Antibacterial peptide based on miraehthes oxycephalus cxcl8 protein and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to antibacterial peptides based on Pseudosciaena crocea CXCL8 protein and application thereof. The application takes Pseudosciaena crocea CXCL8 protein as a template, intercepts a linear polypeptide of 13 amino acids, and obtains an antibacterial peptide AVV13N through carboxyl terminal amidation modification. The amino acid sequence of the antibacterial peptide AVV13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2. The 11th lysine and the 13th valine in the amino acid sequence of the antibacterial peptide AVV13N are exchanged to obtain an antibacterial peptide AVK13N. The amino acid sequence of the antibacterial peptide AVK13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2. The molecular weight of the antibacterial peptides AVV13N and AVK13N is 1669.12 Da, and the isoelectric points thereof are both 12.44. The antibacterial peptides AVV13N and AVK13N are both basic polypeptides. The antibacterial peptides AVV13N and AVK13N have broad-spectrum bactericidal activity and can specifically kill gram-positive bacteria and gram-negative bacteria. In addition, the antibacterial peptides AVV13N and AVK13N both have weak hemolytic activity, low cytotoxicity, and good temperature stability, pH stability and salt ion stability. The antibacterial peptides AVV13N and AVK13N provided by the application can replace antibiotics and be applied to the prevention and treatment of bacterial diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antimicrobial peptide based on large yellow croaker CXCL8 protein and applications thereof. Background Art

[0002] In recent years, the scale of large yellow croaker cage aquaculture in China's southeastern coastal areas has continued to expand, especially in Fujian and Zhejiang, where it has developed into a large-scale pillar industry of aquaculture. However, with seasonal climate changes, temperature fluctuations, and increased aquaculture density, aquaculture diseases have become increasingly prominent. Among them, visceral white spot disease, a serious disease affecting large yellow croaker cage aquaculture, has attracted widespread attention. It is now clear that the disease is caused by Pseudomonas aeruginosa ( Pseudomonas plecoglossicida Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium that causes diseases in a variety of fish, including large yellow croaker, grouper, and rainbow trout. When infected, fish develop distinct white nodules in their spleen, kidneys, and liver, leading to the development of a disease called visceral white spot disease (ICD). This disease causes severe economic losses to the large yellow croaker aquaculture industry, significantly hindering its healthy development. Therefore, finding effective prevention and control measures is crucial.

[0003] Antimicrobial peptides are a class of small polypeptides with antibacterial activity that play a crucial role in protecting organisms from infection by pathogenic microorganisms. Antimicrobial peptides primarily exert their bactericidal effects through two pathways: membrane-targeting and non-membrane-targeting: membrane-permeation and non-membrane-structural mechanisms. In the membrane-targeting mechanism, positively charged antimicrobial peptides preferentially interact with negatively charged components of the bacterial cell membrane. Their hydrophobic moieties then embed into the membrane, triggering the remodeling of the membrane structure and forming transmembrane channels. This disrupts the intracellular ion balance, leading to nutrient loss and ultimately causing bacterial cell rupture and loss of activity. In the non-membrane-targeting mechanism, while antimicrobial peptides also interact with the bacterial cell membrane, their primary mechanism is not to disrupt membrane integrity. Antimicrobial peptides can penetrate the bacterial cell membrane and enter the cell interior, where they then bind to various target molecules. When accumulated to a certain concentration within the bacterial cell, antimicrobial peptides effectively inhibit core bacterial processes such as DNA replication, RNA synthesis, protein translation, and cell wall synthesis, thereby killing the bacteria.

[0004] While antimicrobial peptides have attracted considerable attention due to their broad-spectrum bactericidal activity and promising applications, naturally occurring antimicrobial peptides generally suffer from numerous issues, including weak bactericidal activity, high cytotoxicity, and poor stability. These issues significantly restrict their further application in aquaculture. Therefore, designing and modifying antimicrobial peptides to achieve high bactericidal activity by adding, deleting, or replacing amino acid residues, based on the structure-function relationship, is one of the most effective strategies to overcome the current challenges in their application. Summary of the Invention

[0005] In order to obtain antimicrobial peptides with broad-spectrum antimicrobial activity and good biosafety, the present invention synthesized two antimicrobial peptides AVV13N and AVK13N based on large yellow croaker CXCL8, and clarified their applications.

[0006] The purpose of the present invention is achieved through the following technologies:

[0007] The present invention first provides two antimicrobial peptides, AVV13N and AVK13N. The amino acid sequence of the antimicrobial peptide AVV13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2, and the amino acid sequence of the antimicrobial peptide AVK13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2.

[0008] The present invention further provides a method for preparing the antimicrobial peptides AVV13N and AVK13N, which is as follows:

[0009] (1) Using the large yellow croaker CXCL8 protein sequence as a template, a 13-amino acid linear peptide was extracted and modified by amidation at the carboxyl terminus to obtain the antimicrobial peptide AVV13N. The amino acid sequence of the antimicrobial peptide AVV13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2;

[0010] (2) The lysine at position 11 and the valine at position 13 in the amino acid sequence of the antimicrobial peptide AVV13N were exchanged to obtain the antimicrobial peptide AVK13N. The amino acid sequence of the antimicrobial peptide AVK13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2;

[0011] (3) The complete sequences of antimicrobial peptides AVV13N and AVK13N were synthesized by solid-phase chemical synthesis.

[0012] The present invention also provides the use of the two antimicrobial peptides AVV13N and AVK13N in the preparation of antibacterial drugs, wherein the antibacterial drugs have broad-spectrum antibacterial activity.

[0013] The antimicrobial peptides AVV13N and AVK13N of the present invention have the following advantages and beneficial effects:

[0014] The antimicrobial peptides AVV13N and AVK13N prepared by the present invention have broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria. Furthermore, the antimicrobial peptides AVV13N and AVK13N have weak hemolytic activity, low cytotoxicity, and good temperature, pH, and salt ion stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the mass spectrum of the antimicrobial peptide AVV13N. Figure 2 This is the mass spectrum of the antimicrobial peptide AVK13N.

[0016] Figure 3 The growth curves of Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028, Salmonella enterica subsp. Enteritidis ATCC 9120, Pseudomonas aeruginosa POLYC4) after treatment with antimicrobial peptides AVV13N and AVK13N are shown in Figure 2. Figure 4 Figure 2 shows the growth curves of Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus ATCC 43300, Streptococcus agalactiae ATCC 13813, Bacillus subtilis CMCC 63501, and Micrococcus luteus CMCC 28001) after treatment with antimicrobial peptides AVV13N and AVK13N.

[0017] Figure 5 This is a graph showing the hemolytic activity of antimicrobial peptides AVV13N and AVK13N on erythrocytes.

[0018] Figure 6 The graph shows the cytotoxicity of antimicrobial peptides AVV13N and AVK13N to LYC-FM cells.

[0019] Figure 7 This is a diagram showing the effect of temperature on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N.

[0020] Figure 8 This is a diagram showing the effect of pH on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N.

[0021] Figure 9 This is a diagram showing the effect of salt ions on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to specific embodiments, but the protection content of the present invention is not limited to the following embodiments.

[0023] Example 1:

[0024] The design and synthesis of antimicrobial peptides AVV13N and AVK13N were carried out as follows:

[0025] (1) Using the large yellow croaker CXCL8 protein sequence as a template, a 13-amino acid linear peptide was extracted and modified at the carboxyl end to obtain the antimicrobial peptide AVV13N. The lysine at position 11 and the valine at position 13 in the amino acid sequence of the antimicrobial peptide AVV13N were exchanged to obtain the antimicrobial peptide AVK13N.

[0026] The amino acid sequence of the antimicrobial peptide AVV13N is:

[0027] Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2.

[0028] The amino acid sequence of the antimicrobial peptide AVK13N is:

[0029] Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2.

[0030] The mass spectra of antimicrobial peptides AVV13N and AVK13N are shown in Figures 1 and 2 .

[0031] (2) The antimicrobial peptides AVV13N and AVK13N were synthesized by Shanghai Sangon Biotechnology Co., Ltd. using solid-phase chemical synthesis with a purity of over 95% and were used in subsequent experiments.

[0032] Example 2:

[0033] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptides AVV13N and AVK13N were determined as follows:

[0034] (1) Take out the strain to be tested from the -80℃ freezer, streak inoculate on solid culture medium, and culture in an incubator overnight;

[0035] (2) Pick a single clone and culture it in liquid culture medium overnight in an incubator;

[0036] (3) Inoculate the overnight culture into a new liquid medium and culture until the logarithmic growth phase. Adjust the concentration of the culture to 1.2×10 5 CFU / mL;

[0037] (4) Serial dilution of the antimicrobial peptide to 1280, 640, 320, 160, 80, 40, 20, 10, and 5 μM: 40 μL of the antimicrobial peptide with a starting concentration of 1280 μM was added to 40 μL of 1× PBS (pH = 7.4), pipetted to mix, and then 40 μL of the diluted antimicrobial peptide was taken and serially diluted to 320, 160, 80, 40, 20, 10, and 5 μM in sequence;

[0038] (5) Add 10 µL of the diluted antimicrobial peptide solution to columns 1-9 of a 96-well plate, and then add 90 µL of bacterial solution to each well in turn. Columns 10 and 11 are used as negative and positive controls, respectively. 90 µL of culture medium and 10 µL of 1× PBS (pH = 7.4) are added to the negative control, and 90 µL of bacterial solution and 10 µL of 1× PBS (pH = 7.4) are added to the positive control. Three replicates are set up for each well. The above process must be completed within 15 min.

[0039] (6) Place the 96-well plate in an incubator and culture for 12-18 hours. Take it out and mix it by blowing with a sterilized pipette tip. Measure the OD value with a microplate reader. 600 The values ​​were calculated with the concentration of antimicrobial peptides as the horizontal axis and OD as the 600 The value is plotted as the vertical axis, and the concentration at the inflection point is the minimum inhibitory concentration;

[0040] (7) Take 30 μL of the incubation mixture from each of the three parallel groups and spread it on solid culture medium. Incubate the mixture overnight in an incubator. The minimum antimicrobial peptide concentration that kills at least 99.9% of the bacteria is defined as the MBC.

[0041] Test results such as Figures 3 and 4 As shown, the antimicrobial peptides AVV13N and AVK13N have bactericidal activity against both Gram-negative and Gram-positive bacteria tested, indicating that the antimicrobial peptides AVV13N and AVK13N have broad-spectrum bactericidal activity.

[0042] Example 3:

[0043] The hemolytic activity of antimicrobial peptides AVV13N and AVK13N was determined by the following steps:

[0044] (1) Take 2 mL of whole blood from yellow croaker and immediately add anticoagulant to prevent blood coagulation, 500× g Centrifuge for 10 minutes and discard the supernatant;

[0045] (2) Resuspend the red blood cells in 1 mL of 1× PBS (pH = 7.4), 500× gCentrifuge for 10 min, discard the supernatant, and then wash three times with 1× PBS (pH = 7.4);

[0046] (3) Resuspend the red blood cells in 1×PBS (pH=7.4), count the cells after gradient dilution, and adjust the red blood cell concentration to 1×10 8 cells / mL;

[0047] (4) Serial dilution of the antimicrobial peptide to 1280, 640, 320, 160, 80, 40, 20, and 10 μM;

[0048] (5) 20 μL of the diluted antimicrobial peptide solution was added to columns 1-8 of a 96-well plate, and then 180 μL of red blood cell suspension was added to each well in turn. Columns 9 and 10 were used as negative and positive controls, respectively, as controls for 0% and 100% hemolysis. 180 μL of culture medium and 20 μL of 1× PBS (pH = 7.4) were added to the negative control, and 180 μL of cell suspension and 20 μL of 20% Triton X-100 were added to the positive control. Three replicates were set up for each well.

[0049] (6) Incubate the 96-well plate in a 28°C incubator for 2 h at 500 g After centrifugation for 10 min, 100 μL of supernatant was transferred to a new 96-well plate and the absorbance at 405 nm was read;

[0050] (7) Calculate the hemolytic activity using the following formula:

[0051] Hemolytic activity = [(Apeptide-A0% lysis) / (A100% lysis-A0% lysis)] × 100%,

[0052] Where A is the absorbance at 405 nm.

[0053] Test results such as Figure 5 As shown in the data, the hemolytic rates of the antimicrobial peptides AVV13N and AVK13N at different concentrations on the red blood cells of large yellow croaker were all lower than 1%, indicating that the antimicrobial peptides AVV13N and AVK13N had low hemolytic activity.

[0054] Example 4:

[0055] Cytotoxicity assay of antimicrobial peptides AVV13N and AVK13N, the specific steps are as follows:

[0056] (1) Adjust the number of large yellow croaker macrophages (LYC-FM) to 2×10 5 cells / mL, 100 μL of cell suspension was added to a 96-well plate and cultured overnight in a 28°C incubator;

[0057] (2) Antimicrobial peptide dilution: Serial dilution of antimicrobial peptides to 1280, 640, 320, 160, 80, 40, and 20 μM;

[0058] (3) Remove the culture medium and add 90 µL of fresh culture medium and various concentrations of antimicrobial peptides to each well (the final concentrations of antimicrobial peptides were 2, 4, 8, 16, 32, 64, and 128 µM, respectively). Add 90 µL of fresh culture medium and 10 µL of 1× PBS (pH = 7.4) to the negative control (0% cytotoxicity group), and add 90 µL of fresh culture medium and 10 µL of 20% Triton X-100 to the positive control (100% cytotoxicity group). Set up three replicates for each group.

[0059] (4) After 24 h of static incubation at 28°C, add 10 µL of CCK-8 solution to each well, mix gently, and continue incubation at 28°C for 4 h. Read the absorbance at 450 nm.

[0060] (5) Calculate the viable cell ratio using the following formula:

[0061] Live cell ratio = [(Apeptide-A100% lysis) / (A0% lysis-A100% lysis)] × 100%,

[0062] Where A is the absorbance at 450 nm.

[0063] Test results such as Figure 6 As shown in the figure, the cell viability of large yellow croaker macrophage LYC-FM cells was higher than 90% after treatment with different concentrations of antimicrobial peptides AVV13N and AVK13N, indicating that the antimicrobial peptides AVV13N and AVK13N have low cytotoxicity.

[0064] Example 5:

[0065] The temperature stability test of antimicrobial peptides AVV13N and AVK13N is as follows:

[0066] (1) Add 40 μL of antimicrobial peptide (final concentration of 1×MBC) to a 1.5 mL EP tube and treat it at different temperatures for 30 min. The temperature was set to 5 gradients, including 20°C, 40°C, 60°C, 80°C, and 100°C. Place it on ice immediately after treatment.

[0067] (2) Culture the Pseudomonas aeruginosa to the logarithmic growth phase, measure the OD value and adjust the bacterial solution concentration to 1×10 5 CFU / mL;

[0068] (3) 90 μL of bacterial solution and 10 μL of antimicrobial peptides treated at different temperatures were added to a 96-well plate in sequence. 10 μL of deionized water was added to the positive control. Three replicates were set for each group.

[0069] (4) Place the 96-well plate in a 28°C incubator overnight, perform gradient dilutions in each well, and then apply the diluted solution to TSA solid medium. Count the number of colonies after 14-16 hours.

[0070] (5) Calculate the bacterial killing rate according to the following formula:

[0071] Bacterial sterilization rate = 1-(CFU of each temperature treatment group / CFU of positive control) × 100%

[0072] (6) Plot the temperature on the X-axis and the bacterial killing rate on the Y-axis.

[0073] Test results such as Figure 7 As shown in the figure, after treatment at different temperatures, the bactericidal rates of antimicrobial peptides AVV13N and AVK13N against Pseudomonas aeruginosa can still reach above 99.99%, indicating that the antimicrobial peptides AVV13N and AVK13N have good temperature stability.

[0074] Example 6:

[0075] The pH stability test of antimicrobial peptides AVV13N and AVK13N was carried out as follows:

[0076] (1) Prepare the following buffer solutions of different pH values ​​and filter sterilize them:

[0077] 50 mM glycine-HCl (pH = 2.0);

[0078] 50 mM NaAC-HAC (pH = 4.0);

[0079] 50 mM MES [2-(N-morpholino)ethanesulfonic acid]-NaOH (pH = 6.0);

[0080] 50 mM Tris-HCl (pH = 8.0);

[0081] 50 mM glycine-NaOH (pH=10.0).

[0082] (2) Take 30 µL of the above buffer solutions of different pH values ​​and add them to 1.5 mL EP tubes. Add 10 µL of antimicrobial peptide to each tube to a final concentration of 4 times the MBC. Mix well and place at room temperature for 4 h.

[0083] (3) Inoculate Pseudomonas aeruginosa into TSB medium and culture with shaking until the logarithmic growth phase. Adjust the bacterial solution concentration to 1×10 5 CFU / mL;

[0084] (4) Add 90 μL of bacterial solution to a 96-well plate, add 10 μL of treated antimicrobial peptide solution to each well, and add 10 μL of deionized water to the positive control. Set up 3 replicates for each group.

[0085] (5) Place the 96-well plate in a 28°C incubator overnight, perform gradient dilutions in each well, and then apply the diluted solution to TSA solid medium. Count the number of colonies after 14–16 hours.

[0086] (6) Calculate the bacterial killing rate according to the following formula:

[0087] Bacterial sterilization rate = 1-(CFU of each pH treatment group / CFU of positive control) × 100%

[0088] (7) Draw a line graph with pH as the X-axis and bacterial killing rate as the Y-axis.

[0089] Test results such as Figure 8 As shown in the results, the bactericidal rates of antimicrobial peptides AVV13N and AVK13N against Pseudomonas aeruginosa can still reach above 99.99% under different pH values, indicating that the antimicrobial peptides AVV13N and AVK13N have good pH stability.

[0090] Example 7:

[0091] The salt ion stability test of antimicrobial peptides AVV13N and AVK13N is as follows:

[0092] (1) Prepare the following buffer solutions with different salt ions, adjust the pH to 7.4, and filter sterilize:

[0093] 1500mM NaCl

[0094] 45 mM KCl

[0095] 60µM NH4Cl

[0096] 80µM ZnCl2

[0097] 10mM MgCl2

[0098] 20mM CaCl2

[0099] 40µM FeCl3

[0100] (2) Culture the Pseudomonas aeruginosa to the logarithmic growth phase, measure the OD value and adjust the bacterial solution concentration to 1×10 5 CFU / mL;

[0101] (3) 80 µL of bacterial solution, 10 µL of different salt ion solutions, and 10 µL of antimicrobial peptide were sequentially added to a 96-well plate. The final concentration of the antimicrobial peptide was 1× MBC. 10 µL of different salt ion solutions and 10 µL of 1× PBS (pH = 7.4) were added to the positive control. Three replicates were set for each group.

[0102] (4) Place the 96-well plate in a 28°C incubator overnight, perform gradient dilutions in each well, and then apply the diluted solution to TSA solid medium. Count the number of colonies after 14-16 hours.

[0103] (5) Calculate the bacterial killing rate according to the following formula:

[0104] Bacterial sterilization rate = 1-(CFU of each salt ion solution treatment group / CFU of positive control) × 100%

[0105] (6) Draw a line graph with different salt ion solutions as the X-axis and the bacterial killing rate as the Y-axis.

[0106] Test results such as Figure 9 As shown in the data, except for CaCl2, the bactericidal rates of antimicrobial peptides AVV13N and AVK13N against Pseudomonas aeruginosa can still reach more than 99.9% after treatment with other physiological salts such as NaCl, KCl, NH4Cl, ZnCl2, MgCl2, and FeCl3, indicating that the antimicrobial peptides AVV13N and AVK13N have good salt ion stability.

[0107] In summary, the antimicrobial peptides AVV13N and AVK13N of the present invention exhibit broad-spectrum antimicrobial activity, with strong antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, both antimicrobial peptides AVV13N and AVK13N exhibit weak hemolytic activity and low cytotoxicity, as well as good temperature, pH, and salt ion stability. Therefore, the antimicrobial peptides AVV13N and AVK13N of the present invention possess significant advantages in the preparation of therapeutic agents for bacterial infections.

[0108] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An antimicrobial peptide AVV13N based on the large yellow croaker CXCL8 protein, characterized by: The amino acid sequence of the antimicrobial peptide AVV13N is: Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2.

2. A method for modifying the antimicrobial peptide AVV13N according to claim 1, characterized in that: The 11th lysine and the 13th valine in the amino acid sequence of the antimicrobial peptide AVV13N were exchanged.

3. The antimicrobial peptide AVK13N obtained by the method of claim 2, characterized in that: The amino acid sequence of the antimicrobial peptide AVK13N is: Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2.

4. Use of the antimicrobial peptide AVV13N according to claim 1 or the antimicrobial peptide AVK13N according to claim 3 in the preparation of antibacterial drugs, characterized in that: The bacteria are Gram-negative bacteria or Gram-positive bacteria; the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enterica subspecies enteritidis or Pseudomonas aeruginosa; the Gram-positive bacteria are Staphylococcus aureus, Streptococcus agalactiae, Bacillus subtilis or Micrococcus luteus.

5. The use according to claim 4, characterized in that: The Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

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