High-activity short-chain antibacterial peptide VKLR as well as preparation method and application thereof

Abstract

The invention discloses a high-activity short-chain antibacterial peptide VKLR as well as a preparation method and application thereof, and belongs to the technical field of application of agriculture, animal husbandry and veterinary medicine. The amino acid sequence of the high-activity short-chain antibacterial peptide VKLR disclosed by the invention is as shown in SEQ ID No. 1. The VKLR is a short-chain antibacterial peptide which is composed of natural amino acid and has excellent antibacterial activity, not only has a remarkable antibacterial effect, but also does not show toxicity to cells in an in-vitro cytotoxicity experiment, and has extremely high safety. The antibacterial peptide is mature in preparation process and convenient to obtain, and can be produced through fermentation of an engineering bacterium expression system.

Description

Technical Field

[0001] The invention relates to the field of agricultural animal husbandry and veterinary application technology, and in particular to a highly active short-chain antimicrobial peptide VKLR and a preparation method and application thereof. Background Art

[0002] As one of the most important medical discoveries of the 20th century, antibiotics have saved countless lives and made important contributions to the prevention and treatment of human infectious diseases. About 70% of the antimicrobial drugs currently used in human medicine were obtained during the golden age of antibiotic discovery, and most of these drugs were isolated from actinomycetes and fungi. However, after the 1970s, it has become difficult to obtain antibiotics with development value from microbial metabolites through pure microbial culture under laboratory conditions. At present, the research and development of antimicrobial drugs is mostly focused on the modification or optimization of known compounds. In a period when antibiotic discovery is becoming increasingly difficult, bacterial resistance to antibiotics has increased year by year. Antibiotic resistance can make bacteria tolerate antibiotics at clinical therapeutic concentrations, causing antibiotics to lose their therapeutic effectiveness. It is predicted that by 2050, the number of human deaths caused by multidrug resistance will increase to 10 million, exceeding the number of deaths from cancer and becoming one of the main causes of human death worldwide. Therefore, it is urgent to control the development of drug resistance.

[0003] As a natural biomaterial, antimicrobial peptides have become a popular candidate for antimicrobial preparations, especially alternatives to veterinary antibiotics, in recent years due to their unique mechanism of disrupting pathogen biofilms. However, naturally occurring antimicrobial peptides have a series of problems such as weak biological activity and high toxicity, which seriously limit and hinder their transformation and application. Therefore, it is of great significance to obtain an antimicrobial peptide with high biological activity and low toxicity. Summary of the invention

[0004] The purpose of the present invention is to provide a highly active short-chain antimicrobial peptide VKLR and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention provides an antimicrobial peptide VKLR with high antimicrobial activity, which contains only 13 amino acids, has low synthesis cost, and has excellent broad-spectrum antimicrobial activity. The antimicrobial peptide not only has a significant antibacterial effect, but also does not show toxicity to cells in in vitro cytotoxicity experiments, and has extremely high safety.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: A highly active short-chain antimicrobial peptide VKLR, whose amino acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is as follows: LVKLRVKLRVKLR.

[0007] The present invention adopts the following technical scheme to screen: 1) collect all antimicrobial peptide sequences with antimicrobial function in the antimicrobial peptide database (https: / / aps.unmc.edu / AP / ); 2) count the amino acid proportion, secondary structure, sequence length, positive charge number, hydrophobicity and adjacent amino acid richness of all sequences; 3) rationally design and synthesize a batch of antimicrobial peptides based on the above parameters; 4) screen out an antimicrobial peptide VKLR with excellent antimicrobial activity and lowest cytotoxicity based on in vitro antimicrobial activity, cytotoxicity and hemolytic activity.

[0008] Technical Solution 2: The application of the highly active short-chain antimicrobial peptide VKLR in any of the following items includes:

[0009] (1) Used to prepare broad-spectrum antibacterial agents;

[0010] (2) Used in the preparation of preservatives;

[0011] (3) Used for preparing antibacterial drugs or antibacterial compositions;

[0012] (4) Used for preparing medical dressings.

[0013] Furthermore, the antibacterial activity includes anti-Gram-negative bacteria and / or anti-Gram-positive bacteria.

[0014] Furthermore, the antibacterial agent includes anti-Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and / or Staphylococcus epidermidis.

[0015] Technical solution three: A broad-spectrum antibacterial agent, which contains the highly active short-chain antibacterial peptide VKLR.

[0016] Technical solution 4: A preservative, which contains the highly active short-chain antimicrobial peptide VKLR.

[0017] Technical Solution 5: An antibacterial drug or an antibacterial composition, wherein the antibacterial drug or the antibacterial composition comprises the highly active short-chain antibacterial peptide VKLR.

[0018] Technical Solution 6: A medical dressing, comprising the highly active short-chain antimicrobial peptide VKLR.

[0019] Technical Solution 7: Application of the highly active short-chain antimicrobial peptide VKLR in the preparation of medical imaging agents, feed additives, daily chemical cleaning products and / or medical devices with antimicrobial effects.

[0020] The present invention discloses the following technical effects:

[0021] The present invention is based on an antimicrobial peptide database, and a highly active short-chain antimicrobial peptide VKLR is synthesized by database filtering technology and computer-aided design. By optimizing the amino acid arrangement, the hemolytic activity is minimized while having high antimicrobial activity. The results of the embodiment show that the antimicrobial peptide of the present invention not only has a significant antibacterial effect, but also does not show toxicity to cells in in vitro cytotoxicity experiments, and has extremely high safety. The preparation process of the antimicrobial peptide of the present invention is mature, the acquisition channel is convenient, and it can be produced by fermentation through an engineered bacterial expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the mass spectrum of the antimicrobial peptide VKLR;

[0024] Figure 2 is the liquid chromatogram of the antimicrobial peptide VKLR;

[0025] Figure 3 This is the bactericidal kinetic curve of the antimicrobial peptide VKLR against Pseudomonas aeruginosa;

[0026] Figure 4 This is the bactericidal kinetic curve of the antimicrobial peptide VKLR against Staphylococcus aureus;

[0027] Figure 5 This is a bar graph showing the effect of the antimicrobial peptide VKLR on bacterial outer membrane permeability;

[0028] Figure 6 It is a bar graph of the affinity of the antimicrobial peptide VKLR to LPS / LTA;

[0029] Figure 7 This is the result of the antimicrobial peptide VKLR depolarizing the inner membrane of Pseudomonas aeruginosa;

[0030] Figure 8 This is the result of the antimicrobial peptide VKLR depolarizing the inner membrane of Staphylococcus aureus;

[0031] Fig. 9 This is a graph showing the effect of the antimicrobial peptide VKLR on the hemolysis rate of red blood cells. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Design of antimicrobial peptides

[0038] (1) Collect all antimicrobial peptide sequences with antimicrobial function in the Antimicrobial Peptide Database (https: / / aps.unmc.edu / AP / );

[0039] (2) Count the amino acid percentage, secondary structure, sequence length, positive charge, hydrophobicity, and adjacent amino acid richness of all sequences; (3) Based on the above parameters, rationally design and synthesize a batch of antimicrobial peptides; (4) Based on the in vitro antimicrobial activity, cytotoxicity, and hemolytic activity, screen out an antimicrobial peptide VKLR with excellent antimicrobial activity and the lowest cytotoxicity. Figure 1 is the mass spectrum of the antimicrobial peptide VKLR; Figure 2 The liquid chromatography diagram of the antimicrobial peptide VKLR is shown in Table 1 for the sequence and physicochemical parameters of the antimicrobial peptide VKLR.

[0040] Table 1

[0041]

[0042] Example 2 Synthesis of antimicrobial peptide VKLR by solid phase chemical synthesis

[0043] The synthesis method is as follows:

[0044] The polypeptides of the present invention are all prepared by solid phase synthesis method:

[0045] (1) The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and a resin with side chain protection removed from the Fmoc group is obtained;

[0046] (2) Add a cleavage agent to the peptide resin obtained above, react at 20° C. in the dark for 2 h, filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20° C. for 3 h, precipitate a white powder, centrifuge at 2500 g for 10 min, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0047] (3) Use 0.2M sodium sulfate (adjusted to pH 7.5 with phosphoric acid) to balance the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and use C18 reverse phase atmospheric pressure column, gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate of 1 mL / min, detection wave of 220 nm, collect the main peak, and freeze-dry; then use reverse phase C18 column for further purification, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25% B to 40% B, elution time is 12 minutes, flow rate is 1 mL / min, and the main peak is collected and freeze-dried as above;

[0048] (4) Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weight ( Figure 1 ) is basically consistent with the theoretical molecular weight, and the purity of the antimicrobial peptide is greater than 95%.

[0049] Example 3 Determination of the antimicrobial activity of protease-resistant antimicrobial peptide VKLR

[0050] The minimum inhibitory concentration (μg / mL) of the antimicrobial peptide VKLR was determined by the microdilution method. A diluent containing 0.01% acetic acid and 0.2% fetal bovine serum albumin was added to a 96-well plate, and a series of gradient antimicrobial peptide solutions were prepared in sequence using the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5 CFU / mL) in each well. Set up a positive control (containing bacterial solution but not antimicrobial peptide) and a negative control (containing neither bacterial solution nor antimicrobial peptide) respectively. Incubate at 37°C for 14-18h and read the sample at 492nm (OD 492nm ) to determine the minimum inhibitory concentration. The measured value is less than 0.1, which is considered to be inhibited. Two parallels are tested each time, and repeated three times. The antibacterial activity of peptide VKLR against bacteria is shown in Table 2.

[0051] Table 2

[0052] strains Minimum inhibitory concentration Escherichia coli 25922 2 Escherichia coli K88 2 Pseudomonas aeruginosa 15442 4 Pseudomonas aeruginosa 27853 4 Staphylococcus aureus 25923 2 Staphylococcus aureus 6538 2 Staphylococcus aureus 43300 4 Staphylococcus epidermidis 49134 2

[0053] From the results in Table 2, it can be seen that the antimicrobial peptide VKLR has a low minimum inhibitory concentration against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, showing excellent broad-spectrum antibacterial activity.

[0054] Example 4 Bactericidal kinetics of antimicrobial peptide VKLR

[0055] Pseudomonas aeruginosa ATCC 15442 in the logarithmic growth phase was diluted to (1×10 5 )CFU mL -1 , and in PBS (10×10 -3 M, pH = 7.4) and the concentration was 4 × 10 6 , 8×10 6 and 16×10 6 Similarly, Staphylococcus aureus ATCC 6538 cells in the logarithmic growth phase were diluted to (1×10 6 )CFU mL -1 and with a concentration of 2×10 6 , 4×10 6 and 8×10 6 M of the anti-membrane peptide in PBS (10×10 -3M, pH = 7.4). Samples were taken at different time intervals (0, 3, 5, 15, 30, 60 and 120 minutes) and diluted to appropriate multiples. The diluted bacterial suspension was spread on Mueller-Hinton agar (MHA) plates. After the plates were cultured in an incubator at 37°C for 24 hours, the bacterial colonies were counted. Each test had two parallel items, which were repeated three times independently.

[0056] Depend on Figure 3 It can be seen that the antimicrobial peptide VKLR can kill 99.9% of Pseudomonas aeruginosa within 15 minutes at the bactericidal concentration. Figure 4 It can be seen that at the bactericidal concentration, it takes 120 minutes for the antimicrobial peptide VKLR to kill 99.9% of Staphylococcus aureus. The reason is that Gram-positive bacteria have a dense peptidoglycan layer, which to some extent hinders the antimicrobial peptide VKLR from breaking the membrane. In summary, the antibacterial effect of the antimicrobial peptide VKLR on Gram-negative bacteria is stronger than that on Gram-positive bacteria.

[0057] Example 5 Analysis of the Permeability of the Antimicrobial Peptide VKLR to the Outer Membrane of Pseudomonas aeruginosa

[0058] The outer membrane permeability of Pseudomonas aeruginosa ATCC15442 induced by antimicrobial membrane peptides was measured using the fluorescent probe N-phenyl-1-naphthylamine (NPN). -3 10 × 10 -6 The fluorescent dye NPN was added to the Pseudomonas aeruginosa bacterial suspension. Next, 100 μL of bacterial suspension was mixed with 100 μL of peptide (final concentration range: 2×10 -6 -64×10 -6 M) was mixed into a 96-well plate. The fluorescence value was measured with an ELISA reader at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. 10 μg mL -1 The polymyxin B-treated group was used as a positive control, and the bacterial suspension without peptide was used as a negative control for initial fluorescence. Each test was performed in two parallels and repeated three times independently.

[0059] Depend on Figure 5 It can be seen that the antimicrobial peptide VKLR has a dose-dependent effect on the outer membrane permeability of Pseudomonas aeruginosa. -6 M, it can destroy about 40% of the bacterial outer membrane.

[0060] Example 6 Antimicrobial peptide VKLR binding affinity test for LPS / LTA

[0061] The ability of the antimicrobial peptide VKLR to bind to LPS of Pseudomonas aeruginosa and LTA of Staphylococcus aureus was determined using the fluorescent probe BODIPY-TR cadaverine (BC). -6 M BC and the final concentration was 50 × 10 -6 M of LPS or LTA were incubated at 37°C in the dark for 4 h. Next, the peptide VKLR solution was serially diluted in a 96-well plate to produce 1 × 10 -6 Up to 64×10 -6 The concentration of M. Subsequently, LPS or LTA probes were added to an equal volume of peptide solution and incubated for 1 hour. Finally, the fluorescence intensity was measured by a microplate reader at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. Each test had two parallel items and was repeated three times independently.

[0062] Depend on Figure 6 It can be seen that the binding of VKLR to Pseudomonas aeruginosa LPS and Staphylococcus aureus LTA is dose-dependent, and the binding to LPS is stronger. The results of binding kinetic analysis show that VKLR shows a stronger binding ability to LPS, which is one of the reasons why it has faster bactericidal activity against Pseudomonas aeruginosa.

[0063] Example 7 Effect of the antimicrobial peptide VKLR on the degree of bacterial inner membrane depolarization

[0064] The lipophilic membrane probe 3,3'-dipropylthiodicarbocyanine iodide (DiSC3-5) was used to measure the changes in the cytoplasmic membrane potential of Pseudomonas aeruginosa ATCC15442 and Staphylococcus aureus ATCC6538 induced by peptide VKLR. Logarithmically growing bacteria were resuspended in 5×10 -3 M HEPES buffer (pH 7.4, containing 40×10 -3 M glucose and 200×10 -3 M KCL) and adjusted the OD to 0.1 at 600 nm. Immediately thereafter, 0.8×10 -6 M DiSC3-5 and incubated in the dark for 90 min. Meanwhile, peptide VKLR solution was serially diluted in a 96-well plate to generate a concentration range of 8 × 10 -6 Up to 16×10 -6 M. Next, the peptide solution was mixed with the bacterial suspension (1:1, v / v), and the fluorescence changes at the excitation wavelength of 620 nm and the emission wavelength of 670 nm were immediately detected using a microplate reader.

[0065] Fluorescent probe DiSC3-5 was used as a tracer dye to assess changes in membrane potential. This environmentally sensitive cationic dye diffuses and accumulates in the phospholipid bilayer, resulting in dye self-quenching. When VKLR changes the plasma membrane potential or structure, the fluorescence intensity increases. Figure 7 and Figure 8 It can be seen that the antibiotic membrane peptide VKLR causes cytoplasmic membrane depolarization in a dose-dependent manner. At the same time, Pseudomonas aeruginosa reaches its peak value faster under VKLR than under Staphylococcus aureus, and its fluorescence reaches its peak at 120S.

[0066] Example 8 Determination of hemolytic activity of antimicrobial peptides

[0067] Purchase 1% porcine red blood cells, collect the red blood cells by centrifugation at 3000rpm for 10min; wash with PBS solution for 3 times, and then resuspend with 10mL PBS solution; take 50μL of red blood cell suspension and mix with 50μL of antimicrobial peptide solution of different concentrations, incubate at a constant temperature of 37℃ in an incubator for 1h; then centrifuge at 4℃, 3000rpm for 10min; take out the supernatant and measure the light absorption value at 570nm with an enzyme marker. 50μL of red blood cells plus 50μL of PBS solution is used as a negative control, and 50μL of red blood cells plus 50μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a hemolysis rate of 10%. The test results are shown in Fig. 9 .pass Fig. 9 It can be seen that the antimicrobial peptide VKLR did not show hemolytic activity within the detection range, causing 1% hemolysis of erythrocytes at a concentration of 128 μM and failing to cause 10% hemolysis of erythrocytes, indicating that the antimicrobial peptide VKLR with low hemolytic activity has the potential to be developed as an antibiotic alternative.

[0068] In summary, the peptide VKLR composed of natural amino acids has excellent broad-spectrum antibacterial activity, strong salt stability and protease stability, and has broad prospects for clinical application.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A highly active short-chain antimicrobial peptide VKLR, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the highly active short-chain antimicrobial peptide VKLR according to claim 1 in any of the following items, characterized in that: include: (1) Used to prepare broad-spectrum antibacterial agents; (2) Used in the preparation of preservatives; (3) Used for preparing antibacterial drugs or antibacterial compositions; (4) Used for preparing medical dressings.

3. The use according to claim 2, characterized in that: The antibacterial activity includes anti-Gram-negative bacteria and / or anti-Gram-positive bacteria.

4. The use according to claim 2, characterized in that: The antibacterial agents include anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

5. A broad-spectrum antibacterial agent, characterized in that: The broad-spectrum antibacterial agent comprises the highly active short-chain antibacterial peptide VKLR according to claim 1.

6. A preservative, characterized in that The preservative comprises the highly active short-chain antimicrobial peptide VKLR according to claim 1.

7. An antibacterial drug or antibacterial composition, characterized in that: The antibacterial drug or antibacterial composition comprises the highly active short-chain antibacterial peptide VKLR according to claim 1.

8. The antibacterial drug or antibacterial composition according to claim 7, characterized in that: The antibacterial drug also includes pharmaceutically acceptable excipients.

9. A medical dressing, characterized in that: The medical dressing comprises the highly active short-chain antimicrobial peptide VKLR according to claim 1.

10. Use of the highly active short-chain antimicrobial peptide VKLR according to claim 1 in the preparation of medical imaging agents, feed additives, daily chemical cleaning products and / or medical devices with antimicrobial effects.

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