Antibacterial polypeptides, methods of making and using the same

Abstract

The application discloses an antibacterial polypeptide and a preparation method and application thereof, and the antibacterial polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:1-2. The antibacterial polypeptide prepared by the scheme of the application has good inhibiting and sterilizing effects on pseudomonas aeruginosa and the fungus candida albicans. Meanwhile, the antibacterial polypeptide L18N6P obtained through design has no toxic side effect on cells, can even promote the proliferation of human dermal fibroblasts, has good biocompatibility, has the potential to become a drug for treating diseases caused by pseudomonas aeruginosa or candida albicans infection or to prepare antibacterial medical device materials, is not prone to drug resistance, and has high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an antibacterial polypeptide and a preparation method and application thereof. Background Art

[0002] Overuse of antibiotics has led to the emergence of numerous stubborn, multidrug-resistant bacteria, complicating treatment. Antimicrobial peptides, also known as host defense peptides, have garnered increasing attention from scientists worldwide in recent years and are considered a promising new generation of antimicrobial drugs. Compared to traditional antibiotics, most pathogens exhibit little resistance to antimicrobial peptides. However, the antimicrobial activity of most natural antimicrobial peptides is low, and some are toxic to eukaryotic organisms. Therefore, research on modifying antimicrobial peptides to enhance their antimicrobial properties and reduce their toxicity has become a hot topic and a growing trend. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a polypeptide having good antibacterial activity against Pseudomonas aeruginosa and Candida albicans.

[0004] The present invention also provides a method for preparing the polypeptide.

[0005] The present invention also provides the application of the polypeptide.

[0006] The invention also provides a medicine.

[0007] According to one aspect of the present invention, a polypeptide is provided, wherein the polypeptide is selected from any one of the following (1)-(4):

[0008] (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

[0009] (2) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2;

[0010] (3) A polypeptide having the same function as the polypeptide shown in (1) obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 at the N-terminus and the C-terminus;

[0011] (4) A polypeptide having the same function as the polypeptide shown in (2) obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 at the N-terminus and C-terminus.

[0012] The polypeptide of the present invention changes or replaces the positions of amino acid residues on the spiral wheel projection diagram so that basic amino acid residues are distributed on one side and hydrophobic amino acids are distributed on the other side. At the same time, tryptophan is added to increase the hydrophobic force of the bactericidal effect, arginine provides a positive charge targeting the bacterial membrane, and the targeting effect of the polypeptide on Candida albicans is improved through the synergistic effect of tryptophan and lysine, and proline is used to enhance biological selectivity, and finally the polypeptide sequence of the present application is determined.

[0013] In some embodiments of the present invention, the antimicrobial peptide has a three-dimensional structure with a central helical structure and two coiled ends.

[0014] According to a second aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of 1) to 4):

[0015] 1) A nucleic acid molecule encoding the above polypeptide;

[0016] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0017] 3) a recombinant vector containing 1) the nucleic acid molecule or 2) the expression cassette;

[0018] 4) A recombinant cell containing 1) the nucleic acid molecule, 2) the expression cassette or 3) the recombinant vector.

[0019] The second aspect of the present invention provides a method for preparing the above-mentioned polypeptide, which comprises the following steps: using a solid-phase synthetic resin as a starting material and adopting a solid-phase synthesis method to prepare the polypeptide.

[0020] In some embodiments of the present invention, the antimicrobial peptide can also be artificially synthesized.

[0021] In some embodiments of the present invention, the preparation of the antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus.

[0022] In some embodiments of the present invention, the solid phase synthesis resin comprises Rink resin.

[0023] In some embodiments of the present invention, the method further comprises soaking the solid-phase synthetic resin in a DMF solution to swell the resin.

[0024] In some embodiments of the present invention, the solid phase synthetic resin is immersed in the DMF solution for 30-50 minutes.

[0025] In some embodiments of the present invention, the solid-phase synthesis method includes attaching an amino acid containing a protecting group to a resin, and sequentially performing the steps of washing, deprotection, and coupling until the last amino acid is grafted to obtain a polypeptide chain; the polypeptide chain is sequentially deprotected and washed, and then cut to obtain the polypeptide.

[0026] In some embodiments of the present invention, the protecting group comprises Fmoc-.

[0027] In some embodiments of the present invention, an amino acid containing a protecting group is attached to a resin, and the activating agents used include 0.3-0.5 M HBTU and 1-3 M DIEA.

[0028] In some embodiments of the present invention, the reagent used for flushing includes DMF.

[0029] In some embodiments of the present invention, the deprotection comprises the steps of adding a piperidine deprotection solution with a mass fraction of 15% to 25%, removing the liquid phase after the reaction, and washing the resin.

[0030] In some embodiments of the present invention, the reagent used to clean the resin includes DMF.

[0031] In some embodiments of the present invention, the coupling comprises the steps of dissolving the deprotected resin and then adding HBTU and an amino acid mixture to react.

[0032] In some embodiments of the present invention, the concentration of HBTU is 0.3-0.5M.

[0033] In some embodiments of the present invention, the preparation of the amino acid mixture comprises: introducing nitrogen into the amino acid solution to activate the amino acid solution, and then adding a DIEA solution with a concentration of 1-3 M to react.

[0034] In some embodiments of the present invention, the washing liquid used for washing includes at least one of DMF, methanol and dichloromethane.

[0035] In some embodiments of the present invention, the cutting uses a cutting fluid.

[0036] In some embodiments of the present invention, the cutting fluid comprises the following components: TFA, Tis, EDT, thioanisole, and phenol.

[0037] In some embodiments of the present invention, the method further comprises washing the polypeptide with ether.

[0038] In some embodiments of the present invention, the temperature of the diethyl ether is 0-8°C.

[0039] According to a fourth aspect of the present invention, the use of the above-mentioned polypeptide or biomaterial in the preparation of an antibacterial product is proposed.

[0040] In some embodiments of the present invention, the bacteria include bacteria and fungi.

[0041] In some embodiments of the invention, the bacteria include Gram-negative bacteria.

[0042] In some embodiments of the present invention, the Gram-negative bacteria include Pseudomonas aeruginosa.

[0043] In some embodiments of the invention, the fungus comprises Candida albicans.

[0044] In some embodiments of the present invention, the product comprises a medicament or a formulation.

[0045] According to a fifth aspect of the present invention, a drug is provided, comprising the above-mentioned polypeptide.

[0046] In some embodiments of the present invention, the drug has an antibacterial effect.

[0047] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0048] In some embodiments of the present invention, the pharmaceutical carrier is a conventional pharmaceutical carrier in the pharmaceutical field. In some embodiments of the present invention, the pharmaceutical carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0049] In some embodiments of the invention, the excipient comprises water.

[0050] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0051] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0052] In some embodiments of the invention, the humectant comprises glycerin.

[0053] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0054] In some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0055] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0056] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0057] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0058] In some embodiments of the present invention, the mass fraction of the polypeptide in the drug is 0.01% to 99%.

[0059] According to some preferred embodiments of the present invention, the mass fraction of the polypeptide in the drug is 0.05% to 95%.

[0060] According to some preferred embodiments of the present invention, the mass fraction of the polypeptide in the drug is 0.05% to 20%.

[0061] In some embodiments of the present invention, the dosage form of the drug is various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections or infusions, transdermal agents, and transdermal microneedles.

[0062] In some embodiments of the present invention, the drug may be administered by conventional methods in the art, including but not limited to injection or oral administration.

[0063] The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0064] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the antimicrobial polypeptide prepared according to the present invention, through the rational allocation of basic amino acids, hydrophobic amino acids, tryptophan, and other sites, exhibits excellent inhibitory and bactericidal effects against both Pseudomonas aeruginosa and the fungus Candida albicans. Furthermore, the designed antimicrobial polypeptide L18N6P exhibits no toxic side effects on cells and can even promote the proliferation of human dermal fibroblasts. It has excellent biocompatibility and has the potential to be used as a drug for treating Pseudomonas aeruginosa or Candida albicans infections or as a material for the preparation of antimicrobial medical devices. It is also less likely to develop drug resistance and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0066] Figure 1 : is a helical wheel projection diagram of the polypeptide L18N5 in Example 1 of the present invention;

[0067] Figure 2 A helical wheel projection diagram of L18N6P in Example 1 of the present application;

[0068] Figure 3 A three-dimensional structure diagram of the polypeptide L18N5 in Example 1 of the present application;

[0069] Figure 4 A three-dimensional structure diagram of the polypeptide L18N6P in Example 1 of the present application;

[0070] Figure 5 A reversed-phase high performance liquid chromatography detection result diagram of the polypeptide L18N5 in Example 1 of the present application;

[0071] Figure 6 A reversed-phase high performance liquid chromatography detection result diagram of the polypeptide L18N6P in Example 1 of the present application;

[0072] Figure 7 A result diagram of the influence of different polypeptides on cell activity in Example 1 of the present application;

[0073] Figure 8 A cell morphology diagram observed after staining of live and dead cells in Example 1 of the present application, wherein the scale is 100 μm. DETAILED DESCRIPTION

[0074] The concept and technical effects of the present application will be described below in combination with examples for a clear and complete understanding of the purpose, features and effects of the present application. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0075] Example 1

[0076] In this example, an antibacterial polypeptide is prepared, and the specific process is as follows:

[0077] 1. Design of antibacterial peptide

[0078] By selecting hydrophobic amino acids (leucine, alanine, phenylalanine), basic amino acids (arginine, lysine) and adjusting and replacing specific amino acids on the polypeptide helical wheel, the hydrophobicity of the polypeptide is adjusted so that the hydrophobic amino acids are distributed on one side and the basic amino acids are distributed on the other side of the final helical wheel. The distribution sites of arginine, tryptophan and lysine are reasonably arranged, and the polypeptide L18N5 is designed. By replacing Gly 1 , Gly 4 with Leu 1 , Val 4 , and replacing Arg 2、Arg 3 、Arg 17 Replaced with Lys 2 、Lys 3 、Lys 17 , Ala 18 Replaced with Pro 18 , the polypeptide L18N6P was obtained. The sequences and physicochemical parameters of the two polypeptides are shown in Table 1.

[0079] Table 1 Sequences and physicochemical parameters of antimicrobial peptides

[0080]

[0081] a The average hydrophobicity value was calculated by HeliQuest;

[0082] b The hydrophobic moment was calculated by HeliQuest analysis.

[0083] HeliQuest was used to predict the performance and helical wheel projection of the peptides. The helical wheel projections of peptides L18N5 and L18N6P were shown in Figure 1. Figure 1 、 2 As shown, lysine and arginine residues are distributed on the same side of the helical wheel projection, and other amino acids are distributed on the other side.

[0084] The three-dimensional conformation of the peptide was predicted using the online structure prediction software I-TASSER. The three-dimensional structures of peptides L18N5 and L18N6P are shown in Figure 2. Figure 3 、 4 As shown, it can be seen that both polypeptides have a three-dimensional structure with a helical middle and loops at both ends.

[0085] 2. Synthesis of antimicrobial peptides by solid-phase chemical synthesis

[0086] The preparation of antimicrobial peptides is carried out one by one from C-terminus to N-terminus, and is completed by a peptide synthesizer. The first step is to swell the resin. The Rink resin is soaked in a DMF solution and shaken at a constant speed for 40 minutes to fully swell the resin. Then the first Fmoc-protected amino acid is connected to the resin, and 0.4 moles of HBTU and 2 moles of DIEA are used as activators. The first amino acid is connected, and then the resin is rinsed six times (using 15mL DMF for rinsing), deprotected (15mL of 20% piperidine deprotection solution is added to the reaction bottle, and the waste liquid is discharged after 40 minutes of reaction, and the resin is washed six times with 15mL DMF), coupled (0.51g of Fmoc-Gly-OH is dissolved in 5mL of DMF, 4.3mL of 0.4M HBTU is added thereto, and nitrogen is introduced for 30min to fully mix and activate the amino acid; 1.71mL of the amino acid solution is added After reacting with a 2M DIEA solution through nitrogen for 5 minutes, the amino acid mixture was added to the reaction flask, shaken, and reacted with nitrogen for 70 minutes. For each subsequent amino acid grafted, the resin was rinsed six times, followed by deprotection and coupling, until the last amino acid was grafted. Finally, deprotection was performed and the resin was repeatedly washed with DMF, methanol, and dichloromethane (15 mL of DMF, methanol, and dichloromethane were used each time, and each wash lasted 3 minutes). The peptide was then cleaved with a cutting solution (containing 16.3 mL of TFA, 0.38 mL of Tis, 0.38 mL of EDT, 1.38 mL of thioanisole, 1.46 g of phenol, and 1.46 mL of deionized water), and the product was repeatedly precipitated and washed with cold ether to obtain the initial product.

[0087] The crude product was analyzed and purified using reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column. A 5 μm analytical column and a 10 μm preparative column were used to obtain the purified peptides L18N5 and L18N6P. Two mobile phases were used: phase A contained 99.9% deionized water and 0.1% trifluoroacetic acid, and phase B contained 99.9% acetonitrile (CH3CN) and 0.1% trifluoroacetic acid.

[0088] The reverse phase high performance liquid chromatography of peptides L18N5 and L18N6P were as follows: Figure 5 、 6 The results of reverse phase high performance liquid chromatography showed that the purity of these synthetic peptides was greater than 97%.

[0089] Example 2

[0090] This example measured the antibacterial activity of the antibacterial polypeptide L18N6P prepared in Example 1. The specific process was as follows:

[0091] Determination of minimum inhibitory concentration (MIC):

[0092] Pseudomonas aeruginosa was cultured using LB-Broth and LB agar plates, and Candida albicans was cultured using YPD Broth and YPD agar plates. The polypeptide was dissolved in deionized water and then transferred to LB-broth or YPD Broth to obtain a polypeptide stock solution. The polypeptide was then serially diluted in LB-broth / YPD Broth (final concentrations were 6667μg / mL, 3333μg / mL, 1667μg / mL, 833μg / mL, 417μg / mL, 208μg / mL, 104μg / mL, 52μg / mL, 26μg / mL and 13μg / mL, respectively). LB-broth / YPD Broth without polypeptide was prepared as a control. The concentration of the microbial suspension was adjusted to 1-5×10 6 CFU / mL. Add 100 μL of the microbial suspension to 11 tubes of peptide at varying concentrations. Incubate at 37°C for 24 hours. Visually inspect the tube with the lowest drug concentration to determine if the tube shows no bacterial growth and is clear. This represents the MIC for the test bacteria.

[0093] Determination of minimum bactericidal concentration (MBC):

[0094] After the minimum inhibitory concentration was determined and observed with the naked eye, 0.1 mL of liquid was taken from each dilution tube with no bacterial growth and added to a 90 mm LB-agar / YPD agar plate. The plate was evenly spread with a sterile glass rod. 5 Then, 100 μL of the control bacterial solution was plated on an agar plate as in the experimental group. After incubation at 37°C for 24 hours, the number of colonies on the plate was observed. The lowest drug concentration corresponding to the plate with no more than 5 colonies was the minimum bactericidal concentration of MBC.

[0095] Table 2 Test results of minimum inhibitory concentration and minimum bactericidal concentration of polypeptides

[0096]

[0097] Table 2 shows the peptide MIC and MBC test results. As can be seen from Table 2, the designed peptides L18N5 and L18N6P exhibited significant inhibitory activity against both representative bacterial species. For Pseudomonas aeruginosa, L18N5 achieved a minimum inhibitory concentration of 52 μg / mL and a minimum bactericidal concentration of 104 μg / mL; while L18N6P achieved both a minimum inhibitory concentration and a minimum bactericidal concentration of 104 μg / mL. They also demonstrated good inhibitory and bactericidal activity against the more resistant fungus Candida albicans.

[0098] Due to the differences in the MIC and MBC testing methods, the sterilization rate corresponding to the lowest bactericidal concentration of the peptide for each bacterial species in this result is much higher than 99.99%.

[0099] Example 3

[0100] This example determines the cytotoxicity of the antibacterial polypeptide L18N6P prepared in Example 1, and the procedure is as follows:

[0101] Human dermal fibroblasts are selected as the object for testing the cytotoxicity of the polypeptide, i.e. when the cells grow to about 80%, the culture medium is replaced with a culture medium containing 1 minimum bactericidal concentration of polypeptide. After incubation at 37°C for 24 hours, the cell growth is observed.

[0102] Cell activity test:

[0103] The Alamar Blue method is used to test the cell activity, i.e. after 24 hours, the activity of the cells in each group is tested using the Alamar Blue reagent. The addition ratio of Alamar Blue to the culture medium is 1:10. After incubation in a 37°C incubator for 4 hours, the absorbance of the culture medium in each well is tested using an enzyme-labeled instrument. The complete culture medium without polypeptide solution is the control group (Control), and the 10% DMSO solution is the negative control group (DMSO).

[0104] Figure 7 The results of the Alamar Blue method for testing the effect of different polypeptides on cell activity show that the fluorescence value of the cells cultured with the polypeptide L18N6P solution is the highest, even exceeding that of the complete culture medium control group. The fluorescence value of the cells cultured with the polypeptide L18N5 solution is slightly lower than that of the complete culture medium control group, but is significantly higher than that of the cells cultured with the DMSO solution.

[0105] Cell morphology observation:

[0106] After the Alamar Blue test, the cells are treated with a live / dead staining kit according to the experimental protocol of the Invitrogen company. The cells are rinsed with HBSS, and then stained with a live / dead reagent at room temperature for 15 minutes. After rinsing with HBSS, the cells are fixed with 4% glutaraldehyde / HBSS for 1 hour, and finally the cell growth and morphological changes in each group are observed under a laser confocal scanning electron microscope.

[0107] The results of the cell morphology observation are shown in Figure 8 As can be seen from the figure, the cell morphology after culture with the two polypeptide solutions is almost the same as that of the complete culture medium control group, and the cell morphology is normal. However, the cell proliferation state treated with the polypeptide L18N6P solution is better, and the number of cells treated with the polypeptide L18N5 solution is slightly less than that of the complete culture medium control group, which proves that the polypeptide L18N6P of the present application is completely non-toxic to fibroblasts, and can even promote cell growth.

[0108] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A polypeptide, characterized in that The polypeptide is selected from any one of the following (1)-(2): (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (2) A polypeptide having the amino acid sequence shown in SEQ ID NO:

2.

2. Biomaterial, which is any one of 1) to 4): 1) A nucleic acid molecule encoding the polypeptide according to claim 1; 2) an expression cassette containing the nucleic acid molecule described in 1); 3) a recombinant vector containing 1) the nucleic acid molecule or 2) the expression cassette; 4) A recombinant cell containing 1) the nucleic acid molecule, 2) the expression cassette or 3) the recombinant vector.

3. A method for preparing the polypeptide according to claim 1, characterized in that: The preparation method comprises the following steps: using solid phase synthetic resin as a starting material and adopting a solid phase synthesis method to prepare the polypeptide.

4. Use of the polypeptide according to claim 1 or the biomaterial according to claim 2 in the preparation of an antibacterial product; The bacteria are Pseudomonas aeruginosa and Candida albicans.

5. The use according to claim 4, characterized in that The product includes a drug or a preparation.

6. A drug, characterized in that The drug comprises the polypeptide according to claim 1; and a pharmaceutically acceptable carrier.

7. The drug according to claim 6, characterized in that The dosage form of the drug includes solid, semi-solid or liquid form; And / or, the administration of the drug includes injection or oral administration.

8. The drug according to claim 7, characterized in that The liquid is in the form of an aqueous solution, a non-aqueous solution or a suspension.

9. The drug according to claim 7, characterized in that The dosage form is tablet, capsule, granule, pill, oral liquid, dry suspension, dripping pill, dry extract, injection or transdermal preparation.

Images