Polypeptide from deep sea ecological environment, application of polypeptide in preparation of antibacterial preparation and antibacterial drug

Abstract

The invention belongs to the technical field of biology, and discloses a polypeptide derived from a deep sea ecological environment, an application of the polypeptide in preparation of an antibacterial preparation and an antibacterial drug. According to the application of the polypeptide disclosed by the invention in the preparation of the antibacterial preparation, the polypeptide comprises one or more of amino acid fragments with sequences shown as SEQ ID NO: 1-9, the compound has good bacteriostatic or bactericidal activity on one or more of escherichia coli, halomonas aeruginosa, acinetobacter picoteri, staphylococcus aureus, linear preristeria, klebsiella pneumoniae and vibrio parahaemolyticus, and has extremely low toxicity on mammalian cells under an effective concentration. The compound has excellent bacteriostatic activity, bacteriostatic broad-spectrum property and biocompatibility, and has very excellent application prospects in multiple fields such as food preservation, agricultural disease prevention and control, feed additives and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to polypeptides derived from the deep-sea ecological environment and their application in the preparation of antibacterial agents and antibacterial drugs. Background Technology

[0002] The deep sea generally refers to areas with a depth of more than 1,000 meters, accounting for about 75% of the world's oceans. It mainly includes special ecological environments such as cold seeps, hydrothermal vents, abysses, and seamounts. It has environmental characteristics such as high pressure, low / high temperature, and darkness, and contains rich and unique microbial resources.

[0003] Antimicrobial peptides (AMPs) are an important component of the innate immune system. Currently, over 2000 natural antimicrobial peptides have been discovered, widely found in various organisms including mammals, invertebrates, plants, fungi, and bacteria. Compared to conventional antibiotics, antimicrobial peptides possess broad-spectrum antibacterial, antifungal, antiviral, antispiral, and antiparasitic capabilities, exhibit unique mechanisms of action, and are less likely to induce drug resistance. Therefore, the development of novel broad-spectrum antimicrobial peptides has become a hot topic in the biomedical field and is a crucial strategy for addressing bacterial resistance, with wide applications in food preservation, agricultural disease control, and feed additives.

[0004] Current research has revealed that, compared to terrestrial and surface seawater, deep-sea microorganisms have evolved unique physiological characteristics and metabolic mechanisms over a long period of evolution. These microorganisms can produce novel and highly bioactive natural products to compete for limited resources and space, including a class of functional small proteins with specific origins, broad-spectrum antibacterial effects, and high efficacy. However, antimicrobial peptides are characterized by easy degradation, low abundance, and complex modification. Furthermore, the difficulty in culturing deep-sea microorganisms and the low efficiency of heterologous expression of antimicrobial peptides hinder the effective extraction of antimicrobial peptides from deep-sea microorganisms using traditional methods such as direct isolation, enzymatic hydrolysis, chemical synthesis, and genetic engineering. Current technologies have few reports on antimicrobial peptides derived from deep-sea microorganisms, presenting significant limitations. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of polypeptides in the preparation of antibacterial agents. These polypeptides are derived from deep-sea microorganisms and exhibit good antibacterial or bactericidal activity against one or more of Escherichia coli, Haloxylon ammodendron, Acinetobacter p. petroleum, Staphylococcus aureus, Priscilla filamentosa, Klebsiella pneumoniae, and Vibrio parahaemolyticus. Furthermore, they exhibit extremely low toxicity to mammalian cells at effective concentrations, possessing excellent antibacterial activity, broad-spectrum antibacterial activity, and biocompatibility. They show great promise for applications in multiple fields, including food preservation, agricultural disease control, and feed additives.

[0006] A second objective of this invention is to provide an antibacterial drug.

[0007] A third objective of this invention is to provide a method for screening antimicrobial peptides.

[0008] Specifically, in the application of the polypeptide provided by the present invention in the preparation of antibacterial agents, the polypeptide includes one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:1~9.

[0009] Furthermore, the amino acid sequence of the polypeptide is shown in SEQ ID NO:6, and the polypeptide is a non-membrane-targeting antimicrobial peptide.

[0010] Furthermore, the antibacterial targets of the polypeptide include Gram-positive bacteria and / or Gram-negative bacteria.

[0011] Furthermore, the antibacterial targets of the polypeptide include one or more of Escherichia coli, Haloxylon ammodendron, Acinetobacter p. petroleum, Staphylococcus aureus, Priscilla filamentosa, Klebsiella pneumoniae, and Vibrio parahaemolyticus.

[0012] Furthermore, the concentration of the polypeptide used is not less than 1.25 μM.

[0013] Furthermore, the antibacterial agent is selected from one or more of antibacterial drugs, disinfectants, food preservatives, feed additives, and agricultural antibacterial agents.

[0014] The antibacterial drugs provided by the present invention specifically include polypeptides, wherein the polypeptides include one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:1~9.

[0015] Furthermore, the antimicrobial agent includes pharmaceutically acceptable excipients.

[0016] The antimicrobial peptide screening method provided by this invention includes: mining small protein sequences based on deep-sea ecological environment sediment metagenomic data to obtain a deep-sea sediment smORF database; screening antimicrobial peptides based on the deep-sea sediment smORF database to obtain a candidate antimicrobial peptide set; predicting dominant species in habitats, predicting antimicrobial activity, and verifying solubility based on the candidate antimicrobial peptide set to obtain a high-potential antimicrobial peptide set; and verifying antimicrobial activity based on the high-potential antimicrobial peptide set to obtain antimicrobial peptides that do indeed have antimicrobial activity.

[0017] Furthermore, the deep-sea ecological environment sediment metagenomic data includes one or more of the following: sediment metagenomic sequencing data of abyssal habitats, sediment metagenomic sequencing data of cold seep habitats, sediment metagenomic sequencing data of deep-sea hydrothermal habitats, sediment metagenomic sequencing data of seamount habitats, and sediment metagenomic sequencing data of deep-sea plain habitats.

[0018] Furthermore, the tools used for small protein sequence mining include SmorFinder and / or GMSC-mapper, and the target small protein sequence is no longer than 100aa.

[0019] Furthermore, the tools used for screening the antimicrobial peptides include one or more of Macrel, ampir, amPEPpy, APIN, AI4AMP, and AMPLify.

[0020] Furthermore, the tools used for the source dominant species analysis include MMseqs2, the tools used for the antibacterial activity AI prediction include EvoGradient, and the tools used for the dissolution verification include Protein–Sol.

[0021] Furthermore, the methods for verifying antibacterial activity include one or more of the Oxford cup method, the paper disc diffusion method, and the microbroth dilution method. Attached Figure Description

[0022] Figure 1 This is a simulated three-dimensional structural diagram of the antimicrobial peptide provided in the embodiments of the present invention.

[0023] Figure 2 The figure shows the experimental results of the minimum inhibitory concentration test of the antimicrobial peptide provided in the embodiments of the present invention.

[0024] Figure 3 This is a graph showing the experimental results of the minimum bactericidal concentration test of the antimicrobial peptide provided in the embodiments of the present invention against Priscilla filamentosa.

[0025] Figure 4 This is a graph showing the experimental results of the minimum bactericidal concentration test of the antimicrobial peptide provided in the embodiments of the present invention against Priscilla filamentosa.

[0026] Figure 5 The antimicrobial peptides provided in the embodiments of this invention are effective against Escherichia coli DH5α. α The experimental results of the minimum bactericidal concentration test are shown in the figure.

[0027] Figure 6 The figure shows the experimental results of the minimum bactericidal concentration test of the antimicrobial peptide provided in the embodiments of the present invention against Acinetobacter pituitaria.

[0028] Figure 7 The figure shows the experimental results of the antimicrobial peptide provided in the embodiments of the present invention on the cytotoxicity of HEK293T cells, HepG2 cells and HeLa cells.

[0029] Figure 8 The figure shows the experimental results (scale bar is 10 μm) of the effect of the antimicrobial peptide Ventherin-19 provided in the embodiments of the present invention on the ultrastructure of bacteria. Detailed Implementation

[0030] Based on the gaps in existing research on antimicrobial peptides in the microorganisms of deep-sea hydrothermal vents and cold seeps, the inventors of this invention, through extensive and in-depth thinking and numerous experiments, focused on sediments from abyssal habitats, cold seep habitats, deep-sea hydrothermal vent habitats, seamount habitats, and deep-sea plain habitats. Utilizing a combination of bioinformatics and machine learning techniques, they first assembled metagenomic sequencing data from these sediments to obtain metagenomic data of deep-sea ecological environment sediments. Next, they screened small proteins with antimicrobial potential and high solubility from this data, and finally synthesized corresponding peptides. Through experimental verification of their antimicrobial activity, they creatively discovered nine deep-sea-derived peptide molecules. These peptide molecules possess broad-spectrum antimicrobial activity, exhibiting good inhibitory or bactericidal effects against various strains of Escherichia coli, Haloxylon ammodendron, Acinetobacter p. p., Staphylococcus aureus, Priscilla filamentosa, Klebsiella pneumoniae, and Vibrio parahaemolyticus, with a minimum inhibitory concentration reaching 1.25 μM, demonstrating both excellent antimicrobial activity and broad-spectrum activity. Based on this, the technical solution of the present invention is obtained.

[0031] The present invention relates to the application of the polypeptide in the preparation of antibacterial agents. Specifically, the polypeptide comprises one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:1-9.

[0032] In this invention, when the amino acid sequence of the polypeptide is as shown in SEQ ID NO:6, the polypeptide does not destroy the bacterial cell membrane structure when it acts on bacteria, but achieves the antibacterial effect through a non-membrane-targeting mechanism, that is, the polypeptide is a non-membrane-targeting antimicrobial peptide.

[0033] In this invention, the polypeptide possesses broad-spectrum antibacterial activity, specifically targeting Gram-positive and / or Gram-negative bacteria. More specifically, the polypeptide is effective against one or more of the following: *Escherichia coli*, *Haloxylon ammodendron*, *Acinetobacter p. p.*, *Staphylococcus aureus*, *Priscilla filamentosa*, *Klebsiella pneumoniae*, and *Vibrio parahaemolyticus*. Specifically, the polypeptide with the sequence shown in SEQ ID NO:1 has a minimum inhibitory concentration (MIC) of 1.25 μM against *Priscilla filamentosa*. The polypeptide with the sequence shown in SEQ ID NO:2 has a MIC of 2.5 μM against *Acinetobacter p. p.*. The polypeptide with the sequence shown in SEQ ID NO:3 has a MIC of 5 μM against *Priscilla filamentosa*. The polypeptide with the sequence shown in SEQ ID NO:4 has a MIC of 5 μM against *Acinetobacter p. p.* and a MIC of 10 μM against *Klebsiella pneumoniae* ATCC 13883. The peptide with the sequence shown in SEQ ID NO:5 exhibits a minimum inhibitory concentration (MIC) of 2.5 μM against Vibrio parahaemolyticus VP.1997. The peptide with the sequence shown in SEQ ID NO:6 exhibits a MIC of 1.25 μM against both Priscilla filamentosa and Vibrio parahaemolyticus VP.1997. The peptide with the sequence shown in SEQ ID NO:7 exhibits a MIC of 1.25 μM against Vibrio parahaemolyticus VP.1997. The peptide with the sequence shown in SEQ ID NO:8 exhibits a MIC of 2.5 μM against Vibrio parahaemolyticus VP.1997. The peptide with the sequence shown in SEQ ID NO:9 exhibits a MIC against Escherichia coli DH5α. α Escherichia coli BW25113, Haloxylon ammodendron, and Priestella all exhibit good antibacterial activity.

[0034] In this invention, the antibacterial agent refers to a class of preparations that can selectively inhibit the growth and reproduction of pathogenic microorganisms such as bacteria and fungi or directly kill pathogenic microorganisms. It can be any of the existing options, and specific examples include, but are not limited to, one or more of the following: antibacterial drugs, disinfectants, food preservatives, feed additives, and agricultural antibacterial agents.

[0035] The antibacterial drug provided by the present invention specifically includes a polypeptide, wherein the polypeptide specifically includes one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:1~9.

[0036] In this invention, the antibacterial drug preferably also includes pharmaceutically acceptable excipients for purposes such as achieving, but not limited to, formulation shaping, improving peptide stability, improving route of administration, improving bioavailability, and reducing irritation. These excipients can be of various existing types, and specific examples include, but are not limited to, one or more of solvents, diluents, binders, stabilizers, cosolvents, and protectants.

[0037] The antimicrobial peptide screening method provided by this invention specifically includes: small protein sequence mining based on deep-sea ecological environment sediment metagenomic data to obtain a deep-sea sediment smORF database; screening antimicrobial peptides based on the deep-sea sediment smORF database to obtain a candidate antimicrobial peptide set; predicting dominant species in habitats, predicting antimicrobial activity, and verifying solubility based on the candidate antimicrobial peptide set to obtain a high-potential antimicrobial peptide set; and verifying antimicrobial activity based on the high-potential antimicrobial peptide set to obtain antimicrobial peptides that do indeed have antimicrobial activity.

[0038] In this invention, the deep-sea ecological environment sediment metagenomic data refers to a comprehensive data set containing the genetic material of all microbial communities in the environment obtained by metagenomic analysis of sediment samples from deep sea sources. The method for obtaining the deep-sea ecological environment sediment metagenomic data can be any of the existing options, and this invention does not impose any particular limitation on it.

[0039] In some specific embodiments, the deep-sea ecological environment sediment metagenomic data preferably includes one or more of the following: sediment metagenomic sequencing data of abyssal habitats, sediment metagenomic sequencing data of cold seep habitats, sediment metagenomic sequencing data of deep-sea hydrothermal habitats, sediment metagenomic sequencing data of seamount habitats, and sediment metagenomic sequencing data of deep-sea plain habitats.

[0040] In this invention, small protein sequence mining refers to the analytical process of identifying, screening, and characterizing small protein sequence information with an amino acid length of no more than 100 aa and a stable spatial structure from deep-sea hydrothermal sediment metagenomic data. The tools used in the analysis process can be any existing selection, and specific examples include, but are not limited to, SmorFinder and / or GMSC-mapper.

[0041] In this invention, the antimicrobial peptide screening refers to the analytical process of identifying, isolating, and characterizing small protein sequences with potential antimicrobial activity from the deep-sea hydrothermal sediment smORF database based on the known structural features and functional properties of antimicrobial peptides using bioinformatics methods. The tools used in the analytical process can be of various existing options, and specific examples include, but are not limited to, one or more of: Macrel, ampir, amPEPpy, APIN, AI4AMP, and AMPLify.

[0042] In this invention, the habitat dominance species prediction refers to the process of inferring, through theoretical analysis, statistical models or machine learning, that the species from which the candidate antimicrobial peptide fragments originate will occupy a numerical, biomass or ecological function dominance in the community under current or future habitat conditions, based on information such as the environmental characteristics of a specific habitat, the niche requirements of species, inter-species interactions and historical distribution data. The tools used in the analysis process can be any of the existing options, and specific examples include, but are not limited to, MMseqs2.

[0043] In this invention, the antimicrobial activity prediction refers to the process of predicting the inhibitory or killing ability or antimicrobial spectrum range of candidate antimicrobial peptide fragments based on information such as molecular structure, physicochemical properties, biosynthetic pathways, or genomic characteristics and metabolomics data of microorganisms, through theoretical analysis, statistical models, machine learning algorithms, or molecular simulation. The tools used in the analysis process can be any existing selection, and specific examples include, but are not limited to, EvoGradient.

[0044] In this invention, the solubility verification refers to the analytical process of further identifying, separating and identifying small protein sequences with high solubility potential from a set of candidate antimicrobial peptides based on the sequence characteristics, structural characteristics, physicochemical properties of peptide chains and sequence length of known antimicrobial peptides. The tools used in the analytical process can be any of the existing options, and specific examples include, but are not limited to, Protein-Sol.

[0045] In this invention, the antimicrobial activity verification refers to the experimental verification process of determining the inhibitory, killing, or infection control effects of high-potential antimicrobial peptides concentrated small protein sequences on target pathogenic microorganisms using standardized methods. The methods used can be various existing options, and specific examples include, but are not limited to, one or more of the following: the Oxford cup method, the paper disc diffusion method, and the microbroth dilution method.

[0046] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0047] Example This embodiment illustrates an antimicrobial peptide derived from deep-sea microorganisms, specifically including: 1. Obtaining antimicrobial peptides from deep-sea microorganisms: (1) Assemble metagenomic sequencing data of sediments from abyssal habitats, cold seep habitats, deep-sea hydrothermal vent habitats, seamount habitats and deep-sea plain habitats to obtain deep-sea ecological environment sediment metagenomic data. This deep-sea ecological environment sediment metagenomic data contains 1058734804 contig sequences with a length greater than 200bp.

[0048] (2) Based on the metagenomic data of sediments in the deep-sea ecological environment, the software SmorFinder and GMSC-mapper were used to mine and remove redundancy of small protein (smORF) sequences to obtain a deep-sea sediment smORF database. This deep-sea sediment smORF database contains 88,707,689 smORF sequences with a length of less than 100aa.

[0049] (3) Based on the deep-sea sediment smORF database, antimicrobial peptides were screened using software such as Macrel, ampir, amPEPpy, APIN, AI4AMP and AMPLify to obtain a candidate antimicrobial peptide set, which includes 5,469,335 candidate antimicrobial peptides.

[0050] (4) Based on the candidate antimicrobial peptide set, the software MMseqs2, EvoGradient and Protein-Sol were used in sequence to predict the dominant species in the habitat, predict the antimicrobial activity and verify the solubility of the candidate antimicrobial peptides, and obtain a high-potential antimicrobial peptide set, which includes 131 high-potential antimicrobial peptides.

[0051] (5) Based on the high-potential antimicrobial peptide set, the external biosynthesis company used the Fmoc solid-phase synthesis method to synthesize each high-potential antimicrobial peptide.

[0052] 2. Minimum inhibitory concentration (MIC) of high-potential antimicrobial peptides: (1) Select Escherichia coli DH5α and DH5α. α Escherichia coli BW25113, Haloxylon ammodendron ( Halomonas bluephagenesis TD01, Acinetobacter pitera ( Acinetobacter pittii Staphylococcus aureus Staphylococcus aureus ), Priestella linearis ( Priestia filamentosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC 13883 and Vibrio parahaemolyticus ( Vibrio parahaemolyticusVP.1997 was streaked onto LB solid medium (Staphylococcus aureus inoculated onto MH solid medium; the LB solid medium for culturing Haloxylon ammodendron also contained 60 g / L NaCl) and incubated overnight at 37°C until single colonies formed. Single colonies of each strain were picked and inoculated into 50 mL of LB liquid medium (Staphylococcus aureus inoculated onto MH liquid medium; the LB liquid medium for culturing Haloxylon ammodendron also contained 60 g / L NaCl) and incubated overnight at 37°C and 220 rpm to obtain a seed suspension. The primary seed suspension was diluted 1:100 by volume with fresh LB liquid medium (Staphylococcus aureus inoculated onto MH liquid medium; the LB liquid medium for culturing Haloxylon ammodendron also contained 60 g / L NaCl) and incubated at 37°C and 220 rpm for 3 hours. The cell density was then adjusted to 2 × 10⁻⁶ cells / mL. 5 CFU / mL was used to obtain the bacterial suspension to be tested.

[0053] (2) Each high-potential antimicrobial peptide was serially diluted twofold using LB liquid medium or MH liquid medium to obtain antimicrobial peptide solutions with concentrations of 2.5 μM to 250 μM.

[0054] (3) Mix 50 μL of antimicrobial peptide solution and 50 μL of test bacterial suspension evenly, and incubate at 37℃ and 220 rpm for 16 h. Repeat the experiment in triplicate. Use LB liquid medium or MH liquid medium as blank control, and use an equal volume of LB liquid medium or MH liquid medium instead of antimicrobial peptide solution as control. Use commercially available antimicrobial peptides nisin (Sangon Biotech, catalog number A410681) and melitin (Sangon Biotech, catalog number A410702) as positive controls. Calculate the relative OD according to the following formula. 600 The minimum inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobial peptide at which no visible bacterial growth was observed. Nine antimicrobial peptides with excellent antimicrobial activity were ultimately selected. Their sequence information is shown in Table 1, and their simulated three-dimensional structures are shown in [Table 1]. Figure 1 As shown in the figure (using software AlphaFold3 and PyMOL), the results of the antibacterial experiment are as follows: Figure 2 As shown.

[0055] relative OD 600 Value = (OD) 600,实验组 OD 600,空白 ) / (OD 600,对照 OD 600,空白 ) Table 1.

[0056] Depend on Figure 2The results show that, among the screened antimicrobial peptides, the antimicrobial peptide Abyssin-4 is effective against Escherichia coli DH5α. α It exhibits antibacterial activity against Escherichia coli BW25113, Staphylococcus aureus, and Priscilla filamentosa, with a minimum inhibitory concentration of 1.25 μM against Priscilla filamentosa, demonstrating excellent antibacterial activity.

[0057] Antimicrobial peptide Abyssin-9 against Escherichia coli DH5α α It exhibits antibacterial activity against *Haloxylon ammodendron* TD01, *Acinetobacter pituitaria*, and *Priscilla filamentosa*, with a minimum inhibitory concentration (MIC) as low as 2.5 μM against *Acinetobacter pituitaria*, demonstrating excellent antibacterial activity.

[0058] The antimicrobial peptide Seepin-3 exhibits antimicrobial activity against Acinetobacter pituitaria and Priestella linearis, with a minimum inhibitory concentration (MIC) as low as 5 μM against Priestella linearis, demonstrating excellent antimicrobial activity.

[0059] Antimicrobial peptide Seepin-9 against Escherichia coli DH5α α It exhibits antibacterial activity against Acinetobacter pylori, Staphylococcus aureus, and Klebsiella pneumoniae (ATCC 13883), with a minimum inhibitory concentration (MIC) as low as 5 μM against Acinetobacter pylori and as low as 10 μM against Klebsiella pneumoniae (ATCC 13883), demonstrating excellent antibacterial activity.

[0060] The antimicrobial peptide Ventherin-9 exhibits antimicrobial activity against Haloxylon ammodendron TD01, Priestella linearis, and Vibrio parahaemolyticus VP.1997, with a minimum inhibitory concentration of only 2.5 μM against Vibrio parahaemolyticus VP.1997, demonstrating excellent antimicrobial activity.

[0061] The antimicrobial peptide Ventherin-19 exhibits antimicrobial activity against Escherichia coli BW25113, Staphylococcus aureus, Priscilla filamentosa, and Vibrio parahaemolyticus VP.1997. Furthermore, it demonstrates excellent antimicrobial activity with a minimum inhibitory concentration as low as 1.25 μM against Priscilla filamentosa and Vibrio parahaemolyticus VP.1997.

[0062] The antimicrobial peptide Ventherin-35 exhibits antimicrobial activity against Escherichia coli BW25113 and Vibrio parahaemolyticus VP.1997, with a minimum inhibitory concentration of only 1.25 μM against Vibrio parahaemolyticus VP.1997, demonstrating excellent antimicrobial activity.

[0063] The antimicrobial peptide Ventherin-36 exhibits excellent antimicrobial activity with a minimum inhibitory concentration of only 2.5 μM against Vibrio parahaemolyticus VP.1997.

[0064] Antimicrobial peptide Hadalin-15 against Escherichia coli DH5α α Escherichia coli BW25113, Haloxylon ammodendron D01, and Priestella linearis all exhibit good antibacterial activity.

[0065] 3. Minimum Bacterial Combination Concentration (MBC) of Antimicrobial Peptides: Following the methods provided in the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines for assessing bactericidal activity, the minimum bactericidal concentration (MBC) of each antimicrobial peptide was determined, specifically including: (1) The bacterial suspension and antimicrobial peptide solution were prepared according to the method provided in “2. Minimum inhibitory concentration of high potential antimicrobial peptides”.

[0066] (2) Mix the bacterial suspension and antimicrobial peptide solution at a 1:1 volume ratio, incubate at 37℃ and 220rpm for 16h, then mix with the corresponding liquid culture medium for each strain at a 1:100 volume ratio. Take 5μL of the mixture and spread it on LB solid medium. After incubation at 37℃ for 24h, count the colony forming units (CFU). Repeat the experiment in triplicate, using the bacterial suspension without antimicrobial peptide solution incubation as a blank control. Calculate the bactericidal rate (in %) according to the following formula, and define MBC as the minimum peptide concentration that reduces colony forming units by at least 99.9% compared to the untreated control group. The results are as follows: Figures 3-6 As shown.

[0067] Sterilization rate = (1-CFU) 抗菌肽溶液 / CFU 对照 )×100% Depend on Figure 3 The results show that the antimicrobial peptide Abyssin-4 has an MBC of 1.25 μM against *Priscilla filamentosa*, the antimicrobial peptide Seepin-3 has an MBC of 10 μM against *Priscilla filamentosa*, and the antimicrobial peptide Ventherin-19 has an MBC of 1.25 μM against *Priscilla filamentosa*, all of which have excellent bactericidal effects.

[0068] Depend on Figure 4 The results show that the antimicrobial peptide Seepin-9 is effective against Escherichia coli DH5α. α The MBC is 20 μM, and the antimicrobial peptide Hadalin-15 is effective against Escherichia coli DH5α. α With an MBC of 40μM, it has an excellent bactericidal effect.

[0069] Depend on Figure 5The results show that the antimicrobial peptide Ventherin-9 has an MBC of 2.5 μM against Vibrio parahaemolyticus VP.1997, the antimicrobial peptide Ventherin-19 has an MBC of 1.25 μM against Vibrio parahaemolyticus VP.1997, the antimicrobial peptide Ventherin-35 has an MBC of 1.25 μM against Vibrio parahaemolyticus VP.1997, and the antimicrobial peptide Ventherin-36 has an MBC of 2.5 μM against Vibrio parahaemolyticus VP.1997, all exhibiting excellent bactericidal effects.

[0070] Depend on Figure 6 The results show that the antimicrobial peptide Abyssin-9 has an MBC of 2.5 μM against Acinetobacter pituitaria, the antimicrobial peptide Seepin-3 has an MBC of 80 μM against Acinetobacter pituitaria, and the antimicrobial peptide Seepin-9 has an MBC of 5 μM against Acinetobacter pituitaria, demonstrating excellent bactericidal effects.

[0071] 4. Cytotoxicity of antimicrobial peptides: (1) HEK293T cells, HepG2 cells and HeLa cells were seeded in DMEM liquid medium containing 10% fetal bovine serum and cultured at 37℃ and 5% CO2 for 24 h. The cell density of the solution was then diluted to 1×10⁻⁶ cells using DMEM liquid medium containing 10% fetal bovine serum. 5 Cells / mL were used to obtain cell suspensions.

[0072] (2) The antimicrobial peptides Abyssin-4, Seepin-3, Seepin-9, Ventherin-19 and Ventherin-36 were diluted with DMEM liquid medium to obtain antimicrobial peptide solutions with corresponding concentrations of 1×MIC or 2×MIC.

[0073] (3) Take 50 μL of each cell suspension and mix it with 50 μL of antimicrobial peptide solution, DMEM liquid medium, or DMEM liquid medium containing 1% (v / v) DMSO. Incubate at 37℃ and 5% CO2 for 48 h. Then, use the CCK-8 kit (NCMBiotech, catalog number C6005) to detect cell viability according to the instructions. Perform the experiment in triplicate. Use the cell suspension before adding the antimicrobial peptide solution as the control group. Calculate the relative cell viability (in %) according to the following formula. The results are as follows: Figure 7 As shown.

[0074] Relative cell viability = (A1 - A0) / (A2 - A0) × 100% Wherein, A0 is the absorbance value of the blank group's DMEM liquid culture medium containing 10% fetal bovine serum at 450 nm, A1 is the absorbance value of the experimental group's cell culture medium at 450 nm, and A2 is the absorbance value of the cell culture medium obtained by mixing and incubating the DMEM liquid culture medium containing 10% fetal bovine serum with the cell suspension at 450 nm.

[0075] Depend on Figure 7 The results show that the antimicrobial peptides Abyssin-4, Seepin-3, Ventherin-19, and Ventherin-36 are not cytotoxic to HEK293T, HepG2, and HeLa cells at effective inhibitory concentrations.

[0076] 5. Antimicrobial mechanism of antimicrobial peptide Ventherin-19: (1) A suspension of Priestella linearis and an antimicrobial peptide solution with a concentration of 1.25 μM were prepared by referring to the method provided in "2. Minimum inhibitory concentration of high potential antimicrobial peptides".

[0077] (2) According to the addition amount of 1×MIC, the antimicrobial peptide solution was mixed evenly with the Priestella lineare suspension, incubated at 37℃ for 2h, centrifuged at 4℃ and 6000r / min for 1min, the supernatant was removed, and the bacterial cells were resuspended in PBS buffer (10mM, pH=7.4) to prepare ultrathin sections. The sections were negatively stained with 1% (w / v) uranium acetate for 15s and then air-dried at room temperature. The sections were observed under a transmission electron microscope at 80kV. An equal volume of PBS buffer (10mM, pH=7.4) was used as a control instead of the antimicrobial peptide solution. The results are as follows. Figure 8 As shown.

[0078] Depend on Figure 8 The results show that the antimicrobial peptide Ventherin-19 does not disrupt the cell membrane integrity of Priestella linearis, but rather exerts its bactericidal effect through a non-membrane-targeting pathway.

[0079] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. The application of polypeptides in the preparation of antibacterial agents, characterized in that, The polypeptide comprises one or more segments of amino acid fragments with sequences as shown in SEQ ID NO:1~9.

2. The application of the polypeptide according to claim 1 in the preparation of antibacterial agents, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:6, and the polypeptide is a non-membrane-targeting antimicrobial peptide.

3. The application of the polypeptide according to claim 1 in the preparation of antibacterial agents, characterized in that, The peptide targets Gram-positive and / or Gram-negative bacteria.

4. The application of the polypeptide according to claim 1 in the preparation of antibacterial agents, characterized in that, The antibacterial targets of the polypeptide include one or more of Escherichia coli, Haloxylon ammodendron, Acinetobacter pituitaria, Staphylococcus aureus, Priscilla filamentosa, Klebsiella pneumoniae, and Vibrio parahaemolyticus.

5. The application of the polypeptide according to claim 1 in the preparation of antibacterial agents, characterized in that, The concentration of the polypeptide used is not less than 1.25 μM.

6. The application of the polypeptide according to claim 1 in the preparation of antibacterial agents, characterized in that, The antibacterial agent is selected from one or more of antibacterial drugs, disinfectants, food preservatives, feed additives, and agricultural antibacterial agents.

7. An antibacterial drug, characterized in that, The antimicrobial drug includes a polypeptide, which comprises one or more segments of an amino acid sequence as shown in SEQ ID NO: 1-9.

8. The antibacterial drug according to claim 7, characterized in that, The antimicrobial drug includes pharmaceutically acceptable excipients, which include one or more of solvents, diluents, binders, stabilizers, cosolvents, and protectants.

9. A method for screening antimicrobial peptides, characterized in that, The screening method includes: mining small protein sequences based on deep-sea ecological environment sediment metagenomic data to obtain a deep-sea sediment smORF database; screening antimicrobial peptides based on the deep-sea sediment smORF database to obtain a candidate antimicrobial peptide set; predicting dominant species in habitats, predicting antimicrobial activity, and verifying solubility based on the candidate antimicrobial peptide set to obtain a high-potential antimicrobial peptide set; and verifying antimicrobial activity based on the high-potential antimicrobial peptide set to obtain antimicrobial peptides that do indeed have antimicrobial activity.

10. The method for screening antimicrobial peptides according to claim 9, characterized in that, The screening method includes at least one of the following technical features: (1) The deep-sea ecological environment sediment metagenomic data includes one or more of the following: sediment metagenomic sequencing data of abyssal habitats, sediment metagenomic sequencing data of cold seep habitats, sediment metagenomic sequencing data of deep-sea hydrothermal habitats, sediment metagenomic sequencing data of seamount habitats, and sediment metagenomic sequencing data of deep-sea plain habitats. (2) The tools used for small protein sequence mining include SmorFinder and / or GMSC-mapper, and the target small protein sequence length is no more than 100aa; (3) The tools used for screening the antimicrobial peptides include one or more of Macrel, ampir, amPEPpy, APIN, AI4AMP and AMPLify; (4) The tools used for the source dominant species analysis include MMseqs2, the tools used for the antibacterial activity AI prediction include EvoGradient, and the tools used for the dissolution verification include Protein-Sol; (5) The methods for verifying antibacterial activity include one or more of the Oxford cup method, paper diffusion method and microbroth dilution method.

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