Low-cost antibacterial combined peptide and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a low-cost antibacterial combined peptide and application thereof.The amino acid sequence of the low-cost antibacterial combined peptide is one of Meta219: X3RWRX3X1RX3LRLLR, Meta225: VLRLWRX3X1RRLLX3 and Meta206: RRX2X3RWLIRRLLX1, X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; the Meta219, the Meta225 and the Meta206 are respectively a mixture of all selected sites in the random sites. The combined peptide can have a powerful antibacterial effect on various bacteria, the preparation cost of the antibacterial combined peptide is greatly reduced by 30 times compared with that of a traditional single peptide, the economic feasibility and the popularization value of the antibacterial combined peptide are remarkably improved, and the practical possibility is provided for clinical practice of antibacterial peptide products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a low-cost antibacterial combined peptide and application thereof. BACKGROUND

[0002] The problem of bacterial drug resistance caused by antibiotic abuse has become a major difficulty in modern clinical treatment, which seriously restricts the control effect of infectious diseases. Antibacterial peptides are considered to be a powerful alternative to antibiotics due to their unique antibacterial mechanism of killing bacteria by destroying bacterial cell membranes, and have unique advantages in dealing with drug-resistant bacteria. However, the high synthesis cost of antibacterial peptides limits their widespread use in clinical practice.

[0003] CN 114605496 A discloses a polypeptide and its application, an antibacterial drug and a preparation method thereof. The sequence of the amino acid backbone of the polypeptide is LysLysLysHisLysLysLys or ArgArgLysHisLysArgArg. The sequence and structure of the polypeptide are simple, and the polypeptide can be used for antibacterial. The total number of amino acids is small, the preparation cost is reduced under the premise of ensuring good performance, the hydrophobic beta-fold sequence is used as the side chain, the polypeptide is driven to form a regular assembly structure, and the physiological stability of the assembly is enhanced. After the polypeptide is assembled with metal ions, ligand drugs and other components through coordination, the loading capacity is high, and the drug can be delivered to the deep layer, and the bacteria show active targeting.

[0004] CN103590116A discloses a method for screening antibacterial peptide lead compounds with low cost and high throughput. The method is characterized by: using bacteria as target cells, screening polypeptides that specifically bind to the surface of bacterial cells from a phage display random peptide library, and performing high-throughput screening of antibacterial peptide lead compounds through positive cloning, ssDNA sequencing, synthesis of affinity peptides and antibacterial activity determination. The method uses E. coli cells as target cells, and screens polypeptides that specifically bind to the surface of bacterial cells from a phage display random peptide library. The amino acid sequence of one 10-peptide affinity peptide is QKRPRVRLSA. The present application can screen polypeptides with specific biological activities such as antibacterial peptides from random peptides of a specific length by affinity screening method from a phage peptide library, and can quickly screen antibacterial peptide lead compounds by this strategy, and improve the speed of creating new antibacterial peptide drugs.

[0005] However, the above-mentioned method has a relatively complex preparation process and high cost, and is difficult to be popularized and applied in the market. SUMMARY

[0006] The application provides a kind of combination peptide with broad-spectrum antibacterial efficacy, the combination peptide can have strong antibacterial effect on various bacteria, the preparation cost of antibacterial combination peptide in the application is reduced by 30 times compared with traditional single peptide, significantly improves its economic feasibility and popularization value, provides practical possibility for antibacterial peptide product to enter clinical practice.

[0007] To achieve the above object, the technical scheme adopted by the application is: A kind of low-cost antibacterial combination peptide, characterized in that the amino acid sequence of the low-cost antibacterial combination peptide is one of the following: Meta219: X3RWRX3X1RX3LRLLR Meta225: VLRLWRX3X1RRLLX3 Meta206: RRX2X3RWLIRRLLX1 Wherein, X1, X2 and X3 is a random site, X1 is L or I, X2 is V or L, X3 is I or V; Meta219, Meta225 and Meta206 are mixtures of all selected random sites.

[0008] The application also provides the use of the low-cost antibacterial combination peptide in the preparation of products or drugs for treating and / or preventing bacterial infections.

[0009] Preferably, the product includes one or more of the coating of a medical device, the coating of an implantable medical device, a dressing, a food preservative, a pet disinfectant.

[0010] The bacteria are gram-positive bacteria and / or gram-negative bacteria.

[0011] The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis and Bacillus subtilis.

[0012] The application also provides a nucleotide sequence encoding the low-cost antibacterial combination peptide of claim 1, which includes DNA and RNA.

[0013] The application also provides a kind of expression vector containing the nucleotide sequence.

[0014] The application also provides a kind of composition containing the low-cost antibacterial combination peptide.

[0015] The composition contains any one or more of a pharmaceutically or food acceptable carrier, excipient, diluent, adjuvant or vehicle.

[0016] The application also provides an antibacterial product comprising the low-cost antibacterial combined peptide, the nucleotide sequence or the composition.

[0017] Compared with the prior art, the application has the following beneficial effects: The antibacterial combined peptide in the application has excellent antibacterial efficacy on various bacteria such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis and Bacillus subtilis, and the minimum inhibitory concentration can be reduced to 1 μg / mL; the preparation method is simple, and large-scale and low-cost preparation of the antibacterial peptide can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The ability of Meta219, Meta225 and Meta206 at different concentrations to penetrate the outer membrane of Acinetobacter baumannii pathogen.

[0019] Figure 2 The ability of Meta219, Meta225 and Meta206 at different concentrations to depolarize the outer membrane of Acinetobacter baumannii pathogen. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application. Those skilled in the art can modify or replace equivalently without departing from the spirit and scope of the technical scheme of the application, which should be covered within the protection scope of the application.

[0021] The raw materials used in the following specific embodiments are all purchased from the market.

[0022] Example 1 The application adopts a simultaneous feeding strategy of a group of amino acids, that is, when the amino acid residues are introduced, a plurality of amino acid monomers are introduced simultaneously according to the designed proportion, instead of the traditional step-by-step coupling mode of individual monomers. This strategy can significantly reduce the number of coupling and washing cycles, thereby reducing the amount of reagents and reaction time. In addition, the application simplifies the purification step after synthesis, adopts high-efficiency crude purification combined with necessary fine purification, avoids multiple high-cost purification processes, and significantly reduces production consumables and labor costs.

[0023] The synthesis of the antibacterial combined peptide specifically includes: 1. Swelling of the dendrimer: 2-chlorotrityl chloride resin (2-CTC) resin is weighed, soaked with dichloromethane (DCM) to swell the resin, for 1 hour. Then the resin is washed 3 times with dimethyl sulfoxyl amine (DMF) to remove residual solvents and impurities.

[0024] 2. Attach first amino acid: Take 1 or 2 protected amino acids (e.g., 1 eq of amino acid A and 1 eq of amino acid B, with a 1:1 molar ratio of the two amino acids) and react with 1.5 eq of diisopropylethylamine (DIEA) in DMF for 2 hours to attach the amino acids to the resin.

[0025] 3. Cap: After the resin is drained, wash it 3 times with DMF, then add methanol and DIEA for capping, and react for 1 hour to protect the amino groups on the resin.

[0026] 4. Remove Fmoc protecting group: Use 20% piperidine in DMF to remove the Fmoc protecting group, react for 10 minutes, and repeat 2 times to ensure complete removal of the protecting group.

[0027] 5. Attach new amino acid: After washing the resin, add 3 eq of 1 or 2 amino acids (e.g., amino acid C and amino acid D, with a 1:1 molar ratio of the two amino acids) and 3 eq of HOBT and 3 eq of DIC, and react in DMF for 1.5 hours to attach the amino acids to the peptide chain.

[0028] 6. Repeat steps 4 and 5 until all the desired amino acids are attached and the Fmoc protecting group at the N-terminus is removed, wash the resin and drain it to prepare for the subsequent deprotection step.

[0029] 7. Deprotection and peptide chain cleavage: Use 95% trifluoroacetic acid (TFA), 2% Tis (thiazolone), and 2% EDT (ethylenediaminetetraacetic acid), and 1% water to react for 2 hours to cleave the protecting groups of the resin and the side chains of the polypeptide.

[0030] 8. Filtration and precipitation: Filter the resin, wash the filtrate with ice ethyl ether, centrifuge, and keep the precipitate, which is the crude product, to obtain the final random peptide mixture after freeze-drying.

[0031] Example 2 Analysis of the antibacterial activity of three AMPCs (antibacterial combination peptides) Step 1, 7 strains of bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Acinetobacter baumannii ATCC19606, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 14028, Enterococcus faecalis ATCC29212, Bacillus subtilis ATCC6633) were inoculated into sterilized MHB medium at 37°C, 250 rpm, and cultured overnight.

[0032] Step 2, 3 kinds of antibacterial peptide were respectively prepared with PBS to 128 µg / mL, and diluted with PBS to 128, 64, 32, 16, 8, 4, 2, 1 µg / mL by 2 times of continuous dilution, 50 µL of each concentration was added to A-H wells of 96-well plate. Each antibacterial peptide was repeated 3 groups, and added to column 1-9 wells.

[0033] Step 3, the 7 kinds of bacteria after culture were respectively diluted with PBS to 5×105 CFU / mL. (Because there are many kinds of bacteria, one strain of bacteria was used for each plate, and Staphylococcus aureus was used as an example) and 50 µL of bacterial diluent was added to the A-F wells with antibacterial peptide diluent. At this time, the concentration (µg / mL) of the peptide to be tested is shown in Table 1.

[0034] Table 1 Final concentration of antibacterial peptide Meta219, Meta225 and Meta206 in MIC determination experiment, unit µg / mL Step 4, 100 µL of PBS solution was added to A10-H10 wells as negative control group, and 100 µL of 5×105 CFU / mL bacterial solution was added to A11-H11 wells as positive control. The 96-well plate was sealed with sealing film and placed in a self-sealing bag in a constant temperature incubator at 37°C for 18 hours overnight culture. The OD600 value of A1-G9 wells was measured by enzyme marker, and the minimum OD600 value corresponding to the minimum concentration in each antibacterial peptide group was the minimum inhibitory concentration (MIC) value of the antibacterial peptide corresponding to the bacteria, and the results are shown in Table 2.

[0035] Table 2 MIC of antibacterial peptides Meta219, Meta225 and Meta206 against 7 kinds of pathogens, unit µg / mL The antibacterial combination peptides Meta219, Meta225 and Meta206 are compositions of various sequences of antibacterial peptides, which are directly obtained by synthesis rather than mixing after synthesizing various antibacterial peptides. In the amino acid sequences of the combination peptides in the table, the amino acids in the brackets are randomly one, such as (LV) indicating L or V. For example, the specific sequence of the antibacterial combination peptide can be as follows: Meta219 is a composition of antibacterial peptides with amino acid sequences as shown in SEQ ID NO. 1-16, specifically: SEQ ID NO. 1: IRWRVLRVLRLLR SEQ ID NO. 2: IRWRVLRILRLLR SEQ ID NO. 3: IRWRVIRVLRLLR SEQ ID NO. 4: IRWRVIRILRLLR SEQ ID NO. 5: IRWRILRVLRLLR SEQ ID NO. 6: IRWRILRILRLLR SEQ ID NO. 7: IRWRIIRVLRLLR SEQ ID NO. 8: IRWRIIRILRLLR SEQ ID NO. 9: VRWRVLRVLRLLR SEQ ID NO. 10: VRWRVLRILRLLR SEQ ID NO. 11: VRWRVIRVLRLLR SEQ ID NO. 12: VRWRVIRILRLLR SEQ ID NO. 13: VRWRILRVLRLLR SEQ ID NO. 14: VRWRILRILRLLR SEQ ID NO. 15: VRWRIIRVLRLLR SEQ ID NO. 16: VRWRIIRILRLLR Meta225 is a composition of antibacterial peptides with amino acid sequences as shown in SEQ ID NO. 17-24, specifically: SEQ ID NO. 17: VLRLWRILRRLLI SEQ ID NO. 18: VLRLWRILRRLLV SEQ ID NO. 19: VLRLWRIIRRLLI SEQ ID NO. 20: VLRLWRIIRRLLV SEQ ID NO. 21: VLRLWRVLRRLLI SEQ ID NO. 22: VLRLWRVLRRLLV SEQ ID NO. 23: VLRLWRVIRRLLI SEQ ID NO. 24: VLRLWRVIRRLLV Meta206 is a composition of antibacterial peptides with amino acid sequences as shown in SEQ ID NO. 25-32, specifically: SEQ ID NO. 25: RRLIRWLIRRLLL SEQ ID NO. 26: RRLIRWLIRRLLI SEQ ID NO. 27: RRLVRWLIRRLLL SEQ ID NO. 28: RRLVRWLIRRLLI SEQ ID NO. 29: RRVIRWLIRRLLL SEQ ID NO. 30: RRVIRWLIRRLLI SEQ ID NO. 31: RRVVRWLIRRLLL SEQ ID NO. 32: RRVVRWLIRRLLI Table 2 shows that the three antibacterial combination peptides of the present application all exhibit good bacteriostatic ability against seven different bacteria, with strong efficacy and wide universality. Among them, Meta206 has more obvious bacteriostatic effect on Bacillus subtilis (Bacillus subtilis ATCC6633), with MIC of only 1 µg / mL.

[0036] Example 3 Investigation of the antibacterial mechanism of AMPCs I. NPN (1-naphthyl amino benzene) uptake assay to determine the effect of antibacterial combination peptides on the cell membrane permeability of Acinetobacter baumannii.

[0037] Harvested log-phase microbial cells (5,000 rpm, 5 min) were diluted to OD600=0.2 in 5 mM HEPES buffer (pH=7.4, containing 5 mM glucose). The bacterial suspension was further incubated with 10 mM NPN in the dark for 30 min. Subsequently, different concentrations of peptides prepared in Example 1 were added (final concentration of peptides was 128-1 pg / mL, 2-fold dilution), and NPN fluorescence was detected (excitation λ=350 nm, emission λ=420 nm), and the results are shown in Figure 1 As shown in FIG. 2, the detected fluorescence signal was significantly increased after AMPCs treatment, indicating that the outer membrane permeability of bacteria was significantly enhanced, and AMPCs produced bactericidal efficacy by membrane interaction mechanism.

[0038] II. Cell fluorescence probe DiSC3-5 staining assay for the effect of antibacterial combination peptides on the inner membrane depolarization of Acinetobacter baumannii.

[0039] Harvested mid-log-phase bacteria were washed 3 times with 5 mM HEPES (4- hydroxyethylpiperazine ethanesulfonic acid) buffer, then resuspended in buffer containing 0.1% glucose to OD 600 =0.07. DiSC3-5 was added to a final concentration of 0.4 mM, and the mixture was incubated at 37°C for 90 min in the dark. Subsequently, 300 mM KCl was added and incubated for 20 min, then treated with different concentrations of peptides. Membrane potential changes were evaluated by monitoring fluorescence (excitation wavelength 622 nm, emission wavelength 670 nm) until the fluorescence signal was stable. The results were recorded, background fluorescence was subtracted, and the data were normalized. The results are shown in Figure 2 As shown in FIG. 4, the detected fluorescence signal was significantly increased after AMPCs treatment, indicating that the inner membrane depolarization was obvious, and the potential was lost, and AMPCs produced bactericidal efficacy by membrane interaction mechanism.

Claims

1. A class of low-cost antimicrobial combination peptides, characterized in that, The amino acid sequence of the low-cost antimicrobial combinatorial peptide is one of the following: Meta219: X3RWRX3X1RX3LRLLR Meta225: VLRLWRX3X1RRLLX3 Meta206: RRX2X3RWLIRRLLX1 Among them, X1, X2 and X3 are random sites, X1 is L or I, X2 is V or L, and X3 is I or V; Meta219, Meta225 and Meta206 are mixtures of all selected random sites.

2. The use of the low-cost antimicrobial combination peptide according to claim 1 in the preparation of products or medicines for treating and / or preventing bacterial infections.

3. The application according to claim 2, characterized in that, The products include one or more of the following: coatings for medical devices, coatings for implantable medical devices, dressings, food preservatives, and pet disinfectants.

4. The application according to claim 2, characterized in that, The bacteria are Gram-positive and / or Gram-negative.

5. The application according to claim 2, characterized in that, The bacteria include one or more of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Bacillus subtilis.

6. The nucleotide sequence encoding the low-cost antimicrobial combinatorial peptide of claim 1, characterized in that, The nucleotide sequence includes DNA and RNA.

7. An expression vector containing the nucleotide sequence of claim 6.

8. A composition comprising the low-cost antimicrobial combination peptide of claim 1.

9. The composition according to claim 8, characterized in that, The composition contains one or more of a pharmaceutically or food-acceptable carrier, excipient, diluent, adjuvant, or medium.

10. An antibacterial product, characterized in that, Includes the low-cost antimicrobial combination peptide of claim 1, the nucleotide sequence of claim 7, or the composition of claim 8.

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