ALFPm3 modified antibacterial peptide, molecular design and construction method and application of ALFPm3 modified antibacterial peptide

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to ALFPm3 modified antibacterial peptide, a molecular design and construction method and application of the ALFPm3 modified antibacterial peptide, and the amino acid sequence of the ALFPm3 modified antibacterial peptide is selected from any one of SEQ ID NO: 1-SEQ ID NO: 7. Analysis and design are carried out by combining the structure and action site of vibrio parahaemolyticus and the molecular structure and function relationship of the antibacterial peptide ALFPm3 as a basic template, the antibacterial activity of the antibacterial peptide ALFPm3 is improved, and other biological activities such as hemolysis are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an ALFPm3 modified antimicrobial peptide, a molecular design and construction method, and applications thereof. Background Art

[0002] Vibrio parahaemolyticus is a Gram-negative bacterium widely found in marine and freshwater environments. It has attracted considerable attention due to its high morbidity and potent pathogenicity in aquaculture. V. parahaemolyticus is the primary causative agent of vibriosis in aquatic animals. It grows rapidly, capable of reproducing a generation in 8-9 minutes under favorable conditions. Research has shown that V. parahaemolyticus virulence factors primarily include hemolysins, adhesion factors, proteases, outer membrane proteins, lipopolysaccharide (LPS), and type III (T3SS) and type VI (T6SS) secretion systems. Antibiotics and chemical agents are frequently used to combat bacterial infections. This has led to the emergence of more pathogenic "superbugs." These highly resistant bacteria not only lose sensitivity to traditional antibiotics but also become significantly more difficult to treat. The outer membrane of V. parahaemolyticus cells generally has low permeability to large molecules, which, to a certain extent, hinders the entry of antibiotics into the bacterial cell. Consequently, V. parahaemolyticus is resistant to most antibiotics. In view of the various problems caused by the abuse of antibiotics, and in order to prevent and control Vibrio, finding new antibacterial strategies has become a hot topic in current research.

[0003] Anti-lipopolysaccharide factors (ALFs) were first identified from the blood lymphocytes of two species of horseshoe crabs (Limulus polyphemus). Currently, more than ten ALF subtypes have been discovered. ALFs inhibit both Gram-positive and Gram-negative bacteria through a conserved lipopolysaccharide binding domain (LBD). This domain is typically highly positively charged, generating electrostatic interactions with the negatively charged LPS on the bacterial surface, leading to the accumulation of ALFs on the cell membrane. When ALFs reach a certain threshold, they form membrane-perforating structures that disrupt the cell membrane, causing leakage of cellular contents and cell death. Anti-lipopolysaccharide factor 3 (ALFPm3) is a 98-amino acid cationic antimicrobial peptide isolated from Penaeus monodon. It is composed of three α-helices and four antiparallel β-sheets. Similar to other ALFs, the mature ALFPm3 peptide contains a conserved LBD domain and two cysteine ​​residues. Nuclear magnetic resonance and molecular dynamics simulations reveal that ALFPm3 binds to the phosphate and hydrophobic regions of lipid A via its LBD region: positively charged residues (K / R) bind to the glucosamine region of lipid A, while hydrophobic residues (P40 / Y41, W22, etc.) are embedded in the fatty tail of lipid A. The binding process is dominated by hydrophobic interactions, with K39, T49, and Q70 as key recognition sites. Although in vitro studies have demonstrated its ability to kill bacteria through membrane permeability, the mechanism of differential activity against different pathogens remains unclear, and existing research is largely limited to prediction and simulation analysis. However, natural antimicrobial peptides have low stability in vivo, are easily degraded by proteases, and are sensitive to environmental conditions such as serum and pH.

[0004] Currently, the main methods for antimicrobial peptide molecular design include: 1) amino acid residue design; 2) peptide chain cyclization methods; 3) fragment splicing; 4) biological model design; 5) structural parameter optimization; 6) computer-aided design methods; 7) targeted antimicrobial peptide design; and 8) combinatorial library screening. Among these, structural parameter prediction plays an important guiding role in antimicrobial peptide molecular design. Therefore, structural design of antimicrobial peptides to meet diverse production and living needs may be a new trend in future antimicrobial peptide research and development.

[0005] Based on the above theory, the analysis and design of the basic template were carried out by combining the structure and action site of Vibrio parahaemolyticus and the molecular structure and function relationship of the antimicrobial peptide ALFPm3, which is of great significance for improving its biological expression and antibacterial activity, reducing hemolysis and other biological activities. Summary of the Invention

[0006] The present invention provides an ALFPm3 modified antimicrobial peptide, a molecular design and construction method, and its application. By combining the structure and action site of Vibrio parahaemolyticus and the molecular structure and function relationship of the antimicrobial peptide ALFPm3 as a basic template for analysis and design, its antibacterial activity is improved and other biological activities such as hemolysis are reduced.

[0007] The specific technical solutions are as follows: The first object of the present invention is to provide an ALFPm3 modified antimicrobial peptide, wherein the amino acid sequence of the ALFPm3 modified antimicrobial peptide is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 7.

[0008] The second object of the present invention is to provide a molecular design and construction method for the ALFPm3 modified antimicrobial peptide, comprising the following steps: molecular design of the antimicrobial active region LBD of the natural antimicrobial peptide ALFPm3.

[0009] Furthermore, the molecular design and construction method of the ALFPm3 modified antimicrobial peptide includes the following steps: by replacing amino acids in the antimicrobial active region LBD of the natural antimicrobial peptide ALFPm3 from the aspects of amphiphilicity, hydrophobicity or structural characteristics, the activity of the modified peptide (ALFPm3 modified antimicrobial peptide) is evaluated by combining functional prediction with detection of actual antibacterial effects.

[0010] The third object of the present invention is to provide a use of the ALFPm3 modified antimicrobial peptide in the preparation of antibacterial infection drugs.

[0011] Furthermore, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0012] Furthermore, the Gram-negative bacteria include Vibrio parahaemolyticus.

[0013] Taking the lipopolysaccharide in the cell wall of Vibrio parahaemolyticus as the main target site, the natural antimicrobial peptide ALFPm3 was rationally designed using molecular design schemes such as peptide chain truncation, amino acid replacement, and fusion expression. The physicochemical properties, antimicrobial activity, transmembrane region, and secondary structure of the derived peptide (ALFPm3 modified antimicrobial peptide) were preliminarily judged in combination with antimicrobial peptide computational analysis software and antimicrobial peptide prediction tools.

[0014] The fourth object of the present invention is to provide a nucleic acid molecule encoding the ALFPm3 modified antimicrobial peptide.

[0015] A fifth object of the present invention is to provide a recombinant microorganism, which expresses the ALFPm3 modified antimicrobial peptide.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention studies the molecular interaction mechanism between the natural antimicrobial peptide ALFPm3 and key components of the Vibrio cell membrane and cell wall, and further optimizes the natural antimicrobial peptide ALFPm3 using molecular design and computer-aided methods, thereby further improving the activity of the antimicrobial peptide and reducing cytotoxicity and hemolysis. (2) The present invention uses chemical modification to block both ends of the derivative peptide (ALFPm3 modified antimicrobial peptide) to improve its stability, and then uses solid phase chemical synthesis method, standard Fmoc method, C 18 Purify by reverse phase high performance liquid chromatography, target purity ≥95%; (3) This invention is expected to not only provide a new solution for the prevention and control of Vibrio parahaemolyticus in aquaculture, but also lay a solid theoretical foundation and technical support for the development and application of antimicrobial peptides, thereby promoting the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 3 is a simulation diagram of the helical wheel of LBD and its derivative peptides in the examples of the present invention, where AH are the simulation results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ, respectively. Yellow marks indicate basic amino acids, blue marks indicate polar hydrophobic amino acids, and green marks indicate non-polar hydrophobic amino acids. Glycine (G), serine (T) and glutamine (Q) do not belong to the above categories. Figure 2 This is a diagram showing the effect of molecular docking between the derivative peptide LBD3Y and lipid A of LPS in an embodiment of the present invention; Figure 3 Figures 1 and 2 are circular dichroism spectra of LBD and its derivative peptides according to the present invention, wherein A and H are the circular dichroism spectra of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY, and LBDQ, respectively; Figure 4 This is a graph showing the cytotoxicity of LBD and its derivative peptides in the examples of the present invention. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0019] Example 1: Molecular design and construction of ALFPm3 modified antimicrobial peptide The amino acid sequence of the lipopolysaccharide-binding domain (LBD) of the natural antimicrobial peptide ALFPm3 was intercepted, and the LBD was molecularly designed from three aspects: amphipathicity, hydrophobicity, and structural characteristics. The physicochemical properties of the modified peptide were evaluated using the online prediction website Expasy (https: / / web.expasy.org / cgi-bin / ) and the Antimicrobial Peptide Database (https: / / aps.unmc.edu / ), and its secondary structure was predicted using Alphafold (https: / / alphafoldserver.com / ) to obtain a derivative peptide (ALFPm3 modified antimicrobial peptide) with good performance.

[0020] In terms of amphipathicity, according to the template of Ac-C-(BH)n-KR-(HB)nC-NH2 (B is a basic amino acid, H is a hydrophobic amino acid), the basic amino acids lysine K and arginine R were used to replace threonine T, tyrosine Y, glutamine Q, and tryptophan W in the sequence; By predicting the physicochemical properties, amphiphilicity and binding sites of the designed derivative peptides, three derivative peptides with good prediction results were comprehensively screened out, such as LBD3W, LBD3V and LBD3Y in Table 1.

[0021] In terms of hydrophobicity, hydrophobicity is one of the important parameters affecting the activity of antimicrobial peptides. It affects the degree of interaction between antimicrobial peptides and the phospholipid layer of the cell membrane. Aromatic group modification can not only increase the hydrophobicity of the peptide, but also has a high membrane interface affinity, which is conducive to the penetration of antimicrobial peptides into bacterial cell membranes. Therefore, threonine T and glutamine Q were replaced by lysine K, and tyrosine Y was replaced by the highly hydrophobic tryptophan W to explore the influence of hydrophobicity. The aliphatic valine V was used to replace tyrosine Y to explore the influence of hydrophilicity, so that the structure shows a trend of hydrophilicity at the N-terminus and hydrophobicity at the C-terminus. By predicting the physicochemical properties, hydrophobicity, and secondary structure of the designed derivative peptides, three derivative peptides with good prediction results were comprehensively screened out, such as LBDW, LBDV, and LBDY in Table 1.

[0022] In terms of structural optimization, the disulfide bonds in the LBD sequence play an indispensable role in stabilizing its spatial structure and antibacterial activity. Therefore, the aromatic amino acid tyrosine Tyr was used to replace the cysteine ​​Cys in the sequence to enhance the β-sheet structure. Tyrosine is a strong β-sheet former (Pβ=1.47) and its β-sheet forming ability is greater than that of Cys (Pβ=1.19). The substitution of Tyr can enhance the β-sheet structure of the peptide. At the same time, the hydrophobicity of Tyr (hydrophobicity value 0.26, Eisenberg scale) is less than that of Cys (hydrophobicity value 0.29, Eisenberg scale), which can theoretically reduce the hydrophobicity of the derivative peptide. In addition, the thiol group of Cys is methylated to lose its hydrogen bond forming ability, which can theoretically convert the β-sheet structure of LBD into a random coil.

[0023] By predicting the designed derivative peptides, a derivative peptide with good prediction results was comprehensively screened out, such as LBDQ in Table 1.

[0024] Table 1 shows the derived peptide sequences and their physicochemical properties, where the binding energy is the binding energy between the antimicrobial peptide and lipid A molecules predicted by docking simulation.

[0025] Table 1 Derivative peptide sequences and their physicochemical properties

[0026] The synthesis of the above peptides was commissioned to Shanghai Jier Biochemical Co., Ltd. and confirmed by mass spectrometry and high-performance liquid chromatography.

[0027] The helical wheel simulation diagram of LBD and its derivative peptides is shown in Figure 1 As shown, AH are the simulation results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ, respectively. Yellow marks are basic amino acids, blue marks are polar hydrophobic amino acids, and green marks are non-polar hydrophobic amino acids. Glycine (G), serine (T) and glutamine (Q) do not belong to the above categories.

[0028] The effect of molecular docking of the derivative peptide LBD3Y with lipid A of LPS is shown in the figure Figure 2 shown.

[0029] Example 2: Circular dichroism (CD) determination At room temperature, circular dichroism spectrometry was used to measure the characteristic spectra of the above eight antimicrobial peptides in sterile water, 60 mM SDS solution (simulating the negatively charged bacterial cell membrane), and 50% trifluoroethanol (TFE, simulating the hydrophobic environment of the bacterial cell membrane). The final concentration of the peptide solution was 0.2 mg / mL. A 5 mm thick quartz sample cell was used, and the scanning wavelength was 190-260 nm. Each peptide was measured in parallel three times and the average value was obtained. The results were subtracted from the solvent control. The results are shown in Figure 2. Figure 3 As shown, Figure 3 Circular dichroism spectra of LBD and its derivative peptides, where AH are the circular dichroism detection results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ, respectively.

[0030] Depend on Figure 3 As can be seen, in aqueous solution, all peptides exhibit a distinct negative peak near 200 nm, characteristic of a random coil structure. In SDS and TFE environments, the secondary structures of the peptides undergo significant changes. LBDQ exhibits negative peaks near 209 nm and 222 nm, indicating a tendency toward α-helical formation. The remaining derivative peptides exhibit positive bands between 190 and 200 nm, indicating a tendency toward β-sheet formation for seven derivative peptides. The results for LBDW, LBDY, and LBDV differ from the secondary structures predicted by Alphafold2. This suggests that Alphafold2's predictions, based on existing structures in the database, may contain data errors or errors in the chemical synthesis.

[0031] Example 3: Minimum inhibitory concentration (MIC) test of antimicrobial peptides against Vibrio parahaemolyticus 1. Strain culture: Dip an inoculating loop into a -80℃ frozen Vibrio parahaemolyticus (Vp ATCC17802) and streak it on a Mueller-Hinton Broth (MHB) solid plate. Incubate the plate upside down in a 28℃ constant temperature incubator. After a single colony grows, pick a single colony and place it in 5 mL of fresh MHB liquid medium. Incubate the plate in a shaking incubator at 28℃ / 150 rpm until the logarithmic growth phase. Then dilute the bacterial solution to a concentration of 2×10 5 CFU / mL is reserved.

[0032] 2. Pretreatment of antimicrobial peptide solutions: Place 8 antimicrobial peptide solutions (2 mg / mL) of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY, and LBDQ in a PCR instrument. React at 20°C for 30 min, then quickly remove from the heat and place on ice for 10 min, then allow to equilibrate at room temperature.

[0033] 3. Determination of MIC by 2-fold dilution method: Select a sterile round-bottom 96-well plate, add 90 μL of sterile MH liquid medium to the first row, and add 50 μL of sterile liquid medium to each of the remaining rows. Then, add 10 μL of pre-treated peptide sample solution (2 mg / mL) to each well in the first row, pipette and mix well, then draw 50 μL from the first row of wells and add it to the corresponding wells in the second row. Repeat the operation in sequence, and draw 50 μL from the last well and discard it. Set up 3 parallels for each group. Then, add 50 μL of pre-diluted bacterial solution to each well so that the bacterial solution concentration in each well is 1×10 5 CFU / mL, with a final volume of 100 µL per well. The peptide sample concentrations were ranked from high to low as 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0.78125 μg / mL. An equal volume of bacterial suspension served as a negative control, and the original peptide LBD served as a positive control. The 96-well plate was incubated at 28°C for 10 h. The UV absorbance of each well at 600 nm was measured using a microplate reader. The MIC was calculated as the average of the sum of the peptide sample concentrations in the wells with no bacterial growth and the adjacent wells with bacterial growth. The results are shown in Table 2.

[0034] Table 2 Minimum inhibitory concentration of LBD and its derivative peptides against Vibrio parahaemolyticus

[0035] As shown in Table 2, the minimum inhibitory concentration of the parent peptide LBD against Vibrio parahaemolyticus is 100 μg / mL. The antibacterial activities of LBD3W, LBD3V, and LBD3Y designed by the template against Vibrio parahaemolyticus were increased by approximately 90.625%, 62.5%, and 81.25%, respectively, compared with the parent peptide LBD; the antibacterial activities of LBDW, LBDV, and LBDY with altered spatial structures were all increased by approximately 90.625%; the antibacterial activity of LBDQ without disulfide bonds supporting its spatial structure was increased by approximately 95.31%. The results indicate that the increase in amphiphilicity is beneficial to improving the antibacterial activity of antimicrobial peptides, and disulfide bonds are not necessary for the activity of LBD antimicrobial peptides.

[0036] Example 4: Minimum inhibitory concentration (MIC) test of antimicrobial peptides against Vibrio harveyi 1. Strain culture: Dip an inoculating loop into -80℃ frozen Vibrio harveyi (Vh TS275249) and streak it on a Mueller-Hinton Broth (MHB) solid plate. Incubate the plate upside down in a 30℃ constant temperature incubator. After a single colony grows, pick a single colony and place it in 5 mL of fresh MHB liquid medium. Incubate the plate in a shaking incubator at 30℃ / 150 rpm until the logarithmic growth phase. Then dilute the bacterial solution to a concentration of 2×10 5 CFU / mL is reserved.

[0037] 2. Antimicrobial peptide solution pretreatment: Place the eight antimicrobial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V, and LBD3Y in a PCR instrument. React at 20°C for 30 min, then quickly remove from the heat and place on ice for 10 min, then allow to equilibrate at room temperature.

[0038] 3. Determination of MIC by 2-fold dilution method: Select a sterile round-bottom 96-well plate, add 90 μL of sterile MH liquid medium to the first row, and add 50 μL of sterile liquid medium to each of the remaining rows. Then, add 10 μL of pre-treated peptide sample solution (2 mg / mL) to each well in the first row, pipette and mix well, then draw 50 μL from the first row of wells and add it to the corresponding wells in the second row. Repeat the operation in sequence, and draw 50 μL from the last well and discard it. Set up 3 parallels for each group. Then, add 50 μL of pre-diluted bacterial solution to each well so that the bacterial solution concentration in each well is 1×10 5 CFU / mL, with a final volume of 100 µL per well. The peptide sample concentrations were ranked from high to low as 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL, and 0.390625 μg / mL. An equal volume of bacterial suspension served as a negative control, and the original peptide LBD served as a positive control. The 96-well plate was incubated at 30°C for 14 h. The UV absorbance of each well at 600 nm was measured using a microplate reader. The MIC was calculated as the average of the sum of the peptide sample concentrations in the wells with no bacterial growth and the adjacent wells with bacterial growth. The results are shown in Table 3.

[0039] Table 3 Minimum inhibitory concentration of LBD and its derivative peptides against Vibrio harveyi

[0040] As shown in Table 3, the minimum inhibitory concentration (MIC) of the parent peptide LBD against Vibrio harveyi was 200 μg / mL. The antimicrobial activities of the template-designed peptides LBD3W, LBD3V, and LBD3Y against Vibrio harveyi were all approximately 87.5% higher than those of the parent peptide LBD. The antimicrobial activities of the structurally modified LBDW, LBDV, and LBDY were increased by approximately 93.75%, 96.875%, and 87.5%, respectively. The antimicrobial activity of LBDQ, which lacks disulfide bonds to support its spatial structure, was increased by approximately 93.75%. These results indicate that increased amphiphilicity is beneficial for enhancing the antimicrobial activity of antimicrobial peptides, and that disulfide bonds are not essential for the activity of LBD antimicrobial peptides.

[0041] Example 5: Minimum inhibitory concentration (MIC) test of antimicrobial peptides against Vibrio alginolyticus 1. Strain culture: Dip an inoculation loop into -80℃ frozen Vibrio alginolyticus (Va) and streak it on a Mueller-Hinton Broth (MHB) solid plate. Incubate the plate upside down in a 28℃ constant temperature incubator. After a single colony grows, pick a single colony and place it in 5 mL of fresh MHB liquid medium. Incubate the plate in a shaking incubator at 28℃ / 150rpm until the logarithmic growth phase. Then dilute the bacterial solution to a concentration of 2×10 5 CFU / mL is reserved.

[0042] 2. Antimicrobial peptide solution pretreatment: Place the eight antimicrobial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V, and LBD3Y in a PCR instrument. React at 20°C for 30 min, then quickly remove from the heat and place on ice for 10 min, then allow to equilibrate at room temperature.

[0043] 3. Determination of MIC by 2-fold dilution method: Select a sterile round-bottom 96-well plate, add 90 μL of sterile MH liquid medium to the first row, and add 50 μL of sterile liquid medium to each of the remaining rows. Then, add 10 μL of pre-treated peptide sample solution (2 mg / mL) to each well in the first row, pipette and mix well, then draw 50 μL from the first row of wells and add it to the corresponding wells in the second row. Repeat the operation in sequence, and draw 50 μL from the last well and discard it. Set up 3 parallels for each group. Then, add 50 μL of pre-diluted bacterial solution to each well so that the bacterial solution concentration in each well is 1×10 5CFU / mL, with a final volume of 100 µL per well. The peptide sample concentrations were ranked from high to low as 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL, and 0.390625 μg / mL. An equal volume of bacterial suspension was used as a negative control, and the original peptide LBD was used as a positive control. The 96-well plate was incubated at 28°C for 10 h. The UV absorbance of each well at 600 nm was measured using a microplate reader. The MIC was calculated as the average of the sum of the peptide sample concentrations in the wells with no bacterial growth and the adjacent wells with bacterial growth. The results are shown in Table 4.

[0044] Table 4 Minimum inhibitory concentration of LBD and its derived peptides against Vibrio alginolyticus

[0045] As shown in Table 4, the minimum inhibitory concentration of the parent peptide LBD against Vibrio alginolyticus is 200 μg / mL. The antibacterial activities of LBD3W, LBD3V, and LBD3Y designed by the template against Vibrio alginolyticus were increased by approximately 98.438%, 87.5%, and 87.5%, respectively, compared with the parent peptide LBD. The antibacterial activities of LBDW, LBDV, and LBDY with altered spatial structures were increased by approximately 98.438%, 93.75%, and 96.875%, respectively, and the antibacterial activity of LBDQ without disulfide bonds supporting its spatial structure was increased by approximately 96.875%. The results indicate that the increase in amphiphilicity is beneficial to improving the antibacterial activity of antimicrobial peptides, and disulfide bonds are not necessary for the activity of LBD antimicrobial peptides.

[0046] Example 6: Minimum inhibitory concentration (MIC) test of antimicrobial peptides against Vibrio anguillarum 1. Strain culture: Dip an inoculating loop into -80℃ frozen Vibrio anguillarum (M-TS TS340431) and streak it on a Mueller-Hinton Broth (MHB) solid plate. Incubate the plate upside down in a 28℃ constant temperature incubator. After a single colony grows, pick a single colony and place it in 5 mL of fresh MHB liquid medium. Incubate the plate in a shaking incubator at 28℃ / 150 rpm until the logarithmic growth phase. Then dilute the bacterial solution to a concentration of 2×10 5 CFU / mL is reserved.

[0047] 2. Antimicrobial peptide solution pretreatment: Place the eight antimicrobial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V, and LBD3Y in a PCR instrument. React at 20°C for 30 min, then quickly remove from the heat and place on ice for 10 min, then allow to equilibrate at room temperature.

[0048] 3. Determination of MIC by 2-fold dilution method: Select a sterile round-bottom 96-well plate, add 90 μL of sterile MH liquid medium to the first row, and add 50 μL of sterile liquid medium to each of the remaining rows. Then, add 10 μL of pre-treated peptide sample solution (2 mg / mL) to each well in the first row, pipette and mix well, then draw 50 μL from the first row of wells and add it to the corresponding wells in the second row. Repeat the operation in sequence, and draw 50 μL from the last well and discard it. Set up 3 parallels for each group. Then, add 50 μL of pre-diluted bacterial solution to each well so that the bacterial solution concentration in each well is 1×10 5 CFU / mL, with a final volume of 100 µL per well. The peptide sample concentrations were ranked from high to low as 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL, and 0.390625 μg / mL. An equal volume of bacterial suspension served as a negative control, and the original peptide LBD served as a positive control. The 96-well plate was incubated at 28°C for 14 h. The UV absorbance of each well at 600 nm was measured using a microplate reader. The MIC was calculated as the average of the sum of the peptide sample concentrations in the wells with no bacterial growth and the adjacent wells with bacterial growth. The results are shown in Table 5.

[0049] Table 5 Minimum inhibitory concentration of LBD and its derivative peptides against Vibrio anguillarum

[0050] As shown in Table 5, the parent peptide LBD has good basic antibacterial activity against Vibrio anguillarum. The minimum inhibitory concentration of the parent peptide LBD against Vibrio anguillarum is 25 μg / mL. The antibacterial activities of LBD3V and LBD3Y designed by the template against Vibrio anguillarum are increased by 75% and 50%, respectively, compared with the parent peptide LBD; the antibacterial activities of LBDW and LBDV with altered spatial structures are both increased by 50%; the antibacterial activity of LBDQ without disulfide bonds supporting its spatial structure is increased by 75%.

[0051] Example 7: Minimum bactericidal concentration (MBC) test of antimicrobial peptides against Vibrio parahaemolyticus From each clear well (≥MIC) in the MIC test in Example 3, 20 μL of liquid was pipetted onto MHB solid medium. The plates were incubated upside down in a suitable incubator for 10–12 hours, and bacterial growth was observed. The minimum concentration corresponding to the plate without bacterial growth was considered the MBC for the peptide solution. The results are shown in Table 6.

[0052] Table 6 Minimum bactericidal concentrations of LBD and its derivative peptides against Vibrio parahaemolyticus

[0053] “-” in the table means not detected.

[0054] The minimum bactericidal concentration (MBC) is the lowest concentration of an antimicrobial substance capable of killing a certain percentage (usually 99.9%) of bacteria in a cultured test strain. Comparing the results in Tables 2 and 6, LBD3W, LBD3Y, LBDW, LBDY, and LBDQ effectively kill 99.9% of bacteria at 1x the MIC, while LBD3V and LBDV have a 99.9% kill rate against Vibrio parahaemolyticus at 2x the MIC.

[0055] Example 8: Cytotoxicity test 1. Caco-2 Cell Culture and Passaging: Caco-2 cells frozen at -80°C were rapidly thawed in a 37°C water bath and gently shaken until ice crystals completely melted. In a sterile fume hood, the thawed cell suspension was slowly added dropwise to a pre-prepared centrifuge tube containing an appropriate amount of complete culture medium and gently mixed. The suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in fresh culture medium and transferred to a cell culture flask. Sufficient culture medium was added to cover the cell surface and incubated in a 37°C, 5% CO2 incubator for 48 h. The old culture medium was then discarded, and the cells were washed three times with PBS to remove residual culture medium. 1 mL of trypsin solution was added for digestion and incubated in the incubator for 5 min. The digestion reaction was quickly terminated by adding DMEM complete culture medium (containing 20% ​​fetal bovine serum, 1% streptomycin-penicillin, and 79% DMEM basal medium). The supernatant was discarded by centrifugation at 1000 rpm for 5 min, and the pellet was resuspended in 2 mL of fresh culture medium. After pipetting evenly, the pellet was transferred to a new culture flask and incubated for 24 h. When the cells reached the third generation, they were washed with PBS and centrifuged to discard the supernatant. The cells were diluted to a concentration of 1 × 10 5 The cells were cultured in 96-well cell culture plates for 24 h, with 200 μL per well and 5 replicates per group.

[0056] 2. The peptide samples were diluted with DMEM complete medium to 6 concentration gradients of 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, 20 μg / mL, and 1 μg / mL. The cell culture medium after 24 hours of culture was discarded, and peptide samples of different concentration gradients were added in sequence, 200 μL per well, 5 parallels per group, and an equal volume of DMEM complete medium was added to the blank control. The cells were cultured in the incubator for 24 hours. The peptide solution was then discarded, and 150 μL of MTT (0.5 mg / mL) solution was added to each well. The cells were stained in the incubator for 4 hours, and then DMSO was added to dissolve the crystals. The cell viability was detected by a microplate reader at a UV absorption wavelength of 570 nm. The results are as follows: Figure 4 shown.

[0057] Depend on Figure 4 As shown in the results, Caco-2 cells maintained a good cell survival rate after treatment with antimicrobial peptides at concentrations ranging from 1 to 100 μg / mL for 24 hours. Cell survival rates were relatively high (greater than 90%) at concentrations ranging from 1 to 80 μg / mL, indicating that antimicrobial peptides have a certain promoting effect on cell growth. When the peptide concentration increased to 100 μg / mL, the cell survival rates of LBDW, LBDV, and LBDQ decreased to below 90%, indicating that at high concentrations, these three peptides exerted varying degrees of toxicity on cell growth. This may be because tryptophan W and valine V are highly hydrophobic amino acids, and increased hydrophobicity leads to increased cytotoxicity of the antimicrobial peptides. Furthermore, LBDQ, lacking disulfide bonds to support its structure, transforms into an α-helical structure, enhancing its cell-killing effect.

[0058] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. ALFPm3 modified antimicrobial peptide, characterized in that: The amino acid sequence of the ALFPm3 modified antimicrobial peptide is selected from any one of SEQ ID NO: 1 to SEQ ID NO:

7.

2. A molecular design and construction method for the ALFPm3 modified antimicrobial peptide according to claim 1, characterized in that: The method comprises the following steps: performing molecular design on the antibacterial active region LBD of the natural antibacterial peptide ALFPm3.

3. The molecular design and construction method of the ALFPm3 modified antimicrobial peptide according to claim 2, characterized in that: The method comprises the following steps: performing amino acid substitution on the antibacterial active region LBD of the natural antibacterial peptide ALFPm3 in terms of amphiphilicity, hydrophobicity or structural characteristics.

4. A use of the ALFPm3 modified antimicrobial peptide according to claim 1 in the preparation of antibacterial infection drugs.

5. The use according to claim 4, characterized in that The bacteria include Gram-negative bacteria and Gram-positive bacteria.

6. The use according to claim 5, characterized in that The Gram-negative bacteria include Vibrio parahaemolyticus.

7. A nucleic acid molecule, characterized in that Encodes the ALFPm3 modified antimicrobial peptide as described in claim 1.

8. A recombinant microorganism, characterized in that The recombinant microorganism expresses the ALFPm3 modified antimicrobial peptide according to claim 1.

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