Antibacterial peptide based on pseudosciaena crocea CXCL8 protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to antibacterial peptide based on large yellow croaker CXCL8 protein and application of the antibacterial peptide. The preparation method comprises the following steps: by taking a large yellow croaker CXCL8 protein as a template, intercepting a linear polypeptide of 13 amino acids, and carrying out carboxyl terminal amidation modification, so as to obtain the antibacterial peptide AVV13N, and the amino acid sequence of the antibacterial peptide AVV13N is Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Met-Val-NH2; the antibacterial peptide AVK13N is obtained by interchanging lysine at the 11th site and valine at the 13th site in an amino acid sequence of the antibacterial peptide AVV13N, and 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 weights of the antibacterial peptides AVV13N and AVK13N are both 1669.12 Da, the isoelectric points of the antibacterial peptides AVV13N and AVK13N are both 12.44, and the antibacterial peptides AVV13N and AVK13N are both alkaline polypeptides. The antibacterial peptides AVV13N and AVK13N have broad-spectrum bactericidal activity and can be used for specifically killing gram-positive bacteria and gram-negative bacteria. In addition, the antibacterial peptides AVV13N and AVK13N have relatively weak hemolytic activity, relatively low cytotoxicity and good temperature stability, pH value stability and salt ion stability. The antibacterial peptides AVV13N and AVK13N provided by the invention can replace antibiotics and are applied to prevention and treatment of bacterial diseases.

Description

Technical Field

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

[0002] In recent years, the scale of the large yellow croaker cage farming industry 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 farming density, aquaculture diseases have become increasingly prominent. Among them, visceral white spot disease, as an important disease that seriously affects large yellow croaker cage farming, has attracted widespread attention. It has been confirmed that the disease is caused by Pseudomonas plecoglossicida. Pseudomonas plecoglossicida is a Gram-negative rod-shaped bacterium and a pathogen that causes diseases in many fish, such as large yellow croaker, grouper, and rainbow trout. When fish are infected with the bacteria, obvious white nodules will form in their spleen, kidneys, and liver, and this disease is therefore named visceral white spot disease. Visceral white spot disease causes serious economic losses to the large yellow croaker farming industry and greatly hinders the healthy development of the farming industry. Therefore, it is crucial to seek effective prevention and control measures.

[0003] Antimicrobial peptides are a class of small molecule polypeptides with antibacterial activity. They play an important role in the process of organisms resisting infection by foreign pathogenic microorganisms. Antimicrobial peptides mainly exert their bactericidal function through two pathways: membrane targeting and non-membrane targeting, namely, membrane perforation effect mechanism and non-membrane structure influence mechanism. In the membrane targeting mode, positively charged antimicrobial peptides preferentially interact with the negatively charged components on the bacterial cell membrane, and then their hydrophobic parts are embedded in the membrane, triggering the reconstruction of the cell membrane structure and forming a channel through the membrane, thereby causing the collapse of the intracellular ion balance and the loss of nutrients, and finally causing the bacterial cells to rupture and lose activity. In the non-membrane targeting mechanism, although antimicrobial peptides will also interact with the bacterial cell membrane, they do not use the destruction of membrane integrity as the main means. Antimicrobial peptides can cross the bacterial cell membrane and enter the cell, and then bind to various target molecules in the cell. When the antimicrobial peptides accumulate to a certain concentration in the bacterial cell, they will effectively inhibit the core life activities of the bacteria, such as DNA replication, RNA synthesis, protein translation and cell wall synthesis, thereby achieving the purpose of killing bacteria.

[0004] Although antimicrobial peptides have attracted much attention due to their broad-spectrum bactericidal ability and great application prospects, antimicrobial peptides in nature generally have many problems such as weak bactericidal activity, high cytotoxicity, and poor stability, which greatly restrict their further promotion and application in the breeding process. Therefore, based on the relationship between the structure and function of antimicrobial peptides, designing and modifying them by adding, deleting or replacing amino acid residues to obtain antimicrobial peptides with high bactericidal activity is one of the most effective strategies to overcome the current difficulties in the application of antimicrobial peptides. 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: 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.

[0007] The present invention further provides a method for preparing the antimicrobial peptides AVV13N and AVK13N, which is as follows: (1) Using the large yellow croaker CXCL8 protein sequence as a template, a 13-amino acid linear peptide was extracted and modified by carboxyl terminal amidation 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; (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; (3) The complete sequences of antimicrobial peptides AVV13N and AVK13N were synthesized by solid phase chemical synthesis.

[0008] 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.

[0009] The present invention also provides the use of the two antimicrobial peptides AVV13N and AVK13N in the preparation of feed additives.

[0010] The antimicrobial peptides AVV13N and AVK13N of the present invention have the following advantages and beneficial effects: The antimicrobial peptides AVV13N and AVK13N prepared by the present invention have broad-spectrum bactericidal activity and can kill Gram-positive bacteria and Gram-negative bacteria. In addition, the antimicrobial peptides AVV13N and AVK13N also have weak hemolytic activity, low cytotoxicity and good temperature, pH and salt ion stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the mass spectrum of the antimicrobial peptide AVV13N. Figure 2 This is the mass spectrum of the antimicrobial peptide AVK13N.

[0012] 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 proteus POLYC4) after treatment with antimicrobial peptides AVV13N and AVK13N are shown in Figure 2. Figure 4 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, Micrococcus luteus CMCC 28001) after treatment with antimicrobial peptides AVV13N and AVK13N.

[0013] Figure 5 This is a graph showing the hemolytic activity of antimicrobial peptides AVV13N and AVK13N on erythrocytes.

[0014] Figure 6 The graph shows the cytotoxicity of antimicrobial peptides AVV13N and AVK13N to LYC-FM cells.

[0015] Figure 7 This is a graph showing the effect of temperature on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N.

[0016] Figure 8 This is a graph showing the effect of pH on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N.

[0017] Fig. 9 This is a graph showing the effect of salt ions on the bactericidal activity of antimicrobial peptides AVV13N and AVK13N. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments, but the protection content of the present invention is not limited to the following embodiments.

[0019] Embodiment 1: The design and synthesis of antimicrobial peptides AVV13N and AVK13N are as follows: (1) Using the protein sequence of large yellow croaker CXCL8 as a template, a linear peptide of 13 amino acids 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; The amino acid sequence of the antimicrobial peptide AVV13N is: Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Lys-Met-Val-NH2.

[0020] The amino acid sequence of the antimicrobial peptide AVK13N is: Ala-Arg-Trp-Val-Arg-Arg-Val-Leu-Lys-Lys-Val-Met-Lys-NH2.

[0021] The mass spectra of antimicrobial peptides AVV13N and AVK13N are shown in Figures 1-2 .

[0022] (2) The antimicrobial peptides AVV13N and AVK13N were synthesized by Shanghai Sangon Biotechnology Co., Ltd. using solid phase chemical synthesis with a purity of more than 95% and were used in subsequent experiments.

[0023] Embodiment 2: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides AVV13N and AVK13N were determined by the following specific steps: (1) Take out the strain to be tested from the -80°C refrigerator, streak it onto a solid culture medium, and culture it in an incubator overnight; (2) Pick a single clone and place it in liquid culture medium and culture it in an incubator overnight; (3) Inoculate the overnight culture into new liquid culture medium and culture until the logarithmic growth phase. Adjust the concentration of the culture to 1.2×10 5 CFU / mL; (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; (5) 10 μL of the diluted antimicrobial peptide solution was added to columns 1-9 of a 96-well plate, and then 90 μL of bacterial solution was added to each well in turn. Columns 10 and 11 were used as negative and positive controls, respectively. 90 μL of culture medium and 10 μL of 1×PBS (pH=7.4) were added to the negative control, and 90 μL of bacterial solution and 10 μL of 1×PBS (pH=7.4) were added to the positive control. Three replicates were set up for each well. The above process should be completed within 15 min. (6) Place the 96-well plate in an incubator for 12 to 18 hours, take it out and mix it with a sterile pipette tip, and measure the OD with an enzyme-labeled instrument. 600 The values ​​were calculated with the concentration of antimicrobial peptides as the horizontal axis and OD 600 The value is used as the vertical axis to draw a curve, and the concentration at the inflection point is the minimum inhibitory concentration; (7) From each of the three parallel groups, 30 μL of the incubation mixture was plated on solid culture medium and cultured in an incubator overnight. The minimum antimicrobial peptide concentration that killed at least 99.9% of the bacteria was defined as the MBC.

[0024] Test results such as Figures 3-4 As shown, the antimicrobial peptides AVV13N and AVK13N have bactericidal activity against both the tested Gram-negative bacteria and Gram-positive bacteria, indicating that the antimicrobial peptides AVV13N and AVK13N have broad-spectrum bactericidal activity.

[0025] Embodiment 3: The hemolytic activity of antimicrobial peptides AVV13N and AVK13N was determined by the following specific steps: (1) Take 2 mL of whole blood from large yellow croaker and immediately add anticoagulant to prevent blood coagulation, centrifuge at 500 × g for 10 min, and discard the supernatant; (2) Resuspend the red blood cells in 1 mL of 1× PBS (pH = 7.4), centrifuge at 500×g for 10 min, discard the supernatant, and then wash three times with 1× PBS (pH = 7.4); (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; (4) Serial dilution of the antimicrobial peptide concentration to 1280, 640, 320, 160, 80, 40, 20, and 10 μM; (5) 20 μL of the diluted antimicrobial peptide solution was sequentially added to columns 1-8 of a 96-well plate, and then 180 μL of red blood cell suspension was sequentially added to each well. 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. (6) The 96-well plate was incubated in a 28°C incubator for 2 h. After centrifugation at 500 g for 10 min, 100 μL of the supernatant was transferred to a new 96-well plate and the absorbance was read at 405 nm. (7) The hemolytic activity was calculated using the following formula: Hemolytic activity = [(Apeptide-A0%lysis) / (A100%lysis-A0%lysis)] × 100%, Where A is the absorbance at 405 nm.

[0026] Test results such as Figure 5 As shown, the hemolytic ratios of antimicrobial peptides AVV13N and AVK13N on large yellow croaker red blood cells at different concentrations were all lower than 1%, indicating that the antimicrobial peptides AVV13N and AVK13N had low hemolytic activity.

[0027] Embodiment 4: The cytotoxicity assay of antimicrobial peptides AVV13N and AVK13N, the specific steps are as follows: (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; (2) Antimicrobial peptide dilution: Serial dilution of antimicrobial peptide concentrations to 1280, 640, 320, 160, 80, 40, and 20 μM; (3) Remove the culture medium, 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); add 90 μL of fresh culture medium and 10 μL of 20% TritonX-100 to the positive control (100% cytotoxicity group); set up 3 replicates for each group; (4) After culturing in a 28°C incubator for 24 h, add 10 μL of CCK-8 solution to each well, mix gently, and continue culturing in a 28°C incubator for 4 h. Read the absorbance at 450 nm. (5) Calculate the viable cell ratio using the following formula: Live cell ratio = [(Apeptide-A100% lysis) / (A0% lysis-A100% lysis)] × 100%, Where A is the absorbance at 450 nm.

[0028] Test results such as Figure 6 As shown, the cell survival rate of large yellow croaker macrophage LYC-FM cells treated with different concentrations of antimicrobial peptides AVV13N and AVK13N was higher than 90%, indicating that the antimicrobial peptides AVV13N and AVK13N have lower cytotoxicity.

[0029] Embodiment 5: The temperature stability test of antimicrobial peptides AVV13N and AVK13N, the specific steps are as follows: (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. (2) Pseudomonas aeruginosa was cultured with oscillation until the logarithmic growth phase, the OD value was measured and the bacterial solution concentration was adjusted to 1×10 5 CFU / mL; (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, and 10 μL of deionized water was added to the positive control. Three replicates were set for each group. (4) Place the 96-well plate in a 28°C incubator for overnight culture, apply gradient dilutions to each well and spread on TSA solid medium, and count the number of colonies after 14 to 16 hours; (5) Calculate the bacterial killing rate according to the following formula: Bacterial sterilization rate = 1-(CFU of each temperature treatment group / CFU of positive control) × 100% (6) Plot the temperature on the X-axis and the bacterial killing rate on the Y-axis.

[0030] Test results such as Figure 7 As shown, after treatment at different temperatures, the bactericidal rates of antimicrobial peptides AVV13N and AVK13N against Pseudomonas aeruginosa can still reach more than 99.99%, indicating that the antimicrobial peptides AVV13N and AVK13N have good temperature stability.

[0031] Embodiment 6: The pH stability test of antimicrobial peptides AVV13N and AVK13N was carried out in the following steps: (1) Prepare the following buffer solutions of different pH values ​​and filter sterilize them: 50 mM glycine-HCl (pH = 2.0); 50 mM NaAC-HAC (pH = 4.0); 50 mM MES [2-(N-morpholino)ethanesulfonic acid]-NaOH (pH=6.0); 50 mM Tris-HCl (pH=8.0); 50 mM glycine-NaOH (pH=10.0).

[0032] (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 until the final concentration is 4 times the MBC. Mix well and place at room temperature for 4 h. (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; (4) Add 90 μL of bacterial solution to a 96-well plate, add 10 μL of treated antimicrobial peptide solution to each well, add 10 μL of deionized water to the positive control, and set up 3 replicates for each group; (5) Place the 96-well plate in a 28°C incubator overnight, perform gradient dilutions in each well and apply them to TSA solid medium. Count the number of colonies after 14 to 16 hours. (6) Calculate the bacterial killing rate according to the following formula: Bacterial sterilization rate = 1-(CFU of each pH treatment group / CFU of positive control) × 100% (7) Draw a line graph with pH as the X-axis and bacterial killing rate as the Y-axis.

[0033] Test results such as Figure 8 As shown, the bactericidal rates of antimicrobial peptides AVV13N and AVK13N against Pseudomonas aeruginosa can still reach more than 99.99% under different pH values, indicating that the antimicrobial peptides AVV13N and AVK13N have good pH stability.

[0034] Embodiment 7: The salt ion stability test of antimicrobial peptides AVV13N and AVK13N is as follows: (1) Prepare the following buffer solutions with different salt ions, adjust the pH to 7.4, and filter and sterilize: 1500mM NaCl 45mM KCl60μM NH4Cl80μM ZnCl2 10mM MgCl2 20mM CaCl2 40μM FeCl3 (2) Pseudomonas aeruginosa was cultured with oscillation until the logarithmic growth phase, the OD value was measured and the bacterial solution concentration was adjusted to 1×105 CFU / mL; (3) 80 μL of bacterial solution, 10 μL of different salt ion solutions and 10 μL of antimicrobial peptide were added to a 96-well plate in sequence, where 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, and 3 replicates were set for each group. (4) Place the 96-well plate in a 28°C incubator for overnight culture, apply gradient dilutions to each well and spread on TSA solid medium, and count the number of colonies after 14 to 16 hours; (5) Calculate the bacterial killing rate according to the following formula: Bacterial sterilization rate = 1-(CFU of each salt ion solution treatment group / CFU of positive control) × 100% (6) Draw a line graph with different salt ion solutions as the X-axis and the bacterial killing rate as the Y-axis.

[0035] Test results such as Fig. 9 As shown, 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, FeCl3, etc., indicating that the antimicrobial peptides AVV13N and AVK13N have good salt ion stability.

[0036] In summary, the antimicrobial peptides AVV13N and AVK13N of the present invention have broad-spectrum antimicrobial activity and have strong antimicrobial activity against both Gram-positive bacteria and Gram-negative bacteria. In addition, the antimicrobial peptides AVV13N and AVK13N have 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 have significant application advantages in the preparation of therapeutic drugs for bacterial infections and as animal feed additives.

[0037] 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. Antimicrobial peptide AVV13N based on large yellow croaker CXCL8 protein, characterized in that: 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 lysine at position 11 and the valine at position 13 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 and the antimicrobial peptide AVK13N according to claim 3 in the preparation of antibacterial drugs.

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

6. Use of the antimicrobial peptide AVV13N according to claim 1 and the antimicrobial peptide AVK13N according to claim 3 in the preparation of feed additives.

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