Antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial peptide as well as a preparation method and application thereof. The antibacterial peptide has an amino acid sequence as shown in any one of SEQ ID NO: 1-11. The antibacterial peptide prepared by the scheme provided by the invention has very strong bacteriostasis and sterilization capabilities on methicillin-resistant staphylococcus aureus and multidrug-resistant escherichia coli, has application potential of becoming an antibacterial agent, a preservative or a disinfectant, and is not easy to generate drug resistance.

Description

Technical Field

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

[0002] Infections caused by drug-resistant pathogens are increasing in hospitals and communities, a serious global problem driven by decades of overuse of antibiotics. Due to the development of drug resistance, infections are often difficult to treat, protracted, and even life-threatening. During infection, drug-resistant bacteria produce toxins that cause excessive inflammation, severely compromising human health.

[0003] Compared with antibiotics, antimicrobial peptides are less likely to cause bacteria to develop drug resistance. However, most existing natural antimicrobial peptides do not have high bactericidal activity against drug-resistant bacteria. At the same time, highly effective antimicrobial peptides often require long chains or cyclic structures, resulting in a cumbersome and costly synthesis process, which greatly limits the application of antimicrobial peptides in actual production. Summary of the Invention

[0004] 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 an antimicrobial peptide that has good antibacterial activity against methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

[0005] The present invention also provides biomaterials related to the antimicrobial peptides.

[0006] The present invention also provides a method for preparing the antimicrobial peptide.

[0007] The present invention also provides a conjugate.

[0008] The present invention also provides a method for preparing the conjugate.

[0009] The present invention also provides applications of the antimicrobial peptide and conjugate.

[0010] The invention also provides an antibacterial product.

[0011] The invention also provides a method for inhibiting drug-resistant bacteria.

[0012] According to one aspect of the present invention, an antimicrobial peptide is provided, wherein the antimicrobial peptide is selected from any one of the following (1)-(12):

[0013] (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

[0014] (2) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2;

[0015] (3) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3;

[0016] (4) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4;

[0017] (5) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5;

[0018] (6) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6;

[0019] (7) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 7;

[0020] (8) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 8;

[0021] (9) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 9;

[0022] (10) a polypeptide having the amino acid sequence of SEQ ID NO: 10;

[0023] (11) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 11;

[0024] (12) A polypeptide having the same function as the polypeptide shown in any one of (1) to (11) obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 at the N-terminus and the C-terminus.

[0025] In some embodiments of the present invention, the bacteria include bacteria and fungi.

[0026] In some embodiments of the present invention, the bacteria include Staphylococcus aureus and Escherichia coli.

[0027] In some embodiments of the present invention, the bacteria include drug-resistant bacteria.

[0028] In some embodiments of the present invention, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

[0029] According to a second aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of 1) to 4):

[0030] 1) A nucleic acid molecule encoding the above-mentioned antimicrobial peptide;

[0031] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0032] 3) a recombinant vector containing 1) the nucleic acid molecule or 2) the expression cassette;

[0033] 4) A recombinant cell containing 1) the nucleic acid molecule, 2) the expression cassette or 3) the recombinant vector.

[0034] The third aspect of the present invention provides a method for preparing the antimicrobial peptide, 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 antimicrobial peptide.

[0035] In some embodiments of the present invention, the antimicrobial peptide can also be artificially synthesized.

[0036] 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.

[0037] In some embodiments of the present invention, the solid phase synthesis resin comprises 2-chlorotrityl chloride resin.

[0038] In some embodiments of the present invention, the method further comprises soaking the solid-phase synthetic resin in a dichloromethane solution to swell the resin.

[0039] In some embodiments of the present invention, the solid phase synthetic resin is immersed in the dichloromethane solution for 3-7 minutes.

[0040] 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 cleaved to obtain the antimicrobial peptide.

[0041] In some embodiments of the present invention, the protecting group comprises Fmoc-.

[0042] In some embodiments of the present invention, an amino acid containing a protecting group is attached to a resin using a solvent comprising HBTU at a concentration of 0.3-0.5 M or DIPEA at a concentration of 1-3 M.

[0043] In some embodiments of the present invention, the solvent further comprises DMF.

[0044] In some embodiments of the present invention, the washing liquid used for washing includes at least one of DMF and dichloromethane.

[0045] In some embodiments of the present invention, the cutting is performed using a cutting fluid.

[0046] In some embodiments of the present invention, the cutting fluid comprises trifluoroacetic acid and triisopropylsilane.

[0047] The fourth aspect of the present invention provides a conjugate comprising the above-mentioned antimicrobial peptide and a nonsteroidal anti-inflammatory drug; the nonsteroidal anti-inflammatory drug has a carboxyl group.

[0048] In some embodiments of the present invention, an amide bond is formed between the N-terminus of the antimicrobial peptide and the nonsteroidal anti-inflammatory drug.

[0049] In some embodiments of the present invention, the nonsteroidal anti-inflammatory drug is selected from at least one of flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen and clonixin.

[0050] In some embodiments of the present invention, the conjugate may further include one of a biotin group, a streptavidin group, a radioactive group, a fluorescent group, a Myc tag, glutathione-S-transferase, and protein A.

[0051] The fifth aspect of the present invention provides a method for preparing the above-mentioned conjugate, which comprises the following steps: linking the above-mentioned antimicrobial peptide and non-steroidal anti-inflammatory drug via an amide bond.

[0052] In some embodiments of the present invention, the preparation method specifically comprises the following steps: using a solid phase synthetic resin as a starting material, and adopting a solid phase synthesis method to prepare the conjugate.

[0053] In some embodiments of the present invention, the conjugate can also be artificially synthesized.

[0054] In some embodiments of the present invention, the preparation of the conjugate is performed one by one from the C-terminus to the N-terminus.

[0055] In some embodiments of the present invention, the solid phase synthesis resin comprises 2-chlorotrityl chloride resin.

[0056] In some embodiments of the present invention, the method further comprises soaking the solid-phase synthetic resin in a dichloromethane solution to swell the resin.

[0057] In some embodiments of the present invention, the solid phase synthetic resin is immersed in the dichloromethane solution for 3-7 minutes.

[0058] 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; after adding a non-steroidal anti-inflammatory drug to the polypeptide chain for reaction, washing and cleavage are sequentially performed to obtain the antimicrobial peptide.

[0059] In some embodiments of the present invention, the protecting group comprises Fmoc-.

[0060] 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 HBTU at a concentration of 0.3-0.5 M and DIPEA at a concentration of 1-3 M.

[0061] In some embodiments of the present invention, the activating agent further comprises DMF.

[0062] In some embodiments of the present invention, the washing liquid used for washing includes at least one of DMF and dichloromethane.

[0063] In some embodiments of the present invention, the cutting uses a cutting fluid.

[0064] In some embodiments of the present invention, the cutting fluid comprises trifluoroacetic acid and triisopropylsilane.

[0065] According to a sixth aspect of the present invention, the use of the above-mentioned antimicrobial peptides, biomaterials or conjugates in the preparation of antimicrobial or anti-inflammatory products is proposed.

[0066] In some embodiments of the present invention, the bacteria include bacteria and fungi.

[0067] In some embodiments of the present invention, the bacteria include drug-resistant bacteria.

[0068] In some embodiments of the present invention, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

[0069] In some embodiments of the present invention, the product comprises a medicine, a preservative, or a disinfectant.

[0070] According to a seventh aspect of the present invention, a product is provided, comprising the above-mentioned antimicrobial peptide.

[0071] In some embodiments of the present invention, the product comprises a medicine, a preservative, or a disinfectant.

[0072] In some embodiments of the present invention, the product has an antibacterial effect.

[0073] In some embodiments of the present invention, the bacteria include bacteria and fungi.

[0074] In some embodiments of the present invention, the bacteria include drug-resistant bacteria.

[0075] In some embodiments of the present invention, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

[0076] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0077] In some embodiments of the present invention, the pharmaceutical carrier is a conventional drug carrier in the pharmaceutical field.

[0078] 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.

[0079] In some embodiments of the invention, the excipient comprises water.

[0080] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0081] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, alginate, gelatin, and polyvinyl pyrrolidone.

[0082] In some embodiments of the invention, the humectant comprises glycerin.

[0083] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0084] In some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0085] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0086] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0087] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0088] In some embodiments of the present invention, the mass fraction of the antimicrobial peptide in the drug, preservative or disinfectant is 0.01% to 99%.

[0089] According to some preferred embodiments of the present invention, the mass fraction of the antimicrobial peptide in the drug, preservative or disinfectant is 0.05% to 95%.

[0090] According to some preferred embodiments of the present invention, the mass fraction of the antimicrobial peptide in the drug, preservative or disinfectant is 0.05% to 20%.

[0091] 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, atomizers, sprays, injections or infusions, transdermal agents, and transdermal microneedles.

[0092] 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.

[0093] The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0094] According to the eighth aspect of the present invention, the present invention also proposes a method for inhibiting drug-resistant bacteria for non-disease diagnosis purposes, the method comprising the following steps: using the above-mentioned antimicrobial peptide or conjugate to inhibit drug-resistant bacteria.

[0095] In some embodiments of the present invention, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

[0096] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the antimicrobial peptides prepared by the scheme of the present invention have strong antibacterial and bactericidal abilities against methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli, and have the potential to be used as antimicrobial drugs, preservatives or disinfectants, and are not prone to drug resistance.

[0097] The present invention also couples antimicrobial peptides with non-steroidal anti-inflammatory drugs to obtain conjugates, which, compared with antimicrobial peptides, significantly enhance the killing ability against methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli, have significant effects against drug-resistant bacteria, and have anti-inflammatory effects. They can be effectively used to prepare drugs that are both anti-resistant and anti-inflammatory, and are not prone to drug resistance. DETAILED DESCRIPTION

[0098] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0099] Example 1

[0100] The present embodiment provides an antibacterial peptide 1, the amino acid sequence of which is FLRRIRPKLKW (SEQ ID NO: 1). It can be artificially synthesized by the company or synthesized by a solid-phase chemical synthesis method. If the solid-phase chemical synthesis method is used, the preparation method is as follows:

[0101] (1) Take 0.5 mmol of 2-chlorotrityl chloride resin in a solid-phase synthesizer, add 10 mL of anhydrous dichloromethane, and place it on a shaker for 5 min to allow the 2-chlorotrityl chloride resin to swell fully;

[0102] (2) Use an ear bulb to remove the dichloromethane from the solid-phase synthesizer completely;

[0103] (3) Dissolve 1 mmol of Fmoc-protected amino acid (the first amino acid) in 10 mL of dichloromethane (DCM), add 1.5 mmol of N,N-diisopropylethylamine (DIPEA), and then transfer it to the above-mentioned solid-phase synthesizer. Transfer the solid-phase tube to the shaker and react at room temperature for 1 hour;

[0104] (4) Remove the reaction solution from the solid-phase synthesizer with an ear bulb, and then wash it with 10 mL of dichloromethane, 1 min each time, a total of 5 times. After washing, add a prepared solution of anhydrous dichloromethane:DIEPA:methanol in a volume ratio of 17:1:2, 20 mL, and transfer the solid-phase tube to the shaker and react at room temperature for 10 min;

[0105] (5) Remove the reaction solution from the solid-phase synthesizer with an ear bulb, first wash it with dichloromethane 3 times, and then wash it with N,N-dimethylformamide (DMF) 3 times, 1 min each time. Add 10 mL of DMF containing 20% piperidine by volume to the solid-phase synthesizer and react for 25 min. Then wash it with DMF, 10 mL each time, 1 min each time, a total of 6 times, and proceed to the next reaction;

[0106] (6) Add the second Fmoc-protected amino acid 1 mmol, O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU) 1.5 mmol, DIEPA 2 mmol, and 20 mL of DMF, and add the prepared solution to the above-mentioned solid-phase synthesizer and react for 2 hours;

[0107] (7) Repeat steps 5 and 6, adding the required amino acids in sequence until all amino acids have been added. Then wash with DMF 5 times and dichloromethane 5 times before proceeding to the next step.

[0108] (8) 20 mL of a solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water by volume was added to the solid phase synthesizer and allowed to react for half an hour. The product was cut from the 2-chlorotrityl chloride resin and concentrated in vacuo to remove the solvent to obtain a crude product, which was then separated and purified by high performance liquid chromatography (HPLC).

[0109] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0110] Example 2

[0111] This embodiment provides an antimicrobial peptide 2 having an amino acid sequence of KKFKKKLLK (SEQ ID NO: 2), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0112] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0113] Example 3

[0114] This example provides an antimicrobial peptide 3 having an amino acid sequence of KKLRKRLKKFR (SEQ ID NO: 3), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0115] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0116] Example 4

[0117] This example provides an antimicrobial peptide 4 having an amino acid sequence of RRPKFRKFKL (SEQ ID NO: 4), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0118] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0119] Example 5

[0120] This example provides an antimicrobial peptide 5 having an amino acid sequence of KRFKLYWKGKF (SEQ ID NO: 5), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0121] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0122] Example 6

[0123] This example provides an antimicrobial peptide 6, whose amino acid sequence is FRRPKVRKFKL (SEQ ID NO: 6), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0124] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0125] Example 7

[0126] This embodiment provides an antimicrobial peptide 7 having an amino acid sequence of RKRDWKK (SEQ ID NO: 7), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0127] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0128] Example 8

[0129] This example provides an antimicrobial peptide 8, the amino acid sequence of which is LWRRFNRPLLK (SEQ ID NO: 8), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0130] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0131] Example 9

[0132] This example provides an antimicrobial peptide 9, whose amino acid sequence is RLLKKKTKKQF (SEQ ID NO: 9), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0133] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0134] Example 10

[0135] This embodiment provides an antimicrobial peptide 10 having an amino acid sequence of LFRKLRLFFRR (SEQ ID NO: 10), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0136] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0137] Example 11

[0138] This embodiment provides an antimicrobial peptide 11 having an amino acid sequence of RRKFRRQRPRL (SEQ ID NO: 11), which can be synthesized artificially or by solid-phase chemical synthesis, and the preparation method is the same as that of Example 1.

[0139] The results of HPLC showed that the purity of the synthesized peptide was greater than 97%.

[0140] Example 12

[0141] This example provides a conjugate of the antimicrobial peptide 1 prepared in Example 1 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 1 below.

[0142] Table 1

[0143]

[0144]

[0145] It can be synthesized artificially or by solid-phase chemical synthesis. If solid-phase chemical synthesis is used, the preparation method is as follows:

[0146] (1) Weigh 0.5 mmol of 2-chlorotrityl chloride resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane, and shake on a shaker for 5 min to allow the 2-chlorotrityl chloride resin to fully swell;

[0147] (2) Use an ear bulb to remove the dichloromethane from the solid phase synthesizer;

[0148] (3) Dissolve 1 mmol of Fmoc-protected amino acid in 10 mL of dichloromethane (DCM), add 1.5 mmol of N,N-diisopropylethylamine (DIPEA), and then transfer to the above-mentioned solid phase synthesizer. Transfer the solid phase tube to a shaker and react at room temperature for 1 hour.

[0149] (4) Remove the reaction solution from the solid-phase synthesizer using an ear bulb, then wash with 10 mL of dichloromethane for 1 minute each time, for a total of 5 washes. After washing, add 20 mL of a solution prepared with a volume ratio of anhydrous dichloromethane: DIEPA: methanol = 17:1:2, transfer the solid-phase tube to a shaker, and react at room temperature for 10 minutes.

[0150] (5) Remove the reaction solution from the solid phase synthesizer using an ear bulb. Wash the solid phase synthesizer three times with dichloromethane and then three times with N,N-dimethylformamide (DMF), each washing for 1 minute. Add 10 mL of DMF containing 20% ​​piperidine by volume to the solid phase synthesizer and allow it to react for 25 minutes. Then, wash the solid phase synthesizer with 10 mL of DMF for 1 minute each time, for a total of six washes, and proceed to the next step.

[0151] (6) Add 1 mmol of the second Fmoc-protected amino acid, 1.5 mmol of O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU), 2 mmol of DIEPA, and 20 mL of DMF, and add the prepared solution to the above solid phase synthesizer and react for 2 hours;

[0152] (7) Repeat steps 5 and 6, adding the desired amino acids or 1 mM carboxyl-containing nonsteroidal anti-inflammatory drugs in sequence until all amino acids have been added. Then, wash with DMF five times and dichloromethane five times before proceeding to the next step.

[0153] (8) 20 mL of a solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water by volume was added to the solid phase synthesizer and allowed to react for half an hour. The product was cut from the 2-chlorotrityl chloride resin and concentrated in vacuo to remove the solvent to obtain a crude product, which was then separated and purified by high performance liquid chromatography.

[0154] The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0155] Example 13

[0156] This example provides a conjugate of the antimicrobial peptide 2 prepared in Example 2 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 2.

[0157] Table 2

[0158]

[0159]

[0160] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0161] Example 14

[0162] This example provides a conjugate of the antimicrobial peptide 3 prepared in Example 3 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 3.

[0163] Table 3

[0164]

[0165] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0166] Example 15

[0167] This example provides a conjugate of the antimicrobial peptide 4 prepared in Example 4 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 4.

[0168] Table 4

[0169]

[0170]

[0171] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0172] Example 16

[0173] This example provides a conjugate of the antimicrobial peptide 5 prepared in Example 5 above and a nonsteroidal anti-inflammatory drug. The conjugate is obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 5.

[0174] Table 5

[0175]

[0176]

[0177] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0178] Example 17

[0179] This example provides a conjugate of the antimicrobial peptide 6 prepared in Example 6 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 6.

[0180] Table 6

[0181]

[0182]

[0183] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0184] Example 18

[0185] This example provides a conjugate of the antimicrobial peptide 7 prepared in Example 7 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 7.

[0186] Table 7

[0187]

[0188]

[0189] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0190] Example 19

[0191] This example provides a conjugate of the antimicrobial peptide 8 prepared in Example 8 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 8.

[0192] Table 8

[0193]

[0194]

[0195] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0196] Example 20

[0197] This example provides a conjugate of the antimicrobial peptide 9 prepared in Example 9 above and a nonsteroidal anti-inflammatory drug. The conjugate is obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 9.

[0198] Table 9

[0199]

[0200]

[0201] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0202] Example 21

[0203] This example provides a conjugate of the antimicrobial peptide 10 prepared in Example 10 above and a nonsteroidal anti-inflammatory drug. The conjugate is obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure is shown in Table 10.

[0204] Table 10

[0205]

[0206]

[0207] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0208] Example 22

[0209] This example provides a conjugate of the antimicrobial peptide 11 prepared in Example 11 above and a nonsteroidal anti-inflammatory drug, obtained by linking a carboxyl-containing nonsteroidal anti-inflammatory drug to the N-terminus of a short peptide via an amide bond. The nonsteroidal anti-inflammatory drug is selected from the group consisting of flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen, or clonixin. The conjugate structure differs from that in Table 1 of Example 1 only in that the sequence "FLRRIRPKLKW" is replaced with "RRKFRRQRPRL."

[0210] Table 11

[0211]

[0212]

[0213] The preparation method was the same as that in Example 12. The results of HPLC showed that the purity of the conjugate was greater than 97%.

[0214] Test Case

[0215] In this example, the antibacterial activities of the antimicrobial peptides prepared in Examples 1-11 and the conjugates prepared in Examples 12-22 were determined, and the antibacterial activities of the peptides FLRRIRPKLKW-COOH, the conjugates, flurbiprofen-FLRRIRPKLKW-COOH, bilastine-FLRRIRPKLKW-COOH, levocetirizine hydrochloride-FLRRIRPKLKW-COOH, carprofen-FLRRIRPKLKW-COOH, fenofibric acid-FLRRIRPKLKW-COOH, ibuprofen-FLRRIRPKLKW-COOH, zaltoprofen-FLRRIRPKLKW-COOH, diclofenac sodium-FLRRIRPKLKW-COOH, diclofenac sodium-KKFKKKLLK-COOH, diclofenac sodium-KKFKKKLLK-COOH, and diclofenac sodium-KKFKKKLLK-COOH were determined. As an example, diclofenac sodium-KKLRKRLKKFR-COOH, diclofenac sodium-RRPKFRKFKL-COOH, diclofenac sodium-KRFKLYWKGKF-COOH, diclofenac sodium-FRRPKVRKFKL-COOH, diclofenac sodium-RKRDWKK-COOH, diclofenac sodium-LWRRFNRPLLK-COOH, diclofenac sodium-RLLKKKTKKQF-COOH, diclofenac sodium-LFRKLRLFFRR-COOH, and diclofenac sodium-RRKFRRQRPRL-COOH were used as experimental groups for detection, and flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, fenofibric acid, ibuprofen, zaltoprofen, and diclofenac sodium were used as control groups. The specific process was as follows:

[0216] Determination of minimum inhibitory concentration (MIC):

[0217] The minimum inhibitory concentration (MIC) of the compound was determined using the standard broth microdilution solution provided by the Clinical and Laboratory Standards Institute, which specifically included the following steps: 100 μL of each compound in the experimental group and the control group were added to a 96-well plate at a 2-fold serial dilution solution (512 μM, 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM).

[0218] Methicillin-resistant Staphylococcus aureus (MRSA, purchased from Beina Biotechnology) and multidrug-resistant Escherichia coli (MRE, purchased from Beina Biotechnology) were cultured in LB liquid medium. The bacteria in the middle exponential growth phase of the LB medium were diluted to 1×10 6 CFU / mL. Then, add 100 μL of bacterial solution to each compound in a 96-well plate and incubate at 37°C for 24 hours. Visually inspect the tube with the lowest drug concentration, and the clear, translucent tube showing no bacterial growth is the MIC for the test bacteria.

[0219] Table 12

[0220]

[0221] Table 13

[0222]

[0223]

[0224] The test results of polypeptide MIC are shown in Tables 12-13, and it can be seen from Table 12 that the polypeptide FLRRIRPKLKW has obvious inhibitory effect on methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant Escherichia coli (MRE). Meanwhile, when the polypeptide FLRRIRPKLKW is coupled with non-steroidal anti-inflammatory drugs, the MIC values are significantly lower than those of antibacterial peptides.

[0225] As can be seen from Table 13, the MIC values of the polypeptides provided in Examples 2-11 after being coupled with diclofenac sodium are significantly lower than those of the drug diclofenac sodium, and also show good inhibitory and bactericidal effects.

[0226] Due to the difference in test MIC method, the bactericidal rate corresponding to the minimum bactericidal concentration of each compound for each bacterium in the results is much higher than 99.99%.

[0227] The antibacterial test of the antibacterial peptide prepared in Example 2-11 is also carried out, and the effect is equivalent to that of the polypeptide FLRRIRPKLKW, and it can also be used to inhibit methicillin-resistant Staphylococcus aureus and multi-drug resistant Escherichia coli.

[0228] The above has made a detailed description of the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antimicrobial peptide, characterized in that The antimicrobial peptide is selected from any one of the following (1)-(12): (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (2) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; (3) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3; (4) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4; (5) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5; (6) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6; (7) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 7; (8) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 8; (9) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 9; (10) a polypeptide having the amino acid sequence of SEQ ID NO: 10; (11) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 11; (12) A polypeptide having the same function as the polypeptide shown in any one of (1) to (11) obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 at the N-terminus and the C-terminus.

2. Biomaterial, characterized in that The biological material is any one of 1) to 4): 1) A nucleic acid molecule encoding the antimicrobial peptide 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 antimicrobial peptide 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 antimicrobial peptide.

4. A conjugate, characterized in that The conjugate comprises the antimicrobial peptide according to claim 1 and a nonsteroidal anti-inflammatory drug; the nonsteroidal anti-inflammatory drug has a carboxyl group; Preferably, an amide bond is formed between the N-terminus of the antimicrobial peptide and the nonsteroidal anti-inflammatory drug; Preferably, the nonsteroidal anti-inflammatory drug is selected from at least one of flurbiprofen, bilastine, levocetirizine hydrochloride, carprofen, acetylsalicylic acid, indomethacin, flufenamic acid, fenofibric acid, ibuprofen, ketoprofen, 2-phenyl-4-quinolinecarboxylic acid, tolmetin sodium, zaltoprofen, oxaprozin, fenbufen, tolfenamic acid, loxoprofen, diflunisal, diclofenac sodium, ketoprofen, fenoprofen and clonixin.

5. A method for preparing the conjugate according to claim 4, characterized in that: The antimicrobial peptide according to claim 1 and the nonsteroidal anti-inflammatory drug are linked through an amide bond to obtain the antimicrobial peptide.

6. Use of the antimicrobial peptide according to claim 1, the biomaterial according to claim 2, or the conjugate according to claim 4 in the preparation of antimicrobial or anti-inflammatory products.

7. The use according to claim 6, characterized in that The bacteria include bacteria and fungi; Preferably, the bacteria include drug-resistant bacteria; More preferably, the drug-resistant bacteria include methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

8. A product, characterized in that Comprising the antimicrobial peptide according to claim 1 or the conjugate according to claim 4; Preferably, the product comprises a medicine, a preservative or a disinfectant; More preferably, the drug further comprises a pharmaceutically acceptable carrier.

9. The product according to claim 8, characterized in that The dosage form of the drug is any conventional dosage form in the art; Preferably, it is in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension; More preferably, it is in the form of tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, dripping pills, dry extracts, atomizers, sprays, injections, transdermal preparations or transdermal microneedles; And / or, the administration method of the drug can be a conventional administration method in the art, including but not limited to injection or oral administration.

10. A method for inhibiting drug-resistant bacteria for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: using the antimicrobial peptide according to claim 1 or the conjugate according to claim 4 to inhibit drug-resistant bacteria.

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