A polypeptide, composition and use thereof in the preparation of an antibacterial product

Abstract

The application belongs to the technical field of biological medicine, and relates to a polypeptide, a composition and application of the polypeptide and the composition in preparation of antibacterial products. The amino acid sequence is KWMIKWPRKWFTVL. Experiments show that the polypeptide has broad-spectrum antibacterial activity on gram-positive bacteria and / or gram-negative bacteria, and simultaneously has low cytotoxicity and low hemolyticity. The polypeptide provided by the application is composed of 14 amino acids, the polypeptide chain is short, the molecular weight is small, and the 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 relates to a polypeptide, a composition and application thereof in preparing antibacterial products. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be necessarily taken as an acknowledgement or any form of suggestion that this information forms part of the prior art already known to a person skilled in the art.

[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 the previous research, a new polypeptide is obtained through high-throughput screening. The research shows that the polypeptide has broad-spectrum antibacterial activity on gram-positive bacteria and / or gram-negative bacteria, and has low cytotoxicity and low hemolyticity.

[0005] Based on the above research results, the present application provides a polypeptide, a composition and application thereof in preparing antibacterial products. Specifically, the technical solutions are as follows:

[0006] In a first aspect, a polypeptide has an amino acid sequence of KWMIKWPRKWFTVL, as shown in SEQ ID NO. 1.

[0007] In some embodiments, in the amino acid sequence, K is the N-terminal amino acid and L is the C-terminal amino acid.

[0008] In some embodiments, the C-terminal is subjected to amidation treatment.

[0009] In a second aspect, a method for preparing the polypeptide of the first aspect of the present application comprises:

[0010] synthesizing the anti-angiogenic peptide by a chemical synthesis method; or

[0011] preparing the anti-angiogenic peptide by a genetic engineering method; or

[0012] preparing the anti-angiogenic peptide by a microbial fermentation method.

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

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

[0015] In a third aspect, a composition comprises the polypeptide or derivative thereof according to the first aspect of the present application.

[0016] 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, N-terminal of the polypeptide.

[0017] In some embodiments, the composition further comprises an excipient. The excipient can be a pharmaceutical excipient or a conventional daily chemical excipient. The pharmaceutical excipient includes a pharmaceutical carrier and / or excipient. The pharmaceutical carrier can be serum protein, polyalkyl cyanoacrylate, polylactic acid, etc. The excipient includes binder, filler, disintegrant, solubilizer, stabilizer, etc.

[0018] In a fourth aspect, the polypeptide according to the first aspect of the present application or the composition according to the third aspect of the present application is used for preparing an antibacterial product.

[0019] 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 at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium.

[0020] In some embodiments, the product is a pharmaceutical product or a daily chemical product. The pharmaceutical product is an internal use pharmaceutical product or an external use pharmaceutical product. The internal use pharmaceutical product is an oral preparation or an injection, and the external use pharmaceutical product includes a patch and a smearing preparation. The daily chemical product includes, but is not limited to, a disinfectant and an additive. The additive can be a preservative and a washing additive.

[0021] Since the disinfectant and the additive can be added to the detergent, in a fifth aspect, the polypeptide according to the first aspect of the present application or the composition according to the third aspect of the present application is used for preparing a washing product.

[0022] The present application has the following advantages:

[0023] (1) The polypeptide provided by the application has broad-spectrum antibacterial activity on gram-positive bacteria and / or gram-negative bacteria, and can be used as an antibacterial peptide, and the polypeptide has low cytotoxicity and low hemolyticity.

[0024] (2) The polypeptide provided by the application is composed of 14 amino acids, has a short peptide chain, small molecular weight, and 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 a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] The drawings constituting a part of the specification of the application are used to provide further understanding of the application, and the schematic embodiments of the application and the description thereof are used to explain the application, and do not constitute improper limitation on the application.

[0026] Figure 1 A mass spectrum detection diagram of the polypeptide QLX238 used in the application;

[0027] Figure 2 A hemolytic activity experiment result diagram of the polypeptide QLX238 in Example 2 of the application;

[0028] Figure 3 A cytotoxicity experiment result diagram of the polypeptide QLX238 in Example 3 of the application;

[0029] Figure 4 A treatment result diagram of the polypeptide QLX238 on bacterial infection of mouse skin in Example 4 of the application. DETAILED DESCRIPTION

[0030] It should be pointed out that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the application belongs.

[0031] In order to enable those skilled in the art to more clearly understand the technical solutions of the application, the technical solutions of the application will be described in detail below with specific examples.

[0032] In the following examples, the polypeptide QLX238 used has an amino acid sequence from N segment to C segment KWMIKWPRKWFTVL-NH2, as shown in SEQ ID NO. 1, and is prepared by Jiangsu Jinersu Biological Technology Co., Ltd. It is synthesized by using a polypeptide synthesizer through a solid-phase synthesis method, and the specific steps are as follows:

[0033] (1) The carboxyl group of Fmoc-lysine is connected to Wang resin (as a solid phase carrier) by a covalent bond, and N,N-dimethylformamide (DMF) is used to soak for about 15 min to remove impurities; the Fmoc protection on the resin is removed using DMF containing 20% piperidine (volume ratio of piperidine to DMF is 1:4) for 20 min, and the resin is washed until completely. The piperidine is washed away with DMF, and the remaining solid suspension is deprotected lysine-Wang.

[0034] (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 group of Fmoc-tryptophan to form a peptide bond, and then the Fmoc group is removed by deprotection. Repeat the above peptide bond formation reaction 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 above obtained polypeptide with the cleavage reagent at 20 ℃ for 2 h to cleave the polypeptide from the resin. Evaporate TFA on a rotary evaporator, and then add about 10 times the 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.

[0035] (3) The above crude polypeptide is dissolved in a 90% acetonitrile water solution (volume ratio of acetonitrile to water is 9:1), purified by preparative chromatography column, and the purity is detected by analytical chromatography column. The semi-preparative high performance liquid chromatograph is Waters DeltaPrep 4000, and the preparative chromatography column is Waters X-Bridge Cis, 5Hm reversed phase column. Eluent A is 0.1% TFA aqueous solution, and B is 0.1% TFA acetonitrile aqueous solution; detection wavelength is 220 nm, and elution mode is linear concentration gradient elution of 30%B~65%B, and flow rate is 30 mL / min. Fractions with purity higher than 95% are collected and freeze-dried to obtain polypeptide QLX238. The analytical high performance liquid chromatograph is Agilent 1100, and the analytical chromatography column is Sepax GP-C8 reversed phase column (4.6 mm x 150 mm, 5Hm), eluent A is 0.1% TFA aqueous solution, and B is 0.1% TFA acetonitrile aqueous solution; detection wavelength is 220 nm. Elution mode is linear concentration gradient elution of 50%B~75%B, and flow rate is 1.0 mL / min.

[0036] The mass spectrum of polypeptide QLX238 is as follows: Figure 1As shown, the molecular weight (1915.2) shown in the mass spectrum is consistent with the theoretical molecular weight (1915.41).

[0037] Example 1 Determination of antibacterial activity of polypeptide QLX238

[0038] The method recommended by the American Clinical Laboratory Standardization Institute (CLSI) for determining the minimum inhibitory concentration (MIC) was used, and a series of gradient polypeptide QLX238 solutions were prepared using a two-fold dilution method. The specific steps are as follows:

[0039] (1) Preparation of bacterial cells: Take the bacteria to be tested stored at -20°C, streak inoculate in NB medium and incubate. Pick a single colony and inoculate in 10 mL of NB medium, incubate at 37°C, 200 rpm overnight. Then inoculate the overnight bacterial cells in fresh medium and incubate for 1-2 h until the bacterial cells are in the logarithmic growth phase, with OD600=0.4, adjust the number of bacteria in the obtained bacterial solution to about 10 6 CFU / mL with MH(B).

[0040] (2) Preparation of peptides: Adjust the concentration of polypeptide QLX238 to 512 μg / mL, take 100 μL and add to the first column of the 96-well plate, add 50 μL of MH broth medium to the other wells, then suck out 50 μL of the polypeptide solution from the first well and add it to the second well, and so on, dilute by a factor of 2 to the tenth well, and discard 50 μL.

[0041] (3) Inoculation of bacteria: Take 50 μL of the bacterial solution adjusted in step (1) with a sample gun and add it to the first 11 columns of the 96-well plate, with a final bacterial concentration of 5 x 10 5 CFU / mL per well. Place the 96-well plate on a micro-vibrator and shake for 1 min to mix the liquid in each well, cover the micro-well plate to reduce evaporation during incubation, and incubate at 37°C for 18 h. Set the positive control group as the 11th well: add only 50 μL of MH broth medium and 50 μL of bacterial solution; the negative control is the 12th well, the sterile control group: only add 100 μL of MH broth medium. At this time, the concentration of the antibacterial brain will decrease from the first well to the tenth well.

[0042] (4) Result judgment: The sterile control well should remain clear throughout the entire test, indicating that the entire test was sterile. Compare the bacterial growth characteristics (such as the turbidity of the broth in the micro-wells, the appearance of sediment at the bottom of the wells, etc.) in the growth control well to determine the minimum concentration without visible growth, which is the MIC value of the peptide for the test bacteria.

[0043] The results are shown in Table 1, which shows that polypeptide QLX238 has a broad spectrum of ability to inhibit the growth of gram-negative and gram-positive bacteria.

[0044] Table 1 Minimum inhibitory concentration of polypeptide QLX238

[0045]

[0046] Note: GM stands for the geometric mean of the minimum inhibitory concentration of the gram-positive bacteria and the gram-negative bacteria listed in the table.

[0047] Example 2 Hemolytic activity of polypeptide QLX238

[0048] Determination of the hemolytic activity of polypeptide QLX238. The specific test steps are as follows:

[0049] (1) Collect 1 mL of fresh human blood using a heparin sodium anticoagulant tube, and store at 4°C for standby;

[0050] (2) Centrifuge 1000 g of the above blood for 5 min, discard the supernatant, and collect the red blood cells;

[0051] (3) Wash the collected red blood cells with PBS buffer solution three times, centrifuge at 1000 g for 5 min, discard the supernatant, collect the red blood cells, and finally resuspend the cells with about 10 mL of PBS buffer solution to obtain an 8% (V / V) red blood cell suspension for standby;

[0052] (4) Dilution of polypeptide QLX238: 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. Add 10 μL of polypeptide QLX238 stock solution to the first tube, then mix the polypeptide QLX238 solution in the first tube, aspirate 50 μL, and add it to the second tube. Repeat the dilution in the same manner to the tenth tube, and then discard 50 μL;

[0053] (5) Add 50 μL of the prepared red blood cell suspension to the EP tubes containing different concentrations of polypeptide QLX238 solution, and incubate in a 37°C incubator for 1 h. Add 50 μL of PBS and 50 μL of red blood cell suspension to the 11th well as a negative control, and add 50 μL of 0.1% Triton X-100 and 50 μL of red blood cell suspension to the 12th well as a positive control;

[0054] (6) After 1 h, remove the EP tubes and centrifuge at 100 g for 5 min at 4°C;

[0055] (7) Aspirate the supernatant of the centrifuged solution and transfer it in parallel to a clean 96-well plate, and measure the optical absorption value at 570 nm (OD 570nm ) using a microplate reader.

[0056] Results as shown in Figure 2 Figure 6, polypeptide QLX238 has only 20% hemolysis at 1024 μg / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that polypeptide QLX238 as an antibacterial peptide does not produce hemolysis when it exerts antibacterial effect.

[0057] Example 3 Cytotoxicity of polypeptide QLX238

[0058] Human hepatoma cells MHCC97H were used to detect the cytotoxicity of polypeptide QLX238. 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 polypeptide QLX238 was added to each well containing cells. 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:

[0059] Cell survival rate = (experimental group - blank control) / (negative control group - blank control) x 100%

[0060] As shown in Figure 3 Figure 7, the concentration of polypeptide QLX238 exerting 50% cytotoxicity is above 256 μg / mL, which is much higher than the geometric mean of its minimum antibacterial concentration, indicating that polypeptide QLX238 as an antibacterial peptide does not produce cytotoxicity when it exerts antibacterial effect.

[0061] Example 5

[0062] For the determination of the skin external use anti-infection ability of polypeptide QLX238, the specific test steps are as follows:

[0063] G+ Staphylococcus aureus CMCC26003 bacteria 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 QLX238 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 bacteria solution with a concentration of 10 8 CFU / mL was injected on the wound of each mouse. After 6 hours, the treatment group was treated with different doses of polypeptide QLX238. At 24 hours after modeling, the skin at the infection site of the animals was taken aseptically, and the viable bacteria were detected and counted.

[0064] Results as shown in Figure 4 Figure 8, polypeptide QLX238 as an antibacterial peptide has already shown the ability to clear bacterial infection of mouse skin at a low dose of 0.0375 mg.

[0065] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.

Claims

1. A polypeptide, characterized in that, KWMIKWPRKWFTVL-NH2.

2. A method of producing the polypeptide of claim 1, comprising, The polypeptide is synthesized by a chemical synthesis method.

3. The method of claim 2, wherein, The chemical synthesis method is a solid phase synthesis method.

4. A composition characterized in that, The polypeptide of claim 1.

5. The composition of claim 4, wherein The composition further comprises an excipient.

6. Use of the polypeptide of claim 1 or the composition of any one of claims 4-5 in the preparation of an antibacterial product. The antibacterial product is directed against Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus and / or Enterococcus faecium; the Gram-negative bacteria are at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium; The product is a pharmaceutical product or a daily chemical product; the daily chemical product is a disinfectant or an additive; the additive is a preservative or a washing additive.

7. Use of the polypeptide of claim 1 or the composition of any one of claims 4-5 in the preparation of a washing product.

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