Antibacterial peptide K20NH2 and synthesis method and application thereof

Abstract

The present application relates to the technical field of biopharmacy, and particularly relates to an antibacterial peptide K20NH2 and a synthesis method and application thereof.The antibacterial peptide K20NH2 has an amino acid sequence as shown in SEQ ID NO.1.Experiments show that the antibacterial peptide K20NH2 has broad-spectrum antibacterial activity on gram-positive multi-drug resistant bacteria and / or gram-negative multi-drug resistant bacteria, and simultaneously has low cytotoxicity and low hemolyticity.The antibacterial peptide K20NH2 provided by the present application is composed of 14 amino acids, has a short peptide chain, small molecular weight, and is easy to be artificially synthesized, is a small-molecule polypeptide with high application value, and can be used for preparing a novel antibacterial agent, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to an antibacterial peptide K20NH2 and a synthesis method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is publicly known.

[0003] Since the successful industrialization of penicillin in 1938, the research and development of antibiotics has entered a period of rapid development. The use of antibiotics has treated countless patients with bacterial or fungal infections and has made a great contribution to human health. However, with the improper use of antibiotics and the lack of management of antibiotics, many bacteria have developed resistance to antibiotics, making antibiotic therapy ineffective, and bacterial resistance has seriously threatened human health. At the present stage, the number of deaths caused by drug-resistant bacterial infections worldwide each year reaches 700,000, and if the problem of bacterial resistance is not effectively controlled, the total number of deaths is expected to reach 30 million by 2050. Therefore, while accelerating the development of new antibiotics, it is urgent to develop antibacterial agents with different mechanisms of action and target sites from traditional antibiotics in order to reduce the probability of serious infections caused by drug-resistant bacteria.

[0004] Antimicrobial peptides are a class of active biological molecules with endogenous immune response in most organisms, and are a class of polypeptides with antibacterial, antiviral or tumor inhibiting biological activities. Since the antibacterial and bactericidal mechanisms of antimicrobial peptides are different from those of antibiotics, and the probability of bacterial resistance to antimicrobial peptides as a drug treatment is low, antimicrobial peptides are considered to be a new generation of antibacterial agents that can effectively alleviate the threat of bacterial resistance.

[0005] Currently, the methods for obtaining antimicrobial peptides mainly include isolation of natural antimicrobial peptides, mutation design derived from natural antimicrobial peptides, computer virtual design and random peptide library screening. However, the isolation of natural antimicrobial peptides has problems such as complicated process, tedious process, low content, high price and is not suitable for large-scale production; and various design cycles are usually long and the research and development cost is high. In addition, natural antimicrobial peptides also have many limitations, such as: (1) the antibacterial activity is lower than that of traditional antibacterial agents, and a large dose is required for use as an antibacterial agent, resulting in high application cost; (2) the antibacterial activity is positively correlated with the length of the peptide chain, and the antibacterial activity of the antibacterial peptide with a long peptide chain is strong, and the synthesis cost and immunogenicity are also high; (3) leading to hemolytic destruction of eukaryotic cells; (4) easily degraded in vivo; (5) limited sequence arrangement, while polypeptide molecules with sequence diversity constitute a nearly infinite potential drug reservoir. SUMMARY

[0006] In order to overcome the above problems, the present application provides an antibacterial peptide K20NH2 and a synthesis method and application thereof.

[0007] In order to achieve the above technical purposes, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides an antibacterial peptide K20NH2, which is obtained by amide modification of the C-terminal carboxyl group of the antibacterial peptide K20, and the amino acid sequence of K20NH2 is shown in SEQ ID NO. 1.

[0009] The amino acid sequence shown in SEQ ID NO. 1 is KWKIKWPVRWFTKL-NH2.

[0010] In a second aspect, the present application provides a synthesis method of the above antibacterial peptide K20NH2, which comprises:

[0011] The polypeptide is synthesized by solid-phase synthesis, and the antibacterial peptide K20NH2 is obtained after purification.

[0012] In a third aspect, the present application provides a broad-spectrum antibacterial drug or composition containing the above antibacterial peptide K20NH2.

[0013] In a fourth aspect, the present application provides a disinfectant, a detergent, and a preservative containing the above antibacterial peptide K20NH2.

[0014] In a fifth aspect, the present application provides any one of the following applications of the above antibacterial peptide K20NH2:

[0015] 1) for preparing a broad-spectrum antibacterial drug or composition;

[0016] 2) for preparing a disinfectant;

[0017] 3) for preparing a detergent;

[0018] 4) for preparing a preservative.

[0019] The present application has the following advantages:

[0020] (1) The present application high-throughput screens the antibacterial peptide against specific pathogenic bacteria by introducing the preset functional attribute information against specific pathogenic bacteria. Experiments show that the polypeptide has broad-spectrum antibacterial activity against gram-positive multi-drug resistant bacteria and / or gram-negative multi-drug resistant bacteria, and simultaneously has low cytotoxicity and low hemolyticity.

[0021] (2) The antibacterial peptide K20NH2 provided by the application is composed of 14 amino acids, has a short peptide chain, small molecular weight, is easy to artificially synthesize, is a small molecule polypeptide with high application value, and can be used for preparing a novel antibacterial agent and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings are set to explain the application, and do not constitute an improper limitation to the application.

[0023] Figure 1 It is a mass spectrum detection diagram of the antibacterial peptide K20NH2 prepared in Example 1 of the application;

[0024] Figure 2 It is a hemolytic activity experiment result diagram of the antibacterial peptide K20NH2 in Example 3 of the application;

[0025] Figure 3 It is a cytotoxicity experiment result diagram of the antibacterial peptide K20NH2 in Example 4 of the application;

[0026] Figure 4 It is a treatment result diagram of the antibacterial peptide K20NH2 on bacterial infection of mouse skin in Example 5 of the application. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0029] The first typical embodiment of the application provides an antibacterial peptide K20NH2, which is obtained by amide modification of the C-terminal carboxyl of the antibacterial peptide K20, and the amino acid sequence of K20NH2 is shown in SEQ ID NO. 1.

[0030] The amino acid sequence shown in SEQ ID NO. 1 is KWKIKWPVRWFTKL-NH2.

[0031] The second exemplary embodiment of the present application provides a synthesis method of the antibacterial peptide K20NH2, comprising:

[0032] The polypeptide is synthesized by solid phase synthesis, and the antibacterial peptide K20NH2 is obtained after purification.

[0033] The third exemplary embodiment of the present application provides a broad-spectrum antibacterial drug or composition containing the antibacterial peptide K20NH2.

[0034] In one or more embodiments, the bacteria include gram-positive bacteria and / or gram-negative bacteria.

[0035] Preferably, the gram-positive bacteria include Staphylococcus aureus and Enterococcus faecalis.

[0036] Preferably, the gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella.

[0037] In one or more embodiments, the pharmaceutical composition is an internal medicine or an external medicine; the internal medicine is an oral preparation or an injection; and the external medicine includes a patch and a smearing preparation.

[0038] In one or more embodiments, the carriers include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc.

[0039] The fourth exemplary embodiment of the present application provides a disinfectant, a detergent, and a preservative containing the antibacterial peptide K20NH2.

[0040] The fifth exemplary embodiment of the present application provides any one of the following applications of the antibacterial peptide K20NH2:

[0041] 1) for preparing a broad-spectrum antibacterial drug or composition;

[0042] 2) for preparing a disinfectant;

[0043] 3) for preparing a detergent;

[0044] 4) for preparing a preservative.

[0045] In one or more embodiments, the bacteria include gram-positive bacteria and / or gram-negative bacteria.

[0046] Preferably, the gram-positive bacteria include Staphylococcus aureus and Enterococcus faecalis.

[0047] Preferably, the Gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella typhimurium.

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0049] Example 1

[0050] The amino acid sequence of the antimicrobial peptide K20NH2 is shown in SEQ ID NO.1, specifically: KWKIKWPVRWFTKL-NH2. Its molecular weight is 1915 Daltons.

[0051] The antimicrobial peptide K20NH2 was synthesized using a solid-phase synthesis method via a 12-channel semi-automatic peptide synthesizer. The purity of the synthesized peptide was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (>90%). ESI-MS mass spectrometry confirmed that the molecular weight of the antimicrobial peptide K20NH2 was consistent with the theoretical molecular weight. The mass spectra of the antimicrobial peptide K20NH2 are shown below. Figure 1 As shown.

[0052] Example 2

[0053] Determination of the antibacterial activity of the antimicrobial peptide K20NH2 prepared in Example 1

[0054] The method for determining the minimum inhibitory concentration (MIC) recommended by the Clinical Laboratory Standards Institute (CLSI) was used to prepare a series of gradient solutions of the antimicrobial peptide K20NH2 using a two-fold dilution method. The specific steps are as follows:

[0055] (1) Preparation of bacterial cells: The test bacteria, frozen at -20 ℃, were streaked onto NB medium and incubated. Single colonies were picked and inoculated into 10 mL of NB medium, and cultured overnight at 37 ℃ and 200 rpm. The overnight bacterial cells were then inoculated into fresh medium and cultured for 1-2 h until the cells were in the logarithmic growth phase, with an OD600 of 0.4. The colony count of the obtained bacterial solution was adjusted to approximately 10 using MH(B). 6 Approximately CFU / mL.

[0056] (2) Preparation of peptides: Adjust the concentration of antimicrobial peptide K20NH2 to 512 μg / mL, add 100 μL to the first column of wells in a 96-well plate, add 50 μL of MH broth medium to the other wells, then add 50 μL of the peptide solution in well 1 to well 2, and so on serially dilute to well 10, and discard 50 μL.

[0057] (3) Inoculation: 50 μL of the bacteria solution from step (1) was added to the first 11 wells of the 96-well plate using a pipette gun, and the final concentration of the inoculated bacteria was 5 x 105CFU / mL. The 96-well plate was placed on a micro-vibrator for 1 min to mix the contents of each well, and the plate was covered to reduce evaporation during incubation. The plate was then incubated at 37 °C for 18 h. The 11th well was used as a positive control, i.e., 50 μL of MH broth and 50 μL of the bacteria solution were added. The 12th well was used as a negative control, i.e., 100 μL of MH broth was added. The concentration of the antibacterial peptide in the first 10 wells was then decreased in a stepwise manner. 5 CFU / mL. The 96-well plate was placed on a micro-vibrator for 1 min to mix the contents of each well, and the plate was covered to reduce evaporation during incubation. The plate was then incubated at 37 °C for 18 h. The 11th well was used as a positive control, i.e., 50 μL of MH broth and 50 μL of the bacteria solution were added. The 12th well was used as a negative control, i.e., 100 μL of MH broth was added. The concentration of the antibacterial peptide in the first 10 wells was then decreased in a stepwise manner.

[0058] (4) Result determination: The negative control well should remain clear throughout the entire experiment, indicating that the experiment was performed aseptically. The lowest concentration at which no visible growth occurred was determined by comparing the growth characteristics (e.g., turbidity of the broth, precipitation at the bottom of the well) of the bacteria in the growth control well.

[0059] The results are shown in Table 1. It can be seen that the antibacterial peptide K20NH2 has a broad spectrum of activity against multiple drug-resistant Gram-negative and Gram-positive bacteria.

[0060] Table 1 Minimum inhibitory concentration of antibacterial peptide K20NH2

[0061]

[0062] Note: GM represents the geometric mean of the minimum inhibitory concentrations of the Gram-positive and Gram-negative multiple drug-resistant bacteria listed in the table.

[0063] Example 3

[0064] Hemolytic activity of antibacterial peptide K20NH2

[0065] To determine the hemolytic activity of antibacterial peptide K20NH2. The specific test steps are as follows:

[0066] (1) Collect 1 mL of fresh human blood in a heparin sodium anticoagulant tube and store at 4 °C for later use;

[0067] (2) Centrifuge 1000 g of the above blood for 5 min, discard the supernatant, and collect the red blood cells;

[0068] (3) Wash the collected red blood cells with PBS buffer solution three times, centrifuge at 1000 g for 5 min, discard the supernatant, and collect the red blood cells. Finally, resuspend the cells with about 10 mL of PBS buffer solution to obtain an 8% (V / V) red blood cell suspension for later use;

[0069] (4) Dilution of the antibacterial peptide K20NH2: 90 μL of PBS buffer solution was added to the first tube of each row of 12 EP tubes arranged, and 50 μL of PBS buffer solution was added to the rest of the tubes. Then, 10 μL of the antibacterial peptide K20NH2 stock solution was added to the first tube, and the antibacterial peptide K20NH2 solution in the first tube was mixed and 50 pL was sucked out and added to the second tube. Then, the solution was diluted by a factor of two to the tenth tube, and 50 μL was sucked out and discarded;

[0070] (5) 50 μL of the prepared red blood cell suspension was added to the EP tubes containing different concentrations of antibacterial peptide K20NH2 solution, and incubated in a 37 ℃ incubator for 1 h. Among them, 50 μL of PBS and 50 μL of red blood cell suspension were added to the 11th hole as a negative control, and 50 μL of 0.1% Triton X-100 and 50 μL of red blood cell suspension were added to the 12th hole as a positive control;

[0071] (6) After 1 h, the EP tubes were taken out and centrifuged at 100 g for 5 min at 4 ℃;

[0072] (7) The supernatant of the above centrifuged solution was sucked and transferred to a clean 96-well plate in parallel. The optical absorption value was measured at 570 nm (OD570nm) by an enzyme-labeled instrument.

[0073] The results are shown in Figure 2 From Figure 2 it can be seen that the antibacterial peptide K20NH2 has only 20% hemolysis at 2048 μg / mL, which is much higher than the geometric mean of the minimum antibacterial concentration, indicating that the antibacterial peptide K20NH2 will not produce hemolysis when exerting antibacterial effect.

[0074] Example 4 Cytotoxicity of antibacterial peptide K20NH2

[0075] Mouse embryonic fibroblast NIH / 3T3 was used to detect the cytotoxicity of the antibacterial peptide K20NH2. Cells were seeded in a 96-well plate at 8000 cells per well. The cells were incubated at 37 ℃, 5% CO2 for 24 hours. Then the antibacterial peptide K20NH2 was added to each well containing cells. After 24 hours of incubation, 10 μL of CCK8 was added to each well, and incubated at 37 ℃ for 2 hours. Then the absorbance value at 450 nm was detected, and Prism 8 was used to determine the cell survival rate value according to curve fitting and using the following equation:

[0076] Cell survival rate = (experimental group - blank control) / (negative control group - blank control) x 100%

[0077] As Figure 3As shown in Table 1, the concentration of the antibacterial peptide K20NH2 at which 50% cytotoxicity was exhibited was above 128 μg / mL, which was higher than the geometric mean of the minimum antibacterial concentration, indicating that the antibacterial peptide K20NH2 did not produce cytotoxicity when it was exerting antibacterial action.

[0078] Example 5

[0079] For the determination of the skin external use anti-infective ability of the antibacterial peptide K20NH2, the specific test procedure was as follows:

[0080] The G+ S. aureus CMCC26003 bacterial solution was diluted to 10 8 CFU / mL for standby. Female C57BL / 6 mice were randomly divided by weight into an infection negative control group and an antibacterial peptide K20NH2 treatment group, 5 mice in each group. After the mice were depilated on the back, two pieces of full-thickness skin with a diameter of 5 mm were taken from the center of the back of the mice using a tissue biopsy punch, and the wound area of the mice in each group was uniform, i.e. the model was successfully constructed. 5 μL of bacterial solution with a concentration of 10 8 CFU / mL was injected on the two wounds of each mouse. After 6 h, the treatment group was given 0.2 mg of antibacterial peptide K20NH2 treatment. At 24 h after modeling, the skin at the infection site of the animals was aseptically taken, and viable bacteria were detected and counted. Figure 4 As shown in Table 1, the concentration of the antibacterial peptide K20NH2 at which 50% cytotoxicity was exhibited was above 128 μg / mL, which was higher than the geometric mean of the minimum antibacterial concentration, indicating that the antibacterial peptide K20NH2 did not produce cytotoxicity when it was exerting antibacterial action.

[0081] The above only describes preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibacterial peptide K20NH2, characterized in that, Provided is an antibacterial peptide K20NH2, which is obtained by amidation modification of a carboxyl group at a C-terminal end of the antibacterial peptide K20 with -NH2, and the amino acid sequence of K20 is shown as SEQ ID NO.

1.

2. The method of synthesis of the antibacterial peptide K20NH2 as claimed in claim 1, wherein, The application further provides a use of the antibacterial peptide K20NH2. The polypeptide is synthesized by solid-phase synthesis, and the antibacterial peptide K20NH2 is obtained after purification.

3. A broad-spectrum antimicrobial agent or composition, characterized in that, The application further provides a use of the antibacterial peptide K20NH2.

4. The broad-spectrum antimicrobial agent or composition according to claim 3, wherein The drug is an internal drug or an external drug; the internal drug is an oral preparation or an injection; and the external drug includes a patch and a smearing preparation.

5. A disinfectant, detergent or preservative characterised in that, The application further provides a use of the antibacterial peptide K20NH2.

6. The antibacterial peptide K20NH2 of claim 1 is used in any of the following applications: 1) for preparing a broad-spectrum antibacterial drug or composition; 2) for preparing a disinfectant; 3) for preparing a detergent; 4) for preparing a preservative; The bacteria are gram-positive bacteria and / or gram-negative bacteria; The gram-positive bacteria are Staphylococcus aureus and Enterococcus faecalis; The gram-negative bacteria are at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium.

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