An antibacterial polypeptide-modified protein derivative, and a preparation method and application thereof
By linking antimicrobial peptides to proteins through click chemistry, protein derivatives modified with antimicrobial peptides are formed, solving the problem of antibiotic resistance and achieving highly efficient killing of Gram-negative bacteria and improved biocompatibility.
Patent Information
- Application Number
- CN202110444548.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-04-23
- Publication Date
- 2026-02-17
- Estimated Expiration
- 2041-04-23
AI Technical Summary
The long-term use of existing antibiotics has led to the emergence of drug-resistant strains, necessitating the development of new antibacterial agents to replace antibiotics, especially for the effective treatment of Gram-negative bacteria.
Antimicrobial peptides are linked to proteins via click chemical reactions to form protein derivatives modified with antimicrobial peptides. Specifically, this involves introducing an alkyne group onto the protein and then reacting it with an azide-modified antimicrobial peptide. Cathelicidin-type or Defensin-type antimicrobial peptides are preferred to bind to viral capsid proteins.
The obtained antimicrobial peptide-modified protein derivatives have good biocompatibility and high bactericidal ability, effectively killing Gram-negative bacteria and reducing bacterial resistance.
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Figure CN115232193B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibacterial new materials technology, specifically relating to an antibacterial polypeptide-modified protein derivative, its preparation method, and its application. Background Technology
[0002] Bacterial infections are a major threat to human health. Antibiotics are the primary means of treating bacterial infections due to their low cost and good efficacy. However, the large-scale and long-term use of antibiotics has led to antibiotic abuse and the emergence of drug-resistant bacteria.
[0003] The current mechanism of action of antibiotics is to inhibit bacterial cell wall synthesis, enhance bacterial cell membrane permeability, interfere with bacterial protein synthesis, and inhibit bacterial nucleic acid replication and transcription. Their main targets are cell wall synthesis, inhibition of protein translation, and DNA replication. However, resistance to each of these targets has emerged, so it is necessary to develop new antibacterial agents to replace antibiotics.
[0004] Natural biomolecules typically possess good in vivo degradation capabilities, good biosafety, and in vivo transport capabilities. Natural macromolecules such as protein particles, including serum proteins, are frequently used as drug carriers to deliver drugs to the lesion site for the treatment of corresponding diseases. Summary of the Invention
[0005] To overcome the shortcomings of the prior art, the present invention provides an antibacterial polypeptide-modified protein derivative, its preparation method and uses, which has excellent bactericidal effect and biocompatibility and can be used to kill bacteria, especially Gram-negative bacteria.
[0006] This invention provides the following technical solution:
[0007] An antimicrobial peptide-modified protein derivative comprising a protein and an antimicrobial peptide, wherein the protein and the antimicrobial peptide are linked by a triazole group, for example, by a click chemistry reaction between an alkyne group and an azide group to link the protein and the antimicrobial peptide.
[0008] The antimicrobial peptides include cathelicidin-type antimicrobial peptides and defensin-type antimicrobial peptides, preferably cecropins peptides, magainins peptides, apidaecins peptides, bactenecin peptides, defensins peptides, or nisin peptides, and more preferably R8, CM15, or P7 peptides; the amino acid sequence of R8 is shown in SEQ ID NO.1, the amino acid sequence of CM15 is shown in SEQ ID NO.2, and the amino acid sequence of P7 is shown in SEQ ID NO.3.
[0009] SEQ ID NO.1: RRRRRRRRR.
[0010] SEQ ID NO. 2: KWKLFKKIGAVLKVL.
[0011] SEQ ID NO. 3: GLRRALLRLLRSLRRRLLLRA.
[0012] The protein is a viral capsid protein, serum protein, lactoferrin, transferrin, or collagen. The viral capsid protein is a tobacco mosaic virus capsid protein, a cowpea mosaic virus capsid protein, a bacteriophage capsid protein, or a Bacillus subtilis capsid protein. Preferably, the viral capsid protein is any one of a tobacco mosaic virus capsid protein, a cowpea mosaic virus capsid protein, a T2 bacteriophage capsid protein, or an M13 bacteriophage capsid protein. More preferably, the viral capsid protein is a tobacco mosaic virus capsid protein. Preferably, guanidine groups and / or quaternary ammonium salts can be modified on the surface of the protein to enhance the antibacterial effect.
[0013] The present invention also provides a method for preparing an antimicrobial peptide-modified protein derivative, comprising the following steps: introducing an alkyne group into a protein, and then performing a click chemical reaction with an antimicrobial peptide having an azide group at the end to obtain the protein derivative.
[0014] The protein may be the capsid protein of tobacco mosaic virus, which can be obtained by disassembling tobacco mosaic virus under acidic conditions.
[0015] When the protein is the capsid protein of tobacco mosaic virus, it can also be prepared by the following method:
[0016] 1) The phenolic hydroxyl groups on the surface of tobacco mosaic virus were alkynylated to obtain alkynyl-modified tobacco mosaic virus;
[0017] 2) The alkynyl-modified tobacco mosaic virus obtained in step 1) is disassembled under acidic conditions to obtain the alkynyl-modified tobacco mosaic virus capsid protein.
[0018] 3) The tobacco mosaic virus capsid protein modified with alkyne obtained in step 2) is then subjected to a click chemical reaction with an antimicrobial polypeptide with an azide group at the end to obtain the protein derivative.
[0019] According to the present invention, the alkynylation reaction can be carried out using conventional reactions to introduce alkynyl groups. For example, the alkynylation reaction can be a reaction of tobacco mosaic virus or tobacco mosaic virus capsid protein with 3-ethynylaniline and nitrite (such as NaNO2). Preferably, the reaction is carried out under acidic conditions, for example, the acid can be p-toluenesulfonic acid.
[0020] According to the present invention, the acidic conditions for disassembly refer to conditions with a pH value < 7, for example, mixing the tobacco mosaic virus or alkynyl-modified tobacco mosaic virus with an acid, such as formic acid, acetic acid, hydrochloric acid, or nitric acid, preferably acetic acid. After the virus is disassembled, the RNA precipitate is removed, and then the acid is removed by passing it through a desalting column to obtain the tobacco mosaic virus capsid protein or alkynyl-modified tobacco mosaic virus capsid protein.
[0021] According to the present invention, the protein separation and purification after disassembly can be performed by one or more of dialysis, ultrafiltration, centrifugation, gel filtration or desalting column, preferably using desalting column for separation.
[0022] According to the present invention, the click chemistry reaction is carried out under the catalysis of metal ions (e.g., Cμ(I)), for example in the presence of aminoguanidine hydrochloride, sodium ascorbate and CuSO4.
[0023] According to the present invention, the temperature of the click chemical reaction and the disassembly and reassembly of the virus under acidic conditions are both between 0 and 20°C, preferably at 4°C.
[0024] According to the present invention, after the click reaction is completed, purification is performed, and the purification specifically includes the following steps: dialysis using a dialysis bag for 30-60 hours, preferably 40-50 hours, and most preferably 45-48 hours.
[0025] The use of an antimicrobial peptide-modified protein derivative for killing bacteria, viruses, protozoa, cancer cells and / or fungi, preferably for killing bacteria, and most preferably for killing Gram-negative bacteria.
[0026] The use of an antimicrobial peptide-modified protein derivative for the preparation of a drug with bactericidal effects.
[0027] A bactericidal pharmaceutical composition comprising a protein derivative modified with the said antimicrobial peptide.
[0028] The pharmaceutical composition may also include one or more pharmaceutically acceptable excipients, such as stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients.
[0029] The drug may be an oral tablet, capsule, pill, powder, sustained-release preparation, solution, or suspension; a sterile solution, suspension, or emulsion for parenteral injection; an ointment or cream for external use; or a suppository for rectal administration.
[0030] Beneficial effects
[0031] This invention uses a click reaction to graft antimicrobial peptides with antimicrobial activity onto proteins, particularly onto the capsid protein of tobacco mosaic virus, to obtain protein derivatives modified with antimicrobial peptides. These protein derivatives have good biocompatibility and stronger antimicrobial activity than the pure antimicrobial peptides.
[0032] The antibacterial peptide-modified protein derivatives described in this invention not only have good bactericidal effects (broad-spectrum and highly efficient, capable of killing superbugs), but also can prevent bacteria from developing drug resistance. Attached Figure Description
[0033] Figure 1 A schematic diagram of the structure of the R8-modified tobacco mosaic virus capsid protein prepared in Example 1;
[0034] Figure 2 SDS-PAGE image of the R8-modified tobacco mosaic virus capsid protein prepared in Example 1;
[0035] Figure 3 The image shows the test results of the R8-modified tobacco mosaic virus capsid protein prepared in Example 1 against Escherichia coli ATCC25922. Detailed Implementation
[0036] Example 1
[0037] S101. 800 μL of 0.3 M p-toluenesulfonic acid aqueous solution, 150 μL of 0.68 M acetonitrile solution of 3-ethynylaniline, and 50 μL of 3 M NaNO2 aqueous solution were mixed and stirred in the dark at 4 °C for 1 h to obtain a bright yellow pre-synthesized salt.
[0038] S102. Add 800 μL of the pre-synthesized salt to 4.8 mL of the prepared TMV (tobacco mosaic virus) solution (100 mM borate buffer solution with pH 9.0, containing 2 mg / mL TMV and 0.1 M sodium chloride). After mixing, stir and react in the dark at 4 °C for 1 h. Then dialyze the product for two days using a dialysis bag (Mw = 1,000,000) to obtain alkynyl-functionalized TMV.
[0039] S103. Mix 2 mg / mL of the above-mentioned alkyne-functionalized TMV with acetic acid at a volume ratio of 1:2, react at 4℃ for 30 min for disassembly, centrifuge at 10000g at 4℃ to remove the RNA precipitate generated during disassembly, then add 2.5 mL of ultrapure water to wash the PD-10 pre-loaded Sephadex G-25 desalting column three times. After each wash, add 2.5 mL of disassembly solution for multiple washes. After the solution has completely passed through the desalting column, add 500 μL of ultrapure water each time. Collect the eluent using a 1.5 mL centrifuge tube without RNase and detect it with Coomassie Brilliant Blue G250 until the Coomassie solution no longer forms a bright blue color. Perform the next round of elution to remove acetic acid, and finally obtain an ultrapure aqueous solution of alkyne-modified tobacco mosaic virus capsid protein. The concentration of alkyne-modified tobacco mosaic virus capsid protein is measured using Lowry's assay (a method for determining protein content).
[0040] S104. At 4°C, 20 μL of 0.2 M aminoguanidine hydrochloride aqueous solution, 20 μL of 0.2 M sodium ascorbate aqueous solution, and 20 μL of 0.1 M CuSO4 aqueous solution were mixed thoroughly. The mixture was then added to 467 μL of a mixture of 5 mg / mL alkyne-modified tobacco mosaic virus capsid protein and 1.2 eq of azide-modified polypeptide R8 (RRRRRRRRRR). The mixture was stirred overnight at 4°C. After the reaction was complete, 20 μL of 1 M EDTA buffer solution with a pH of 6.0–8.0 was added, and the mixture was reacted at 4°C for 30 minutes. The product was dialyzed for two days using a dialysis bag (Mw = 3000–5000) to obtain R8-modified tobacco mosaic virus capsid protein (hereinafter referred to as TMVCP-R8).
[0041] The SDS spectrum of TMVCP-R8 is as follows: Figure 2 As shown, R8 was successfully grafted onto the capsid protein of tobacco mosaic virus. Two bands appeared on the TMVCP-R8 electrophoresis channel, with the upstream band having a larger molecular weight of Mw(TMVCP) + Mw(R8) and a reaction rate (grafting rate of the polypeptide) of 39%.
[0042] Example 2
[0043] S201. This step is the same as S101.
[0044] S202. This step is the same as S102.
[0045] S203, This step is the same as S103.
[0046] S204. At 4°C, 20 μL of 0.2 M aminoguanidine hydrochloride solution, 20 μL of 0.2 M sodium ascorbate solution, and 20 μL of 0.1 M CuSO4 solution were mixed thoroughly. This mixture was then added to 467 μL of a 5 mg / mL aqueous solution of alkyne-modified tobacco mosaic virus capsid protein and 1.2 eq of azide-modified CM15 (KWKLFKKIGAVLKVL). The mixture was stirred overnight at 4°C. After the reaction was complete, 20 μL of 1 M EDTA buffer solution with a pH of 6.0–8.0 was added, and the mixture was reacted at 4°C for 30 minutes. The product was then dialyzed for two days using a dialysis bag (Mw = 3000–5000) to obtain CM15-modified tobacco mosaic virus capsid protein.
[0047] Example 3
[0048] S301, This step is the same as S101.
[0049] S302. This step is the same as S102.
[0050] S303, This step is the same as S103.
[0051] S304. At 4°C, 20 μL of 0.2 M aminoguanidine hydrochloride aqueous solution, 20 μL of 0.2 M sodium ascorbate aqueous solution, and 20 μL of 0.1 M CuSO4 aqueous solution were mixed thoroughly. The mixture was then added to a mixture of 467 μL of 5 mg / mL alkyne-modified tobacco mosaic virus capsid protein aqueous solution and 1.2 eq of azide-modified antimicrobial peptide P7 (GLRRALLRLLRSLRRLLLRA). The mixture was stirred overnight at 4°C. Then, 20 μL of 1 M EDTA buffer solution with a pH of 6.0–8.0 was added, and the mixture was reacted at 4°C for 30 minutes. The product was dialyzed for two days using a dialysis bag (Mw = 3000–5000) to obtain P7-modified tobacco mosaic virus capsid protein.
[0052] Example 4
[0053] S401. 800 μL of 0.3 M p-toluenesulfonic acid aqueous solution, 150 μL of 0.68 M 3-ethynylaniline acetonitrile solution, and 50 μL of 3 M NaNO2 aqueous solution were mixed and stirred in the dark at 4 °C for 1 h to obtain a bright yellow pre-synthesized salt.
[0054] S402. Add 800 μL of the above-mentioned pre-synthesized salt to 4.8 mL of the prepared tobacco mosaic virus capsid protein solution (100 mM borate buffer solution with pH 9.0, containing 2 mg / mL tobacco mosaic virus capsid protein and 0.1 M sodium chloride). After mixing, stir and react in the dark at 4 °C for 1 h. Then dialyze the product for two days using a dialysis bag (Mw = 1,000,000) to obtain the alkyne-functionalized tobacco mosaic virus capsid protein.
[0055] S403. At 4°C, mix 20 μL of 0.2 M aminoguanidine hydrochloride aqueous solution, 20 μL of 0.2 M sodium ascorbate aqueous solution, and 20 μL of 0.1 M CuSO4 aqueous solution. Add the above mixture to 467 μL of a mixture of 5 mg / mL alkyne-modified tobacco mosaic virus capsid protein and 1.2 eq of azide-modified antimicrobial peptide R8 (RRRRRRRRRR). Stir overnight at 4°C. After the reaction is complete, add 20 μL of 1 M EDTA buffer solution with a pH of 6.0–8.0 and react at 4°C for 30 minutes. Then dialyze the product for two days using a dialysis bag (Mw = 3000–5000) to obtain R8-modified tobacco mosaic virus capsid protein.
[0056] Example 5
[0057] S501. Mix 2 mg / mL tobacco mosaic virus with acetic acid at a volume ratio of 1:2, react at 4℃ for 30 min, and centrifuge at 10000g at 4℃ to remove the RNA precipitate generated during disassembly.
[0058] S502. Add 2.5 mL of ultrapure water to wash the PD-10 pre-loaded Sephadex G-25 desalting column three times. After each wash, add 2.5 mL of disassembly solution. After the solution has completely passed through the desalting column, add 500 μL of ultrapure water each time. Collect the eluent using a 1.5 mL centrifuge tube without RNase and detect it with Coomassie Brilliant Blue G250 until the Coomassie solution no longer forms a bright blue color. Perform the next round of elution to remove acetic acid. Finally, obtain an ultrapure aqueous solution of tobacco mosaic virus capsid protein. The concentration of tobacco mosaic virus capsid protein is measured using Lowry's method.
[0059] S503: 800 μL of 0.3 M p-toluenesulfonic acid aqueous solution, 150 μL of 0.68 M 3-ethynylaniline acetonitrile solution, and 50 μL of 3 M NaNO2 aqueous solution were mixed and stirred in the dark at 4 °C for 1 h to obtain a bright yellow pre-synthesized salt.
[0060] S504. Add 800 μL of the above-mentioned pre-synthesized salt to 4.8 mL of the prepared tobacco mosaic virus capsid protein solution (100 mM borate buffer solution with pH 9.0, containing 2 mg / mL tobacco mosaic virus capsid protein solution and 0.1 M sodium chloride). After mixing, stir and react in the dark at 4 °C for 1 h. Then dialyze the product for two days using a dialysis bag (Mw = 1,000,000) to obtain the alkyne-functionalized tobacco mosaic virus capsid protein.
[0061] S505. At 4°C, 20 μL of 0.2 M aminoguanidine hydrochloride aqueous solution, 20 μL of 0.2 M sodium ascorbate aqueous solution, and 20 μL of 0.1 M CuSO4 aqueous solution were mixed thoroughly. The mixture was then added to a mixture of 467 μL of 5 mg / mL alkyne-modified tobacco mosaic virus capsid protein aqueous solution and 1.2 eq of azide-modified antimicrobial peptide R8. The mixture was stirred overnight at 4°C. After the reaction was completed, 20 μL of 1 M EDTA buffer solution with a pH of 6.0–8.0 was added, and the mixture was reacted at 4°C for 30 minutes. The product was dialyzed for two days using a dialysis bag (Mw = 3000–5000) to obtain R8-modified tobacco mosaic virus capsid protein.
[0062] Test case
[0063] A. Pick Escherichia coli ATCC25922 colonies from MH agar plates and place them in TSB (tryptone soy broth). Pick Pseudomonas aeruginosa colonies and place them in LB broth (Luria-Bertani lysate broth). Incubate in a shaker at 37°C for 16-24 hours. Then, use M9 medium to serially dilute the bacterial suspension to a bacterial concentration of 1×10⁶ CFU / mL.
[0064] B. In a 96-well plate, set up 3 parallel groups: initial group, first sample group and second sample group. Add 200 μL of TMVCP-R8 prepared in Example 1 with a concentration of 250 mg / mL to each well of the initial group, add 200 μL of TMVCP-R8 diluted by 1 to each well of the first sample group, and add 200 μL of TMVCP-R8 diluted by 2 to each well of the second sample group.
[0065] C. Add 100 μL of the diluted bacterial solution from step A to each sample well, mix well with a pipette, and place in an incubator at 37°C for 24 hours. Determine the inhibition rate by measuring the OD value.
[0066] See Figure 3It can be seen that the antibacterial rate of TMVCP-R8 is significantly higher than that of R8. When the concentration of TMVCP-R8 is greater than 16 μg / mL, the antibacterial rate is >60%, and when the concentration reaches 30 μg / mL, the antibacterial rate is >99%. In contrast, the antibacterial effect of R8 at the same concentration is significantly worse than that of TMVCP-R8. It can be concluded that modifying TMVCP with R8 significantly improves its antibacterial effect.
[0067] Because proteins have better biocompatibility, modifying proteins with antimicrobial peptides such as R8 improves their biocompatibility, allowing more antimicrobial peptides to enter the body and interact with the lesion site for sterilization, thus achieving a good bactericidal effect. The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A protein derivative modified with an antibacterial polypeptide, characterized in that, It consists of a protein and an antimicrobial peptide, which are linked by a click chemical reaction between an alkyne group and an azide group; the antimicrobial peptide is a cathelicidin-type antimicrobial peptide or a defensin-type antimicrobial peptide, and the protein is the capsid protein of tobacco mosaic virus.
2. The antimicrobial peptide-modified protein derivative as described in claim 1, characterized in that, The antimicrobial peptide is cecropins peptide, magainins peptide, apidaecins peptide, bactenecin peptide, defensins peptide, or nisin peptide.
3. The antimicrobial peptide-modified protein derivative as described in claim 2, characterized in that, The antimicrobial polypeptide is R8 polypeptide, CM15 polypeptide or P7 polypeptide; The amino acid sequence of R8 is shown in SEQ ID NO.1, the amino acid sequence of CM15 is shown in SEQ ID NO.2, and the amino acid sequence of P7 is shown in SEQ ID NO.
3. SEQ ID NO.1: RRRRRRRRR; SEQ ID NO.2: KWKLFKKIGAVLKVL; SEQ ID NO. 3: GLRRALLRLLRSLRRRLLLRA.
4. A method for preparing an antimicrobial polypeptide-modified protein derivative according to any one of claims 1 to 3, comprising the following steps: 1) The phenolic hydroxyl groups on the surface of tobacco mosaic virus were alkynylated to obtain alkynyl-modified tobacco mosaic virus; 2) The alkynyl-modified tobacco mosaic virus obtained in step 1) is disassembled under acidic conditions to obtain the alkynyl-modified tobacco mosaic virus capsid protein. 3) The tobacco mosaic virus capsid protein modified with alkyne obtained in step 2) is then subjected to a click chemical reaction with an antimicrobial polypeptide with an azide group at the end to obtain the protein derivative.
5. The method for preparing the antimicrobial peptide-modified protein derivative as described in claim 4, characterized in that, The alkynylation reaction is carried out by reacting tobacco mosaic virus with 3-ethynylaniline and NaNO2.
6. The method for preparing the antimicrobial peptide-modified protein derivative as described in claim 4, characterized in that, The disassembly involves mixing the alkyne-modified tobacco mosaic virus with an acid, wherein the acid is any one of formic acid, acetic acid, hydrochloric acid, or nitric acid. After the virus is disassembled, the RNA precipitate is removed, and then the acid is removed by passing the virus through a desalting column to obtain the alkyne-modified tobacco mosaic virus capsid protein.
7. The method for preparing the antibacterial polypeptide-modified protein derivative as described in claim 6, characterized in that, The acid is acetic acid.
8. The method for preparing the antimicrobial peptide-modified protein derivative as described in claim 4, characterized in that, The click chemical reaction is carried out under the catalysis of metal ions.
9. The method for preparing the antimicrobial polypeptide-modified protein derivative as described in claim 8, characterized in that, The metal ion is Cu(I).
10. The method for preparing the antibacterial polypeptide-modified protein derivative as described in claim 8, characterized in that, The click chemistry reaction is carried out in the presence of aminoguanidine hydrochloride, sodium ascorbate aqueous solution, and CuSO4.
11. The method for preparing the antibacterial polypeptide-modified protein derivative as described in claim 4, characterized in that, The click chemical reaction and the temperature at which the virus disassembles under acidic conditions are 0-20°C.
12. The method for preparing the antimicrobial peptide-modified protein derivative as described in claim 11, characterized in that, The click chemical reaction and the temperature at which the virus disassembles under acidic conditions are both 4°C.
13. The method for preparing the antimicrobial polypeptide-modified protein derivative as described in claim 4, characterized in that, After the click reaction is complete, purification is performed, which specifically includes the following steps: dialyzing with a dialysis bag for 30-60 hours.
14. Use of a protein derivative modified with an antimicrobial peptide according to any one of claims 1-3 or a protein derivative modified with an antimicrobial peptide prepared by the method according to any one of claims 4-13, characterized in that, It is used to prepare drugs with bactericidal effects.
15. A bactericidal drug composition, characterized in that, It includes protein derivatives modified with antimicrobial peptides as described in any one of claims 1-3 or protein derivatives modified with antimicrobial peptides prepared by the method described in any one of claims 4-13.
16. The bactericidal pharmaceutical composition according to claim 15, characterized in that, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.
17. The bactericidal pharmaceutical composition according to claim 16, characterized in that, The excipients are stabilizers, diluents, dispersants, suspending agents, and / or thickeners.
18. The bactericidal pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition is an oral tablet, capsule, pill, powder, sustained-release preparation, solution, or suspension; a sterile solution, suspension, or emulsion for parenteral injection; an ointment or cream for external use; or a suppository for rectal administration.