Antibacterial peptide mb217 and modified peptide and application thereof

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to an antibacterial peptide mb217 as well as a modified peptide and application thereof, and researches find that the antibacterial peptide mb217 and the modified peptide thereof have a remarkable inhibition effect on various marine pathogenic vibrios, the infection rate of the vibrios is effectively reduced, and diseases in aquaculture are reduced; according to the two modified peptides of the antibacterial peptide mb217 provided by the invention, the molecular structure of the antibacterial peptide is optimized, so that the stability of the antibacterial peptide in a high-salt environment is improved, the antibacterial peptide can effectively play an antibacterial role in seawater or a high-salt water body in the future, the common high-salt environment in aquaculture is effectively dealt with, and a more stable biological prevention and control means is provided for the breeding industry.

Description

Technical Field

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

[0002] Vibrio ( Vibrio spp. ) is a common and highly pathogenic pathogenic microorganism in the process of aquaculture, which can pose a serious threat to aquatic animals such as fish, shrimps and shellfishes. Vibrio parahaemolyticus ( V. parahaemolyticus ) is the main pathogenic bacterium of Acute Hepatopancreatic Necrosis Disease (AHPND) in crustaceans, which can cause pathological changes such as body surface ulcers, gill lesions and muscle necrosis in aquatic animals such as shrimps, crabs and shellfishes. Similarly, Vibrio harveyi ( V. harveyi ) and Vibrio owensii ( V. owensii ) can also cause AHPND. Vibrio alginolyticus ( V. alginolyticus ) is known to cause bacterial diseases in various aquatic animals such as shrimps, fish and shellfishes, and in some cases, it has also been reported as a potential pathogenic factor of AHPND. Vibrio natriegens ( V. natriegens ) mainly infects bivalves and shrimps, and has been reported to be able to cause Translucent Post-larvae Disease (TPD) in shrimps. Vibrio mediterranei ( V. mediterranei ) can cause disease infections in aquatic animals such as oysters, razor clams and mussels, and lead to their large-scale deaths. Therefore, Vibrio infection not only brings significant economic losses to the aquaculture industry, but also poses a threat to the health of the water ecosystem.

[0003] Currently, high-frequency antibiotic prevention and control means are commonly used in the field of aquaculture. This practice is prone to the generation of drug-resistant bacteria, and the problem of antibiotic resistance has become an urgent problem to be solved in this field.

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

[0005] With the continuous development of seawater aquaculture and aquaculture in high-salt environments, the demand for the salt tolerance of antimicrobial peptides is increasing day by day. Since antimicrobial peptides usually carry positive charges, high concentrations of salt ions (such as sodium ions) may interfere with the charge distribution within their molecules, resulting in unstable structures. The mechanism of action of antimicrobial peptides depends on their specific three-dimensional structure binding to the bacterial membrane. High salt concentrations may cause structural changes, weakening their binding force and antibacterial effect with pathogenic bacteria. In addition, the presence of salts may also affect the solubility of antimicrobial peptides, leading to their aggregation or precipitation, thereby reducing their effectiveness.

[0006] In view of this, it is of great significance to the aquaculture industry to develop a peptide inhibitor with good salt tolerance and broad-spectrum vibrio inhibitory effect. Summary of the Invention

[0007] The purpose of the present invention is to provide an antibacterial peptide mb217, its modified peptide and application in view of the problems existing in the above-mentioned prior art. The present invention provides an antibacterial peptide mb217 and its modified peptide derived from the marine biofilm Flavobacterium, which have a significant inhibitory effect on a variety of marine pathogenic vibrios (such as Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, etc.), especially in the prevention and treatment of drug-resistant marine vibrios, showing excellent effects. This antibacterial peptide will effectively reduce the infection rate of vibrios, reduce the occurrence of diseases in aquaculture, and reduce economic losses.

[0008] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows: An antibacterial peptide mb217, the amino acid sequence of the antibacterial peptide mb217 is shown in SEQ ID No.1.

[0009] The amino acid sequence SEQ ID No.1 of the antibacterial peptide mb217 is as follows: MSRSLRNLLKYVWVKVKGE.

[0010] An antibacterial peptide mb217cap, the antibacterial peptide mb217cap is a modified peptide of the above antibacterial peptide mb217, and the amino acid sequence of the antibacterial peptide mb217cap is shown in SEQ ID No.2.

[0011] The amino acid sequence SEQ ID No.2 of the antibacterial peptide mb217cap is as follows: APKAMMSRSLRNLLKYVWVKVKGELQKKGI.

[0012] An antibacterial peptide mb217C, the antibacterial peptide mb217C is a modified peptide of the above antibacterial peptide mb217, and the modification method includes amidation modification at the C-terminus of the antibacterial peptide mb217 sequence, and the amino acid sequence of the antibacterial peptide mb217C is shown in SEQ ID No.3.

[0013] The amino acid sequence SEQ ID No.3 of the antibacterial peptide mb217C is as follows: MSRSLRNLLKYVWVKVKGE-NH2.

[0014] Another object of the present invention is to provide the application of the above antibacterial peptide.

[0015] The application of the antibacterial peptide mb217 as described above, and / or, the antibacterial peptide mb217cap as described above, and / or, the antibacterial peptide mb217C as described above in the preparation of antibacterial drugs.

[0016] Further, the antibacterial agent is used to inhibit or kill pathogenic Vibrio.

[0017] Further, the pathogenic Vibrio includes at least one of Vibrio natriegens, Vibrio owensii, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mediterranei, and Vibrio alginolyticus.

[0018] Another object of the present invention is to provide another application of the above antibacterial peptide.

[0019] Use of the antibacterial peptide mb217 as described above, and / or the antibacterial peptide mb217cap as described above, and / or the antibacterial peptide mb217C as described above in the preparation of a medicament for aquaculture in seawater or a high-salt environment.

[0020] Preferably, use of the antibacterial peptide mb217cap as described above, and / or the antibacterial peptide mb217C as described above in the preparation of a medicament for aquaculture in seawater or a high-salt environment. Research has found that the salt tolerance performance of the modified peptide is better. By optimizing the molecular structure of the antibacterial peptide, its stability in a high-salt environment is improved, enabling it to have the potential to effectively exert antibacterial effects in seawater or high-salt water bodies. The improved antibacterial peptide can still maintain strong antibacterial effects under different salinity conditions, effectively coping with the common high-salt environment in aquaculture and providing a more stable biological prevention and control means for the aquaculture industry.

[0021] Another object of the present invention is to provide an antibacterial agent containing the above antibacterial peptide.

[0022] An antibacterial agent, the active ingredient of which includes at least one of the antibacterial peptide mb217 as described above, the antibacterial peptide mb217cap as described above, and the antibacterial peptide mb217C as described above.

[0023] Another object of the present invention is to provide an application of the above antibacterial agent.

[0024] Use of the above antibacterial agent in inhibiting or killing pathogenic Vibrio.

[0025] Use of the above antibacterial agent in seawater aquaculture or aquaculture in a high-salt environment.

[0026] The high-salt environment refers to a water environment where the concentration of NaCl reaches 100 mM or more.

[0027] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: 1. The present invention provides an antibacterial peptide mb217 and its modified peptides derived from marine biofilm Flavobacterium, which have significant inhibitory effects on various pathogenic Vibrio species (such as Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, etc.). Especially in the prevention and control of drug-resistant Vibrio, it shows excellent effects. This antibacterial peptide will effectively reduce the infection rate of Vibrio, reduce the occurrence of diseases in aquaculture, and reduce economic losses.

[0028] 2. The antibacterial peptide mb217 and its modified peptides of the present invention have unique amino acid sequences and molecular structures, which enable the antibacterial peptide to remain stable in a high-salt environment and effectively inhibit the growth of various pathogenic Vibrio species.

[0029] 3. The present invention provides two modified peptides of antibacterial peptide mb217. By optimizing the molecular structure of the antibacterial peptide, its stability in a high-salt environment is improved, enabling it to effectively exert its antibacterial effect in seawater or high-salt water bodies in the future. The improved antibacterial peptide can still maintain a strong antibacterial effect under different salinity conditions, effectively coping with the common high-salt environment in aquaculture, and providing a more stable biological prevention and control means for the aquaculture industry.

[0030] 4. The antibacterial peptide mb217 and its modified peptides provided by the present invention are derived from natural organisms, avoiding the use of antibiotics, solving the problem of antibiotic resistance, and meeting the current green environmental protection requirements of the aquaculture industry. During its use, it will not cause pollution to the water environment, has high safety, and reduces the environmental burden brought by traditional chemical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a wheel diagram of antibacterial peptide mb217 and its modified peptides.

[0032] Figure 2 It is a predicted three-dimensional structure diagram of antibacterial peptide mb217 and its modified peptides.

[0033] Figure 3 It is a graph of the minimum inhibitory concentration data of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under 0 mM NaCl conditions.

[0034] Figure 4 It is a graph of the minimum inhibitory concentration data of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under 100 mM NaCl conditions.

[0035] Figure 5 It is a graph of the minimum inhibitory concentration data of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under 150 mM NaCl conditions.

[0036] Figure 6It is a data graph of the minimum inhibitory concentration of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under the condition of 200 mM NaCl.

[0037] Figure 7 It is a data graph of the minimum inhibitory concentration of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under the condition of 250 mM NaCl.

[0038] Figure 8 It is a data graph of the minimum inhibitory concentration of antibacterial peptide mb217 and its modified peptides against 6 species of marine pathogenic Vibrio under the condition of 300 mM NaCl. Detailed implementation manners

[0039] The present invention will be described in detail below with reference to the accompanying drawings.

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer, 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 used to limit the present invention.

[0041] Embodiment I. Synthesis of antibacterial peptides 1) Synthesis of antibacterial peptides: The sequence of antibacterial peptide mb217 is derived from a bacterium of the genus Olleya in marine biofilms, which was isolated from the biofilm community on the surface of rocks in the coastal waters of Qingdao, China. By establishing a deep learning model to predict all small open reading frames in the genome of this bacterium, the sequence information of antibacterial peptide mb217 was screened. The antibacterial peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. through solid-phase chemical synthesis method, and the purity was greater than 95%. After the product was synthesized, it was purified by HPLC and detected by ESI mass spectrometry to ensure the correct polypeptide sequence. The antibacterial peptide mb217cap sequence was modified on the basis of the antibacterial peptide mb217 sequence, and the peptide sequences APKAM and LQKKGI were added to the N-terminus and C-terminus of mb217 respectively to form helix caps at both ends of the sequence, and its synthesis steps were the same as those of mb217. The antibacterial peptide mb217C sequence was amide-modified at the C-terminus of the antibacterial peptide mb217 sequence, and its synthesis steps were the same as those of mb217.

[0042] 2) Characterization of antibacterial peptides: DBAASP (Database of Antimicrobial Activity and Structure–Property Relationships of Peptides) is a database for studying antimicrobial peptides and other bioactive peptides, and provides functions for predicting the physicochemical properties of peptides. Enter the DBAASP website (https: / / dbaasp.org / tools?page=property-calculation), select the MF–Moon and Fleming scale at Hydrophobicity scale, input the sequence in FASTA format, click submit, and obtain physicochemical properties such as the net charge number and isoelectric point of the sequence.

[0043] HeliQuest is an online tool for analyzing and predicting the structure of peptide chains (especially focusing on the properties of α-helical peptides), and 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. Enter the HeliQuest website (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParamsV2.py), select α helix at Helix type, and select FULL at Window size. Input the sequence, keep other options default, click Process, and obtain the 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 arrow represent the predicted hydrophobic moment and strength.

[0044] AlphaFold is a deep learning model developed by DeepMind for predicting the three-dimensional structure of proteins. AlphaFold3 is its updated version, which uses a multi-layer neural network to predict the structure of proteins. Enter the AlphaFold3 website (https: / / golgi.sandbox.google.com / about), select Protein, input the sequence, keep other options default, click Continue and preview job, and obtain the three-dimensional structure of the peptide chain.

[0045] Antimicrobial peptide mb217 The sequence of antimicrobial peptide mb217 is MSRSLRNLLKYVWVKVKGE, and its molecular formula is C 105 H 176 N 30 O 26S1 has a molecular weight (MW) of 2306.80. The net charge and isoelectric point of the antimicrobial peptide mb217 were predicted using the DBAASP online tool. Its predicted net charge is 4, carrying 4 positive charges; its predicted isoelectric point is 10.97. The wheel-like diagram of mb217 containing hydrophilic and hydrophobic surfaces was predicted using the online tool HeliQuest ( Figure 1 ), and the wheel-like diagram shows their amphiphilic structure. The three-dimensional structure of mb217 was predicted using the online tool AlphaFold3, and the results shown indicate that mb217 is a typical α-helical peptide ( Figure 2 ). Therefore, the antimicrobial peptide mb217 is an amphiphilic α-helical cationic peptide.

[0046] Antimicrobial peptide mb217cap The sequence of the antimicrobial peptide mb217cap is APKAMMSRSLRNLLKYVWVKVKGELQKKGI, and its molecular formula is C 158 H 271 N 45 O 38 S2 has a molecular weight (MW) of 3473.29. Its N-terminal modification is to add a hydrophobic short peptide APKAM at the N-terminus of the original sequence. The hydrophobic short peptide is designed according to the sequence pattern of the Extended capping box, and its main function is to form a "hydrophobic nail" between the side chains of alanine and methionine to stabilize the structure of the α-helix. Its C-terminal modification is to add a Schellman motif LQKKGI at the C-terminus of the original sequence, that is, to enhance the salt resistance of the helical antimicrobial peptide by introducing a helix-capping motif. Hydrogen bonds are formed between the amide group of leucine and the carbonyl group of isoleucine, and between the amide group of the glutamine backbone and the carbonyl group of glycine in the C-terminal modified sequence. There is a hydrophobic interaction between leucine and isoleucine in the C-terminal modified sequence. Both hydrogen bonds and hydrophobic interactions contribute to stabilizing the structure of the α-helix. Therefore, the stability of the helical structure of the antimicrobial peptide mb217 is enhanced by introducing helix-capping motifs, thereby enhancing the salt resistance of the antimicrobial peptide mb217. The antimicrobial peptide modified with helix caps is mb217cap. The net charge and isoelectric point of the antimicrobial peptide mb217cap were predicted using the DBAASP online tool. Its predicted net charge is 7, carrying 7 positive charges; its predicted isoelectric point is 11.28. The wheel-like diagram of mb217cap containing hydrophilic and hydrophobic surfaces was predicted using the online tool HeliQuest ( Figure 1 ), and the wheel-like diagram shows their amphiphilic structure. The three-dimensional structure of mb217cap was predicted using the online tool AlphaFold3, and the results shown indicate that mb217cap is also a typical α-helical peptide ( Figure 2). Therefore, the antimicrobial peptide mb217cap is also an amphiphilic α-helical cationic peptide.

[0047] Antimicrobial peptide mb217C The sequence of antimicrobial peptide mb217C is MSRSLRNLLKYVWVKVKGE-NH2, and its modification is amidation at the C-terminus of the antimicrobial peptide mb217 sequence, that is, converting -COOH into an amide bond (-CONH2). The molecular formula of antimicrobial peptide mb217C is C 105 H 177 N 31 O 25 S1, and the molecular weight (MW) is 2305.78. The net charge number and isoelectric point of antimicrobial peptide mb217C were predicted by the online tool DBAASP. The predicted net charge number is 5, carrying 5 positive charges; the predicted isoelectric point is 11.52. Since mb217C is similar in structure to antimicrobial peptide mb217 (see Figure 1 and Figure 2 ), the conformation of mb217C should also be α-helical and is an amphiphilic cationic peptide.

[0048] II. Determination of the antibacterial effects of antimicrobial peptide mb217 and its modified peptides against 6 pathogenic Vibrio species Determination of the minimum inhibitory concentration MIC: The marine pathogenic Vibrio parahaemolyticus ( V. parahaemolyticus ), Vibrio anguillarum ( V. owensii ), Vibrio harveyi ( V. harveyi ), Vibrio alginolyticus ( V. alginolyticus ), Vibrio natriegens ( V. alginolyticus ), and Vibrio mediterranei ( V. mediterranei ) used in the experiment were all isolated from diseased mariculture animals. The MIC determination of the antimicrobial peptide was performed by the broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, P. A. Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991.

[0049] The 6 marine pathogenic Vibrio species were inoculated into sterile 2216E medium and cultured overnight with shaking at 25°C. The 6 marine pathogenic Vibrio species were inoculated into fresh CAMHB medium at an inoculum size of 1%, cultured to the exponential phase, and the cell concentration was adjusted to 1×10 5c.f.u. / mL obtained bacterial solution. Then, 180 μL of the bacterial solution was transferred into a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted into an antimicrobial peptide solution with serial two-fold dilutions. 20 μL of the solution was added to the bacterial solution in the 96-well plate to make the antimicrobial peptide concentration in the bacterial solution range from 4 to 64 μg / mL. After incubating the 96-well plate at 25 °C for 12 hours, the bacterial growth was detected by a microplate reader (OD 600 ), and three replicates were set for the experiment. MIC (Minimum Inhibitory Concentration) was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. The lower the MIC value, the stronger the antibacterial effect of the antimicrobial peptide.

[0050] The experimental results are as Figure 3 shown. Under the condition of not adding extra NaCl (0 mM NaCl), the MIC of antimicrobial peptide mb217 against Vibrio natriegens and Vibrio owensii was 4 μg / mL, the MIC against Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio mediterranei was 8 μg / mL, and the MIC against Vibrio alginolyticus was 16 μg / mL. While the MIC of the modified peptides mb217cap and mb217C of antimicrobial peptide mb217 against the six marine pathogenic Vibrio species was only 4 μg / mL. It shows that under this condition, the three antimicrobial peptides all exhibit strong antibacterial activities against the six Vibrio species, and the antibacterial activities of the modified peptides of mb217 against Vibrio are stronger than that of the unmodified mb217 itself.

[0051] III. Determination of the antibacterial effects of antimicrobial peptide mb217 and its modified peptides against six pathogenic Vibrio species under different salinity conditions Marine pathogenic Vibrio species, as the pathogenic bacteria of aquaculture animals such as prawns, are mainly colonized in the water environment with high salinity. It has been reported that Na + has the greatest impact on the antibacterial activity of antimicrobial peptides and can even cause the complete loss of activity of antimicrobial peptides. Therefore, the present invention detected the antibacterial effects of antimicrobial peptide mb217 and its modified peptides against marine pathogenic Vibrio species under different salinity conditions (i.e., 100 - 300 mM NaCl).

[0052] The six marine pathogenic Vibrio species were inoculated into sterile 2216E medium and cultured overnight with shaking at 25 °C. With an inoculation amount of 1%, the above six marine pathogenic Vibrio species were inoculated into fresh CAMHB medium containing different salinities (different concentrations of NaCl), cultured until the exponential phase, and the cell concentration was adjusted to 1×10 5The obtained bacterial suspension with c.f.u. / mL is ready for use. The NaCl concentration gradients are set at 100 mM (5.844‰), 150 mM (8.766‰), 200 mM (11.688‰), 250 mM (14.610‰), and 300 mM (17.532‰). Then, 180 μL of the bacterial suspension is transferred into a 96-well plate. The antimicrobial peptide powder is dissolved in sterile water and diluted into an antimicrobial peptide solution with serial two-fold dilutions. 20 μL of the solution is added to the bacterial suspension in the 96-well plate, such that the antimicrobial peptide concentration range in the bacterial suspension is 4 - 64 μg / mL. After incubating the 96-well plate at 25 °C for 12 hours, the bacterial growth is detected by a microplate reader (OD 600 ), and the experiment is set with 3 replicates.

[0053] The experimental results are as Figures 3 - 7 shown. When additional NaCl is added to the medium (100 mM - 300 mM NaCl), the MIC values of the antimicrobial peptide mb217 and its modified peptides against 5 vibrios except Vibrio natriegens increase with the increase in salinity, indicating that the antibacterial effects of the antimicrobial peptide mb217 and its modified peptides are affected by salinity. The antibacterial effects of the three peptides against Vibrio natriegens remain consistent under low-salt and high-salt conditions, and the MIC is relatively low (both are 4 μg / mL), indicating that the antibacterial effects of the antimicrobial peptide mb217 and its modified peptides against Vibrio natriegens are not significantly affected by salinity changes, suggesting that these peptides may have a high specificity for Vibrio natriegens. Additionally, the MIC of the three peptides against Vibrio mediterranei only increases by 2 - 4 times. Among them, mb217cap exhibits a strong antibacterial effect against Vibrio mediterranei under high-salt conditions, with the MIC only increasing by 2 times to 8 μg / mL under the condition of 300 mM NaCl; while the MIC under the conditions of 0 - 250 mM NaCl remains 4 μg / mL. For the 4 vibrios of Vibrio parahaemolyticus, Vibrio owensii, Vibrio harveyi, and Vibrio alginolyticus, the salt tolerance effect of mb217C is significantly stronger than that of mb217 and mb217cap.

[0054] Under the salinity condition of 100 mM NaCl (5.844‰), the experimental results are as Figure 4 shown. The MIC values of mb217 and mb217cap against Vibrio parahaemolyticus, Vibrio owensii, Vibrio harveyi, and Vibrio alginolyticus all increase to 16 μg / mL. While the MIC values of mb217C against Vibrio parahaemolyticus, Vibrio owensii, and Vibrio alginolyticus all increase to 8 μg / mL, and the MIC against Vibrio harveyi remains 4 μg / mL. The MIC of the three peptides against Vibrio mediterranei remains consistent with that under the condition of 0 mM NaCl.

[0055] Under the salinity condition of 150 mM NaCl (8.766‰), the experimental results are as Figure 5As shown, the MIC of antibacterial peptide mb217 against Vibrio parahaemolyticus and Vibrio owensii increased to 32 μg / mL, and the MIC against the other 4 Vibrio strains was consistent with that under 100 mM NaCl condition. The MIC of antibacterial peptide mb217cap against Vibrio owensii increased to 32 μg / mL, and the MIC against the other 5 Vibrio strains was consistent with that under 100 mM NaCl condition. The MIC of antibacterial peptide mb217C against Vibrio owensii and Vibrio alginolyticus increased to 16 μg / mL, the MIC against Vibrio harveyi and Vibrio mediterranei increased to 8 μg / mL, and the MIC against Vibrio parahaemolyticus and Vibrio natriegens was consistent with that under 100 mM NaCl condition.

[0056] Under the salinity condition of 200 mM NaCl (11.688‰), the experimental results are as Figure 6 shown. The MIC of antibacterial peptide mb217 against Vibrio owensii has increased to 64 μg / mL, the MIC against Vibrio harveyi and Vibrio alginolyticus has increased to 32 μg / mL, and the MIC against Vibrio mediterranei has increased to 16 μg / mL. The MIC of antibacterial peptide mb217cap against Vibrio owensii has also increased to 64 μg / mL, the MIC against Vibrio parahaemolyticus and Vibrio alginolyticus has increased to 32 μg / mL, and the MIC against the other 3 Vibrio strains is consistent with that under 150 mM NaCl condition. The MIC of antibacterial peptide mb217C against Vibrio parahaemolyticus has increased to 16 μg / mL, and the MIC against the other 5 Vibrio strains has not increased.

[0057] Under the salinity condition of 250 mM NaCl (14.61‰), the experimental results are as Figure 7 shown. The MIC of antibacterial peptide mb217 and antibacterial peptide mb217cap against 6 Vibrio strains is consistent with that under 200 mM NaCl condition. The MIC of antibacterial peptide mb217C against Vibrio owensii has increased to 32 μg / mL, and the MIC against the other 5 Vibrio strains has not increased.

[0058] Under the salinity condition of 300 mM NaCl (17.532‰), the experimental results are as Figure 8As shown, the MIC of antibacterial peptide mb217 against Vibrio parahaemolyticus increased to 64 μg / mL, the MIC against Vibrio owensii exceeded 64 μg / mL, and the MIC against the other 4 Vibrio strains remained unchanged. The MIC of antibacterial peptide mb217cap against Vibrio parahaemolyticus and Vibrio alginolyticus increased to 64 μg / mL, the MIC against Vibrio harveyi increased to 32 μg / mL, the MIC against Vibrio mediterranei only increased to 8 μg / mL, and the MIC against the other 2 Vibrio strains remained unchanged. The MIC of antibacterial peptide mb217C against Vibrio parahaemolyticus increased to 32 μg / mL, the MIC against Vibrio mediterranei increased to 16 μg / mL, and the MIC against the other 4 Vibrio strains was consistent with that under the condition of 250 mM NaCl. Under the condition of 300 mM NaCl, the salt tolerance and antibacterial activity of antibacterial peptide mb217C against the 4 Vibrio strains, namely Vibrio parahaemolyticus, Vibrio owensii, Vibrio harveyi, and Vibrio alginolyticus, were significantly stronger than those of mb217 and mb217cap; the MIC of antibacterial peptide mb217cap against Vibrio mediterranei only increased by 2-fold, demonstrating its strong antibacterial effect against Vibrio mediterranei under high-salt conditions. The antibacterial effects of the three peptides against sodium-requiring Vibrio strains were consistent under low-salt and high-salt conditions and the MIC was relatively low, indicating that these peptides may have high specificity for sodium-requiring Vibrio strains.

[0059] In summary, antibacterial peptide mb217 and its modified peptides have certain antibacterial effects against 6 marine pathogenic Vibrio strains at different salinities. The three peptides have strong antibacterial effects against sodium-requiring Vibrio strains under the salinity condition of 0 - 300 mM NaCl (the MICs are all 4 μg / mL), indicating that their antibacterial effects are not significantly affected by salinity changes and have high specificity. In contrast, the modified peptide mb217cap showed excellent antibacterial effects against Vibrio mediterranei under high-salt conditions, and its MIC value only increased to 8 μg / mL. The antibacterial activity of the modified peptide mb217C against Vibrio parahaemolyticus, Vibrio owensii, Vibrio harveyi, and Vibrio alginolyticus under high-salt conditions was significantly better than that of the other two peptides, showing strong salt tolerance and potential for inhibiting multiple Vibrio strains. These results indicate that after modification of antibacterial peptide mb217, its salt tolerance and antibacterial activity against Vibrio have been significantly improved. This shows that by rationally designing and modifying antibacterial peptides, their stability and antibacterial effects in complex environments can be enhanced, providing an important theoretical basis and practical reference for the development of new antibacterial agents, especially inhibitors against marine pathogenic Vibrio strains.

[0060] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

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

1.

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

2.

3. An antibacterial peptide mb217C, characterized in that, The antimicrobial peptide mb217C is a modified peptide of the antimicrobial peptide mb217 as described in claim 1. The modification method includes amidation modification at the C-terminus of the antimicrobial peptide mb217 sequence, and the amino acid sequence of the antimicrobial peptide mb217C is shown in SEQ ID No.

3.

4. Use of the antimicrobial peptide mb217 as described in claim 1, and / or the antimicrobial peptide mb217cap as described in claim 2, and / or the antimicrobial peptide mb217C as described in claim 3 in the preparation of an antibacterial drug.

5. The application according to claim 4, characterized in that, The antibacterial drug is used to inhibit marine pathogenic Vibrio.

6. The application according to claim 5, wherein The marine pathogenic Vibrio includes at least one of Vibrio natriegens, Vibrio owensii, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mediterranei, and Vibrio alginolyticus.

7. Use of the antimicrobial peptide mb217 as described in claim 1, and / or the antimicrobial peptide mb217cap as described in claim 2, and / or the antimicrobial peptide mb217C as described in claim 3 in the preparation of a medicament for aquaculture in seawater or a high-salt environment.

8. An antibacterial drug, characterized in that, The active ingredient includes at least one of the antimicrobial peptide mb217 as described in claim 1, the antimicrobial peptide mb217cap as described in claim 2, and the antimicrobial peptide mb217C as described in claim 3.

9. Use of the antibacterial drug as described in claim 8 in inhibiting marine pathogenic Vibrio.

10. Use of the antibacterial drug as described in claim 8 in seawater aquaculture or aquaculture in a high-salt environment.

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