A novel antimicrobial peptide from Japanese eel, AjCath2b 143-174 and its applications

Abstract

A new antibacterial peptide of Japanese eel A1 Cath2b 143‑174 and its application, belong to the field of biotechnology. The antibacterial peptide is a Cathelicidin family antibacterial peptide of Japanese eel A1 The 143-174 amino acid fragment of Cathelicidin 2b is named as Cath2b A1 Cath2b 143‑174 , and the amino acid sequence is shown as SEQ ID NO. 3. The antibacterial peptide has high inhibition / killing activity on various aquatic pathogenic bacteria, has excellent thermal stability and salt ion tolerance, and has no obvious toxicity to host cells; it plays a bactericidal role by destroying the bacterial cell membrane, and can also attract host immune cells to enhance immunity. The antibacterial peptide can be applied in the preparation of aquatic animal bacterial disease prevention and control drugs. It can solve the problems of drug resistance and drug residue caused by the abuse of traditional antibiotics, and is convenient to prepare, has strong adaptability, and is suitable for the prevention and control of bacterial diseases of aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to eels derived from Japanese eels (Eel spp.) Japanese eel Cathelicidin family of antimicrobial peptides Aj Cath2b 143-174 , and its application in combating various aquatic pathogens. Background Technology

[0002] eel ( Anguilla Eels (spp.), commonly known as eels, are a typical migratory spawning fish. With the continuous expansion of aquaculture scale and density, eel diseases have become increasingly prominent, especially bacterial diseases, which frequently break out, causing huge economic losses to aquaculture farmers. Currently, prevention and control mainly rely on antibiotics, but their overuse has led to the emergence of drug-resistant strains and excessive drug residues, threatening not only the quality and safety of aquatic products but also posing a potential risk to public health. Therefore, developing highly effective and safe new antibacterial drugs has become an urgent need to promote the sustainable development of the eel industry.

[0003] Antimicrobial peptides (AMPs), also known as defensive peptides, are a class of cationic polypeptides with antibacterial activity produced by the animal body, serving as an important natural barrier against pathogen infection. AMPs possess advantages such as natural origin, broad antibacterial spectrum, and low susceptibility to drug resistance in pathogens, leading to their widespread application in various fields. In recent years, with the increasing demand for antibiotic alternatives, antimicrobial peptides have attracted significant attention; their unique bactericidal mechanism—by disrupting the bacterial cell membrane or killing bacteria intracellularly—not only exhibits significant antibacterial effects but also is less likely to induce drug resistance mutations in pathogens, making them a promising candidate for a new generation of effective "antibiotics" for controlling pathogens.

[0004] Cathelicidin is a family of antimicrobial peptides unique to vertebrates. Its mature peptide is an amphiphilic cationic peptide that can directly target bacterial membranes to exert antimicrobial activity, while also possessing certain immunomodulatory functions. Since the first isolation and identification of cathelicidin from the intestines of hagfish in 2003, which exhibited significant inhibitory activity against both Gram-negative and Gram-positive bacteria, research on cathelicidin in fish has made significant progress. Researchers have subsequently observed its activity in rainbow trout (…). rt CATH-1 / 2), Atlantic cod ( cod CATH), Atlantic salmon as CATH-1 / 2), sweetfish ( a This type of antimicrobial peptide has been identified in CATH and various salmonid fish, indicating that Cathelicidin is widely distributed in the fish immune system and has important functions.

[0005] Although research on fish cathelicidins has made some progress, there are still significant gaps in the improvement and screening of specific cathelicidins for Japanese eels. Existing reports on fish cathelicidins are mostly full-length peptides or non-specific truncated fragments, failing to target specific pathogens prevalent in eel farming (such as Edwardsiella tarda). Some natural cathelicidins exhibit an imbalance between antibacterial activity and stability, potential cytotoxicity at high concentrations, and lack adaptability verification for aquaculture environments (such as high temperature and high ionic strength), limiting their practical application. Furthermore, current antimicrobial peptide screening largely relies on traditional separation and purification methods, which are cumbersome and inefficient. Techniques for precise prediction and targeted screening of highly active fragments using bioinformatics have not yet been reported in Japanese eel cathelicidin research, making it difficult to quickly obtain highly efficient antimicrobial peptide candidates suitable for eel farming scenarios.

[0006] Based on the aforementioned technological gaps, this invention uses bioinformatics analysis to predict the Japanese eel. Aj The amino acid fragment from position 143 to 174 of Cath2b Aj Cath2b 143-174 It may possess highly efficient and stable antibacterial activity. High purity can be obtained through chemical synthesis. Aj Cath2b 143-174 The peptide fragment was found to have significant inhibitory / killing effects on both Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Edwardsiella tarda) and Gram-positive bacteria (Staphylococcus aureus), exhibiting excellent thermal stability and salt ion tolerance, while showing no significant toxicity to host cells. Further observation using membrane permeability staining and transmission electron microscopy revealed… Aj Cath2b 143-174 This antimicrobial peptide exerts its bactericidal effect by disrupting the bacterial cell membrane structure. It exhibits highly effective inhibitory and bactericidal activity against a variety of aquatic pathogens and holds promise as a replacement for traditional antibiotics in the prevention and control of bacterial diseases in aquatic animals. Summary of the Invention

[0007] The primary objective of this invention is to address the problems of antibiotic resistance and drug residues caused by the overuse of antibiotics in the prevention and control of diseases in eel farming, as well as the shortcomings of existing antimicrobial peptides such as poor targeting, weak environmental adaptability, and potential toxicity, by providing a novel antimicrobial peptide for Japanese eels. Aj Cath2b 143-174 It possesses highly efficient antibacterial activity, high thermal stability, high salt ion tolerance, low cytotoxicity, and immunotactic activity, and can be used as a novel antibiotic alternative in the field of aquaculture.

[0008] The second objective of this invention is to provide the novel antimicrobial peptide from Japanese eel.Aj Cath2b 143-174 Its application in the preparation of drugs for the prevention and treatment of various aquatic pathogens solves the technical problems of low efficiency, poor safety and insufficient environmental adaptability of antibacterial agents in aquaculture.

[0009] A third objective of this invention is to provide a product containing an antimicrobial peptide. Aj Cath2b 143-174 An antibacterial composition for aquaculture is available. This composition is highly safe, environmentally friendly, and does not easily induce drug resistance. It can be used for the prevention and treatment of aquatic animal diseases and for the inhibition of bacteria in aquaculture water.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention discloses an antimicrobial peptide from Japanese eel. Aj Cath2b 143-174 Japanese eel (Eel) Anguilla japonica Cathelicidin family peptides Aj The amino acid sequence from position 143 to position 174 of Cathelicidin 2b. Aj Cath2b 143-174 The amino acid sequence is shown in SEQ ID NO.3; encoding Aj The nucleic acid sequence of Cathelicidin2b is shown in SEQ ID NO.1. Aj The amino acid sequence of Cathelicidin2b is shown in SEQ ID NO.2.

[0012] SEQ ID NO.1:

[0013] ATGAGAAGTGAGACACATAAGATGAAGAGCTCTGTTGGACCTCTGCTGCTGCTCTCCCTTGTTGCTTTTGTCTCTGTGACATTGGCCAGGAGCGTCTTCACCTTTACAGATGTCCTTGCCGCGGCCACTGCAGACTTCAACCAGAAAAGCCAGGAGACAAAAGCTTTTGGACCTCCAAAGCAGGGCGCTTTGCGGTCAATGTCGGTGTTTGAGCCAGGAGATGGCTCCGTCATGATCAAG TCCATTACGTTTACGCTTAAGGAGACGGTGTGCCCCAAATCAGAAGACTACCTAAAGGAAGAGTGCGTCTTCAAGGAAAATGGGTCTCTGAAGAAGTGCTCCAGTACAGCTACAGTCCTCAAGTCACAGCCAGGAGAGGCAGCATCTCTGACAGTGTCCTGTCAGGAGGTCACAGACCCAGAGGAGCGCAAGGTACAGGCTTTACATCTGATTAGCCTTCATGGAACACCTAAAATATAT

[0014] SEQ ID NO.2:

[0015] MRSETHKMKSSVGPLLLLSLVAFVSVTLARSVFTFTDVLAAATADFNQKSQETKAFGPPKQGALRSMSVFEPGDGSVMIKSITFTLKETVCPKSEDYLKEECVFKENGSLKKCSSTATVLKSQPGEAASLTVSCQEVTDPEERKVQALHLISLHGTPKIYSQTKKYVSMWTKSI

[0016] SEQ ID NO.3:

[0017] RKVQVLHLISLHGTPKIYSQTKKYVSMWTKSI

[0018] The antimicrobial peptide Aj Cath2b 143-174 The peptides were obtained through bioinformatics prediction and screening. Specifically, the active fragments were identified by analyzing the APD3 and CAMPR3 antimicrobial peptide databases using online tools such as SMART and SignalP. The peptides were then chemically synthesized using solid-phase synthesis and purified by high-performance liquid chromatography with a purity of ≥98%.

[0019] The antimicrobial peptide Aj Cath2b 143-174 For Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Edwardsiella tarda ( ), Edwardsiella tarda ( Edwardsiella tarda This synthetic peptide exhibits highly efficient inhibitory / killing activity. Specifically, its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus and Escherichia coli are both 2 μM; against Pseudomonas aeruginosa, both the MIC and MBC are 8 μM; against Edwardsiella tarda, the MIC is 16 μM and the MBC is 32 μM; and against Aeromonas hydrophila, the MIC is 64 μM and the MBC is greater than 64 μM. Experiments show that this synthetic peptide can effectively inhibit the growth of pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Edwardsiella tarda.

[0020] The antimicrobial peptide Aj Cath2b 143-174 It exhibits good thermal stability and retains its antibacterial activity even after treatment at 100℃; it also has strong salt ion tolerance and retains its antibacterial activity in the concentration range of 10~160 mM NaCl, making it suitable for high-salt environments, especially for aquaculture and related applications.

[0021] The antimicrobial peptide Aj Cath2b 143-174 This antimicrobial peptide causes bacterial cell death by disrupting the integrity of bacterial cell membranes and increasing membrane permeability. It also has no significant toxicity to Japanese eel kidney cells. Based on its mechanism of action, this antimicrobial peptide can be used for the prevention and control of bacterial diseases in aquatic animals.

[0022] The antimicrobial peptide Aj Cath2b 143-174 This synthetic peptide can efficiently chemotact with peripheral blood leukocytes of eels. In particular, at a concentration of 10 μM, it has a significant chemotactic effect on eel lymphocytes and myeloid cells.

[0023] The antimicrobial peptide Aj Cath2b 143-174 It can be used in the preparation of products for the prevention and treatment of bacterial diseases in aquatic animals.

[0024] The aquatic animals include eels; the bacterial diseases are caused by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, or Aeromonas hydrophila; the prevention and treatment products include at least one of the following: drugs for the prevention and treatment of bacterial diseases in aquatic animals and antibacterial agents for aquaculture water.

[0025] A composition for the prevention and treatment of bacterial diseases in aquatic animals, comprising the aforementioned novel antimicrobial peptide from Japanese eel. Aj Cath2b 143-174 And pharmaceutically or aquaculture-acceptable carriers or excipients. The carriers or excipients may be selected from at least one of sterile saline, PBS buffer, starch, dextrin, sodium alginate, chitosan, defatted soybean meal, and fishmeal carriers.

[0026] The aforementioned preventive composition can be used for the prevention and control of bacterial diseases in aquatic animals and for the inhibition of bacteria in aquaculture water.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The antimicrobial peptide of this invention Aj Cath2b 143-174 Based on the characteristics of highly pathogenic bacteria in eel farming, the active fragments were obtained through precise prediction and screening using bioinformatics, rather than being randomly truncated. They have a significant inhibitory effect on eel-specific pathogens such as Edwardsiella tarda, and their antibacterial activity is superior to most natural full-length Cathelicidin peptides.

[0029] 2. Antimicrobial peptides Aj Cath2b 143-174 It possesses outstanding thermal stability and salt ion tolerance, and can adapt to high-temperature environments (such as water temperature rise in summer) and waters with different salinity (such as freshwater and brackish water aquaculture) in the process of aquaculture, solving the core problem that the activity of existing antimicrobial peptides is easily reduced in aquatic environments.

[0030] 3. Antimicrobial peptides Aj Cath2b 143-174 It has no obvious toxicity to the host cells of Japanese eel and is a natural antimicrobial peptide fragment that can be naturally degraded in water and organisms, without causing drug residues, thus avoiding food safety and environmental pollution problems caused by antibiotic abuse.

[0031] 4. In addition to direct sterilization, antimicrobial peptides Aj Cath2b 143-174 It can also chemotactically attract host immune cells, enhance the aquatic animals' own immunity, and prevent and control diseases from two aspects: killing pathogens and improving host resistance, resulting in a more lasting prevention and control effect. Attached Figure Description

[0032] Figure 1 Antimicrobial peptide Aj The open reading frame sequence (ORF, SEQ ID NO.2) and the predicted polypeptide sequence (SEQ ID NO.3) of Cath2b.

[0033] Figure 2 Antimicrobial peptide AjCath2b 143-174 Bactericidal kinetic curves against Escherichia coli and Staphylococcus aureus; where A represents Escherichia coli and B represents Staphylococcus aureus.

[0034] Figure 3 for Aj Cath2b 143-174 Thermal stability analysis; where A is Escherichia coli; B is Staphylococcus aureus;

[0035] Figure 4 for Aj Cath2b 143-174 Salt tolerance analysis; where A is Escherichia coli; B is Staphylococcus aureus;

[0036] Figure 5 for Aj Cath2b 143-174 Toxicity analysis of Japanese eel kidney cells;

[0037] Figure 6 for Aj Cath2b 143-174 Effects on bacterial cell membrane permeability; where A is Escherichia coli and B is Staphylococcus aureus;

[0038] Figure 7 for Aj Cath2b 143-174 Effects on bacterial ultrastructure; where A represents untreated E. coli; B represents... Aj Cath2b 143-174 C represents treated Escherichia coli; D represents untreated Staphylococcus aureus; Aj Cath2b 143-174 Staphylococcus aureus after treatment;

[0039] Figure 8 for Aj Cath2b 143-174 Chemotactic activity analysis of peripheral blood leukocytes from Japanese eels; where A is the chemotactic activity curve of lymphocytes and B is the chemotactic activity curve of myeloid cells. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. For matters not described in detail, conventional technical means in the art can be used.

[0041] 1. Experimental Materials

[0042] 1.1 Experimental strains

[0043] Staphylococcus aureus ( Staphylococcus aureusATCC25923, Escherichia coli ( Escherichia coli ATCC25922, purchased from the Microbial Culture Collection Center of the Chinese Academy of Sciences. Edwardsiella tarda ( Edwardsiella tarda ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Aeromonas hydrophila ( ) Aeromonas hydrophila This strain is a common pathogenic bacterium in eels. It was isolated from diseased Japanese eels and its identity was confirmed by conventional microbial isolation and culture methods and 16S rRNA genome sequencing. The purity and pathogenicity of the strain were verified to meet the experimental requirements.

[0044] 1.2 Experimental cells: Japanese eel kidney cells.

[0045] 1.3 Main reagents: Enhanced Cell Counting Kit-8 (CCK-8) kit (Shanghai Beyotime), propidium iodide (1 mg / mL) (Beijing Solarbio), fetal bovine serum 10270106 (Gibco), Percoll lymphocyte separation medium (Beijing Solarbio).

[0046] 1.4 Main instruments: DK-6005 constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd., China), PYX-280S-B biochemical incubator (Guangdong Keli Experimental Equipment Co., Ltd.), QuantStudio™ 3D multi-functional microplate reader (Berten Instruments, Inc., USA), CytoFLEX flow cytometer (Beckman Coulter, USA).

[0047] 2 Experimental Methods

[0048] 2.1 Synthesis of antimicrobial peptides

[0049] The antimicrobial peptides of this eel were determined by analysis using the online tools SMART (http: / / smart.embl-heidelberg.de / ) and SignalP (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), combined with the antimicrobial peptide databases APD3 (https: / / aps.unmc.edu / AP / ) and CAMPR3 (http: / / www.camp3.bicnirrh.res.in / ). Aj The target peptide is the amino acid fragment from positions 143 to 174 of Cathelicidin2b, named... Aj Cath2b 143-174Its amino acid sequence is shown in SEQ ID NO.3. It was synthesized by Shanghai Sangon Biotech Co., Ltd. using solid-phase synthesis and purified by high performance liquid chromatography. The purity of the peptide after purification is ≥98%.

[0050] 2.2 In vitro antibacterial activity assay

[0051] Activated Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Aeromonas hydrophila were adjusted to a concentration of 1×10⁻⁶ with sterile physiological saline. 5 ~10 6 CFU / mL. Add the following to a 96-well cell culture plate sequentially: Aj Cath2b 143-174 Synthetic peptides (128, 64, 32, 16, 8, 4 μM) and diluted bacterial suspensions (1×10⁻⁶ μM) 5 ~10 6 50 µL of each of the following were prepared: CFU / mL. A negative control (50 μL sterile ddH2O and 50 μL culture medium) and a positive control (50 μL bacterial suspension and 50 μL culture medium) were also prepared. Each sample was tested in triplicate. The cultures were incubated at 28°C for 18–24 h, and OD was measured. 600 Results: After 18–24 h of culture, visual observation showed no significant change in turbidity of the bacteria in the micropores after adding the synthetic peptide compared to before culture. Meanwhile, the OD... 600 There was no significant change compared to the negative control, and its corresponding lowest Aj Cath2b 143-174 The concentration was used as the minimum inhibitory concentration (MIC). To determine the minimum bactericidal concentration (MBC), the culture mixture from wells with concentrations at or above the MIC was taken, mixed by pipetting, transferred to fresh liquid culture medium, centrifuged at 3000 g for 5 min, most of the supernatant was discarded, the remaining precipitate was resuspended with the supernatant and mixed, plated, and incubated at 28°C for 24 h. After counting the colonies, the lowest peptide concentration that killed 99.9% of the original inoculum was taken as the minimum bactericidal concentration (MBC).

[0052] 2.3 Aj Cath2b 143-174 bactericidal kinetics determination

[0053] Activated Staphylococcus aureus and Escherichia coli were adjusted to a concentration of 1×10⁻⁶ with sterile physiological saline. 5 ~10 6 CFU / mL. Experimental and control groups were set up in sterile EP tubes: the experimental group was treated with 10 μL of bacterial suspension and 10 μL of... Aj Cath2b 143-174The synthetic peptide (4 µM) was prepared by adding 10 μL of bacterial suspension and 10 μL of sterile water to the control group, mixing thoroughly, and incubating at 28 °C. Immediately after mixing, 10 μL of the mixed bacterial suspension was taken every 30 min and added to 5 mL of fresh liquid culture medium. The mixture was then centrifuged at 3000 g for 5 min at room temperature, and 4.8 mL of supernatant was discarded. The remaining 200 μL of supernatant was thoroughly resuspended with the precipitate and mixed well. The entire mixture was then spread onto LB agar plates and incubated at 28 °C for 24 h. After incubation, colonies were observed and counted, and a time-colony count curve was plotted to characterize its bactericidal kinetics.

[0054] 2.4 Aj Cath2b 143-174 thermal stability

[0055] Activated Staphylococcus aureus and Escherichia coli were adjusted to a concentration of 1×10⁻⁶ with sterile physiological saline. 5 ~10 6 CFU / mL. Aj Cath2b 143-174 The synthetic peptide was prepared into a 4 µM solution using sterile water, aliquoted, and then heat-treated at 100 °C for 0, 5, 10, 15, 30, and 60 min, respectively. After heat treatment, experimental and control groups were set up in sterile EP tubes: the experimental group was added with 10 μL of bacterial culture and 10 μL of the above synthetic peptide solution, and the control group was added with 10 μL of bacterial culture and 10 μL of sterile water. After mixing, the tubes were incubated at 28 °C, and the OD was measured every 12 h. 600 The value was continuously monitored for 48 hours. The thermal stability of the synthetic peptide was evaluated by comparing the antibacterial activity of the peptide solution after heat treatment for different times with that of the untreated peptide solution.

[0056] 2.5 Aj Cath2b 143-174 Salt ion tolerance

[0057] The bacterial suspension of the test strain was adjusted to a concentration of 1×10⁻⁶ using sterile physiological saline. 5 ~10 6 CFU / mL. Aj Cath2b 143-174 A 4 µM synthetic peptide solution was mixed with NaCl solutions of different concentrations to achieve final concentrations of 10, 20, 40, 80, and 160 mM. The experimental group received 10 μL of bacterial culture mixed with 10 μL of the synthetic peptide solution, while the control group received 10 μL of bacterial culture mixed with 10 μL of sterile water. The mixture was incubated at 28 °C, and the OD (oxidative stress) was measured every 12 h. 600 The salt ion tolerance of the synthetic peptide was assessed by comparing the antibacterial activity of peptide solutions treated with different ion concentrations with that of untreated peptide solutions.

[0058] 2.6 Aj Cath2b 143-174 cytotoxicity

[0059] The cytotoxicity of synthetic peptides to Japanese eel kidney cells was assessed using the Enhanced Cell Counting Kit-8 (CCK-8) kit (Beyotime Biotechnology Co., Ltd.). Kidney cells in logarithmic growth phase were seeded at a density of 5000 cells per well in 96-well plates. The synthetic peptides were diluted to final concentrations of 2, 4, 8, 16, 32, and 64 μM using complete culture medium. Experimental setups included: a blank control group (containing only 200 μL of complete culture medium), a negative control group (cells + 200 μL of complete culture medium without the synthetic peptide), and experimental treatment groups (cells + 200 μL of complete culture medium containing different concentrations of the synthetic peptide). After gentle mixing, the plates were incubated at 28 °C in a 5% CO2 incubator for 24 h. After incubation, 20 μL of CCK-8 was added to each well, and incubation continued for 1 h. The OD values ​​of each well were measured using a microplate reader. 450 Cytotoxicity is calculated using the following formula:

[0060] Cytotoxicity (%) = [1 - (OD)] 处理 - OD 空白 ) / (OD 对照 -OD 空白 )]× 100%

[0061] 2.7 Membrane permeability analysis

[0062] The concentration of the test strain was adjusted to 1×10⁻⁶ using sterile physiological saline. 5 CFU / mL, using 4 μM Aj Cath2b 143-174 The synthetic peptides were incubated for 4 h, stained with propidium iodide (PI), and analyzed by flow cytometry. Data were analyzed using FlowJo software, and the effect of the synthetic peptides on bacterial cell membrane permeability was quantitatively assessed by comparing the percentage of PI-positive cells in the experimental and control groups.

[0063] 2.8 Transmission Electron Microscopy (TEM) Analysis

[0064] TEM was used to observe the destructive effects of synthetic peptides on bacterial microstructure. The test strain was adjusted to 1×10⁻⁶ saline solution. 7CFU / mL, kept on ice for later use. The synthetic peptide solution was prepared to twice the MBC concentration of the test strain. Experimental and control groups were set up in sterile EP tubes: the experimental group was added with 50 μL of high-concentration bacterial suspension and 50 μL of synthetic peptide solution, and the control group was added with 50 μL of bacterial suspension and 50 μL of sterile water. After mixing, the mixture was incubated at 28 ℃ for 4 h. After incubation, the mixture was centrifuged at 4000 g for 5 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 1 mL of PBS buffer and washed. The mixture was centrifuged again at 4000 g for 5 min at room temperature, the supernatant was discarded, and the bacterial precipitate was thoroughly resuspended and fixed in 500 μL of 4% paraformaldehyde solution. The samples were stored at 4 ℃ until sent for testing. The fixed samples were subjected to ultrathin sectioning and transmission electron microscopy to compare the differences in cell wall, cell membrane, and internal structure between the synthetic peptide-treated and untreated bacteria.

[0065] 2.9 Aj Cath2b 143-174 Chemotaxis of peripheral blood leukocytes in Japanese eels

[0066] The chemotactic effect of synthetic peptides on peripheral blood leukocytes of Japanese eel was investigated using the Transwell assay. The lower chamber pore of the Transwell chamber was rinsed with 1 mL of fetal bovine serum (FBS), and excess serum was discarded. 1 mL of heparin-anticoagulated whole blood from Japanese eel was mixed with 9 mL of culture medium and gently stacked in a centrifuge tube containing 6 mL of 60% Percoll separation solution. The mixture was centrifuged at 400 g for 20 min at room temperature to separate the peripheral blood lymphocyte population. The supernatant plasma was discarded, and the lymphocyte-rich white cell layer was aspirated, transferred to a new tube, resuspended in 4 times its volume of complete culture medium, centrifuged at 400 g for 5 min, washed, resuspended, and the cell density adjusted to 1 × 10⁻⁶ cells / mL. 6 cells / mL. Different concentrations of synthetic peptide solutions (0 nM, 100 nM, 1000 nM, 10000 nM) were added to the lower chamber of a Transwell chamber, covered with a polycarbonate membrane, and 100 μL of cell suspension was added to the upper chamber. After incubation at 28°C for 28 h, the solution from the lower chamber was collected, centrifuged at 400 g for 5 min, the cells were resuspended, washed once with PBS, and the cell count was determined by flow cytometry. Cell migration was analyzed using FlowJo software. One-way ANOVA was performed using GraphPad Prism 8.0 software to compare the chemotactic activity of different concentrations of synthetic peptides.

[0067] 2.10 Validation of the antibacterial effect of the composition

[0068] Will Aj Cath2b 143-174A 2 mg / mL aquatic soaking composition was prepared by mixing the composition with sterile physiological saline. The composition was used to treat artificially infected juvenile eels with Edwardsiella tarda by bathing once a day for 3-5 days. A sterile physiological saline bath group without antimicrobial peptides was set up as a blank control. Each group was set up in 3 replicates. The mortality rate of eels was recorded to verify the actual prevention and control effect of the composition.

[0069] 3 Results and Analysis

[0070] 3.1 Synthetic Peptides Aj Cath2b 143-174 Related information

[0071] Based on what has been obtained Aj Cath2b amino acid sequence ( Figure 1 ), Figure 1 In the diagram, underlined areas represent signal peptides, gray areas represent CLD (Cathelin-like) domains, and bolded areas represent mature peptides. express Aj The termination marker in the Cath2b amino acid sequence corresponds to the polypeptide chain termination site encoded by the stop codon in its nucleic acid sequence (SEQ ID NO.2), indicating that the amino acid sequence ends at this site and there are no additional amino acid residues following it. It should be noted that... Aj The full-length Cath2b peptide has no obvious antibacterial activity, while the mature peptide (amino acids 143-174) marked in bold above is the core region of its antibacterial activity.

[0072] Using the online tools SMART (http: / / smart.embl-heidelberg.de / ) and SignalP (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), and in conjunction with the antimicrobial peptide databases APD3 (https: / / aps.unmc.edu / AP / ) and CAMPR3 (http: / / www.camp3.bicnirrh.res.in / ), the mature peptide sequence was predicted to be amino acids 143 (arginine) to 174 (isoleucine), and named... Aj Cath2b 143-174 The peptides were synthesized in solid phase by Shanghai Sangon Biotech Co., Ltd. and purified by HPLC. The purity of the peptides was above 98%. The sequence information and physicochemical parameters are shown in Table 1.

[0073] Table 1 Aj Cath2b 143-174 Sequence information and its physicochemical parameters

[0074]

[0075] 3.2 Aj Cath2b 143-174 It has antibacterial / bactericidal activity

[0076] Staphylococcus aureus (Gram-positive) and four Gram-negative bacteria—Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Aeromonas hydrophila—were selected and detected using a microwell method. Aj Cath2b 143-174 It has antibacterial and bactericidal effects. Results showed that... Aj Cath2b 143-174 The MIC and MBC against Staphylococcus aureus and Escherichia coli were both 2 μM; the MIC and MBC against Pseudomonas aeruginosa were both 8 μM; and the MIC against Edwardsiella tarda was 16 μM and the MBC was 32 μM. Aj Cath2b 143-174 The results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against the five bacteria are shown in Table 2.

[0077] Table 2 Aj Cath2b 143-174 Antibacterial activity

[0078]

[0079] 3.3 Aj Cath2b 143-174 bactericidal kinetic curve

[0080] Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) were used as the test strains. Aj Cath2b 143-174 The concentration was 4 µM. The results showed that... Aj Cath2b 143-174 After incubation with E. coli for 12 hours, the survival rate was 0%. Figure 2 (A in the text); after incubation with Staphylococcus aureus for 7 hours, the survival rate was 0% ( Figure 2 (B) indicates that... Aj Cath2b 143-174 It has a bactericidal effect on both Escherichia coli and Staphylococcus aureus, and its bactericidal speed is faster against Staphylococcus aureus.

[0081] 3.4 Aj Cath2b 143-174 It has thermal stability

[0082] 4 µM Aj Cath2b 143-174After incubation in a metal bath at 100℃ for 5, 10, 15, 30, and 60 min, respectively, the bacteria were co-incubated with Escherichia coli and Staphylococcus aureus, and OD was measured every 12 h. 600 Value. The results showed that after treatment at 100℃... Aj Cath2b 143-174 It retains antibacterial activity against Escherichia coli and Staphylococcus aureus, but its antibacterial activity against Escherichia coli has decreased. Figure 3 (A in the text); while the antibacterial activity against Staphylococcus aureus showed no significant change, and it could completely inhibit and kill Staphylococcus aureus ( ). Figure 3 (B in the middle).

[0083] 3.5 Aj Cath2b 143-174 Salt ion tolerance

[0084] Will Aj Cath2b 143-174 The bacteria were placed in NaCl solutions with final concentrations of 10, 20, 40, 80, and 160 mM and co-incubated with *Escherichia coli* and *Staphylococcus aureus*. OD was measured every 12 hours. 600 Value. The results showed that in the presence of sodium ions, Aj Cath2b 143-174 It maintains antibacterial activity against Escherichia coli, but the activity is somewhat reduced. Figure 4 (A in the text); while at low salt concentrations (10, 20, and 40 mM NaCl), it can completely inhibit and kill Staphylococcus aureus, and its antibacterial activity decreases with increasing salt ion concentration (80 and 160 mM). Figure 4 (B in the middle).

[0085] 3.6 Aj Cath2b 143-174 No cytotoxicity to Japanese eel kidney cells

[0086] Different concentrations (2, 4, 8, 16, 32, and 64 µM) Aj Cath2b 143-174 Cell viability was assessed by co-incubation with Japanese eel kidney cells for 24 hours using the CCK-8 assay. Results showed that... Aj Cath2b 143-174 No significant cytotoxicity to Japanese eel kidney cells ( Figure 5 ).

[0087] 3.7 Aj Cath2b 143-174 Enhance the permeability of bacterial membranes

[0088] Will Aj Cath2b 143-174The bacteria were co-incubated with Escherichia coli and Staphylococcus aureus, respectively. After PI staining, flow cytometry analysis showed that in the Escherichia coli group, the percentage of PI-positive bacteria increased from 1.63% before treatment to 67.7% after treatment. Figure 6 A); In the Staphylococcus aureus group, the percentage of PI-positive bacteria was 1.16% before treatment; after treatment, it increased to 30.3% ( Figure 6 B). Indicates Aj Cath2b 143-174 Treatment can significantly increase the cell membrane permeability of Escherichia coli and Staphylococcus aureus.

[0089] 3.8 Aj Cath2b 143-174 It can damage the bacterial plasma membrane.

[0090] Will Aj Cath2b 143-174 The samples were incubated with Escherichia coli and Staphylococcus aureus, respectively. After processing, the samples were observed using TEM. The results showed that: without... Aj Cath2b 143-174 Synthetic peptide-treated E. coli ( Figure 7 A in the middle) and Staphylococcus aureus ( Figure 7 C) The bacteria are morphologically intact, have smooth surfaces, and are of normal cell size; Aj Cath2b 143-174 After treatment with synthetic peptides, Escherichia coli ( Figure 7 B) and Staphylococcus aureus ( Figure 7 In the case of D) shrinkage, the bacterial cell membrane perforates, and in severe cases, the entire bacterial cell ruptures. (Explanation) Aj Cath2b 143-174 It can cause damage to the membrane structure of Escherichia coli and Staphylococcus aureus, leading to leakage of cytoplasm and bacterial death.

[0091] 3.9 Aj Cath2b 143-174 It has a significant chemotactic effect on peripheral blood cells of Japanese eel.

[0092] Peripheral blood leukocytes (PBMCs) were isolated in vitro and separated using the Percoll density centrifugation method. Chemotaxis experiments were performed using Trans Well chambers, with 1.0 × 10⁻⁶ ppm added above the nested membrane. 5 PBMC, with different concentrations (10, 100, 1000, 10000 nM) added below. Aj Cath2b 143-174 Synthetic peptides were incubated at 28°C for 6 h using a medium containing 5% FBS as a positive control and a complete medium as a negative control. Flow cytometry was used to record 2-minute events, and the data were analyzed using Flowjo software. Results showed:Aj Cath2b 143-174 At 10000 nM, it can significantly promote the activity of Japanese eel lymphocytes ( Figure 8 A) and myeloid cells ( Figure 8 The chemotaxis of B in B).

[0093] 3.10 (inclusive) Aj Antibacterial efficacy verification of Cath2b143-174 aquatic composition

[0094] Compared with the blank control group, containing Aj Cath2b 143-174 The soaking composition can significantly reduce the mortality rate of artificially infected juvenile eels with Edwardsiella tarda, with a prevention and control efficacy rate of over 80%, indicating that the composition has a significant prevention and control effect on bacterial diseases in eels and can be used for the practical prevention and control of bacterial diseases in aquatic animals.

[0095] This invention uses bioinformatics analysis to accurately predict the Japanese eel. Aj The active fragment of Cathelicidin2b was used to obtain an antimicrobial peptide through chemical synthesis. Aj Cath2b 143-174 Experiments have verified that this antimicrobial peptide has significant inhibitory / killing effects on various aquatic pathogens, and possesses excellent thermal stability, ion tolerance, and biosafety, with no obvious toxicity to host cells. Its bactericidal mechanism is clear: it exerts its bactericidal effect by disrupting the bacterial cell membrane, and simultaneously enhances immunity by chemotactically attracting host immune cells. This antimicrobial peptide can be used in the preparation of drugs for the prevention and control of bacterial diseases in aquatic animals. It can solve the problems of drug resistance and drug residues caused by the overuse of traditional antibiotics. Its preparation is convenient and highly adaptable, and it can be widely used in the prevention and control of bacterial diseases in aquatic animals, possessing significant economic and social value. It holds promise for replacing traditional antibiotics in the prevention and control of bacterial diseases in aquatic animals, filling the gap in research on antimicrobial peptides specific to Japanese eels.

[0096] In this invention Aj Cath2b 143-174 The MIC / MBC values ​​against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Aeromonas hydrophila were 2 / 2, 2 / 2, 8 / 8, 16 / 32, and 64µM, respectively. Previous studies have shown that Atlantic hagfish (… Myxine glutinosa The truncated peptide of cathelicidin 37 has a MIC of 8 µM against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Enterobacter aerogenes (T. Uzzell et al., 2003). Rainbow trout ( Oncorhynchus mykissThe truncated peptide of Cathelicidin2 has a MIC of 2.5 µM and an MBC of 5 µM against E. coli, exhibiting moderate ion tolerance (F.D' Este et al., 2016). Atlantic cod ( Gadus morhua The MICs of cathelicidin1 against different strains of Staphylococcus aureus range from 0.36 to 3.6 µM, against Escherichia coli it is 5 µM, and against Aeromonas hydrophila and Aeromonas salmonicidae both are 10 µM. This peptide is sensitive to salt ions, and its antibacterial activity is significantly higher under low-salt conditions than under high-salt conditions (DCBroekman et al., 2011). In summary, the antibacterial spectrum, antibacterial intensity, and bactericidal ability of cathelicidin family antimicrobial peptides from different fish sources vary significantly, and their MICs and MBCs are affected by factors such as peptide sequence composition, truncation method, and environmental ionic conditions. The present invention... Aj Cath2b 143-174 It exhibits excellent and stable antibacterial activity against a variety of important aquatic pathogens, further demonstrating its research and application value as a novel antimicrobial peptide.

[0097] The specific embodiments described above are merely examples of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent modifications, improvements, and alterations made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An antimicrobial peptide from Japanese eel Aj Cath2b 143-174 Its characteristics are, Its amino acid sequence is shown in SEQ ID NO. 3 of the sequence listing; this antimicrobial peptide Aj Cath2b 143-174 Japanese eel ( Anguilla japonica Cathelicidin family peptides Aj The amino acid sequence from position 143 to position 174 of Cathelicidin2b encodes... Aj The nucleic acid sequence of Cathelicidin2b is shown in SEQ ID NO.

1. Aj The full-length amino acid sequence of Cathelicidin2b is shown in SEQ ID NO.

2.

2. The Japanese eel antimicrobial peptide according to claim 1 Aj Cath2b 143-174 Its characteristics are, The antimicrobial peptides were obtained through bioinformatics prediction and screening, chemically synthesized using solid-phase synthesis, and purified by high-performance liquid chromatography, with a purity ≥98% after purification.

3. The Japanese eel antimicrobial peptide according to claim 1 Aj Cath2b 143-174 Its characteristics are, It exhibits inhibitory or bactericidal activity against aquatic pathogens, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Aeromonas hydrophila. Specifically, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Staphylococcus aureus and Escherichia coli are both 2 μM; the MIC and MBC against Pseudomonas aeruginosa are both 8 μM; the MIC against Edwardsiella tarda is 16 μM, and the MBC is 32 μM; and the MIC against Aeromonas hydrophila is 64 μM, with an MBC greater than 64 μM.

4. The Japanese eel antimicrobial peptide according to claim 1 Aj Cath2b 143-174 Its characteristics are, It exhibits excellent thermal stability and salt ion tolerance; it retains its antibacterial activity after treatment at 100℃ for 0–60 min; and it maintains its antibacterial activity within the concentration range of 10–160 mM NaCl.

5. The Japanese eel antimicrobial peptide according to claim 1 Aj Cath2b 143-174 Its characteristics are, It achieves its bactericidal effect by disrupting the integrity of bacterial cell membranes and enhancing membrane permeability; and the antimicrobial peptide has no significant cytotoxicity to Japanese eel kidney cells in the concentration range of 2~64μM.

6. The Japanese eel antimicrobial peptide according to claim 1 Aj Cath2b 143-174 Its characteristics are, It exhibits chemotactic activity against peripheral blood lymphocytes and myeloid cells of Japanese eel, with significant chemotactic activity at a concentration of 10 μM.

7. The Japanese eel antimicrobial peptide according to any one of claims 1 to 6 Aj Cath2b 143-174 Application in the preparation of products for the prevention and treatment of bacterial diseases in aquatic animals.

8. The application according to claim 7, characterized in that, The aquatic animals include eels; the bacterial diseases are caused by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, or Aeromonas hydrophila; the prevention and treatment products include at least one of the following: drugs for the prevention and treatment of bacterial diseases in aquatic animals and antibacterial agents for aquaculture water.

9. A composition for the prevention and treatment of bacterial diseases in aquatic animals, characterized in that, Contains Japanese eel antimicrobial peptides as described in any one of claims 1 to 6 Aj Cath2b 143-174 And pharmaceutically or aquaculture-acceptable carriers or excipients.

10. The application of the preventive composition according to claim 9 in the prevention and control of bacterial diseases in aquatic animals and in the inhibition of bacteria in aquaculture water.

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