A Jilin Nonomura antimicrobial peptide SR25 and its preparation method and medical use

Abstract

The present invention discloses an antimicrobial peptide SR25 from Jilin Nonomura, a preparation method and medical use. The antimicrobial peptide SR25 is an antimicrobial peptide with a dual antibacterial mechanism. The antimicrobial peptide SR25 can change the morphology of Escherichia coli, affect the permeability of the cell membrane, and cause the leakage of intracellular substances. At the same time, it targets the succinate dehydrogenase of Escherichia coli, inhibits the activity of succinate dehydrogenase, and affects bacterial metabolism. The antimicrobial peptide SR25 has a different antibacterial mechanism from traditional antibiotics and is not easy to induce bacterial resistance. In addition, the SR25 hydrogel preparation shows excellent therapeutic effects in a diabetic mouse wound infection model, providing a new direction for the development of new antimicrobial drugs and laying a foundation for clinical treatment. The antimicrobial peptide of the present invention also has low cytotoxicity and hemolytic activity; in particular, it shows a dual antibacterial mechanism against Escherichia coli, and can be used to prepare antimicrobial agents and additives for use in food, medicine, feed and other industries.

Description

Technical Field

[0001] The invention discloses a Jilin Nonomura antimicrobial peptide SR25 and a preparation method thereof, which is a new antimicrobial peptide and an antimicrobial peptide targeting bacterial succinate dehydrogenase. The invention also provides medical uses of the antimicrobial peptide and belongs to the technical field of microbial medicine. Background Art

[0002] Currently, aquatic environments have become a reservoir for antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes, and serve as a channel for the maintenance and enhancement of antibiotic residues and ARB and their genetic transformation, enabling bacteria to acquire resistance in the presence of antibiotics. Therefore, in order to overcome the increasingly serious phenomenon of bacterial resistance, it is necessary to continuously explore and find new antimicrobial drugs.

[0003] Antimicrobial peptides (AMPs) have attracted much attention as a new generation of antibiotics. Unlike most traditional antibiotics that act on specific intracellular targets, AMPs primarily fight bacteria through electrostatic interactions and physical disruption of the microbial lipid bilayer, and the nature of the membrane-active mechanism minimizes the likelihood that bacteria will evolve resistance to AMPs. Currently, more than 3,000 AMPs have been documented in the Antimicrobial Peptide Database, however, only seven of them have been approved by the U.S. Food and Drug Administration for clinical use. Reduced efficacy in clinically relevant settings, high toxicity to mammalian cells, and proteolytic instability of natural AMPs have slowed their clinical implementation and application in the food industry. Therefore, new potent AMPs with improved in vivo antimicrobial properties and reduced cytotoxicity are urgently needed to overcome the limitations of natural AMPs. Summary of the invention

[0004] The purpose of the present invention is to provide an actinomycete Jilin Nonomura antimicrobial peptide SR25, which is a new antimicrobial peptide mined from the actinomycete genome, an antimicrobial peptide targeting bacterial succinate dehydrogenase, and has a broad-spectrum antibacterial medical use.

[0005] The present invention provides a Jilin Nonomura antimicrobial peptide SR25, which is a hydrophilic cationic polypeptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0006] Furthermore, the screening method of the Jilin Nonomura antimicrobial peptide SR25 described in the present invention comprises the following steps:

[0007] 1. Screening of potential antimicrobial peptides from Nonomura jilinensis: Searching for open reading frames in the sequenced whole genome sequence of Nonomura jilinensis, and using the antimicrobial peptide prediction server to search for peptides that may have antimicrobial activity, and obtaining potential antimicrobial peptide sequences from Nonomura jilinensis;

[0008] 2. Synthesis of potential Jilin Nonomura antimicrobial peptide: According to the potential peptide sequence obtained in step 1), artificial synthesis of potential Jilin Nonomura antimicrobial peptide SR25;

[0009] 3. Antimicrobial activity testing: The potential antimicrobial peptides from Jilin Nonomura were artificially synthesized in step 2) and the minimum inhibitory concentration, hemolytic activity and cytotoxicity were determined.

[0010] The Jilin Nonomura antimicrobial peptide SR25 of the present invention has antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella pullorum, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Listeria monocytogenes, Staphylococcus epidermidis and Candida albicans.

[0011] The Jilin Nonomura antimicrobial peptide SR25 of the present invention can be prepared into antimicrobial drugs, additives and preservatives.

[0012] The positive effects of the present invention are:

[0013] The provided Jilin Nonomura antimicrobial peptide SR25 is a new antimicrobial peptide and an antimicrobial peptide with a dual antimicrobial mechanism. The antimicrobial peptide SR25 can change the morphology of Escherichia coli, affect the permeability of the cell membrane, and cause the leakage of intracellular substances. At the same time, it targets Escherichia coli succinate dehydrogenase, inhibits the activity of succinate dehydrogenase, and affects bacterial metabolism. The antimicrobial peptide SR25 has a different antimicrobial mechanism from traditional antibiotics and is not easy to induce bacterial resistance. In addition, the SR25 hydrogel preparation showed excellent therapeutic effects in the diabetic mouse wound infection model, providing a new direction for the development of new antimicrobial drugs and laying the foundation for clinical treatment. The antimicrobial peptide of the present invention also has low cytotoxicity and hemolytic activity; in particular, it exhibits a dual antimicrobial mechanism against Escherichia coli, and can be used to prepare antimicrobial agents and additives for use in food, medicine, feed and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the cytotoxicity of the antimicrobial peptide SR25 against mouse macrophage cell lines and mouse fibroblast cell lines;

[0015] Figure 2 It is the hemolytic activity of the antimicrobial peptide SR25 on mouse erythrocytes;

[0016] Figure 3 It is the result of the experimental study on the development of resistance of Escherichia coli to the antimicrobial peptide SR25;

[0017] Figure 4 is a scanning electron micrograph of the effect of the antimicrobial peptide SR25 on the morphology of Escherichia coli;

[0018] Figure 5 is the effect of the antimicrobial peptide SR25 on the outer membrane permeability of Escherichia coli;

[0019] Figure 6 is the effect of the antimicrobial peptide SR25 on the inner membrane permeability of Escherichia coli;

[0020] Figure 7 is the effect of antimicrobial peptide SR25 on the leakage of intracellular nucleic acid and protein in Escherichia coli (A is the change of nucleic acid content, B is the change of protein content);

[0021] Figure 8 is the effect of antimicrobial peptide SR25 on the ATP content of E. coli;

[0022] Fig. 9 is the effect of antimicrobial peptide SR25 on ROS release from E. coli;

[0023] Fig.10 It is the molecular docking result of antimicrobial peptide SR25 and Escherichia coli succinate ubiquinone oxidoreductase (SQR);

[0024] Fig.11 It is the molecular dynamics simulation result of the antimicrobial peptide SR25 and Escherichia coli succinate ubiquinone oxidoreductase (SQR);

[0025] Fig.12 is the effect of antimicrobial peptide SR25 on the activity of succinate dehydrogenase in Escherichia coli;

[0026] Fig.13 is the effect of antimicrobial peptide SR25 on the activity of succinate ubiquinone oxidoreductase of Escherichia coli;

[0027] Fig.14 is the morphological diagram of the antimicrobial peptide SR25 hydrogel preparation;

[0028] Fig.15 It is the therapeutic effect of the antimicrobial peptide SR25 hydrogel preparation on wound infection in diabetic mice;

[0029] Fig.16 This is the effect of the antimicrobial peptide SR25 hydrogel preparation on the bacterial load in wound infection of diabetic mice (A is Escherichia coli, B is methicillin-resistant Staphylococcus aureus). DETAILED DESCRIPTION

[0030] Below, the present invention is described by giving examples, but the present invention is not limited to the following examples. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are all commercially available products. All the raw and auxiliary materials selected in the present invention, as well as the selected bacterial strain culture methods are well known in the art, and the % involved in the present invention are all mass volume percentages.

[0031] Example 1: Screening of antimicrobial peptides from Nonomura, Jilin

[0032] The whole genome sequence of Jilin Nonomura DL99 was imported into the EditSeq function of Lasergene software to find the open reading frame sequence below 200 bp, and then the nucleic acid sequence was translated into an amino acid sequence. The method of using the antimicrobial peptide prediction server to find peptides that may have antimicrobial activity is as follows: first, use the ADP3 (http: / / aps.unmc.edu / AP / main.php) program to make a preliminary prediction of the polypeptide sequence found in the previous step to evaluate whether it is likely to become an antimicrobial peptide. The indicators include: ① The polypeptide chain is positively charged; ② Whether there is a helical structure or whether the disulfide bond is paired; ③ The Berman index (kcal / mol), the larger the value, the higher the possibility of becoming an antimicrobial peptide. The antimicrobial peptide sequences that meet the above indicators and have a Berman index greater than 2 were screened and uploaded to the CAMPR3 (http: / / www.camp3.bicnirrh.res.in / predict / ) analysis program. Four calculation rules, Support Vector Machine, Random Forest, Artificial Neural Network, and Discriminant Analysis, were selected to evaluate the probability of the pre-selected peptides becoming antimicrobial peptides. The antimicrobial peptides with scores greater than 0.9 from the four algorithms were predicted to have toxicity using the Antimicrobial Peptide Toxicity Prediction Server (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), and the Design Peptide program in ToxinPred was used to predict the toxicity of potential antimicrobial peptides. The peptide sequence of potential antimicrobial peptides and no cytotoxicity obtained by computer screening was artificially synthesized and named: Jilin Nonomura Antimicrobial Peptide SR25, and its amino acid sequence is shown in SEQ ID No. 1.

[0033] Experimental Example 1: Antibacterial activity of Jilin Nonomura antimicrobial peptide SR25

[0034] Inoculate Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella pullorum, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Listeria monocytogenes, Bacillus subtilis, Staphylococcus epidermidis and Candida albicans until the logarithmic growth phase, and dilute the bacterial solution to 2×10 6 CFU / mL, different concentrations of antimicrobial peptide SR25, ampicillin and polymyxin E were added as positive controls, and after incubation at 37°C for 18 hours, the absorbance at 600nm of each well was detected by an ELISA instrument, and the minimum inhibitory concentration was calculated. The test results are shown in Table 1 below:

[0035] Table 1.

[0036]

[0037] Experimental Example 2: Cytotoxicity of Jilin Nonomura Antimicrobial Peptide SR25

[0038] The density is 5×10 4 100 μL of mouse macrophages RAW264.7 and mouse fibroblasts L929 were inoculated into 96-well cell culture plates at 5% CO2 and 37°C for 12 h. Antimicrobial peptides with final concentrations of 800 μM, 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM and 3.13 μM were added according to the half-dilution method and incubated in a constant temperature incubator at 5% CO2 and 37°C for 24 h. 10 μL of CCK-8 reagent was added to each well, mixed and cultured for 3 h in dark conditions. The absorbance at 450 nm of each well was detected by an enzyme-labeled instrument to calculate the cell survival rate. The specific results are shown in the attached figure. Figure 1 .

[0039] By the attached Figure 1 The results show that SR25 in the Jilin Nonomura antimicrobial peptide provided by the present invention has weak cytotoxicity to mouse macrophages RAW264.7 and mouse fibroblasts L929. At a concentration of 800 μM antimicrobial peptide, the cell survival rate is above 80%, indicating that the peptide has low cytotoxicity.

[0040] Experimental Example 3: Hemolytic activity of Jilin Nonomura antimicrobial peptide SR25

[0041] Prepare 0.9% saline, dilute the collected fresh mouse eyeball blood into 2% red blood cell suspension, add three antimicrobial peptides, PBS and 0.1% TritonX-100 as negative and positive controls according to the half-dilution method, and incubate the above groups in a 37°C water bath for 1 h. After incubation, take out and centrifuge at 1500 rpm / min for 10 min. Take 200 μL of the supernatant and add it to a 96-well cell culture plate, and detect the absorbance of each well at 414 nm by an enzyme marker. For specific results, see the attached Figure 2 .

[0042] By the attached Figure 2 The results show that SR25 in the Jilin Nonomura antimicrobial peptide of the present invention has little damage to mouse erythrocytes, indicating that the peptide has a low hemolytic effect.

[0043] Experimental Example 4: Development of drug resistance experiment of Jilin Nonomura antimicrobial peptide SR25

[0044] Collect E. coli in the logarithmic growth phase and dilute the bacterial solution to 2×10 6CFU / mL, different concentrations of antibiotics ampicillin, polymyxin E and antimicrobial peptide SR25 were added by half dilution method, incubated at 37℃ for 18 hours, and the MIC value of each group was detected. Then, Escherichia coli with sublethal concentration of 1 / 2×MIC was taken for continuous inoculation. After culturing to logarithmic growth, different concentrations of antibiotics ampicillin, polymyxin E and antimicrobial peptide SR25 were added to test the minimum inhibitory concentration, and the culture was continued in this way. The growth of MIC value was used to evaluate the development of drug resistance of antimicrobial peptides and antibiotics. For specific results, please refer to the attached Figure 3 .

[0045] By the attached Figure 3 The results show that after the antimicrobial peptide SR25 of the present invention continuously acts on Escherichia coli, Escherichia coli is not easy to develop drug resistance. When the Escherichia coli treated with the antibiotic ampicillin grows to the 11th generation, the MIC value increases by 20 times, and the Escherichia coli develops drug resistance to the antibiotic ampicillin.

[0046] Experimental Example 5: Effect of Jilin Nonomura Antimicrobial Peptide SR25 on Escherichia coli Morphology

[0047] Escherichia coli bacterial suspension (10 6 CFU / mL) was washed 3 times with filtered PBS for later use. Experimental group: Peptides with a final concentration of 1×MIC were added to 1 mL of bacterial solution, and incubated at 37°C for 1 h and 5 h, respectively. After incubation, the samples were centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the samples were washed 3 times with filtered PBS to remove the antimicrobial peptides. The bacteria were fixed with 2.5% glutaraldehyde and kept overnight at 4°C. After the samples were centrifuged, they were washed 3 times with PBS to wash away the glutaraldehyde. The samples were dehydrated by ethanol stepwise dehydration, and 30%, 50%, 70%, 80%, 90% and 100% ethanol were prepared. After 15 min of each step, the supernatant was discarded and the next step of ethanol was added until 100% ethanol was added. The samples were evenly coated on a glass slide and dried at room temperature. The samples were placed on an ion sputtering instrument for gold spraying and then observed under a scanning electron microscope. The specific results are shown in the attached Figure 4 .

[0048] By the attached Figure 4 The results show that the antimicrobial peptide SR25 provided by the present invention caused a significant change in the morphology of Escherichia coli after treating Escherichia coli. The surface of normal Escherichia coli cells is smooth and the morphology is intact. After the antimicrobial peptide SR25 acts on Escherichia coli for 1 hour, the bacterial surface appears wrinkled. After 5 hours of action, it can be observed that the cell membrane ruptures and the contents leak out.

[0049] Experimental Example 6: Effect of Jilin Nonomura antimicrobial peptide SR25 on the outer membrane permeability of Escherichia coli

[0050] Escherichia coli in logarithmic growth phase were centrifuged at 4000 r / min for 10 min, resuspended in HEPES buffer containing sodium azide and 1×10 8 CFU / mL for later use; NPN dye was first dissolved in 20 μL DMSO and then diluted with HEPES buffer to a final concentration of 10 μM for later use; the antimicrobial peptide was diluted from a final concentration of 50 μM to 1.57 μM according to the half-dilution method, 90 μL bacterial solution, 20 μL NPN dye, and 10 μL antimicrobial peptide were added to a 96-well plate, mixed and incubated in the dark for 3 h, and the well plate was placed on a fluorescence microplate reader to detect the fluorescence value of each well (Ex=350 nm, Em=420 nm). The specific results are shown in the attached Figure 5 .

[0051] By the attached Figure 5 The results show that the antimicrobial peptide SR25 provided by the present invention can change the permeability of the outer membrane of Escherichia coli and destroy the integrity of the outer membrane by increasing the permeability of the outer membrane.

[0052] Experimental Example 7: Effect of Jilin Nonomura antimicrobial peptide SR25 on the inner membrane permeability of Escherichia coli

[0053] The E. coli strain obtained after lactose induction was activated and cultured to the logarithmic growth phase, and the strain concentration was adjusted to 1×10 8 CFU / mL. Use a desktop centrifuge at 1000 g for 10 min, discard the supernatant, resuspend the bacterial solution with 0.9% NaCl solution, and use a multifunctional microplate reader to detect the absorbance value at 420 nm to make OD420=1.2±0.02. Use 0.5% NaCl solution to dilute the polypeptide to 4×MIC, 2×MIC, 1×MIC and 1 / 2×MIC, add 90 μL of polypeptide compound, 100 μL of the above bacterial solution, and 10 μL (30 mM) of o-nitrophenyl-β-D-pyranogalactoside (ONPG) to each well of a new 96-well plate. Among them, 0.9% NaCl solution was used as the negative control group of the experiment, and 3 parallels were set up for each group. After all the above components were added, the absorbance value at 420 nm was detected with a multifunctional microplate reader. The entire detection process was carried out every 10 minutes. The specific results are shown in the attached. Figure 6 .

[0054] By the attached Figure 6 The results show that as the concentration of the antimicrobial peptide SR25 provided by the present invention increases, the level of extracellular β-galactosidase increases significantly, proving that the antimicrobial peptide SR25 can target the bacterial cell membrane and enhance membrane permeability.

[0055] Experimental Example 8: Effects of Jilin Nonomura antimicrobial peptide SR25 on intracellular nucleic acid and protein leakage in Escherichia coli

[0056] Peptides at a concentration of 1×MIC were mixed with bacterial suspension (10 6 CFU / mL). The cultures were then incubated at 37°C for 3 hours and centrifuged at 3500×g for 5 minutes. The bacterial suspension was filtered through a 0.22 μm membrane, and the amount of protein and nucleic acid released from the cytoplasm into the supernatant was estimated by measuring the absorbance at 280 nm and 260 nm. Figure 7 .

[0057] By the attached Figure 7 The results show that as the action time of the antimicrobial peptide SR25 provided by the present invention increases, the nucleic acids and proteins in the Escherichia coli cells gradually leak into the extracellular environment, indicating that the cell membrane of the Escherichia coli is damaged.

[0058] Experimental Example 9: Effect of Jilin Nonomura Antimicrobial Peptide SR25 on ATP Content in Escherichia coli

[0059] To 1×10 8 Different concentrations of antimicrobial peptides were added to E. coli with CFU / mL for different treatment times, and the bacteria were collected by centrifugation and the bacterial density was adjusted to a uniform level. The bacterial cells were fully lysed using ATP detection lysis buffer and the sample was prepared for determination. The ATP content in the bacterial cells was determined and analyzed according to the instructions of the kit. The specific results are shown in the attached Figure 8 .

[0060] By the attached Figure 8 The results show that after the antimicrobial peptide SR25 provided by the present invention acts on Escherichia coli, the intracellular ATP level of the bacteria decreases significantly, proving that the antimicrobial peptide SR25 affects the synthesis of ATP in Escherichia coli.

[0061] Experimental Example 10: Effect of Jilin Nonomura antimicrobial peptide SR25 on ROS release from Escherichia coli

[0062] The reactive oxygen species detection kit was used to measure the reactive oxygen species level of antimicrobial peptides acting on bacterial cells. Escherichia coli in the logarithmic growth phase were collected by centrifugation, and the bacterial cells were washed three times with PBS buffer and the OD was adjusted. 600nm = 0.5, 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added at a final concentration of 10 μM, and the cells were incubated at 37°C for 30 min. The cells were washed three times with PBS to remove the fluorescent probes that did not enter the cells. After oscillation and mixing, different concentrations of antimicrobial peptides were added for treatment, and the fluorescence intensity of each group was immediately monitored in real time at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. For specific results, see the attached Fig. 9 .

[0063] By the attached Fig. 9The results show that the antimicrobial peptide SR25 provided by the present invention affects the accumulation level of reactive oxygen species (ROS) in Escherichia coli cells, triggers bacterial oxidative damage, and affects the energy metabolism of Escherichia coli.

[0064] Experimental Example 11: Molecular docking of Jilin Nonomura antimicrobial peptide SR25 with Escherichia coli succinate ubiquinone oxidoreductase

[0065] The protein structure of Escherichia coli succinate ubiquinone oxidoreductase was downloaded from the Protein Data Bank (www.rcsb.org) with the PDB number 2WDV; the three-dimensional structure of SR25 was predicted by CI-TASSER (https: / / seq2fun.dcmb.med.umich.edu / CI-TASSER / ). The succinate ubiquinone oxidoreductase protein was depleted of water molecules and extra ligands in the initial structure using Pymol software, polar hydrogen and Coleman charges were added, and the protein coordinates were saved in the PDBQT file format. Molecular docking simulation was performed using AutoDock software to obtain the lowest binding energy conformation, which was -1661.36 kcal / mol. The specific results are shown in the attached. Fig.10 .

[0066] By the attached Fig.10 The results show that the antimicrobial peptide SR25 provided by the present invention binds tightly to Escherichia coli succinate ubiquinone oxidoreductase, mainly to the membrane-anchored subunit sdhD of succinate dehydrogenase, and the interacting residues between the antimicrobial peptide and Escherichia coli succinate ubiquinone oxidoreductase mostly form hydrogen bonds.

[0067] Experimental Example 12: Molecular dynamics simulation of Jilin Nonomura antimicrobial peptide SR25 and Escherichia coli succinate ubiquinone oxidoreductase

[0068] Gromacs 2022.3 version software was used for molecular dynamics simulation. AmberTools22 was used for peptide pretreatment to add the GAFF force field to SR25, and Gaussian 16W was used to add hydrogen and calculate the RESP potential. The potential data will be added to the molecular dynamics system topology file. The simulation conditions were carried out at a static temperature of 300K and normal pressure (1 Bar). The force field used was Amber99sb-ildn. Each simulation system was placed in the TIP3P water box model. In order to maintain the total charge of the simulation system, an appropriate number of sodium ions were randomly added to the simulation system. Before the molecular dynamics simulation, the steepest descent method was used to minimize the energy of the protein complex molecules to eliminate the repulsion between molecules. After that, 100,000 steps of isothermal isochoric ensemble (NVT) equilibrium and isothermal isobaric ensemble (NPT) equilibrium were performed, respectively, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, the free molecular dynamics simulation was run, with a total of 5,000,000 steps, a step length of 2 fs, and a total duration of 100 ns. After the simulation was completed, the software's own tools were used to calculate the trajectory analysis, and the root mean square deviation (RMSD) and binding free energy (MMPBSA) of the trajectory of each amino acid were calculated. The free binding energy of the antimicrobial peptide SR25 provided by the present invention and SQR was -126.7 kcal / mol. The RMSD analysis showed that the two were stably bound. The specific results are shown in Table 2 and the attached Fig.11 .

[0069] Table 2.

[0070]

[0071] Experimental Example 13: Effect of Jilin Nonomura antimicrobial peptide SR25 on the activity of Escherichia coli succinate dehydrogenase

[0072] The crude membrane extract of Escherichia coli was prepared by referring to the succinate dehydrogenase activity kit, the bacteria were collected, 1 mL of reagent 1 and 10 uL of reagent 3 were added, homogenized with an ice bath homogenizer, centrifuged at 600 g, 4°C for 5 min, the precipitate was discarded, the supernatant was transferred to another centrifuge tube, centrifuged at 11000 g, 4°C for 10 min, 200 uL of reagent 2 and 2 uL of reagent 3 were added to the precipitate, and ultrasonically disrupted (ice bath, 200 W, ultrasonic for 3 s, interval of 10 s, repeated 30 times), different concentrations of antimicrobial peptides were added to the crude membrane extract, and the succinate dehydrogenase activity was detected according to the instructions of the kit. The specific results are shown in the attached. Fig.12 .

[0073] By the attached Fig.12 The results show that the antimicrobial peptide SR25 provided by the present invention can affect the activity of succinate dehydrogenase, but cannot completely inhibit its activity.

[0074] Experimental Example 14: Effect of Jilin Nonomura antimicrobial peptide SR25 on the activity of Escherichia coli succinate ubiquinone oxidoreductase

[0075] The crude membrane extract of Escherichia coli was prepared by referring to the succinate ubiquinone oxidoreductase activity kit. The bacteria were collected, 1 mL of reagent 1 and 10 uL of reagent 3 were added, and the mixture was homogenized with an ice bath homogenizer. The mixture was centrifuged at 600 g and 4°C for 5 min. The precipitate was discarded and the supernatant was transferred to another centrifuge tube. The mixture was centrifuged at 11,000 g and 4°C for 10 min. 200 uL of reagent 2 and 2 uL of reagent 3 were added to the precipitate, and the mixture was ultrasonically disrupted (ice bath, 200 W, ultrasonication for 3 s, interval of 10 s, repeated 30 times). Different concentrations of antimicrobial peptides were added to the crude membrane extract. The activity of succinate ubiquinone oxidoreductase was detected by referring to the instructions of the kit. The specific results are shown in the attached figure. Fig.13 .

[0076] By the attached Fig.13 The results show that the antimicrobial peptide SR25 provided by the present invention can completely inhibit the activity of Escherichia coli succinate ubiquinone oxidoreductase at 1 μM.

[0077] Experimental Example 15: Preparation of Jilin Nonomura Antimicrobial Peptide SR25 Hydrogel Preparation

[0078] 0.05 g of sodium alginate (SA) powder was weighed and added to 3 mL of antimicrobial peptide SR25 solution (200 ug / mL) for pre-swelling for 1 h and stirred for 30 min. At the same time, 0.05 g of gelatin particles were added to deionized water for pre-swelling for 40 min and then stirred at 60 °C for 30 min. Bubbles were eliminated from all the above solutions by using an ultrasonic cleaner. Then, 0.3% (w / v) CaCO3 powder was ultrasonically dispersed in the gelatin solution. The gelatin solution was mixed and stirred with the SA / SR25 solution for 5 min, and 0.75% GDL (w / v) was added and stirred thoroughly, and bubbles were removed by an ultrasonic cleaner. Finally, the solution (the mass concentration of SA and gelatin was 1% w / v) was poured into the mold to form a hydrogel.

[0079] Experimental Example 16: Treatment of wound infection in diabetic mice with Jilin Nonomura antimicrobial peptide SR25 hydrogel preparation

[0080] Thirty-six male C57bl / 6 mice (5 weeks old) were purchased and fasted for 12 h before intraperitoneal injection of streptozotocin STZ (100 mg / kg) for 5 days and 2 days after STZ was dissolved in sterile citrate buffer (0.05 mol / L sodium citrate, pH 4.5, 70 mg / kg). Blood glucose was monitored after 2 weeks, and mice with blood glucose levels above 16.7 mM were considered diabetic. After anesthetizing the diabetic mice with chloral hydrate, the dorsal hair of the mice was completely removed with a depilatory cream, and then a full-thickness wound with a diameter of 8 mm was formed with a punch, and a mixed bacterial solution of Escherichia coli and resistant Staphylococcus aureus (1× 10 each) was dripped into the wound. 8 CFU / mL, 10 μL, 20 μL in total), and 24 hours later the mice were randomly divided into 3 / 4 groups (n=9): the wounds were treated with equal volumes of PBS, SR25 hydrogel (50 μL in the treatment group: SR25 content 20 μg / mL), simple hydrogel material (50 μL) and Kangfuxin (50 μL), respectively. After 3, 7 and 14 days, photographs were taken to record the wound recovery process of diabetic wounds. Throughout the process, the dressing was changed every 2 days. The appearance of all wounds was photographed, and the degree of wound closure was assessed based on the size of the wound remaining on the skin; specific results are in the attached. Fig.15 .

[0081] By the attached Fig.15 The results showed that the infected wounds of diabetic mice were difficult to heal after 14 days of PBS treatment, while the wounds treated with the antimicrobial peptide SR25 hydrogel preparation showed obvious healing effects, which were significantly better than the clinical drug Kangfuxin for diabetic wound infection.

[0082] On the third day, three mice were taken from each group to evaluate the viability of bacteria in the wounds of diabetic mice. The solution of the ground wound was diluted and spread on the agar plate. The dilution was divided into two parts, one of which was spread on mannitol sodium chloride agar medium (Staphylococcus aureus selection medium) and eosin methylene blue agar medium (Escherichia coli selection medium). The specific results are shown in the attached Fig.16 .

[0083] By the attached Fig.16 The results showed that there was still a large amount of bacterial infection in the mouse wounds on the third day, and treatment with the antimicrobial peptide SR25 hydrogel preparation could effectively inhibit the bacterial proliferation in wound infection.

[0084] In summary, the above embodiments are merely descriptions of the preferred implementation methods of this test, and do not limit the scope of this test. Without departing from the spirit of the design of this test, various modifications and improvements made to the technical solutions of this test by ordinary technicians in this field should fall within the protection scope determined by this test.

Claims

1. A Jilin Nonomura antimicrobial peptide SR25, which is a hydrophilic cationic polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The use of the Jilin Nonomura antimicrobial peptide SR25 in the preparation of antibacterial preparations as claimed in claim 1, wherein the bacteria are selected from: one or more of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans.

3. An antibacterial drug comprising the Jilin Nonomura antibacterial peptide SR25 as claimed in claim 1.

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