Novel antibacterial peptide aiming at marine pathogen infection and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and discloses a novel antibacterial peptide aiming at marine pathogen infection and application thereof. The method comprises the following steps: by taking aurelia as a research object, analyzing the differential expression condition of an aurelia gene after bacterial infection by virtue of a multi-omics technology, predicting candidate antibacterial peptides by virtue of an AI model, and carrying out activity verification on MIC of bacteria by measuring polypeptides, so as to obtain the high-activity aurelia antibacterial peptide. The in-vitro activity of the antibacterial peptide is deeply explored. And then determining the hemolytic activity, cytotoxicity and stability, and carrying out preliminary research on the antibacterial mechanism. The safety and antibacterial activity of the antibacterial peptide in vivo are further explored through a vibrio vulnificus abdominal cavity infected animal model. The obtained antibacterial peptide 21546 can possibly become a powerful leading peptide for developing a novel antibacterial drug aiming at marine pathogen infection after being modified and optimized, and a novel candidate drug is provided for treating marine organism injury, marine battle injury secondary infection and clinical pathogenic bacterium infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and more specifically, relates to a novel antimicrobial peptide targeting marine pathogen infection and application thereof. Background Art

[0002] Marine organisms often harbor complex microbial communities on their surfaces and in their mouths. When stung, punctured, bitten, or otherwise injured by marine organisms, infections often develop. On the ocean surface, open wounds, such as skin wounds or marine organism wounds, exposed to seawater can rapidly trigger localized tissue inflammation, edema, severe pain, and even progressive tissue necrosis due to the combined effects of hypertonic fluid infiltration and microbial invasion. This not only impairs wound healing but can also be life-threatening if severe sepsis develops. Testing of marine environmental samples has revealed the presence of over 20 bacterial species with significant pathogenicity in humans, the vast majority belonging to the halophilic Vibrio genus. Vibrio vulnificus is the most common and causes the most severe symptoms. According to epidemiological statistics, the mortality rate from sepsis caused by Vibrio vulnificus infection can be as high as 50%. Furthermore, with the widespread use and misuse of antibiotics in recent years, drug-resistant and multidrug-resistant bacteria that cannot be controlled by conventional drugs are emerging, causing serious health and medical problems, including marine pathogens. As early as 2013, a study analyzing the antibiotic susceptibility of marine bacteria in Chinese waters showed that these bacteria exhibit varying degrees of resistance to some commonly used clinical antibiotics. To address this issue, there is an urgent need to find new antimicrobial drugs that can replace traditional antibiotics to combat drug-resistant bacteria.

[0003] Antimicrobial peptides (AMPs) are an important component of the innate immune system of many organisms. They generally consist of 10-50 amino acids and are also known as host defense peptides. They have good inhibitory activity against bacteria, fungi, and viruses. Positively charged peptides interact electrostatically with negatively charged bacterial cell membranes, causing changes in the permeability of the cell membrane and rupturing the cell membrane, allowing intracellular ions and metabolites to leak out. This unique mechanism of action makes it difficult for bacteria to evolve drug resistance, and antimicrobial peptides have a rapid bactericidal rate, making them promising candidates for preventing and controlling bacterial infections. Marine organisms are an important source for the discovery of antimicrobial active molecules. Marine invertebrates mainly rely on a nonspecific immune system. In the long-term process of adapting to the highly competitive and dynamically changing marine ecosystem, they are likely to have evolved a unique set of immune defense mechanisms to ensure the survival and reproduction of their populations. Many studies have isolated antimicrobial peptides from marine organisms.

[0004] Jellyfish, belonging to the phylum Cnidaria, are ancient marine invertebrates that have undergone hundreds of millions of years of evolution. Jellyfish are numerous and found in a variety of marine environments. Their body cavities are open and immersed in seawater, creating a large surface area of ​​contact with the water. Prolonged contact with a variety of pathogens on the body surface may have led to the evolution of a unique innate immune system and antimicrobial molecules. Studies have previously discovered antimicrobial peptides from the moon jellyfish (Aurelia aurita). However, due to the difficulty and low efficiency of directly isolating and enriching active ingredients from jellyfish, the antimicrobial peptide resources in jellyfish remain a potential treasure trove of untapped pharmaceuticals. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention aims to provide novel antimicrobial peptides against marine pathogen infections.

[0006] The second object of the present invention is to provide the use of the antimicrobial peptide.

[0007] The third object of the present invention is to provide a preparation containing the above antimicrobial peptide.

[0008] The fourth object of the present invention is to provide the use of the above antimicrobial peptide in the preparation of drugs for treating diseases caused by marine pathogen infection.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] An antimicrobial peptide targeting marine pathogen infection, wherein the sequence of the antimicrobial peptide is: GYGHLLRYLARNFISKVRLN.

[0011] This study combined transcriptomics and artificial intelligence technologies to analyze the innate immune response of the moon jellyfish. Bioinformatics analysis revealed that differentially expressed genes were significantly enriched in pathways related to nucleic acid metabolism, suggesting that pathogen stimulation may enhance host defense by activating nucleotide-mediated cell proliferation signaling and transcriptional regulatory mechanisms. A novel antimicrobial peptide 21546, derived through transcriptomics analysis combined with artificial intelligence prediction and experimental validation, showed low homology to previously reported antimicrobial peptides. These antimicrobial peptides were derived from differentially expressed genes in a moon jellyfish infection model and may participate in the jellyfish's innate immune response and exert antimicrobial activity.

[0012] Therefore, the present invention also protects a preparation containing the antimicrobial peptide.

[0013] The present invention also protects the preparation method of the antimicrobial peptide.

[0014] Antimicrobial activity was assessed using the broth microdilution method. The MICs of antimicrobial peptide 21546 against Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa ranged from 3.16 μM to 104.5 μM. Further studies revealed that the antimicrobial peptide exhibited a rapid bactericidal effect at a concentration 5 times the MIC, effectively killing E. coli, Vibrio vulnificus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa within 30 minutes. This rapid killing property significantly shortens the bacterial stress response time, thereby effectively reducing the probability of bacterial resistance mutations.

[0015] Therefore, the present invention also protects the use of the antimicrobial peptide in inhibiting pathogen infection in vitro.

[0016] Preferably, the pathogens include Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Pseudomonas aeruginosa.

[0017] More preferably, the dosage of the antimicrobial peptide is less than 50 μM. Studies of the present invention have shown that when the dosage of the antimicrobial peptide is less than 50 μM, the safety of the antimicrobial peptide to cells can be ensured, and hemolysis caused by cells can be avoided.

[0018] As one of the key factors in the mechanism of bacterial resistance, the formation of biofilm significantly enhances the drug resistance of microorganisms. The biofilm experiment of the present invention shows that the antimicrobial peptide 21546 can effectively inhibit the formation of bacterial biofilms at the MIC concentration and has a significant destructive effect on mature biofilms. The resistance induction experiment further confirmed that Vibrio vulnificus is not easy to develop resistance to antimicrobial peptides. Through these preliminary biological activity experiments, it was found that the antimicrobial peptide 21546 has better antimicrobial activity against Gram-negative bacteria than Gram-positive bacteria, and its antimicrobial activity against marine Vibrio is higher than that against traditional strains. This may be closely related to the adaptive evolution of moon jellyfish. Marine jellyfish live in the marine environment and frequently come into contact with marine pathogenic microorganisms. Since Vibrio vulnificus is a common marine-specific Vibrio that can cause serious infection or even death in the host, the innate immune system of moon jellyfish may produce specific defense responses and antimicrobial molecules to resist the invasion of Vibrio vulnificus.

[0019] Therefore, the present invention also protects the use of the antimicrobial peptides in disrupting biofilm formation and / or already formed biofilms of pathogens.

[0020] Preferably, the pathogens include Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Pseudomonas aeruginosa.

[0021] More preferably, the dosage of the antimicrobial peptide is less than 50 μM. Studies of the present invention have shown that when the dosage of the antimicrobial peptide is less than 50 μM, the safety of the antimicrobial peptide to cells can be ensured, and hemolysis caused by cells can be avoided.

[0022] The in vivo antimicrobial activity of antimicrobial peptide 21546 was evaluated using an intraperitoneal infection model in ICR mice. Treatment with antimicrobial peptide 21546 significantly reduced bacterial burdens in mouse organs, with the most pronounced effect in the lungs. This phenomenon is hypothesized to be related to its pharmacokinetic properties: after intraperitoneal absorption, the antimicrobial peptide enters the bloodstream, resulting in high drug concentrations in the lungs. However, subsequent metabolic consumption in other organs reduces effective drug concentrations, likely due to its short half-life and poor stability in vivo. Serum levels of the inflammatory cytokines IL-6, IL-10, IFN-γ, and TNF-α were also significantly decreased. Furthermore, the extent of damage to tissues such as the liver, kidney, spleen, and lung, as well as the infiltration of inflammatory cells, were alleviated.

[0023] Therefore, the present invention also protects the use of the antimicrobial peptide in preparing a drug for treating and / or alleviating diseases caused by pathogen infection.

[0024] The present invention also protects the use of the antimicrobial peptide in preparing a drug for inhibiting inflammatory response caused by pathogen infection.

[0025] Preferably, in the above application, the pathogen is Vibrio vulnificus.

[0026] Preferably, in the above application, the disease is sepsis caused by Vibrio vulnificus infection.

[0027] More preferably, in the treatment of the above diseases, the dosage of the antimicrobial peptide is less than 15 mg / kg.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This study, focusing on the moon jellyfish (Aesculus truncatus), employed multi-omics techniques to analyze the differential expression of Aesculus genes following bacterial infection. An AI model was used to predict candidate antimicrobial peptides, which were then validated by measuring their MICs against bacteria, resulting in the identification of highly active Aesculus truncatus antimicrobial peptides. The in vitro activity of the antimicrobial peptides was further investigated using bactericidal kinetics and biofilm assays. The antimicrobial activity, hemolytic activity, cytotoxicity, and stability were then measured, and the antimicrobial mechanism was preliminarily investigated using electron microscopy and flow cytometry. The safety and antimicrobial activity of the antimicrobial peptides in vivo were further investigated using an animal model of intraperitoneal infection with Vibrio vulnificus.

[0030] The obtained antimicrobial peptide 21546, after modification and optimization, may become a potent lead peptide for the development of new antimicrobial drugs for marine pathogen infections, providing a new candidate drug for the treatment of marine biological injuries, secondary infections of naval combat injuries and clinical pathogenic bacteria infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Analysis of differentially expressed genes in moon jellyfish, including (A) statistical histogram of differentially expressed genes; (B) cluster diagram of each differential group;

[0032] Figure 2 Functional enrichment analysis of differentially expressed genes in the jellyfish infection model, including (A) GO enrichment; (B) KEEG enrichment;

[0033] Figure 3 The bactericidal kinetic curves of different concentrations of antimicrobial peptides against bacteria, including (A) Escherichia coli; (B) Staphylococcus aureus; (C) Vibrio vulnificus; (D) Vibrio parahaemolyticus; (E) Vibrio alginolyticus; (F) Pseudomonas aeruginosa; the control group is bacteria without drug;

[0034] Figure 4 Figure 3 Inhibitory activities of different concentrations of antimicrobial peptides and ampicillin on bacterial biofilm formation, including (A)-(C) Effects of 21546 on biofilm formation of Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus; (D)-(F) Effects of ampicillin on biofilm formation of Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus; Control group is the bacterial solution group without drug addition, n=3, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001;

[0035] Figure 5 Figure 3 Destructive activity of different concentrations of antimicrobial peptides and ampicillin on biofilms formed by bacteria. (A)-(C) Effects of 21546 on biofilm formation of Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus; (D)-(F) Effects of ampicillin on biofilm formation of Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus. Control group refers to the bacterial solution group without drug, n=3, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0036] Figure 6 The induction of resistance of Vibrio vulnificus to antimicrobial peptides and ampicillin, where antimicrobial peptide 21546 and ampicillin were treated with Vibrio vulnificus for 30 consecutive days. The MIC multiple corresponding to each day in the figure is the ratio of the MIC after each passage to the initial bacterial MIC before the first passage;

[0037] Figure 7 Cytotoxicity of antimicrobial peptides against RAW264.7 cells, n = 3, *, P < 0.05; **, P < 0.01; ****, P < 0.0001;

[0038] Figure 8is the hemolytic activity of antimicrobial peptides on rabbit erythrocytes, where Triton X-100 is the positive control; n = 3, *, P < 0.05; ****, P < 0.0001;

[0039] Figure 9 Effects of temperature and pH on antimicrobial peptide 21546, with pH = 7 as the control; n = 3, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001;

[0040] Figure 10 Scanning electron micrographs of Vibrio vulnificus treated with antimicrobial peptides. Note: Control: Vibrio vulnificus not treated with antimicrobial peptides. Scale bars are 5 μm and 1 μm (magnification).

[0041] Figure 11 Transmission electron micrographs of Vibrio vulnificus treated with antimicrobial peptides. Note: Control: Vibrio vulnificus not treated with antimicrobial peptides. Scale bars are 1 μm and 200 nm (magnification).

[0042] Figure 12 Flow cytometry analysis of membrane permeability of Vibrio vulnificus treated with different concentrations of antimicrobial peptides. (A) Blank; (B) MIC 21546; (C) 2MIC 21546; (D) PBS control group. The abscissa represents PI fluorescence intensity, and the ordinate represents cell counts. (E) Quantitative analysis of PI fluorescence-positive bacteria in different groups, n = 3, **, P < 0.01; ****, P < 0.0001.

[0043] Figure 13 The weight changes of mice treated with different concentrations of antimicrobial peptides;

[0044] Figure 14 The levels of inflammatory factors in the serum of mice in different drug treatment groups: (A) TNF-α level; (B) IL-6 level; (C) IL-10 level; (D) IFN-γ level. n = 5, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001;

[0045] Figure 15 Bacterial loads in the liver, kidney, spleen, and lung of mice in different drug treatment groups, including (A) liver; (B) kidney; (C) spleen; (D) lung; n = 6, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001;

[0046] Figure 16 H&E staining of the liver, kidney, spleen, and lung tissues of mice in different drug treatment groups. Scale bar: 100 nm. DETAILED DESCRIPTION

[0047] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0048] All moon jellyfish used in this experiment were captive-cultured and purchased from Qingdao, Shandong Province. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Vibrio vulnificus ATCC 27562, Pseudomonas aeruginosa ATCC 27853, Vibrio algaelyticus ATCC 33787, Vibrio parahaemolyticus ATCC 17802, and Vibrio harvey ATCC BAA-1117 were purchased from Shanghai Fuxiang Biological. All peptides used in this experiment were synthesized by Shanghai Jier Biochemical Co., Ltd. with a purity of >95%.

[0049] In this study, GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used to perform statistical analysis of the data. The results are presented as mean ± standard deviation (mean ± SD). Differences among multiple groups of data were analyzed using one-way ANOVA test, and the significance of the differences was analyzed using the Tukey test. P < 0.05 was considered statistically significant.

[0050] Example 1 Screening of new antimicrobial peptides from Aequorea jellyfish based on multi-omics and AI

[0051] 1. Collection of Moon Jellyfish Tissue Samples

[0052] All consumables that came into contact with samples in this experiment (including micropipette tips, centrifuge tubes, tissue disruption beads, and surgical instruments) were soaked in 0.1% diethyl pyrocarbonate (DEPC) solution and then sterilized at high temperature and high pressure to inactivate residual DEPC to prevent RNase contamination.

[0053] (1) Inoculate Vibrio vulnificus onto Luria-Bertani (LB) agar medium and culture at 37°C for 16-24 h. Pick a single colony and inoculate it into 10 mL of LB medium. Culture overnight at 37°C and 200 rpm / min. Dilute to 1 × 108 CFU / mL, centrifuge at 5000 rpm / min for 5 min, discard the supernatant, and resuspend in seawater for later use.

[0054] (2) Randomly select jellyfish of uniform size and inflict identical wounds on the oral arms. The wounded jellyfish are cultured for 8 h in water with a salinity of 30‰ at a bacterial concentration of 4.1×105 CFU / mL and in control seawater under the same conditions. Five jellyfish from each of the wounded and control seawater groups are rinsed with purified water, and tissue samples are cut and quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0055] 2. Transcriptome Sequencing and Bioinformatics Analysis

[0056] First, total RNA was extracted from the moon jellyfish tissue and stored at -80°C for later use.

[0057] In this study, transcriptome libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Library quality was subsequently tested using an Agilent 2100 Bioanalyzer to ensure compliance with standards. Sequencing was then performed on an Illumina Novasek 6000 sequencing platform, generating 150-bp paired-end reads. The entire transcriptome sequencing and analysis process was performed by Shanghai Ouyi Biotechnology Co., Ltd. Fastq-format raw data (rawreads) were processed using Trimmomatic. After data filtering, reads containing poly-N and low-quality reads were removed to obtain high-quality clean reads. These clean reads were then assembled to form expressed sequence tag clusters (contigs) by removing adapter sequences and low-quality regions. De novo transcript sequence assembly was performed using Trinity software. Based on sequence similarity and length alignment, the longest transcripts were selected as unigenes for subsequent bioinformatics analysis. Unigenes were compared with NCBI non-redundant (NR), Swiss-Prot, and gene evolutionary lineage databases to annotate the functions of Unigenes: diamond software was used for comparison, and e<1e-5 was used as the screening criterion. Unsupervised orthologous group analysis was performed through the eggNOG database, and orthologous group cluster annotation of the complete genome of eukaryotes was performed through the KOG database. Through comparison analysis, the protein sequence with the highest similarity to the Unigenes sequence was selected to obtain the corresponding functional annotation results. These Unigenes were also mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway annotation. Gene Ontology (GO) classification was performed by mapping the relationship between Swiss-Prot and GO terms. After Unigenes were annotated, bowtie2 software was used to count the number of reads aligned to Unigenes in each sample, and then eXpress

[29] The tool was used to quantitatively analyze Unigenes and obtain FPKM expression values. DESeq2 software was used to calculate the expression difference fold, and the statistical significance of differential expression was evaluated by negative binomial distribution test. The default conditions for screening differences were q < 0.05 and the difference fold (FoldChange) > 2. R software (v3.2.0) was used to perform hierarchical clustering of the screened differentially expressed genes (DEGs) to visualize the gene expression characteristics between different groups and samples. Subsequently, based on the principle of hypergeometric distribution, GO functional annotation and KEGG pathway enrichment analysis were performed using R software to explore the biological functions and metabolic pathway characteristics of the differentially expressed genes.

[0058] Based on the Illumina NovaSeq 6000 sequencing platform, this study conducted a comparative transcriptomics study on the Cnidaria species, the moon jellyfish, and compared the gene expression differences between normal moon jellyfish and those exposed to Vibrio vulnificus for 8 hours. Pathogen stimulation caused significant changes in jellyfish gene expression ( Figure 1 ), 1652 differentially expressed genes (DEGs) were identified by bioinformatics analysis, including 525 up-regulated genes and 1127 down-regulated genes.

[0059] The results of differential gene enrichment analysis are as follows Figure 2 As shown, 8 hours after bacterial challenge, differentially expressed genes were enriched in GO terms primarily related to biological processes such as glycogen metabolism, inflammatory response, fibrinolysis, and the intrinsic coagulation pathway. At the cellular level, these differentially expressed genes were primarily involved in components such as cells, organelles, membranes, and extracellular regions. Their molecular functions primarily included nutritional activity, molecular structural activity, and stimulus response. KEGG pathway enrichment analysis further revealed that the differentially expressed genes were primarily involved in pathways related to metabolism, ribosomes, thermogenesis, disease pathways, and cell proliferation, as well as pathways closely associated with immune regulation, such as the nuclear factor κB (NF-κB) signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and the interleukin-17 (IL-17) signaling pathway. This suggests that the moon jellyfish may be undergoing bacterial infection and cell proliferation, while also engaging in immune responses and tissue repair.

[0060] 3. Prediction of potential antimicrobial peptides from jellyfish

[0061] CAMPR4, an AI model, integrates multiple machine learning algorithms, including support vector machines (SVM), random forests (RF), artificial neural networks (ANN), discriminant analysis (DA), and hidden Markov models (HMM). It provides tools for AMP prediction and rational design (natural and synthetic AMPs), sequence analysis (BLAST and clustal omega), structure analysis (VAST), and family analysis (PRATT, ScanProsite, CAMPSign). Drawing on previous research, this study identified full-length protein sequences corresponding to differentially expressed genes in the moon jellyfish (Aequorea spp.) in FASTA format. Using CAMPR4's predition tool with a 20aa window size, the probability of all candidate protein sequences becoming functional antimicrobial peptides was predicted. The ANN reports either AMP or NAMP, with AMP indicating the sequence is predicted to be antimicrobial and NAMP indicating the sequence is not. SVM, RF, and DA assign predicted probability scores to the peptides, screening for peptide sequences with a probability greater than 0.9. Next, we evaluated the antimicrobial activity of the peptides against Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus using DBAASP, using machine learning (ML) methods and strain-specific antimicrobial prediction methods based on peptide sequence and bacterial genome data. The V. vulnificus genome ID (SAMN05196200) was used. Sequences showing antimicrobial activity against all three bacteria were ranked by average score, and the peptides with the highest scores were selected for solid-phase synthesis.

[0062] Analysis of transcriptome data from the moon jellyfish revealed that some differentially expressed genes may be involved in regulating the innate immune response, particularly encoding peptides with antimicrobial activity. To identify these potential antimicrobial molecules, this study employed high-throughput screening using machine learning-based AI models, such as CAMPR4 and DBAASP, to identify candidate peptide sequences with potential antimicrobial activity. CAMPR4 was first used to predict the likelihood of all candidate genes becoming functional antimicrobial peptides, screening for peptide sequences with a probability greater than 0.9. DBAASP was then used to predict the antimicrobial activity of these peptides against Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus. Eight peptides with the highest scores were identified, demonstrating antimicrobial activity against all three bacteria.

[0063] 4. Detection of MIC and MBC of Candidate Antimicrobial Peptides

[0064] In this paper, MIC was assessed by broth microdilution. Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi were cultured on LB agar plates to activate the strains and then incubated in cation adjusted Mueller Hinton broth (CAMHB) at 37°C and 200 rpm until the logarithmic growth phase. The culture medium was used to dilute the 10 6 A bacterial suspension containing 100 CFU / mL of antimicrobial peptides was prepared. Antimicrobial peptides were dissolved in PBS to a concentration of 10 mg / mL and diluted to 4000 μg / mL in culture medium. 50 μL of CAMHB broth was added to each well of a 96-well plate, covering ten columns. 50 μL of the prepared peptides was added to the first column. The drug concentration in each subsequent well was then diluted two-fold. Finally, 50 μL of the diluted bacterial suspension was pipetted into each well. The final antimicrobial peptide concentrations obtained were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 μg / mL, respectively. Standard antibiotics ampicillin and doxycycline served as positive controls, and wells containing bacterial suspension without antimicrobial peptides served as negative controls. The final volume per well was 100 μL. The 96-well plate was incubated at 37°C for 18 h, and the absorbance at a wavelength of 600 nm was measured. Three parallel experiments were set up for each group of experiments.

[0065] The minimum bactericidal concentration (MBC) is determined as follows: Following the previously described MIC determination method, the plate containing the sample is incubated at 37°C for 18 hours. Subsequently, 100 μL of bacterial suspension is aspirated from each well and evenly spread onto a plate containing a selective culture medium for the target bacteria. The inoculated plates are incubated in a 37°C incubator overnight. The next day, the growth of colonies on each plate is observed and recorded. The lowest concentration of the drug that completely kills the bacteria in the culture medium is the MBC value for the target strain.

[0066] To validate the antibacterial activity of the predicted antimicrobial peptides, eight peptide sequences were synthesized by solid-phase synthesis and their antimicrobial activity was verified using Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus as test strains. The MICs for 21546 against E. coli were 26.13 μM, 6.53 μM against S. aureus, and 13.06 μM against V. vulnificus. Since lower MIC values ​​indicate stronger antimicrobial activity, 21546 was selected as a candidate antimicrobial peptide for further investigation. Its MICs against three marine Vibrio species and Pseudomonas aeruginosa were determined (Table 1). The results showed that the MICs for 21546 against Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa were 52.25 μM, 6.53 μM, 3.27 μM, and 104.5 μM, respectively.

[0067] Table 1 MIC of antimicrobial peptide 21546 against four bacteria

[0068]

[0069] The MBC of 21546 against four marine Vibrio and three common strains was determined by plate bioassay (Table 2). The results showed that the MBC of antimicrobial peptide 21546 against Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Pseudomonas aeruginosa were 104.5 μM, 13.06 μM, 13.06 μM, 52.25 μM, 26.13 μM, 13.06 μM and 418 μM, respectively.

[0070] Table 2 MBC of antimicrobial peptide 21546 against four marine Vibrio and three common strains

[0071]

[0072]

[0073] The two antimicrobial peptides were then compared against the CAMPR4 antimicrobial peptide database, which contains a rich collection of antimicrobial peptide data, including 24,243 AMP sequence information, 933 structural data, 2,143 patent records, and characteristic information for 263 different AMP families. The results in Table 3 show that the antimicrobial peptide from the moon jellyfish is a novel antimicrobial peptide sequence.

[0074] Table 3 Comparison results of antimicrobial peptides from Aequorea truncatula and CAMPR4 database

[0075]

[0076] Example 2 Physicochemical properties, in vitro activity and mechanism studies of antimicrobial peptides

[0077] 1. Determination of time-kill curve

[0078] Bactericidal kinetics were determined according to the method of Wei et al., with minor modifications. The activated strain was cultured in fresh LB medium at 37°C and 200 rpm / min until the logarithmic growth phase. The final bacterial concentration was adjusted to 1 × 106 CFU / mL using the culture medium. Antimicrobial peptide solution was added to final concentrations of 1 × MIC, 5 × MIC, and 10 × MIC, respectively. After mixing, the mixture was incubated in a 37°C incubator. A bacterial solution without antimicrobial peptide was used as a control. Samples were collected at 0, 5, 10, 30, 60, 180, and 240 min, diluted, and plated on LB agar plates. The colony count was counted after overnight incubation at 37°C.

[0079] This study selected three clinical common strains and three marine Vibrio as experimental strains and evaluated the inhibitory effect of antimicrobial peptide 21546 on the growth activity of the strains. Figure 3 Experimental data showed that the antimicrobial peptide exhibited an inhibitory effect on Staphylococcus aureus. When the concentration reached or exceeded 5×MIC, it exhibited significant bactericidal effects against Escherichia coli, Vibrio vulnificus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa, killing these strains within 30 minutes.

[0080] 2. Biofilm Experiment

[0081] In order to determine the biofilm inhibition ability of antimicrobial peptide 21546, experiments on inhibiting biofilm formation and destroying formed biofilm were carried out using crystal violet staining. 5 To determine the CFU / mL, 180 μL of bacterial culture was added to 20 μL of a serially two-fold diluted antimicrobial peptide solution in a 96-well plate. The final concentrations of the antimicrobial peptide serial dilutions were 0.125×MIC, 0.25×MIC, 0.5×MIC, and 1×MIC. A bacterial culture without antimicrobial peptide served as a control, and a LB medium group served as a blank. After 24 hours of incubation, the samples were washed with sterile phosphate-buffered saline to remove supernatant bacteria. The cultures were then fixed in 99% methanol for 15 minutes. The methanol was then removed, and the 96-well plates were air-dried. 200 μL of 0.1% crystal violet stain was added to each well and allowed to stand for 15 minutes to complete the staining process. Excess stain was then gently rinsed with distilled water to remove. Finally, the stained samples were immersed in 95% ethanol to dissolve the stain, and absorbance was measured at 600 nm.

[0082] In order to determine the activity of antimicrobial peptides in destroying the formed biofilm, 200 μL of 1×10 5CFU / mL bacterial suspension was added to a 96-well plate and incubated in a 37°C incubator for 24 hours to form a biofilm. After rinsing the bacteria with PBS, a serial dilution of the antimicrobial peptide was prepared using fresh culture medium, and 200 μL of the suspension was then transferred to the well plate containing the biofilm. 200 μL of culture medium was added to each well of the control group and the blank group. After the culture plate was incubated at 37°C for another 24 hours, the crystal violet staining method described above was used. The percentage of biofilm formed = (OD 样品 –OD 空白 ) / (OD 对照 –OD 空白 )×100%.

[0083] Biofilm inhibition test results ( Figure 4 ) showed that when the antimicrobial peptide concentration was ≥0.5 times the MIC, it significantly inhibited biofilm formation in the three tested strains. Further analysis showed that at the MIC concentration, antimicrobial peptide 21546 inhibited biofilm formation by 69.57%, 61.44%, and 53.7% against Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus, respectively. In comparison, ampicillin had a lower inhibitory effect against these strains, with inhibition rates of 42.11%, 40.93%, and 42.12%, respectively.

[0084] The destructive effect of antimicrobial peptides on bacterial biofilms Figure 5 As shown in the figure, at the MIC concentration, the antimicrobial peptide 21546 had a biofilm destruction rate of 60.61%, 56.65%, and 43.86% against Escherichia coli, Staphylococcus aureus, and Vibrio vulnificus, respectively. The traditional antibiotic ampicillin had a relatively low effect on these bacterial biofilms, with destruction rates of 35.98%, 38.14%, and 28.76%, respectively.

[0085] 3. Resistance induction assay

[0086] The strain was cultured in a shaking incubator at 37°C overnight, and the bacterial suspension was diluted to 1×10 5 CFU / mL. The bacterial suspension was mixed with the antimicrobial peptide solution in a volume ratio of 9:1 to make the final concentration of the peptide substance reach the sub-MIC level. The mixed system was placed in a 37°C constant temperature shaker and cultured overnight at a speed of 180 rpm / min. Subsequently, the MIC value of the antimicrobial peptide against Vibrio vulnificus was determined by microdilution method. In subsequent experiments, the cultured bacterial suspension was adjusted to the same concentration gradient with fresh culture medium every day and transferred to a sterile test tube, and an appropriate amount of antimicrobial peptide was added to maintain its final concentration at the sub-MIC level. The culture was continued for 30 generations, and the ratio of the MIC after each passage to the initial MIC before the first generation was taken as the fold change of MIC.

[0087] In this study, a 30-generation continuous subculture experiment was conducted on Vibrio vulnificus. In the initial culture, Vibrio vulnificus showed different sensitivities to the three antimicrobial agents ( Figure 6 The MICs of 21546 and ampicillin were 13.06 μM and 8.94 μM, respectively. After 30 consecutive passages, the MIC of ampicillin increased significantly, by 8-fold. In contrast, the MIC of the antimicrobial peptide remained stable throughout the passages, showing no significant changes. These findings suggest that V. vulnificus is less likely to develop resistance to the antimicrobial peptide 21546 than to ampicillin.

[0088] IV. Cytotoxicity Assay

[0089] Rapidly transfer the RAW264.7 cells frozen in liquid nitrogen to a 37°C constant temperature water bath and gently shake the cryovials for rapid recovery. Transfer the cell suspension to 5 mL of DMEM complete medium containing 10% fetal bovine serum (FBS) and centrifuge at 2000 rpm / min for 5 minutes to remove the supernatant. Resuspend the cell pellet in 1 mL of fresh DMEM medium and then transfer it to a culture flask pre-added with complete medium. Gently mix to evenly distribute the cells and culture in a 37°C, 5% CO2 constant temperature incubator. After the cells have reached a stable growth state after 2-3 generations of subculture, adjust the cell density to 2×10 5 cells / mL, 100 μL per well was inoculated into a 96-well culture plate and cultured at 37°C for 24 hours. After removing the culture supernatant, 100 μL of culture medium containing gradient concentrations of antimicrobial peptides was added to the experimental wells, 100 μL of complete culture medium was added to the control wells, and another set of complete culture medium without cells was set up as a blank well and cultured for another 24 hours. Finally, 10 μL of CCK-8 solution was added to each well, incubated in the dark for 0.5-4 hours, and the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. Three parallel wells were set up for each group.

[0090] Cell survival rate (%) = [(A 样品 -A 空白 ) / (A 对照 -A 空白 )]×100, A is the absorbance at 450 nm.

[0091] In this study, we selected the mouse-derived RAW264.7 cell line as the research object and evaluated the potential toxic effects of different concentrations of antimicrobial peptides on cells using the CCK-8 assay. Figure 7 It was shown that when the concentration of antimicrobial peptide 21546 reached or exceeded 50 μM, it exhibited a certain cytotoxic effect on RAW264.7 cells.

[0092] 5. Hemolytic activity detection

[0093] Following the method of Wu et al. with slight modifications, serial dilutions of the antimicrobial peptide were prepared in PBS buffer at concentrations ranging from 3.125 to 100 μM. An equal volume of a 4% rabbit erythrocyte suspension was added and mixed. Fifty μL of the erythrocyte suspension was mixed with equal volumes of 1% Triton X-100 and PBS buffer to serve as 100% hemolysis and 0% hemolysis controls, respectively, with three replicates per group. After incubation at 37°C for 1 hour, the cells were centrifuged at 1000 rpm / min for 10 minutes. The supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm.

[0094] Hemolytic activity calculation formula

[0095] Hemolytic activity (%) = [(A 样品 -A 0％lysis ) / (A 100％lysis -A 0％lysis )]×100, where A is the absorbance at 540 nm.

[0096] Hemolytic activity is one of the key indicators for measuring the safety of antimicrobial peptides. In order to evaluate the hemolytic activity of antimicrobial peptide 21546, we used a 4% rabbit red blood cell suspension to measure the hemolytic rate of these two antimicrobial peptides at different concentrations. Figure 8 As shown in the figures, when the concentration of antimicrobial peptide 21546 was no more than 50 μM, the hemolytic rate of antimicrobial peptide 21546 on rabbit erythrocytes was less than 10%, indicating that it had no significant hemolytic activity.

[0097] 6. Temperature detection

[0098] Effect of Temperature on Antimicrobial Peptide Stability: Culture the strain overnight at 37°C, 200 rpm / min, and dilute the bacterial suspension to a concentration of 106 CFU / mL with fresh CAMHB medium. Prepare a 10 mg / mL solution of the antimicrobial peptide in PBS buffer and treat at -20, 30, 70, and 100°C for 30 min. MICs of the strain were determined using the same method as in 1.2.5. Three replicates were performed for each test.

[0099] Effect of pH on the stability of antimicrobial peptides: PBS buffer was adjusted to pH 2, 5, 7, and 9, and antimicrobial peptide solutions were prepared using PBS buffers of different pH values. The MIC of the antimicrobial peptides against bacteria was determined at these pH values.

[0100] This study used Vibrio vulnificus as an indicator strain and systematically investigated the effects of temperature and pH on the activity of antimicrobial peptides by measuring the changes in MIC. Figure 9), under high temperature conditions of 100°C, the antibacterial activity of antimicrobial peptides against Vibrio vulnificus decreased. Further analysis of the effect of pH value found that the activity of antimicrobial peptide 21546 decreased in a strongly acidic environment (pH = 2). These findings also provide an important reference basis for further optimizing the structure of antimicrobial peptides and improving their environmental stability.

[0101] 7. Electron microscopy observation and analysis

[0102] Scanning electron microscopy: Vibrio vulnificus was cultured to the logarithmic growth phase, removed, and centrifuged at 3000 rpm / min for 5 minutes until mung bean-sized cells remained at the bottom of the tube. The supernatant was discarded and the tube was washed three times with PBS. The antimicrobial peptide solution was added to the test bacteria to a final concentration of the MIC. The PBS-treated bacteria served as the control and were incubated at 37°C, 120 rpm / min, for 1 hour. After treatment, the bacteria were centrifuged at 3000 rpm / min for 5 minutes, and the supernatant was discarded. Glutaraldehyde fixative was added and fixed at 4°C for at least 6 hours. Dehydration was performed using a gradient of 50%, 70%, 80%, and 90% ethanol for 15 minutes each, followed by dehydration three times with 100% ethanol for 30 minutes each. The samples were dried using a freeze-vacuum dryer, the bacterial surface was gold-coated, and the samples were observed under a scanning electron microscope.

[0103] Transmission Electron Microscopy: Bacteria were prepared using the same conditions as those described for scanning electron microscopy. The antimicrobial peptide solution was added to the test bacteria to a final concentration of the MIC. A PBS-treated group served as a control and the cells were incubated at 37°C, 120 rpm / min, for 1 hour. Following treatment, the cells were centrifuged at 3000 rpm / min for 5 minutes, and the supernatant discarded. Glutaraldehyde fixative was added and fixed at 4°C for 2-4 hours. The cells were washed three times with 0.1 M PBS for 15 minutes each. The cells were then fixed with 1% osmium hydroxide in 0.1 M PBS for 2 hours at room temperature and washed three times with PBS. The samples were dehydrated using the aforementioned ethanol gradient and then dehydrated with 100% acetone for 15 minutes. The cells were treated with a 1:1 ratio of acetone to 812 embedding medium for 2 hours and a 2:1 ratio overnight. The cells were then transferred to pure 812 embedding medium for 4 hours and incubated overnight at 37°C in embedding plates. The specimens were sliced ​​using a microtome, stained with a 2% saturated aqueous solution of uranyl acetate and lead citrate for 15 min, and observed under a transmission electron microscope.

[0104] This study used Vibrio vulnificus as the research object and observed the effect of antimicrobial peptide 21546 on the morphological structure of bacterial cells using scanning electron microscopy. The experiment set up an antimicrobial peptide treatment group with MIC concentration and a blank control group, and sample preparation and observation were performed after 1 hour of treatment. Figure 10As shown, untreated Vibrio vulnificus maintained a complete, long rod-shaped morphology, with intact cell structure and evenly distributed intracellular material, with no leakage of contents. In contrast, bacteria treated with antimicrobial peptides exhibited significant morphological changes: noticeable wrinkles and depressions appeared on the cell surface, the integrity of the cell membrane was compromised, and a large amount of cytoplasmic contents leaked out, ultimately resulting in a deflated cell state. These morphological changes confirm that antimicrobial peptides exert their antimicrobial effects by disrupting bacterial cell membrane structure.

[0105] The ultrastructure of the treated Vibrio vulnificus was observed using a transmission electron microscope. Figure 11 As shown, the bacterial cells in the control group exhibited typical ultrastructural features: intact and continuous cell membrane structure, uniform distribution of cytoplasmic substances, and high electron density. In stark contrast, bacteria treated with antimicrobial peptide 21546 exhibited severe membrane damage: local perforation and rupture of the cell membrane, and membrane structural collapse accompanied by a significant decrease in electron density. These morphological evidences suggest that antimicrobial peptides may cause the outflow of intracellular substances by altering the permeability of the plasma membrane.

[0106] 8. Flow cytometry to detect the permeability of your bacterial cell membrane

[0107] Collect Vibrio vulnificus in the logarithmic growth phase and dilute it to 10 8 Bacterial suspensions were prepared at 100 CFU / mL and mixed with different concentrations of antimicrobial peptides at a 1:1 volume ratio to achieve final concentrations of 1 and 2 times the MIC, respectively. The suspensions were incubated at 37°C for 1 hour at 3000 rpm, centrifuged for 5 minutes, and the supernatant discarded. After washing three times with PBS, the suspensions were stained with 200 μL of propidium iodide (PI) and incubated in the dark for 30 minutes. The effects of the antimicrobial peptides on the membrane permeability of V. vulnificus were analyzed using flow cytometry.

[0108] The experiment used propidium iodide (PI) fluorescence labeling method for quantitative detection. Flow cytometry detection data ( Figure 12 ) showed that under the treatment condition of 2 times MIC concentration, the membrane permeability of Vibrio vulnificus treated with antimicrobial peptide 21546 was significantly improved compared with the control group. The quantitative results were highly consistent with the morphological changes observed by electron microscopy, which jointly confirmed that antimicrobial peptides can achieve antibacterial effects by destroying the bacterial cell membrane structure.

[0109] Example 3 In vivo antimicrobial effect of antimicrobial peptides

[0110] The Vibrio vulnificus of this example was purchased from Shanghai Fuxiang Biological Co., Ltd., and SPF-grade ICR female mice were purchased from Shanghai Bikekeyi Biotechnology Co., Ltd. The breeding environment was subjected to a light-dark cycle every 12 hours.

[0111] Prior to in vivo experiments, this study evaluated the biosafety of the antimicrobial peptide 21546. Twenty-four female ICR mice weighing 26-27 g were randomly divided into four groups: a blank control group and groups receiving low, medium, and high doses of the antimicrobial peptide. Mice were intraperitoneally injected daily for seven consecutive days at doses of 7.5 mg / kg, 15 mg / kg, and 30 mg / kg. The blank control group received saline. The mice were weighed daily, and their behavior and mental status were observed.

[0112] The safety of antimicrobial peptide 21546 in vivo was preliminarily evaluated by short-term continuous intraperitoneal injection of ICR mice and monitoring of body weight change rate and behavioral characteristics. The experimental data showed that all mice in the treatment group showed a dose-dependent weight loss trend. Figure 13 As shown. After each administration, mice in each dose group showed slow movement and returned to normal state 1 hour after administration. It is speculated that this may be related to the local pain response caused by peritoneal irritation. It is worth noting that the low-dose group (7.5 mg / kg) did not show significant physiological abnormalities in the later stage of the experimental period (days 5-7), and its hair condition was not significantly different from that of the control group. The medium and high-dose groups (≥15 mg / kg) continued to show toxicity symptoms such as decreased autonomous activity, chills, and dull hair. After seven consecutive days of administration, the body weight fluctuation of the 15 mg / kg group was maintained within 5%, which is in line with the ethical requirements of experimental animals. Comprehensive analysis shows that subsequent in vivo studies should choose a dosage of ≤15 mg / kg to ensure experimental safety.

[0113] 1. Analysis of Serum Inflammatory Factors in Mice

[0114] The in vivo activity of antimicrobial peptide 21546 was determined using a Vibrio vulnificus infection mouse model. Twenty female ICR mice weighing 21-22 g were used. The mice were divided into an infection model group, an antimicrobial peptide 21546-treated group, an ampicillin-treated group, and a blank group, with five mice in each group. All mice were fasted for 12 hours prior to inoculation with the bacterial solution. The mice were then intraperitoneally injected with 0.1 mL / 10 g (approximately 1.5 × 10 8 CFU / mL) was used for infection modeling. 2 hours after infection, the antimicrobial peptide 21546 group was injected with 5 mg / kg, the ampicillin group was injected with 32 mg / mL, the model group was injected with the same volume of normal saline, and the blank control group was not treated. 4 hours after infection, all mice were killed and orbital blood was collected. Serum was collected by centrifugation at 3000 rpm / min for 15 minutes, and the expression levels of inflammatory cytokines interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ (IFN-γ), and TNF-α in the serum were evaluated using a mouse ELISA kit.

[0115] The immunomodulatory properties of antimicrobial peptides were evaluated by dynamic monitoring of inflammatory factors. Figure 14 As shown in Figure 2, infection with Vibrio vulnificus significantly induced abnormal increases in serum levels of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ, as well as the anti-inflammatory cytokine IL-10 in mice. Notably, administration of antimicrobial peptide 21546 and ampicillin 2 hours after infection significantly downregulated cytokine levels in all four groups. These results suggest that antimicrobial peptides can inhibit excessive inflammatory responses.

[0116] 2. Analysis of bacterial burden in multiple organs of mice

[0117] Eighteen female ICR mice weighing 21-22 g were randomly selected and divided into an infection model group, an antimicrobial peptide 21546-administered group, and an ampicillin-administered group, with six mice in each group. The treatment method was the same as in 3.2.2, and liver, kidney, spleen, and lung tissues were collected for bacterial quantitative analysis. After the wet weight of the tissue samples was accurately weighed using an electronic balance, 500 μL of pre-chilled PBS buffer was added, and a uniform suspension was prepared using a low-temperature tissue homogenizer. The homogenate was diluted in a gradient, and 100 μL of the dilution was evenly inoculated on the surface of LB agar medium. After constant temperature incubation at 37°C for 24 hours, the colony-forming units were counted.

[0118] In this study, a mouse model of intraperitoneal infection with Vibrio vulnificus was established to systematically evaluate the effects of antimicrobial peptides on the biodistribution of the pathogen. Two hours after intraperitoneal infection, mice were intraperitoneally injected with 5 mg / kg 21546 and 32 mg / kg ampicillin, respectively, while the control group was injected with normal saline. Two hours after administration, the mouse organs were harvested and the colony counts in the liver, kidney, spleen, and lungs were counted. The bacterial load in each organ was as follows: Figure 15 The results showed that compared with the control group, the number of colonies in the organs of mice in all drug-treated groups decreased to varying degrees, among which the antibacterial effect in lung tissue was the most prominent.

[0119] 3. Histopathological Analysis of Mice

[0120] Twelve ICR female mice weighing 21-22 g were randomly divided into four groups, each containing three mice: an infection model group, an antimicrobial peptide 21546-treated group, an ampicillin-treated group, and a blank control group. Treatment of each group was identical to that described in step 1. The liver, kidney, spleen, and lungs were then removed, washed with PBS, and fixed in 5 mL of 4% paraformaldehyde. Following tissue fixation, tissue sections were routinely embedded in paraffin. Sections were sequentially treated with the following procedure: immersion in xylene I for 20 minutes, followed by transfer to xylene II for another 20 minutes, followed by treatment with anhydrous ethanol I (5 minutes), anhydrous ethanol II (5 minutes), and 75% ethanol (5 minutes), followed by rinsing with running tap water. Sections were stained with hematoxylin for 3-5 minutes, rinsed with tap water, treated with differentiation solution, and rinsed again. Sections were then treated with bluing solution and rinsed thoroughly with running water. Dehydration was performed sequentially through 85% and 95% graded ethanol treatments for 5 minutes each, followed by staining in eosin solution for 5 minutes. The samples were then placed in anhydrous ethanol I, II, and III for 5 minutes each, followed by clearing in xylene I and II for 5 minutes each. Finally, the slides were mounted with neutral gum and analyzed under a laser confocal microscope.

[0121] The protective effect of antimicrobial peptides on organ damage was evaluated by histopathology. Mice were intraperitoneally infected with Vibrio vulnificus for 2 hours. Two hours after the drug treatment, the liver, kidney, spleen and lung of the mice were obtained and observed by H&E staining. Figure 16 The results showed that compared with the blank control group, the model group showed disorganized hepatocytes, extensive hydropic degeneration, and nuclear lysis. In the groups treated with antimicrobial peptide 21546 and ampicillin, the hepatic cord structure returned to normal, and the areas of cell necrosis were reduced. Compared with the blank control group, the model group showed interstitial hemorrhage in the renal medullary tissue and vacuolar degeneration of the renal tubular epithelium. Hemorrhage and granular degeneration decreased in the kidneys of all treatment groups, with the antimicrobial peptide 21546 group showing a more pronounced effect. Compared with the blank control group, the red and white pulp structures of the spleen in the model group showed blurred boundaries, with disorganized and loosely arranged tissue cells. After treatment, the tissue arrangement returned to normal. Compared with the blank control group, the model group showed thickening of the alveolar walls, infiltration of numerous inflammatory cells, and desquamation of the alveolar epithelium in the lungs. After treatment, the alveolar wall thickening and inflammatory cell infiltration were alleviated.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antimicrobial peptide for marine pathogen infection, characterized in that: The sequence of the antimicrobial peptide is: GYGHLLRYLARNFISKVRLN.

2. A preparation containing the antimicrobial peptide according to claim 1.

3. A method for preparing the antimicrobial peptide according to claim 1.

4. Use of the antimicrobial peptide according to claim 1 in inhibiting pathogen infection in vitro.

5. Use of the antimicrobial peptide according to claim 1 in destroying pathogen biofilm formation and / or existing biofilm.

6. The use according to claim 4 or 5, characterized in that The pathogens include Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Pseudomonas aeruginosa.

7. Use of the antimicrobial peptide according to claim 1 in the preparation of a medicament for treating and / or alleviating diseases caused by pathogen infection.

8. Use of the antimicrobial peptide according to claim 1 in the preparation of a medicament for inhibiting inflammatory response caused by pathogen infection.

9. The use according to claim 7 or 8, characterized in that The pathogen is Vibrio vulnificus.

10. The use according to claim 9, characterized in that The disease is sepsis caused by Vibrio vulnificus infection.

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