Antibacterial peptide MA25 with broad-spectrum antibacterial activity and application thereof

Abstract

The invention relates to the technical field of antibacterial peptide application, in particular to an antibacterial peptide MA25 with broad-spectrum antibacterial activity and application of the antibacterial peptide MA25. The amino acid sequence of the antibacterial peptide MA25 is as shown in SEQ ID NO. 1. The antibacterial peptide MA25 disclosed by the invention has antibacterial activity on various bacteria (staphylococcus aureus, escherichia coli, acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae) and drug-resistant strains thereof, is excellent in biological safety and physiological stability, and has a wide development prospect; the compound can be used for alternative treatment of bacterial infection or used for application scenarios of bacteriostasis or sterilization.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide applications, and specifically relates to an antimicrobial peptide MA25 with broad-spectrum antibacterial activity and its applications. Background Art

[0002] Antimicrobial peptides (AMPs) are an important part of the innate immune system in organisms, providing the first line of defense against microbial infections. As a class of active small-molecule polypeptides, AMPs are not only active against bacteria, fungi, and viruses, but are also less likely to induce the evolution of bacterial drug resistance compared to traditional small-molecule antibiotics. In addition, when combined with clinically used antibiotics, AMPs can restore the antibacterial or bactericidal activity of antibiotics, effectively reduce the dosage of highly toxic drugs, and reduce the adverse reactions of drugs. These unique activity advantages make AMPs promising candidates as ideal replacements for the next generation of antibiotics and are expected to alleviate the AMR crisis caused by drug-resistant bacterial infections.

[0003] However, most of the existing antimicrobial peptides are narrow-spectrum antimicrobial peptides and do not have antibacterial effects on a variety of bacteria. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an antimicrobial peptide MA25 with broad-spectrum antibacterial activity and its applications.

[0005] The present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides an antimicrobial peptide MA25 with broad-spectrum antibacterial activity, and the amino acid sequence of the antimicrobial peptide MA25 is shown as SEQ ID NO.1.

[0007] The present invention screened out a highly efficient and low-toxic cationic active AMP molecule. Experiments confirmed that the antimicrobial peptide MA25 exhibits broad-spectrum antibacterial activity against the tested standard strains (Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and their drug-resistant strains. In addition, the antimicrobial peptide MA25 also has good biocompatibility (hemolytic activity / cytotoxicity) and physiological stability. These biological characteristics indicate that the antimicrobial peptide MA25 has good development prospects and can be used for alternative treatment of bacterial infections or for antibacterial or bactericidal application scenarios.

[0008] In the second aspect, the present invention provides the application of the antimicrobial peptide MA25 in the preparation of antimicrobial products, and the antimicrobial products are used to inhibit or kill Staphylococcus aureus and its drug-resistant strains, Escherichia coli and its drug-resistant strains, Acinetobacter baumannii and its drug-resistant strains, Pseudomonas aeruginosa and its drug-resistant strains, and Klebsiella pneumoniae and its drug-resistant strains.

[0009] Furthermore, the antibacterial product uses the antibacterial peptide MA25 as the sole active ingredient.

[0010] Furthermore, the antibacterial product is a drug or a bacteriostatic agent.

[0011] Specifically, the antibacterial peptide can be used as a broad-spectrum antibacterial drug for the treatment of various diseases caused by drug-resistant bacterial infections, and is particularly suitable for combating Staphylococcus aureus and its drug-resistant strains, Escherichia coli and its drug-resistant strains, Acinetobacter baumannii and its drug-resistant strains, Pseudomonas aeruginosa and its drug-resistant strains, and Klebsiella pneumoniae and its drug-resistant strains; the bacteriostatic agent can be used in the fields of medical and health, food preservation, personal care products, and environmental disinfection.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipient is an excipient required for preparing one of a water infusion, powder, lotion, tincture, oil, emulsion, ointment, plaster, or aerosol.

[0014] In a third aspect, the present invention provides the use of the antibacterial peptide MA25 in the preparation of a drug for treating cancer.

[0015] Furthermore, the cancer includes lung cancer and cervical cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention provides a polypeptide with diverse functions and excellent performance in the field of antibacterial peptides, providing important technical support and alternative resources for the development of antibacterial drugs and the treatment of multi-drug resistant bacterial infections. At the same time, it provides support for the development of anti-cancer drugs. Description of the Drawings

[0018] Figure 1 It is the protein structure of the antibacterial peptide MA25. A is the helical wheel analysis diagram of the antibacterial peptide MA25, and B is the protein structure modeling diagram.

[0019] Figure 2 It is the antibacterial activity of the antibacterial peptide MA25 against the bacteria to be tested. A: The bacteria to be tested is S. aureus ATCC25923, B: The bacteria to be tested is MRSA_WM, C: The bacteria to be tested is E. coli ATCC25922, D: The bacteria to be tested is A. baumannii ATCC19606, E: The bacteria to be tested is P. aeruginosa CMCC10104, F: The bacteria to be tested is K. pneumoniae ATCC700603.

[0020] Figure 3Results of biocompatibility and cytotoxicity tests of antimicrobial peptide MA25. A: Results of hemolysis assay. B: Results of cytotoxicity test of antimicrobial peptide MA25 on LO2 cells. B: Results of cytotoxicity test of antimicrobial peptide MA25 on HK-2 cells. C: Results of cytotoxicity test of antimicrobial peptide MA25 on A549 cells. D: Results of cytotoxicity test of antimicrobial peptide MA25 on HeLa cells.

[0021] Figure 4 Results of physiological stability test of antimicrobial peptide MA25. Detailed implementation manners

[0022] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0023] Example 1: Preparation and characterization of antimicrobial peptide MA25

[0024] The amino acid sequence of the antimicrobial peptide MA25 of the present invention is shown in SEQ ID NO.1 and was prepared by GL Biochem (Shanghai) Ltd. using solid-phase chemical synthesis. The synthesis steps include: ① connecting the first amino acid to an insoluble high-molecular resin carrier; ② protecting the active functional groups that do not participate in peptide chain elongation; ③ activating the functional groups that participate in peptide chain elongation and then introducing the next amino acid; ④ cutting the polypeptide from the resin carrier after the polypeptide sequence is extended to the specified length, deprotecting the protected functional groups and purifying the obtained crude peptide; ⑤ Subsequently, HPLC-MS mass spectrometry was used to detect the purity of the polypeptide and identify the actual molecular weight of the polypeptide.

[0025] To study whether the activity of antimicrobial peptide MA25 is correlated with structural factors, prediction and analysis were carried out through helical wheel diagrams, three-dimensional structure projections and ProtParam software. Figure 1 A of shows the helical wheel diagram of antimicrobial peptide MA25; Figure 1 B of shows the protein crystal structure of antimicrobial peptide MA25, both presenting an α-helical structure; Table 1 details the molecular weight, isoelectric point, net charge, half-life, hydrophobicity and hydrophobic moment of antimicrobial peptide MA25. These results indicate that antimicrobial peptide MA25 belongs to a typical cationic α-helical peptide. SEQ ID NO.1: MAVYPWGRLFLKKILHLHSRFKKVI.

[0026] Table 1 Analysis results of physicochemical properties of antimicrobial peptide MA25

[0027] Physical and chemical indexes Characteristics Relative molecular mass 3080.83 Structural formula <![CDATA[C 150 H 238 N 40 O 28 S1 <!-- 2 -->]]> Isoelectric point 11.22 Net charge +6 Half-life In vitro half-life: 30 h / In vivo half-life: > 20 h Hydrophobicity 0.645 Hydrophobic moment 0.409

[0028] Example 2: Verification of the antibacterial activity of antibacterial peptide MA25

[0029] To clarify the drug sensitivity of antibacterial peptide MA25 to clinical Gram-positive and Gram-negative bacteria, the minimum inhibitory concentration (MIC) was detected by the micro-liquid dilution method. The MIC is expressed in the form of molar concentration / mass concentration. The unit of molar concentration is μM, and the unit of mass concentration is μg / mL. The specific method is as follows: The concentration of the test bacterial liquid in the logarithmic phase was adjusted to 2.0×10 6 CFU / mL; then an equal volume of the bacterial liquid was added to a 96-well plate with different concentrations of antibacterial peptide MA25. An equal volume of 100 μL MHB culture medium and 100 μL of the test bacterial liquid were set as the blank control and the growth control respectively; the above 96-well plate was cultured at 37 °C for 24 h, and then the results were observed. The drug concentration corresponding to the well that was clear or showed no growth when observed with the naked eye compared with the negative control group was the MIC value of the test drug.

[0030] The results are shown in Table 2. Antibacterial peptide MA25 showed good in vitro antibacterial effects against the test bacteria (Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae), and the MIC values were between 1.30 / 4 and 2.60 / 8. To further clarify the activity of antibacterial peptide MA25 against clinical drug-resistant bacteria, we determined the MIC values of antibacterial peptide MA25 against 3 strains of MRSA, 3 strains of CRAB, 3 strains of ESBL-EC, 3 strains of CRPA, and 3 strains of CRKP. The results were still exciting: Antibacterial peptide MA25 also showed good antibacterial activity against the above drug-resistant bacteria (Table 3). To further verify the activity of antibacterial peptide MA25, we observed the change in the MIC value after treating with antibacterial peptide MA25 for 24 h. The results are as Figure 2 shown. At the MIC concentration, antibacterial peptide MA25 could effectively kill the test bacteria. The above results indicate that antibacterial peptide MA25 is a novel antibacterial peptide with high efficiency and broad spectrum and has good research value.

[0031] Table 2 In vitro antibacterial spectrum of antibacterial peptide MA25

[0032]

[0033] Note: Polymyxin B: PMB; Vancomycin: Van; "NA" means not detected, the same below.

[0034] Table 3 In vitro activity of antibacterial peptide MA25 against drug-resistant bacteria

[0035]

[0036] Example 3: Biocompatibility and cytotoxicity of antibacterial peptide MA25

[0037] To investigate the safety of antimicrobial peptide MA25, we detected its hemolytic activity and cytotoxicity. The specific methods are as follows: ① Hemolytic activity detection: 8% human red blood cells (hRBCs) were incubated with different concentrations of MA25 for 24 h, and then the supernatant was taken to detect the absorbance value at 540 nm in a multifunctional microplate reader. 10 mM PBS and 1% Triton-X 100 were used as negative and positive controls respectively. ② Cytotoxicity detection: 2×10 4 cells / well were transferred to a 96-well plate and cultured overnight; the next day, different concentrations of MA25 were added to the 96-well plate and co-cultured for 24 h. Among them, only cells and only culture medium were used as negative and blank control groups respectively, and a CCK-8 detection kit was used for determination.

[0038] The hemolytic activity results showed that antimicrobial peptide MA25 did not show obvious hemolysis on human red blood cells at a concentration of 32 μM ( Figure 3 A); the cytotoxicity results also showed that antimicrobial peptide MA25 had low cytotoxicity on human normal liver cells LO2 and human renal proximal tubular epithelial cells HK-2 at a concentration of 32 μM ( Figure 3 B and C); while it showed anti-cancer activity against cancer cells (A549 and HeLa) ( Figure 3 D and E). The above results indicate that antimicrobial peptide MA25 has good biocompatibility and the potential to be developed into an anti-cancer drug.

[0039] Example 4: Physiological stability of antimicrobial peptide MA25

[0040] To evaluate the physiological stability of antimicrobial peptide MA25, in this study, the micro liquid dilution method was used to detect the change degree of MIC value of the new peptide under different environmental conditions. The specific method is as follows: Different concentrations of antimicrobial peptide MA25 were added to the test bacterial solution containing different concentrations of NaCl, KCl, CaCl2, serum and proteases (trypsin, pepsin, proteinase K). For the detection of enzyme stability, the enzyme activity needed to be eliminated in a 60 °C water bath. MHB culture medium was used as the control group. After culturing at 37 °C for 24 h, the change of MIC value of MA25 was detected.

[0041] The results are as Figure 4 shown. 5 mM NaCl, 5 mM KCl, 5 mM CaCl2 and 10% serum had no significant effect on the activity of antimicrobial peptide MA25. Surprisingly, the activity of antimicrobial peptide MA25 only increased by 2-4 times in the pepsin and proteinase K environments; like other polypeptides, antimicrobial peptide MA25 was sensitive to trypsin and was easily inactivated in the trypsin environment.

[0042] Although the preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

Claims

1. An antibacterial peptide MA25 with broad-spectrum antibacterial activity, characterized in that, The amino acid sequence of the antimicrobial peptide MA25 is shown in SEQ ID NO.

1.

2. Use of the antibacterial peptide MA25 according to claim 1 in the preparation of antibacterial products, characterized in that, The antimicrobial product is used to inhibit or kill Staphylococcus aureus and its drug-resistant strains, Escherichia coli and its drug-resistant strains, Acinetobacter baumannii and its drug-resistant strains, Pseudomonas aeruginosa and its drug-resistant strains, and Klebsiella pneumoniae and its drug-resistant strains.

3. Use of the antimicrobial peptide MA25 according to claim 2 in the preparation of antimicrobial products, characterized in that, The antimicrobial product uses the antimicrobial peptide MA25 as the sole active ingredient.

4. Use of the antimicrobial peptide MA25 according to claim 2 in the preparation of antimicrobial products, characterized in that, The antimicrobial product is a drug or a bacteriostatic agent.

5. Use of the antibacterial peptide MA25 according to claim 4 in the preparation of antibacterial products, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. Use of the antibacterial peptide MA25 according to claim 5 in the preparation of antibacterial products, characterized in that, The excipients are the excipients required for preparing one of a water infusion, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

7. Use of the antimicrobial peptide MA25 according to claim 1 in the preparation of a drug for treating cancer.

8. Use of the antibacterial peptide MA25 according to claim 7 in the preparation of a medicament for treating cancer, characterized in that, The cancer includes lung cancer and cervical cancer.

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