptide of claim 1, the conjugate of claim 2 or the fusion protein of claim 3; b2) an expression cassette comprising the nucleic acid molecule of b1); b3) a vector comprising the nucleic acid molecule of b1); b4) a vector comprising the expression cassette of b2); b5) a transgenic cell line comprising the nucleic acid molecule of b1); b6) a transgenic cell line comprising the expression cassette of b2); b7) a transgenic cell line comprising the vector of b3); b8) a transgenic cell line comprising the vector of b4). 5.A composition comprising at least one of c1) to c3): c1) the antibacterial peptide or a salt thereof of claim 1; c2) the conjugate of claim 2; c3) the fusion protein of claim 3. Preferably, the composition further comprises other active antibacterial ingredients. Preferably, the other active antibacterial ingredients comprise at least one of penicillin antibiotics, cephalosporin antibiotics, carbapenem antibiotics, lipopeptide antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, lincosamide antibiotics, macrolide antibiotics, fluoroquinolone antibiotics, fusidic acid. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Preferably, the other active antibacterial ingredient comprises at least one of cinnamaldehyde, eugenol, berberine, berberine, quercetin, baicalein, thymol, andrographolide, curcumin, kaempferol, apigenin, soy isoflavones, gallic acid, protocatechuic acid, chlorogenic acid, emodin, juglone, ellagic tannin, condensed tannin, propolis, lysozyme, carvacrol, menthol, eucalyptol, cinnamaldehyde, linalool, artemisinin, berberine, sanguinarine, quinine, allicin, allyl sulfide, sulforaphane, agglutinin.

6. Use of the antibacterial peptide according to claim 1 or a salt thereof, the conjugate according to claim 2, the fusion protein according to claim 3, the biomaterial according to claim 4 and / or the composition according to claim 5 in any one of e1) to e3): e1) antibacterial; e2) preparation of an antibacterial product; e3) preparation of a product for preventing and treating a disease caused by infection of a pathogenic microorganism; Preferably, the product in e2) comprises at least one of a reagent, a drug, a feed, a feed additive, a preservative, a daily chemical product, a decontamination product, a fabric, a paper product, a medical material, a cell culture medium; Preferably, the daily chemical product comprises at least one of a hand sanitizer, a body wash, a shampoo, a mouthwash, a toothpaste, a soap, a cosmetic, a feminine care wash, a laundry soap, a laundry liquid, a laundry powder, a detergent, a disinfectant, a toilet cleaner; Preferably, the fabric comprises at least one of a clothing, a bedding, a disinfection paper towel, a dressing, a bandage; Preferably, the paper product comprises at least one of a sanitary napkin, a sanitary pad, a diaper, a urine pad; Preferably, the medical material comprises at least one of a tissue regeneration material, a tissue repair material, a medical hydrogel material, a medical antibacterial material; Preferably, the product in e3) comprises at least one of a drug, a daily chemical product, a medical device, a health product.

7. A product comprising at least one of d1) to d4): d1) the antibacterial peptide according to claim 1 or a salt thereof; d2) the conjugate according to claim 2; d3) the fusion protein according to claim 3; d4) the composition according to claim 5; The product is any one of f1) to f2): f1) an antibacterial product; f2) a product for preventing and treating a disease caused by infection of a pathogenic microorganism.

8. The product according to claim 7, wherein: The product in f1) comprises at least one of a reagent, a drug, a feed, a feed additive, a preservative, a daily chemical product, a decontamination product, a fabric, a paper product, a medical material, a cell culture medium; Preferably, the daily chemical product comprises at least one of a hand sanitizer, a body wash, a shampoo, a mouthwash, a toothpaste, a soap, a cosmetic, a feminine care wash, a laundry soap, a laundry liquid, a laundry powder, a detergent, a disinfectant, a toilet cleaner; Preferably, the fabric comprises at least one of a clothing, a bedding, a disinfection paper towel, a dressing, a bandage; Preferably, the paper product comprises at least one of a sanitary napkin, a sanitary pad, a diaper, a urine pad; Preferably, the medical material comprises at least one of tissue regeneration material, tissue repair material, medical hydrogel material, medical antibacterial material; Preferably, the product in f2) comprises at least one of drugs, daily chemical products, medical devices, health products.

9. A method for inhibiting the growth of pathogenic microorganisms, comprising treating a sample to be treated with the antibacterial peptide or salt thereof according to claim 1, the conjugate according to claim 2, the fusion protein according to claim 3, the biomaterial according to claim 4, the composition according to claim 5, the product according to claim 7; the sample to be treated comprises pathogenic microorganisms or samples containing pathogenic microorganisms. Preferably, the pathogenic microorganisms comprise at least one of bacteria, fungi; Preferably, the pathogenic microorganisms comprise at least one of Fusobacterium nucleatum, Streptococcus gordonii, Escherichia coli, Salmonella enteritidis, Streptococcus suis, Staphylococcus aureus, Candida guilliermondii, Enterococcus faecalis, Porphyromonas gingivalis.

10. A method for producing the antibacterial peptide or salt thereof according to claim 1, the method comprising chemical synthesis or by culturing the transgenic cell line according to claim 4. Preferably, the chemical synthesis comprises polypeptide solid-phase synthesis.

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