Antibacterial peptide rrl14 based on igf ih of p. major and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an antibacterial peptide RRL14 based on IFNh synthesis of Pseudosciaena crocea and application thereof. The full sequence of the antibacterial peptide RRL14 is Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu, the molecular weight is 1933.36 Da, the isoelectric point is 12.12, and the antibacterial peptide RRL14 belongs to an alkaline polypeptide. The antibacterial peptide RRL14 has broad-spectrum and high-efficiency bactericidal activity, weak hemolytic activity, small cytotoxicity, high temperature, pH, salt ion and serum stability. The antibacterial peptide RRL14 is expected to replace antibiotics and be applied to the prevention and treatment of bacterial diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antimicrobial peptide RRL14 synthesized from IFNh of large yellow croaker and its applications. Background Technology

[0002] Antimicrobial peptides are small molecule polypeptides with antimicrobial activity, widely found in prokaryotes and eukaryotes, and involved in the body's fight against microbial infections. The diversity of amino acid sequences and structures of antimicrobial peptides enables them to efficiently kill both Gram-negative and Gram-positive bacteria. The mechanisms of action of antimicrobial peptides mainly include membrane-targeting and non-membrane-targeting mechanisms. Studies have shown that cationic antimicrobial peptides can exert their bactericidal function through interaction with bacterial cell membranes.

[0003] While natural antimicrobial peptides possess bactericidal activity, they suffer from drawbacks such as instability under physiological conditions, sensitivity to high temperatures, strong acids, and strong alkalis, high cytotoxicity and hemolytic activity, and difficulty in obtaining large quantities from organisms, hindering industrial production and clinical application. Artificial synthesis methods can be used to design or modify antimicrobial peptides, addressing these issues. The most common methods for designing antimicrobial peptides based on their structure-function relationships include site-directed mutagenesis, novel design, synthetic peptide libraries, template-assisted design, mechanism-based design, and database-assisted design. Template-assisted design, using natural peptide sequences as templates, designs and synthesizes novel peptides through amino acid substitution, reducing synthesis costs while maintaining the peptide sequence.

[0004] Antibiotics can prevent and treat bacterial diseases, but their overuse can lead to antibiotic resistance, making the control of bacterial diseases increasingly difficult. Antimicrobial peptides possess broad-spectrum antimicrobial activity and are less likely to induce antibiotic resistance, making them suitable as alternatives to antibiotics for the prevention and treatment of bacterial diseases. Furthermore, antimicrobial peptides can also be applied in animal husbandry and aquaculture to develop antibiotic-reduced and antibiotic-alternative feed additives. Summary of the Invention

[0005] In order to obtain an antimicrobial peptide with broad-spectrum and high-efficiency antimicrobial activity and good biosafety, this invention designs and synthesizes an antimicrobial peptide RRL14 based on the IFNh of large yellow croaker.

[0006] The objective of this invention is achieved through the following techniques: The present invention first provides an antimicrobial peptide RRL14, the amino acid sequence of which is: Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu.

[0007] This invention further provides a method for preparing the above-mentioned antimicrobial peptide RRL14, as follows: (1) Using the IFNh protein sequence of large yellow croaker as a template, a linear polypeptide of 14 amino acids was extracted and named SRL14. The amino acid sequence of the linear polypeptide SRL14 is Ser-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Ser-Thr-Leu. (2) By replacing the 1st and 12th serine (Ser) in SRL14 with positively charged arginine (Arg) and the 13th threonine (Thr) with the hydrophobic amino acid isoleucine (Ile), the antimicrobial peptide RRL14 is obtained. The amino acid sequence of the antimicrobial peptide RRL14 is Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu.

[0008] (3) The antimicrobial peptide RRL14 was synthesized by solid-phase chemical synthesis.

[0009] The present invention also provides the application of the above-mentioned antimicrobial peptide RRL14 in the preparation of drugs for preventing and treating bacterial infections.

[0010] This invention also provides the application of the above-mentioned antimicrobial peptide RRL14 in the preparation of feed additives.

[0011] The antimicrobial peptide RRL14 of this invention has the following advantages and beneficial effects: the antimicrobial peptide RRL14 prepared by this invention has broad-spectrum and highly efficient bactericidal activity, weak hemolytic activity, low cytotoxicity, and high stability under temperature, pH, salt ion and serum conditions. Attached Figure Description

[0012] Figure 1 This is the mass spectrum of the antimicrobial peptide RRL14.

[0013] Figure 2 The graph shows the bactericidal activity and bactericidal kinetics of the antimicrobial peptide RRL14 against Pseudomonas proteus.

[0014] Figure 3 The growth curves of Gram-negative and Gram-positive bacteria after treatment with the antimicrobial peptide RRL14 are shown.

[0015] Figure 4 Figure showing the cytotoxic and hemolytic activity of the antimicrobial peptide RRL14 against LYC-FM cells.

[0016] Figure 5 The graph shows the effects of temperature, pH, salt ion concentration, and serum on the bactericidal activity of the antimicrobial peptide RRL14. Detailed Implementation

[0017] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0018] Example 1: Design and synthesis of antimicrobial peptide RRL14, the specific steps are as follows: (1) Using the IFNh protein sequence of large yellow croaker as a template, a linear polypeptide of 14 amino acids was extracted and named SRL14. By replacing the serine (Ser) at positions 1 and 12 in the linear polypeptide SRL14 with positively charged arginine (Arg) and the threonine (Thr) at position 13 with the hydrophobic amino acid isoleucine (Ile), the antimicrobial peptide RRL14 was obtained. The amino acid sequence of the linear polypeptide SRL14 is as follows: Ser-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Ser-Thr-Leu; The amino acid sequence of the antimicrobial peptide RRL14 is as follows: Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu.

[0019] Compared to SRL14, RRL14 has a theoretical molecular weight that changes from 1783.09 Da to 1933.36 Da, a net charge that increases from +5 to +7, and an isoelectric point that changes from 11.7 to 12.12.

[0020] (2) The antimicrobial peptide RRL14 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase chemical synthesis.

[0021] The mass spectrum of the antimicrobial peptide RRL14 is shown below. Figure 1 .

[0022] Example 2: Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide RRL14, the specific steps are as follows: (1) Pick test strains and place them in liquid culture medium, shake and culture until the logarithmic growth phase, then adjust the bacterial concentration to 1×10⁻⁶ using liquid culture medium. 5 CFU / mL; (2) The antimicrobial peptide RRL14 was serially diluted to the following concentrations: 1280 μM, 640 μM, 320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM and 5 μM. (3) Take 90 μL of test bacterial solution and 10 μL of each gradient of antimicrobial peptide RRL14 and add them to columns 2-10 of a 96-well plate, respectively. Add 90 μL of culture medium and 10 μL of PBS to the negative control in column 1, and add 90 μL of test bacterial solution and 10 μL of PBS to the positive control in column 11. Set up 3 replicates for each. (4) Place the 96-well plate in a constant temperature incubator and incubate for 12-18 hours; (5) After mixing the 96-well plate, measure the OD using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Plotting antimicrobial peptide concentration on the x-axis, OD 600 The value is plotted on the ordinate to create growth curves for different test bacteria, with the inflection point representing the minimum inhibitory concentration.

[0023] like Figure 2 As shown in Figure A, the antimicrobial peptide RRL14 exhibits strong antibacterial activity against *Pseudomonas proteus*, with a minimum inhibitory concentration (MIC) of 1 μM and a minimum bactericidal concentration (MBC) of 32 μM. Figure 3 As shown, the antimicrobial peptide RRL14 exhibits bactericidal activity against both Gram-negative and Gram-positive bacteria tested. This demonstrates that the antimicrobial peptide RRL14 of this invention possesses broad-spectrum bactericidal activity.

[0024] Example 3: Determination of the bactericidal kinetic curve of antimicrobial peptide RRL14, the specific steps are as follows: (1) The *Pseudomonas proteus* was cultured with shaking until it reached the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶. 5 CFU / mL; (2) Mix 90 μL of Pseudomonas proteus bacterial culture with 10 μL of antimicrobial peptide RRL14 (final concentration of antimicrobial peptide RRL14 is 1×MBC) and incubate for different times (0 min, 5 min, 10 min, 20 min, 30 min, 60 min). (3) After incubation, the bacterial culture was diluted and spread onto a solid culture medium. The culture was then incubated at 28°C for 18–24 h. The bacterial survival rate at each time point was then calculated. Wells containing 10 μL PBS were used as controls. Three replicates were set up for each time point.

[0025] like Figure 2 As shown in Figure B, the antimicrobial peptide RRL14 can kill all Pseudomonas proteus bacteria within 5 minutes. This demonstrates that the antimicrobial peptide of the present invention has highly efficient bactericidal activity.

[0026] Example 4: Cytotoxicity assay of antimicrobial peptide RRL14, the specific steps are as follows: (1) Take 100 μL of a concentration of 2 × 10 5 Add LYC-FM cell suspension at 100 cells / mL to 96-well plates and incubate overnight at 28°C. (2) Remove the culture medium and label wells 1 to 9 in the 96-well plate. Add 60 μL of phenol red-free L-15 medium to each well from 1 to 9. Add 40 μL of antimicrobial peptide RRL14 solution at concentrations of 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM to wells 2 to 8, respectively, as experimental wells (i.e., A). peptide (well); 40 μL of PBS was added to well 1, resulting in a final concentration of 0 μM for the antimicrobial peptide, serving as a negative control for 0% cytotoxicity (i.e., A). 0％ (lysis well); add 40 μL of 5% Triton X-100 mixed culture medium to well 9 as a 100% cytotoxicity positive control (i.e., A). 100％ Each lysis well was set to have 3 replicates. (3) After incubating at 28℃ for 24 hours, add 10 μL of LCK-8 solution to each well, mix well, continue incubating at 28℃ for 4 hours, and read the absorbance value at 450 nm. (4) Calculate the live cell ratio: viable cell ratio = [(A peptide –A 100％ lysis) / (A 0％ lysis-A 100％ [lysis)]×100%, where A is the absorbance at 450 nm.

[0027] like Figure 4 As shown in Figure A, the cell survival rate of antimicrobial peptide RRL14 at each detected concentration in large yellow croaker macrophage LYC-FM was higher than 90%, indicating that the antimicrobial peptide RRL14 of this invention has very low cytotoxicity.

[0028] Example 5: Determination of the hemolytic activity of the antimicrobial peptide RRL14, the specific steps are as follows: (1) After adding anticoagulant to the blood of large yellow croaker, centrifuge at 500g for 10min; (2) Discard the supernatant, resuspend the blood cells in PBS, then centrifuge at 500g for 10min, and repeat the washing 3 times; (3) Resuspend the cells in PBS and adjust the cell concentration to 1×10⁻⁶. 8 cells / mL; (4) Add 120 μL of cell suspension to a 96-well plate, and then add 80 μL of antimicrobial peptide RRL14 at different concentrations to make the final concentrations of antimicrobial peptide 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM and 128 μM. Use 80 μL of 20% Triton-X100 and 80 μL of PBS as 100% and 0% hemolysis controls, respectively. Set up 3 replicates for each group. (5) Incubate the 96-well plate at 28°C for 2 hours, centrifuge, and then measure the absorbance at 540 nm. (6) Calculate hemolytic activity: Hemolytic activity = [(A peptide -A 0％ lysis) / (A 100％ lysis-A 0％ [lysis)]×100%, where A is the absorbance at 540 nm.

[0029] like Figure 4 As shown in B, the hemolysis rate of antimicrobial peptide RRL14 at each tested concentration was less than 4% on the red blood cells of large yellow croaker, indicating that the antimicrobial peptide RRL14 of the present invention has a small hemolytic effect on red blood cells.

[0030] Example 6: Determination of the effect of temperature on the bactericidal activity of antimicrobial peptide RRL14, the specific steps are as follows: (1) Adjust the concentration of *Pseudomonas proteus* in the logarithmic growth phase to 1×10⁻⁶ using culture medium. 5 CFU / mL; (2) Take 40 μL of antimicrobial peptide RRL14 with a final concentration of 80 μM (10×MBC) into a 1.5 mL centrifuge tube and incubate at 20℃, 40℃, 60℃, 80℃ and 100℃ respectively. (3) Add 90 μL of the above bacterial culture to a 96-well plate, and then add 10 μL of the treated antimicrobial peptide RRL14 solution to each well. Add 10 μL of PBS to the positive control. Set up 3 replicates for each temperature gradient. (4) Incubate overnight at 28℃, and after gradient dilution of each well, plate the cells and count the number of colonies. Plot the cells with temperature on the x-axis and bacterial sterilization rate on the y-axis.

[0031] Bacterial mortality rate = (1 - CFU in temperature treatment group / CFU in positive control group) × 100% like Figure 5 As shown in Figure A, the bactericidal rate of antimicrobial peptide RRL14 against Pseudomonas mutans was higher than 99% under different temperature treatments, indicating that the antimicrobial peptide RRL14 of the present invention has good temperature stability.

[0032] Example 7: Determination of the effect of pH on the bactericidal activity of antimicrobial peptide RRL14, the specific steps are as follows: (1) Adjust the concentration of *Pseudomonas proteus* in the logarithmic growth phase to 1×10⁻⁶ using culture medium. 5 CFU / mL; (2) Prepare buffer solutions with different pH values: 50mM glycine-HCl (pH 2.0), 50mM NaAC-HAC (pH 4.0), 50mM MMES[2-(N-morpholino)ethanesulfonic acid]-NaOH (pH 6.0), 50mM M Tris-HCl (pH 8.0), 50mM glycine-NaOH (pH 10.0), and filter for sterilization; (3) Take 30 μL of the above buffer solution into a 1.5 mL centrifuge tube, add 10 μL of antimicrobial peptide RRL14 to each tube, so that the final concentration of antimicrobial peptide RRL14 is 10×MBC, mix well and let stand at room temperature for 4 h. (4) Add 90 μL of the above bacterial culture to a 96-well plate, and then add 10 μL of antimicrobial peptide RRL14 solution treated with different pH values ​​to each well. Add 10 μL of PBS to the positive control. Set up 3 replicates for each. (5) Incubate overnight at 28°C, and after serial dilution of each well, plate the cells and count the number of colonies. Plot the cells with pH on the x-axis and bacterial mortality rate on the y-axis.

[0033] Bacterial mortality rate = (1 - CFU in pH treatment group / CFU in positive control group) × 100% like Figure 5 As shown in Figure B, the bactericidal rate of antimicrobial peptide RRL14 against Pseudomonas mutans was higher than 99% under different pH treatments, indicating that the antimicrobial peptide RRL14 of the present invention has good pH stability.

[0034] Example 8: Determination of the effect of salt ions on the bactericidal activity of antimicrobial peptide RRL14, the specific steps are as follows: (1) Prepare 1500mM NaCl, 45mM KCl, 60μM NH4Cl, 80μM ZnCl2, 10MmMgCl2, 20mM CaCl2 and 40μM FeCl3 respectively, and sterilize at high temperature; (2) Adjust the concentration of *Pseudomonas proteus* in the logarithmic growth phase to 1.3 × 10⁻⁶ using culture medium. 5 CFU / mL; (3) Add 80 μL of bacterial culture to a 96-well plate, then add 10 μL of antimicrobial peptide RRL14 and 10 μL of the above salt solution in sequence, so that the final concentration of antimicrobial peptide RRL14 is 1×MBC. Add 10 μL of PBS and 10 μL of the above different salt solutions to the positive control. All are set to 3 repetitions; (4) Incubate overnight at 28°C, and after serial dilution of each well, plate the cells and count the number of colonies. Plot a graph with different salt solutions as the X-axis and bacterial survival rate as the Y-axis.

[0035] Bacterial survival rate = (CFU in each salt solution treatment group / CFU in the positive control) × 100% like Figure 5As shown in Figure C, the bactericidal rate of antimicrobial peptide RRL14 against Pseudomonas mutans was higher than 99% under different salt solution treatments, indicating that the antimicrobial peptide RRL14 of the present invention has good salt ion stability.

[0036] Example 10: Determination of the effect of serum on the bactericidal activity of antimicrobial peptide RRL14, the specific steps are as follows: (1) Heat inactivation of serum: The serum was placed in a 56°C water bath and heat-inactivated for 30 min. The serum was shaken once every 10 min. After inactivation, the serum was centrifuged for 10 min. (2) Adjust the concentration of *Pseudomonas proteus* in the logarithmic growth phase to 1 × 10⁻⁶ using culture medium. 5 CFU / mL; (3) Add 40 μL of bacterial culture, serum of different concentrations (12.5%, 25%, 50%) and antimicrobial peptide RRL14 solution to each well of a 96-well plate in sequence, so that the final concentration of antimicrobial peptide RRL14 is 1×MBC. PBS is used instead of serum as a positive control group. Three replicates are set up for each. (4) Incubate overnight at 28°C, and after serial dilution in each well, plate the cells and count the number of colonies. Plot a graph with different serums as the X-axis and bacterial survival rate as the Y-axis.

[0037] Bacterial survival rate = (CFU in each serum treatment group / CFU in the positive control group) × 100% like Figure 5 As shown in D, serum can affect the bactericidal activity of the antimicrobial peptide RRL14, but the bactericidal rate of the antimicrobial peptide RRL14 against Pseudomonas mucosa remains above 60%, indicating that the RRL14 of the present invention has serum stability.

[0038] In summary, the antimicrobial peptide RRL14 of this invention possesses broad-spectrum and highly effective bactericidal activity, relatively weak hemolytic activity, low cytotoxicity, and high stability under temperature, pH, salt ion, and serum conditions. Therefore, the antimicrobial peptide RRL14 of this invention can be well applied in the preparation of antibacterial drugs and feed additives.

[0039] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An antimicrobial peptide RRL14 synthesized based on IFNh from large yellow croaker, characterized in that: The amino acid sequence of the antimicrobial peptide RRL14 is: Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu.

2. A method for preparing the antimicrobial peptide RRL14 as described in claim 1, characterized in that: By extracting 14 amino acids from the IFNh protein of large yellow croaker, a linear polypeptide SRL14 containing 5 positive charges and an isoelectric point of 11.7 was obtained. The amino acid sequence of the linear polypeptide SRL14 is Ser-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Ser-Thr-Leu. The serine residues at positions 1 and 12 of the linear polypeptide SRL14 were replaced with positively charged arginine, and the threonine residue at position 13 was replaced with the hydrophobic amino acid isoleucine, resulting in the antimicrobial peptide RRL14 containing 7 positive charges and an isoelectric point of 12.

12. The amino acid sequence of the antimicrobial peptide RRL14 is Arg-Leu-Arg-Lys-Tyr-Tyr-Arg-Arg-Leu-Ala-Arg-Arg-Ile-Leu. The complete sequence of the antimicrobial peptide RRL14 was synthesized using solid-phase chemical synthesis.

3. The use of the antimicrobial peptide RRL14 as described in claim 1 in the preparation of a drug for preventing and treating bacterial infections, characterized in that: The bacteria are selected from at least one of Pseudomonas proteus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella pullorum, Streptococcus agalactiae, and Micrococcus luteus.

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