Antibacterial peptide cAMP-1116nc with broad-spectrum antibacterial effect and application thereof

Abstract

The invention relates to the technical field of antibacterial peptide application, in particular to an antibacterial peptide cAMP-1116nc with a broad-spectrum antibacterial effect and application of the antibacterial peptide cAMP-1116nc. The antibacterial peptide cAMP-1116nc is obtained by carrying out N-terminal acetylation modification and C-terminal amidation modification on a polypeptide as shown in SEQ ID NO. 1. The antibacterial peptide cAMP-1116nc is an antibacterial peptide cAMP-1116nc. The antibacterial peptide cAMP-1116nc disclosed by the invention shows remarkable antibacterial activity in a plurality of gram-positive bacteria and gram-negative bacteria. The antibacterial peptide cAMP-1116nc can efficiently kill clinically common gram-negative pathogenic bacteria acinetobacter baumannii under the conditions that the pH is equal to 2, the pH is equal to 7.4 and the pH is equal to 8.4, has relatively low cytotoxicity, has the potential of preparing antibacterial drugs, and is suitable for developing antibacterial products with multiple purposes, including the antibacterial drugs, disinfectants and biological preservatives. The invention provides a novel functional polypeptide for the field of antibacterial peptides, and the novel functional polypeptide has important practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide application, and in particular to an antimicrobial peptide cAMP-1116nc with a broad-spectrum antibacterial effect and an application thereof. Background Art

[0002] In recent years, with the increasing problem of antibiotic resistance, antimicrobial peptides have gradually become an important research direction for new anti-infective drugs due to their broad antimicrobial spectrum, strong antibacterial activity, and low resistance to drug resistance. Unlike traditional antibiotics, antimicrobial peptides have a unique mechanism of action, such as destroying the integrity of cell membranes, and thus exhibit high antibacterial capacity against a variety of pathogenic microorganisms.

[0003] Although antimicrobial peptides have good antibacterial properties, they still face several challenges in practical applications, such as insufficient environmental stability, high toxicity, and low selectivity for certain pathogens. In order to solve these problems, scientists have developed a variety of antimicrobial peptide modification technologies in recent years, including N-terminal acetylation, C-terminal amidation, cyclization modification, introduction of non-natural amino acids, etc. These technologies have achieved remarkable results in improving the environmental stability of antimicrobial peptides, reducing toxicity, and enhancing targeting. However, most of the existing antimicrobial peptides have a relatively limited antimicrobial spectrum. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an antimicrobial peptide cAMP-1116nc with a broad-spectrum antibacterial effect and its application.

[0005] The present invention adopts the following technical solution: First, the present invention provides an antimicrobial peptide cAMP-1116nc with a broad-spectrum antibacterial effect, wherein the antimicrobial peptide cAMP-1116nc is obtained by modifying the polypeptide shown in SEQ ID NO.1 by acetylation at the N-terminus and amidation at the C-terminus. The molecular formula of the antimicrobial peptide cAMP-1116nc is C 75 H 125 O 13 N 21 S1, molecular weight is 1559.95.

[0006] The present invention provides an antimicrobial peptide cAMP-1116nc. The antimicrobial peptide not only has a broad-spectrum antimicrobial activity, but also exhibits excellent stability in a variety of extreme environments. The antimicrobial peptide cAMP-1116nc provided by the present invention has a significant antibacterial effect on a variety of Gram-positive and Gram-negative pathogens, and the minimum inhibitory concentrations for common clinical resistant strains such as Staphylococcus aureus and Acinetobacter baumannii are as low as 8μg / mL and 16μg / mL, respectively. At the same time, the antimicrobial peptide has good stability under pepsin conditions and exhibits efficient antimicrobial activity in environments of pH=2, pH=7.4 and pH=8.4. In addition, the antimicrobial peptide of the present invention has low cytotoxicity to human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B, showing excellent safety.

[0007] Secondly, the present invention provides the use of the antimicrobial peptide cAMP-1116nc in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae.

[0008] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0009] Specifically, the antimicrobial peptide can be used as a broad-spectrum antimicrobial drug for treating a variety of diseases caused by drug-resistant bacterial infections, and is particularly suitable for combating multi-drug resistant strains, including Staphylococcus aureus, multi-drug resistant Staphylococcus aureus, Acinetobacter baumannii, multi-drug resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibacterial agent can be used in the fields of medical care, food preservation, personal care products and environmental disinfection.

[0010] Furthermore, the antibacterial product contains the antibacterial peptide cAMP-1116nc as the only active ingredient.

[0011] Furthermore, the minimum inhibitory concentrations of the antimicrobial peptide cAMP-1116nc against Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae are 8 μg / mL, 16 μg / mL, 64 μg / mL, 16 μg / mL, 16 μg / mL and 32 μg / mL, respectively.

[0012] Furthermore, the antibacterial agent is obtained by dissolving the antimicrobial peptide cd77AHP-2 in sterile water.

[0013] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the dosage form of the drug is one of aqueous infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Broad spectrum and high efficiency: The antimicrobial peptide cAMP-1116nc has significant antibacterial activity against both Gram-positive and Gram-negative pathogens, especially against clinical multidrug-resistant strains of Staphylococcus aureus and multidrug-resistant Acinetobacter baumannii.

[0016] Environmental stability: The antimicrobial peptide cAMP-1116nc can maintain excellent activity under pepsin conditions and in different pH environments.

[0017] Low cytotoxicity: Toxicity experiments show that the antimicrobial peptide of the present invention has low toxicity to human skin keratinocytes and bronchial epithelial cells and is safer.

[0018] Broad application prospects: The antimicrobial peptide cAMP-1116nc is not only suitable for the development of anti-infective drugs, but also can be used in the preparation of food preservatives, disinfectants and other antibacterial products.

[0019] In summary, the present invention provides a functional polypeptide with novel source and excellent performance in the field of antimicrobial peptides, and provides important technical support and alternative resources for the development of antimicrobial drugs and the treatment of multidrug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The amino acid composition and structure of the antimicrobial peptide cAMP-1116nc, A is the amino acid composition, and B is the amino acid structure.

[0021] Figure 2 These are the MIC test results of the antimicrobial peptide cAMP-1116nc against various pathogens. In the figure, S. aureus 1 is Staphylococcus aureus 1, S. aureus 2 is Staphylococcus aureus 2, S. aureus 3 is Staphylococcus aureus 3, Pseudomonas aeruginosa is Pseudomonas aeruginosa, Baumannii 1 is Acinetobacter baumannii 1, Baumannii 2 is Acinetobacter baumannii 2, and Pneumoniae is Klebsiella pneumoniae.

[0022] Figure 3 These are the stability test results of the antimicrobial peptide cAMP-1116nc.

[0023] Figure 4 These are the results of the cytotoxicity experiment of the antimicrobial peptide cAMP-1116nc. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0025] The pathogenic bacteria numbers and sources used in the following examples are: Staphylococcus aureus 1:ATCC12600.

[0026] Staphylococcus aureus 2: ATCC33591, a multidrug-resistant strain.

[0027] Staphylococcus aureus 3: ATCC33592, a multidrug-resistant strain.

[0028] Klebsiella pneumoniae: ATCC17978.

[0029] Acinetobacter baumannii 1:ATCC19606.

[0030] Acinetobacter baumannii 2: bio-53272, a multidrug-resistant strain.

[0031] Staphylococcus aureus 1, Staphylococcus aureus 2, Staphylococcus aureus 3, Klebsiella pneumoniae, and Acinetobacter baumannii 1 were purchased from Beina Chuanglian Biotechnology Co., Ltd., Acinetobacter baumannii 2 was purchased from Beijing Biobowei Biological Co., Ltd., and Pseudomonas aeruginosa came from the laboratory.

[0032] The present invention provides a broad-spectrum antimicrobial peptide cAMP-1116nc for multidrug-resistant bacteria, and its amino acid sequence is shown in SEQ ID NO.1. The antimicrobial peptide cAMP-1116nc exhibits high antimicrobial activity under the conditions of Ph=7.4 and Ph=2 of simulated gastric acid, respectively, and can kill a variety of Gram-positive bacteria including Staphylococcus aureus and Gram-negative bacteria such as Acinetobacter baumannii. The minimum inhibitory concentration value MIC of the antimicrobial peptide against Staphylococcus aureus is 8 μg / mL. The antimicrobial peptide cAMP-1116nc has extremely high antimicrobial value of medical drugs and can provide alternative medicinal resources for clinical combating multidrug-resistant pathogens.

[0033] Example 1: An antimicrobial peptide cAMP-1116nc and a preparation method thereof.

[0034] The antimicrobial peptide cAMP-1116nc is obtained by modifying the polypeptide shown in SEQ ID NO.1 by acetylation at the N-terminus and amidation at the C-terminus. Its molecular formula is C 75 H 125 O 13 N 21 S1, molecular weight is 1559.95Da.

[0035] SEQ ID NO.1: ILKFPRKVFRCI.

[0036] The amino acid structure and related schematics are from the peptide website: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool AlphaFold3 cannot predict the structure of modified peptides, the protein structure prediction of the unmodified peptide of cAMP-1116nc was performed to indirectly reflect the structure of cAMP-1116nc. The prediction results are as follows: Figure 1 As shown, the peptide is a typical α-helical peptide with amphipathic characteristics, which helps it interact with bacterial membranes. The website of AlphaFold3 is: https: / / golgi.sandbox.google.com / about. Preparation method: The antimicrobial peptide cAMP-1116nc was prepared by Sangon Biotech (Shanghai) Co., Ltd. using solid phase chemical synthesis, with a purity of >95%.

[0037] Example 2: MIC determination of antimicrobial peptide cAMP-1116nc.

[0038] The MIC of antimicrobial peptides was determined by broth microdilution method, referring to the guidelines of the Clinical and Laboratory Standards Institute: Wayne, PA Performance Standards for Antimicrobial Disk Susceptibility Tests, Clinical and Laboratory Standards Institute, 1991. The above pathogens were inoculated into sterile LB medium and cultured at 37°C overnight with shaking. Each pathogen was inoculated into fresh LB medium at a 1% inoculum and cultured until the exponential phase, and the cell concentration was adjusted to 1×10 5 cfu / mL to obtain the bacterial solution. Then transfer 180μL of the bacterial solution to a 96-well plate. The antimicrobial peptide powder was dissolved in sterile water and diluted to a 2-fold serial dilution of the antimicrobial peptide solution. 20μL was added to the bacterial solution in the 96-well plate to make the antimicrobial peptide concentration in the bacterial solution range from 0.5μg / mL to 256μg / mL. After incubating the 96-well plate at 37°C for 24 hours, the bacterial growth was detected by an enzyme reader. MIC was defined as the minimum antimicrobial peptide concentration for detecting bacterial growth. The experiment was set up with 3 replicates.

[0039] like Figure 2As shown, the MICs of the antimicrobial peptide cAMP-1116nc against Staphylococcus aureus 1, Staphylococcus aureus 2, Staphylococcus aureus 3, Pseudomonas aeruginosa, Acinetobacter baumannii 1, Acinetobacter baumannii 2, and Klebsiella pneumoniae were tested to be 8 μg / mL, 16 μg / mL, 16 μg / mL, 64 μg / mL, 16 μg / mL, 16 μg / mL and 32 μg / mL, respectively.

[0040] Example 3: Determination of the stability of the antimicrobial peptide cAMP-1116nc.

[0041] Sterile PBS solution was added with 1 mol / L HCl and NaOH solution to prepare PBS solutions of pH=2, pH=7.4 and pH=8.4, then the antimicrobial peptide was dissolved in PBS solutions of different pH values ​​and incubated at 37°C for 1 h, and then the MIC changes of the antimicrobial peptide under different pH treatments were determined according to the method described in Example 2. The results showed that Figure 2 As shown, pH has almost no effect on antimicrobial peptides. At pH = 2 and pH = 7.4, the MIC of the peptide did not change, and at pH = 8.4, the MIC of the peptide only changed to 2 times the MIC, with a small change range.

[0042] For the pepsin stability assay, pepsin was dissolved in a sterile PBS solution at pH 2 to a final concentration of 100 μg / ml, and then the peptide was dissolved in the pepsin solution and treated at 37°C for 1 h, and then boiled at 100°C for 15 min to inactivate pepsin. Then, the MIC changes of the antimicrobial peptide against Acinetobacter baumannii 1 under different pH treatments were determined according to the method described in Example 2. The results showed that Figure 3 As shown in the figure, when the concentration of pepsin was as high as 100 μg / ml, the MIC of the antimicrobial peptide did not change and was very stable.

[0043] Example 4: Toxicity determination of the antimicrobial peptide cAMP-1116nc.

[0044] Human bronchial epithelial cells BEAS-2B and human keratinocytes HaCat were used to conduct cytotoxicity experiments on the antimicrobial peptide cAMP-1116nc. BEAS-2B and HACAT cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin, and cultured in a cell culture incubator at 37°C with 5% CO2. Cell subculture: When the cells grow to 80%-90%, remove the medium and add PBS to wash 1-2 times. After removing PBS, add 1ml of trypsin to digest for 1-3min, add 3ml of complete medium to neutralize the trypsin to terminate the digestion, transfer the digested cells to a 15ml centrifuge tube, and centrifuge at 1000rpm for 5min. After discarding the supernatant, add 3ml of medium to resuspend the cells, and subculture them in a culture dish at a ratio of 1:3. Digest BEAS-2B and HACAT cells that have grown to 80% to 90% of the culture dish with trypsin, centrifuge at 1000rpm, remove the supernatant, add complete culture medium to resuspend the cells, and plant the cells in 96-well plates, with 5000 cells in each well. After the cells adhere to the wall, add drugs at concentrations of 0μg / mL, 64μg / mL, 96μg / mL, and 128μg / mL, respectively, mix gently, add to the incubator and cultivate for 24h, add 10μl of MTT at a concentration of 5mg / ml, and continue to culture in the cell culture incubator for about 3h. Remove the culture medium, add 150μl of DMSO to each well, shake evenly, and measure the absorbance with an enzyme plate reader. Use an enzyme plate reader to read the OD value of each well at a wavelength of 570nm, and calculate the cell survival rate. The formula is as follows: Cell survival rate (%) = (OD sample group / OD blank control group) × 100.

[0045] After testing, the cytotoxicity results of the antimicrobial peptide cAMP-1116nc are as follows Figure 4 As shown, when the antimicrobial peptide concentration was as high as 96 μg / ml, the survival rate of human bronchial epithelial cells BEAS-2B and human keratinocytes HaCat was still greater than 90%, indicating that the antimicrobial peptide cAMP-1116nc has extremely low cytotoxicity and has great medical therapeutic potential.

[0046] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. An antimicrobial peptide cAMP-1116nc with a broad-spectrum antibacterial effect, characterized in that: The antimicrobial peptide cAMP-1116nc is obtained by modifying the polypeptide shown in SEQ ID NO.1 by acetylation at the N-terminus and amidation at the C-terminus.

2. The use of the antimicrobial peptide cAMP-1116nc according to claim 1 in the preparation of antimicrobial products, characterized in that: The antibacterial product is used to inhibit Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae.

3. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 2, characterized in that: The antibacterial product is a medicine or a bacteriostatic agent.

4. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 2, characterized in that: The antibacterial product uses the antibacterial peptide cAMP-1116nc as the only active ingredient.

5. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 2, characterized in that: The minimum inhibitory concentrations of the antimicrobial peptide cAMP-1116nc against Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae are 8 μg / mL, 16 μg / mL, 64 μg / mL, 16 μg / mL, 16 μg / mL and 32 μg / mL, respectively.

6. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 3, characterized in that: The antibacterial agent is obtained by dissolving the antimicrobial peptide cd77AHP-2 in sterile water.

7. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use of the antimicrobial peptide cAMP-1116nc in the preparation of antimicrobial products according to claim 7, characterized in that: The dosage form of the medicine is one of water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster or aerosol.

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