An antimicrobial peptide mb217 and its modified peptide and application

Abstract

The present invention relates to the technical field of antimicrobial peptides, and specifically to an antimicrobial peptide mb217, a modified peptide thereof, and applications thereof. Studies have found that the antimicrobial peptide mb217 and its modified peptides have significant inhibitory effects on a variety of marine pathogenic Vibrio, effectively reducing the infection rate of Vibrio and the occurrence of diseases in aquaculture. The two modified peptides of the provided antimicrobial peptide mb217 improve its stability in high-salt environments by optimizing its molecular structure, enabling it to effectively exert its antimicrobial effect in seawater or high-salt water bodies in the future, effectively coping with the high-salt environment commonly found in aquaculture, and providing a more stable biological control method for the aquaculture industry.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and in particular to an antimicrobial peptide mb217 and its modified peptides and applications. Background Art

[0002] Vibrio ( Vibrio spp. ) is a common and highly pathogenic microorganism in aquaculture, which can pose a serious threat to aquatic animals such as fish, shrimp and shellfish. V. parahaemolyticus ) is the main pathogen of Acute Hepatopancreatic Necrosis Disease (AHPND) in crustaceans, which can cause pathological changes such as body ulcers, gill lesions and muscle necrosis in aquatic animals such as shrimp, crabs and shellfish. Similarly, Vibrio harveyi ( V. harveyi ) and Vibrio erwiniae ( V. owensii ) can also cause AHPND. V. alginolyticus ) is known to cause bacterial diseases in a variety of aquatic animals such as shrimp, fish and shellfish, and has also been reported as a potential causative agent of AHPND in some cases. V. natriegens ) mainly infects bivalves and shrimps, and has been reported to cause translucent post-larvae disease (TPD) in shrimp. V. mediterranei Vibrio infections can cause illness and mass mortality in aquatic animals such as oysters, clams, and mussels. Therefore, Vibrio infections not only cause significant economic losses to the aquaculture industry but also pose a threat to the health of aquatic ecosystems.

[0003] At present, high-frequency antibiotic prevention and control measures are widely used in the aquaculture field. This practice easily leads to the production of drug-resistant bacteria. The problem of antibiotic resistance has become a difficult problem that needs to be solved urgently in this field.

[0004] As a new type of natural antimicrobial substance, antimicrobial peptides have low potential for drug resistance development and good biocompatibility, and show great application potential in the research and development of antimicrobial drugs for aquaculture.

[0005] With the continued development of marine aquaculture and aquaculture in high-salt environments, the demand for salt-tolerant antimicrobial peptides is increasing. Because antimicrobial peptides typically carry a positive charge, high concentrations of salt ions (such as sodium ions) can disrupt the charge distribution within their molecules, causing structural instability. The mechanism of action of antimicrobial peptides relies on their specific three-dimensional structure to bind to bacterial membranes. High salt concentrations can induce structural changes, weakening their binding to pathogens and their antibacterial effects. Furthermore, the presence of salt can affect the solubility of antimicrobial peptides, causing them to aggregate or precipitate, thereby reducing their effectiveness.

[0006] In view of this, the development of a peptide inhibitor with good salt tolerance and broad-spectrum anti-Vibrio inhibition is of great significance to the aquaculture industry. Summary of the Invention

[0007] The present invention aims to address the problems of the prior art by providing an antimicrobial peptide, MB217, and its modified peptides and applications. The present invention provides an antimicrobial peptide, MB217, derived from the marine biofilm bacterium Flavobacterium, and its modified peptides. These peptides exhibit significant inhibitory effects against various marine pathogenic Vibrio species (such as Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio alginolyticus), demonstrating particularly excellent efficacy against drug-resistant marine Vibrio species. These antimicrobial peptides effectively reduce Vibrio infection rates, mitigate disease incidence in aquaculture, and mitigate economic losses.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] An antimicrobial peptide mb217, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] The amino acid sequence of the antimicrobial peptide mb217, SEQ ID No. 1, is as follows: MSRSLRNLLKYVWVKVKGE.

[0011] An antimicrobial peptide mb217cap is a modified peptide of the antimicrobial peptide mb217. The amino acid sequence of the antimicrobial peptide mb217cap is shown in SEQ ID No. 2.

[0012] The amino acid sequence of the antimicrobial peptide mb217cap, SEQ ID No. 2, is as follows: APKAMMSRSLRNLLKYVWVKVKGELQKKGI.

[0013] An antimicrobial peptide mb217C is a modified peptide of the antimicrobial peptide mb217. The modification method comprises amidation modification at the C-terminus of the antimicrobial peptide mb217 sequence. The amino acid sequence of the antimicrobial peptide mb217C is shown in SEQ ID No. 3.

[0014] The amino acid sequence of the antimicrobial peptide mb217C, SEQ ID No. 3, is as follows: MSRSLRNLLKYVWVKVKGE-NH2.

[0015] Another object of the present invention is to provide the application of the above antimicrobial peptide.

[0016] Use of the antimicrobial peptide mb217 as described above, and / or the antimicrobial peptide mb217cap as described above, and / or the antimicrobial peptide mb217C as described above in the preparation of antimicrobial drugs.

[0017] Furthermore, the antibacterial drug is used to inhibit or kill pathogenic Vibrio.

[0018] Furthermore, the pathogenic Vibrio includes at least one of Vibrio natriureticus, Vibrio erwinia, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mediterranei and Vibrio alginolyticus.

[0019] Another object of the present invention is to provide another application of the antimicrobial peptide.

[0020] Use of the antimicrobial peptide mb217 as described above, and / or the antimicrobial peptide mb217cap as described above, and / or the antimicrobial peptide mb217C as described above in the preparation of a medicament for aquaculture in seawater or a high-salt environment.

[0021] Preferably, the antimicrobial peptide mb217cap and / or the antimicrobial peptide mb217C described above are used in the preparation of a pharmaceutical agent for use in seawater or high-salinity aquaculture. Studies have shown that modified peptides exhibit enhanced salt tolerance. By optimizing the antimicrobial peptide's molecular structure, its stability in high-salinity environments has been improved, potentially enabling effective antimicrobial activity in seawater or other high-salinity environments. These improved antimicrobial peptides maintain a strong antimicrobial effect under varying salinity conditions, effectively addressing the high-salinity environments commonly found in aquaculture and providing a more stable biocontrol strategy for the aquaculture industry.

[0022] Another object of the present invention is to provide an antibacterial drug comprising the above antibacterial peptide.

[0023] An antimicrobial drug, the active ingredient of which includes at least one of the above-mentioned antimicrobial peptide mb217, the above-mentioned antimicrobial peptide mb217cap and the above-mentioned antimicrobial peptide mb217C.

[0024] Another object of the present invention is to provide the application of the above-mentioned antibacterial drug.

[0025] The use of the above-mentioned antibacterial drugs in inhibiting or killing pathogenic Vibrio.

[0026] The application of the above-mentioned antibacterial drugs in marine aquaculture or aquaculture in a high-salt environment.

[0027] A high-salinity environment refers to a water environment where the concentration of NaCl reaches 100 mM or more.

[0028] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0029] 1. This invention provides an antimicrobial peptide, MB217, derived from the marine biofilm bacterium Flavobacterium, and its modified peptides. These peptides exhibit significant inhibitory effects against various pathogenic Vibrio species (such as Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio alginolyticus), particularly effective against drug-resistant Vibrio species. These antimicrobial peptides are expected to effectively reduce Vibrio infection rates, mitigate disease incidence in aquaculture, and mitigate economic losses.

[0030] 2. The antimicrobial peptide mb217 and its modified peptides of the present invention have unique amino acid sequences and molecular structures, which enable the antimicrobial peptides to remain stable in a high-salt environment and effectively inhibit the growth of various pathogenic Vibrio.

[0031] 3. This invention provides two modified peptides of the antimicrobial peptide mb217. By optimizing its molecular structure, the peptide improves its stability in high-salinity environments, enabling it to effectively exert its antimicrobial effects in seawater and other high-salinity water bodies. These modified peptides maintain strong antimicrobial efficacy under varying salinity conditions, effectively addressing the high-salinity environments common in aquaculture and providing a more stable biocontrol tool for the aquaculture industry.

[0032] 4. The antimicrobial peptide mb217 and its modified peptides provided by this invention are derived from natural organisms, avoiding the use of antibiotics and addressing the issue of antibiotic resistance. This meets the current environmental protection needs of the aquaculture industry. Their use does not pollute the aquatic environment, ensuring high safety and reducing the environmental burden of traditional chemical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a wheel diagram of the antimicrobial peptide mb217 and its modified peptides.

[0034] Figure 2 This is the predicted three-dimensional structure of the antimicrobial peptide mb217 and its modified peptides.

[0035] Figure 3 This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 0 mM NaCl conditions.

[0036] Figure 4 This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 100 mM NaCl conditions.

[0037] Figure 5 This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 150mM NaCl conditions.

[0038] Figure 6This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 200 mM NaCl conditions.

[0039] Figure 7 This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 250mM NaCl conditions.

[0040] Figure 8 This is the minimum inhibitory concentration data of the antimicrobial peptide mb217 and its modified peptides against six marine pathogenic Vibrio under 300mM NaCl conditions. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below with reference to the accompanying drawings.

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example

[0044] 1. Antimicrobial Peptide Synthesis

[0045] 1) Synthesis of antimicrobial peptides:

[0046] The antimicrobial peptide mb217 sequence is derived from a marine biofilm bacterium, the genus Olleya, isolated from a biofilm community on a rock surface near Qingdao, China. A deep learning model was developed to predict all small reading frames in the bacterial genome, screening and identifying the sequence information for the antimicrobial peptide mb217. The antimicrobial peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. via solid-phase chemical synthesis with a purity exceeding 95%. The product was purified by HPLC and analyzed by ESI mass spectrometry to ensure sequence accuracy. The antimicrobial peptide mb217cap sequence is modified from the mb217 sequence by adding the peptide sequences APKAM and LQKKGI to the N- and C-termini of mb217, respectively, to form helical caps at both ends of the sequence. The synthesis steps for the antimicrobial peptide mb217 are similar to those for mb217. The antimicrobial peptide mb217C sequence is modified from the mb217 sequence by amidation at the C-terminus. The synthesis steps for the antimicrobial peptide mb217 are similar to those for mb217.

[0047] 2) Characterization of antimicrobial peptides:

[0048] DBAASP (Database of Antimicrobial Activity and Structure–Property Relationships of Peptides) is a database used to study antimicrobial and other bioactive peptides and provides a function for predicting their physicochemical properties. Visit the DBAASP website (https: / / dbaasp.org / tools?page=property-calculation), select the MF–Moon and Fleming scale for Hydrophobicity scale, enter the sequence in FASTA format, and click Submit to obtain physicochemical properties such as net charge and isoelectric point.

[0049] HeliQuest is an online tool for analyzing and predicting peptide chain structures (with a particular focus on the properties of α-helical peptides). It can calculate some important structural features of peptide chains, such as helicity, hydrophobicity, and hydrogen bonds. HeliQuest predicts which parts may trigger the formation of helical structures by evaluating the hydrophobicity of amino acids on the peptide chain. Go to the HeliQuest website (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParamsV2.py), select α-helix in Helix type, and select FULL in Window size. Enter the sequence, keep other options as default, and click Process to obtain a wheel diagram of the peptide chain. Cationic residues, anionic residues, and hydrophobic residues are displayed in blue, red, and yellow, respectively, and the direction and length of the arrows indicate the predicted hydrophobic moment and strength.

[0050] AlphaFold is a deep learning model developed by DeepMind for predicting the 3D structure of proteins. AlphaFold3 is an updated version that uses a multi-layer neural network to predict protein structure. Go to the AlphaFold3 website (https: / / golgi.sandbox.google.com / about), select Protein, enter the sequence, keep all other options as default, click Continue and preview the job, and the 3D structure of the peptide chain will be obtained.

[0051] antimicrobial peptide mb217

[0052] The sequence of antimicrobial peptide mb217 is MSRSLRNLLKYVWVKVKGE, and the molecular formula is C 105 H 176 N 30 O 26S1, molecular weight (MW) 2306.80. The net charge and isoelectric point of the antimicrobial peptide mb217 were predicted using the DBAASP online tool. Its net charge was predicted to be 4, with four positive charges; its isoelectric point was predicted to be 10.97. The wheel diagram containing the hydrophilic and hydrophobic faces of mb217 was predicted using the online tool HeliQuest ( Figure 1 ), the wheel diagram shows their amphipathic structure. The three-dimensional structure of mb217 was predicted by the online tool AlphaFold3. The results show that mb217 is a typical α-helical peptide ( Figure 2 ). Therefore, the antimicrobial peptide mb217 is an amphipathic α-helical cationic peptide.

[0053] Antimicrobial peptide mb217cap

[0054] The sequence of the antimicrobial peptide mb217cap is APKAMMSRSLRNLLKYVWVKVKGELQKKGI, and the molecular formula is C 158 H 271 N 45 O 38 S2, with a molecular weight (MW) of 3473.29, was modified at the N-terminus by adding a short hydrophobic peptide, APKAM, to the N-terminus of the original sequence. This short hydrophobic peptide, designed based on the sequence pattern of the extended capping box, primarily functions to form a "hydrophobic spike" between the side chains of alanine and methionine, stabilizing the α-helical structure. The C-terminus was modified by adding a Schellman motif, LQKKGI, to the C-terminus of the original sequence. This helix-capping motif enhances the salt resistance of the helical antimicrobial peptide. Hydrogen bonds are formed between the amide group of the leucine in the C-terminal modification sequence and the carbonyl group of isoleucine, and between the amide group of the glutamine backbone and the carbonyl group of glycine. Hydrophobic interactions also occur between the leucine and isoleucine in the C-terminal modification sequence. Both hydrogen bonds and hydrophobic interactions contribute to stabilizing the α-helical structure. Therefore, the introduction of helix-capping motifs enhances the stability of the helical structure of the antimicrobial peptide MB217, thereby enhancing its salt resistance. The antimicrobial peptide modified with a helical cap is mb217cap. The net charge and isoelectric point of the antimicrobial peptide mb217cap were predicted using the DBAASP online tool. Its net charge was predicted to be 7, with 7 positive charges; its isoelectric point was predicted to be 11.28. The wheel diagram containing the hydrophilic and hydrophobic faces of mb217cap was predicted using the online tool HeliQuest ( Figure 1 ), the wheel diagram shows their amphiphilic structure. The three-dimensional structure of mb217cap was predicted by the online tool AlphaFold3, and the results showed that mb217cap is also a typical α-helical peptide ( Figure 2). Therefore, the antimicrobial peptide mb217cap is also an amphipathic α-helical cationic peptide.

[0055] Antimicrobial peptide mb217C

[0056] The sequence of the antimicrobial peptide mb217C is MSRSLRNLLKYVWVKVKGE-NH2, and the modification thereof is an amidation modification at the C-terminus of the antimicrobial peptide mb217 sequence, that is, converting -COOH into an amide bond (-CONH2). The molecular formula of the antimicrobial peptide mb217C is C 105 H 177 N 31 O 25 S1, molecular weight (MW) is 2305.78. The net charge and isoelectric point of the antimicrobial peptide mb217C were predicted using the DBAASP online tool. The net charge was predicted to be 5, with 5 positive charges; the isoelectric point was predicted to be 11.52. Figure 1 and Figure 2 ), so the conformation of mb217C should also be α-helical and it is an amphipathic cationic peptide.

[0057] 2. Determination of the antibacterial effect of antimicrobial peptide mb217 and its modified peptides against six pathogenic Vibrio species

[0058] Determination of minimum inhibitory concentration (MIC):

[0059] The marine pathogen Vibrio parahaemolyticus used in the experiment V. parahaemolyticus ), Vibrio erwiniae ( V. owensii harveyi ( V. harveyi )、Vibrio alginolyticus( V. alginolyticus ), Vibrio natriuresis ( V. alginolyticus ), Vibrio mediterranei ( V. mediterranei ) were isolated from diseased marine aquaculture animals. MICs of antimicrobial peptides were determined using the broth microdilution method, following the Clinical and Laboratory Standards Institute guidelines: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests (Clinical and Laboratory Standards Institute, 1991).

[0060] Six marine pathogenic Vibrio species were inoculated into sterile 2216E medium and cultured overnight at 25°C with shaking. Six marine pathogenic Vibrio species were inoculated into fresh CAMHB medium at a 1% inoculum volume and cultured until the exponential phase. The cell concentration was adjusted to 1×10 5cfu / mL to obtain bacterial solution. Then transfer 180μL of bacterial solution to a 96-well plate. Dissolve the antimicrobial peptide powder in sterile water and dilute it to a serial dilution of 2-fold antimicrobial peptide solution. Take 20μL and add it to the bacterial solution in the 96-well plate to make the antimicrobial peptide concentration in the bacterial solution range from 4 to 64μg / mL. Place the 96-well plate at 25℃ and incubate for 12 hours. Then use a microplate reader to detect bacterial growth (OD 600 The experiment was repeated three times. MIC (Minimum Inhibitory Concentration) is defined as the minimum concentration of antimicrobial peptide that detects bacterial growth. Lower MIC values ​​indicate stronger antibacterial effects.

[0061] The experimental results are as follows Figure 3 As shown. Without the addition of NaCl (0 mM NaCl), the MIC of antimicrobial peptide mb217 against Vibrio natriuresis and Vibrio erwiniae was 4 μg / mL, against Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio mediterranei was 8 μg / mL, and against Vibrio alginolyticus was 16 μg / mL. However, the MICs of the modified peptides mb217cap and mb217C against the six marine pathogenic Vibrio species were only 4 μg / mL. This indicates that under these conditions, the three antimicrobial peptides exhibited strong antibacterial activity against all six Vibrio species, and the antibacterial activity of the modified mb217 peptides against Vibrio was stronger than that of the unmodified mb217 itself.

[0062] 3. Determination of the antibacterial effect of antimicrobial peptide mb217 and its modified peptides against six pathogenic Vibrio species under different salinity conditions

[0063] Marine pathogenic Vibrio, as a pathogen of aquatic animals such as shrimp, mainly colonizes in high-salinity water environments. + The greatest impact on the antimicrobial activity of antimicrobial peptides can even lead to complete loss of activity. Therefore, the present invention tested the antimicrobial activity of the antimicrobial peptide mb217 and its modified peptides against the marine pathogen Vibrio at different salinity levels (i.e., 100-300 mM NaCl).

[0064] Six marine pathogenic Vibrio species were inoculated into sterile 2216E medium and cultured overnight at 25°C with shaking. The six marine pathogenic Vibrio species were inoculated into fresh CAMHB medium containing different salinities (different NaCl concentrations) at a 1% inoculum and cultured until the exponential phase. The cell concentration was adjusted to 1×10 5cfu / mL to obtain bacterial solution for use. The NaCl concentration gradient was set to 100mM (5.844‰), 150mM (8.766‰), 200mM (11.688‰), 250mM (14.610‰) and 300mM (17.532‰). Then 180μL of bacterial solution was transferred to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted to a 2-fold serial dilution of the antimicrobial peptide solution. 20μL was added to the bacterial solution in the 96-well plate to make the antimicrobial peptide concentration in the bacterial solution range from 4-64μg / mL. The 96-well plate was incubated at 25°C for 12 hours, and the bacterial growth (OD 600 ), the experiment was set up with 3 replicates.

[0065] The experimental results are as follows Figure 3-Figure 7 As shown. When NaCl was added to the culture medium (100 mM-300 mM NaCl), the MICs of the antimicrobial peptide mb217 and its modified peptides against five Vibrio species, except V. natrii, increased with increasing salinity, indicating that the antibacterial activity of the antimicrobial peptide mb217 and its modified peptides against V. natrii was affected by salinity. The three peptides maintained consistent antibacterial activity against V. natrii under both low and high salinity conditions, with low MICs (all 4 μg / mL). This indicates that the antimicrobial activity of the antimicrobial peptide mb217 and its modified peptides against V. natrii was not significantly affected by salinity, suggesting that these peptides may have high specificity for V. natrii. Furthermore, the MICs of the three peptides against V. mediterranei only increased 2-4-fold. MB217cap demonstrated strong antibacterial activity against V. mediterranei under high salinity conditions, with its MIC increasing only 2-fold to 8 μg / mL at 300 mM NaCl and remaining at 4 μg / mL across 0-250 mM NaCl. For four types of Vibrio, namely Vibrio parahaemolyticus, Vibrio Erwiniae, Vibrio harveyi and Vibrio alginolyticus, the salt tolerance of mb217C is significantly stronger than that of mb217 and mb217cap.

[0066] Under the salinity condition of 100 mM NaCl (5.844‰), the experimental results are as follows Figure 4 As shown in Figure 2, the MICs of mb217 and mb217cap against Vibrio parahaemolyticus, Vibrio Erwiniae, Vibrio harveyi, and Vibrio alginolyticus all increased to 16 μg / mL. In contrast, the MICs of mb217C against Vibrio parahaemolyticus, Vibrio Erwiniae, and Vibrio alginolyticus all increased to 8 μg / mL, while the MIC against Vibrio harveyi remained at 4 μg / mL. The MICs of the three peptides against Vibrio mediterranei remained consistent with those under 0 mM NaCl conditions.

[0067] Under the salinity condition of 150 mM NaCl (8.766‰), the experimental results are as follows Figure 5As shown. The MIC of antimicrobial peptide mb217 against Vibrio parahaemolyticus and Vibrio Erwiniae increased to 32 μg / mL, while the MIC against the other four Vibrio strains remained consistent with the MIC under 100 mM NaCl conditions. The MIC of antimicrobial peptide mb217cap against Vibrio Erwiniae increased to 32 μg / mL, while the MIC against the other five Vibrio strains remained consistent with the MIC under 100 mM NaCl conditions. The MIC of antimicrobial peptide mb217C against Vibrio Erwiniae and Vibrio alginolyticus increased to 16 μg / mL, and the MIC against Vibrio harveyi and Vibrio mediterranei increased to 8 μg / mL, while the MIC against Vibrio parahaemolyticus and Vibrio natriuresis remained consistent with the MIC under 100 mM NaCl conditions.

[0068] Under the salinity condition of 200 mM NaCl (11.688‰), the experimental results are as follows Figure 6 As shown in the table, the MIC of antimicrobial peptide mb217 against Vibrio Erwiniae increased to 64 μg / mL, the MIC against Vibrio harveyi and Vibrio alginolyticus increased to 32 μg / mL, and the MIC against Vibrio mediterranei increased to 16 μg / mL. The MIC of antimicrobial peptide mb217cap against Vibrio Erwiniae also increased to 64 μg / mL, and the MIC against Vibrio parahaemolyticus and Vibrio alginolyticus increased to 32 μg / mL. The MICs against the other three Vibrio strains remained consistent with the MICs under 150 mM NaCl. The MIC of antimicrobial peptide mb217C against Vibrio parahaemolyticus increased to 16 μg / mL, but did not increase against the other five Vibrio strains.

[0069] Under the salinity condition of 250 mM NaCl (14.61‰), the experimental results are as follows Figure 7 The MICs of antimicrobial peptides mb217 and mb217cap against the six Vibrio strains remained consistent with those in 200 mM NaCl. The MIC of antimicrobial peptide mb217C against Vibrio erwinii increased to 32 μg / mL, but did not increase against the other five Vibrio strains.

[0070] Under the salinity condition of 300 mM NaCl (17.532‰), the experimental results are as follows Figure 8As shown. The MIC of antimicrobial peptide mb217 against Vibrio parahaemolyticus increased to 64 μg / mL and exceeded 64 μg / mL against Vibrio erwiniae, while the MIC against the other four Vibrio strains remained unchanged. The MIC of antimicrobial peptide mb217cap against Vibrio parahaemolyticus and Vibrio alginolyticus increased to 64 μg / mL, and against Vibrio harveyi to 32 μg / mL. The MIC against Vibrio mediterranei only increased to 8 μg / mL, while the MIC against the other two Vibrio strains remained unchanged. The MIC of antimicrobial peptide mb217C against Vibrio parahaemolyticus increased to 32 μg / mL and against Vibrio mediterranei to 16 μg / mL, while the MIC against the other four Vibrio strains remained consistent with the MIC under 250 mM NaCl conditions. At 300 mM NaCl, the antimicrobial peptide mb217C exhibited significantly greater salt tolerance and antibacterial activity against four strains of Vibrio species, including Vibrio parahaemolyticus, Vibrio Erwiniae, Vibrio harveyi, and Vibrio alginolyticus, than mb217 and mb217cap. The MIC of the antimicrobial peptide mb217cap against Vibrio mediterranei increased only 2-fold, demonstrating its potent inhibitory activity against Vibrio mediterranei under high-salt conditions. The three peptides maintained consistent inhibitory activity against Vibrio natriuresis under both low and high-salt conditions, with lower MICs, suggesting that these peptides may possess high specificity for Vibrio natriuresis.

[0071] In summary, the antimicrobial peptide mb217 and its modified peptides demonstrated robust antibacterial activity against six strains of marine pathogenic Vibrio at varying salinities. All three peptides demonstrated robust antibacterial activity against Vibrio natriuresis (MICs of 4 μg / mL) across salinities of 0–300 mM NaCl, demonstrating high specificity and unaffected by salinity. In contrast, the modified peptide mb217cap exhibited excellent antibacterial activity against Vibrio mediterranei under high-salinity conditions, with its MIC increasing only to 8 μg / mL. The modified peptide mb217C exhibited significantly superior antibacterial activity against Vibrio parahaemolyticus, Vibrio erwinii, Vibrio harveyi, and Vibrio alginolyticus under high-salinity conditions, demonstrating robust salt tolerance and potential to inhibit a wide range of Vibrio species. These results demonstrate that the salt tolerance and antibacterial activity of the antimicrobial peptide mb217 against Vibrio spp. have been significantly enhanced after modification. This shows that through rational design and modification of antimicrobial peptides, their stability and antibacterial effect in complex environments can be enhanced, providing an important theoretical basis and practical basis for the development of new antimicrobial agents, especially inhibitors against marine pathogenic Vibrio.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antimicrobial peptide mb217, characterized in that The amino acid sequence of the antimicrobial peptide mb217 is shown in SEQ ID No.

1.

2. An antimicrobial peptide mb217cap, characterized in that The antimicrobial peptide mb217cap is a modified peptide of the antimicrobial peptide mb217 according to claim 1, and the amino acid sequence of the antimicrobial peptide mb217cap is shown in SEQ ID No.

2.

3. An antimicrobial peptide mb217C, characterized in that The antimicrobial peptide mb217C is a modified peptide of the antimicrobial peptide mb217 according to claim 1, wherein the modification method comprises amidation modification at the C-terminus of the antimicrobial peptide mb217 sequence; the amino acid sequence of the antimicrobial peptide mb217C is MSRSLRNLLKYVWVKVKGE-NH2.

4. Use of the antimicrobial peptide mb217 according to claim 1, and / or the antimicrobial peptide mb217cap according to claim 2, and / or the antimicrobial peptide mb217C according to claim 3 in the preparation of antimicrobial drugs, characterized in that: The antibacterial drug is used to inhibit marine pathogenic Vibrio; marine pathogenic Vibrio includes at least one of Vibrio natriureticus, Vibrio erwiniae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mediterranei and Vibrio alginolyticus.

5. Use of the antimicrobial peptide mb217 according to claim 1, and / or the antimicrobial peptide mb217cap according to claim 2, and / or the antimicrobial peptide mb217C according to claim 3 in the preparation of a medicament for aquaculture in seawater or a high-salt environment, characterized in that: A high-salinity environment refers to a water environment where the concentration of NaCl reaches 100 mM or more.

6. An antibacterial drug, characterized in that: The active ingredient comprises at least one of the antimicrobial peptide mb217 according to claim 1, the antimicrobial peptide mb217cap according to claim 2, and the antimicrobial peptide mb217C according to claim 3.

7. Use of the antibacterial drug according to claim 6 in inhibiting marine pathogenic Vibrio, characterized in that: The marine pathogenic Vibrio includes at least one of Vibrio natriureticus, Vibrio erwinia, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mediterranei and Vibrio alginolyticus.

8. Use of the antimicrobial drug according to claim 6 in marine aquaculture or aquaculture in a high-salt environment, characterized in that: A high-salinity environment refers to a water environment where the concentration of NaCl reaches 100 mM or more.

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