Broad-spectrum bacterial-source antibacterial peptide cAMP-919nc and application thereof

Abstract

The invention relates to the technical field of application of antibacterial peptides, in particular to a broad-spectrum bacterial-source antibacterial peptide cAMP-919nc and application of the broad-spectrum bacterial-source antibacterial peptide cAMP-919nc. The antibacterial peptide cAMP-919nc 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 invention further discloses a preparation method of the antibacterial peptide cAMP-919nc. The antibacterial peptide shows an excellent antibacterial effect in various bacteria, and particularly, the minimum inhibitory concentration of the antibacterial peptide to staphylococcus aureus is as low as 4 mu g / mL. Under different pH conditions, the antibacterial peptide also shows remarkable bactericidal activity on gram-negative pathogenic bacteria acinetobacter baumannii. In addition, the peptide has extremely low cytotoxicity and shows good safety. The invention provides the antibacterial peptide with excellent performance, and the antibacterial peptide has wide application potential and can be used for developing various antibacterial products such as antibacterial drugs, disinfectants and biological preservatives. The invention opens up a new direction for the research and application of the antibacterial peptide, and has important scientific and practical values.

Description

Technical Field

[0001] The invention relates to the technical field of antimicrobial peptide application, and in particular to a broad-spectrum bacterial antimicrobial peptide cAMP-919nc and an application thereof. Background Art

[0002] Antimicrobial peptides, as a naturally occurring antimicrobial molecule, have become an important research direction in recent years to solve the problem of antibiotic resistance due to their broad-spectrum antimicrobial activity and low resistance to inducing drug resistance. Unlike traditional antibiotics, antimicrobial peptides have a unique mechanism of action, such as destroying cell membrane integrity or interfering with intracellular functions, and exhibit significant antibacterial and bactericidal effects on a variety of pathogenic microorganisms.

[0003] Despite the superior antimicrobial properties of antimicrobial peptides, they still face many challenges in practical applications, such as insufficient environmental stability, high potential toxicity to host cells, and relatively limited selectivity for specific pathogens. To overcome these limitations, recent studies have developed a variety of modification methods, including acetylation, amidation, cyclization, and non-natural amino acid substitution, which have significantly improved the stability, biological activity, and targeting of antimicrobial peptides. However, the antimicrobial spectrum of most known antimicrobial peptides is relatively limited. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a broad-spectrum bacterial antimicrobial peptide cAMP-919nc and its application.

[0005] The present invention adopts the following technical solution: First, the present invention provides a broad-spectrum bacterial antimicrobial peptide cAMP-919nc, which 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-919nc is C 111 H 175 O 20 N 31 , molecular weight is 2262.36.

[0006] The present invention provides a broad-spectrum antimicrobial peptide cAMP-919nc derived from bacteria, which not only exhibits broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, but also maintains good activity under the conditions of pH=2, pH=7.4 and pH=8.4. In addition, toxicity evaluation shows that the toxicity of cAMP-919nc to human skin keratinocytes HaCat and bronchial epithelial cells BEAS-2B is much lower than that of conventional antimicrobial peptides, providing a good safety basis for its clinical application. In the fields of anti-infective drugs, disinfectants and biopreservatives, antimicrobial peptides have broad application prospects.

[0007] The minimum inhibitory concentration of the antimicrobial peptide cAMP-919nc in the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Acinetobacter baumannii is as low as 4 μg / mL and 16 μg / mL, respectively, showing significant antibacterial potential. As a functional peptide derived from bacteria, the cAMP-919nc of the present invention has excellent performance and wide application value, providing new ideas and technical support for key issues in the clinical transformation of antimicrobial peptides.

[0008] Secondly, the present invention provides the use of the antimicrobial peptide cAMP-919nc in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

[0009] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0010] Specifically, the antimicrobial peptide cAMP-919nc 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 multidrug-resistant strains, including Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. The antimicrobial peptide cAMP-919nc of the present invention can still maintain high antimicrobial performance in extreme environments, significantly superior to the clinical performance of existing antimicrobial peptides or antibiotics.

[0011] Specifically, the antibacterial agent can be used in the fields of medical care, food preservation, personal care products and environmental disinfection.

[0012] Furthermore, the antibacterial product contains the antibacterial peptide cAMP-919nc as the only active ingredient.

[0013] Furthermore, the minimum inhibitory concentrations of the antimicrobial peptide cAMP-919nc against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii are 4 μg / mL, 32 μg / mL, 16 μg / mL and 16 μg / mL, respectively.

[0014] Furthermore, the antibacterial agent is obtained by dissolving the antimicrobial peptide cd77AHP-2 in sterile water.

[0015] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the auxiliary material is an auxiliary material required for preparing an aqueous extract, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Broad spectrum and high efficiency: The antimicrobial peptide cAMP-919nc has significant antibacterial activity against both Gram-positive and Gram-negative pathogens, especially against clinical multidrug-resistant strains of Staphylococcus aureus and Acinetobacter baumannii.

[0018] Environmental stability: The antimicrobial peptide cAMP-919nc can maintain excellent activity in different pH environments.

[0019] 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.

[0020] Broad application prospects: The antimicrobial peptide cAMP-919nc 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.

[0021] 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

[0022] Figure 1 is the amino acid composition and structure of the antimicrobial peptide cAMP-919nc, A is the amino acid composition, and B is the amino acid structure.

[0023] Figure 2 These are the MIC test results of the antimicrobial peptide cAMP-919nc against various pathogens. In the figure, S. aureus is Staphylococcus aureus, Pseudomonas aeruginosa is Pseudomonas aeruginosa, Baumannii 1 is Acinetobacter baumannii 1, and Baumannii 2 is Acinetobacter baumannii 2.

[0024] Figure 3 These are the stability test results of the antimicrobial peptide cAMP-919nc.

[0025] Figure 4 These are the results of the cytotoxicity experiment of the antimicrobial peptide cAMP-919nc. DETAILED DESCRIPTION

[0026] 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.

[0027] The pathogenic bacteria deposit numbers and sources used in the following examples are: Staphylococcus aureus: ATCC12600.

[0028] Acinetobacter baumannii 1:ATCC19606.

[0029] Acinetobacter baumannii 2: ATCC53272, a multidrug-resistant strain.

[0030] Staphylococcus aureus and Acinetobacter baumannii 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.

[0031] The present invention provides a broad-spectrum antimicrobial peptide cAMP-919nc against multidrug-resistant bacteria, which is obtained by modifying the polypeptide shown in SEQ ID NO.1 by N-terminal acetylation and C-terminal amidation. The antimicrobial peptide cAMP-919nc exhibits high antimicrobial activity under the conditions of pH=2, pH=7.4 and pH=8.4, 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 4μg / mL. The antimicrobial peptide cAMP-919nc has extremely high antimicrobial value as a medical drug and can provide an alternative medicinal resource for clinical combating multidrug-resistant pathogens.

[0032] Example 1: An antimicrobial peptide cAMP-919nc and a preparation method thereof.

[0033] The antimicrobial peptide cAMP-919nc 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 111 H 175 O 20 N 31 , with a molecular weight of 2262.36Da and an amino acid composition such as Figure 1 As shown in A.

[0034] SEQ ID NO. 1: KFLSLRRKLKVFAGFHKF.

[0035] The amino acid structure and related schematics are from the peptide website: https: / / www.allpeptide.com / jiegoutu.html. Since the online tool acetylated AlphaFold3 cannot predict the structure of modified peptides, the protein structure prediction of the unmodified peptide of cAMP-919nc was performed to indirectly reflect the structure of cAMP-919nc. The prediction results showed that the peptide was a typical α-helical peptide, such as Figure 1As shown in B, it has amphiphilic 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-919nc was prepared by Acetylation Biotech (Shanghai) Co., Ltd. using solid phase chemical synthesis, and the purity reached acetylation>95%.

[0036] Example 2: MIC determination of antimicrobial peptide cAMP-919nc.

[0037] 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 0.5 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.

[0038] like Figure 2 As shown, the MICs of the antimicrobial peptide cAMP-919nc against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii 1, and Acinetobacter baumannii 2 were detected to be 4 μg / mL, 32 μg / mL, 16 μg / mL, and 16 μg / mL, respectively.

[0039] Example 3: Stability determination of the antimicrobial peptide cAMP-919nc.

[0040] 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. Figure 3The results shown in the figure show that pH has no effect on the action of antimicrobial peptides. At pH = 2, pH = 7.4 and pH = 8.4, the MIC of the peptides did not change. The antimicrobial peptides can maintain stability and function under different pH environments.

[0041] Example 4: Toxicity determination of the antimicrobial peptide cAMP-919nc.

[0042] Human bronchial epithelial cells BEAS-2B and human keratinocytes HaCat were used to conduct cytotoxicity experiments on the antimicrobial peptide cAMP-919nc. 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, 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.

[0043] After testing, the cytotoxicity results of the antimicrobial peptide cAMP-919nc 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 was still greater than 90%. When the antimicrobial peptide concentration was increased from 0 to as high as 128 μg / ml, the antimicrobial peptide had no negative impact on the growth of human keratinocytes HaCat, indicating that the antimicrobial peptide cAMP-919nc has extremely low cytotoxicity and has great medical therapeutic potential.

[0044] 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. A broad-spectrum bacterial antimicrobial peptide cAMP-919nc, characterized in that: The antimicrobial peptide cAMP-919nc 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-919nc according to claim 1 in the preparation of antimicrobial products, characterized in that: The antibacterial product is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

3. The use of the antimicrobial peptide cAMP-919nc 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-919nc in the preparation of antimicrobial products according to claim 2, characterized in that: The antibacterial product uses the antibacterial peptide cAMP-919nc as the only active ingredient.

5. The use of the antimicrobial peptide cAMP-919nc in the preparation of antimicrobial products according to claim 2, characterized in that: The minimum inhibitory concentrations of the antimicrobial peptide cAMP-919nc against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii are 4 μg / mL, 32 μg / mL, 16 μg / mL and 16 μg / mL, respectively.

6. The use of the antimicrobial peptide cAMP-919nc 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-919nc 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-919nc in the preparation of antimicrobial products according to claim 7, characterized in that: The auxiliary material is an auxiliary material required for preparing an aqueous extract, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

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