Antibacterial polypeptides, compositions, and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to an antibacterial polypeptide, a composition and application thereof. The amino acid sequence of the antibacterial polypeptide is shown in SEQ ID NO. 1: KWKIKWPVKWFKM-NH2. Experiments show that the antibacterial polypeptide has broad-spectrum antibacterial activity on gram-positive bacteria and / or gram-negative bacteria, and simultaneously has low cytotoxicity and low hemolyticity. The antibacterial polypeptide provided in the application is composed of 14 amino acids, the polypeptide chain is short, the molecular weight is small, and the antibacterial polypeptide is easy to be artificially synthesized, is a small-molecule polypeptide with high application value, and can be used for preparing a novel antibacterial agent, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an antibacterial polypeptide, a composition and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art with respect to the present application.

[0003] Antimicrobial peptides are a kind of active biological molecules with endogenous immune response in most organisms, and are a kind of polypeptides with biological activities such as antibacterial, antiviral or tumor inhibition. Since the antibacterial and bactericidal mechanism of antimicrobial peptides is different from that of antibiotics, and the probability of bacterial production is low when used as a drug, and antimicrobial peptides have broad-spectrum antibacterial effect, antimicrobial peptides are considered to be a new generation of antibacterial agents, which can effectively alleviate the threat of bacteria. SUMMARY

[0004] In order to solve the problems of the prior art, the present application aims to provide an antibacterial polypeptide, a composition and application thereof. The present application obtains a new polypeptide through high-throughput screening, and researches show that the polypeptide has broad-spectrum antibacterial activity against gram-positive bacteria and / or gram-negative bacteria, and also has low cytotoxicity and low hemolyticity.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] In the first aspect, the present application provides an antibacterial polypeptide, and 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 the N-terminal amino acid, and L is the C-terminal amino acid. The -NH2 connected to the C-terminal is obtained by carboxyl amidation modification.

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

[0010] The antibacterial polypeptide is prepared by a chemical synthesis method;

[0011] Or, the antibacterial polypeptide is prepared by a genetic engineering method;

[0012] Or, the antibacterial polypeptide is prepared by a microbial fermentation method.

[0013] Since the antibacterial polypeptide provided by the present application is only composed of 14 amino acids, it is easy to be obtained by chemical synthesis method, and the efficiency of obtaining the antibacterial polypeptide by chemical synthesis method is higher, therefore, the antibacterial polypeptide is preferably synthesized by chemical synthesis method.

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

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

[0016] The carboxyl group of Fmoc-lysine is connected to the resin, impurities are removed, and the Fmoc protection on the resin is removed to obtain a deprotected lysine-resin;

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

[0018] The polypeptide is cut from the resin by using a cleavage reagent (the cleavage reagent comprises trifluoroacetic acid (TFA), triisopropylchlorosilane (TIS) and water, which are mixed and configured in a volume ratio of (94-96):(2-3):(2-3)), the cleavage reagent components are removed, and the crude polypeptide is precipitated and dried to obtain a crude polypeptide;

[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 application provides a composition comprising the above-mentioned antibacterial polypeptide or derivative thereof.

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

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

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

[0024] In some embodiments, the bacteria against which the antibacterial product is directed are gram-positive bacteria and / or gram-negative bacteria. The gram-positive bacteria include Staphylococcus aureus and / or Enterococcus faecium, and 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, against Enterococcus faecium is 4 μg / mL, against Escherichia coli is 4 μg / mL, against Klebsiella pneumoniae is 4 μg / mL, against Acinetobacter baumannii is 4 μg / mL, against Salmonella typhimurium is 4 μg / mL, and against Pseudomonas aeruginosa is 4 μg / mL.

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

[0027] Since the disinfectant and the additive can be added to the detergent, in a fifth aspect, the present application provides a use of the antibacterial polypeptide or the composition in the preparation of a detergent.

[0028] The present application has the following beneficial effects:

[0029] (1) The polypeptide provided by the present application has broad-spectrum antibacterial activity against gram-positive bacteria and / or gram-negative bacteria, and can be used as an antibacterial peptide. In addition, the polypeptide has low cytotoxicity and low hemolytic activity.

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

[0031] The drawings constituting a part of the specification of the present application are used to provide a further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application.

[0032] Figure 1 Mass spectrum detection diagram of the antibacterial polypeptide R2 used in the present application;

[0033] Figure 2Figure of the hemolysis activity test result of the polypeptide R2 in the embodiment 2 of the present application;

[0034] Figure 3 Figure of the cytotoxicity test result of the polypeptide R2 in the embodiment 3 of the present application;

[0035] Figure 4 Figure of the treatment result of the polypeptide R2 on the bacterial infection of the mouse skin in the embodiment 4 of the present application. DETAILED DESCRIPTION

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

[0037] In the following embodiments, the adopted antibacterial polypeptide R2 has the amino acid sequence from N segment to C segment as KWKIKWPVKWFKML-NH2, which is shown in SEQ ID NO. 1, and is prepared by Jiangsu Jinerswai Biological Technology Co., Ltd. by solid-phase synthesis method using a polypeptide synthesizer. The specific steps are as follows:

[0038] (1) The carboxyl of Fmoc-lysine is connected to Wang resin (as a solid-phase carrier) by a covalent bond structure, and N,N-dimethylformamide (DMF) is used to soak for about 15 min to remove impurities; Fmoc protection on the resin is removed using DMF containing 20% piperidine (the volume ratio of piperidine to DMF is 1:4), and the reaction is carried out for 20 min, and the resin is washed until completely. The remaining solid suspension of deprotected lysine-Wang is washed with DMF.

[0039] (2) Fmoc-tryptophan is synthesized by the same synthesis method, and the amino group of the deprotected lysine-Wang in (1) is condensed with the carboxyl of Fmoc-tryptophan to form a peptide bond, and then the Fmoc group is removed by deprotection, and the above-mentioned peptide bond formation reaction is 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 is deprotected, wash with DMF for 8 times, and then cross-wash with ethanol and dichloromethane (DCM) for 8 times. Mix trifluoroacetic acid (TFA), triisopropylsilyl chloride (TIS) and water in a volume ratio of 95:2.5:2.5 to prepare a cleavage reagent, and react the polypeptide obtained above with the cleavage reagent at 20 ℃ for 2 h to cleave the polypeptide from the resin. Evaporate TFA by rotary evaporator, and then add about 10 times volume of pre-cooled anhydrous ether to precipitate the polypeptide for 3 h to obtain white powder solid. Vacuum drying to obtain the crude polypeptide.

[0040] (3) The above crude polypeptide was dissolved in 90% acetonitrile water (volume ratio of acetonitrile to water was 9:1), purified by preparative chromatography column, and the purity was detected by analytical chromatography column. The semi-preparative high performance liquid chromatography was Waters DeltaPrep 4000, and the preparative chromatography column was Waters X-Bridge Cis, 5Hm reversed phase column. The eluent A was 0.1% TFA aqueous solution, and the B was 0.1% TFA acetonitrile aqueous solution; the detection wavelength was 220 nm, the elution mode was linear concentration gradient elution of 30%B~65%B, and the flow rate was 30 mL / min. The fraction with purity higher than 95% was collected and freeze-dried to obtain the polypeptide, which was then subjected to amidation treatment to obtain the antibacterial polypeptide R2. The analytical high performance liquid chromatography was Agilent 1100, and the analytical chromatography column was Sepax GP-C8 reversed phase column (4.6 mm x 150 mm, 5Hm). The eluent A was 0.1% TFA aqueous solution, and the B was 0.1% TFA acetonitrile aqueous solution; the detection wavelength was 220 nm. The elution mode was linear concentration gradient elution of 50%B~75%B, and the flow rate was 1.0 mL / min.

[0041] The mass spectrum of the polypeptide R2 is shown in Figure 1 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 antibacterial polypeptide R2

[0043] The method recommended by the American Clinical Laboratory Standardization Institute (CLSI) for determining the minimum inhibitory concentration (MIC) was used, and the polypeptide R2 solution was prepared in series by the double dilution method. The specific steps are as follows:

[0044] (1) Preparation of bacterial cells: the bacteria to be tested stored at -20°C were streaked and inoculated in NB medium for incubation. Single colonies were picked and inoculated in 10 mL NB medium, and incubated at 37°C, 200 rpm overnight. Then the overnight bacterial cells were inoculated in fresh culture medium and cultured for 1-2 h until the bacterial cells were in the logarithmic growth phase, and the OD600 was adjusted to about 10 6 CFU / mL.

[0045] (2) Preparation of peptides: the concentration of polypeptide R2 was adjusted to 512 μg / mL, 100 μL of which was added to the first column of the 96-well plate, and 50 μL of MH broth medium was added to the other wells. Then 50 μL of polypeptide solution in the first well was removed and added to the second well, and the same was repeated for the other wells, and 50 μL was removed and discarded.

[0046] (3) Inoculation: 50 μL of the bacteria solution adjusted in step (1) was added into the first 11 wells of the 96-well plate using a sample gun, and the final concentration of the bacteria was 5 x 105CFU / mL per well. The 96-well plate was shaken for 1 min on a microshaker to mix the liquid in each well, and the microplate was covered to reduce evaporation during incubation. The plate was incubated at 37 °C for 18 h. The 11th well was set as a positive control, i.e., 50 μL of MH broth medium and 50 μL of the bacteria solution were added; the 12th well was set as a negative control, i.e., 100 μL of MH broth medium was added. The concentration of the antibacterial peptide in the first 10 wells was decreased successively. 5 CFU / mL. The 96-well plate was shaken for 1 min on a microshaker to mix the liquid in each well, and the microplate was covered to reduce evaporation during incubation, and incubated at 37 °C for 18 h. The 11th well was set as a positive control, i.e., 50 μL of MH broth medium and 50 μL of the bacteria solution were added; the 12th well was set as a negative control, i.e., 100 μL of MH broth medium was added. The concentration of the antibacterial peptide in the first 10 wells was decreased successively.

[0047] (4) Result judgment: The sterile control well should remain clear throughout the entire test, indicating that the entire test was aseptic. The minimum concentration without visible growth was determined as the MIC of the peptide against the test bacteria by comparing the growth characteristics (e.g., turbidity of the broth in the micro-well, precipitation at the bottom of the well, etc.) with those of the growth control well.

[0048] The results are shown in Table 1. It can be seen that the antibacterial peptide R2 has a broad spectrum of inhibitory activity against the growth of gram-negative and gram-positive bacteria. Specifically, the minimum inhibitory concentration of the antibacterial peptide R2 against S. aureus was 4 μg / mL, against E. faecium was 4 μg / mL, against E. coli was 4 μg / mL, against K. pneumoniae was 4 μg / mL, against A. baumannii was 4 μg / mL, against S. typhimurium was 4 μg / mL, and against P. aeruginosa was 4 μg / mL.

[0049] In addition, the concentration of the antibacterial peptide R2 for complete killing of S. aureus, E. faecium, E. coli, K. pneumoniae, A. baumannii, S. typhimurium, and P. aeruginosa was ≥ 4, 8, 4, 4, 4, 4, and 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 the gram-positive and gram-negative bacteria listed in the table.

[0053] Example 2 Hemolytic activity of antibacterial peptide R2

[0054] For the determination of the hemolytic activity of the antibacterial peptide R2, the following specific test steps were used:

[0055] (1) Collect 1 mL fresh human blood with heparin sodium anticoagulation tube, and store at 4 °C for standby;

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

[0057] (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 8% (V / V) red blood cell suspension for standby;

[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 rest of the tubes. Then add 10 μL of polypeptide R2 stock solution to the first tube, mix the polypeptide R2 solution in the first tube, and aspirate 50 μL, add it to the second tube, and sequentially dilute by a factor of two to the tenth tube, and then aspirate 50 μL and discard it;

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

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

[0061] (7) Aspirate the supernatant of the above centrifuged solution and transfer it in parallel to a clean 96-well plate corresponding to the well, and measure the optical absorption value at 570 nm (OD 570nm ) with a microplate reader.

[0062] The results are shown in Figure 2 , and the antibacterial polypeptide R2 has only 12% hemolysis at 256 μg / mL, which is much higher than the geometric mean of the minimum antibacterial concentration, indicating that the polypeptide R2 as an antibacterial peptide will not produce hemolysis when exerting antibacterial effect.

[0063] Example 3 Cytotoxicity of antibacterial polypeptide R2

[0064] Human hepatocarcinoma cell line MHCC97H was used to detect the cytotoxicity of polypeptide R2. Cells were seeded in a 96-well plate at 8000 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 cell-containing well, with a final concentration of 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, respectively. After 24 hours of incubation, 10 μL of 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:

[0065] Cell survival rate = (experimental group - blank control) / (negative control group - blank control) x 100%

[0066] As shown in Figure 3 , the concentration of antibacterial polypeptide R2 that exerts 50% cytotoxicity is above 128 μg / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that antibacterial polypeptide R2 as an antibacterial peptide does not produce cytotoxicity when exerting antibacterial effect.

[0067] Example 5

[0068] For the determination of the skin external anti-infection ability of antibacterial polypeptide R2, the specific test steps are as follows:

[0069] G+ Staphylococcus aureus CMCC26003 bacterial solution was diluted to 10 8 CFU / mL for standby. Female C57BL / 6 mice were randomly divided by weight into infection negative control group and polypeptide R2 treatment group, 5 in each group. After the mice were depilated on the back, a full-thickness skin with a diameter of 5 mm was taken from the center of the back of the mice with a tissue biopsy instrument, and the wound area of each group of mice was uniform, i.e. the model was successfully constructed. 5 μL of bacterial solution with a concentration of 10 8 CFU / mL was injected onto the wound of each mouse. After 6 hours, the treatment group was treated with 0.1 mg of polypeptide R2. At 24 hours after modeling, the skin at the infection site of the animals was taken aseptically, and viable bacteria were detected and counted.

[0070] As shown in Figure 4 , antibacterial polypeptide R2 as an antibacterial peptide has obvious clearing ability against bacterial infection of mouse skin at 0.1 mg.

[0071] 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 principles of the present application shall be included in the protection scope of the present application.

Claims

1. An antimicrobial polypeptide, characterized in that, The amino acid sequence of the antimicrobial polypeptide is shown in SEQ ID NO.1, and the carboxyl group at the C-terminus of the sequence is modified by -NH2 amidation.

2. A method for preparing the antimicrobial polypeptide of claim 1, characterized in that, Antimicrobial peptides were prepared by chemical synthesis.

3. The method as described in claim 2, characterized in that, The chemical synthesis method is a solid-phase synthesis method.

4. A composition, characterized in that, Includes the antimicrobial polypeptide as described in claim 1.

5. The composition according to claim 4, characterized in that, It also includes excipients, including pharmaceutical excipients or excipients for daily chemical products.

6. The use of the antimicrobial polypeptide of claim 1 or the composition of any one of claims 4 to 5 in the preparation of antimicrobial products; The antibacterial products target Gram-positive and / or Gram-negative bacteria. The Gram-positive bacteria are Staphylococcus aureus and / or Enterococcus faecalis; The Gram-negative bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella typhimurium. The antibacterial products are pharmaceuticals or daily chemical products; The daily chemical products mentioned are disinfectants or additives; the additives are preservatives and detergent additives.

7. The use of the antimicrobial polypeptide of claim 1 or the composition of any one of claims 4 to 5 in the preparation of detergents.

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