Antibacterial peptide from aurelia and application thereof

Abstract

The invention discloses an antibacterial peptide derived from aurelia and application of the antibacterial peptide. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. According to the invention, a multi-omics technology is utilized to analyze the differential expression condition of a gene of the aurelia after bacterial infection, an AI model is utilized to predict candidate antibacterial peptides, and activity verification is carried out on MIC of bacteria by determining polypeptides, so that the high-activity aurelia antibacterial peptide is obtained. Compared with the traditional antibiotic drug ampicillin, the high-activity antibacterial peptide disclosed by the invention has lower possibility of generating drug resistance, is expected to be developed into a novel antibacterial drug aiming at marine pathogen infection, and provides a novel candidate drug for treating marine organism injury, marine battle injury secondary infection and clinical pathogenic bacterium infection.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical and antimicrobial peptide technology, specifically relating to an antimicrobial peptide derived from the moon jellyfish and its applications. Background Technology

[0002] With the widespread use and abuse of antibiotics in recent years, drug-resistant and multidrug-resistant bacteria that cannot be controlled by conventional drugs are constantly emerging, causing serious health and medical problems, including marine pathogens. As early as 2013, a study analyzed the antibiotic sensitivity of marine bacteria in Chinese waters, and the results showed that these marine bacteria exhibited varying degrees of resistance to some commonly used clinical antibiotics. To solve this problem, there is an urgent need to find new antibacterial drugs that can replace traditional antibiotics to combat drug-resistant bacteria.

[0003] Antimicrobial peptides (AMPs) are important components of the innate immune system in many organisms. They typically contain 10-50 amino acids and are also known as host defense peptides, exhibiting good inhibitory activity against bacteria, fungi, and viruses. Antimicrobial peptides are widely distributed in nature. Since Swedish scientists Boman et al. first isolated the first specific antimicrobial active peptide, cecropin, from silkworm pupae in 1980, researchers have successfully isolated polypeptides with antimicrobial activity from various evolutionary groups. Marine organisms are an important source for the discovery of antimicrobial molecules, and many studies have isolated antimicrobial peptides from marine organisms. These peptides not only possess strong antimicrobial activity against various bacteria, fungi, and viruses, but also exhibit anti-inflammatory, anticancer, immunomodulatory, and wound-healing biological functions, playing an important role in medicine, food processing, aquaculture, and animal husbandry.

[0004] Jellyfish, belonging to the phylum Cnidaria, are ancient marine invertebrates that have evolved over hundreds of millions of years. Diverse jellyfish species inhabit various marine environments, their open coeloms immersed in seawater, resulting in a large surface area in contact with the water. Prolonged contact with various pathogens on their body surface may have led to the evolution of unique innate immune systems and antimicrobial molecules. However, due to the difficulty and low efficiency of directly isolating and enriching active ingredients from jellyfish, antimicrobial peptides in jellyfish remain a largely untapped potential drug resource. With the rapid development of high-throughput sequencing technology and bioinformatics, scientists have performed genome, transcriptome, and proteome sequencing on many species, including jellyfish, and established multi-omics databases. Simultaneously, the rise of artificial intelligence (AI) technology in recent years has led to the creation of numerous AI models for predicting antimicrobial peptides, including CAMPR4 and DBAASP, which integrate machine learning algorithms, and ADP3, which has protein three-dimensional structure prediction capabilities. These AI platforms provide researchers with abundant reference information, significantly improving the efficiency of rational antimicrobial peptide design.

[0005] This invention mines potential antimicrobial active molecules in the marine invertebrate moon jellyfish based on multi-omics databases and AI prediction models, clarifies their in vivo and in vitro activities and mechanisms of action, and screens out a class of novel antimicrobial peptides targeting marine pathogenic microorganisms, providing novel candidate drugs for the treatment of marine biological wounds, secondary infections of naval combat wounds and clinical pathogenic bacterial infections. Summary of the Invention

[0006] The purpose of this invention is to provide a novel antimicrobial peptide drug derived from the moon jellyfish. This aims to address the drug resistance problem associated with traditional antibiotics in the prior art and to provide a novel antimicrobial drug.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO: 1 (the sequence is: FPKNFLTIAKTILKRLFRVY). The antimicrobial peptide is derived from the moon jellyfish.

[0009] As a preferred embodiment, the antimicrobial peptide is obtained by a solid-phase synthesis method.

[0010] The present invention also provides the use of the described antimicrobial peptide in the preparation of medicaments for the prevention and / or treatment of bacterial infections.

[0011] As a preferred embodiment, the bacteria are one or more of Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa.

[0012] As a preferred embodiment, when the antimicrobial peptide is used as a drug for the prevention and / or treatment of bacterial infections, it is used under conditions of -20 to 70°C and pH 4 to 8.

[0013] The present invention also provides an antimicrobial peptide gene, wherein the gene encodes the antimicrobial peptide shown in SEQ ID NO: 1.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention utilizes multi-omics technology to analyze the differential expression of genes in moon jellyfish after bacterial infection, uses an AI model to predict candidate antimicrobial peptides, and verifies the activity of the peptides by measuring their MIC against bacteria, thereby obtaining highly active moon jellyfish antimicrobial peptides. Compared with the traditional antibiotic ampicillin, the highly active antimicrobial peptides discovered in this invention have a lower likelihood of inducing drug resistance, and hold promise for development into novel antimicrobial drugs targeting marine pathogen infections, providing new drug candidates for treating secondary infections from marine organism wounds, naval combat wounds, and clinically pathogenic bacterial infections. Attached Figure Description

[0016] Figure 1 The image shows the results of differential gene expression analysis in moon jellyfish in Example 1. In the image, A is a bar chart of differentially expressed genes, and B is a cluster diagram of each differential group.

[0017] Figure 2 The figure shows the results of functional enrichment analysis of differentially expressed genes in Example 1, where (A) is GO enrichment and (B) is KEGG enrichment.

[0018] Figure 3 The images show the bactericidal kinetic curves of different concentrations of antimicrobial peptide 20259 against bacteria in Example 1, where (A) Escherichia coli; (B) Staphylococcus aureus; (C) Vibrio vulnificus; (D) Vibrio parahaemolyticus; (E) Vibrio alginolyticus; (F) Pseudomonas aeruginosa; and the control group consisted of bacteria without the drug.

[0019] Figure 4 This is a schematic diagram illustrating the resistance induction of antimicrobial peptide 20259 and ampicillin against Vibrio vulnificus in Example 2, where the daily MIC fold is the ratio of the MIC after each passage to the initial bacterial MIC before the first generation.

[0020] Figure 5 To illustrate the effect of temperature on antimicrobial peptide 20259 in Example 3, a temperature of -20℃ was used as a control; n=3, ****, P<0.0001.

[0021] Figure 6To illustrate the effect of pH on antimicrobial peptide 20259 in Example 3, pH=7 was used as a control; n=3, ***, P<0.001; ****, P<0.0001. Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, all reagents and biological materials used below are commercial products.

[0023] Example 1: Screening of novel antimicrobial peptides from moon jellyfish

[0024] All moon jellyfish used in this invention were artificially cultured populations purchased from Qingdao, Shandong Province. The following bacteria were also used: *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. All peptides used in the experiments were synthesized by Shanghai Jier Biochemical Co., Ltd., with a purity >95%.

[0025] The preparation methods for the main buffer solutions and culture media used in this invention are as follows:

[0026]

[0027] 1. Collection of tissue samples from moon jellyfish

[0028] In this invention, all consumables that come into contact with samples (including micropipette tips, centrifuge tubes, tissue disruption beads, and surgical instruments) are soaked in a 0.1% diethyl pyrocarbonate (DEPC) solution and then sterilized under high temperature and high pressure to inactivate residual DEPC, thereby preventing RNase contamination.

[0029] Vibrio vulnificus was inoculated onto Luria-Bertani (LB) agar and incubated at 37°C for 16–24 h. Single colonies were then picked and inoculated into 10 mL of LB medium and incubated overnight at 37°C and 200 rpm / min. The culture was then diluted to 1 × 10⁻⁶. 8CFU / mL, centrifuged at 5000 rpm / min for 5 min, discarded the supernatant, and resuspended in seawater for later use; another set of the same seawater was used as a control group. Uniformly sized jellyfish were randomly selected and subjected to identical wounds at their oral arms. The wounded jellyfish were then placed in water with a salinity of 30‰, and seawater-resuspended Vibrio vulnificus was added to achieve a bacterial concentration of 4.1 × 10⁻⁶. 5 CFU / mL, with the same volume of control seawater added under the same conditions as the control group, and cultured for 8 h respectively. Five jellyfish from the wound group and five jellyfish from the control group soaked in bacterial solution were taken, rinsed with purified water, and tissue samples were cut, flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0030] 2. Extraction of total RNA from moon jellyfish

[0031] (1) Take an appropriate amount of sodium dodecyl sulfate (SDS) lysis buffer and add β-mercaptoethanol to prepare an extract with a final concentration of 10% β-mercaptoethanol;

[0032] (2) Take jellyfish tissue and place it into a 2 mL RNase-free centrifuge tube. Then add 400 μL of 10% β-mercaptoethanol extraction solution. Add grinding beads to each centrifuge tube at a sample-to-grinding-bead volume ratio of 3:1. Use a tissue homogenizer to break the tissue at a frequency of 60 Hz for 60 s to ensure thorough tissue disruption.

[0033] (3) Add 300 μL of SDS extract, and quickly add 600 μL of saturated phenol-chloroform-isoamyl alcohol (25:24:1). Place the mixture in a shaker under ice bath conditions and mix for 10 minutes. Then transfer it to a centrifuge at 4℃ and centrifuge at 12000 rpm / min for 10 minutes.

[0034] (4) Mix the supernatant with an equal amount of isopropanol, shake thoroughly for 15 seconds, place on ice and let stand for 15 minutes, then centrifuge at 12000 rpm / min for 15 minutes at 4°C.

[0035] (5) Discard the supernatant, collect the RNA, add 100 μL of DEPC water to dissolve the precipitate, then add 1 mL of Trizol reagent, mix quickly and place on ice for 5 minutes;

[0036] (6) Add 200 μL of chloroform, shake for 15 s to mix, place on ice for 3 minutes, and centrifuge at 12000 rpm / min for 15 minutes at 4℃;

[0037] (7) Transfer 400-600 μL of supernatant to a new centrifuge tube, add an equal amount of ice-cold isopropanol, mix quickly, place on ice for 8 minutes, centrifuge at 12000 rpm / min for 10 minutes at 4℃, and discard the supernatant.

[0038] (8) Add 1 mL of 75% ethanol to the precipitate for washing, centrifuge at 7500 rpm / min at 4℃ for 5 minutes, discard the supernatant, repeat the above operation, discard the supernatant, run at 7500 rpm / min at 4℃ for 1 minute, and remove excess ethanol with a pipette tip.

[0039] (9) Resuspend the RNA in 30-80 μL of DEPC water; take 10 μL for detection. Detect whether the RNA is degraded by 2% agarose gel electrophoresis, and detect the RNA purity (OD260 / 280) and concentration by a Multiskan 1550 microplate reader. Store at -80°C for later use.

[0040] 3. Transcriptome sequencing and bioinformatics analysis

[0041] Total RNA was sequenced and analyzed in both the wound group and the control group after immersion in bacterial solution to identify differentially expressed genes in the wound group compared to the control group. The specific methods are as follows:

[0042] Transcriptome libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep kit. The library quality was then assessed using an Agilent 2100 bioanalyzer to ensure compliance with standards. Sequencing was then performed using an Illumina Novaseq 6000 sequencing platform, yielding 150 bp paired-end sequences. The entire transcriptome sequencing and analysis process was performed by Shanghai Ouyi Biotechnology Co., Ltd. Trimmomatic was used to process the fastq format raw data (raw reads). After data filtering, reads containing poly-N and low-quality reads were first removed, resulting in high-quality clean reads. Subsequently, adapter sequences and low-quality regions were removed, and these clean reads were assembled to form expression sequence tag clusters (contigs). De novo assembly was performed using Trinity software to obtain transcript sequences. Based on sequence similarity and length alignment results, the longest transcript was selected as the Unigenes for subsequent bioinformatics analysis. Unigenes were aligned with NCBI Non-Redundant (NR), Swiss-Prot, and phylogenetic databases to annotate their functions. Diamond software was used for alignment, with e < 1e-5 as the screening criterion. Unsupervised ortholog analysis was performed using the eggNOG database, and ortholog cluster annotation of complete eukaryotic genomes was conducted using the KOG database. Protein sequences with the highest sequence similarity to the unigenes were selected through alignment analysis to obtain corresponding functional annotation results. These unigenes were also mapped to the Kyoto Encyclopedia of Genomes and Genomes (KEGG) for pathway annotation. Gene Ontology (GO) classification was performed using the mapping relationship between Swiss-Prot and GO terms. After unigene annotation, bowtie2 software was used to count the number of reads aligned to unigenes in each sample. Subsequently, eXpress was used to perform quantitative analysis of unigenes to obtain FPKM expression values. DESeq2 software was used to calculate the fold change in expression, and the statistical significance of differential expression was assessed using the negative binomial distribution test. The default criteria for screening differentially expressed genes (DEGs) are q < 0.05 and fold change > 2. Hierarchical clustering of the screened differentially expressed genes (DEGs) was performed using R software (v3.2.0) to visualize gene expression characteristics between different groups and samples. See [link to R software]. Figure 1 Figure 1 shows the results of differential gene expression analysis in moon jellyfish, where A is a bar chart of differentially expressed genes and B is a cluster diagram of each differential group. The results indicate that the wound group immersed in bacterial solution showed significant changes in jellyfish gene expression due to pathogen stimulation.

[0043] Subsequently, based on the hypergeometric distribution principle, GO functional annotation and KEGG pathway enrichment analysis were performed using R software to explore the biological functions and metabolic pathway characteristics of differentially expressed genes. See also Figure 2 The image shows the results of functional enrichment analysis of differentially expressed genes, where (A) represents GO enrichment and (B) represents KEGG enrichment. Figure 2 As shown, 8 hours after bacterial challenge, differentially expressed genes enriched in GO terms mainly involved biological processes such as glycogen metabolism, inflammatory response, fibrinolysis, and intrinsic coagulation pathways. From a cellular compositional perspective, these genes primarily involved cells, organelles, membranes, and extracellular regions. Their molecular functions included nutritional activity, molecular structural activity, and responsiveness to stimuli. KEGG pathway enrichment analysis further revealed that the differentially expressed genes were involved in signaling pathways related to metabolism, ribosomes, thermogenic processes, disease pathways, and cell proliferation, as well as the nuclear factor κB (NF-κB) signaling pathway, tumor necrosis factor (TNF) signaling pathway, and interleukin-17 (IL-17) signaling pathway, all closely related to the immune regulatory system. This suggests that the moon jellyfish may be undergoing bacterial infection and cell proliferation, and is initiating an immune response and tissue repair.

[0044] 4. Prediction of antimicrobial peptides from moon jellyfish

[0045] Analysis of transcriptome data from the moon jellyfish revealed that some differentially expressed genes may be involved in regulating its innate immune response mechanism, particularly those encoding peptides with antibacterial activity. To identify these potential antibacterial molecules, this invention employed AI models based on machine learning algorithms, such as CAMPR4 and DBAASP, for high-throughput screening, ultimately obtaining candidate peptide sequences with potential antibacterial activity. First, the corresponding full-length protein sequences in FASTA format were identified based on the differentially expressed genes in the moon jellyfish. CAMPR4 was then used with a window size of 20 aa to predict the probability of all candidate protein sequences becoming functional antibacterial peptides, screening out peptide sequences with a probability > 0.9. Then, the antibacterial activity of the peptides against *Escherichia coli*, *Staphylococcus aureus*, and *Vibrio vulnificus* was predicted using DBAASP. Seven peptides with the highest scores and antibacterial activity against all three bacteria were obtained, as shown in Table 1.

[0046] Table 1

[0047]

[0048] 5. Analysis of MIC and MBC of candidate antimicrobial peptides

[0049] The minimum inhibitory concentration (MIC) is the lowest concentration of antimicrobial peptide that can effectively inhibit bacterial growth. In this invention, the MIC is assessed using a microbroth dilution method. *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. They were then cultured in cation-adjusted mueller hintion broth (CAMHB) at 37°C and 200 rpm / min until the logarithmic growth phase. The culture was diluted to a concentration of 10... 6 CFU / mL bacterial suspension. The antimicrobial peptide was dissolved in PBS to a concentration of 10 mg / mL, and the culture medium was diluted to 4000 μg / mL. 50 μL of CAMHB broth was added to each well of a 96-well plate (ten columns total). 50 μL of the prepared antimicrobial peptide was added to the first column, and then the concentration of the antimicrobial peptide in each subsequent well was sequentially diluted twofold. Finally, 50 μL of the diluted bacterial suspension was pipetted into each well. The final concentrations of the antimicrobial peptide were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 μg / mL. Standard antibiotics ampicillin and doxycycline were used as positive controls, and the wells containing the antimicrobial peptide were used as negative controls. The final volume of each well was 100 μL. The 96-well plate was incubated at 37℃ for 18 h, and the absorbance value at a wavelength of 600 nm was measured. Each experiment was set up in triplicate.

[0050] The minimum bactericidal concentration (MBC) was determined as follows: Following the previous MIC determination method, the prepared ELISA plate was incubated at 37°C for 18 h. Subsequently, 100 μL of bacterial suspension was taken from each well and evenly spread onto the corresponding selective culture medium plate. The inoculated plates were then incubated overnight at 37°C. The colony growth on each plate was observed and recorded the next day. The lowest drug concentration that completely killed the bacteria in the culture medium was the MBC value of the drug against the target strain.

[0051] To verify the antimicrobial activity of the predicted antimicrobial peptides, the seven peptide sequences in Table 1 were synthesized in a solid-phase manner. *Escherichia coli*, *Staphylococcus aureus*, and *Vibrio vulnificus* were used as test strains to verify the antimicrobial activity of the peptides. The results are shown in Table 2, indicating that 20259 and 20111 exhibited certain antimicrobial activity against all three strains within the tested concentration range, and 24909 also showed certain antimicrobial activity against *Escherichia coli* and *Vibrio vulnificus*. Specifically, the MIC of 20259 against *Escherichia coli* was 12.66 μM, against *Staphylococcus aureus* was 3.16 μM, and against *Vibrio vulnificus* was 6.33 μM. The lower the MIC value, the stronger the antimicrobial activity, indicating that the antimicrobial activity of 20259 was superior to that of 20111 and 24909.

[0052] Table 2: MICs of candidate peptides against three bacteria

[0053] 20259 was selected as a candidate antimicrobial peptide for further research, and its MICs against three marine Vibrio species and Pseudomonas aeruginosa were determined (see Table 3). The results showed that the MICs of 20259 against Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa were 12.66 μM, 6.33 μM, 3.16 μM, and 50.63 μM, respectively.

[0054] Table 3: MICs of 20259 against four bacteria

[0055] The mechanotoxicity (MBC) of antimicrobial peptide 20259 against four types of marine Vibrio and three conventional strains was determined by plate bioassay (see Table 4). The results showed that antimicrobial peptide 20259 could kill bacteria at relatively low concentrations. The MBCs against Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa were 12.66 μM, 6.33 μM, 6.33 μM, 25.31 μM, 12.66 μM, 6.33 μM, and 101.25 μM, respectively.

[0056] Table 4: MBC of 20259 against four types of Vibrio vulnificus and three common bacteria

[0057]

[0058] Next, the antimicrobial peptide 20259 was searched and compared with the CAMPR4 antimicrobial peptide database, which contains a wealth of antimicrobial peptide-related data, including 24,243 AMP sequence records, 933 structural data, 2,143 patent records, and 263 different AMP family characteristics. Table 5 shows that the moon jellyfish antimicrobial peptide 20259 is a novel antimicrobial peptide sequence.

[0059] Table 5: Comparison results of antimicrobial peptides from moon jellyfish with the CAMPR4 database

[0060] 6. Bactericidal kinetics of antimicrobial peptide 20259

[0061] The activated bacterial strain was cultured in fresh LB medium at 37°C and 200 rpm / min until the logarithmic growth phase, and the final bacterial concentration was adjusted to 1×10⁻⁶ using the culture medium. 6 CFU / mL. Antimicrobial peptide solution was added to achieve final concentrations of 1×MIC, 5×MIC, and 10×MIC, respectively. After mixing, the mixture was incubated at 37°C, with the bacterial culture without antimicrobial peptide serving as a control. Samples were taken at 0, 5, 10, 30, 60, 180, and 240 min, diluted, and plated on LB agar plates. After incubation at 37°C overnight, the colony count was calculated.

[0062] Three clinically common bacterial strains and three Vibrio marinum strains were selected as experimental strains to evaluate the inhibitory effect of antimicrobial peptide 20259 on the growth activity of these strains. (See [link to relevant documentation]). Figure 3 The figures show the bactericidal kinetic curves of different concentrations of antimicrobial peptide 20259 against bacteria, including (A) Escherichia coli; (B) Staphylococcus aureus; (C) Vibrio vulnificus; (D) Vibrio parahaemolyticus; (E) Vibrio alginolyticus; (F) Pseudomonas aeruginosa; and the control group consisted of bacteria without the drug. Figure 3 The results showed that antimicrobial peptide 20259 only exhibited inhibitory activity against Staphylococcus aureus. At concentrations reaching or exceeding 5×MIC, it demonstrated significant bactericidal effects against Escherichia coli, Vibrio vulnificus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa, killing these strains within 30 minutes, exhibiting near-instantaneous bactericidal characteristics. Because it can rapidly kill bacteria, it reduces the chance of bacteria developing resistance.

[0063] Example 2: Drug resistance induction of antimicrobial peptide 20259

[0064] To assess the potential risk of antimicrobial peptides inducing bacterial resistance, Vibrio vulnificus was subjected to 30 consecutive passages of culture. Two antimicrobial agents (antimicrobial peptide 20259 and ampicillin) were added during the culture process. The specific methods are as follows:

[0065] The bacterial strain was cultured overnight in a shaker at 37°C, and the bacterial suspension was diluted to 1×10⁻⁶ using freshly prepared LB liquid medium. 5CFU / mL. The bacterial suspension was mixed with two antibacterial agent solutions at a volume ratio of 9:1 to achieve a final antibacterial agent concentration below the MIC level. The mixture was incubated overnight at 180 rpm / min in a 37°C constant-temperature shaker. Subsequently, the MIC values ​​of the two antibacterial agents against Vibrio vulnificus were determined using the microdilution method. In subsequent experiments, the cultured bacterial suspension was adjusted to the same concentration gradient with fresh culture medium daily, transferred to sterile test tubes, and an appropriate amount of antibacterial agent was added to maintain the final concentration below the MIC level. The culture was continuously cultured for 30 generations, and the ratio of the MIC after each passage to the initial MIC before the first passage was used as the fold change in MIC.

[0066] At initial culture, Vibrio vulnificus exhibited different sensitivities to the two antimicrobial agents, with MICs of 6.33 μM and 8.94 μM for antimicrobial peptide 20259 and ampicillin, respectively. See also Figure 4 The diagram illustrates the resistance induction of antimicrobial peptide 20259 and ampicillin against Vibrio vulnificus, where the daily MIC fold is the ratio of the MIC after each passage to the initial bacterial MIC before the first generation. Figure 4 The results showed that after 30 consecutive passages, the MIC value of ampicillin increased significantly, by 8-fold. In contrast, the MIC value of antimicrobial peptide 20259 remained stable throughout the passages, without significant change. These findings suggest that Vibrio vulnificus is less likely to develop resistance to antimicrobial peptide 20259 compared to ampicillin.

[0067] Example 3: Stability of antimicrobial peptide 20259

[0068] The effect of temperature on the stability of antimicrobial peptides was investigated: the strain was cultured overnight at 37°C and 200 rpm, and the bacterial concentration was diluted to 10% with fresh CAMHB medium. 6 CFU / mL. The antimicrobial peptide 20259 was prepared into a solution with a concentration of 10 mg / mL using PBS buffer and treated at -20, 30, 70, and 100°C for 30 min, respectively. The MIC of the strain was determined using the same method as the MIC determination in Example 1. Each group of experiments was set up in triplicate.

[0069] Effect of pH on the stability of antimicrobial peptides: PBS buffer was prepared at pH values ​​of 2, 5, 7, and 9, and antimicrobial peptide solutions were prepared using PBS buffers at different pH values. The MIC of the antimicrobial peptides against bacteria was then determined.

[0070] To assess the stability of antimicrobial peptides under different environmental conditions, 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 changes in MIC. See also Figure 5 and Figure 6 , Figure 5 To investigate the effect of temperature on antimicrobial peptide 20259, a temperature of -20℃ was used as a control; n=3, ****, P<0.0001. Figure 6 To investigate the effect of pH on antimicrobial peptide 20259, pH=7 was used as a control; n=3, ***, P<0.001; ****, P<0.0001. Experimental results showed that antimicrobial peptide 20259 exhibited good antibacterial effects within the temperature range of -20℃ to 70℃; however, at a high temperature of 100℃, the antimicrobial activity of antimicrobial peptide 20259 against Vibrio vulnificus decreased. Further analysis of the effect of pH revealed that antimicrobial peptide 20259 showed good antibacterial effects at pH 5 and 7, while its antimicrobial activity decreased to varying degrees under strongly acidic (pH=2) and strongly alkaline (pH=9) conditions.

[0071] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of these embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the present invention's technical solutions, and any such changes and modifications are within the protection scope of the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide is derived from the moon jellyfish.

3. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide was obtained by solid-phase synthesis.

4. The use of the antimicrobial peptide according to any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of bacterial infections.

5. The application according to claim 4, characterized in that: The bacteria are one or more of the following: Escherichia coli, Staphylococcus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Pseudomonas aeruginosa.

6. The application according to claim 4, characterized in that: When the antimicrobial peptide is used as a drug for the prevention and / or treatment of bacterial infections, it is used under conditions of -20 to 70°C and pH 4 to 8.

7. An antimicrobial peptide gene, characterized in that, The gene encodes the antimicrobial peptide as described in claim 1.

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