Antibacterial peptide ajaponin from anguilla japonica and application thereof

Abstract

The application discloses an antibacterial peptide Ajaponin of Japanese eel and application thereof. The antibacterial peptide Ajaponin is composed of 21 amino acids and has a molecular weight of 2693.29 Dalton. The antibacterial peptide Ajaponin contains 7 positively charged amino acid residues, has a hydrophobicity of 0.316 and an isoelectric point of 11.45. The antibacterial peptide Ajaponin is a novel cationic short peptide, has good water solubility, high antibacterial activity, low toxicity and other advantages, is composed of limited amino acid residues, has compact structure, is easy to synthesize and purify, and is convenient for large-scale production and application. In addition, the antibacterial peptide Ajaponin contains multiple positively charged amino acid residues, is beneficial to interaction with negative charges of bacterial cell membranes, destroys the membrane integrity, and thus plays a role in rapid bacteriostasis and sterilization.

Description

Technical Field

[0001] This invention belongs to the field of marine molecular biology technology, specifically relating to Ajaponin, an antimicrobial peptide from Japanese eels, and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of small-molecule polypeptides produced by the innate immune system in response to invasion by pathogenic microorganisms. They possess broad-spectrum antimicrobial activity. These peptides are widely distributed in nature, including bacteria, fungi, plants, insects, fish, amphibians, and mammals. The main mechanisms of action of antimicrobial peptides include disrupting the cell membranes of pathogens, interfering with their metabolic processes, or inhibiting biofilm formation, thereby exhibiting inhibitory or killing effects on Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and parasites. As an important component of the organism's defense system, antimicrobial peptides have evolved diverse structures and functions, exhibiting rapid response, low drug resistance, and low toxicity, making them a hot area for the development of novel anti-infective drugs.

[0003] In aquaculture, bacterial diseases, particularly vibriosis, pose a significant threat to economic losses. Vibrio bacteria, such as Vibrio parahaemolyticus, Vibrio harzianum, Vibrio fluvialis, and Vibrio alginolyticus, are common pathogens in aquatic animals. These bacteria infect hosts through virulence factors such as adhesins, lipopolysaccharides, hemolysins, and outer membrane proteins, leading to symptoms in fish such as skin ulcers, fin damage, muscle erosion, and internal bleeding, sometimes causing mass mortality. Vibriosis has a high incidence rate and spreads rapidly, often breaking out in intensive aquaculture systems due to factors such as aquatic environment, stocking density, and seasonal changes, posing a huge challenge to the global aquaculture industry. Currently, the main strategies for controlling vibriosis include improving water quality, strengthening disease prevention management, and using vaccines, but these measures often have limited effectiveness during outbreaks. Controlling infection typically relies on chemical drugs and antibiotics, such as fluoroquinolones, tetracyclines, and sulfonamides. However, long-term overuse of antibiotics has led to bacterial resistance, and the emergence of resistant strains increases the difficulty of treatment. Meanwhile, drug residues accumulate in fish and the aquatic environment, threatening food safety and ecological balance, disrupting aquatic microbial communities, and even affecting human health through the food chain. Internationally, numerous regulations have restricted the use of antibiotics in aquaculture, prompting the search for green and sustainable alternatives.

[0004] As aquatic organisms, fish have evolved highly efficient natural defense mechanisms in complex environments, making them a valuable resource for developing novel antimicrobial peptides. This is especially true for migratory fish like the Japanese eel (Anguilla japonica), whose life cycle involves switching between freshwater and marine environments, making them susceptible to various microbial infections and thus leading to the development of abundant innate immune factors. These factors include a variety of antimicrobial peptides that effectively resist bacterial, fungal, and viral invasions. While research on antimicrobial peptides from the Japanese eel has made progress, current reports primarily focus on full-length proteins or long-chain peptides, with limited development of short-chain cationic peptides. Existing antimicrobial peptides have shortcomings in terms of water solubility, antimicrobial spectrum, and safety. For example, some peptides have poor solubility, are easily degraded by proteases, or exhibit certain toxicity to host cells, limiting their application in aquatic feed additives and antimicrobial compositions. Furthermore, existing fish-derived antimicrobial peptides are mostly targeted at specific bacteria, and their broad-spectrum and stability need further improvement. To address these issues, it is necessary to screen novel short peptides from fish genomes and optimize their structures to improve antibacterial efficiency and biocompatibility, thereby developing highly effective and low-toxicity antibacterial agents to replace traditional antibiotics and promote the sustainable development of aquaculture. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide, Ajaponin, from Japanese eel.

[0006] Another object of the present invention is to provide the application of the above-mentioned eel antimicrobial peptide Ajaponin.

[0007] One of the technical solutions of the present invention is as follows:

[0008] A Japanese eel antimicrobial peptide, Ajaponin, has the amino acid sequence shown in SEQ ID NO. 01.

[0009] The application of the aforementioned Japanese eel antimicrobial peptide Ajaponin in the preparation of antimicrobial compositions.

[0010] In a preferred embodiment of the present invention, the antibacterial composition has inhibitory and bactericidal effects on *Pseudomonas fluorescens*, *Pseudomonas putida*, *Vibrio parahaemolyticus*, *Vibrio harzianum*, *Vibrio fluvialis*, and *Vibrio alginolyticus*.

[0011] An antibacterial composition comprising the aforementioned Japanese eel antimicrobial peptide Ajaponin.

[0012] In a preferred embodiment of the present invention, the active ingredient is the aforementioned Japanese eel antimicrobial peptide Ajaponin.

[0013] The above-mentioned application of Ajaponin, an antimicrobial peptide from Japanese eel, in the preparation of aquatic feed additives.

[0014] An aquatic feed additive whose active ingredient includes the aforementioned Japanese eel antibacterial polypeptide Ajaponin.

[0015] In a preferred embodiment of the present invention, the active ingredient is the aforementioned Japanese eel antimicrobial peptide Ajaponin.

[0016] The beneficial effects of this invention are:

[0017] 1. The Japanese eel antimicrobial peptide Ajaponin of the present invention is composed of 21 amino acids with a molecular weight of 2693.29 Daltons, including 7 positively charged amino acid residues. It has a hydrophobicity of 0.316 and an isoelectric point of 11.45. It is a novel cationic short peptide with advantages such as good water solubility, high antibacterial activity and low toxicity. It is composed of a limited number of amino acid residues, has a compact structure, is easy to synthesize and purify, and is convenient for large-scale production and application. Moreover, it contains multiple positively charged amino acid residues, which are beneficial for interacting with the negative charge of bacterial cell membranes, disrupting membrane integrity, and thus exerting rapid antibacterial and bactericidal effects.

[0018] 2. The Japanese eel antimicrobial peptide Ajaponin of the present invention exhibits broad-spectrum antimicrobial activity against a variety of common aquatic pathogens, including Pseudomonas and Vibrio bacteria, and can effectively inhibit bacterial growth, reproduction and pathogenic processes, making it suitable for the prevention and control of bacterial diseases in aquaculture.

[0019] 3. The Japanese eel antimicrobial peptide Ajaponin of the present invention has no obvious cytotoxicity to fish and mammalian cells, has high safety, does not interfere with the normal physiological functions of the host, and can be used as a green alternative to reduce the use of antibiotics, reduce drug resistance and environmental pollution risks.

[0020] 4. The Japanese eel antimicrobial peptide Ajaponin of the present invention can be used as an active ingredient and combined with other adjuvants to form a stable formula for the prevention and treatment of aquatic animal diseases, thereby improving aquaculture efficiency and product quality.

[0021] 5. The application of the Japanese eel antimicrobial peptide Ajaponin of the present invention in aquatic feed additives can enhance the antimicrobial properties of feed, improve animal immunity and growth performance, reduce disease occurrence, and promote sustainable aquaculture. Attached Figure Description

[0022] Figure 1 The image shows the bactericidal kinetic curves of the Japanese eel antimicrobial peptide Ajaponin against Vibrio parahaemolyticus, Vibrio harzianum, Vibrio fluvialis, and Vibrio alginolyticus in Example 3 of this invention.

[0023] Figure 2 This is a diagram showing the morphological changes induced by treatment with the Japanese eel antimicrobial peptide Ajaponin in Example 4 of the present invention on Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, and Vibrio alginolyticus.

[0024] Figure 3 The graph shows the results of the MTS-PMS assay for detecting the cytotoxicity of the Japanese eel antimicrobial peptide Ajaponin against ZF4 and HEK-293T cells in Example 5 of this invention. The horizontal axis represents the Ajaponin protein concentration (μM), and the vertical axis represents the cell viability (%). Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0026] Example 1

[0027] The amino acid sequence of the Japanese eel antimicrobial peptide Ajaponin in this embodiment is SEQ ID NO. 01: RRMLVCVKNIKLRWNYRNARC.

[0028] In this embodiment, Nanjing GenScript Co., Ltd. was commissioned to synthesize Ajaponin, an antimicrobial peptide from Japanese eel with a purity of over 95%. The company provided information on peptide molecular weight, HPLC and other detection data. HeliQuest was used to predict its charge and hydrophobicity, and ProtParam was used to predict other physicochemical parameters. The physicochemical parameters of the antimicrobial peptide Ajaponin are shown in Table 1.

[0029] Table 1 Physicochemical parameters of antimicrobial peptide Ajaponin

[0030]

[0031] Example 2: Determination of the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) of the Japanese eel antimicrobial peptide Ajaponin

[0032] The strains involved in this embodiment are: *Pseudomonas fluorescens*, *Pseudomonas putida*, *Vibrio parahaemolyticus*, *Vibrio harzianum*, *Vibrio fluvialis*, and *Vibrio alginolyticus*. All of these strains were purchased from the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences.

[0033] The specific method is as follows:

[0034] (1) Activate the bacterial strain by streaking the preserved bacteria onto a plate and incubating it upside down in a 28°C incubator overnight.

[0035] (2) Once the colonies have grown to a suitable size, the bacteria are picked and cloned into a liquid culture medium and cultured at 28°C and 180 rpm in a shaker until the logarithmic phase.

[0036] (3) Collect the bacteria by centrifugation and dilute them with liquid culture medium to make the final bacterial concentration approximately 5 × 10⁻⁶. 5 cfu / mL.

[0037] (4) Dissolve the synthesized Ajaponin powder in sterile Milli-Q water and serially dilute the protein concentration to 3 μM, 6 μM, 12 μM, 24 μM, 48 μM and 96 μM, and place on ice for later use.

[0038] (5) In a 96-well cell culture plate, a blank control group, a negative control group, and a test experimental group were set up for each test bacterium, and three replicates were set up for each group:

[0039] a. Blank control group: 50 μL of the protein sample to be tested and 50 μL of culture medium;

[0040] b. Negative control group: 50 μL sterile MilliQ water and 50 μL bacterial suspension;

[0041] c. Test group: 50 μL of the protein sample to be tested and 50 μL of bacterial suspension.

[0042] The 96-well cell culture plate was placed in an incubator at 28°C and cultured for 24 h. The MIC results of the experimental group were then observed.

[0043] After mixing the experimental groups under test by pipetting, take an appropriate amount of bacterial solution and drop it onto the corresponding solid culture medium plate. Incubate at a suitable temperature with the plate upside down for 24 h and observe the MBC results.

[0044] The antibacterial activity results of the Japanese eel antimicrobial peptide Ajaponin in this embodiment are shown in Table 2. It can be seen that it has strong bactericidal activity against common pathogenic bacteria in aquatic animals.

[0045] Table 2. Antibacterial activity of Ajaponin, an antimicrobial peptide from Japanese eel.

[0046]

[0047] Note: MIC: Minimum inhibitory concentration (μM), denoted by ab. a: The highest protein concentration at which bacterial growth is visible to the naked eye; b: The lowest protein concentration at which no bacterial growth is visible to the naked eye. MBC: Minimum bactericidal concentration (μM), denoted by ab. a: The highest protein concentration at which colonies are visible on agar plates; b: The lowest protein concentration at which no colonies are visible on agar plates.

[0048] Example 3: Kinetic curve of Ajaponin, an antimicrobial peptide from Japanese eel.

[0049] The strains involved in this embodiment are: Vibrio parahaemolyticus, Vibrio halys, Vibrio fluvialis, and Vibrio alginolyticus.

[0050] The specific method of this embodiment is as follows: The specific method is similar to the method for determining antibacterial activity. After the antimicrobial peptide and bacteria are co-incubated for a certain period of time, the co-incubated mixture is serially diluted at different time points, spread on plates, and after static incubation, colony counting is performed.

[0051] In this embodiment, the bactericidal kinetic curves of the Japanese eel antimicrobial peptide Ajaponin against Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, and Vibrio alginolyticus are shown in the figure below. Figure 1 As shown.

[0052] Example 4: Scanning electron microscopy observation of morphological and structural changes in bacteria treated with the antimicrobial peptide Ajaponin from Japanese eels.

[0053] The strains involved in this embodiment are: Vibrio parahaemolyticus, Vibrio halys, Vibrio fluvialis, and Vibrio alginolyticus.

[0054] The specific experimental method is as follows:

[0055] (1) Scanning electron microscopy sample preparation: Activate the bacterial strain, wait for the clones to grow to a suitable size, randomly pick single clones and transfer them to the corresponding culture medium, shake to the logarithmic growth phase, centrifuge to remove the supernatant, and adjust to 5×10⁻⁶ medium. 7 Add an equal volume of antimicrobial peptide (cfu / mL) at a concentration of 1×MBC for the corresponding bacteria, incubate for 30 min, centrifuge to remove the supernatant, wash once with PBS, and collect the bacterial cells.

[0056] (2) Fixation, washing, and slide preparation: Fix the bacterial cells collected in the previous step for more than 2 hours with 2.5% glutaraldehyde at 4℃. After washing the bacterial cells three times with PBS, prepare a high-concentration suspension and drop it onto a glass slide. After the bacterial cells adhere, perform gradient dehydration with ethanol.

[0057] (3) After the critical point drying, sputter gold at a current of 10 mA for 60 s; observe and record the results using a scanning electron microscope.

[0058] In this embodiment, the morphological and structural changes of the Japanese eel antimicrobial peptide Ajaponin against Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, and Vibrio alginolyticus are as follows: Figure 2 As shown.

[0059] Example 5: Detection and evaluation of the cytotoxicity of the Japanese eel antimicrobial peptide Ajaponin using MTS-PMS.

[0060] Zebrafish embryonic cells (ZF4) and human kidney epithelial cells (HEK-293T) were selected to determine the cytotoxicity of the eel antimicrobial peptide Ajaponin. The x-axis represents the Ajaponin protein concentration (μM), and the y-axis represents the cell viability (%).

[0061] The specific method is as follows:

[0062] (1) Collect cells in good growth condition and adjust the concentrations of ZF4 and HEK-293T cells to 1×10⁻⁶. 5 Add 100 μL of the above cell suspension to each well of a 96-well cell culture plate and incubate at a suitable temperature until more than 80% of the cells adhere to the plate.

[0063] (2) Remove the culture medium, add culture medium containing different concentrations of Ajaponin, and incubate in a suitable temperature incubator for 24 h.

[0064] (3) Add 20 μL of MTS-PMS mixed solution to each well, and after 4 h in the dark, measure the OD value of the microplate reader. 492 Read the plate and calculate the cell viability.

[0065] The results are as follows Figure 3 As shown, the Japanese eel antimicrobial peptide Ajaponin has no cytotoxicity against ZF4 and HEK-293T cells.

[0066] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A Japanese eel antimicrobial peptide, Ajaponin, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The use of the Japanese eel antimicrobial peptide Ajaponin according to claim 1 in the preparation of antimicrobial compositions, characterized in that: The antibacterial composition has inhibitory and bactericidal effects on *Pseudomonas fluorescens*, *Pseudomonas putida*, *Vibrio parahaemolyticus*, *Vibrio harzianum*, *Vibrio fluvialis*, and *Vibrio alginolyticus*.

3. An antibacterial composition, characterized in that: Its active ingredient includes Ajaponin, the Japanese eel antimicrobial peptide described in claim 1.

4. The antibacterial composition according to claim 3, characterized in that: Its active ingredient is the Japanese eel antimicrobial peptide Ajaponin as described in claim 1.

5. The application of the Japanese eel antimicrobial peptide Ajaponin as described in claim 1 in the preparation of aquatic feed additives.

6. An aquatic feed additive, characterized in that: Its active ingredient includes Ajaponin, the Japanese eel antimicrobial peptide described in claim 1.

7. An aquatic feed additive as described in claim 6, characterized in that: Its active ingredient is the Japanese eel antimicrobial peptide Ajaponin as described in claim 1.

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