Antibacterial peptide MM19 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 MM19 with broad-spectrum antibacterial activity and application of the antibacterial peptide MM19. The amino acid sequence of the antibacterial peptide MM19 is as shown in SEQ ID NO. 1. The antibacterial peptide MM19 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 particularly relates to an antimicrobial peptide MM19 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 and provide the first line of defense against microbial infections for the body. 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 commonly used antibiotics, AMPs can restore antibacterial or bactericidal activity, effectively reduce the dosage of drugs with strong toxicity, 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 against a variety of bacteria. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an antimicrobial peptide MM19 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 MM19 with broad-spectrum antibacterial activity, and the amino acid sequence of the antimicrobial peptide MM19 is as shown in 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 MM19 exhibited 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 MM19 also had good biocompatibility (hemolytic activity / cytotoxicity) and physiological stability. These biological characteristics indicate that the antimicrobial peptide MM19 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 MM19 in the preparation of antibacterial products, and the antibacterial 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 antimicrobial product uses the antimicrobial peptide MM19 as the sole active ingredient.

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

[0011] Specifically, the antimicrobial peptide can be used as a broad-spectrum antimicrobial drug for treating 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 antimicrobial peptide MM19 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 antimicrobial peptides, providing important technical support and alternative resources for the development of antimicrobial drugs and the treatment of multi-drug resistant bacterial infections. At the same time, it provides support for the development of anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the protein structure of the antimicrobial peptide MM19. A is the helical wheel analysis diagram of the antimicrobial peptide MM19, and B is the protein structure modeling diagram.

[0019] Figure 2 It is the antibacterial activity of the antimicrobial peptide MM19 against the test bacteria. A: The test bacteria is S.aureus ATCC25923, B: The test bacteria is MRSA_WM, C: The test bacteria is E.coli ATCC25922, D: The test bacteria is A.baumanniiATCC19606, E: The test bacteria is P.aeruginosa CMCC10104, F: The test bacteria is K.pneumoniaeATCC700603.

[0020] Figure 3Biocompatibility test results of antimicrobial peptide MM19. A: Hemolysis test results. B: Toxicity test results of antimicrobial peptide MM19 on LO2 cells. C: Toxicity test results of antimicrobial peptide MM19 on HK-2 cells. D: Toxicity test results of antimicrobial peptide MM19 on A549 cells. E: Toxicity test results of antimicrobial peptide MM19 on HeLa cells.

[0021] Figure 4 Physiological stability test results of antimicrobial peptide MM19. Specific 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 channels unless otherwise specified.

[0023] Example 1: Preparation and characterization of antimicrobial peptide MM19

[0024] The amino acid sequence of the antimicrobial peptide MM19 of the present invention is shown in SEQ ID NO.1 and was prepared by Gil Biochemical (Shanghai) Co., 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 After the polypeptide sequence is extended to the specified length, cutting it from the resin carrier, deprotecting the protected functional groups and purifying the obtained crude peptide; ⑤ Subsequently, using HPLC-MS mass spectrometry 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 MM19 is correlated with structural factors, predictive analysis was carried out through helical wheel diagrams, three-dimensional structure projections and ProtParam software. Figure 1 A shows the helical wheel diagram of antimicrobial peptide MM19; Figure 1 B shows the protein crystal structure of antimicrobial peptide MM19, 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 MM19. These results indicate that antimicrobial peptide MM19 belongs to a typical cationic α-helical peptide.

[0026] SEQ ID NO.1: MMLRRVVTLIYKKLRKLLK.

[0027] Table 1 Analysis results of the physicochemical properties of antimicrobial peptide MM19

[0028] Physical and chemical indexes Characteristics Relative molecular mass 2402.15 Structural formula <![CDATA[C 111 H 204 N 32 O 22 S2]]> Isoelectric point 11.75 Net charge +7 Half-life Half-life in vitro: 30 h / Half-life in vivo: > 20 h Hydrophobicity 0.496 Hydrophobic moment 0.561

[0029] Example 2: Verification of the antibacterial activity of antimicrobial peptide MM19

[0030] In order to clarify the drug sensitivity of antimicrobial peptide MM19 to clinical Gram-positive and Gram-negative bacteria, the minimum inhibitory concentration (MIC) was detected by the micro-liquid dilution method. MIC is expressed in the form of molar concentration / mass concentration, with the molar concentration unit being μM and the mass concentration unit being μg / mL. The specific method is as follows: the concentration of the bacterial culture to be tested in the logarithmic phase is adjusted to 2.0×10 6 CFU / mL; then take an equal volume of bacterial solution and add it to a 96-well plate with different concentrations of antimicrobial peptide MM19, and set an equal volume of 100 μL MHB culture medium and 100 μL of the test bacterial solution as blank control and growth control, respectively; the above 96-well plate was cultured at 37°C for 24 hours and the results were observed. Compared with the negative control group, the drug concentration corresponding to the clear or no growth wells observed by naked eye was the MIC value of the test drug.

[0031] The results are shown in Table 2. The antimicrobial peptide MM19 showed good in vitro antibacterial effects on the tested bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae), and the MIC values ​​ranged from 1.67 / 4 to 3.34 / 8. In order to further clarify the activity of the antimicrobial peptide MM19 against clinical drug-resistant bacteria, we determined the MIC values ​​of the antimicrobial peptide MM19 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 are still encouraging: the antimicrobial peptide MM19 also showed good antibacterial activity against the above-mentioned drug-resistant bacteria (Table 3). In order to further verify the activity of the antimicrobial peptide MM19, we used the changes in the MIC value after 24 hours of action of the antimicrobial peptide MM19. The results are as follows. Figure 2 As shown in the figure, the antimicrobial peptide MM19 can effectively kill the tested bacteria at the MIC concentration. The above results show that the antimicrobial peptide MM19 is a new type of antimicrobial peptide with high efficiency and broad spectrum, and has good research value.

[0032] Table 2 In vitro antimicrobial spectrum of antimicrobial peptide MM19

[0033]

[0034] Note: Polymyxin B: PMB; Vancomycin: Van; “NA” means not detected, the same below.

[0035] Table 3 In vitro activity of antimicrobial peptide MM19 against drug-resistant bacteria

[0036]

[0037] Example 3: Biocompatibility of antimicrobial peptide MM19

[0038] To investigate the safety of antimicrobial peptide MM19, we detected its hemolytic activity and cytotoxicity. The specific methods are as follows: ① Hemolytic activity detection: 8% hRBCs were incubated with different concentrations of MM19 for 24 h, and then the supernatant was taken to detect the absorbance value at A 540 nm in a multi-functional 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 MM19 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 CCK8 detection kit was used for determination.

[0039] The hemolytic activity results showed that antimicrobial peptide MM19 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 MM19 had low cytotoxic effects on human normal liver cells LO2 and human renal proximal tubular epithelial cells HK-2 at a concentration of 32 μM ( Figure 3 B-C); while it showed anti-cancer activity against cancer cells (A549 and HeLa) ( Figure 3 D-E). The above results indicate that antimicrobial peptide MM19 has good biocompatibility and the potential to be developed into an anti-cancer drug.

[0040] Example 4: Physiological stability of antimicrobial peptide MM19

[0041] To evaluate the physiological stability of antimicrobial peptide MM19, 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 MM19 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. Using MHB culture medium as the control group, after culturing at 37 °C for 24 h, the change of MIC value of MM19 was detected.

[0042] 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 MM19. Surprisingly, the activity of antimicrobial peptide MM19 only increased by 2-4 times in the pepsin and proteinase k environments; like other polypeptides, antimicrobial peptide MM19 was sensitive to trypsin and was easily inactivated in the trypsin environment.

[0043] Although the preferred embodiments of the present invention have been described, additional changes and modifications 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 MM19 with broad-spectrum antibacterial activity, characterized in that, The amino acid sequence of the antimicrobial peptide MM19 is shown as SEQ ID NO.

1.

2. Use of the antimicrobial peptide MM19 according to claim 1 in the preparation of an antimicrobial product, 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 antibacterial peptide MM19 according to claim 2 in the preparation of antibacterial products, characterized in that, The antimicrobial product takes the antimicrobial peptide MM19 as the only active ingredient.

4. Use of the antibacterial peptide MM19 according to claim 2 in the preparation of antibacterial products, characterized in that, The antimicrobial product is a drug or a bacteriostatic agent.

5. Use of the antimicrobial peptide MM19 according to claim 2 in the preparation of antimicrobial products, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. Use of the antibacterial peptide MM19 according to claim 5 in the preparation of antibacterial products, characterized in that, The excipients are the excipients required for preparing one of aqueous extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

7. Use of the antimicrobial peptide MM19 according to claim 1 in the preparation of a drug for treating cancer.

8. Use of the antibacterial peptide MM19 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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