Antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial peptide as well as a preparation method and application thereof. The amino acid sequence of the antibacterial peptide is LKVVFKVLFKRRR. The antibacterial peptide disclosed by the invention has relatively excellent antibacterial activity and metabolic stability and relatively low hemolytic activity and cytotoxicity, and is expected to become a novel antibacterial candidate drug. In addition, the antibacterial peptide is short in polypeptide sequence and only contains 13 amino acids, and all the amino acids are L-type amino acids, so that the antibacterial peptide is convenient to produce, low in production cost and beneficial to application and popularization. Generally speaking, the antibacterial peptide provided by the invention lays a foundation for further development of novel antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an antimicrobial peptide and a preparation method and application thereof. Background Art

[0002] Bacteria are one of the factors that cause diseases. The indiscriminate use of antibiotics can make bacteria resistant and even produce superbugs. Therefore, the development and production of new bactericidal substances are very important for improving human health and quality of life. Antimicrobial peptides are a class of peptides with antimicrobial activity that scientists first discovered in insects. Because of their broad-spectrum antimicrobial activity and unique antimicrobial mechanism of action, they are expected to become a new alternative to traditional antibiotics. However, natural antimicrobial peptides often have disadvantages such as limited sources, weak activity, metabolic instability, long polypeptide sequences leading to high production costs, hemolytic and cytotoxic properties, which have led to many restrictions on their development and application. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an artificially synthesized antimicrobial peptide and a preparation method and application thereof. The antimicrobial peptide has relatively excellent antimicrobial activity and metabolic stability as well as low hemolytic and cytotoxicity, and is expected to become a new antimicrobial candidate drug. In addition, the polypeptide sequence of the antimicrobial peptide is relatively short, comprising only 13 amino acids, and all amino acids are L-type amino acids, which is easy to produce and has low production cost, which is conducive to application and promotion.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] The present invention provides an antimicrobial peptide, the amino acid sequence of which is: LKVVFKVLFKRRR.

[0006] The present invention also provides a method for preparing the antimicrobial peptide, which comprises synthesizing the antimicrobial peptide by a standard Fmoc solid phase synthesis method.

[0007] Preferably, the method comprises the following:

[0008] S1. Place dichlororesin in a synthesis column and add DCM to swell it; then add the first amino acid activated by ACT activator, and then shake on a shaker for reaction; after completion, wash with DMF; then add DEP solution for shaking reaction; after completion, wash with DMF, and use ninhydrin detection solution to detect the connection of amino acids. After the reaction is complete, connect the next amino acid;

[0009] S2. The second amino acid is activated by shaking with HBTU and ACT activator on a shaker, and then added to the synthesis column and shaken on a shaker; after the reaction, it is washed with DMF; then DEP solution is added for shaking reaction; after the reaction, it is washed with DMF, and the connection of the amino acid is detected with ninhydrin detection solution. After the reaction is complete, the next amino acid is connected;

[0010] S3. Connect the subsequent amino acids according to the method described in S2 until the last amino acid is completely connected; after the last amino acid is completely connected, add methanol to wash; then wash with DCM to form a fine sand; then add a cutting agent to carry out a cutting reaction; after the end, add icy ether to precipitate and centrifuge, discard the supernatant, wash the precipitate with icy ether and centrifuge to obtain a crude antimicrobial peptide;

[0011] S4. Purify the crude antimicrobial peptide by HPLC to obtain a pure antimicrobial peptide with a purity of more than 95%.

[0012] Preferably, the amount of the first amino acid added in S1 is 1 times by weight, the amount of the second amino acid added in S2 is 3 times by weight, and the amount of HBTU added is 3 times by weight.

[0013] Preferably, the ACT activator in S1 and S2 consists of N-methylmorpholine and DMF, wherein the volume ratio of N-methylmorpholine to DMF is 4:87.

[0014] Preferably, the cutting agent in S3 is composed of trifluoroacetic acid, methyl phenyl sulfide, water, phenol and ethanedithiol, wherein the volume ratio of trifluoroacetic acid, methyl phenyl sulfide, water, phenol and ethanedithiol is 82.5:5:5:5:2.5.

[0015] Preferably, in S4, the HPLC purification method is: using a C18 semi-preparative column; mobile phase: phase A: deionized water containing 0.1% TFA (m / v); phase B: acetonitrile solution containing 0.1% TFA (m / v); flow rate: 1 ml / min; elution gradient: phase A changes from 95% to 5% at a rate of 6% per minute, and phase B changes from 5% to 95% at a rate of 6% per minute.

[0016] The present invention also provides the use of the antimicrobial peptide in the preparation of antimicrobial infection drugs.

[0017] As a preferred embodiment, the bacteria are Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis or Staphylococcus aureus.

[0018] The present invention also provides an anti-bacterial infection drug, wherein the active ingredient of the drug includes the antimicrobial peptide.

[0019] The beneficial effects of the present invention are:

[0020] The present invention discloses an antimicrobial peptide, the amino acid sequence of which is: LKVVFKVLFKRRR. The antimicrobial peptide of the present invention has relatively excellent antimicrobial activity and metabolic stability as well as low hemolytic activity and cytotoxicity, and is expected to become a new antimicrobial candidate drug. In addition, the polypeptide sequence of the antimicrobial peptide of the present invention is relatively short, comprising only 13 amino acids, and all amino acids are L-type amino acids, which is convenient for production and has low production cost, and is conducive to application and promotion. In general, the antimicrobial peptide of the present invention lays a foundation for further development of new antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC diagram for the preparation of antimicrobial peptides;

[0022] Figure 2 To prepare mass spectra of antimicrobial peptides;

[0023] Figure 3 The results of enzyme stability experiments for the preparation of antimicrobial peptides;

[0024] Figure 4 The hemolytic toxicity results of the prepared antimicrobial peptides at different concentrations are shown;

[0025] Figure 5 To prepare the results of the effects of antimicrobial peptides on the survival of RAW264.7 cells;

[0026] Figure 6 To prepare the results of the effects of antimicrobial peptides on the survival of 293T cells;

[0027] Figure 7 To prepare the results of the effects of antimicrobial peptides on the survival of THP-1 cells. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods; the instruments and equipment used in the following embodiments, unless otherwise specified, are conventional laboratory instruments and equipment; the test materials used in the following embodiments, unless otherwise specified, are all from conventional biochemical reagents and can be purchased commercially. It should be noted that in the present invention, DCM is an abbreviation for dichloromethane, TFA is an abbreviation for trifluoroacetic acid, HBTU is a peptide coupling reagent, the Chinese name is O-benzotriazole-tetramethyluronium hexafluorophosphate, DMF is an abbreviation for N,N-dimethylformamide, and DEP is an abbreviation for diethyl phthalate.

[0029] Example 1 Preparation of antimicrobial peptides

[0030] The antimicrobial peptide with the amino acid sequence of LKVVFKVLFKRRR was synthesized using a standard solid phase synthesis method, and the structural formula is as follows:

[0031]

[0032] The specific steps are as follows: First, weigh 600 mg of dichlororesin into the synthesis column, add 5 mL of DCM to swell the resin for one hour. Take 1 times the mass of the first amino acid, add 4 mL of ACT activator (ACT activator consists of N-methylmorpholine and DMF, where the volume ratio of N-methylmorpholine and DMF is 4:87), and shake on a shaker for 6 minutes at a speed of 200 r / min. Pour the first amino acid after activation into the synthesis column, and shake the shaker at 200 r / min for 2 hours. After the end, open the synthesis column and wash it with DMF 5-6 times, 5 mL each time. Add 3 mL of methanol and 3 mL of ACT to the column, and the reaction time is 2 hours. Open the synthesis column, wash it with DMF 5-6 times, 5 mL each time, add 5 mL of DEP solution, and shake it on a shaker for 6 minutes at a speed of 200 r / min. Wash it with DMF 5-6 times, 5 mL each time. Use ninhydrin detection solution to detect the connection of amino acids, and after the reaction is complete, connect the next amino acid.

[0033] Weigh 3 times the mass of the second amino acid, add 3 times the mass of HBTU, add 4mL ACT activator (ACT activator consists of N-methylmorpholine and DMF, wherein the volume ratio of N-methylmorpholine and DMF is 4:87), shake on a shaker for 6min at a speed of 200r / min. Pour the activated amino acid into the synthesis column and shake on a shaker for 1h at a speed of 200r / min. After the end, open the synthesis column and wash it with DMF 5-6 times, 5mL each time. Add 5mL DEP solution, shake on a shaker for 6min at a speed of 200r / min, and wash it with DMF 5-6 times. Use ninhydrin detection solution to detect the connection of the amino acid. After the reaction is complete, the connection of the next amino acid is carried out. The reaction steps are the same as the second amino acid, and the reaction is continued until the last amino acid. According to the above steps, the antibacterial peptide Leu-Lys-Val-Val-Phe-Lys-Val-Leu-Phe-Lys-Arg-Arg-Arg of the present invention was synthesized continuously from the carbon end to the nitrogen end of the peptide chain.

[0034] After the last amino acid is coupled, wash with DMF 5-6 times, 5 mL each time. Add 5 mL of DEP solution, shake on a shaker for 6 minutes at a speed of 200 r / min, wash with DMF 5-6 times, 5 mL each time, and pump for 5 minutes. Wash with methanol 5-6 times, 5 mL each time, and pump until the resin is dry. Wash with DCM 5-6 times, 5 mL each time, and pump until the resin is dry. The final state is fine sand. Add a cutting agent and cut for 10 hours (the cutting agent is composed of trifluoroacetic acid, methyl phenyl sulfide, water, phenol, and ethanedithiol, wherein the volume ratio of trifluoroacetic acid: methyl phenyl sulfide: water: phenol: ethanedithiol is 82.5:5:5:5:2.5). Then add about 35 mL of ice ether to the centrifuge tube for precipitation, and a white precipitate is precipitated. Centrifuge at 7500 rmp for 5 minutes; discard the supernatant, wash the precipitate with 30 mL of ice ether, centrifuge twice; pour out the ice ether, and dry at room temperature for 3-5 hours. The crude antimicrobial peptide is obtained.

[0035] Purification and identification of antimicrobial peptides. Antimicrobial peptides were purified and identified by HPLC. A C18 semi-preparative column was used, with the mobile phase being: Phase A (aqueous phase): deionized water (containing 0.1% TFA (m / v)); Phase B (organic phase): acetonitrile solution (containing 0.1% TFA (m / v)); flow rate: 1 ml / min; elution gradient: Phase A decreased from 95% to 5% at a rate of 6% per minute, and Phase B increased from 5% to 95% at a rate of 6% per minute. A rotary evaporator was used to remove the organic phase acetonitrile from the eluent, and the remaining antimicrobial peptide aqueous solution was then placed in a refrigerator and frozen into ice cubes. Finally, a freeze dryer was used to remove moisture to obtain a fluffy solid powder. The purity of the antimicrobial peptide was determined by HPLC, and the purity reached more than 95%. Figure 1 This is the HPLC result of the pure antimicrobial peptide. Figure 2 Its mass spectrum.

[0036] Example 2: Antimicrobial peptide antibacterial activity test

[0037] Take out the freeze-dried powdered peptide from the -20℃ refrigerator, place it at room temperature for 1h, then add sterile water to dissolve and mix evenly to prepare a 512μg / mL peptide solution. Reduce the concentration of the peptide sample solution by half, and the concentrations are 256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, and 8μg / mL; store it in aliquots at -20℃ for later use.

[0038] The Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis or Staphylococcus aureus used in the experiment were all strains preserved in this laboratory.

[0039] The formula of broth culture medium (liquid): peptone 10g, beef extract 5g, NaCl 5g, add distilled water 800mL, adjust the pH to 7.2 with 5M NaOH, make up to 1000mL, sterilize at 121℃ for 20-30min, and store at room temperature.

[0040] Formula of broth medium (solid): weigh 23.5g plate count agar, add 1000mL distilled water, heat and boil until completely dissolved, then sterilize at 121℃ for 20-30min, cool to about 46℃ for use. First, dissolve the 4 bacteria (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus) stored at -80℃ at 4℃, then take them out and balance them to room temperature, then activate them in small amounts (1mL broth liquid culture medium + 50μL strains), and culture them at 37℃ constant temperature shaker for 18h until the logarithmic growth phase; then, inoculate the activated bacterial liquid onto the broth solid slant medium, incubate at 37℃, grow single colonies, and place the selected bacteria in liquid broth medium and culture them at 37℃ constant temperature shaker until the logarithmic growth phase. When the bacterial solution is in the logarithmic growth phase, dilute it 200 times; take 100 μL of the diluted bacterial solution and mix it evenly with 100 μL of the peptide sample solution of different components, make 3 parallels for each group, and add it to a 96-well plate. After culturing in a 37℃ bacterial incubator for 24 hours, measure the OD value with an enzyme marker and take pictures. The negative control is sterile culture medium; the positive control is the diluted bacterial solution.

[0041] The antibacterial rate was calculated according to the following formula: 1-(OD 试验 -OD 阴性 ) / OD 阳性 *100%

[0042] Table 1 Inhibition rate of antimicrobial peptides against four pathogenic bacteria (%)

[0043]

[0044] From Table 1, we can see that the inhibition rate of the polypeptide against Enterobacter, Pseudomonas aeruginosa, Bacillus subtilis or Staphylococcus aureus and the antibacterial effect diagram show that although the inhibition rate does not show regularity, it can still be explained that the polypeptide LKVVFKVLFKRRR has an antibacterial effect on these bacteria.

[0045] According to the minimum inhibitory concentration (MIC) of the polypeptide, the minimum inhibitory concentration of the antimicrobial peptide of the present invention can be obtained by observing the turbidity of the sample, as shown in Table 2 below:

[0046] Table 2 Minimum inhibitory concentration

[0047]

[0048] Example 3: Trypsin degradation experiment

[0049] Mix 0.25% trypsin used for cell digestion with the polypeptide sample at a molar ratio of 1:200, incubate at 37°C, take 50uL samples at 4, 6, 8, 10, 12, and 14h, then dilute the sample 1-fold with a polypeptide solvent, filter with a 0.22um filter membrane, and take 20μL to determine the residual amount of the polypeptide sample by reverse HPLC. Phase A uses pure water containing 0.1% trifluoroacetic acid (TFA), and phase B uses acetonitrile containing 0.1% TFA for gradient elution to obtain the elution peaks and integrated areas at different time points after the polypeptide sample LKVVFKVLFKRRR is mixed with trypsin, and then the software Origin 2021 is used to draw the graph. The results are as follows Figure 3 As shown in the figure, the antimicrobial peptide LKVVFKVLFKRRR began to be degraded at 6 hours, but there were still undegraded entities within 14 hours, indicating that LKVVFKVLFKRRR was highly stable.

[0050] Example 4: Evaluation of hemolytic toxicity of the antimicrobial peptide LKVVFKVLFKRRR

[0051] The biosafety of antimicrobial peptides was evaluated by assessing hemolytic toxicity using mouse erythrocytes.

[0052] Take 200μL of blood, add it to an EP tube with the bottle wall moistened with sodium heparin, centrifuge at 3000r / min for 10min, discard the supernatant, wash with PBS, and centrifuge twice under the same conditions until the supernatant is no longer red. After discarding the supernatant, take 0.1mL and add 9.9mL PBS buffer to obtain a red blood cell suspension. First, prepare antimicrobial peptide dispersions of different concentrations, with concentrations of 4, 8, 16, 32, 64, 128, and 256μg / mL respectively. Add 200μl of red blood cell suspension to a 1.5mL centrifuge tube, and add 10μL of antimicrobial peptide dispersions of different concentrations to each centrifuge tube. Incubate in a 37℃ water bath for 1h, and then centrifuge at 5000r / min for 10min. Take 100μL of the supernatant and add it to a 96-well plate. Each group is repeated three times in parallel. Use the pure DMSO group as a blank control, Triton-x100 as a complete hemolysis group, and test the OD in the well. 595 The results are shown in Table 3. Figure 4 As shown, the hemolysis rate was less than 15%, and the antimicrobial peptides exhibited low hemolytic toxicity.

[0053] Table 3 Hemolytic toxicity of antimicrobial peptides at different concentrations

[0054]

[0055] Example 5: Cytotoxicity study of antimicrobial peptides

[0056] The cytotoxicity of antimicrobial peptides in RAW264.7, THP-1, and 293T cells was determined by MTT and CCK-8 (Cell Counting Kit-8) experimental methods. The cells were cultured to the logarithmic phase, and 5000 cells were plated in a 96-well plate with 100 μL of cell suspension per well, and placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, except for THP-1 cells, the culture supernatant was discarded from all other cells, and then antimicrobial peptides containing concentration gradients were added respectively, and placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the cytotoxicity to THP-1 cells was determined by the CCK-8 method, and the cytotoxicity to RAW264.7 and other cells was determined by the MTT method. The cytotoxicity of the antimicrobial peptide LKVVFKVLFKRRR was then evaluated by measuring the absorbance of each well at 490 nm using an enzyme reader. Three replicate wells were set for each drug concentration, and each data was expressed as the mean + standard deviation of three independent experiments. Figure 5-7 As shown, the results showed that the antimicrobial peptides had no significant effect on cell survival rate, and even at high concentrations, the cell survival rate was still above 70%.

Claims

1. An antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is: LKVVFKVLFKRRR.

2. The method for preparing the antimicrobial peptide according to claim 2, characterized in that: The method is to synthesize the antimicrobial peptide by a standard Fmoc solid phase synthesis method.

3. The method according to claim 2, characterized in that The method comprises the following: S1. Place dichlororesin in a synthesis column and add DCM to swell it; then add the first amino acid activated by ACT activator, and then shake on a shaker for reaction; after completion, wash with DMF; then add DEP solution for shaking reaction; after completion, wash with DMF, and use ninhydrin detection solution to detect the connection of amino acids. After the reaction is complete, connect the next amino acid; S2. The second amino acid is activated by shaking with HBTU and ACT activator on a shaker, and then added to the synthesis column and shaken on a shaker; after the reaction, it is washed with DMF; then DEP solution is added for shaking reaction; after the reaction, it is washed with DMF, and the connection of the amino acid is detected with ninhydrin detection solution. After the reaction is complete, the next amino acid is connected; S3. Connect the subsequent amino acids according to the method described in S2 until the last amino acid is completely connected; after the last amino acid is completely connected, add methanol to wash; then wash with DCM to form a fine sand; then add a cutting agent to carry out a cutting reaction; after the end, add icy ether to precipitate and centrifuge, discard the supernatant, wash the precipitate with icy ether and centrifuge to obtain a crude antimicrobial peptide; S4. Purify the crude antimicrobial peptide by HPLC to obtain a pure antimicrobial peptide with a purity of more than 95%.

4. The method according to claim 3, characterized in that In S1, the amount of the first amino acid added was 1 times by weight, in S2, the amount of the second amino acid added was 3 times by weight, and the amount of HBTU added was 3 times by weight.

5. The method according to claim 3, characterized in that: The ACT activator in S1 and S2 consists of N-methylmorpholine and DMF, wherein the volume ratio of N-methylmorpholine to DMF is 4:

87.

6. The method according to claim 3, characterized in that The cutting agent in S3 is composed of trifluoroacetic acid, methyl phenyl sulfide, water, phenol, and ethanedithiol, wherein the volume ratio of trifluoroacetic acid, methyl phenyl sulfide, water, phenol, and ethanedithiol is 82.5:5:5:5:2.

5.

7. The method according to claim 3, characterized in that In S4, the HPLC purification method is: using a C18 semi-preparative column; mobile phase: phase A: deionized water containing 0.1% TFA (m / v); phase B: acetonitrile solution containing 0.1% TFA (m / v); flow rate: 1 ml / min; elution gradient: phase A decreases from 95% to 5% at a rate of 6% per minute, and phase B increases from 5% to 95% at a rate of 6% per minute.

8. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial infection drugs.

9. The use according to claim 8, characterized in that: The bacteria are Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis or Staphylococcus aureus.

10. An antibacterial infection drug, characterized in that: The active ingredient of the drug includes the antimicrobial peptide according to claim 1.

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