A Pseudomonas aeruginosa targeted antimicrobial peptide RAK18 and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide RAK18 targeting Pseudomonas aeruginosa and its application. The full sequence of the antimicrobial peptide RAK18 is Arg‑Lys‑Lys‑Met‑Thr‑Tyr‑Gln‑Lys‑Met‑Ala‑Arg‑Ala‑Leu‑Arg‑Asn‑Tyr‑Gly‑Lys, with a molecular weight of 2243.71 Da and an isoelectric point of 11.5, and it is a basic polypeptide. The antimicrobial peptide RAK18 of the present invention can target and kill Pseudomonas aeruginosa, and at the same time has weak hemolytic activity and less cytotoxicity. The antimicrobial peptide RAK18 is expected to replace antibiotics and be used in the prevention and treatment of visceral white spot disease in farmed fish.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Pseudomonas aeruginosa targeted antimicrobial peptide RAK18 and an application thereof. Background Art

[0002] Antimicrobial peptides are a class of natural small-molecule peptides with antimicrobial activity of biological origin, which are positively charged. As an important component of the innate immune system, antimicrobial peptides constitute the first line of defense of the body in the process of resisting infection by external pathogenic microorganisms. Antimicrobial peptides have high-efficiency and broad-spectrum bactericidal activity, and also have a killing effect on a variety of antibiotic-resistant bacteria. Antimicrobial peptides mainly exert their bactericidal effects through membrane-targeted and non-membrane-targeted effects. In the process of membrane-targeted action, the main target of antimicrobial peptides is the cell membrane of microorganisms. By changing the membrane potential, it induces the depolarization of the microbial cell membrane, changes the membrane structure, and then forms transmembrane holes in the cell membrane, causing the leakage of intracellular substances, thereby killing the target cells. In the process of non-membrane-targeted action, antimicrobial peptides interact with the microbial cell membrane, enter the cell, act on the intracellular components of the bacteria, and interfere with the normal metabolism of the bacteria, but this process does not change the permeability of the cell membrane. After entering the cell, the antimicrobial peptides bind to related molecules in the cell. The large amount of antimicrobial peptides in the bacterial cytoplasm inhibits the synthesis of bacterial DNA, RNA, protein and cell wall, causing bacterial death.

[0003] In order to make antimicrobial peptides play a better role in practical applications, it is necessary to artificially design and develop new synthetic peptides. Natural antimicrobial peptides have problems such as weak biological activity, high toxicity, and poor stability, which limit the practical application of antimicrobial peptides. Artificially designed antimicrobial peptides can solve these problems in a targeted manner, improve the biological activity of antimicrobial peptides, reduce their toxicity, and enhance their stability, thereby broadening their scope of application. In order to reduce the damage of antimicrobial peptides to probiotics, artificially designed antimicrobial peptides should have strong killing activity against target pathogenic microorganisms and have no or very low killing activity against other microorganisms. This can reduce the killing of antimicrobial peptides to probiotics and improve the safety of the use of antimicrobial peptides.

[0004] Pseudomonas aeruginosa ( Pseudomonas plecoglossicida ) is a Gram-negative bacterium with a rod-shaped morphology and polar flagella, which can infect fish such as large yellow croaker, grouper and rainbow trout. After Pseudomonas aeruginosa infects fish, it can cause white nodules in the spleen, kidneys and liver of the fish, so the disease is called visceral white spot disease. Visceral white spot disease caused by Pseudomonas aeruginosa is a key disease that plagues the large yellow croaker breeding industry. It has a high morbidity and mortality rate, causing huge economic losses to the aquaculture industry and seriously restricting the development of the aquaculture industry. The development of antimicrobial peptides that can specifically kill Pseudomonas aeruginosa can provide potential therapeutic drugs for the prevention and treatment of visceral white spot disease in aquaculture animals. Summary of the invention

[0005] In order to obtain an antimicrobial peptide that can target and kill Pseudomonas aeruginosa and has good biosafety, the present invention synthesized a Pseudomonas aeruginosa targeting antimicrobial peptide RAK18 based on large yellow croaker PU.1 (NCBI accession numbers XP_010746449.1 and XP_010746450.1).

[0006] The amino acid sequence of the antimicrobial peptide RAK18 is: Arg-Lys-Lys-Met-Thr-Tyr-Gln-Lys-Met-Ala-Arg-Ala-Leu-Arg-Asn-Tyr-Gly-Lys.

[0007] The preparation method of the antimicrobial peptide RAK18 is as follows:

[0008] (1) Using the protein sequence of large yellow croaker PU.1 as a template, a linear peptide of 18 amino acids was extracted to obtain the peptide RAK18;

[0009] (2) The antimicrobial peptide RAK18 was synthesized by solid phase chemical synthesis.

[0010] The present invention also provides the use of the antimicrobial peptide RAK18 in the preparation of a drug for preventing and treating Pseudomonas aeruginosa infection.

[0011] The present invention also provides the use of the antimicrobial peptide RAK18 in the preparation of feed additives.

[0012] The antimicrobial peptide RAK18 of the present invention has the following advantages and beneficial effects: the antimicrobial peptide RAK18 prepared by the present invention can target and kill Pseudomonas aeruginosa, and has no bactericidal activity or only very weak antibacterial activity against other tested Gram-negative bacteria and Gram-positive bacteria. It also has weak hemolytic activity, and the hemolytic rate of the antimicrobial peptide RAK18 on the red blood cells of large yellow croaker at each detection concentration is lower than 0%, and the cytotoxicity is relatively small. When the detection concentration reaches 128µM, the cell survival rate of the antimicrobial peptide RAK18 on the large yellow croaker macrophage LYC-FM is still higher than 75%. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the mass spectrum of the antimicrobial peptide RAK18.

[0014] Figure 2 The bactericidal activity (A) and bactericidal kinetic curve (B) of the antimicrobial peptide RAK18 against Pseudomonas aeruginosa.

[0015] Figure 3 Growth curves of Gram-negative bacteria (A) and Gram-positive bacteria (B) after treatment with the antimicrobial peptide RAK18.

[0016] Figure 4Figure 2 shows the cytotoxicity (A) and erythrocyte hemolytic activity (B) of the antimicrobial peptide RAK18 on LYC-FM. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0018] Example 1: Design and synthesis of antimicrobial peptide RAK18, the specific steps are:

[0019] (1) Using the protein sequence of large yellow croaker PU.1 as a template, a linear peptide of 18 amino acids was extracted and named RAK18;

[0020] The amino acid sequence of the linear peptide RAK18 is:

[0021] Arg-Lys-Lys-Met-Thr-Tyr-Gln-Lys-Met-Ala-Arg-Ala-Leu-Arg-Asn-Tyr-Gly-Lys

[0022] The theoretical molecular weight of RAK18 is 2243.71, the net charge is +7, and the isoelectric point is 11.5.

[0023] (2) The antimicrobial peptide RAK18 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the solid-phase chemical synthesis method.

[0024] The mass spectrum of the antimicrobial peptide RAK18 is shown in Figure 1 .

[0025] Example 2: Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide RAK18, the specific steps are:

[0026] (1) Pick a single colony of the test strain and place it in liquid culture medium. Cultivate it in a shaking manner until the logarithmic growth phase. Use culture medium to adjust the concentration of the bacterial solution to 1.2×10 5 CFU / mL;

[0027] (2) Take the peptide at a concentration of 1280 µM and serially dilute it to a final concentration of 5, 10, 20, 40, 80, 160, 320, and 640 µM;

[0028] (3) Add 10 µL of the diluted peptide solution (5–1280 µM) to columns 1-9 of the 96-well plate, then add 90 µL of bacterial solution to each well. Add 90 µL of bacterial solution and 10 µL of PBS to the 10th column as the positive control, and add 90 µL of culture medium and 10 µL of PBS to the 11th column as the negative control. Set up 3 replicates for each well. The above process should be completed within 15 min.

[0029] (4) Place the 96-well plate in an incubator for 12-18 hours. After taking it out, observe with the naked eye that the minimum peptide concentration at which there is no turbidity at the bottom of the well is the minimum inhibitory concentration (MIC).

[0030] (5) Place the 96-well plate in a spectrophotometer and read the absorbance at 600 nm. Use the polypeptide concentration as the horizontal axis and the absorbance value as the vertical axis to draw the growth curves of different test bacteria. The inflection point value is the minimum inhibitory concentration.

[0031] Test results such as Figure 2 As shown in A, the antimicrobial peptide RAK18 has bactericidal activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration and a minimum bactericidal concentration of 64 μM. Figure 3 As shown, the antimicrobial peptide RAK18 has no bactericidal activity or only very weak antibacterial activity against other tested Gram-negative bacteria and Gram-positive bacteria, indicating that the antimicrobial peptide RAK18 of the present invention has targeted bactericidal activity against Pseudomonas aeruginosa.

[0032] Example 3: Determination of the bactericidal kinetics of the antimicrobial peptide RAK18 against Pseudomonas aeruginosa, the specific steps are as follows:

[0033] (1) Pick a single colony of Pseudomonas aeruginosa and place it in liquid culture medium. Cultivate it in a shaking manner until the logarithmic growth phase. Use culture medium to adjust the concentration of the bacterial solution to 1.2×10 5 CFU / mL;

[0034] (2) Mix 90 µL of bacterial culture and 10 µL of peptide in a 96-well plate. The final peptide concentration is 1×MBC. The negative control group is 90 µL of liquid culture medium plus 10 µL PBS, and the positive control group is 90 µL of bacterial culture and 10 µL PBS.

[0035] (3) Samples were collected at different time points (0 h, 0.5 h, 2.5 h, 3.5 h, 4.5 h, 5.5 h, 6.5 h, 8.5 h, 10.5 h, and 12.5 h), diluted in multiples, and then spread on solid culture medium. o After culturing in a C incubator for 12-18 h, the cells were counted and the bacterial survival rate at each time point was calculated.

[0036] Test results such as Figure 2As shown in B, the antimicrobial peptide RAK18 showed a certain bactericidal effect at 0.5h of action and could kill all Pseudomonas aeruginosa within 4.5 hours.

[0037] Example 4: Determination of hemolytic activity of antimicrobial peptide RAK18, the specific steps are:

[0038] (1) Take whole blood of yellow croaker and add anticoagulant to 1000× g Centrifuge at room temperature for 10 minutes and discard the supernatant;

[0039] (2) After resuspending in PBS, 1000× g Centrifuge for 10 minutes, discard the supernatant, and repeat three times;

[0040] (3) Resuspend the cells in PBS and adjust the cell concentration to 1×10 8 cells / mL;

[0041] (4) Add 120 µL of cell suspension to each well of a 96-well plate, add 80 µL of different concentrations of antimicrobial peptide RAK18 to a final concentration of 2, 4, 8, 16, 32, 64, and 128 µM, set up three replicates, and add 80 µL of 5% Triton-X 100 and 80 µL of PBS to the control wells as controls for 100% and 0% hemolysis, respectively;

[0042] (5) The 96-well plate was incubated at 28°C for 2 h at 1000 × g Centrifuge for 10 min, take 100 µL of supernatant into a 96-well plate, and read the absorbance at 405 nm;

[0043] (6) Calculation of hemolytic activity:

[0044] Hemolytic activity = [(A peptide - A 0% lysis) / (A 100% Lysis - A 0% lysis)] × 100%, where A is the absorbance at 405 nm.

[0045] Test results such as Figure 4 As shown in A, the hemolytic rate of the antimicrobial peptide RAK18 at each detected concentration on the red blood cells of large yellow croaker was lower than 0%, indicating that the antimicrobial peptide RAK18 of the present invention has little hemolytic effect on red blood cells.

[0046] Example 5: Cytotoxicity determination of antimicrobial peptide RAK18, the specific steps are:

[0047] (1) Adjust the number of large yellow croaker macrophage LYC-FM cells to 2×10 5cells / mL, 100 µL of cell suspension was added to each well of a 96-well plate and cultured overnight at 28°C;

[0048] (2) Remove the culture medium and add 90 µL of fresh culture medium and 10 µL of different concentrations of antimicrobial peptide RAK18 to each well. The final concentrations of the antimicrobial peptide are 2, 4, 8, 16, 32, 64, and 128 µM. Add 90 µL of fresh culture medium and 10 µL of 20% Triton X-100 to the positive control wells (100% cytotoxicity group), and add 90 µL of fresh culture medium and 10 µL of PBS to the negative control wells (0% cytotoxicity group). Set up 3 replicates for each gradient.

[0049] (3) After 24 h of static incubation at 28°C, add 10 µL of CCK-8 solution to each well, mix well, continue incubation at 28°C for 4 h, and read the absorbance at 450 nm.

[0050] (4) Calculate the live cell ratio:

[0051] Live cell ratio = [(A peptide – A 100% lysis) / (A 0% lysis-A 100% lysis)] × 100%, where A is the absorbance at 450 nm.

[0052] Test results such as Figure 4 As shown in B, when the detection concentration reached 128 µM, the cell survival rate of the antimicrobial peptide RAK18 on large yellow croaker macrophage LYC-FM was still higher than 75%, indicating that the antimicrobial peptide of the present invention has low cytotoxicity.

[0053] In summary, the antimicrobial peptide RAK18 of the present invention has the activity of targeted killing of Pseudomonas aeruginosa, weak hemolytic activity, and small cytotoxicity. The antimicrobial peptide of the present invention can replace antibiotics and be applied to the prevention and treatment of fish visceral white spot disease caused by Pseudomonas aeruginosa.

[0054] 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. A Pseudomonas aeruginosa targeted antimicrobial peptide RAK18, characterized in that: The amino acid sequence of the antimicrobial peptide RAK18 is shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide RAK18 as claimed in claim 1 in the preparation of a drug for preventing and treating Pseudomonas aeruginosa infection.

3. Use of the antimicrobial peptide RAK18 as claimed in claim 1 in the preparation of feed additives.

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