Broad-spectrum antibacterial peptide 910302 and application thereof

Abstract

The invention relates to a broad-spectrum antibacterial peptide 910302 and application thereof, and relates to the technical field of biology. The antibacterial peptide has broad-spectrum antibacterial activity, can inhibit gram-positive bacteria such as staphylococcus aureus, gram-negative bacteria such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa, and fungi such as candida auriculata and candida albicans, and has high antibacterial activity. The compound has a relatively strong inhibition effect on clinically separated drug-resistant strains with multiple drug resistance. In addition, the antibacterial peptide is low in hemolytic activity, small in toxicity, short in synthetic sequence, small in molecular weight and easy to chemically synthesize. And the in-vivo safety is ensured while efficient sterilization is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a broad-spectrum antimicrobial peptide 910302 and an application thereof. Background Art

[0002] Antibiotic resistance is one of the severe challenges facing modern medicine. In recent decades, the number of pathogenic microorganisms that develop antibiotic resistance has continued to increase, and multidrug-resistant bacteria and super-resistant bacteria have emerged in an endless stream. The number of deaths caused by infections caused by resistant bacteria reaches 700,000 worldwide each year.

[0003] Antimicrobial peptides stand out due to their mechanism of action and have become a powerful weapon against drug-resistant bacterial infections. Antimicrobial peptides are small peptides with antimicrobial activity that are widely present in nature. They have the advantages of being difficult to develop drug resistance and acting quickly. At the same time, because they are easily degraded, they will not cause persistent pollution to the environment. Antimicrobial peptides can achieve the effect of inhibiting pathogenic microorganisms through a variety of mechanisms of action. Among them, the more common mechanism of action is to bind to the cell membrane of pathogenic microorganisms and disrupt the cell membrane structure; or directly form micropores in the cell membrane to allow the cell contents to flow out, ultimately killing the pathogenic microorganisms. In recent years, antimicrobial peptides that can resist multi-drug resistant bacteria and are not easy to develop drug resistance have been considered to be the next generation of antimicrobial agents to replace traditional antibiotics.

[0004] The antimicrobial peptides reported so far have the disadvantages of low biological activity, high toxicity, and poor stability. The antimicrobial strength is generally expressed by the minimum inhibitory concentration (MIC). If the MIC value of the antimicrobial peptide is too high, a large dose is required to achieve effectiveness, and such a product is worthless. In addition, increasing the drug concentration will also cause problems such as cytotoxicity and cell hemolysis. As a polypeptide substance, antimicrobial peptides are not particularly resistant to high temperatures and acid and alkali. Antimicrobial peptides are biologically active polypeptides. During the biological fermentation process, the host bacteria will produce a lot of proteases. These proteases have the activity of degrading polypeptides, so that the antimicrobial peptides are degraded and lost during the fermentation process, and lose their production value. Natural antimicrobial peptides or antimicrobial peptides obtained by genetic engineering technology account for a very small proportion of the separation and purification matrix, and there are a large number of impurities and other substances, and the separation and purification cost is high. Therefore, it is urgent to design and develop antimicrobial peptides with simple structure, low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, high biological activity, and synthesized using solid-phase chemical methods through artificial intelligence to deal with the emergence of drug-resistant bacteria. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In order to solve the above technical problems, the present invention provides a broad-spectrum antimicrobial peptide 910302 and its application.

[0007] (II) Technical solution

[0008] Based on this, according to one aspect of the present invention, a broad-spectrum antimicrobial peptide 910302 is provided, wherein the antimicrobial peptide is an antimicrobial peptide 910302 having an amino acid sequence shown in SEQ ID: KLAGLAKKLAGLAKKLAGLAK; the sequence length of the antimicrobial peptide is 21, the charge number is 5.995, the hydrophilicity is 0.57, and the isoelectric point is 10.70.

[0009] The design steps of the broad-spectrum antimicrobial peptide 910302 are as follows:

[0010] Step S1: Synthesis of antimicrobial peptides, using a chemical organic solid phase synthesis method, condensing the sequence from the C-terminus to the N-terminus to complete the peptide chain, using a cutting solution to cut the polypeptide from the resin, and at the same time cut off the side chain protecting group, the crude peptide was precipitated with ice ether, and the crude polypeptide was purified by preparative high performance liquid chromatography to a purity of more than 95% and freeze-dried.

[0011] According to another aspect of the present invention, there is provided an application of a broad-spectrum antimicrobial peptide 910302, wherein the antimicrobial peptide is used to prepare an inhibitor for antibacterial infection, wherein the minimum inhibitory concentration of the antimicrobial peptide 910302 against Staphylococcus aureus is 0 μg / mL, the minimum inhibitory concentration against Escherichia coli is 3.9 μg / mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg / mL, the minimum inhibitory concentration against Acinetobacter baumannii is 1.95 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 62.5 μg / mL.

[0012] Preferably, the antimicrobial peptide is used to prepare an inhibitor against fungal infection, the fungi are Candida auris and Candida albicans, and the minimum inhibitory concentration of the antimicrobial peptide 910302 against Candida auris is 125 μg / mL, and the minimum inhibitory concentration against Candida albicans is 1.95 μg / mL.

[0013] (III) Beneficial effects

[0014] Compared with the prior art, the present invention provides a broad-spectrum antimicrobial peptide 910302 and its application, which has the following beneficial effects:

[0015] The invention discloses a broad-spectrum antimicrobial peptide 910302 and its application. The antimicrobial peptide is an antimicrobial peptide 910302 having an amino acid sequence shown in SEQ ID: KLAGLAKKLAGLAKKLAGLAK. The antimicrobial peptide has a broad-spectrum antimicrobial activity, and can inhibit Gram-positive bacteria such as Staphylococcus aureus, Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, and fungi such as Candida auris and Candida albicans, and has high antimicrobial activity, and has a strong inhibitory effect on multidrug-resistant strains isolated from clinical sites. In addition, the antimicrobial peptide has low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, and is easy to chemically synthesize. It ensures in vivo safety while achieving high bactericidal efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the drug sensitivity test results of the clinical drug-resistant bacteria of the present invention;

[0017] Figure 2 Schematic diagram of the cytotoxicity of the antimicrobial peptide of the present invention;

[0018] Figure 3 Schematic diagram of the hemolytic activity of the antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0020] As analyzed in the background technology of this application, the natural antimicrobial peptides or antimicrobial peptides obtained by genetic engineering technology in the prior art account for a very small proportion in the separation and purification matrix, and there are a large number of impurities and other substances, and the separation and purification cost is high. In order to solve this problem, this application provides a broad-spectrum antimicrobial peptide 910302 and its application.

[0021] In a typical embodiment of the present application, a broad-spectrum antimicrobial peptide 910302 is provided, wherein the antimicrobial peptide is an antimicrobial peptide 910302 having an amino acid sequence shown in SEQ ID: KLAGLAKKLAGLAKKLAGLAK; the antimicrobial peptide has a sequence length of 21, a charge number of 5.995, a hydrophilicity of 0.57, and an isoelectric point of 10.70.

[0022] Using the technical solution of the present application, the present application provides a broad-spectrum antimicrobial peptide 910302, the synthesis steps of which are as follows:

[0023] The antimicrobial peptide sequence of the present invention is: KLAGLAKKLAGLAKKLAGLAK; the synthesis of the antimicrobial peptide is completed by using a chemical organic solid phase synthesis method, the sequence is sequentially condensed from the C-terminus to the N-terminus to form a peptide chain, the polypeptide is cut off from the resin with a cutting liquid, and the side chain protecting group is cut off at the same time, the crude peptide is precipitated with ice ether, and the crude polypeptide is purified by a preparative high performance liquid chromatography to a purity of more than 95% and then freeze-dried.

[0024] In the following, in conjunction with the examples, the activity of the antimicrobial peptides, the minimum inhibitory concentration of multidrug-resistant strains, the cytotoxicity of the antimicrobial peptides, and the hemolytic activity of the antimicrobial peptides are measured to further illustrate the beneficial effects of the present application.

[0025] Example 1

[0026] This example provides an activity assay for antimicrobial peptides, and the activity assay protocol is as follows:

[0027] 1. Determination of minimum inhibitory concentration of standard strains.

[0028] Bacteria: Staphylococcus aureus (ATCC25923) was streaked into TSB medium, Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC 43816), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC27853) were streaked into LB medium, and placed in a 37°C CO2 incubator for inversion culture for 24 hours. After a single colony is formed on the plate, pick a single colony and transfer it to the corresponding liquid culture medium, Staphylococcus aureus to TSB liquid culture medium, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa to LB liquid culture medium. Place in a 37°C incubator and shake for 4 hours until OD 600 =0.6, dilute the bacterial solution into CAMHB liquid culture medium and adjust the bacterial volume to 10 5 CFU / mL, save for later use;

[0029] Fungi: Candida auris (ATCC 12766), Candida albicans (ATCC 10231). The strain was inoculated on a Sabouraud dextrose solid culture plate (SDA) and cultured at 25°C for 24 hours, and activated at least twice in a row. Then a single colony was picked and placed in Sabouraud dextrose liquid medium (SDB), and placed in a shaker at 37°C and 200rpm for overnight culture. The overnight culture solution was diluted 10 times twice with Sabouraud liquid medium (SDB). Take a piece of blood cell counting, soak it in 75% alcohol, wipe it dry with lens paper, and cover the counting area with a coverslip. Pipette 10μL of homogenized bacterial solution from the diluted bacterial solution and inject it from the lower edge of the coverslip to fill the counting area with the bacterial suspension to avoid bubbles. After 1 minute of rest, under a microscope with a 40x objective lens, it can be seen that the bacteria basically no longer float with the liquid. Use a manual counter to count the number of spores in the four squares in the upper left, lower left, upper right, lower right and the five squares in the center according to the diagonal direction, which is N (for budding spores, when the bud reaches half the size of the mother body, it is counted as two bacteria). Bacterial liquid concentration = N / 5×25×10 4 CFU / mL. The bacterial solution concentration was calculated and serially diluted within ten times using RPMI-1640 medium to adjust the bacterial solution concentration to 2×10 3 CFU / ml. Since the final volume of the bacterial solution doubles when it is added to the drug sensitivity plate, it is equivalent to being diluted by one time, so the final concentration of the bacterial solution in the drug sensitivity plate is 1×10 3 CFU / m;

[0030] Dissolve 1 mg of antimicrobial peptide in 1 mL of saline to a concentration of 1 mg / mL. Add 100 μL of the two-fold gradient dilution of antimicrobial peptide to a 96-well plate, and then add 100 μL of 10 5 CFU / mL of bacterial solution. At this time, the concentrations of antimicrobial peptides were 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, 31.25μg / mL, 15.63μg / mL, 7.81μg / mL, 3.91μg / mL and 1.95μg / mL. Record the OD 600 100 μL bacterial solution and 100 μL saline solution mixture were used as negative control. The 96-well plate was placed in a 37°C CO2 incubator and cultured for 20 h. The OD was recorded again. 600 The minimum inhibitory concentration (MIC) is the lowest concentration at which the growth of the strain is completely inhibited. The calculation formula for the antimicrobial peptide inhibition rate is as follows;

[0031]

[0032] The results in Table 1 show that the antimicrobial peptides of the present invention have obvious inhibitory effects on bacteria: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, and have obvious inhibitory effects on fungi: Candida auris and Candida albicans. The minimum inhibitory concentration for Escherichia coli is 3.9μg / mL, the minimum inhibitory concentration for Klebsiella pneumoniae is 125μg / mL, the minimum inhibitory concentration for Acinetobacter baumannii is 1.95μg / mL, the minimum inhibitory concentration for Pseudomonas aeruginosa is 62.5μg / mL, the minimum inhibitory concentration for Candida auris is 125μg / mL, and the minimum inhibitory concentration for Candida albicans is 1.95μg / mL. It can be seen that the antibacterial effect of the antimicrobial peptides of the present invention on Escherichia coli, Acinetobacter baumannii and Candida albicans is particularly obvious;

[0033] Table 1 Minimum inhibitory concentration of antimicrobial peptides against standard strains

[0034]

[0035] (2) Determination of minimum inhibitory concentration of multidrug-resistant strains

[0036] In order to verify the antibacterial effect against multidrug-resistant bacteria, 4 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa were isolated from the clinic, a total of 16 multidrug-resistant bacteria, and their resistance was as follows Figure 1 As shown;

[0037] The minimum inhibitory concentration determination method was used to determine the minimum inhibitory concentration of antimicrobial peptides against the above 16 clinically isolated multidrug-resistant bacteria. The results are shown in the following table:

[0038] Table 2 Minimum inhibitory concentration for multidrug-resistant bacteria

[0039]

[0040] Combined with the above results, it can be seen that the antimicrobial peptide of the present invention has excellent antibacterial effects on different clinical multidrug-resistant bacteria: the minimum inhibitory concentration for multidrug-resistant strains of Acinetobacter baumannii is 1.95 μg / mL; the minimum inhibitory concentration for multidrug-resistant strains of Escherichia coli is 7.8 μg / Ml; the minimum inhibitory concentration for multidrug-resistant strains of Klebsiella pneumoniae is 31.3 μg / mL;

[0041] Example 2

[0042] This example provides a cytotoxicity assay for antimicrobial peptides, as follows:

[0043] Prepare 100 μL of cell suspension in a 96-well plate (10 cells per well). 4). Pre-culture the culture plate in an incubator for 24 hours (at 37°C, 5% CO2). Add 100 μL of the peptide to be tested with a concentration range of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 μg / mL to the culture plate. Incubate in an incubator for 24 hours. Add 10 μL of CCK-8 solution to each well. Incubate the culture plate in an incubator for 4 hours. Measure the absorbance at 450 nm with an enzyme-labeled instrument. The formula for cell activity detection is as follows;

[0044]

[0045] A (drug added): absorbance of wells with cells, CCK-8 solution, and drug solution;

[0046] A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells;

[0047] A(0 drug addition): absorbance of the wells with cells and CCK-8 solution but no drug solution;

[0048] Cell viability: cell proliferation activity or cytotoxic activity;

[0049] Cytotoxicity test is an experimental method widely used in the field of biological research to evaluate the degree of toxicity of compounds to cells in organisms. This test aims to reveal whether the antimicrobial peptides of the present invention will affect the structure and function of cells, so as to evaluate the potential risks of antimicrobial peptides to the health of organisms, thereby providing important data for drug development and safety assessment;

[0050] Specific as Figure 2 As shown, the cell proliferation activity of the antimicrobial peptide of the present invention is 100% when the concentration is 100 μg / mL, which is consistent with the result of culturing cells in the negative control cell culture medium DMEM, that is, the antimicrobial peptide has no toxic effect on cells at this concentration.

[0051] Example 3

[0052] This example provides a hemolytic assay of antimicrobial peptides, as follows:

[0053] Take a 96-well plate and add 100 μL of the peptide to be tested in the concentration range of 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 μg / mL. Pipette 100 μL of 2% red blood cells into the 96-well plate and add it to the drug solution and mix well. Incubate at 37℃ for 1 hour, centrifuge at 5000 rpm / min for 5 minutes; carefully pipette 100 μL of supernatant into the enzyme-linked well, set up four parallel control wells, and measure OD 570nm with an enzyme-linked well reader. The negative control is saline, and the positive control is 10% Triton-saline. Physiological saline was selected as the negative control and TritionX-100 as the positive control. The results met the experimental requirements: TritionX-100 (positive control) has the effect of increasing the permeability of the cell membrane, making the system non-isotonic, causing the red blood cells to rupture, hemolysis to occur, and a clear red solution to appear; while the physiological saline solution control tube (negative control tube) had no hemolysis or red blood cell agglutination. After comparing each experimental well with the negative control tube and the positive control tube, the red blood cells in all experimental wells sank, the supernatant was colorless and clear, no hemolysis occurred, and the precipitated red blood cells dispersed after shaking, and no red blood cell agglutination occurred. The absorbance of the supernatant was detected by an enzyme marker at OD570nm, and the hemolysis rate of the polypeptide was calculated. The formula is as follows:

[0054]

[0055] Hemolysis refers to reactions such as extravascular or intravascular hemolysis and erythrocyte aggregation caused by drug preparations. This experiment aims to investigate whether the antimicrobial peptides of the present invention can cause hemolysis and erythrocyte aggregation in vitro. The active ingredients of the drug and its metabolites, excipients, related substances and physicochemical properties (such as pH value, osmotic pressure, etc.) may cause hemolysis, and severe toxicity will affect the safety and effectiveness of the drug. Therefore, before the clinical application of the drug, the local and / or systemic toxicity caused by its preparation at the administration site should be studied to indicate the possible toxic reactions, toxic target organs, safety range, clinical research monitoring indicators during clinical application, and provide reference for clinical detoxification or rescue measures to ensure the safety and effectiveness of clinical drug use.

[0056] like Figure 3 As shown, the hemolysis rate of the antimicrobial peptide of the present invention is 0% when the concentration is 100 μg / mL, which is consistent with the result of culturing red blood cells in the negative control saline, that is, the antimicrobial peptide does not cause hemolysis at this concentration.

[0057] From the data obtained in Example 1, Example 2, and Example 3, it can be seen that the antimicrobial peptide has a broad-spectrum antimicrobial activity, can inhibit Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, as well as fungi such as Candida auris and Candida albicans, and has high antimicrobial activity, and has a strong inhibitory effect on multidrug-resistant strains isolated from clinical sites. In addition, the antimicrobial peptide has low hemolytic activity, low toxicity, short synthetic sequence, small molecular weight, and is easy to chemically synthesize. It ensures in vivo safety while efficiently killing bacteria.

[0058] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A broad-spectrum antimicrobial peptide 910302, characterized in that: The antimicrobial peptide is antimicrobial peptide 910302 having an amino acid sequence shown in SEQ ID: KLAGLAKKLAGLAKKLAGLAK; The antimicrobial peptide has a sequence length of 21, a charge number of 5.995, a hydrophilicity of 0.57, and an isoelectric point of 10.

70.

2. A use of a broad-spectrum antimicrobial peptide 910302 as claimed in claim 1, characterized in that: The antimicrobial peptide is used for preparing an inhibitor against bacterial infection.

3. A use of a broad-spectrum antimicrobial peptide 910302 as claimed in claim 1, characterized in that: The antimicrobial peptide is used for preparing an inhibitor against fungal infection.

4. The use of a broad-spectrum antimicrobial peptide 910302 according to claim 2, characterized in that: The bacteria are Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa.

5. The use of a broad-spectrum antimicrobial peptide 910302 according to claim 3, characterized in that: The fungi are Candida auris and Candida albicans.

6. The use of a broad-spectrum antimicrobial peptide 910302 according to claim 4, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide 910302 against Staphylococcus aureus is 0 μg / mL, the minimum inhibitory concentration against Escherichia coli is 3.9 μg / mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 125 μg / mL, the minimum inhibitory concentration against Acinetobacter baumannii is 1.95 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 62.5 μg / mL.

7. The use of a broad-spectrum antimicrobial peptide 910302 according to claim 5, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide 910302 against Candida auris is 125 μg / mL, and the minimum inhibitory concentration against Candida albicans is 1.95 μg / mL.

8. The broad-spectrum antimicrobial peptide 910302 according to claim 1, characterized in that: The design steps are as follows: Step S1: Synthesis of antimicrobial peptides, using a chemical organic solid phase synthesis method, condensing the sequence from the C-terminus to the N-terminus to complete the peptide chain, and using a cutting solution to cut the peptide from the resin; Step S2: the side chain protecting groups are removed, the crude peptide is precipitated with ice ether, the crude peptide is purified by preparative high performance liquid chromatography to a purity of more than 95% and then freeze-dried.

Images