Alfpm3 modified antibacterial peptide, molecular design and construction method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to an ALFPm3 modified antibacterial peptide, a molecular design construction method and application thereof, and an amino acid sequence of the ALFPm3 modified antibacterial peptide is selected from any one of SEQ ID NO:1-SEQ ID NO:7. The application is designed based on a template analysis of the structure and action site of Vibrio parahaemolyticus and the functional relationship between the molecular structure and function of the antibacterial peptide ALFPm3, so that the antibacterial activity is improved, and other biological activities such as hemolysis are reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to ALFPm3 modified antibacterial peptide, a molecular design construction method and application thereof. BACKGROUND

[0002] Vibrio parahaemolyticus is a gram-negative bacterium widely existing in marine and freshwater environments, which has attracted much attention due to its high morbidity and strong pathogenicity in aquaculture. Vibrio parahaemolyticus is the main pathogen of vibrio disease in aquatic animals, and grows rapidly, which can reproduce one generation in 8-9 min in a suitable environment. Studies have shown that the virulence factors of Vibrio parahaemolyticus mainly include hemolysin, adhesion factor, protease, outer membrane protein, lipopolysaccharide (LPS), type III (T3SS) and type VI (T6SS) secretion systems, etc. In order to overcome the problem of bacterial infection, antibiotics and chemical drugs are frequently used. This situation has brought more pathogenic “super bacteria”, which are highly resistant to drugs. These bacteria not only lose sensitivity to traditional antibiotics, but also greatly increase the difficulty of treatment. The outer membrane of Vibrio parahaemolyticus cell membrane has generally low permeability to macromolecular substances, which will affect the entry of antibiotics into the interior of bacterial cells to some extent, and therefore, Vibrio parahaemolyticus has resistance to most antibiotics. In view of the various problems caused by the abuse of antibiotics, and in order to prevent and treat vibrio, finding new antibacterial strategies has become a hot research topic at present.

[0003] Anti-lipopolysaccharide factors (ALFs) were first identified from the hemocytes of two species of Limulus polyphemus, and more than ten subtypes of ALFs have been found. ALFs inhibit Gram-positive and Gram-negative bacteria through the conserved lipopolysaccharide binding domain (LBD) in the structure. The domain is usually rich in high positive charge, which can interact with the negatively charged LPS on the surface of bacteria to make AFLs enriched on the cell membrane. When AFLs reach a certain threshold, they will form a membrane-penetrating structure to destroy the cell membrane, causing the contents of the cell to leak out and the cell to die. Anti-lipopolysaccharide factor 3 (ALFPm3) is a cationic antibacterial peptide isolated from Penaeus monodon, which consists of 98 amino acids, three alpha-helices and four reverse parallel beta-sheets. Similar to other ALFs, the mature peptide of ALFPm3 contains a LBD conserved domain and two cysteine residues. Nuclear magnetic resonance and molecular dynamics simulation show that ALFPm3 binds to the phosphate group and hydrophobic region of lipid A through the LBD region: the positively charged residues (K / R) bind to the glucosamine region of lipid A, and the hydrophobic residues (P40 / Y41, W22, etc.) are embedded in the tail of the lipid A fatty chain. The binding process is dominated by hydrophobic interaction, in which K39, T49 and Q70 are key recognition sites. Although in vitro experiments have confirmed that it can kill bacteria by membrane permeability destruction, the mechanism of activity difference against different pathogens is still unclear, and existing research is mostly limited to prediction and simulation analysis. However, natural antibacterial peptides have low stability in vivo and are easily degraded by proteases, and are sensitive to environmental conditions such as serum and pH.

[0004] Currently, the methods of antibacterial peptide molecular design mainly include: 1) amino acid residue design; 2) peptide chain cyclization method; 3) fragment splicing; 4) simulation of biological model design; 5) structure parameter optimization; 6) computer-aided design method; 7) targeted antibacterial peptide design; 8) combinatorial library screening, etc. Among them, the prediction of structure parameters plays an important guiding role in the molecular design of antibacterial peptides. Therefore, it is possible that the structural design of antibacterial peptides to meet different production and life needs may be a new trend in the future research and development of antibacterial peptides.

[0005] Based on the above theory, the structure and action site of Vibrio parahaemolyticus and the relationship between the molecular structure and function of antibacterial peptide ALFPm3 are analyzed and designed based on the template, which is of great significance to improve the biological expression and antibacterial activity of ALFPm3 and reduce hemolysis and other biological activities. SUMMARY

[0006] The application provides an ALFPm3 modified antibacterial peptide, a molecular design construction method and application thereof.

[0007] The specific technical scheme is as follows:

[0008] A first object of the application is to provide an ALFPm3 modified antibacterial peptide, wherein the amino acid sequence of the ALFPm3 modified antibacterial peptide is selected from any one of SEQ ID NO: 1-SEQ ID NO: 7.

[0009] A second object of the application is to provide a molecular design construction method of the ALFPm3 modified antibacterial peptide, comprising the following steps: performing molecular design on an antibacterial activity region LBD of a natural antibacterial peptide ALFPm3.

[0010] Further, the molecular design construction method of the ALFPm3 modified antibacterial peptide comprises the following steps: performing amino acid replacement on the antibacterial activity region LBD of the natural antibacterial peptide ALFPm3 from the aspects of amphiphilicity, hydrophobicity or structural characteristics, and evaluating the activity of the modified peptide (the ALFPm3 modified antibacterial peptide) by combining function prediction with actual antibacterial effect detection.

[0011] A third object of the application is to provide application of the ALFPm3 modified antibacterial peptide in preparation of antibacterial infection drugs.

[0012] Further, the bacteria include gram-negative bacteria and gram-positive bacteria.

[0013] Further, the gram-negative bacteria include Vibrio parahaemolyticus.

[0014] Taking lipopolysaccharide in a cell wall of Vibrio parahaemolyticus as a main targeting site, the natural antibacterial peptide ALFPm3 is reasonably designed by using molecular design schemes such as peptide chain truncation, amino acid replacement and fusion expression, and the physicochemical properties, antibacterial activity, transmembrane region and secondary structure of the derived peptide (the ALFPm3 modified antibacterial peptide) are preliminarily judged by combining antibacterial peptide calculation analysis software and antibacterial peptide prediction tools.

[0015] A fourth object of the application is to provide a nucleic acid molecule encoding the ALFPm3 modified antibacterial peptide.

[0016] A fifth object of the application is to provide a recombinant microorganism expressing the ALFPm3 modified antibacterial peptide.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] (1) The application further optimizes the natural antibacterial peptide ALFPm3 by molecular design and computer assistance, further improves the activity of the antibacterial peptide, and reduces the cytotoxicity, hemolysis and the like, through the research on the molecular interaction mechanism of the natural antibacterial peptide ALFPm3 and key components of Vibrio cell membrane and cell wall.

[0019] (2) The two ends of the derivative peptide (ALFPm3 modified antibacterial peptide) are closed by a chemical modification method to improve the stability, and then a solid-phase chemical synthesis method, a standard Fmoc method, C 18 Reversed-phase column high performance liquid chromatography purification, target purity ≥95%;

[0020] (3) The application not only provides a new solution for the prevention and control of Vibrio parahaemolyticus in aquaculture, but also lays a solid theoretical foundation and technical support for the development and application of antibacterial peptides, thereby promoting the sustainable development of aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a helical wheel simulation diagram of LBD and derivative peptides in the embodiments of the application, wherein A-H are simulation results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ respectively, the yellow mark is a basic amino acid, the blue mark is a polar hydrophobic amino acid, and the green mark is a non-polar hydrophobic amino acid, glycine (G), serine (T) and glutamine (Q) do not belong to the above categories;

[0022] Figure 2 It is an effect diagram of molecular docking of derivative peptide LBD3Y and LPS lipid A in the embodiments of the application;

[0023] Figure 3 It is a circular dichroism spectrum of LBD and derivative peptides in the embodiments of the application, wherein A-H are circular dichroism detection results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ respectively;

[0024] Figure 4 It is a cytotoxicity diagram of LBD and derivative peptides in the embodiments of the application. DETAILED DESCRIPTION

[0025] The principles and characteristics of the application are described below in conjunction with examples, and the examples are only used to explain the application and are not used to limit the scope of the application.

[0026] Example 1: Molecular design and construction of ALFPm3 modified antibacterial peptide

[0027] The amino acid sequence of the lipopolysaccharide binding domain LBD of the natural antibacterial peptide ALFPm3 was intercepted, and LBD was designed from three aspects of amphiphilicity, hydrophobicity and structural characteristics. The physicochemical properties of the modified polypeptide were evaluated by using the online prediction website Expasy (https: / / web.expasy.org / cgi-bin / ) and Antimicrobial Peptide Database (https: / / aps.unmc.edu / ), and the secondary structure was predicted by Alphafold (https: / / alphafoldserver.com / ) to obtain a derivative peptide (ALFPm3 modified antibacterial peptide) with good performance.

[0028] In terms of amphiphilicity, according to the template Ac-C-(BH)n-KR-(HB)n-C-NH2 (B is a basic amino acid, H is a hydrophobic amino acid), threonine T, tyrosine Y, glutamine Q and tryptophan W in the sequence are replaced with basic amino acids lysine K and arginine R;

[0029] By predicting the physicochemical properties, amphiphilicity and binding site of the designed derivative peptide, three derivative peptides with better prediction results were screened out, such as LBD3W, LBD3V and LBD3Y in Table 1.

[0030] In terms of hydrophobicity, hydrophobicity is one of the important parameters affecting the activity of antibacterial peptides, which affects the degree of interaction between antibacterial peptides and cell membrane phospholipid layer; aromatic group modification not only can increase the hydrophobicity of the peptide, but also has high membrane interface affinity, which is beneficial to the penetration of antibacterial peptides through bacterial cell membrane; therefore, lysine K is used to replace threonine T and glutamine Q, and hydrophobic tryptophan W is used to replace tyrosine Y to explore the influence of hydrophobicity; valine V, which is aliphatic, is used to replace tyrosine Y to explore the influence of hydrophilicity, so that the structure shows the trend of N-terminal hydrophilicity and C-terminal hydrophobicity;

[0031] By predicting the physicochemical properties, hydrophobicity and secondary structure of the designed derivative peptide, three derivative peptides with better prediction results were screened out, such as LBDW, LBDV and LBDY in Table 1.

[0032] In terms of structure optimization, the disulfide bond in the LBD sequence plays an essential role in stabilizing its spatial structure and antibacterial activity. Therefore, the aromatic amino acid tyrosine Tyr is used to replace the cysteine Cys in the sequence to enhance the β-pleated structure; tyrosine is a strong β-pleated structure former (Pβ=1.47), and its β-pleated structure formation ability is greater than that of Cys (Pβ=1.19), and the substitution of Tyr can enhance the β-pleated structure of the peptide; at the same time, the hydrophobicity of Tyr (hydrophobic value 0.26, Eisenberg scale) is less than that of Cys (hydrophobic value 0.29, Eisenberg scale), which can theoretically reduce the hydrophobicity of the derived peptide; in addition, the thiol group of Cys is methylated to lose the hydrogen bond association ability, which can theoretically convert the β-pleated structure of LBD to random coil.

[0033] Through prediction of the designed derived peptide, one derived peptide with better prediction results was screened out, which was LBDQ in Table 1.

[0034] Table 1 is a table of derived peptide sequences and their physicochemical properties, wherein the binding energy is the binding energy of the antibacterial peptide and the lipid A molecule predicted by docking simulation.

[0035] Table 1 is a table of derived peptide sequences and their physicochemical properties, wherein the binding energy is the binding energy of the antibacterial peptide and the lipid A molecule predicted by docking simulation.

[0036]

[0037] The above polypeptide synthesis was entrusted to Shanghai Gil Biochemical Co., Ltd., and was confirmed by mass spectrometry and high performance liquid chromatography.

[0038] The helical wheel simulation diagram of LBD and derived peptides is shown in Figure 1 , wherein A-H are the simulation results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ, respectively. The yellow label is a basic amino acid, the blue label is a polar hydrophobic amino acid, and the green label is a non-polar hydrophobic amino acid. Glycine (G), serine (T) and glutamine (Q) do not belong to the above categories.

[0039] The effect diagram of molecular docking of derived peptide LBD3Y and lipid A of LPS is shown in Figure 2 .

[0040] Example 2: Circular dichroism (CD) determination

[0041] At room temperature, the characteristic spectra of the above eight antimicrobial peptides were determined using circular dichroism spectroscopy in sterile water, 60 mM SDS solution (simulating a negatively charged bacterial cell membrane), and 50% trifluoroethanol (TFE, simulating the hydrophobic environment of the bacterial cell membrane). The final peptide concentration was 0.2 mg / mL. A 5 mm thick quartz sample cell was used, with scanning wavelengths from 190 to 260 nm. Each peptide was measured in triplicate, and the average value was taken. The results were subtracted from the solvent control. The results are as follows: Figure 3 As shown, Figure 3 The image shows circular dichroism chromatograms of LBD and its derivative peptides, where A and H represent the circular dichroism detection results of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY, and LBDQ, respectively.

[0042] Depend on Figure 3 As observed, in aqueous solution, all peptides exhibit a distinct negative peak near 200 nm, a characteristic peak of random coil structures. Under SDS and TFE conditions, the secondary structures of the peptides underwent significant changes. Specifically, the negative peaks of LBDQ appeared near 209 nm and 222 nm, indicating a tendency for LBDQ to form α-helical structures. The remaining derived peptides showed positive bands in the 190–200 nm range, indicating a tendency for seven derived peptides to form β-sheet structures. The results for LBDW, LBDY, and LBDV differed from the secondary structures predicted by Alphafold2. Preliminary inferences suggest that Alphafold2's predictions were based on existing structures in its database, resulting in data errors, or that errors occurred during chemical synthesis.

[0043] Example 3: Determination of the minimum inhibitory concentration (MIC) of antimicrobial peptides against Vibrio parahaemolyticus

[0044] 1. Strawberry Culture: Using an inoculation loop, streak Vibrio parahaemolyticus (Vp ATCC17802) frozen at -80℃ onto a Mueller-Hinton Broth (MHB) agar plate. Incubate upside down in a 28℃ incubator. Once single colonies have grown, pick a single colony and transfer it to 5 mL of fresh MHB liquid medium. Incubate at 28℃ / 150 rpm with a shaking incubator until the logarithmic growth phase. Then, dilute the bacterial culture to a concentration of 2 × 10⁻⁶ using fresh MHB liquid medium. 5 CFU / mL available for use.

[0045] 2. Antimicrobial peptide solution pretreatment: The above 8 antimicrobial peptide solutions (2 mg / mL) of LBD, LBD3W, LBD3V, LBD3Y, LBDW, LBDV, LBDY and LBDQ were placed in a PCR instrument and reacted at 20℃ for 30 min. After that, they were quickly removed and placed on ice to cool for 10 min, and then placed at room temperature to equilibrate.

[0046] 3. 2-fold dilution method for determining MIC: 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 the pretreated peptide sample solution (2 mg / mL) to each well in the first row, mix well by blowing, and then transfer 50 μL from the first row of wells to the corresponding second row of wells, and repeat the operation in turn. The last well is discarded after 50 μL is taken. Three parallel samples are set for each group. Then add 50 μL of the pre-diluted bacterial solution to each well, so that the concentration of the bacterial solution in each well is 1×10 5 CFU / mL, and the final volume of the liquid in each well is 100 μL. The final concentration of the peptide sample is 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, respectively. An equal volume of bacterial suspension is used as a negative control, and the original peptide LBD is used as a positive control. Place the 96-well plate in a 28°C incubator for 10 h, and use a microplate reader to detect the ultraviolet absorption value at 600 nm for each well. The average value of the concentration of the polypeptide sample in the wells where no bacterial growth was detected and the wells adjacent to them is taken as the MIC. The results are shown in Table 2.

[0047] Table 2 Minimum inhibitory concentration of LBD and derived peptides against Vibrio parahaemolyticus

[0048]

[0049] As can be seen from Table 2, the minimum inhibitory concentration of the parent peptide LBD against Vibrio parahaemolyticus is 100 μg / mL. The antibacterial activity of LBD3W, LBD3V, and LBD3Y designed from the template is increased by about 90.625%, 62.5%, and 81.25%, respectively, compared with the parent peptide LBD. The antibacterial activity of LBDW, LBDV, and LBDY with altered spatial structure is increased by about 90.625%. The antibacterial activity of LBDQ without disulfide bond support for its spatial structure is increased by about 95.31%. The results show that the increase in amphiphilicity is beneficial to improving the antibacterial activity of the antibacterial peptide, and disulfide bond is not necessary for the LBD antibacterial peptide to exert activity.

[0050] Example 4: Determination of the minimum inhibitory concentration MIC of the antibacterial peptide against Vibrio harveyi

[0051] 1. Strain culture: inoculate loop -80℃ frozen V. harveyi (V. h TS275249) on Mueller-Hinton Broth (MHB) solid plate, inverted culture in 30℃ constant temperature incubator, after single colony grows, pick single colony in 5 mL fresh MHB liquid medium, 30℃ / 150 rpm shaking incubator culture to logarithmic growth phase, then use fresh MHB liquid medium to dilute the concentration of bacterial solution to 2×10 5 CFU / mL standby.

[0052] 2. Antimicrobial peptide solution pretreatment: place the above 8 kinds of antimicrobial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V and LBD3Y in the PCR instrument, after 20℃ reaction for 30 min, take out quickly and place on ice for 10 min, then place at room temperature for balance.

[0053] 3. MIC determination by 2-fold dilution method: select sterile round-bottom 96-well plates, add 90 μL sterile MH liquid medium in the first row, and add 50 μL sterile liquid medium in each row. Then add 10 μL of pretreated peptide sample solution (2 mg / mL) to each well in the first row, mix well by blowing, and then take 50 μL from the first row of wells and add to the corresponding second row of wells, repeat the operation in turn, and discard the last well. Each group sets 3 parallels. Then add 50 μL of pre-diluted bacterial solution to each well, so that the concentration of bacterial solution in each well is 1×10 5 CFU / mL, and the final volume of each well is 100 μL, and the final concentration of the peptide sample is 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, 0.390625 μg / mL, respectively. Use equal volume of bacterial suspension as negative control, and use original peptide LBD as positive control. Place the 96-well plate in a 30℃ incubator for 14 h, use a microplate reader to detect the ultraviolet absorption value of each well at 600 nm, take the average value of the concentration of the polypeptide sample of the wells with no bacterial growth and the wells with bacterial growth adjacent to them as the MIC, and the results are shown in Table 3.

[0054] Table 3 Minimum inhibitory concentration of LBD and derived peptides against V. harveyi

[0055]

[0056] As shown in Table 3, the minimum inhibitory concentration of the parent peptide LBD against Vibrio harveyi was 200 μg / mL. The antibacterial activity of LBD3W, LBD3V and LBD3Y designed from the template was increased by about 87.5% compared with that of the parent peptide LBD. The antibacterial activity of LBDW, LBDV and LBDY with changed spatial structure was increased by about 93.75%, 96.875% and 87.5%, respectively. The antibacterial activity of LBDQ without disulfide bond support for its spatial structure was increased by about 93.75%. The results showed that the increase of amphiphilicity was conducive to improving the antibacterial activity of the antibacterial peptide, and disulfide bond was not necessary for the LBD antibacterial peptide to exert activity.

[0057] Example 5: Determination of the minimum inhibitory concentration MIC of the antibacterial peptide against Vibrio alginolyticus

[0058] 1. Strain culture: inoculate the Vibrio alginolyticus (V. a) stored at -80°C into Mueller-Hinton Broth (MHB) solid plates, and then incubate in an inverted incubator at 28°C. After single colonies grow, pick single colonies into 5 mL of fresh MHB liquid medium, and then incubate in a 28°C / 150 rpm shaking incubator until the logarithmic growth phase. Then dilute the bacterial liquid to 2×10 5 CFU / mL with fresh MHB liquid medium.

[0059] 2. Pretreatment of antibacterial peptide solution: place the above-mentioned eight antibacterial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V and LBD3Y in a PCR instrument, and then react at 20°C for 30 min. After that, quickly take them out and place on ice for 10 min, and then place at room temperature for equilibration.

[0060] 3. Determination of MIC by 2-fold dilution method: select a sterile round-bottom 96-well plate, and then add 90 μL of sterile MH liquid medium into the first row, and add 50 μL of sterile liquid medium into each row. Then add 10 μL of the pretreated peptide sample solution (2 mg / mL) into each well of the first row, mix well by blowing, and then take 50 μL from each well of the first row and add into the corresponding wells of the second row, and then repeat the operation in turn. Finally, discard 50 μL from the last well, and set three parallels for each group. Then add 50 μL of the pre-diluted bacterial liquid into each well, so that the concentration of the bacterial liquid in each well is 1×10 5CFU / mL, the final liquid volume of each well was 100 μL, and the final concentration of the peptide sample was 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, 0.390625 μg / mL from high to low. 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 placed in a 28°C incubator for 10 h, and the ultraviolet absorbance value at 600 nm of each well was detected using an enzyme-labeled instrument. The average value of the polypeptide sample concentration of the wells in which no bacterial growth was detected and the wells adjacent to them was taken as the MIC, and the results are shown in Table 4.

[0061] Table 4 Minimum inhibitory concentration of LBD and derived peptides against Vibrio alginolyticus

[0062]

[0063] As can be seen from Table 4, the minimum inhibitory concentration of the parent peptide LBD against Vibrio alginolyticus is 200 μg / mL. The antibacterial activity of LBD3W, LBD3V and LBD3Y designed from the template is increased by about 98.438%, 87.5% and 87.5% respectively compared with the parent peptide LBD; the antibacterial activity of LBDW, LBDV and LBDY with changed spatial structure is increased by about 98.438%, 93.75% and 96.875% respectively; the antibacterial activity of LBDQ without disulfide bond support for its spatial structure is increased by about 96.875%. The results show that the increase of amphiphilicity is conducive to improving the antibacterial activity of the antibacterial peptide, and disulfide bond is not necessary for the LBD antibacterial peptide to exert activity.

[0064] Example 6: Determination of minimum inhibitory concentration MIC of antibacterial peptide against V. anguillarum

[0065] 1. Strain culture: inoculate the V. anguillarum (M-TS TS340431) stored at -80°C with a loop on Mueller-Hinton Broth (MHB) solid plate, and invert culture in a 28°C constant temperature incubator. After single colonies grow, pick single colonies in 5 mL fresh MHB liquid medium, and culture in a 28°C / 150 rpm shaking incubator to the logarithmic growth phase. Then dilute the bacterial liquid to 2×10 5 CFU / mL for standby.

[0066] 2. Pretreatment of antimicrobial peptide solution: The above-mentioned eight antimicrobial peptide solutions (4 mg / mL) of LBD, LBD3W, LBDW, LBDV, LBDY, LBDQ, LBD3V and LBD3Y were placed in a PCR instrument, and after 30 min of reaction at 20°C, they were quickly taken out and placed on ice for 10 min, and then placed at room temperature for equilibration.

[0067] 3. Determination of MIC by 2-fold dilution method: A sterile round-bottom 96-well plate was selected, 90 μL of sterile MH liquid medium was added to the first row, and 50 μL of sterile liquid medium was added to each of the remaining rows. Then 10 μL of the pretreated peptide sample solution (2 mg / mL) was added to each well of the first row, which was mixed by blowing and then 50 μL was taken from the first row of wells and added to the corresponding second row of wells, and the operation was repeated in turn, and the last well was discarded after taking 50 μL, and three parallel groups were set. Then 50 μL of the pre-diluted bacterial solution was added to each well, so that the concentration of the bacterial solution in each well was 1×10 5 CFU / mL, and the final volume of the liquid in each well was 100 μL, and the final concentration of the peptide sample was 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, respectively. 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 placed in a 28°C incubator for 14 h, and the ultraviolet absorbance value at 600 nm of each well was detected using an enzyme-labeled instrument, and the average value of the concentration of the polypeptide sample of the well in which no bacterial growth was detected and the well adjacent to it with bacterial growth was taken as the MIC, and the results are shown in Table 5.

[0068] Table 5 Minimum inhibitory concentration of LBD and derived peptides against Vibrio anguillarum

[0069]

[0070] As can be seen from Table 5, the parent peptide LBD has good basic antimicrobial activity against Vibrio anguillarum, and the minimum inhibitory concentration of the parent peptide LBD against Vibrio anguillarum is 25 μg / mL, and the antimicrobial activity of LBD3V and LBD3Y obtained by template design is increased by 75% and 50% respectively compared with the parent peptide LBD; the antimicrobial activity of LBDW and LBDV with changed spatial structure is increased by 50%; the antimicrobial activity of LBDQ without disulfide bond support for its spatial structure is increased by 75%.

[0071] Example 7: Determination of minimum bactericidal concentration MBC of antimicrobial peptides against Vibrio parahaemolyticus

[0072] From each well of the MIC test in Example 3 (≥ MIC), 20 μL of the liquid was spread on MHB solid medium and incubated in an appropriate incubator for 10-12 h, and the growth of bacteria was observed. The minimum concentration corresponding to the plate without colony growth was considered as the MBC of the peptide solution, and the results are shown in Table 6.

[0073] Table 6 Minimum bactericidal concentration of LBD and derived peptides against Vibrio parahaemolyticus

[0074]

[0075] “-” in the table indicates not detected.

[0076] The minimum bactericidal concentration MBC refers to the lowest concentration of an antibacterial substance that can kill a certain proportion (usually 99.9%) of bacteria in the cultured test strain. As can be seen from the comparison of the results in Table 2 and Table 6, LBD3W, LBD3Y, LBDW, LBDY and LBDQ can effectively kill 99.9% of bacteria at 1xMIC, and LBD3V and LBDV have a 99.9% killing effect on Vibrio parahaemolyticus at 2xMIC.

[0077] Example 8: Cytotoxicity test

[0078] 1. Culture and subculture of Caco-2 cells: The Caco-2 cells stored at -80°C were quickly thawed in a 37°C water bath, and gently shaken until the ice crystals completely melted. In a sterile fume hood, the melted cell suspension was slowly added to a pre-prepared centrifuge tube containing an appropriate amount of complete medium, and gently mixed, centrifuged at 1000 rpm for 5 min to discard the supernatant, the cell pellet was resuspended with fresh medium, transferred to a cell culture flask, added with sufficient medium to cover the cell surface, and incubated in a 37°C, 5% CO2 incubator for 48 h. Then the old medium was discarded, the cells were washed with PBS three times to remove residual medium, 1 mL trypsin solution was added for digestion, and incubated in the incubator for 5 min. The digestion reaction was terminated by adding DMEM complete medium (containing 20% fetal bovine serum, 1% streptomycin-penicillin and 79% DMEM basal medium), centrifuged at 1000 rpm for 5 min to discard the supernatant, and resuspended with 2 mL of fresh medium. After uniform blowing, it was transferred to a new culture flask for incubation for 24 h. When the cells were subcultured to the 3rd generation, the cells were washed with PBS and centrifuged to discard the supernatant, and the cell concentration was diluted to 1×10 5 6 / mL with fresh DMEM complete medium, and incubated in a 96-well cell culture plate for 24 h, 200 μL per well, and 5 replicates per group.

[0079] 2. Peptide samples were diluted with DMEM complete medium to six concentration gradients: 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, 20 μg / mL, and 1 μg / mL. The cell culture medium from the previous 24-hour incubation was discarded. Peptide samples of different concentration gradients were added sequentially, 200 μL per well, with five replicates per group. An equal volume of DMEM complete medium was added to the blank control. Cells were co-cultured for 24 hours. The peptide solution was then discarded, and 150 μL of MTT (0.5 mg / mL) solution was added to each well. Staining was performed in an incubator for 4 hours. DMSO was then added to dissolve and crystallize the cells. Cell viability was measured using a microplate reader at 570 nm UV absorption wavelength. The results are shown below. Figure 4 As shown.

[0080] Depend on Figure 4 As observed, Caco-2 cells maintained good cell viability after treatment with antimicrobial peptides ranging from 1 to 100 μg / mL for 24 h. The cell viability was relatively high (greater than 90%) at concentrations of 1–80 μg / mL, indicating that the antimicrobial peptides promoted cell growth. When the peptide concentration increased to 100 μg / mL, the cell viability of LBDW, LBDV, and LBDQ decreased to below 90%, indicating that these three peptides had varying degrees of toxicity to cell growth under high concentrations. This may be because tryptophan W and valine V are amino acids with high hydrophobicity, and increased hydrophobicity leads to increased cytotoxicity of the antimicrobial peptides; while LBDQ, lacking disulfide bonds to support its structure, transformed into an α-helix structure, increasing its cytotoxic effect.

[0081] 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 within the protection scope of the present invention.

Claims

1. An 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-SEQ ID NO:

3.

2. A method for molecularly designing and constructing an ALFPm3-modified antimicrobial peptide as described in claim 1, characterized in that, The steps include: molecular design of the LBD region, the antimicrobial activity region of the natural antimicrobial peptide ALFPm3; Specifically, the steps include: replacing the amino acid in the LBD region of the natural antimicrobial peptide ALFPm3 from an amphiphilic perspective; Following the template Ac-C-(BH)n-KR-(HB)nC-NH2, where B is a basic amino acid and H is a hydrophobic amino acid, threonine (T), tyrosine (Y), glutamine (Q), and tryptophan (W) in the sequence were replaced with the basic amino acids lysine (K) and arginine (R). Based on predictions of the physicochemical properties, amphiphilicity, and LPS binding sites of the designed derived peptides, three derived peptides with good prediction results were comprehensively screened: LBD3W, LBD3V, and LBD3Y. The amino acid sequence of LBD3W is SEQ ID NO.1, the amino acid sequence of LBD3V is SEQ ID NO.2, and the amino acid sequence of LBD3Y is SEQ ID NO.

3.

3. The application of the ALFPm3 modified antimicrobial peptide as described in claim 1 in the preparation of antibacterial infection drugs, characterized in that, The amino acid sequence of the ALFPm3 modified antimicrobial peptide is selected from any one of SEQ ID NO:1-SEQ ID NO:3, and the bacteria are Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio alginolyticus.

4. The application according to claim 3, characterized in that, The amino acid sequence of the ALFPm3 modified antimicrobial peptide is selected from SEQ ID NO:2 or SEQ ID NO:3, and the bacteria are Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio anguillarum.

5. A nucleic acid molecule, characterized in that, The ALFPm3 modified antimicrobial peptide as described in claim 1 is encoded.

6. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the ALFPm3 modified antimicrobial peptide of claim 1.

Images