A self-assembling antibacterial peptide against drug-resistant bacteria, its preparation method and application

Abstract

The present invention discloses a self-assembling antibacterial peptide against drug-resistant bacteria, its preparation method and application, belonging to the field of biotechnology. It includes a branched chain A and a branched chain B. The amino acid sequence of the branched chain A is: RRRCFFF, and the amino acid sequence of the branched chain B is: FFFCRRR. The branched chain A and the branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues. The antibacterial peptide 3RF of the present invention self-assembles into a nanofiber structure in PB solution, and at the same time has strong antibacterial activity against drug-resistant bacteria, and the antibacterial peptide 3RF has almost no hemolytic toxicity, and has extremely high application potential in the treatment of drug-resistant bacterial infections.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a self-assembling antibacterial peptide against drug-resistant bacteria, a preparation method thereof, and an application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are regarded as an attractive and promising strategy to combat the formidable multi-drug resistant bacterial infections. Different from traditional antibiotics that act on limited fixed targets, antimicrobial peptides mainly cause bacterial death by physically disrupting the integrity of the bacterial membrane. This antibacterial mechanism reduces the probability of bacteria developing resistance to antimicrobial peptides. Since the ordered nanostructures of self-assembling antimicrobial peptides contribute to improving the activity and stability of antimicrobial peptides, thereby enhancing the activity against drug-resistant bacteria. In addition, self-assembling nano-delivery systems can alleviate the pharmacokinetic / pharmacodynamic defects of antimicrobial peptides, improve their shelf life, stability, and bioavailability, and prolong the half-life of antimicrobial peptides. Therefore, self-assembling antimicrobial peptides have great potential in the treatment of bacterial infections, especially drug-resistant bacterial infections. However, most chemically modified self-assembling antimicrobial peptides have toxic effects that cannot be ignored, while self-assembling antimicrobial peptides driven by intermolecular forces of amino acids often have weak activities, making them unable to be used as antibacterial agents against drug-resistant bacteria. Therefore, it is particularly necessary to develop non-toxic and highly effective antimicrobial peptides against drug-resistant bacteria. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide a self-assembling antibacterial peptide 3RF against drug-resistant bacteria, a preparation method thereof, and an application thereof, which solves the problems of high toxicity of existing self-assembling antibacterial peptides and poor activity against drug-resistant bacteria, and has the ability to inhibit drug-resistant bacteria with low toxicity and high efficiency.

[0004] The technical solution adopted by the present invention is as follows: A self-assembling antibacterial peptide 3RF against drug-resistant bacteria, including a branched chain A and a branched chain B. The amino acid sequence of the branched chain A is: RRRCFFF, and the amino acid sequence of the branched chain B is: FFFCRRR. The branched chain A and the branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues.

[0005] Further, the molecular formula of the antibacterial peptide 3RF as described above is shown in formula (I):

[0006]

[0007] Further, the antibacterial peptide 3RF as described above is dissolved in a PB buffer solution with a concentration of 10 mM and a pH of 7.4. The concentration of the antibacterial peptide 3RF is 32 - 256 μM, and it is incubated at room temperature for 12 hours to self-assemble into a nanostructure.

[0008] Another object of the present invention is to provide a method for preparing a self-assembled antibacterial peptide 3RF against drug-resistant bacteria as described above, as follows: Design two branched chains. The sequence of branched chain A is: RRRCFFF, and the sequence of branched chain B is: FFFCRRR. Branched chain A and branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues, and the self-assembly between branched chain A and branched chain B is driven by cation-π interaction; Solid-phase chemical synthesis method is used to synthesize branched chain A and branched chain B respectively, and then branched chain A and branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues. The polypeptide is identified by mass spectrometry and purified by reverse-phase high-performance liquid chromatography to obtain the polypeptide, and then the self-assembled morphology of the polypeptide is observed, the antibacterial activity is measured, and the hemolytic toxicity is measured. Finally, it is named antibacterial peptide 3RF.

[0009] Another object of the present invention is to provide the use of a self-assembled antibacterial peptide 3RF against drug-resistant bacteria as described above in the preparation of a drug for treating infectious diseases caused by drug-resistant bacteria, and the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus, kanamycin-resistant Escherichia coli or spectinomycin-resistant Pseudomonas aeruginosa.

[0010] The present invention has the following advantages and beneficial effects: The self-assembled antibacterial peptide 3RF of the present invention can form a stable fibrous structure, has excellent inhibitory effects on drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, kanamycin-resistant Escherichia coli and spectinomycin-resistant Pseudomonas aeruginosa, and has almost no toxicity to human red blood cells. Description of the Drawings

[0011] Figure 1 It is the mass spectrum of antibacterial peptide 3RF;

[0012] Figure 2 It is the high-performance liquid chromatography of antibacterial peptide 3RF;

[0013] Figure 3 It is the minimum aggregation concentration diagram (CAC) of antibacterial peptide 3RF;

[0014] Figure 4 It is the hemolytic activity diagram of antibacterial peptide 3RF. Detailed Embodiments

[0015] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0016] Example 1

[0017] Design of Antibacterial Peptide

[0018] Design principle: Design two branched chains, branched chain A with the sequence: RRRCFFF, and branched chain B with the sequence: FFFCRRR. Branched chain A and branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues. The polypeptide sequence drives its self-assembly through cation-π interaction (R-F); then, branched chain A and branched chain B are synthesized separately by solid-phase chemical synthesis method, and then branched chain A and branched chain B are connected by a disulfide bond formed between the thiol groups of two cysteine residues.

[0019] The amino acid sequence of antimicrobial peptide 3RF is as follows:

[0020]

[0021] Table 1 Amino acid sequences of antimicrobial peptides

[0022]

[0023] The molecular formula of self-assembled antimicrobial peptide 3RF is shown in formula (I),

[0024]

[0025] Example 2

[0026] I. Synthesis of antimicrobial peptide by solid-phase chemical synthesis method

[0027] 1. First, Fmoc-Arg(pbf)-OH is attached to Rink resin. After 30 minutes of piperidine deprotection reaction, piperidine is removed and washed with dimethylformamide (DMF), and the deprotection color is detected by ninhydrin. Then the subsequent linear amino acids are connected in sequence until Fmoc-Trp(boc)-OH at the N-terminus is reached. After removing the Fmoc at the N-terminus, FFFC(Trt)R(pbf)R(pbf)R(pbf) is obtained. The above resin is used to cleave the monomeric peptide from the resin with 95% TFA, and at the same time, all side-chain protecting groups of the sequence are removed. The crude product of FFFC(Trt)R(pbf)R(pbf)R(pbf) is obtained, purified by liquid phase, and freeze-dried.

[0028] 2. Prepare R(pbf)R(pbf)R(pbf)C(Trt)FFF by the same method as in step 1. The monomeric peptide is cleaved from the resin, and at the same time, all side-chain protecting groups of the sequence are removed. The crude product of the monomer R(pbf)R(pbf)R(pbf)C(Trt)FFF is obtained, purified by liquid phase, and freeze-dried.

[0029] 3. Oxidative synthesis: Dissolve the two monomeric peptides, adjust the pH to 7.5 - 8 with ammonium bicarbonate solution after mixing, stir for 1 hour, monitor the oxidation situation by mass spectrometry and liquid phase, and after complete oxidation, purify by HPLC and freeze-dry to obtain the target product.

[0030] II. Purification and Identification:

[0031] 1. Detection of the crude product by MS: Take a small amount of the crude product, dissolve it, and use LC-MS to determine the molecular weight. After the molecular weight (as Figure 1 shown) is basically consistent with the theoretical molecular weight in Table 1, then proceed with purification.

[0032] 2. Purification: Purify the polypeptide using a high-performance liquid chromatograph to obtain a polypeptide with a purity > 95%; as Figure 2 shown, the high-performance liquid chromatogram of the antimicrobial peptide 3RF.

[0033] Example 3

[0034] Determine the critical aggregation concentration (CAC) of the nanopolypeptide using the 1-anilino-8-naphthalenesulfonate (ANS) fluorescent probe. Preparation of the dye: Dissolve the ANS powder in N,N-dimethylformamide (DMF) to a concentration of 40 mM, and store it in the dark at low temperature for later use. Dissolve different concentrations of the antimicrobial peptide 3RF (1 - 256 μM) in PB (10 mM, pH 7.4) buffer, and incubate at room temperature for 12 hours. Subsequently, mix different concentrations of the antimicrobial peptide 3RF with ANS. Use a microplate reader to monitor the change in fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 420 - 670 nm, with a slit width of 2 nm; as Figure 3 shown, it can be observed that there are obvious fluctuations in the fluorescence intensity, indicating that the antimicrobial peptide 3RF has a certain ability of aggregation and self-assembly.

[0035] Example 4

[0036] Determination of the antibacterial activity of the antimicrobial peptide 3RF

[0037] The minimum inhibitory concentration (MIC) of the peptide was determined using the standard microbroth dilution method. Dilute the bacteria in the logarithmic phase to ~2×10 5 CFU / mL. Add 50 μl of different concentrations of the antimicrobial peptide 3RF (final concentration 1 - 128 μM) and an equal volume of the bacterial suspension to each well of a 96-well plate. At the same time, set up a negative control (only medium) and a positive control (bacteria and medium), and then place the 96-well plate in a 37 °C constant temperature incubator for 18 - 20 hours. Use a microplate reader to measure the absorbance at 492 nm. Set two parallels for each test, and these tests were repeated at least three times. The results are shown in Table 2.

[0038] Table 2 Minimum inhibitory concentration (μM) of the antimicrobial peptide 3RF

[0039]

[0040] As can be seen from Table 2, the antimicrobial peptide 3RF has excellent activity against methicillin-resistant Staphylococcus aureus (MRSA), kanamycin-resistant Escherichia coli M15, and spectinomycin-resistant Pseudomonas aeruginosa 109004, with a minimum inhibitory concentration of 2 - 4 μM.

[0041] Example 5

[0042] Determination of the hemolytic activity of antimicrobial peptide 3RF

[0043] Preparation of blood: Fresh human blood was drawn and centrifuged at 3000 - 3500 rpm for 10 minutes at 4°C. The supernatant was aspirated. Phosphate buffer solution (PBS, pH = 7.4) was filtered through a 0.22 μM water-based filter membrane and then added to the red blood cells. The red blood cells were centrifuged and washed three times, and finally resuspended in 10 volumes of PBS. Dilution of antimicrobial peptide 3RF: PBS was added to columns 1 - 12 in a 96-well plate, with 90 μL added to the first column and 50 μL added to the other columns. 10 μL of the dissolved nanoparticle peptide solution was aspirated and added to the first column of the 96-well plate, and serially diluted to the tenth column. Addition of red blood cells: The resuspended red blood cells were added to columns 1 - 12 of the 96-well plate, 50 μL per well. 0.1% Triton X-100 was added to column 12 as a positive control (100% hemolysis), and column 11 was used as a negative control. The 96-well plate was placed in an incubator at 37°C for 1 hour, and then centrifuged at 1000 × g for 5 minutes at 4°C. 50 μL of the supernatant from each well was aspirated and transferred to a new 96-well plate. Result determination: The absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Two parallels were set for each test, and these tests were repeated at least three times.

[0044] As Figure 4 shown, antimicrobial peptide 3RF exhibited negligible hemolytic effects at all tested concentrations, indicating that antimicrobial peptide 3RF has good biocompatibility.

[0045] In summary, antimicrobial peptide 3RF has excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), kanamycin-resistant Escherichia coli M15, and spectinomycin-resistant Pseudomonas aeruginosa 109004, has little toxicity to human red blood cells, and has extremely high application potential.

Claims

1. A self-assembling antimicrobial peptide 3RF for resisting drug-resistant bacteria, characterized in that: Its molecular formula is shown in formula (I), including branch A and branch B, the amino acid sequence of branch A is: RRRCFFF, the amino acid sequence of branch B is: FFFCRRR, and the branch A and branch B are connected by a disulfide bond formed between the thiol groups in two cysteine ​​residues.

2. The self-assembly method of a self-assembling antimicrobial peptide 3RF against drug-resistant bacteria according to claim 1, characterized in that: The nano self-assembly conditions are as follows: dissolving the antimicrobial peptide 3RF in a PB buffer solution with a concentration of 10 mM and a pH of 7.4, wherein the concentration of the antimicrobial peptide 3RF is 32-256 μM, incubating at room temperature for 12 hours, and self-assembling into a nanostructure.

3. The method for preparing a self-assembling antimicrobial peptide 3RF against drug-resistant bacteria according to claim 1, characterized in that: The method is as follows: two branches are designed, the sequence of branch A is: RRRCFFF, the sequence of branch B is: FFFCRRR, branch A and branch B are connected through a disulfide bond formed between thiol groups in two cysteine ​​residues, and the self-assembly of branch A and branch B is driven by cation-π interaction; branch A and branch B are synthesized separately by solid phase chemical synthesis, and then branch A and branch B are connected through a disulfide bond formed between thiol groups in two cysteine ​​residues, the polypeptide is identified by mass spectrometry and purified by reversed-phase high performance liquid chromatography to obtain the polypeptide, and then the polypeptide self-assembly morphology is observed, the antibacterial activity is determined, and the hemolytic toxicity is determined, and finally it is named antimicrobial peptide 3RF.

4. Use of the self-assembling antimicrobial peptide 3RF against drug-resistant bacteria according to claim 1 in the preparation of a drug for treating infectious diseases caused by drug-resistant bacteria, wherein the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus, kanamycin-resistant Escherichia coli or spectinomycin-resistant Pseudomonas aeruginosa.

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