Antibacterial polypeptide, composition and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to an antibacterial polypeptide, a composition and application thereof. The amino acid sequence of the antibacterial polypeptide is as shown in SEQ ID NO. 1: KWKIKWPVKWFKM-NH2. The invention also discloses a preparation method of the antibacterial polypeptide. Experiments show that the antibacterial polypeptide disclosed by the invention has broad-spectrum antibacterial activity on gram-positive bacteria and / or gram-negative bacteria, and also has low cytotoxicity and low hemolysis. The antibacterial polypeptide provided by the invention consists of 14 amino acids, is relatively short in peptide chain, small in molecular weight and easy to artificially synthesize, is a micromolecular polypeptide with extremely high application value, can be used for preparing a novel antibacterial agent, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antibacterial polypeptide, a composition and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Antimicrobial peptides are a class of active biological molecules that most organisms have endogenous immune responses. They are a class of polypeptides with biological activities such as antibacterial, antiviral or tumor inhibition. Since the antibacterial and bactericidal mechanisms of antimicrobial peptides are different from those of antibiotics, and the probability of bacterial infection is low when used as drug treatment, and they have a broad spectrum of antibacterial effects, antimicrobial peptides are considered to be a new generation of antibacterial agents that can effectively alleviate the threat posed by bacterial infection. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an antibacterial polypeptide, a composition and its application. The present invention obtains a new polypeptide through high-throughput screening, and studies have shown that the polypeptide has a broad-spectrum antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria, and has low cytotoxicity and low hemolytic activity.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides an antibacterial polypeptide, the amino acid sequence of the antibacterial polypeptide is shown as SEQ ID NO.1.

[0007] The amino acid sequence shown in SEQ ID NO.1 is: KWKIKWPVKWFKML-NH2.

[0008] In some embodiments, in the amino acid sequence, K is used as the N-terminal amino acid and L is used as the C-terminal amino acid, wherein the -NH2 connected to the C-terminus is obtained by amidation modification of the carboxyl group.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned antimicrobial polypeptide, comprising the following steps:

[0010] Preparation of antimicrobial peptides by chemical synthesis;

[0011] Or, preparing antimicrobial peptides by genetic engineering methods;

[0012] Alternatively, the antimicrobial peptides may be prepared by microbial fermentation.

[0013] Since the antimicrobial polypeptide provided by the present invention consists of only 14 amino acids, it is easy to synthesize by chemical synthesis, and the efficiency of obtaining antimicrobial polypeptides by chemical synthesis is higher, therefore, the present invention preferably synthesizes the anti-angiogenic peptide by chemical synthesis.

[0014] In some embodiments, the chemical synthesis method is a solid phase synthesis method, wherein the resin used in the solid phase synthesis method is Wang resin.

[0015] Specifically, the solid phase synthesis method comprises the following steps:

[0016] Connecting the carboxyl group of Fmoc-lysine to the resin, removing impurities and removing the Fmoc protection on the resin to obtain a deprotected lysine-resin;

[0017] The amino group of the deprotected lysine-resin and the carboxyl group of the Fmoc-tryptophan are condensed and deprotected, and methionine, isoleucine, lysine, tryptophan, proline, arginine, lysine, tryptophan, phenylalanine, threonine, valine and leucine are repeatedly connected to the resin, and the deprotected structure is repeatedly condensed and deprotected, and then washed to obtain a resin containing a polypeptide;

[0018] Using a cleavage reagent (the cleavage reagent includes trifluoroacetic acid (TFA), triisopropylsilyl chloride (TIS) and water, mixed in a volume ratio of (94-96):(2-3):(2-3)), the polypeptide is cleaved from the resin, the cleavage reagent component is removed, and the crude polypeptide is precipitated and dried;

[0019] The crude polypeptide is purified to obtain an antibacterial polypeptide R2 with a purity higher than 95%.

[0020] In a third aspect, the present invention provides a composition comprising the above-mentioned antimicrobial polypeptide or its derivatives.

[0021] In some embodiments, the derivative is a derivative peptide obtained by amidation, carbonylation, phosphorylation, hydroxylation, esterification, glycosylation, methylation, carboxylation and / or acetylation based on the side chain group, C-terminus, and N-terminus of the antibacterial polypeptide.

[0022] In some embodiments, the composition further comprises an auxiliary material. The auxiliary material may be a pharmaceutical auxiliary material or an auxiliary material of a conventional daily chemical product. The pharmaceutical auxiliary material comprises a pharmaceutical carrier and / or an excipient. The pharmaceutical carrier may be serum protein, polycyanoacrylate, polylactic acid, etc. The excipient comprises a binder, a filler, a disintegrant, a solubilizer, a stabilizer, etc.

[0023] In a fourth aspect, the present invention provides the use of the above-mentioned antibacterial polypeptide or composition in the preparation of antibacterial products.

[0024] In some embodiments, the bacteria targeted by the antibacterial products are Gram-positive bacteria and / or Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus and / or Enterococcus faecium; the Gram-negative bacteria include one or more of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella typhimurium.

[0025] Specifically, the minimum inhibitory concentration of the antibacterial product against Staphylococcus aureus is 4 μg / mL, the minimum inhibitory concentration against Enterococcus faecium is 4 μg / mL, the minimum inhibitory concentration against Escherichia coli is 4 μg / mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 4 μg / mL, the minimum inhibitory concentration against Acinetobacter baumannii is 4 μg / mL, the minimum inhibitory concentration against Salmonella typhimurium is 4 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 4 μg / mL.

[0026] In some embodiments, the antibacterial product is a medicine or a daily chemical. The medicine is an internal medicine or an external medicine. The internal medicine is an oral preparation or an injection; the external medicine includes a patch and an smear preparation. The daily chemical includes but is not limited to a disinfectant and an additive. The additive can be a preservative, a washing additive, or a feed additive.

[0027] Since the above disinfectants and additives can be added to detergents and feeds, in a fifth aspect, the present invention provides a use of the above antimicrobial polypeptide or the above composition in the preparation of detergents or feeds.

[0028] The beneficial effects of the present invention are:

[0029] (1) The polypeptide provided by the present invention has a broad-spectrum antibacterial activity against Gram-positive bacteria and / or Gram-negative bacteria and can be used as an antibacterial peptide. At the same time, the polypeptide has the characteristics of low cytotoxicity and low hemolysis.

[0030] (2) The polypeptide provided by the present invention is composed of 14 amino acids, has a short peptide chain, a small molecular weight, and is easy to synthesize artificially. It is a small molecule polypeptide with great application value and can be used to prepare a new antibacterial agent with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 This is a mass spectrometry detection diagram of the antibacterial polypeptide R2 used in the present invention;

[0033] Figure 2This is a graph showing the hemolytic activity test results of polypeptide R2 in Example 2 of the present invention;

[0034] Figure 3 This is a diagram showing the cytotoxicity test results of polypeptide R2 in Example 3 of the present invention;

[0035] Figure 4 This is a graph showing the therapeutic results of polypeptide R2 in Example 4 of the present invention on bacterial skin infection in mice. DETAILED DESCRIPTION

[0036] The reagents of the present invention can all be purchased through commercial channels.

[0037] In the following examples, the amino acid sequence of the antibacterial peptide R2 from segment N to segment C is KWKIKWPVKWFKML-NH2, as shown in SEQ ID NO.1, and was prepared by entrusting Jiangsu GenScript Biotechnology Co., Ltd. It was synthesized by solid phase synthesis using a peptide synthesizer, and the specific steps are as follows:

[0038] (1) The carboxyl group of Fmoc-lysine is connected to Wang resin (as a solid phase carrier) by a covalent bond structure, and impurities are removed by soaking in N,N-dimethylformamide (DMF) for about 15 minutes; the Fmoc protection on the resin is removed by using DMF containing 20% ​​piperidine (the volume ratio of piperidine to DMF is 1:4), reacting for 20 minutes, and washing the resin until it is completely washed. The piperidine is washed away with DMF, and the remaining solid suspension is deprotected lysine-Wang.

[0039] (2) Fmoc-tryptophan was synthesized by the same synthesis method. The amino group of the deprotected lysine-Wang in (1) was condensed with the carboxyl group of Fmoc-tryptophan to form a peptide bond, and then the Fmoc group was removed by deprotection. The above peptide bond formation reaction was repeated to gradually connect methionine, isoleucine, lysine, tryptophan, proline, arginine, lysine, tryptophan, phenylalanine, threonine, valine and leucine to the resin. After the last amino acid was deprotected, it was washed with DMF 8 times, and then cross-washed with ethanol and dichloromethane (DCM) 8 times. Trifluoroacetic acid (TFA), triisopropylsilyl chloride (TIS) and water were mixed in a volume ratio of 95:2.5:2.5 to prepare a cleavage reagent, and reacted with the above-obtained polypeptide at 20°C for 2 hours to cleave the polypeptide from the resin. TFA was evaporated by a rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether was added to precipitate the polypeptide for 3 hours, and a white powder solid was precipitated. The crude polypeptide was obtained by vacuum drying.

[0040] (3) The crude polypeptide was dissolved in 90% acetonitrile-water solution (the volume ratio of acetonitrile to water was 9:1), purified using a preparative chromatographic column, and the purity was detected using an analytical chromatographic column. The semi-preparative HPLC was Waters DeltaPrep 4000, and the preparative chromatographic columns were Waters X-Bridge Cis, 5Hm reverse phase columns. Eluent A was an aqueous solution containing 0.1% TFA, and B was an acetonitrile aqueous solution containing 0.1% TFA; the detection wavelength was 220 nm, the elution method was a linear concentration gradient elution of 30% B to 65% B, and the flow rate was 30 mL / min. The fractions with a purity higher than 95% were collected and freeze-dried to obtain the polypeptide, which was then amidated to obtain the antibacterial polypeptide R2. The analytical high performance liquid chromatograph was Agilent 1100, the analytical chromatographic column was Sepax GP-C8 reverse phase column (4.6 mm × 150 mm, 5 Hm), the eluent A solution was 0.1% TFA in water, and the eluent B solution was 0.1% TFA in acetonitrile in water: the detection wavelength was 220 nm. The elution method was a linear concentration gradient elution of 50% B to 75% B, and the flow rate was 1.0 mL / min.

[0041] The mass spectrum of peptide R2 is as follows Figure 1 As shown, the molecular weight (1917.4) shown in the mass spectrum is consistent with the theoretical molecular weight (1917.49).

[0042] Example 1 Determination of antibacterial activity of antimicrobial polypeptide R2

[0043] The method for determining the minimum inhibitory concentration (MIC) recommended by the Clinical Laboratory Standards Institute (CLSI) of the United States was used to prepare a series of gradient peptide R2 solutions in sequence using the two-fold dilution method. The specific steps are as follows:

[0044] (1) Preparation of bacteria: Take the test bacteria frozen at -20℃, streak inoculate in NB medium and incubate. Pick a single colony and inoculate it in 10mL NB medium, and culture it at 37℃ and 200rpm overnight. Then inoculate the overnight bacteria into fresh medium and culture it for 1-2h until the bacteria are in the logarithmic growth phase and the OD600 is 0.4. Use MH(B) to adjust the colony count of the obtained bacterial solution to about 10 6 CFU / mL or so.

[0045] (2) Preparation of peptides: The concentration of peptide R2 was adjusted to 512 μg / mL, 100 μL was pipetted and added to the first column of the 96-well plate. 50 μL of MH broth was added to the other wells. Then 50 μL of the peptide solution in well No. 1 was pipetted and added to well No. 2. The dilution was repeated in the same manner until the well No. 10 was reached. 50 μL was pipetted and discarded.

[0046] (3) Inoculation: Use a pipette to take 50 μL of the bacterial solution adjusted in step (1) and add it to the first 11 wells of the 96-well plate in sequence. The final inoculation bacterial concentration is 5×10 5 CFU / mL. Place the 96-well plate on a micro-oscillator and oscillate for 1 minute to mix the liquid in each well. Cover the microplate to reduce evaporation during incubation and incubate at 37°C for 18 hours. The positive control group is set as the 11th well: only 50μL MH broth medium and 50μL bacterial solution are added; the negative control is the sterile control group in the 12th well: only 100μL MH broth medium is added. At this time, the concentration of antimicrobial peptides will decrease from the 1st well to the 10th well.

[0047] (4) Result judgment: The sterile control wells should remain clear throughout the test, indicating that the entire test was performed aseptically. The lowest concentration without visible growth is the MIC value of the peptide against the test bacteria, which is compared with the bacterial growth characteristics in the growth control wells (such as turbid broth in the microwells and precipitation at the bottom of the wells).

[0048] The test results are shown in Table 1, and it can be seen that the antibacterial polypeptide R2 has a broad spectrum of ability to inhibit the growth of Gram-negative and Gram-positive bacteria. Specifically, the minimum inhibitory concentration of the antibacterial polypeptide R2 against Staphylococcus aureus is 4 μg / mL, the minimum inhibitory concentration against Enterococcus faecium is 4 μg / mL, the minimum inhibitory concentration against Escherichia coli is 4 μg / mL, the minimum inhibitory concentration against Klebsiella pneumoniae is 4 μg / mL, the minimum inhibitory concentration against Acinetobacter baumannii is 4 μg / mL, the minimum inhibitory concentration against Salmonella typhimurium is 4 μg / mL, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 4 μg / mL.

[0049] In addition, when Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella typhimurium and Pseudomonas aeruginosa were completely killed, the concentrations of the antibacterial polypeptide R2 were ≥4, 8, 4, 4, 4, 4, 4 μg / mL, respectively.

[0050] Table 1 Minimum inhibitory concentration of antibacterial peptide R2

[0051]

[0052] Note: GM represents the geometric mean of the minimum inhibitory concentrations of Gram-positive and Gram-negative bacteria listed in the table.

[0053] Example 2 Hemolytic activity of antimicrobial polypeptide R2

[0054] For the determination of the hemolytic activity of the antimicrobial peptide R2, the specific test steps are as follows:

[0055] (1) Collect 1 mL of fresh human blood using a sodium heparin anticoagulant tube and store at 4°C for later use;

[0056] (2) Centrifuge the blood at 1000 g for 5 min, discard the supernatant, and collect the red blood cells;

[0057] (3) Wash the collected red blood cells three times with PBS buffer solution, centrifuge at 1000g 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 use;

[0058] (4) Dilution of polypeptide R2: Add 90 μL of PBS buffer solution to the first tube of each row of 12 EP tubes, and add 50 μL of PBS buffer solution to the remaining tubes. Then add 10 μL of polypeptide R2 stock solution to the first tube, and then mix the polypeptide R2 solution in the first tube, aspirate 50 μL, add it to the second tube, and dilute it in multiples to the tenth tube, then aspirate 50 μL and discard it;

[0059] (5) Take 50 μL of the prepared red blood cell suspension and add it to the EP tubes containing different concentrations of peptide R2 solution, and incubate at a constant temperature of 37°C in an incubator for 1 hour. Among them, 50 μL PBS and 50 μL red blood cell suspension are added to the 11th well as a negative control, and 50 μL 0.1% Triton X-100 and 50 μL red blood cell suspension are added to the 12th well as a positive control;

[0060] (6) After 1 h, remove the EP tube and centrifuge at 100 g for 5 min at 4 °C;

[0061] (7) Take the supernatant of the centrifuged solution and transfer it to the corresponding wells of a clean 96-well plate in parallel. Use an ELISA reader to read the plate at 570 nm (OD 570nm ) to measure the light absorption value.

[0062] The results are as follows Figure 2 As shown, the antimicrobial peptide R2 had only 12% hemolysis at 256 μg / mL, which was much higher than the geometric mean of its minimum antimicrobial concentration, indicating that the peptide R2, as an antimicrobial peptide, would not produce hemolysis when exerting its antimicrobial effect.

[0063] Example 3 Cytotoxicity of antimicrobial peptide R2

[0064] Human hepatoma cells MHCC97H were used to detect the cytotoxicity of polypeptide R2. The cells were seeded in a 96-well plate at 8,000 cells per well. The cells were incubated at 37°C, 5% CO2 for 24 hours. Then different concentrations of polypeptide R2 were added to each well containing cells, so that the final concentrations were 32μg / mL, 64μg / mL, 128μg / mL, and 256μg / mL, respectively. After incubation for 24 hours, 10μL CCK8 was added to each well and incubated at 37°C for 2 hours. The absorbance value at 450nm was then detected, and Prism 8 was used to determine the cell viability value by curve fitting and applying the following equation:

[0065] Cell survival rate = (experimental group - blank control) / (negative control group - blank control) × 100% Figure 3 As shown, the concentration of antimicrobial peptide R2 at which it exerts 50% cytotoxicity is above 128 μg / mL, which is much higher than the geometric mean of its minimum antimicrobial concentration, indicating that antimicrobial peptide R2, as an antimicrobial peptide, does not produce cytotoxicity when exerting its antimicrobial effect.

[0066] Example 5

[0067] For the determination of the skin external anti-infection ability of the antimicrobial peptide R2, the specific test steps are as follows:

[0068] G+ Staphylococcus aureus CMCC26003 bacterial solution was diluted to 10 8 CFU / mL for later use. Female C57BL / 6 mice were randomly divided into infection negative control group and peptide R2 treatment group according to body weight, with 5 mice in each group. After depilating the back of the mice, a piece of full-thickness skin with a diameter of 5 mm was removed from the center of the back of the mice using a tissue biopsy device. The wound area of ​​each group of mice was uniform, indicating that the model was successfully established. 5 μL of 10 8 CFU / mL of bacterial solution. 6 hours later, the treatment group was given 0.1 mg of peptide R2. 24 hours after modeling, the skin of the infected part of the animal was aseptically taken to detect and count the live bacteria.

[0069] The results are as follows Figure 4 As shown, the antimicrobial peptide R2 as an antimicrobial peptide has a significant ability to clear bacterial infection on mouse skin at 0.1 mg.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antimicrobial polypeptide, characterized in that: The amino acid sequence of the antibacterial polypeptide is shown in SEQ ID NO.

1.

2. A method for preparing the antimicrobial polypeptide according to claim 1, characterized in that: include: Preparation of antimicrobial peptides by chemical synthesis; Or, preparing antimicrobial peptides by genetic engineering methods; Alternatively, the antimicrobial peptides may be prepared by microbial fermentation.

3. The method according to claim 2, characterized in that The chemical synthesis method is a solid phase synthesis method.

4. A composition, characterized in that It comprises the antibacterial polypeptide or its derivatives as described in claim 1.

5. The composition according to claim 4, characterized in that The derivative is a derivative peptide obtained by amidation, carbonylation, phosphorylation, hydroxylation, esterification, glycosylation, methylation, carboxylation and / or acetylation modification of the side chain group, C-terminus and N-terminus of the antibacterial polypeptide.

6. The composition according to claim 4, characterized in that Also included are auxiliary materials, which include pharmaceutical auxiliary materials or auxiliary materials of daily chemical products.

7. Use of the antibacterial polypeptide according to claim 1 or the composition according to any one of claims 4 to 6 in the preparation of antibacterial products.

8. The use according to claim 7, characterized in that The bacteria targeted by the antibacterial products are Gram-positive bacteria and / or Gram-negative bacteria.

9. The use according to claim 8, characterized in that The Gram-positive bacteria include Staphylococcus aureus and / or Enterococcus faecium; Preferably, the Gram-negative bacteria include one or more of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium; Preferably, the antibacterial product is a medicine or a daily chemical; Preferably, the daily chemical is a disinfectant or an additive; further preferably, the additive is a preservative, a detergent additive or a feed additive.

10. Use of the antimicrobial polypeptide according to claim 1 or the composition according to any one of claims 4 to 6 in the preparation of detergents or feed.

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