A novel antibacterial peptide against marine pathogen infection and application thereof

Abstract

The application belongs to the technical field of antibacterial peptides, and discloses a novel antibacterial peptide for marine pathogen infection and application thereof. The application takes Aequorea victoria as a research object, analyzes the differential expression of genes of Aequorea victoria after bacterial infection by means of multi-omics technology, predicts candidate antibacterial peptides by using an AI model, verifies the activity by determining the MIC of the polypeptide on bacteria, and thus obtains a high-activity Aequorea victoria antibacterial peptide. The in-vitro activity of the antibacterial peptide is further explored. Then, the hemolytic activity, cytotoxicity and stability of the antibacterial peptide are determined, and the antibacterial mechanism thereof is preliminarily studied. The safety and antibacterial activity of the antibacterial peptide in vivo are further explored through an animal model of Vibrio vulnificus peritoneal infection. The obtained antibacterial peptide 21546, after modification and optimization, is likely to become a powerful lead peptide for developing a novel antibacterial drug for marine pathogen infection, and provides a novel candidate drug for treating secondary infection of marine biological injury, sea combat injury and clinical pathogenic bacteria infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial peptides, and more particularly relates to a novel antibacterial peptide against marine pathogen infection and application thereof. BACKGROUND

[0002] There are often complex microbial colonization on the surface of marine organisms and in the oral cavity. When the human body is stung, stabbed, bitten or wounded by marine organisms, it often causes infection. On the sea surface, the open wound exposed to seawater environment after skin trauma or marine organism injury can quickly cause local tissue inflammatory edema and severe pain reaction due to the dual effects of high osmotic fluid infiltration and microbial invasion. In severe cases, even progressive tissue necrosis occurs, which not only affects wound healing, but also threatens life if severe sepsis occurs. Marine environment sample detection shows that there are more than 20 kinds of bacteria that have obvious pathogenic effects on the human body, most of which belong to halophilic Vibrio, among which Vibrio vulnificus causes the most common and most severe infection. According to epidemiological statistics, the mortality rate of sepsis caused by Vibrio vulnificus infection can be as high as 50%. On the other hand, with the widespread use and abuse of antibiotics in recent years, drug-resistant bacteria and multi-drug resistant bacteria that cannot be controlled by conventional drugs continue to appear, causing serious health and medical problems, including marine pathogenic bacteria. As early as 2013, a study analyzed the antibiotic sensitivity of marine bacteria in Chinese waters, and the results showed that these marine bacteria showed varying degrees of resistance to some commonly used clinical antibiotics. In order to solve this problem, it is urgent to find new antibacterial drugs that can replace traditional antibiotics to combat drug-resistant bacteria.

[0003] Antibacterial peptides (AMPs) are an important part of the natural immune system of many organisms, generally have 10-50 amino acids, and are also known as host defense peptides. They have good inhibitory activity against bacteria, fungi and viruses. The positively charged peptides interact with the negatively charged bacterial cell membrane through electrostatic interaction, leading to changes in the permeability of the cell membrane, rupture of the cell membrane, and leakage of intracellular ions and metabolites. This unique mechanism makes it difficult for bacteria to evolve drug resistance, and the rapid bactericidal rate of antibacterial peptides makes them promising candidates for preventing and controlling bacterial infections. Marine organisms are an important source of discovery of antibacterial active molecules. Marine invertebrates mainly rely on the non-specific immune system, and in the process of long-term adaptation to the high-competitive and dynamic marine ecosystem, they have likely evolved a unique immune defense mechanism to protect their population from survival and reproduction. Many studies have isolated antibacterial peptides from marine organisms.

[0004] Jellyfish belong to the phylum Cnidaria, which is an ancient marine invertebrate that has evolved for hundreds of millions of years. Jellyfish are diverse in species and are distributed in various marine environments, and their body cavity is open and immersed in seawater, with a large contact area with seawater. The body surface is in contact with various pathogens for a long time, which may evolve unique innate immune systems and antibacterial active molecules, and studies have found antibacterial peptides in Aurelia aurita. However, due to the difficulty and low efficiency of directly separating and enriching effective components from jellyfish, the antibacterial peptide resources in jellyfish are still a potential treasure trove of underdeveloped potential drugs. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a novel antibacterial peptide against marine pathogen infection.

[0006] The second purpose of the present application is to provide the use of the above antibacterial peptide.

[0007] The third purpose of the present application is to provide a preparation containing the above antibacterial peptide.

[0008] The fourth purpose of the present application is to provide the use of the above antibacterial peptide in the preparation of a drug for treating diseases caused by marine pathogen infection.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] An antibacterial peptide against marine pathogen infection, the sequence of the antibacterial peptide is: GYGHLLRYLARNFISKVRLN.

[0011] In this study, transcriptomics and artificial intelligence technology were used to jointly analyze the innate immune response of Aurelia aurita. Bioinformatics analysis revealed that differentially expressed genes were significantly enriched in nucleic acid metabolism-related pathways, suggesting that pathogen stimulation may enhance host defense ability by activating nucleotide-mediated cell proliferation signals and transcriptional regulation mechanisms. Through transcriptomic analysis combined with artificial intelligence prediction and experimental verification, a novel antibacterial peptide 21546 was obtained, which has low homology with the reported antibacterial peptides. The antibacterial peptides are derived from differentially expressed genes of the Aurelia aurita infection model, and may be involved in the innate immune response of jellyfish and exert antibacterial activity.

[0012] Therefore, the present application also protects the preparation containing the antibacterial peptide.

[0013] The present application also protects the preparation method of the antibacterial peptide.

[0014] The anti-bacterial peptide 21546 has MIC ranging from 3.16 μM to 104.5 μM against E. coli, S. aureus, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. harveyi and P. aeruginosa by micro-broth dilution method. Further study shows that the anti-bacterial peptide exhibits rapid bactericidal effect at 5 times of MIC concentration, and can effectively kill E. coli, V. vulnificus, V. parahaemolyticus and P. aeruginosa within 30 minutes. The rapid killing property can significantly shorten the stress response time of bacteria, thereby effectively reducing the probability of drug resistance mutation of bacteria.

[0015] Therefore, the present application also protects the use of the anti-bacterial peptide in inhibiting pathogen infection in vitro.

[0016] Preferably, the pathogen includes E. coli, S. aureus, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. harveyi and P. aeruginosa.

[0017] More preferably, the anti-bacterial peptide is used in an amount less than 50 μM. The present application shows that when the anti-bacterial peptide is used in an amount less than 50 μM, the safety of the anti-bacterial peptide to cells can be ensured, and hemolysis caused by cells can be avoided.

[0018] As one of the key factors of bacterial drug resistance mechanism, the formation of biofilm significantly enhances the drug resistance of microorganisms. The biofilm experiment of the present application shows that the anti-bacterial peptide 21546 can effectively inhibit the formation of bacterial biofilm at MIC concentration and has a significant destructive effect on mature biofilm. Drug resistance induction experiment further confirms that V. vulnificus is not prone to drug resistance to the anti-bacterial peptide. Through these preliminary bioactivity experiments, it is found that the anti-bacterial activity of the anti-bacterial peptide 21546 to gram-negative bacteria is better than that to gram-positive bacteria, and the anti-bacterial activity to marine vibrio is higher than that to traditional strains. This may be closely related to the adaptive evolution of A. japonica. A. japonica lives in the marine environment and frequently contacts with marine pathogenic microorganisms. Since V. vulnificus is a common marine specific vibrio, which can cause severe infection and even death of the host, the innate immune system of A. japonica may produce specific defense response and anti-bacterial molecules to resist the invasion of V. vulnificus.

[0019] Therefore, the present application also protects the use of the anti-bacterial peptide in destroying the formation of pathogen biofilm and / or the formed biofilm.

[0020] Preferably, the pathogen includes E. coli, S. aureus, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. harveyi and P. aeruginosa.

[0021] More preferably, the anti-bacterial peptide is used in an amount less than 50 μM. The present application shows that when the anti-bacterial peptide is used in an amount less than 50 μM, the safety of the anti-bacterial peptide to cells can be ensured, and hemolysis caused by cells can be avoided.

[0022] The in vivo antibacterial activity of the antibacterial peptide 21546 was evaluated by an ICR mouse intraperitoneal infection model. After treatment with the antibacterial peptide 21546, the bacterial load in the organs of the mice was significantly reduced, especially in the lungs. It is speculated that this phenomenon may be related to the pharmacokinetic characteristics: after intraperitoneal absorption, the antibacterial peptide enters the blood circulation, resulting in a higher drug concentration in the lungs, while the effective drug concentration in the subsequent organs is reduced due to metabolic consumption, which may be closely related to its short half-life and poor stability in vivo. At the same time, the levels of inflammatory factors IL-6, IL-10, IFN-gamma and TNF-alpha in the serum were also significantly decreased, in addition, the degree of damage and inflammatory cell infiltration in tissues such as liver, kidney, spleen and lung were also reduced.

[0023] Therefore, the application also protects the use of the antibacterial peptide in the preparation of a drug for treating and / or alleviating a disease caused by pathogen infection.

[0024] The application also protects the use of the antibacterial peptide in the preparation of a drug for inhibiting inflammatory response caused by pathogen infection.

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

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

[0027] More preferably, in the above disease treatment, the amount of antibacterial peptide is less than 15 mg / kg.

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

[0029] In this study, A. australis was used as the research object, and the differential expression of genes in A. australis after bacterial infection was analyzed by multi-omics technology. The AI model was used to predict candidate antibacterial peptides, and the activity of the polypeptides was verified by determining the MIC of the polypeptides on bacteria, thereby obtaining high-activity A. australis antibacterial peptides. The in vitro activity of the antibacterial peptides was further explored by bactericidal kinetics test, biofilm experiment and other methods. Then, the hemolytic activity, cytotoxicity and stability of the antibacterial peptides were determined, and electron microscopy and flow cytometry analysis were used for preliminary study on the antibacterial mechanism. The safety and antibacterial activity of the antibacterial peptides in vivo were further explored by an animal model of Vibrio vulnificus intraperitoneal infection.

[0030] The obtained antibacterial peptide 21546 can be a powerful lead peptide for developing new antibacterial drugs against marine pathogen infection after optimization, and can provide a new candidate drug for treating secondary infection of marine biological injury and sea combat injury and clinical pathogenic bacteria infection. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1(A) Differential expression gene statistical column chart; (B) Cluster diagram of each differential group for Aequorea victoria differential expression gene analysis;

[0032] Figure 2 Functional enrichment analysis of differential expression genes of jellyfish infection model, wherein (A) GO enrichment; (B) KEEG enrichment;

[0033] Figure 3 Bactericidal kinetics curves of antibacterial peptides of different concentrations on bacteria, wherein (A) Escherichia coli; (B) Staphylococcus aureus; (C) Vibrio vulnificus; (D) Vibrio parahaemolyticus; (E) Vibrio alginolyticus; (F) Pseudomonas aeruginosa; the control group is bacteria without drugs;

[0034] Figure 4 Inhibitory activity of antibacterial peptides and ampicillin of different concentrations on bacterial biofilm formation, wherein (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; the Control group is the bacterial solution group without drugs, n = 3, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001;

[0035] Figure 5 Destructive activity of antibacterial peptides and ampicillin of different concentrations on bacterial biofilm, wherein (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; the Control group is the bacterial solution group without drugs, n = 3, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001;

[0036] Figure 6 Drug resistance induction of antibacterial peptides and ampicillin on Vibrio vulnificus, wherein the antibacterial peptide 21546 and ampicillin treat Vibrio vulnificus for 30 consecutive days, and the MIC ratio of 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 antibacterial peptides on RAW264.7, wherein n = 3, *, P < 0.05; **, P < 0.01; ****, P < 0.0001;

[0038] Figure 8Hemolytic activity of antimicrobial peptides on rabbit red blood cells, Triton X-100: positive control; n = 3, *, P < 0.05; ****, P < 0.0001;

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

[0040] Figure 10 Scanning electron micrograph of V. vulnificus treated with antimicrobial peptides, Note: Control: V. vulnificus without antimicrobial peptide treatment, scale bar 5 μm and 1 μm (magnification);

[0041] Figure 11 Transmission electron micrograph of V. vulnificus treated with antimicrobial peptides, Note: Control: V. vulnificus without antimicrobial peptide treatment, scale bar 1 μm and 200 nm (magnification);

[0042] Figure 12 Membrane permeability of V. vulnificus treated with different concentrations of antimicrobial peptides detected by flow cytometry, (A) blank without staining; (B) MIC 21546; (C) 2MIC 21546; (D) PBS control group; the abscissa represents the PI fluorescence intensity, and the ordinate represents the cell count; (E) quantitative analysis of PI fluorescence positive bacteria in different groups, n = 3, **, P < 0.01; ****, P < 0.0001;

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

[0044] Figure 14 Inflammatory factor levels in the serum of mice treated with different drugs, (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 load in the liver, kidney, spleen and lung of mice treated with different drugs, (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 liver, kidney, spleen and lung tissues of mice treated with different drugs, scale bar: 100 nm. DETAILED DESCRIPTION

[0047] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0048] The Aequorea victoria used in this experiment is an artificial breeding population, which is purchased from Qingdao, Shandong. Escherichia coli ATCC 25922, Staphylococcus aweus 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 Biology. The polypeptides used in the 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 for statistical analysis of data, and the results were shown as mean ± standard deviation (mean ± SD). The difference between multiple groups of data was analyzed by one-way ANOVA test, and the significant difference was analyzed by Tukey test. P<0.05 was considered statistically significant.

[0050] Example 1 Screening of new antibacterial peptides of Aequorea victoria based on multi-omics and AI

[0051] I. Collection of Aequorea victoria tissue samples

[0052] All consumables (including micro pipette gun heads, centrifuge tubes, tissue crushing beads and surgical instruments) of the contact samples in this experiment were soaked in 0.1% diethyl pyrocarbonate (DEPC) solution, and then high-temperature high-pressure sterilization was used to inactivate the residual DEPC, so as to prevent RNAse contamination.

[0053] (1) Vibrio vulnificus was inoculated into Luria-Bertani (LB) agar medium, cultured at 37°C for 16-24h, then single colonies were picked and inoculated into 10 mL LB medium, cultured at 37°C, 200 rpm / min overnight, then diluted to 1x108 CFU / mL, centrifuged at 5000 rpm / min for 5 min, the supernatant was discarded, and the bacteria were resuspended in seawater for use.

[0054] (2) Randomly selected uniform-sized jellyfish were wounded at the oral arm, and the wounded jellyfish were cultured in water with a salinity of 30‰ at a bacterial concentration of 4.1x105 CFU / mL and in control seawater under the same conditions for 8h. Five jellyfish from each of the wound group soaked in bacterial solution and the control seawater group were rinsed clean with purified water, and tissue samples were quickly frozen in liquid nitrogen and stored at -80°C.

[0055] II. Transcriptome sequencing and bioinformatics analysis

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

[0057] The VAHTS Universal V6 RNA-seq Library Prep Kit was used to construct the transcriptome library. Subsequently, the library quality was detected by Agilent 2100 Bioanalyzer to ensure that it met the standard. On this basis, the Illumina Novaseq 6000 sequencing platform was used to carry out sequencing work, and 150 bp double-end sequences were obtained. The whole transcriptome sequencing and analysis process was completed by Shanghai Euilight Biotechnology Co., Ltd. Trimmomatic was used to process fastq format Rawdata (raw reads). After data filtering, the reads containing ploy-N and low-quality reads were removed to obtain high-quality clean reads. Subsequently, by removing the adapter sequence and low-quality region, the clean reads were assembled to form expression sequence tag clusters (contigs). Trinity software was used for de novo assembly to obtain transcript sequences. Based on the sequence similarity and length comparison results, the longest transcript was selected as Unigenes for subsequent bioinformatics analysis. Unigenes were aligned with NCBI non-redundant (NR), Swiss-Prot, and gene evolutionary lineage database to annotate the function of Unigenes: diamond software was used for alignment, e<1e-5 was used as the screening standard, and non-supervised orthologous group analysis was performed by eggNOG database, and KOG database was used to carry out eukaryotic complete genome orthologous group cluster annotation. Through alignment 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. The mapping relationship between Swiss-Prot and GO terms was used for gene ontology (GO) classification. After Unigenes annotation, bowtie2 software was used to count the number of reads aligned to Unigenes in each sample, and then eXpress

[29] tool was used to quantitatively analyze Unigenes to 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 screening condition for difference was q<0.05 and difference fold (FoldChange)>2. R software (v3.2.0) was used to perform hierarchical clustering on the screened differential expression genes (DEGs) to visualize the gene expression characteristics between different groups and samples. Subsequently, based on the hypergeometric distribution principle, R software was used to carry out GO function annotation and KEGG pathway enrichment analysis to explore the biological function and metabolic pathway characteristics of differential expression genes.

[0058] Based on Illumina NovaSeq 6000 sequencing platform, comparative transcriptomic analysis was performed on the species Aequorea victoria in the phylum Cnidaria. The gene expression differences between normal A. victoria and Vibrio harveyi exposure group for 8 hours were compared and analyzed. Pathogen stimulation led to significant changes in gene expression in the jellyfish (P<0.05) and 1652 differentially expressed genes (DEGs) were identified by bioinformatics analysis, including 525 up-regulated genes and 1127 down-regulated genes. Figure 1

[0059] The results of differential gene enrichment analysis are shown in Figure 2 After 8 hours of bacterial attack, the differential expression genes were enriched in GO terms, mainly including glycogen metabolic process, inflammatory response, fibrinolysis, endogenous coagulation pathway and other biological processes; from the perspective of cell composition, it mainly involved cell, organelle, membrane and extracellular region components; the molecular functions mainly included nutritional activity, molecular structure activity, response to stimulation activity, etc. KEGG pathway enrichment analysis further revealed that the differential genes were mainly involved in metabolic pathways, ribosomes, thermogenesis, disease pathways and cell proliferation signaling pathways, and were also involved in the nuclear factor kappa B (NF-κB) signaling pathway, tumor necrosis factor (TNF) signaling pathway and interleukin-17 (IL-17) signaling pathway closely related to immune regulation system. It showed that A. victoria may be suffering from bacterial infection and cell proliferation, and was undergoing immune response and tissue repair.

[0060] III. Prediction of potential A. victoria antibacterial peptides

[0061] ​CAMPR4, as an AI model, integrates multiple machine learning algorithms including support vector machine (SVM), random forest (RF), artificial neural network (ANN), discriminant analysis (DA) and hidden Markov model (HMM), for AMP prediction and tools for rational design (natural and synthetic AMPs), sequence (BLAST and clustal omega), structure (VAST) and family analysis (PRATT, ScanProsite, CAMPSign). According to previous studies, based on the differentially expressed genes of A. digitifera, the corresponding full-length protein sequences in FASTA format were found, and the prediction tool of CAMPR4 was used to predict the possibility of all candidate protein sequences becoming functional antibacterial peptides with a window size of 20 aa. ANN gives the results of AMP or NAMP, AMP is the sequence predicted to be antibacterial, NAMP is the sequence predicted to be non-antibacterial, SVM, RF and DA give the predicted probability score for polypeptides, and polypeptide sequences with probability > 0.9 are screened out. Then, we evaluated the antibacterial effect of polypeptides on E. coli, S. aureus and V. vulnificus in DBAASP based on machine learning (ML) methods and strain-specific antibacterial prediction methods based on peptide sequences and bacterial genome data. The Vibrio vulnificus genome ID number (SAMN05196200) was used. Sequences with antibacterial activity against the three bacteria were ranked by the average score, and the polypeptide with the highest score was selected for solid-phase synthesis.

[0062] Based on the analysis of the transcriptome data of A. digitifera, it was found that some differentially expressed genes may be involved in the regulation of its innate immune response mechanism, especially those encoding polypeptides with antibacterial activity. To identify these potential antibacterial molecules, we used AI models based on machine learning algorithms such as CAMPR4 and DBAASP for high-throughput screening, and finally obtained candidate peptide sequences with potential antibacterial activity. First, CAMPR4 was used to predict the possibility of all candidate genes becoming functional antibacterial peptides, and polypeptide sequences with probability > 0.9 were screened out. Then, the antibacterial activity of polypeptides against E. coli, S. aureus and V. vulnificus was predicted in DBAASP. Eight polypeptides with the highest scores were obtained, which had antibacterial activity against the three bacteria.

[0063] IV. Detection of MIC and MBC of candidate antibacterial peptides

[0064] MIC was evaluated by micro-broth dilution method. E. coli, S. aureus, V. vulnificus, P. aeruginosa, V. alginolyticus, V. parahaemolyticus and V. harveyi were cultured on LB agar plates to activate the strains, and then incubated in cation adjusted mueller hintion broth (CAMHB) medium at 37℃, 200 rpm / min to the logarithmic growth phase. The bacterial suspension was diluted to 10 6 CFU / mL with the medium. The antibacterial peptides were dissolved in PBS to a concentration of 10 mg / mL, and the medium was diluted to 4000 μg / mL. In a 96-well plate, 50 μL of CAMHB broth was added to each well in ten columns, and 50 μL of the prepared polypeptide was added to the first column, then the drug concentration in each subsequent well was diluted by two, and finally, 50 μL of the diluted bacterial solution was added to each well. The final concentration of the antibacterial peptides was 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 μg / mL, respectively. The standard antibiotics ampicillin and doxycycline were positive controls in the experiment, and the wells without antibacterial peptides were negative controls. The final volume in each well was 100 μL. The 96-well plate was incubated at 37℃ for 18 h, and the absorbance value at 600 nm was measured. Three replicates were set for each test group.

[0065] The minimal bactericidal concentration (MBC) was determined as follows: according to the previous MIC determination method, after the enzyme-labeled plate was incubated at 37℃ for 18 h, 100 μL of bacterial suspension was then removed from each well and evenly spread on the corresponding selective medium plate. The inoculated plates were incubated in a 37℃ incubator overnight. The next day, the growth of bacterial colonies on each plate was observed and recorded, and the lowest drug concentration that completely killed the bacteria in the medium was the MBC value of the drug for the target strain.

[0066] To verify the predicted antibacterial activity of the peptides, eight peptide sequences were synthesized by solid phase synthesis, and E. coli, S. aureus and V. vulnificus were used as test strains to verify the antibacterial activity of the peptides. Among them, the MIC of 21546 against E. coli was 26.13 μM, the MIC of 21546 against S. aureus was 6.53 μM, and the MIC of 21546 against V. vulnificus was 13.06 μM. The smaller the MIC value, the stronger the antibacterial activity. Therefore, 21546 was selected as a candidate antibacterial peptide for further study, and the MIC of 21546 against three marine Vibrio and P. aeruginosa was determined, as shown in Table 1. The results showed that the MIC of 21546 against V. parahaemolyticus, V. alginolyticus, V. harveyi and P. aeruginosa was 52.25 μM, 6.53 μM, 3.27 μM and 104.5 μM, respectively.

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

[0068]

[0069] The MBC of 21546 against four marine Vibrio and three conventional strains was determined by plate growth method (Table 2). The results showed that the MBC of 21546 against E. coli, S. aureus, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. harveyi and P. aeruginosa was 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 antibacterial peptide 21546 against four marine Vibrio and three conventional strains

[0071]

[0072]

[0073] Then the two antibacterial peptides were searched and compared with the CAMPR4 antibacterial peptide database, which contains rich antibacterial peptide related data, including 24243 AMP sequence information, 933 structure data, 2143 patent records and 263 different AMP family characteristic information. The results in Table 3 show that the A. australis antibacterial peptide is a new antibacterial peptide sequence.

[0074] Table 3 Comparison of A. australis antibacterial peptide with CAMPR4 database

[0075]

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

[0077] I. Determination of time-kill curve

[0078] The bactericidal kinetics assay was performed according to Wei et al. with slight modification. The activated strains were cultured in fresh LB medium at 37°C, 200 rpm / min to the logarithmic growth phase, and the final concentration of the bacteria was adjusted to 1 x 106 CFU / mL with the medium. The final concentration of the antimicrobial peptide solution was 1 x MIC, 5 x MIC and 10 x MIC, respectively. After mixing, the mixture was incubated in a 37°C incubator. The bacterial solution without the addition of the antimicrobial peptide was used as a control. Samples were taken at 0, 5, 10, 30, 60, 180 and 240 min, diluted and plated on LB agar plates, and incubated at 37°C overnight before counting the number of colonies.

[0079] In this study, three clinical routine strains and three marine Vibrio strains were selected as experimental strains to evaluate the inhibitory time-effect of the antimicrobial peptide 21546 on the growth activity of the strains. The relevant data are shown in Table 1. Figure 3 The experimental data showed that the antimicrobial peptide exhibited inhibitory effect on Staphylococcus aureus. When the concentration reached or exceeded 5 x MIC, it exhibited significant bactericidal effect on Escherichia coli, Vibrio vulnificus, Vibrio parahaemolyticus and Pseudomonas aeruginosa, and could kill the above-mentioned strains within 30 minutes.

[0080] II. Biofilm experiment

[0081] To determine the biofilm inhibition ability of the antimicrobial peptide 21546, the inhibition of biofilm formation and the destruction of the formed biofilm were tested by crystal violet staining. The bacterial solution was diluted to 10 5 CFU / mL with fresh LB medium, and 180 μL of the bacterial solution was added to 20 μL of the antimicrobial peptide solution in a 96-well plate, which was serially diluted by 2 times. The final concentration of the antimicrobial peptide was 0.125 x MIC, 0.25 x MIC, 0.5 x MIC and 1 x MIC. The bacterial solution without the addition of the antimicrobial peptide was used as a control, and the LB medium was used as a blank control. After 24 hours of culture, the samples were washed with sterile phosphate buffered saline to remove the bacteria floating on the surface. Then, the culture was fixed in 99% methanol for 15 minutes. Subsequently, the methanol was removed, and the 96-well plate was naturally air-dried. 200 μL of 0.1% crystal violet staining solution was added to each well, and the staining process was completed after 15 minutes of standing. Excess staining agent was removed by gently washing with distilled water. Finally, the stained samples were immersed in 95% ethanol to dissolve the colorant, and the absorbance was measured at a wavelength of 600 nm.

[0082] To determine the activity of the antimicrobial peptide in destroying the formed biofilm, 200 μL of 1 x 10 5CFU / mL of bacterial solution was added to 96-well plates and incubated in a 37°C incubator for 24 hours to form biofilm. After rinsing the bacteria with PBS, serial dilutions of the antimicrobial peptide were prepared in fresh identical medium and 200 μL of the suspension was transferred to the well plate containing the biofilm. 200 μL of medium was added to the control and blank wells. After incubating the plates at 37°C for another 24 hours, the same crystal violet staining method described above was used. The percentage of biofilm formation = (OD 样品 - OD 空白 ) / (OD 对照 - OD 空白 ) x 100%.

[0083] The results of the biofilm inhibition experiment Figure 4 indicate that the antimicrobial peptide can significantly inhibit the biofilm formation of the three test strains when the concentration of the antimicrobial peptide is greater than or equal to 0.5 times the MIC. Further analysis shows that at the MIC concentration, the biofilm formation inhibition rates of the antimicrobial peptide 21546 on E. coli, S. aureus and V. vulnificus are 69.57%, 61.44% and 53.7% respectively; in contrast, the inhibition effect of ampicillin on these strains is lower, with inhibition rates of 42.11%, 40.93% and 42.12% respectively.

[0084] The destruction effect of the antimicrobial peptide on the biofilm formed by the bacteria is shown in Table 3 Figure 5 . At the MIC concentration, the biofilm destruction rates of the antimicrobial peptide 21546 on E. coli, S. aureus and V. vulnificus are 60.61%, 56.65% and 43.86% respectively. The destruction effect of the traditional antibiotic ampicillin on the biofilm of these bacteria is relatively low, with destruction rates of 35.98%, 38.14% and 28.76% respectively.

[0085] III. Drug resistance induction determination

[0086] The strains were cultured overnight at 37°C on a shaker, and the bacterial suspension was diluted to 1 x 10 5 CFU / mL using freshly prepared LB liquid medium. The bacterial suspension was mixed with the antimicrobial peptide solution in a volume ratio of 9:1 to achieve a final concentration of the peptide at a sub-MIC level. The mixed system was incubated in a 37°C constant temperature shaker at a speed of 180 rpm / min overnight. Subsequently, the MIC value of the antimicrobial peptide on V. vulnificus was determined by microdilution method. In subsequent experiments, the bacterial suspension after incubation was adjusted to the same concentration gradient with fresh medium and transferred to sterile test tubes, and an appropriate amount of antimicrobial peptide was added to maintain the final concentration at a sub-MIC level. After 30 generations of continuous culture, the ratio of the MIC after each passage to the initial MIC before the first passage was taken as the fold change of the MIC.

[0087] This study conducted a 30-generation subculture experiment on Vibrio vulnificus. At the initial culture stage, Vibrio vulnificus exhibited different sensitivities to three antimicrobial agents. Figure 6 The MICs of antimicrobial peptide 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 without significant change. These findings suggest that Vibrio vulnificus is less likely to develop resistance to antimicrobial peptide 21546 compared to ampicillin.

[0088] IV. Cytotoxicity Detection

[0089] RAW264.7 cells frozen in liquid nitrogen were rapidly transferred to a 37°C constant temperature water bath, and the cryovials were gently shaken to achieve rapid thawing. The cell suspension was transferred to 5 mL of DMEM complete medium containing 10% fetal bovine serum (FBS), and centrifuged at 2000 rpm / min for 5 minutes to remove the supernatant. The cell pellet was resuspended in 1 mL of fresh DMEM medium, and then transferred to a culture flask pre-filled with complete medium. The cells were gently mixed to ensure even distribution and incubated in a 37°C, 5% CO2 incubator. After 2-3 passages to achieve stable growth, the cell density was adjusted to 2 × 10⁶ cells / mL. 5 Cells / mL were seeded at 100 μL per well in a 96-well plate and incubated 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 each experimental well, and 100 μL of complete culture medium was added to each control well. A separate set of cell-free complete culture medium was set up as a blank. Incubation continued for 24 hours. Finally, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 0.5–4 hours. The absorbance of each well was measured at 450 nm using a microplate reader. Each group was divided into triplicate.

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

[0091] In this study, we used the mouse-derived RAW264.7 cell line as the research subject and evaluated the potential toxic effects of different concentrations of antimicrobial peptides on cells using the CCK-8 assay. The experimental results are as follows: Figure 7 The study showed that when the concentration of antimicrobial peptide 21546 reached or exceeded 50 μM, it exhibited a certain cytotoxic effect on RAW264.7 cells.

[0092] V. Hemolytic Activity Detection

[0093] The antibacterial peptides were diluted in PBS buffer to a concentration range of 3.125-100 μM, and the same volume of 4% rabbit red blood cell suspension was added and mixed. 50 μL of the red blood cell suspension was mixed with equal volume of 1% Triton X-100 and PBS buffer, respectively, as 100% hemolysis and 0% hemolysis controls, and each group was set in triplicate. After incubation at 37°C for 1 h, centrifugation was performed at 1000 rpm / min for 10 min, and 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 )] x 100, where A is the absorbance at 540 nm.

[0096] Hemolytic activity is one of the key indicators of the safety of antibacterial peptides. To evaluate the hemolytic activity of antibacterial peptide 21546, we used 4% rabbit red blood cell suspension to determine the hemolysis rate of the two antibacterial peptides at different concentrations. The experimental results are shown in Table 2. Figure 8 As shown in Table 2, when the concentration of antibacterial peptide 21546 does not exceed 50 μM, its hemolysis rate on rabbit red blood cells is less than 10%, indicating that it has no significant hemolytic activity.

[0097] Six, temperature detection

[0098] Effect of temperature on the stability of antibacterial peptides: the strain was cultured at 37°C at 200 rpm / min overnight, and the bacterial solution was diluted to 106 CFU / mL with fresh CAMHB medium. The antibacterial peptide was prepared into a solution with a concentration of 10 mg / mL using PBS buffer, and was treated at -20, 30, 70, and 100°C for 30 min, respectively. The MIC of the strain was determined, and the method was the same as 1.2.5, and each test was set in triplicate.

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

[0100] In this study, Vibrio vulnificus was used as the indicator strain, and the effects of temperature and pH on the activity of antibacterial peptides were systematically investigated by determining the changes in MIC. The experimental results showed that Figure 9), the antibacterial activity of the antibacterial peptide on Vibrio vulnificus decreased under high temperature conditions at 100℃. Further analysis of the effect of pH showed that the activity of the antibacterial peptide 21546 decreased in a strong acidic environment (pH = 2). These findings provide an important reference for further optimizing the structure of the antibacterial peptide and improving its environmental stability.

[0101] Seven, electron microscope observation and analysis

[0102] Scanning electron microscope: Vibrio vulnificus was cultured to the logarithmic growth phase, and then centrifuged at 3000 rpm / min for 5 min to obtain bacteria of mung bean size at the bottom of the tube. The supernatant was discarded, and the bacteria were washed with PBS three times. The antibacterial peptide solution was added to the bacteria solution to be tested, and the final concentration was MIC. The bacteria solution treated with PBS was used as a control, and incubated at 37℃ at 120 rpm / min for 1 h. After treatment, the bacteria solution was centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. Glutaraldehyde fixing solution was added, and the bacteria were fixed at 4℃ for more than 6 h. The bacteria were dehydrated using a gradient of 50%, 70%, 80%, and 90% ethanol for 15 min each, and 100% ethanol for 30 min each. The samples were dried using a freeze vacuum dryer, the bacterial surface was coated with gold, and the samples were observed under a scanning electron microscope.

[0103] Transmission electron microscope: the bacteria were prepared under the same conditions described in the scanning electron microscope. The antibacterial peptide solution was added to the bacteria solution to be tested, and the final concentration was MIC. The bacteria solution treated with PBS was used as a control, and incubated at 37℃ at 120 rpm / min for 1 h. After treatment, the bacteria solution was centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. Glutaraldehyde fixing solution was added, and the bacteria were fixed at 4℃ for 2-4 h. The bacteria were washed with 0.1M PBS three times for 15 min each. Then, the bacteria were fixed with 1% osmium acid and 0.1M PBS at room temperature for 2 h, and washed with PBS three times. The samples were dehydrated using a series of ethanol concentrations as described above, and dehydrated with 100% acetone for 15 min. The samples were treated with acetone and 812 embedding agent at a ratio of 1:1 for 2 h, and then treated with acetone and 812 embedding agent at a ratio of 2:1 overnight. Then, the samples were transferred to pure 812 embedding agent for 4 h, and incubated in the embedding plate at 37℃ overnight. The samples were sectioned using a microtome, and stained with 2% uranyl acetate saturated aqueous solution and citric acid lead for 15 min. The samples were observed under a transmission electron microscope.

[0104] In this study, Vibrio vulnificus was used as the research object, and the effect of the antibacterial peptide 21546 on the morphological structure of the bacteria was observed using a scanning electron microscope. The MIC concentration of the antibacterial peptide treatment group and the blank control group were set up, and the samples were prepared and observed after 1 hour of action. The results are as follows Figure 10As shown, Vibrio vulnificus untreated with antimicrobial peptides maintained its intact rod-shaped morphology, with complete cell structure, uniform distribution of intracellular material, and no leakage of contents. In contrast, the bacteria in the antimicrobial peptide-treated group exhibited significant morphological changes: obvious wrinkles and depressions appeared on the cell surface, cell membrane integrity was disrupted, and a large amount of cytoplasmic contents leaked out, ultimately resulting in shriveled cells. These morphological changes confirm that antimicrobial peptides exert their antibacterial effect by disrupting the bacterial cell membrane structure.

[0105] The ultrastructure of the treated Vibrio vulnificus was observed using transmission electron microscopy. For example... Figure 11 As shown, the control group bacterial cells exhibited typical ultrastructural characteristics: intact and continuous cell membrane structure, uniform distribution of intracellular substances, and high electron density. In stark contrast, bacteria treated with antimicrobial peptide 21546 showed severe membrane damage: localized perforations and ruptures in the cell membrane, and the disintegration of the membrane structure was accompanied by a significant decrease in electron density. This morphological evidence suggests that the antimicrobial peptide may alter plasma membrane permeability, leading to the outflow of intracellular substances.

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

[0107] Collect Vibrio vulnificus in the logarithmic growth phase and dilute with PBS to 10⁻⁶. 8 Bacterial suspensions were prepared at CFU / mL. The bacteria were mixed with different concentrations of antimicrobial peptides at a 1:1 volume ratio to achieve final antimicrobial peptide concentrations of 1-fold and 2-fold MIC. The suspensions were incubated at 37°C for 1 hour, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. After washing three times with PBS, 200 μL of propidium iodide (PI) was added for staining. The suspensions were incubated in the dark for 30 minutes. Flow cytometry was used to analyze the effect of the antimicrobial peptides on the membrane permeability of Vibrio vulnificus.

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

[0109] Example 3: In vivo antibacterial activity of antimicrobial peptides

[0110] Vibrio vulnificus in this embodiment was purchased from Shanghai Fuxiang Biotechnology Co., Ltd., and SPF-grade ICR female mice were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. The housing environment was subjected to light and dark cycles every 12 hours.

[0111] Before the in vivo experiment, the biological safety of the antimicrobial peptide 21546 in vivo was evaluated. Twenty-four female ICR mice, weighing 26-27 g, were randomly divided into four groups: a blank control group and low, medium, and high-dose antimicrobial peptide administration groups. The mice were injected intraperitoneally with 7.5 mg / kg, 15 mg / kg, and 30 mg / kg of the antimicrobial peptide daily for 7 consecutive days, and the blank control group was injected with normal saline. The mice were weighed daily, and their behavior and mental state were observed.

[0112] The safety of the antimicrobial peptide 21546 in vivo was preliminarily evaluated by monitoring the body weight change rate and behavioral characteristics of ICR mice after short-term continuous intraperitoneal injection. The experimental data showed that all the mice in the administration groups exhibited a dose-dependent weight loss trend, as shown in FIG. 1. Figure 13 After each administration, the mice in each dose group exhibited slow movement, which returned to normal 1 h after administration. It was speculated that this might be related to the local pain response caused by peritoneal irritation. Notably, the low-dose group (7.5 mg / kg) did not exhibit significant physiological abnormalities in the late experimental period (days 5-7), and its hair condition was not significantly different from that of the control group. However, the medium and high-dose groups (≥15 mg / kg) continuously exhibited reduced voluntary activity, chills, and dull hair, among other toxic signs. After seven days of continuous administration, the body weight of the 15 mg / kg group fluctuated within 5%, which met the requirements of animal ethics experiments. Comprehensive analysis indicated that a dose of ≤15 mg / kg should be selected for subsequent in vivo studies to ensure experimental safety.

[0113] I. Serum Inflammatory Factor Analysis of Mice

[0114] The in vivo activity of the antimicrobial peptide 21546 was determined using a Vibrio vulnificus-infected 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 administration group, an ampicillin administration group, and a blank control group, with five mice in each group. Before inoculation, all mice were fasted for 12 h and injected intraperitoneally with 0.1 mL / 10 g (about 1.5 x 10 8 CFU / mL) of Vibrio vulnificus to establish the infection model. Two hours after infection, the antimicrobial peptide 21546 administration group was injected with 5 mg / kg, the ampicillin administration 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. All mice were sacrificed and their eyes were bled 4 h after infection. The serum was obtained by centrifugation at 3000 rpm / min for 15 min, and the expression levels of the 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 immune regulation characteristics of the antibacterial peptide were evaluated by dynamic monitoring of inflammatory factors, and the experimental results are shown in Figure 14 The infection of V. vulnificus significantly induced the abnormal increase of serum pro-inflammatory factors TNF-a, IL-6, IFN-g and anti-inflammatory factor IL-10 in mice. It is worth noting that the levels of the four cytokines in the four groups showed a significant downward trend after 2 hours of infection with the administration of antibacterial peptide 21546 and ampicillin. The results showed that the antibacterial peptide had the effect of inhibiting excessive inflammatory response.

[0116] II. Analysis of bacterial load in multiple organs of mice

[0117] Randomly take 18 ICR female mice, weighing 21-22g. Divide them into infection model group, antibacterial peptide 21546 administration group, ampicillin administration group, 6 in each group. The treatment method is the same as 3.2.2, and liver, kidney, spleen, lung tissues are collected for bacterial quantification analysis. After the tissue samples are accurately weighed by an electronic balance, 500μL of pre-cooled PBS buffer is added, and a uniform suspension is prepared using a low-temperature tissue homogenizer. The homogenate is gradient diluted, 100μL of the diluted solution is evenly inoculated on the surface of LB agar medium, and after 24 hours of constant temperature culture at 37℃, the colony forming units are counted.

[0118] In this study, a mouse model of V. vulnificus peritoneal infection was established to systematically evaluate the effect of antibacterial peptide on the biological distribution of pathogens. After 2 hours of peritoneal infection, 5mg / kg of 21546 and 32mg / kg of ampicillin were injected intraperitoneally, and the control group was injected with normal saline. Two hours after administration, the mouse organs were taken, and the number of colonies in the liver, kidney, spleen and lung of the mice was counted. The bacterial load in each organ is shown in Figure 15 As shown in

[0119] III. Histopathological analysis of mice

[0120] Select 12 ICR female mice with body weight of 21-22 g, and randomly divide them into 4 groups, 3 mice in each group, namely, infection model group, antibacterial peptide 21546 administration group, ampicillin administration group and blank control group. The mice in each group are treated according to the method in step one. Then, the liver, kidney, spleen and lung of the mice are taken, washed with PBS buffer and put into 5 mL 4% paraformaldehyde fixing solution. After the tissue fixation is completed, the conventional paraffin embedding treatment is performed. The section is subjected to the following treatment process in turn: dimethylbenzene I solution immersion for 20 min, transfer into dimethylbenzene II solution for continuous treatment for 20 min, then subjected to anhydrous ethanol I (5 min), anhydrous ethanol II (5 min) and 75% ethanol (5 min) in turn, and finally washed with flowing tap water. Hematoxylin staining solution is used to stain the section for 3-5 min, and after washing with tap water, differentiation solution is used for treatment and water washing again. Then, back blue solution is used for treatment, and the section is washed with flowing water. The dehydration process is subjected to 85% and 95% gradient ethanol for 5 min in turn, and then dyed in eosin staining solution for 5 min. The sample is put into anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III in turn, each for 5 min, and then put into dimethylbenzene I and dimethylbenzene II, each for 5 min, to complete the transparent treatment. Finally, neutral balsam is used for section sealing, and the section is observed and analyzed under a laser confocal microscope.

[0121] The protective effect of the antibacterial peptide on the organ damage is evaluated by histopathology. The mice are infected with Vibrio vulnificus in the abdominal cavity for 2 h, and the liver, kidney, spleen and lung of the mice are taken 2 h after the administration treatment and subjected to H&E staining observation. Figure 16 It is shown that, compared with the blank control group, the liver cells in the model group are arranged in disorder, present extensive hydropic degeneration and nuclear lysis, and the liver cord structure in the antibacterial peptide 21546 and ampicillin administration groups recovers regularity and the cell necrosis area is reduced. Compared with the blank control group, the kidney medulla interstitium of the model group is hemorrhagic, and the renal tubular epithelial vacuolar degeneration is present. The hemorrhagic phenomenon of the kidney of each administration group is reduced, and the granular degeneration phenomenon is reduced, and the antibacterial peptide 21546 group is more obvious. Compared with the blank control group, the boundary of the red pulp and white pulp structure of the spleen of the model group is blurred, the tissue cells are arranged in disorder and loose, and the tissue arrangement tends to be normal after administration. Compared with the blank control group, the lung alveolar wall of the model group is thickened, the lung tissue is infiltrated by a large number of inflammatory cells, and the lung alveolar epithelium is exfoliated, and after administration, the lung alveolar wall thickening phenomenon is reduced, and the inflammatory cell infiltration is reduced.

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

Claims

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

2. A preparation comprising the antibacterial peptide of claim 1.

3. A method for preparing the antibacterial peptide of claim 1.

4. Use of the antibacterial peptide according to claim 1 for the preparation of a preparation for inhibiting a pathogen infection in vitro, characterized in that, The pathogen is Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Pseudomonas aeruginosa.

5. Use of the antibacterial peptide according to claim 1 for the preparation of a preparation for the disruption of the formation of a biofilm and / or of an already formed biofilm of a pathogenic organism, characterized in that, The pathogen is Escherichia coli, Staphylococcus aureus, Vibrio vulnificus.

6. Use of the antibacterial peptide of claim 1 in the preparation of a medicament for treating and / or alleviating sepsis caused by Vibrio vulnificus infection.

7. Use of the antibacterial peptide of claim 1 in the preparation of a medicament for inhibiting inflammation caused by Vibrio vulnificus infection.

Images