An antibacterial peptide QLX-227-13, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to an antibacterial peptide QLX-227-13 and a preparation method and application thereof. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 13. The antibacterial peptide of the application is derived from computer high-throughput screening, introduces preset functional attribute information, can generate antibacterial peptides with specific attributes, and is different from existing antibacterial peptides in nature, which makes up for the defects of limited arrangement of natural antibacterial peptide amino acid sequences. After strict detection and screening of antibacterial performance, hemolytic toxicity and cytotoxicity in vitro and in vivo, the antibacterial peptide has broad-spectrum antibacterial activity on gram-positive multi-drug resistant bacteria and / or gram-negative multi-drug resistant bacteria and good safety. The antibacterial peptide of the application is composed of 14 amino acids, the peptide chain is short, the preparation method and technology are mature, and the synthesis cost is low.

Description

[0001] This application is a divisional application of application number 202410318620.3, filed on March 20, 2024, entitled "Antibacterial Peptide and Preparation Method and Application Thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and specifically relates to an antibacterial peptide QLX-227-13 and a preparation method and application thereof. BACKGROUND

[0003] The information disclosed in this background section is only intended to increase the understanding of the general background of the application and should not necessarily be taken as an acknowledgment or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

[0004] Antibiotics are important drugs that can inhibit or kill pathogenic microorganisms. However, with the widespread use of antibiotics, the drug resistance of pathogenic microorganisms to traditional antibiotics has become a more and more serious problem. Antibacterial peptides have the advantages of rapid bactericidal effect, wide antibacterial spectrum, small toxic and side effects, and no immunogenicity. Moreover, due to its special bactericidal mechanism, the inhibited or killed pathogenic microorganisms will not produce resistant strains, and the bactericidal effect will not be weakened due to drug resistance. Therefore, antibacterial peptide drugs have become an ideal substitute for antibiotics and have received much attention from the medical community.

[0005] Although antibacterial peptides have high medical health efficacy and economic value, the existing exploration and production technology methods have some problems, such as long cycle of antibacterial peptide exploration method, high research and development cost; the extraction and separation of existing polypeptides in nature are usually complicated, tedious, low in content, and expensive, which is not suitable for large-scale production.

[0006] In addition, natural antibacterial peptides also have many limitations, such as: (1) the antibacterial activity is lower than that of traditional antibacterial agents, and a large dose is required for use as an antibacterial agent, resulting in high application cost; (2) the antibacterial activity is positively correlated with the length of the peptide chain, and the peptide chain of antibacterial peptides with strong antibacterial activity is longer, resulting in high synthesis cost and immunogenicity; (3) leading to hemolytic destruction of eukaryotic cells; (4) easily degradable in vivo; (5) limited sequence arrangement, while polypeptide molecules with sequence diversity constitute a nearly unlimited potential drug reservoir. SUMMARY

[0007] In order to solve the problems of the prior art, the purpose of the present application is to provide an antibacterial peptide QLX-227-13 and a preparation method and application thereof. The antibacterial peptide of the present application is composed of 14 amino acids, and the peptide chain is short, the preparation method and technology are mature, and the synthesis cost is low.

[0008] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0009] In a first aspect, the present application provides an antibacterial peptide, the amino acid sequence of which is shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 13, wherein the leucine at position 14 in SEQ ID NO. 2 is in D configuration, the lysines at positions 1 and 5 in SEQ ID NO. 3 are in D configuration, the lysines at positions 12 and 13 in SEQ ID NO. 9 are in D configuration, the lysine at position 3 in SEQ ID NO. 10 is in D configuration, and all the amino acids in SEQ ID NO. 11 are in D configuration.

[0010] The specific names and sequences of the antibacterial peptides are as follows:

[0011] QLX-227-1: the amino acid sequence is shown in SEQ ID NO. 1;

[0012] QLX-227-2: the amino acid sequence is shown in SEQ ID NO. 2, and the leucine (L) at position 14 is in D configuration;

[0013] QLX-227-3: the amino acid sequence is shown in SEQ ID NO. 3, and the lysines (K) at positions 1 and 5 are in D configuration;

[0014] QLX-227-4: the amino acid sequence is shown in SEQ ID NO. 4;

[0015] QLX-227-5: the amino acid sequence is shown in SEQ ID NO. 5;

[0016] QLX-227-6: the amino acid sequence is shown in SEQ ID NO. 6;

[0017] QLX-227-7: the amino acid sequence is shown in SEQ ID NO. 7;

[0018] QLX-227-8: the amino acid sequence is shown in SEQ ID NO. 8;

[0019] QLX-227-9: the amino acid sequence is shown in SEQ ID NO. 9, and the lysines (K) at positions 12 and 13 are in D configuration;

[0020] QLX-227-10: the amino acid sequence is shown as SEQ ID NO. 10, the lysine (K) at the 3rd position is in D configuration;

[0021] QLX-227-11: the amino acid sequence is shown as SEQ ID NO. 11, all the amino acids are in D configuration;

[0022] QLX-227-12: the amino acid sequence is shown as SEQ ID NO. 12;

[0023] QLX-227-13: the amino acid sequence is shown as SEQ ID NO. 13.

[0024] In a second aspect, the present application provides a preparation method of the antibacterial peptide according to the first aspect, and the preparation method comprises the following steps:

[0025] The polypeptide is synthesized by using a polypeptide synthesizer through solid-phase synthesis, and purified by using reverse-phase high performance liquid chromatography, so as to obtain the antibacterial peptide.

[0026] In a third aspect, the present application provides an application of the antibacterial peptide according to the first aspect in the preparation of a medicine for resisting gram-positive bacteria and / or gram-negative bacteria.

[0027] Preferably, the gram-positive bacteria include Staphylococcus aureus or Enterococcus faecalis.

[0028] Preferably, the gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium.

[0029] Preferably, the medicine comprises the antibacterial peptide according to the first aspect and at least one pharmaceutically acceptable carrier and / or additive.

[0030] Preferably, the medicine is an internal medicine or an external medicine; the internal medicine is an oral preparation or an injection; and the external medicine includes a patch and a smearing preparation.

[0031] In a fourth aspect, the present application provides an application of the antibacterial peptide according to the first aspect in the preparation of an antibacterial product, wherein the antibacterial product comprises at least one of a disinfectant, a detergent, a preservative and a feed additive.

[0032] The above-mentioned one or more technical solutions of the present application have the following beneficial effects:

[0033] (1) The antibacterial peptide of the present application is derived from computer high-throughput screening, and the preset functional attribute information is introduced, so that the antibacterial peptide with specific attributes can be generated, and the generated antibacterial peptide is different from the existing antibacterial peptides in nature, which makes up for the defect of limited arrangement of natural antibacterial peptide amino acid sequences.

[0034] (2) Based on the computer high-throughput screening results, D-type amino acid substitution modification is carried out on the antibacterial peptide sequence, so as to obtain an antibacterial peptide with better safety.

[0035] (3) After strict detection and screening of antibacterial performance, hemolytic toxicity and cytotoxicity in vitro and in vivo, the antibacterial peptide has broad-spectrum antibacterial activity on gram-positive multi-drug resistant bacteria and / or gram-negative multi-drug resistant bacteria, and has better safety.

[0036] (3) The antibacterial peptide is composed of 14 amino acids, the peptide chain is short, the preparation method and technology are mature, and the synthesis cost is low. BRIEF DESCRIPTION OF DRAWINGS

[0037] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

[0038] Figure 1 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-1 of the present application;

[0039] Figure 2 It is a circular dichroism spectrum of the antibacterial peptide QLX-227-1 in PBS of the present application, wherein Helix represents helix, Antiparallel & Parallel represents fold, Beta-turn represents corner, and Random coil represents irregular coil;

[0040] Figure 3 It is a circular dichroism spectrum of the antibacterial peptide QLX-227-1 in 50% TFE of the present application, wherein Helix represents helix, Antiparallel & Parallel represents fold, Beta-turn represents corner, and Random coil represents irregular coil;

[0041] Figure 4 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-2 of the present application;

[0042] Figure 5 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-3 of the present application;

[0043] Figure 6 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-4 of the present application;

[0044] Figure 7 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-5 of the present application;

[0045] Figure 8 It is a mass spectrometric detection diagram of the antibacterial peptide QLX-227-6 of the present application;

[0046] Figure 9 Mass spectrum detection chart of the antibacterial peptide QLX-227-7 of the application;

[0047] Figure 10 Mass spectrum detection chart of the antibacterial peptide QLX-227-8 of the application;

[0048] Figure 11 Mass spectrum detection chart of the antibacterial peptide QLX-227-9 of the application;

[0049] Figure 12 Mass spectrum detection chart of the antibacterial peptide QLX-227-10 of the application;

[0050] Figure 13 Mass spectrum detection chart of the antibacterial peptide QLX-227-11 of the application;

[0051] Figure 14 Mass spectrum detection chart of the antibacterial peptide QLX-227-12 of the application;

[0052] Figure 15 Mass spectrum detection chart of the antibacterial peptide QLX-227-13 of the application;

[0053] Figure 16 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-1 of the application;

[0054] Figure 17 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-2 of the application;

[0055] Figure 18 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-3 of the application;

[0056] Figure 19 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-4 of the application;

[0057] Figure 20 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-5 of the application;

[0058] Figure 21 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-6 of the application;

[0059] Figure 22 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-7 of the application;

[0060] Figure 23 Hemolytic activity experiment result chart of the antibacterial peptide QLX-227-8 of the application;

[0061] Figure 24Figure of hemolysis activity experiment result of the antibacterial peptide QLX-227-9 of the present application;

[0062] Figure 25 Figure of hemolysis activity experiment result of the antibacterial peptide QLX-227-10 of the present application;

[0063] Figure 26 Figure of hemolysis activity experiment result of the antibacterial peptide QLX-227-11 of the present application;

[0064] Figure 27 Figure of hemolysis activity experiment result of the antibacterial peptide QLX-227-12 of the present application;

[0065] Figure 28 Figure of hemolysis activity experiment result of the antibacterial peptide QLX-227-13 of the present application;

[0066] Figure 29 Figure of cytotoxicity experiment result of the antibacterial peptide QLX-227-1 of the present application;

[0067] Figure 30 Figure of treatment result of the antibacterial peptide QLX-227-1 of the present application on bacterial infection of mouse skin, wherein a is the treatment result on G+ Staphylococcus aureus CMCC26003 infection, and b is the treatment result on G- Acinetobacter baumannii ATCC19606 infection. DETAILED DESCRIPTION

[0068] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0069] The materials used in the embodiments of the present application, such as bacteria, cells, mice, etc., can be purchased, and the purchase channels are as follows.

[0070] MH Broth Medium (liquid): Qingdao hopebio, HB6231.

[0071] NB Nutrient Broth Medium (liquid): Qingdao hopebio, HB0108.

[0072] Escherichia coli CMCC44102: Qingdao hopebio, HBJZ069.

[0073] Staphylococcus aureus CICC25138, Enterococcus faecalis CICC24243, Klebsiella pneumoniae ATCC BAA-1705, Acinetobacter baumannii ATCC19606, Salmonella typhimurium ATCC14028: China Industrial Microbial Culture Collection Center (CICC).

[0074] Pseudomonas aeruginosa PAO1: Ningbo Mingzhou Bio CO., Ltd, BMZ114547.

[0075] G+ Staphylococcus aureus CMCC26003: Qingdao Haibo Biohope, HBJZ061.

[0076] HEK-293T human embryonic kidney cells: Savy Bio, STCC10301G-1.

[0077] Mice: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0078] Example 1

[0079] Synthesis of antibacterial peptide QLX-227-1

[0080] 1. Choose a modified resin to start synthesizing polypeptides, add pip / DMF to the reactor, and shake the reactor for some time.

[0081] 2. Filter out the solvent, add DMF to the system, shake the reactor for 1 min, and filter out the liquid; this operation is performed three times.

[0082] 3. Take a small amount of test reagents A and B and a little resin into the test tube, put the test tube into a 100 °C environment for 20 s, and check if the resin has color change. If the color changes, it means that Fmoc removal is successful.

[0083] 4. Add the prepared amino acid solution to the reactor, and record it in the record table. Add 1 mL of DIC / DMF solution, and shake the reactor for some time.

[0084] 5. Operation as 3, if the color does not change, it means that the coupling is successful.

[0085] 6. Repeat operation 2 to wash the resin.

[0086] 7. Add pip / DMF to the reactor, and shake the reactor for some time.

[0087] 8. Repeat operation 2 to wash the resin.

[0088] 9. Repeat the above operations 3-8, and add the corresponding amino acids to the polypeptide synthesis.

[0089] The retention time of the target peptide was determined by analyzing the obtained crude product and recorded on the table. The solubilization method of the polypeptide was determined according to the hydrophobicity and pi of the polypeptide, and the polypeptide was dissolved. The solution was filtered through a 0.45 um organic filter membrane into a 50 mL centrifuge tube. The preparation method of the polypeptide was determined according to the amount, purity and isoelectric point of the polypeptide, the solution was injected into the instrument, the fraction was collected according to the HPLC and purity, and whether it was qualified was analyzed by MS and HPLC. The qualified fraction was freeze-dried, and the antibacterial peptide QLX-227-1 was obtained as a freeze-dried powder.

[0090] The mass spectrum of the antibacterial peptide QLX-227-1 is shown in Figure 1 The molecular weight shown in the mass spectrum is consistent with the theoretical molecular weight.

[0091] Alpha-helix coiled coil and anti-parallel beta-sheet are the most common structures in antibacterial peptides, and amphipathic alpha peptides are usually more active than peptides with ambiguous secondary structure. Increasing the content of alpha-helix is related to improving the antibacterial activity. Beta-sheet antibacterial peptides contain stable structure disulfide bonds and help antibacterial peptides to pass through the cell membrane. In order to determine the alpha-helix of the peptide, the secondary structure of the antibacterial peptide molecule in aqueous environment (PBS) and simulated cell membrane hydrophobic environment (50% TFE) was monitored by circular dichroism (CD). The analysis chart of the secondary structure of the antibacterial peptide QLX-227-1 is shown in Figure 2 and Figure 3 It can be seen that the alpha-helix structure of the antibacterial peptide QLX-227-1 in the aqueous environment (PBS) and the simulated cell membrane hydrophobic environment (50% TFE) is 6.4% and 8.1% respectively, and the beta-sheet structure is 17.0% and 18.9% respectively.

[0092] The antibacterial peptides QLX-227-2 to QLX-227-13 were synthesized by the above synthesis method. The mass spectra of the antibacterial peptides QLX-227-2 to QLX-227-13 are shown in Figures 4 to 15 The molecular weight shown in the mass spectrum is consistent with the theoretical molecular weight.

[0093] Example 2 Determination of antibacterial activity of antibacterial peptides

[0094] The method recommended by the American Clinical Laboratory Standardization Institute (CLSI) for determining the minimum inhibitory concentration (MIC) was used, and a series of gradient antibacterial peptide solutions were prepared by double dilution method. The specific steps are as follows:

[0095] (1) Preparation of bacterial cells: Take the bacteria to be tested stored at -20 ℃, inoculate on NB nutrient broth medium and incubate. Pick single colonies, inoculate in 10 mL NB nutrient broth medium, and incubate at 37 ℃, 200 rpm overnight. Then inoculate the overnight bacterial cells in fresh culture medium, and incubate for 1-2 h until the bacterial cells are in the logarithmic growth phase, with OD600 = 0.4. Adjust the number of bacterial cells in the obtained bacterial solution to about 10 6 CFU / mL with MH broth medium.

[0096] (2) Preparation of peptides: Adjust the concentration of the antibacterial peptides to 512 ug / mL, and take 100 uL and add to the first column of holes in the 96-hole plate. Add 50 uL of MH broth medium to the other holes, then take out 50 uL of the peptide solution in the first hole and add to the second hole, and so on, diluting by a factor of two to the tenth hole, and discard 50 uL.

[0097] (3) Inoculation of bacteria: Take 50 uL of the bacterial solution adjusted in step (1) with a sample gun and add to the first 11 columns of holes in the 96-hole plate, with a final bacterial concentration of 5X10 5 CFU / mL per hole. Place the 96-hole plate on a micro-vibrator and shake for 1 min to mix the liquid in each hole. Cover the micro-holes to reduce evaporation during incubation, and incubate at 37 ℃ for 18 h. Set the positive control group as the eleventh hole, i.e., add only 50 uL of MH broth medium and 50 uL of bacterial solution; the negative control is the twelfth hole, i.e., only add 100 uL of MH broth medium. The above settings make the concentration of antibacterial peptides in the first to tenth holes decrease by a factor of two.

[0098] (4) Result judgment: The sterile control hole should remain clear throughout the entire test, indicating that the entire test was sterile. Compare the bacterial growth characteristics (such as the turbidity of the broth in the micro-holes, the appearance of sediment at the bottom of the hole, etc.) in the growth control hole to determine the minimum concentration without visible growth, which is the MIC value of the peptide for the test bacteria.

[0099] The results of the antibacterial peptide inhibition activity determination are shown in Tables 1 and 2. It can be seen that the antibacterial peptides QLX-227-1 to QLX-227-13 of the present application all have the ability to inhibit the growth of multiple drug-resistant gram-positive and gram-negative bacteria.

[0100] Table 1 Minimum inhibitory concentration of antibacterial peptides against gram-positive multiple drug-resistant bacteria, in units of ug / mL

[0101] Staphylococcus aureus CICC25138 Enterococcus faecalis CICC24243 QLX-227-1 64 128 QLX-227-2 128 128 QLX-227-3 64 256 QLX-227-4 64 256 QLX-227-5 32 128 QLX-227-6 64 128 QLX-227-7 32 64 QLX-227-8 16 256 QLX-227-9 128 256 QLX-227-10 128 256 QLX-227-11 32 256 QLX-227-12 32 256 QLX-227-13 16 256

[0102] Table 2 Minimum inhibitory concentration of antibacterial peptides against gram-negative multiple drug-resistant bacteria, in units of ug / mL

[0103] Escherichia coli CMCC44102 (non-resistant) Klebsiella pneumoniae ATCC BAA-1705 Acinetobacter baumannii ATCC19606 Salmonella typhimurium ATCC14028 Pseudomonas aeruginosa PAO1 QLX-227-1 16 32 16 32 128 QLX-227-2 32 256 32 64 256 QLX-227-3 32 64 16 32 128 QLX-227-4 16 32 32 32 64 QLX-227-5 8 32 16 32 16 QLX-227-6 4 8 4 16 16 QLX-227-7 16 32 16 16 16 QLX-227-8 8 16 16 8 32 QLX-227-9 16 64 32 64 64 QLX-227-10 8 16 8 32 64 QLX-227-11 32 16 8 16 32 QLX-227-12 16 8 16 16 32 QLX-227-13 8 8 16 8 16

[0104] Example 3 Hemolytic activity of antibacterial peptides

[0105] For the determination of hemolytic activity of antibacterial peptides, for example, antibacterial peptide QLX-227-1, the specific test steps are as follows:

[0106] (1) Collect 1 mL of fresh human blood in a heparin sodium anticoagulant tube and store at 4 °C for standby;

[0107] (2) Centrifuge 1000 g of the above blood for 5 min, discard the supernatant, and collect the red blood cells;

[0108] (3) Wash the collected red blood cells with PBS buffer solution for three times, centrifuge at 1000 g for 5 min, discard the supernatant, collect the red blood cells, and finally resuspend the cells with about 10 mL of PBS buffer solution to obtain an 8% (V / V) red blood cell suspension for standby;

[0109] (4) Dilution of antibacterial peptides: add 90 uL of PBS buffer solution to the first tube of each row of 12 EP tubes, and add 50 uL of PBS buffer solution to the rest of the tubes. Then add 10 uL of antibacterial peptide QLX-227-1 stock solution (concentration of 2048 ug / mL) to the first tube, mix the antibacterial peptide QLX-227-1 solution in the first tube, and then add 50 uL to the second tube. Repeat the dilution process to the tenth tube, and then discard 50 uL;

[0110] (5) Take 50 uL of the prepared red blood cell suspension and add it to the EP tubes containing different concentrations of antibacterial peptide QLX-227-1 solution, and incubate in a 37 °C incubator for 1 h. Among them, the 11th hole adds 50 uL of PBS and 50 uL of red blood cell suspension as negative control, and the 12th hole adds 50 uL of 01% Triton X-100 and 50 uL of red blood cell suspension as positive control;

[0111] (6) After 1 h, take out the EP tubes and centrifuge at 100 g for 5 min at 4 °C;

[0112] (7) Take the supernatant of the above centrifuged solution and transfer it to a clean 96-well plate in parallel, and measure the optical absorption value at 570 nm (OD570nm) using a microplate reader.

[0113] For example, Figure 16As shown, the antibacterial peptide QLX-227-1 has 50% hemolytic activity at 1024 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-1 will not produce hemolysis when exerting antibacterial effect.

[0114] As shown, the antibacterial peptide QLX-227-2 has 50% hemolytic activity at 256 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-2 will not produce hemolysis when exerting antibacterial effect. Figure 17 As shown, the antibacterial peptide QLX-227-3 has 50% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-3 will not produce hemolysis when exerting antibacterial effect.

[0115] Figure 18 As shown, the antibacterial peptide QLX-227-4 has 35% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-4 will not produce hemolysis when exerting antibacterial effect.

[0116] As shown, the antibacterial peptide QLX-227-5 also does not produce hemolytic activity at 2048 ug / mL, indicating that the antibacterial peptide QLX-227-5 will not produce hemolysis when exerting antibacterial effect. Figure 19 As shown, the antibacterial peptide QLX-227-6 has 50% hemolytic activity at 512 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-6 will not produce hemolysis when exerting antibacterial effect.

[0117] Figure 20 As shown, the antibacterial peptide QLX-227-7 has 50% hemolytic activity at 256 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-7 will not produce hemolysis when exerting antibacterial effect.

[0118] As shown, the antibacterial peptide QLX-227-8 has 30% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-8 will not produce hemolysis when exerting antibacterial effect. Figure 21 As shown, the antibacterial peptide QLX-227-9 has 50% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-9 will not produce hemolysis when exerting antibacterial effect.

[0119] Figure 22 As shown, the antibacterial peptide QLX-227-10 has 50% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-10 will not produce hemolysis when exerting antibacterial effect.

[0120] As shown, the antibacterial peptide QLX-227-11 has 50% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-11 will not produce hemolysis when exerting antibacterial effect. Figure 23 As shown, the antibacterial peptide QLX-227-12 has 50% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentrations, indicating that the antibacterial peptide QLX-227-12 will not produce hemolysis when exerting antibacterial effect.

[0121] Figure 24 ​​​​As shown, the antibacterial peptide QLX-227-9 has no hemolytic activity at 2024 ug / mL, indicating that the antibacterial peptide QLX-227-9 will not produce hemolysis when exerting antibacterial effect.

[0122] As shown, the antibacterial peptide QLX-227-10 has 30% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-10 will not produce hemolysis when exerting antibacterial effect. Figure 25 As shown, the antibacterial peptide QLX-227-11 has 40% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-11 will not produce hemolysis when exerting antibacterial effect.

[0123] Figure 26 As shown, the antibacterial peptide QLX-227-12 has 5% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-11 will not produce hemolysis when exerting antibacterial effect.

[0124] As shown, the antibacterial peptide QLX-227-13 has 15% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-11 will not produce hemolysis when exerting antibacterial effect. Figure 27 Example 4 Cytotoxicity of antibacterial peptides

[0125] Figure 28 HEK-293T human embryonic kidney cells were used to detect the cytotoxicity of antibacterial peptides. Cells were seeded in 96-well plates at 8000 cells per well. Cells were incubated at 37 °C, 5% CO2 for 24 hours. Then antibacterial peptide QLX-227-2 or QLX-227-1 was added to each well containing cells. After 24 hours of incubation, 10 uL CCK8 was added to each well and incubated at 37 °C for 2 hours. Then the absorbance value at 450 nm was detected, and Prism 8 was used to determine the cell survival rate value according to curve fitting and application of the following equation:

[0126] Cell survival rate = (experimental group - blank control) / (negative control group - blank control) x 100%

[0127] The blank control is culture medium containing only CCK8, and the negative control group is cells without the addition of antibacterial peptides.

[0128] As shown, the antibacterial peptide QLX-227-10 has 30% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-10 will not produce hemolysis when exerting antibacterial effect.

[0129] The blank control is culture medium containing only CCK8, and the negative control group is cells without the addition of antibacterial peptides.

[0130] As shown, the antibacterial peptide QLX-227-11 has 40% hemolytic activity at 2048 ug / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-11 will not produce hemolysis when exerting antibacterial effect. Figure 29 ​​As shown, the concentration of the antibacterial QLX-227-1 exerting 50% cytotoxicity is above 256 ug / mL, higher than the geometric mean of its minimum antibacterial concentration, indicating that the antibacterial peptide QLX-227-1 does not produce cytotoxicity when exerting antibacterial effect.

[0131] Example 5 Skin external use anti-infection ability of antibacterial peptide

[0132] For the skin external use anti-infection ability of antibacterial peptide, the specific test steps are as follows:

[0133] G+ Staphylococcus aureus CMCC26003 and G- Acinetobacter baumannii ATCC19606 were diluted to 10 8 CFU / mL for standby. Female C57BL / 6 mice were randomly divided by weight into infection negative control group and antibacterial peptide treatment group, 6 in each group. After the mice were depilated on the back, two pieces of full-thickness skin with a diameter of 5 mm were taken from the center of the back of the mice with a tissue biopsy, and the wound area of each group of mice was uniform, that is, the model was successfully constructed. 5 uL of bacteria solution with a concentration of 10 8 CFU / mL was injected on the two wounds of each mouse. After 6 h, the treatment group was given 0.3 mg of antibacterial peptide QLX-227-1 treatment. At 24 h after modeling, the skin at the infection site of the animals was taken aseptically, and viable bacteria were detected and counted. Figure 30 As shown, the antibacterial peptide QLX-227-1 has the ability to clear G+ and G- bacterial infections in mice skin.

[0134] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antibacterial peptide QLX-227-13, characterized in that, The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.

13.

2. A method for preparing the antibacterial peptide QLX-227-13 according to claim 1, characterized by, The method comprises the following steps: The polypeptide is synthesized by using a polypeptide synthesizer through solid-phase synthesis, and purified by using reverse-phase high performance liquid chromatography to obtain the antibacterial peptide QLX-227-13.

3. The use of the antibacterial peptide QLX-227-13 according to claim 1 in the preparation of a drug against Gram-positive bacteria and / or Gram-negative bacteria, characterized in that, The gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis, and the gram-negative bacteria are at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium.

4. The use according to claim 3, wherein the compound is ###0002### The medicine comprises the antibacterial peptide QLX-227-13 and at least one pharmaceutically acceptable carrier and / or additive.

5. The use according to claim 3, wherein the compound is ###0002### The medicine is an internal medicine or an external medicine; the internal medicine is oral preparation or injection; the external medicine is a patch and a smearing preparation.

6. Use of the antibacterial peptide QLX-227-13 according to claim 1 for the preparation of an antibacterial product, characterized in that, The antibacterial product is at least one of a disinfectant, a detergent and a preservative.

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