Antibacterial peptide Mp-gc21, precursors and uses thereof

Abstract

The application belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide Mp-GC21, a precursor thereof and application. The amino acid sequence of the antibacterial peptide Mp-GC21 is shown as SEQ ID NO:1, wherein the C terminal is subjected to amidation modification, and two cysteines are connected through a disulfide bond; and the amino acid sequence of the precursor is shown as SEQ ID NO:3. The antibacterial peptide Mp-GC21 is separated from the flower frog, has broad-spectrum antibacterial activity, and has good bacteriostatic and bactericidal effects on standard strains and clinically derived drug-resistant strains of Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, and can target to destroy the cell membrane of bacteria, remove the biofilm and inhibit the formation of the biofilm. Moreover, the antibacterial peptide Mp-GC21 has good stability, does not have hemolytic activity, cytotoxicity and in-vivo toxicity, and is not easy to induce drug resistance of strains.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide Mp-GC21, its precursor, and its applications. Background Technology

[0002] The widespread use and abuse of broad-spectrum antibiotics and antimicrobial drugs have led to a surge in drug-resistant bacteria. Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of structurally diverse short peptides, typically composed of fewer than 100 amino acids. Due to their ability to kill pathogens, including bacteria and fungi, and their broad-spectrum antimicrobial activity, AMPs have become one of the most promising alternatives to antibiotics. Furthermore, because bacteria or fungi often develop resistance to AMPs more slowly than to traditional antibiotics, they can serve as a valuable supplement to the current antibiotic repertoire, alleviating the problem of antibiotic resistance. The discovery and development of AMPs hold great promise.

[0003] Flower frog ( Microhyla pulchra The spotted frog (Rana spp.) is an amphibian belonging to the genus Rana of the family Ranaidae. Adults, when soaked in baijiu (Chinese white liquor) or pounded with alcohol and applied topically, can be used to treat various ailments such as fractures, back pain, rheumatic pain, weakness, injuries from falls and blows, suppuration of sores and carbuncles, and slow-healing wounds. Due to the small size of the spotted frog and the difficulty in capturing it, there is currently limited research on the structure and function of its skin pharmacologically active substances, as well as the material basis for its efficacy in traditional Chinese medicine. Antimicrobial peptides developed from the spotted frog are even rarer. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide Mp-GC21, its precursor, and its applications, which exhibits good bactericidal and bacteriostatic effects against Gram-negative bacteria, including Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae, Gram-positive bacteria, including Staphylococcus aureus, and clinically derived drug-resistant strains. It targets and destroys bacterial cell membranes, inhibits biofilm formation, and removes existing biofilms. Furthermore, it does not possess hemolytic activity or cytotoxicity and has good safety.

[0005] The present invention provides an antimicrobial peptide Mp-GC21, the amino acid sequence of which is shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds.

[0006] Preferably, the C-terminus of the antimicrobial peptide Mp-GC21 is modified by amidation.

[0007] The present invention also provides a precursor of the antimicrobial peptide Mp-GC21 described in the above technical solution, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] The present invention also provides a DNA sequence encoding the antimicrobial peptide Mp-GC21 or its precursor described in the above technical solution, wherein the DNA sequence has the nucleotide sequence shown in SEQ ID NO:3.

[0009] The present invention also provides the application of the antimicrobial peptide Mp-GC21 or its precursor described in the above technical solution in one or more of the following:

[0010] (1) Preparation of bactericides; (2) Preparation of bacteriostatic agents; (3) Preparation of antimicrobial drugs; (4) Preparation of antibiofilm drugs.

[0011] Preferably, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria; the Gram-negative bacteria include one or more of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae; the Gram-positive bacteria include Staphylococcus aureus.

[0012] The anti-biofilm drug is used to break down the membrane of Acinetobacter baumannii.

[0013] Preferably, the Acinetobacter baumannii includes a standard strain of Acinetobacter baumannii and / or carbapenem-resistant Acinetobacter baumannii;

[0014] The Klebsiella pneumoniae include carbapenem-resistant Klebsiella pneumoniae;

[0015] The Staphylococcus aureus includes standard strains of Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

[0016] Preferably, the antimicrobial agent includes drugs for the prevention and / or treatment of bacterial infectious diseases;

[0017] The anti-biofilm drug is used to remove biofilms and / or inhibit biofilm formation.

[0018] The present invention also provides a reagent with bactericidal and / or bacteriostatic functions, wherein the active ingredient of the reagent includes the antimicrobial peptide Mp-GC21 or its precursor as described in the above technical solution.

[0019] The present invention also provides an antibacterial and / or antibiofilm drug, the drug comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprising the antimicrobial peptide Mp-GC21 or its precursor as described in the above technical solution.

[0020] Beneficial effects:

[0021] This invention provides an antimicrobial peptide Mp-GC21, with the amino acid sequence shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds, and the amino acid sequence of its precursor is shown in SEQ ID NO:3. The antimicrobial peptide Mp-GC21 provided by this invention was isolated from the flower frog and possesses broad-spectrum antimicrobial activity. It exhibits good antibacterial and bactericidal effects against standard strains and clinically resistant strains of Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus. Furthermore, it can target and disrupt bacterial cell membranes, clearing biofilms and inhibiting biofilm formation. Considering factors such as stability, hemolytic activity, cytotoxicity, extracellular membrane permeability, cellular resistance, and in vivo toxicity, the antimicrobial peptide Mp-GC21 demonstrates good stability and lacks hemolytic activity, cytotoxicity, and in vivo toxicity, making it unlikely to induce drug resistance in bacterial strains. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 This is a helical wheel diagram of the peptide Mp-GC21;

[0024] Figure 2 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) of the polypeptide Mp-GC21 against different bacterial strains;

[0025] Figure 3 The bactericidal kinetics of peptide Mp-GC21 against Acinetobacter baumannii ATCC 19606;

[0026] Figure 4 Salt sensitivity of peptide Mp-GC21;

[0027] Figure 5 The thermal stability of peptide Mp-GC21;

[0028] Figure 6 The graph shows the hemolytic activity of peptide Mp-GC21 against hRBCs.

[0029] Figure 7 The image shows the hemolytic activity of peptide Mp-GC21 against hRBCs; from left to right, the samples are PBS, 1% Triton X-100, 16×MIC, 8×MIC, 4×MIC, 2×MIC, 1×MIC, and 0.5×MIC.

[0030] Figure 8 The graph shows the cytotoxicity test results of peptide Mp-GC21 on different cells.

[0031] Figure 9The effect of peptide Mp-GC21 on intracellular reactive oxygen species in Acinetobacter baumannii;

[0032] Figure 10 The effect of peptide Mp-GC21 on biofilm formation in Acinetobacter baumannii;

[0033] Figure 11 The effect of peptide Mp-GC21 on the removal of biofilm from Acinetobacter baumannii;

[0034] Figure 12 The effect of peptide Mp-GC21 on the biomembrane permeation of Acinetobacter baumannii;

[0035] Figure 13 The image shows the results of the detection of Acinetobacter baumannii's resistance to the peptide Mp-GC21.

[0036] Figure 14 The results of in vivo toxicity assays for peptide Mp-GC21;

[0037] Where NS indicates no significant difference express P <0.005; express P <0.01; express P <0.001; express P＜ 0.0001. Detailed Implementation

[0038] The present invention provides an antimicrobial peptide Mp-GC21, the amino acid sequence of which is shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds.

[0039] In one embodiment, the C-terminus of the antimicrobial peptide Mp-GC21 of the present invention is modified by amidation. This amidation modification of the C-terminus of the antimicrobial peptide Mp-GC21 further improves its stability.

[0040] The present invention also provides a precursor of the antimicrobial peptide Mp-GC21 described in the above technical solution, the amino acid sequence of which is shown in SEQ ID NO:2, specifically: MFTLKKTMLLLFFLGTISLSLCEQERNAEEERRDEEVAKIEEVKRGILKGILGMGKNLVCGLSGLC.

[0041] The present invention also provides a DNA sequence encoding the antimicrobial peptide Mp-GC21 or its precursor described in the above technical solution, wherein the DNA sequence has the nucleotide sequence shown in SEQ ID NO:3, specifically: ATGTTCACCTTGAAGAAAACCATGTTACTCCTTTTCTTTCTTGGAACCATCTCCTTATCTCTCTGTGAGCAAGAGAGAAACGCCGAAGAAGAAAGAAGAGATGAAGAAGTTGCTAAAATAGAAGAGGTAAAACGCGGTATTTTAAAGGGCATCCTCGGTATGGGGAAGAACTTAGTATGTGGACTTAGCGGGCTGTGC.

[0042] The present invention also provides the application of the antimicrobial peptide Mp-GC21 or its precursor described in the above technical solution in one or more of the following: (1) preparation of bactericides; (2) preparation of bacteriostatic agents; (3) preparation of antimicrobial drugs; (4) preparation of antibiofilm drugs.

[0043] In one embodiment, the bacteria described in this invention include Gram-negative bacteria and / or Gram-positive bacteria. In one embodiment, the Gram-negative bacteria described in this invention include one or more of *Escherichia coli*, *Acinetobacter baumannii*, and *Klebsiella pneumoniae*. In one embodiment, the Gram-positive bacteria described in this invention include *Staphylococcus aureus*. In one embodiment, the anti-biofilm drug described in this invention is used to disrupt the biofilm of *Acinetobacter baumannii*.

[0044] In one embodiment, the *Acinetobacter baumannii* of the present invention includes a standard strain of *Acinetobacter baumannii* and / or carbapenem-resistant *Acinetobacter baumannii*. In another embodiment, the *Klebsiella pneumoniae* of the present invention includes carbapenem-resistant *Klebsiella pneumoniae*. In yet another embodiment, the *Staphylococcus aureus* of the present invention includes a standard strain of *Staphylococcus aureus* and / or methicillin-resistant *Staphylococcus aureus*.

[0045] In one embodiment, the antibacterial drug of the present invention includes drugs for the prevention and / or treatment of bacterial infectious diseases. In another embodiment, the antibiofilm drug of the present invention is used to remove biofilms and / or inhibit biofilm formation.

[0046] The present invention also provides a reagent with bactericidal and / or bacteriostatic functions, wherein the active ingredient of the reagent includes the antimicrobial peptide Mp-GC21 or its precursor as described in the above technical solution.

[0047] This invention also provides an antibacterial and / or antibiofilm drug, the drug comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprising the antimicrobial peptide Mp-GC21 or its precursor described in the above-described technical solution. This invention does not impose strict requirements on the type of excipients; they can be conventionally selected based on the drug's dosage form.

[0048] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an antimicrobial peptide Mp-GC21, its precursor, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] The polypeptide sequence described in SEQ ID NO:1 (GILKGILGMGKNLVCGLSGLC) was synthesized artificially. Its carboxyl terminus was amidated and designated Mp-GC21. Two cysteine ​​residues are linked by disulfide bonds. The helical wheel diagram is shown below. Figure 1 As shown in Table 1, the physicochemical properties were predicted using the EXPASY online webpage (https: / / www.expasy.org / ).

[0051] Table 1 Physicochemical properties of peptide Mp-GC21

[0052]

[0053] Example 2

[0054] Antibacterial activity of peptide Mp-GC21

[0055] 1. Test strain

[0056] Escherichia coli ( E. coil ATCC 8739, Acinetobacter baumannii ( A. baumannii ATCC 19606, carbapenem-resistant Acinetobacter baumannii CRAb (obtained from the Fourth Affiliated Hospital of Guangzhou Medical University), carbapenem-resistant Klebsiella pneumoniae CRKp (obtained from the Fourth Affiliated Hospital of Guangzhou Medical University), Staphylococcus aureus ( S. Aureus ATCC 6538 and methicillin-resistant Staphylococcus aureus (MRSA) (taken from the Third Affiliated Hospital of Sun Yat-sen University).

[0057] 2. Determination of minimum inhibitory concentration (MIC)

[0058] The antibacterial activity of peptide Mp-GC21 was determined using the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). First, the cryopreserved solution of the test strain was removed from -80°C and inoculated into LB liquid medium at a ratio of 1:200 (v:v), and cultured overnight on a shaker (37°C, 220 rpm). Then, an appropriate amount of the bacterial culture was transferred to LB medium and cultured at 37°C, 220 rpm until the logarithmic growth phase. The bacterial culture was then diluted with LB medium to a final volume of 2 × 10⁻⁶. 6 CFU / mL. Prepare a 100 µM stock solution of peptide Mp-GC21 and store it at 4°C. Transfer 200 µL of the peptide Mp-GC21 stock solution to the first column of a 96-well plate and serially dilute using a two-fold dilution method to obtain peptide solutions of different concentrations. Each sample should have at least three replicates. Then, add 100 µL of bacterial suspension to each well, mix thoroughly, and incubate at 37°C for 16 h. Use the uninoculated bacterial group (different concentrations of peptide Mp-GC21 + LB liquid medium) as a blank control, the group without peptide Mp-GC21 (physiological saline + bacterial suspension) as a negative control, and melittin and polymyxin B as positive controls. After incubation, measure the OD value at 630 nm using a microplate reader. The lowest concentration that completely inhibits bacterial growth is defined as the minimum inhibitory concentration (MIC).

[0059] The results are shown in Table 2 and Figure 2 As shown, the minimum inhibitory concentrations (MICs) of peptide Mp-GC21 against Escherichia coli ATCC 8739, Acinetobacter baumannii ATCC 19606, carbapenem-resistant Acinetobacter baumannii CRAb, carbapenem-resistant Klebsiella pneumoniae CRKp, Staphylococcus aureus ATCC 6538, and methicillin-resistant Staphylococcus aureus MRSA were 2.3438 μM, 4.6875 μM, 4.6875 μM, 37.5 μM, 2.3438 μM, and 4.6875 μM, respectively.

[0060] Table 2. Results of minimum inhibitory concentration (μM) detection

[0061]

[0062] 3. Minimum bactericidal concentration (MBC) determination

[0063] After overnight culture in step 2, all wells where no sterile growth was observed by visual inspection were mixed by pipetting. 10 µL of bacterial culture was then spotted onto the corresponding culture medium plates using a pipette. The plates were incubated upside down in a constant temperature incubator for 16 h, and no microbial growth was observed and recorded. The minimum bactericidal concentration (MBC) was defined as the highest concentration of peptide with visible microbial growth minus the concentration of peptide with no visible microbial growth.

[0064] The results are shown in Table 3 and Figure 2 As shown, the minimum bactericidal concentrations of peptide Mp-GC21 against Escherichia coli ATCC 8739, Acinetobacter baumannii ATCC 19606, carbapenem-resistant Acinetobacter baumannii CRAb, carbapenem-resistant Klebsiella pneumoniae CRKp, Staphylococcus aureus ATCC 6538, and methicillin-resistant Staphylococcus aureus MRSA are 4.6875 μM, 4.6875 μM, 9.375 μM, >50 μM, 4.6875 μM, and 9.375 μM, respectively.

[0065] Table 3. Minimum bactericidal concentration test results (μM)

[0066]

[0067] Example 3

[0068] bactericidal kinetics of peptide Mp-GC21

[0069] Acinetobacter baumannii ATCC 19606 in logarithmic growth phase was diluted to 2 × 10⁻⁶ using fresh liquid LB medium. 6 CFU / mL, peptide Mp-GC21, melittin, and polymyxin B were added to the diluted bacterial solution at a 1:1 volume ratio to achieve final concentrations of 1×MIC, 2×MIC, and 4×MIC, respectively. The final concentration of Acinetobacter baumannii ATCC19606 was 1×10⁻⁶. 6 CFU / mL, with the corresponding volume of physiological saline added to the negative control. The bacterial suspension added to the sample was immediately placed in a 37°C incubator. At 0 min, 1 min, 10 min, 30 min, 60 min, 120 min, and 180 min, 5 µL of the bacterial suspension was taken and diluted 100-fold with sterile physiological saline. 10 µL of each solution was then spread onto solid culture plates using a multi-channel pipette. The plates were then incubated upside down at 37°C for 16 h, and colony counts were performed. Each group was performed in triplicate, and the mean ± SEM values ​​were calculated.

[0070] The results are as follows Figure 3 As shown, at a concentration of 1×MIC, peptide Mp-GC21 completely killed Acinetobacter baumannii ATCC 19606 within 60 min, while the positive control Melittin required a concentration of 2×MIC to completely kill Acinetobacter baumannii ATCC19606. Specifically, at a concentration of 2×MIC, peptide Mp-GC21 completely killed the bacteria within 30 min, indicating that peptide Mp-GC21 has a rapid, dose-dependent bactericidal ability against Acinetobacter baumannii.

[0071] Example 4

[0072] Stability of peptide Mp-GC21

[0073] 1. Salt sensitivity

[0074] Physiological salts affect the bioactivity of antimicrobial peptides by interfering with the electrostatic interaction between the peptides and the bacterial membrane. This study investigated the antimicrobial activities of peptides Mp-GC21, melittin, and polymyxin B against *Acinetobacter baumannii* ATCC 19606 in the presence of different physiological salt solutions (150 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, and 2.5 mM CaCl2). Different physiological salts were dissolved in LB broth, and the bacterial culture was diluted to 2 × 10⁻⁶ with LB broth containing physiological salts. 6 A bacterial suspension of CFU / mL was prepared and the peptide Mp-GC21 was serially diluted twofold. 100 µL of each bacterial suspension and peptide Mp-GC21 solution were added to a 96-well plate and incubated at 37°C for 16 h. The minimum inhibitory concentration (MIC) was then determined.

[0075] The results are shown in Table 4 and Figure 4 As shown, the activity of peptide Mp-GC21 was not significantly affected in solutions containing ions of various physiological concentrations, indicating that it can maintain antibacterial activity in complex physiological environments.

[0076] Table 4 Salt sensitivity of peptide Mp-GC21

[0077]

[0078] 2. Thermal stability

[0079] The antibacterial activities of peptide Mp-GC21, melittin, and polymyxin B against Acinetobacter baumannii ATCC 19606 were investigated under different temperature treatments (4℃, 40℃, 60℃, 80℃, 100℃). The peptides Mp-GC21, melittin, and polymyxin B were treated at 4℃, 40℃, 60℃, 80℃, and 100℃ for 1 h, respectively, and then cooled to room temperature. The bacterial suspensions were then diluted to 2×10⁻⁶. 6 A bacterial suspension of CFU / mL was prepared and serially diluted twofold (the initial concentration of peptide Mp-GC21 was 100 µM). 100 µL of each bacterial suspension and peptide solution were added to a 96-well plate and incubated at 37°C for 16 h. The minimum inhibitory concentration (MIC) was then determined.

[0080] The results are shown in Table 5 and Figure 5As shown, the antibacterial activity of peptide Mp-GC21 remained unchanged after being treated at different temperatures (from 4°C to 100°C), demonstrating its excellent thermal stability.

[0081] Table 5 Thermal stability of peptide Mp-GC21

[0082]

[0083] Example 5

[0084] Hemolytic activity of peptide Mp-GC21

[0085] The hemolytic activity of peptide Mp-GC21 was determined by measuring the amount of hemoglobin released from lysed human erythrocytes. Fresh human erythrocytes (hRBCs) were centrifuged at 2500 rpm for 15 min at 4 °C. The concentrated hRBCs were washed three times with PBS until the supernatant was clear and colorless, and then resuspended in physiological saline to obtain a 4% (v / v) erythrocyte suspension. Next, 400 μL of the suspension was incubated with 400 μL of the drug (peptide Mp-GC21 and melittin) solution (5 replicates) in PBS prepared by double dilution, at concentrations of 0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC for Acinetobacter baumannii ATCC 19606, at 37 °C for 1 h. After centrifugation at 3000 rpm for 10 min, the absorbance (OD) of the supernatant at 540 nm was measured using a microplate reader. hRBCs in PBS and 1% (v / v) Triton X-100 were used as negative and positive controls, respectively. The test was repeated three times, and the average of the three independent measurements was used for data analysis. Hemolytic activity (%) was calculated as follows: Hemolytic activity (%) = (OD) / (HRBCs in PBS and 1% (v / v) Triton X-100) 多肽 -OD PBS ) / (OD Triton X-100 -OD PBS ) × 100%.

[0086] The results are shown in Table 6 and Figures 6-7 As shown, peptide Mp-GC21 exhibited almost no hemolytic activity within the concentration range of 2.3438–9.375 µM (0.5–2 × MIC). Even at a concentration of 18.75 µM (4 × MIC), the hemolytic activity of peptide Mp-GC21 remained low; however, further increases in the concentration of Mp-GC21 significantly increased the hemolytic activity. These results indicate that, at effective concentrations, peptide Mp-GC21 exhibits low hemolytic activity against human erythrocytes.

[0087] Table 6. Hemolytic activity of peptide Mp-GC21 against hRBC (%)

[0088]

[0089] Example 6

[0090] Cytotoxicity of peptide Mp-GC21

[0091] The toxicity of peptide Mp-GC21 to various cell types was determined using the CCK-8 assay. The cell density in the 96-well plate was approximately 1 × 10⁻⁶. 5 Cells / well, final concentration of peptide Mp-GC21: 2.3438–75 µM. First, cells were resuscitated, collected by centrifugation, resuspended in DMEM complete medium, and then transferred to T25 culture flasks and incubated in a cell culture incubator (37°C, 5% CO2). After a certain period of culture, cell growth was observed using a fluorescence inverted microscope. Once cells adhered, the medium was changed. When the cell density reached 80%–90%, the cells were passaged. After 2–4 passages, cell counts were performed using a cell counter, and the cells were diluted to 1 × 10⁻⁶ with DMEM complete medium. 5 Cells / mL. Transfer 100 µL of cell suspension into a 96-well plate; the cell density at this point is approximately 1 × 10⁻⁶ cells / mL. 4 Cells / wells were incubated in a cell culture incubator for 24 h. After discarding the culture medium, the cells were washed twice with PBS, and 100 µL of different concentrations of peptide solution was added to each well, with 6 parallel wells for each concentration. Cells were then incubated for another 24 h. After the culture period, 100 µL of CCK-8 solution (V...) was added to each well. 培养基 V CCK-8 =10:1), cultured in the dark for about 1 h. Then, the OD value at 450 nm was measured using a microplate reader. The unseeded cell group (different concentrations of peptide + serum-free DMEM medium) served as the blank group, and the group without added peptide (PBS + cell suspension) served as the control group. According to the formula (OD... 多肽组 -OD 空白组 ) / (OD 对照组 -OD 空白组 ) × 100%, calculate cell viability.

[0092] The results are as follows Figure 8 As shown, at effective concentrations, the peptide exhibits low cytotoxicity to a variety of cell types.

[0093] Example 7

[0094] Effects of peptide Mp-GC21 on intracellular reactive oxygen species in Acinetobacter baumannii

[0095] Acinetobacter baumannii ATCC 19606 in the logarithmic growth phase was collected, washed twice with PBS buffer, resuspended in LB medium, and the bacterial concentration was adjusted to 2 × 10⁻⁶. 7CFU / mL. Add 5 µM DCFH-DA solution to the final concentration, mix well, and incubate at 37 °C for 30 min in the dark. Collect the bacterial cells and wash twice with PBS. Then, serially dilute the peptide to different concentrations. Take 1 mL of bacterial suspension and mix it with an equal volume of peptide to final concentrations of 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC. The positive control Rousp is the reagent provided with the kit, and the negative control is the same volume of PBS. Then, incubate at 37 °C for 150 min. Every 30 min, take an appropriate amount of bacterial solution and measure the fluorescence value (Ex / Em=488 / 525nm) using a multi-mode microplate reader.

[0096] The results are as follows Figure 9 As shown, compared with the negative control group, the intracellular fluorescence intensity of Acinetobacter baumannii ATCC 19606 was enhanced after exposure to a peptide solution of 0.5×MIC. The fluorescence intensity continued to increase with increasing peptide concentration and incubation time. This result suggests that the peptide may induce intracellular oxidative stress, leading to excessive production of reactive oxygen species in the bacteria, ultimately resulting in cell death.

[0097] Example 8

[0098] Effects of peptide Mp-GC21 on biofilm of Acinetobacter baumannii

[0099] 1. Effect of peptide Mp-GC21 on biofilm formation in Acinetobacter baumannii

[0100] Collect Acinetobacter baumannii ATCC 19606 in the logarithmic growth phase, wash twice with PBS, and then dilute to 2×10⁻⁶ with LB medium. 7 CFU / mL. The peptide and the positive controls melittin and polymyxin B were diluted with physiological saline to a final concentration of 8×MIC. 200 µL of the 8×MIC sample was added to the first well of a 96-well plate, and 100 µL of physiological saline was added to the remaining wells. 100 µL of the sample was added to the second well, and the concentrations were diluted sequentially to 0.5×, 1×, 2×, and 4×MIC. Then, 100 µL of bacterial suspension was added to each well, and the plate was incubated at 37°C for 48 h. After incubation, the culture medium and suspended bacterial cells were discarded, and the plate was gently washed with PBS. The 96-well plate was dried in a 60°C oven for 1 h, and then incubated with 0.1% crystal violet aqueous solution for 15 min. After washing with ddH2O and drying, the biofilm was dissolved in 95% ethanol, and the absorbance was measured at 600 nm using a microplate reader. The blank control was not treated, and the negative control was added with the same volume of physiological saline. The absorbance was measured according to the formula (OD). 多肽组 -OD 空白对照 ) / (OD 阴性对照组 -OD空白对照 ) × 100%, calculate the biofilm inhibition rate (%).

[0101] The results are as follows Figure 10 As shown, the peptide Mp-GC21 inhibits biofilm formation in Acinetobacter baumannii ATCC 19606 in a dose-dependent manner.

[0102] 2. Effect of peptide Mp-GC21 on biofilm formation in Acinetobacter baumannii

[0103] Collect Acinetobacter baumannii ATCC 19606 in the logarithmic growth phase, wash twice with PBS, and then dilute to 1×10⁻⁶ with LB medium. 7 CFU / mL. Add 200 µL of bacterial suspension to each well of a 96-well plate and incubate at 37°C for 48 h. After incubation, discard the culture medium and suspended bacterial cells, and wash three times with PBS. Dilute the polypeptide and the positive controls melittin and polymyxin B to 1×MIC, 2×MIC, and 4×MIC concentrations with physiological saline, respectively, and then add 100 µL of sample to each well, incubating at 37°C for 24 h. After incubation, wash the plate 2-3 times with PBS. Dry the 96-well plate in a 60°C oven for 1 h, add 0.1% crystal violet aqueous solution and let stand for 15 min. Wash with ddH2O and air dry, then dissolve the biofilm in 95% ethanol and measure the absorbance at 600 nm using a microplate reader. The blank control was not treated, and the negative control was added with the same volume of physiological saline. Measure the absorbance according to the formula (OD... 多肽组 -OD 空白对照 ) / (OD 阴性对照组 -OD 空白对照 ) × 100%, calculate the biofilm inhibition rate (%).

[0104] The results are as follows Figure 11 As shown, the peptide Mp-GC21 removes the biofilm formed by Acinetobacter baumannii ATCC 19606 in a dose-dependent manner.

[0105] Example 9

[0106] Effect of peptide Mp-GC21 on bacterial outer membrane permeability

[0107] The effect of the peptide on bacterial outer membrane permeability was detected using the hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN). *Acinetobacter baumannii* ATCC 19606 in the logarithmic growth phase was collected by centrifugation at 3000 rpm for 5 min, washed three times with HEPES buffer (5 mM HEPES, 5 mM glucose, pH 7–7.5), and resuspended to 1 × 10⁻⁶. 7CFU / mL. Peptides at concentrations of 1×, 2×, and 4× MIC were mixed with bacterial culture and incubated at 37°C for 1 h. Then, NPN was added to a final concentration of 10 µM, and the mixture was incubated at room temperature for 30 min. The mixture was then washed with HEPES buffer, and the fluorescence intensity at 420 nm (excitation wavelength 350 nm) was measured using a fluorescence spectrometer. An equal volume of HEPES treatment served as a blank control, and a positive control was 0.1 mg / mL Polymyxin B.

[0108] The results are as follows Figure 12 As shown, after treating Acinetobacter baumannii ATCC 19606 with different concentrations of peptide Mp-GC21 and 0.1 mg / mL Polymyxin B for 1 h, the NPN fluorescence intensity increased in a concentration-dependent manner, indicating that peptide Mp-GC21 has the ability to disrupt the outer membrane.

[0109] Example 10

[0110] Resistance of Acinetobacter baumannii to peptide Mp-GC21

[0111] Acinetobacter baumannii ATCC 19606 was activated overnight, then transferred at a ratio of 1:200, and the bacterial count was adjusted to 1×10⁶. 6 CFU / mL, with a peptide or positive control (melittin and polymyxin B) at a concentration of 0.5 × MIC, were co-incubated with Acinetobacter baumannii. After 24 h of culture, the MIC of the surviving bacteria was measured. Then, a portion of the bacteria was taken and co-incubated with the peptide or positive control at a concentration of 0.5 × MIC again, and this process was repeated until the 15th generation. The MIC values ​​of the bacterial cultures of the 1st, 5th, 10th, and 15th generations were measured, and the changes in the MIC values ​​of each group were analyzed. The group without peptide or positive control was used as the negative control group (Nc).

[0112] The results are shown in Table 7 and Figure 13 As shown, after the 15th passage, the MIC value of Acinetobacter baumannii ATCC 19606 against Polymyxin B increased 16-fold, indicating that the antibacterial performance of Polymyxin B gradually decreased after continuous stress. However, after 15 consecutive treatments, the MIC value of Mp-GC21 against Acinetobacter baumannii ATCC 19606 did not change, suggesting that Polymyxin B easily induces drug resistance in Acinetobacter baumannii.

[0113] Table 7. Results of resistance testing of Acinetobacter baumannii to peptide Mp-GC21

[0114]

[0115] Example 11

[0116] In vivo toxicity assay of peptide Mp-GC21

[0117] Acute toxicity of the peptide in mice was assessed by a single tail vein injection of either a peptide dissolved in saline at a specified dose or a positive control (polymyxin B), and the LD50 was calculated. Eight-week-old female C57BL / 6j mice were randomly assigned to each group (n=6 mice per group). The peptide treatment group consisted of three subgroups treated with 5, 10, and 20 mg / kg of peptide Mp-GC21. The positive control group consisted of three subgroups treated with 5, 10, and 20 mg / kg of polymyxin B. The negative control group received an equal volume of saline. Survival rates of mice in each group were assessed within 14 days.

[0118] The results are as follows Figure 14 As shown, when the concentration of Polymyxin B reached 10 mg / kg, all mice died within 1 minute, indicating that Polymyxin B has acute toxicity to mice. In contrast, when the concentration of peptide Mp-GC21 was as high as 20 mg / kg, all mice survived during the observation period, indicating that peptide Mp-GC21 did not have acute toxicity to mice at this concentration.

[0119] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of antimicrobial peptide Mp-GC21 or its precursor in one or more of the following: (1) Preparation of bactericides; (2) Preparation of bacteriostatic agents; (3) Preparation of antimicrobial drugs; (4) Preparation of antibiofilm drugs; The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria mentioned are Escherichia coli (Escherichia coli) Escherichia coli Acinetobacter baumannii ( Acinetobacter baumnnii ) and Klebsiella pneumoniae ( Klebsiella Pneumoniae One or more of the following; The Gram-positive bacteria is Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus ); The anti-biofilm drug is used to break down the membrane of Acinetobacter baumannii; The amino acid sequence of the antimicrobial peptide Mp-GC21 is shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds; the C-terminus of the antimicrobial peptide Mp-GC21 is modified by amidation.

2. The application according to claim 1, characterized in that, The amino acid sequence of the precursor of the antimicrobial peptide Mp-GC21 is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The nucleotide sequence encoding the DNA sequence of the antimicrobial peptide Mp-GC21 or its precursor is shown in SEQ ID NO:

3.

4. The application according to claim 1, characterized in that, The Acinetobacter baumannii includes standard strains of Acinetobacter baumannii and / or carbapenem-resistant Acinetobacter baumannii; The Klebsiella pneumoniae include carbapenem-resistant Klebsiella pneumoniae; The Staphylococcus aureus includes standard strains of Staphylococcus aureus and / or methicillin-resistant Staphylococcus aureus.

5. The application according to any one of claims 1 to 4, characterized in that, The antimicrobial drugs include drugs for the prevention and / or treatment of bacterial infectious diseases; The anti-biofilm drug is used to remove biofilms and / or inhibit biofilm formation.

6. A reagent with bactericidal and / or bacteriostatic functions, characterized in that, The active ingredient of the reagent includes the antimicrobial peptide Mp-GC21 or its precursor; The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria are one or more of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae. The Gram-positive bacteria is Staphylococcus aureus; The amino acid sequence of the antimicrobial peptide Mp-GC21 is shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds; the C-terminus of the antimicrobial peptide Mp-GC21 is modified by amidation.

7. A drug with antibacterial and / or antibiofilm properties, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises the antimicrobial peptide Mp-GC21 or its precursor. The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria are one or more of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae. The Gram-positive bacteria is Staphylococcus aureus; The anti-biofilm is a membrane ruptured by Acinetobacter baumannii; The amino acid sequence of the antimicrobial peptide Mp-GC21 is shown in SEQ ID NO:1, wherein two cysteine ​​residues in the amino acid sequence are linked by disulfide bonds; the C-terminus of the antimicrobial peptide Mp-GC21 is modified by amidation.

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