Antibacterial peptide based on pseudosciaena crocea RPL39 protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to antibacterial peptide based on pseudosciaena crocea RPL39 protein and application of the antibacterial peptide. The preparation method comprises the following steps: by taking a pseudosciaena crocea RPL39 protein as a template, intercepting a linear polypeptide of 12 amino acids, and carrying out amidation modification on a carboxyl terminal, so as to obtain the antibacterial peptide KRK12N: Lys-Thr-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2; the method comprises the following steps: replacing threonine at the second site in an amino acid sequence of KRK12N with tryptophan, so as to obtain KRK12N-1: Lys-Trp-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2; the method comprises the following steps: carrying out interchange on lysine at the sixth site and phenylalanine at the eighth site in an amino acid sequence of KRK12N-1, so as to obtain KRK12N-2: Lys-Trp-Phe-Arg-Ile-Phe-Arg-Lys-Leu-Ala-Lys-Lys-NH2, and carrying out interchange on lysine at the sixth site and phenylalanine at the eighth site in the amino acid sequence of KRK12N-1. The antibacterial peptide KRK12N, the antibacterial peptide KRK12N-1 and the antibacterial peptide KRK12N-2 have broad-spectrum bactericidal activity and can be used for specifically killing gram-positive bacteria and gram-negative bacteria. In addition, the antibacterial peptides KRK12N, KRK12N-1 and KRK12N-2 all have relatively weak hemolytic activity, relatively low cytotoxicity and good temperature, acid-base and salt ion stability. The antibacterial peptides KRK12N, KRK12N-1 and KRK12N-2 provided by the invention can be used for replacing antibiotics and are applied to prevention and treatment of bacterial diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to antimicrobial peptides based on the large yellow croaker RPL39 (ribosomal protein L39) protein and their applications. Background Technology

[0002] Antimicrobial peptides are a class of polypeptides with molecular weights below 10 kDa. Cationic antimicrobial peptides are characterized by the simultaneous presence of both positively charged and hydrophobic amino acid residues, exhibiting significant amphiphilic and cationic properties. These peptides demonstrate significant bactericidal or inhibitory effects against a variety of microorganisms, including bacteria, fungi, and viruses. Unlike traditional antibiotics, antimicrobial peptides exert their bactericidal function through multiple pathways, including membrane-targeting (direct disruption of cell membrane integrity) and non-membrane-targeting (such as intracellular targeting). Notably, antimicrobial peptides also possess bactericidal activity against various drug-resistant bacteria, a characteristic that makes them valuable for applications in the treatment of bacterial infections.

[0003] Antimicrobial peptides, as an important component of the innate immune system, not only have a direct killing effect on pathogens but also exert anti-infective immune effects through immunomodulation. However, natural antimicrobial peptides suffer from a series of problems, such as weak biological activity, strong cytotoxicity, and poor stability, which severely limit and hinder their application. Therefore, based on in-depth research into the structure-function relationship of antimicrobial peptides, the artificial design and synthesis of novel antimicrobial peptides is the best way to overcome the bottlenecks in their application.

[0004] Pseudomonas proteus ( Pseudomonas plecoglossicida *Pseudomonas proteus*, belonging to the family Pseudomonasceae and the genus *Pseudomonas*, is a Gram-negative, aerobic rod-shaped bacterium that moves via polar flagella. *Pseudomonas proteus* can infect fish such as large yellow croaker, grouper, and rainbow trout, causing white nodules on the spleen, kidneys, and liver, hence the name "visceral white spot disease." Visceral white spot disease causes significant economic losses to aquaculture and severely restricts its development. Developing antimicrobial peptides that can kill *Pseudomonas proteus* could provide a potential therapeutic agent for the prevention and treatment of visceral white spot disease in farmed animals. Summary of the Invention

[0005] In order to obtain antimicrobial peptides with broad-spectrum antimicrobial activity, as well as good biosafety and stability, this invention designed and synthesized three antimicrobial peptides, KRK12N, KRK12N-1 and KRK12N-2, based on large yellow croaker RPL39, and clarified their applications.

[0006] The objective of this invention is achieved through the following technical solution: This invention first provides three antimicrobial peptides: KRK12N, KRK12N-1, and KRK12N-2. The amino acid sequence of antimicrobial peptide KRK12N is Lys-Thr-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2, the amino acid sequence of antimicrobial peptide KRK12N-1 is Lys-Trp-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2, and the amino acid sequence of antimicrobial peptide KRK12N-2 is Lys-Trp-Phe-Arg-Ile-Phe-Arg-Lys-Leu-Ala-Lys-Lys-NH2.

[0007] The present invention further provides a method for preparing the above-mentioned antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2, as follows: (1) Using the RPL39 protein sequence of large yellow croaker as a template, a linear polypeptide of 12 amino acids was extracted and its carboxyl terminus was modified by amidation to obtain the antimicrobial peptide KRK12N; the amino acid sequence of the antimicrobial peptide KRK12N is Lys-Thr-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2; (2) By replacing the threonine at position 2 in the amino acid sequence of the antimicrobial peptide KRK12N with tryptophan, the antimicrobial peptide KRK12N-1 was obtained; the amino acid sequence of the antimicrobial peptide KRK12N-1 is Lys-Trp-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2; (3) By exchanging the lysine at position 6 and the phenylalanine at position 8 in the amino acid sequence of the antimicrobial peptide KRK12N-1, the antimicrobial peptide KRK12N-2 was obtained; the amino acid sequence of the antimicrobial peptide KRK12N-2 is Lys-Trp-Phe-Arg-Ile-Phe-Arg-Lys-Leu-Ala-Lys-Lys-NH2; (4) The complete sequences of antimicrobial peptides KRK12N, KRK12N-1 and KRK12N-2 were synthesized by solid-phase chemical synthesis.

[0008] The present invention also provides the application of the above-mentioned antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 in the preparation of antibacterial drugs, which have broad-spectrum antibacterial activity.

[0009] The aforementioned antibacterial agents include those against Gram-negative bacteria and those against Gram-positive bacteria.

[0010] The aforementioned Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella pullorum, Pseudomonas proteus, and Vibrio harveyi; the aforementioned Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae, Micrococcus luteus, and Bacillus subtilis.

[0011] This invention also provides the application of the above-mentioned antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 in the preparation of feed additives.

[0012] The significant advantages of this invention are: The antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 provided by this invention have broad-spectrum bactericidal activity and can kill both Gram-positive and Gram-negative bacteria. Furthermore, KRK12N, KRK12N-1, and KRK12N-2 also exhibit weak hemolytic activity, low cytotoxicity, and good stability under temperature, acid-base, and salt ion conditions. Attached Figure Description

[0013] Figure 1 This is the mass spectrum of the antimicrobial peptide KRK12N.

[0014] Figure 2 This is the mass spectrum of the antimicrobial peptide KRK12N-1.

[0015] Figure 3 This is the mass spectrum of the antimicrobial peptide KRK12N-2.

[0016] Figure 4 Growth curves of Gram-negative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC14028, Salmonella pullorum ATCC 9120, Pseudomonas proteus PQLYC4, Vibrio harveyi W2023) after treatment with antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2.

[0017] Figure 5 Growth curves of Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Streptococcus agalactiae ATCC 13813, Micrococcus luteus CMCC 28001, and Bacillus subtilis CMCC 63501) after treatment with antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2.

[0018] Figure 6 The graph shows the hemolytic activity of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 against erythrocytes.

[0019] Figure 7The graph shows the cytotoxicity of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 on LYC-FM cells.

[0020] Figure 8 Figure showing the effect of temperature on the bactericidal activity of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2.

[0021] Figure 9 The effect of pH on the bactericidal activity of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 is shown in the figure.

[0022] Figure 10 The effect of salt ions on the bactericidal activity of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 is shown in the figure. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0024] Example 1: The design and synthesis of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 are carried out through the following steps: (1) Using the RPL39 protein sequence of large yellow croaker as a template, a linear polypeptide of 12 amino acids was extracted and modified by carboxyl-terminal amidation to obtain the antimicrobial peptide KRK12N. The threonine at position 2 in the antimicrobial peptide KRK12N polypeptide sequence was replaced with tryptophan to obtain the antimicrobial peptide KRK12N-1. Furthermore, the lysine at position 6 and the phenylalanine at position 8 in the antimicrobial peptide KRK12N-1 polypeptide sequence were interchanged to obtain the antimicrobial peptide KRK12N-2.

[0025] The amino acid sequence of the antimicrobial peptide KRK12N is as follows: Lys-Thr-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2 The amino acid sequence of the antimicrobial peptide KRK12N-1 is as follows: Lys-Trp-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2 The amino acid sequence of the antimicrobial peptide KRK12N-2 is as follows: Lys-Trp-Phe-Arg-Ile-Phe-Arg-Lys-Leu-Ala-Lys-Lys-NH2 Mass spectra of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 are shown below. Figures 1-3 .

[0026] (2) The antimicrobial peptides KRK12N, KRK12N-1 and KRK12N-2 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase chemical synthesis.

[0027] Example 2: The determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 is performed using the following steps: (1) Pick the test strain into liquid culture medium, shake and culture until the logarithmic growth phase, and adjust the bacterial concentration to 1.2 × 10⁻⁶ using liquid culture medium. 5 CFU / mL; (2) Dissolve the antimicrobial peptides KRK12N, KRK12N-1 and KRK12N-2 in 1×PBS (pH=7.4) to a concentration of 1280 μM. Then, take 50 μL of the diluted antimicrobial peptides and serially dilute them to a final concentration of 5, 10, 20, 40, 80, 160, 320, 640 and 1280 μM. (3) Add 10µL of the diluted antimicrobial peptide solution (5-1280µM) to columns 1-9 of the 96-well plate in sequence. Then add 90µL of the test strain culture to each well in sequence. Add 90µL of the test strain culture and 10µL of 1×PBS (pH=7.4) to the positive control in column 10. Add 90µL of liquid culture medium and 10µL of 1×PBS (pH=7.4) to the negative control in column 11. Set up 3 replicates for each. The above process should be completed within 15min. (4) Place the 96-well plate in an incubator and incubate for 12-18 hours. After taking it out, the minimum concentration of antimicrobial peptide that does not show any turbidity at the bottom of the well is the minimum inhibitory concentration (MIC). (5) Take 30µL of each of the three parallel incubation mixtures and spread it on a solid culture medium. Incubate overnight in an incubator. The minimum bactericidal concentration (MBC) is determined by the lowest polypeptide concentration that can kill more than 99.9% of bacteria.

[0028] Test results as follows Figure 4 and 5 As shown, the antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 are effective against the tested Gram-negative bacteria ( E. coli ATCC 25922 P. aeruginosa ATCC 27853 P. aeruginosa ATCC 9027 S. typhimurium ATCC 14028 S. pullorum ATCC 9120 P. plecoglossicida PQLYC4 V. harveyiW2023) and Gram-positive bacteria ( S. aureus ATCC 25923 S. agalactiae ATCC13813 M. luteus CMCC 28001 B. subilis CMCC 63501) all exhibit bactericidal activity. This indicates that the antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 all possess broad-spectrum bactericidal activity.

[0029] Example 3: The hemolytic activity assay of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 was performed using the following steps: (1) After adding anticoagulant to the blood of large yellow croaker, 500 g Centrifuge for 10 minutes; (2) Discard the supernatant, resuspend in 1×PBS (pH=7.4) for 500 minutes. g Centrifuge for 10 minutes, repeat three times; (3) Resuspend the cells in 1×PBS (pH=7.4) and adjust the cell concentration to 1×10⁻⁶. 8 cell / mL; (4) Add 180 μL of cell suspension to a 96-well plate, add 20 μL of antimicrobial peptides of different concentrations to a final concentration of 2, 4, 8, 16, 32, 64, 128 μM, and add 20 μL of 2% Triton-X100 and 20 μL of 1×PBS (pH=7.4) to the control wells respectively, as 100% and 0% hemolysis controls, respectively. Set up 3 replicates for each group; (5) Incubate the cells at 28°C for 2 hours, centrifuge, and transfer 100 μL of the supernatant to a 96-well plate. Read the absorbance value at 405 nm. (6) Calculate hemolytic activity: Hemolytic activity = [(A peptide -A 0% ) / (A 100% -A 0% )]×100%, where A is the absorbance at 405nm.

[0030] Test results as follows Figure 6 As shown, the hemolytic rates of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 on large yellow croaker erythrocytes at each tested concentration were all less than 2%, indicating that antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 have low hemolytic activity.

[0031] Example 4: The cytotoxicity assays for the antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 were performed using the following steps: (1) Adjust the suspension of large yellow croaker macrophages LYC-FM to 2×105 Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight at 28°C. (2) Remove the culture medium and add 100µL of fresh culture medium containing antimicrobial peptides KRK12N, KRK12N-1 or KRK12N-2 to each well. The final concentrations of the antimicrobial peptides are 2, 4, 8, 16, 32, 64 and 128µM. Add 90µL of blank culture medium mixed with 10µL of 2% Triton X-100 and 90µL of blank culture medium mixed with 10µL of 1×PBS (pH=7.4) to the control wells as 100% and 0% cytotoxicity controls, respectively. Each group has 3 replicates. (3) After incubating at 28℃ for 24 hours, add 10µL of CCK-8 solution to each well, mix well, continue incubating at 28℃ for 4 hours, and read the absorbance value at 450nm. (4) Calculate the live cell ratio: viable cell ratio = [(A peptide –A 100% ) / (A 0% -A 100% )]×100%, where A is the absorbance at 450nm.

[0032] Test results as follows Figure 7 As shown, the cell viability of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 at all tested concentrations was higher than 90% in large yellow croaker macrophages LYC-FM. This indicates that antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 have low cytotoxicity.

[0033] Example 5: The temperature stability of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 was determined using the following steps: (1) After diluting the antimicrobial peptide, take 40 μL of the antimicrobial peptide into a 1.5 mL centrifuge tube and treat it at 20℃, 40℃, 60℃, 80℃ and 100℃ for 30 min respectively, and then immediately place it on ice; (2) The concentration of *Pseudomonas proteus* cultured to the logarithmic growth phase was adjusted to 1 × 10⁻⁶ using fresh TSB. 5 CFU / mL; (3) Take 90 μL of Pseudomonas proteus bacterial culture and add it to a 96-well plate. Then add 10 μL of the antimicrobial peptides KRK12N, KRK12N-1 or KRK12N-2 treated in step (1) to each well. Add 10 μL of 1×PBS (pH=7.4) to the positive control. Set up 3 replicates for each temperature treatment group and incubate at 28℃ for 16-18h. (4) After serial dilution, each well was plated on a TSA plate and the number of colonies was counted. (5) Sterilization rate of antimicrobial peptides = (1 - CFU in each temperature treatment group / CFU in the positive control) × 100% Test results as follows Figure 8 As shown, the antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2, after treatment at different temperatures, all exhibited bactericidal rates higher than 99%. This indicates that the antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 possess good temperature stability.

[0034] Example 6: The pH stability of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 was determined using the following steps: (1) Prepare the following buffer solutions with different pH values ​​and filter them aseptically: 50mM glycine-HCl buffer (pH=2.0); 50mM NaAC-HAC buffer (pH=4.0); 50mM MES-NaOH buffer (pH=6.0); 50 mM Tris-HCl buffer (pH=8.0); 50mM glycine-NaOH buffer (pH=10.0).

[0035] (2) Take 30µL of pH buffer and add it to a 1.5mL EP tube. Add 10µL of antimicrobial peptide solution to each tube to make the final concentration 10×MBC. Mix well and incubate at room temperature for 4 hours. (3) Inoculate *Pseudomonas proteus* into TSB liquid medium, shake and culture until the logarithmic growth phase, then adjust the bacterial concentration to 1×10⁻⁶. 5 CFU / mL; (4) Add 90µL of Pseudomonas proteus bacterial solution to each well of a 96-well plate, and then add 10µL of the antimicrobial peptide solution after step (2) to each well. Add 10µL of deionized water to the positive control well. Set up 3 replicate wells for each group. (5) Place the 96-well plate in an incubator at 28°C and incubate overnight. Take the culture from each well, perform serial dilutions, and spread it on TSA plates. Count the number of colonies after 14-16 hours. (6) Calculate the sterilization rate using the following formula: Sterilization rate = [1 - (CFU in the treatment group / CFU in the positive control group)] × 100% Test results as follows Figure 9As shown, except for a slight decrease in the bactericidal activity of KRK12N-2 at pH=10, the bactericidal rates of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 against *Pseudomonas proteus* remained above 99.99% under other test conditions. This indicates that antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 possess good pH stability.

[0036] Example 7: The salt ion stability determination of antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 was performed using the following steps: (1) Preparation of salt solutions: Prepare 1500mM NaCl, 45mM KCl, 60μM NH4Cl, 80μM ZnCl2, 10mM MgCl2, 20mM CaCl2 and 40μM FeCl3 with ultrapure water respectively, adjust pH=7.4, and sterilize at high temperature; (2) Shake the *Pseudomonas proteus* to the logarithmic growth phase, measure the OD value and convert it into bacterial concentration, then adjust the bacterial concentration to 1×10⁻⁶ using culture medium. 5 CFU / mL; (3) Add 80 μL of Pseudomonas proteus bacterial culture to a 96-well plate, then add 10 μL of antimicrobial peptide and 10 μL of salt solution in sequence to make the final concentration of antimicrobial peptide 1×MBC. Add 10 μL of 1×PBS (pH=7.4) and 10 μL of salt solution to the positive control. Set up 3 replicates for each. (4) Incubate overnight at 28°C, and count the number of colonies after serial dilution in each well; (5) Antimicrobial peptide bactericidal rate = (1 - CFU in the salt solution treatment group / CFU in the positive control group) × 100% Test results as follows Figure 10 As shown, antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 retain over 90% of their bactericidal activity after exposure to different salt ions. Antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 exhibit good salt ion stability.

[0037] In summary, antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 exhibit broad-spectrum antimicrobial activity, showing good inhibitory effects against both Gram-positive and Gram-negative bacteria. Furthermore, they possess low hemolytic activity, low cytotoxicity, and good stability to temperature, pH, and different salt ions. Therefore, antimicrobial peptides KRK12N, KRK12N-1, and KRK12N-2 hold promise for applications in the preparation of antibacterial drugs and feed additives.

[0038] 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. The antimicrobial peptide KRK12N based on RPL39 protein of large yellow croaker, characterized in that: The amino acid sequence of the antimicrobial peptide KRK12N is: Lys-Thr-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2.

2. The method for modifying the antimicrobial peptide KRK12N according to claim 1, characterized in that: Replace the threonine at position 2 in the amino acid sequence of the antimicrobial peptide KRK12N with tryptophan.

3. The antimicrobial peptide KRK12N-1 obtained by the method described in claim 2, characterized in that: The amino acid sequence of the antimicrobial peptide KRK12N-1 is: Lys-Trp-Phe-Arg-Ile-Lys-Arg-Phe-Leu-Ala-Lys-Lys-NH2.

4. The method for modifying the antimicrobial peptide KRK12N-1 according to claim 3, characterized in that: The lysine at position 6 and the phenylalanine at position 8 in the amino acid sequence of the antimicrobial peptide KRK12N-1 were interchanged.

5. The antimicrobial peptide KRK12N-2 obtained by the method described in claim 4, characterized in that: The amino acid sequence of the antimicrobial peptide KRK12N-2 is: Lys-Trp-Phe-Arg-Ile-Phe-Arg-Lys-Leu-Ala-Lys-Lys-NH2.

6. The use of the antimicrobial peptide KRK12N of claim 1, the antimicrobial peptide KRK12N-1 of claim 3, and the antimicrobial peptide KRK12N-2 of claim 5 in the preparation of antibacterial drugs.

7. The application according to claim 6, characterized in that: The bacteria include Gram-negative bacteria and Gram-positive bacteria.

8. The application according to claim 7, characterized in that: The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella pullorum, Pseudomonas proteus, and Vibrio harveyi; the Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae, Micrococcus luteus, and Bacillus subtilis.

9. The application of the antimicrobial peptide KRK12N of claim 1, the antimicrobial peptide KRK12N-1 of claim 3, and the antimicrobial peptide KRK12N-2 of claim 5 in the preparation of feed additives.

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