CN118930607B

CN118930607B - Self-assembled antibacterial peptide for resisting drug-resistant bacteria, and preparation method and application thereof

Info

Publication number: CN118930607B
Application number: CN202410971569.6A
Authority: CN
Inventors: 单安山; 李国雨; 陈雯雯; 邵长轩
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2024-07-19
Filing date: 2024-07-19
Publication date: 2025-06-10
Anticipated expiration: 2044-07-19
Also published as: CN118930607A

Abstract

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

Description

Self-assembled antibacterial peptide for resisting drug-resistant bacteria, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a self-assembled antibacterial peptide for resisting drug-resistant bacteria, and a preparation method and application thereof.

Background

Antibacterial peptides (AMPs) are regarded as an attractive and promising strategy to combat terrible multi-drug resistant bacterial infections. Unlike traditional antibiotics which act on limited immobilized targets, antibacterial peptides lead to bacterial death mainly by physically destroying the bacterial membrane integrity. This antibacterial mechanism results in a reduced probability of bacterial resistance to antibacterial peptides. The ordered nanostructure of the self-assembled antibacterial peptide is beneficial to improving the activity and stability of the antibacterial peptide, so that the activity on drug-resistant bacteria is enhanced. In addition, the self-assembled nano drug delivery system can alleviate the pharmacokinetics/pharmacodynamics defects of the antibacterial peptide, improve the shelf life, stability and bioavailability of the antibacterial peptide and prolong the half-life of the antibacterial peptide. Self-assembled antimicrobial peptides therefore have great potential in the treatment of bacterial infections, particularly drug resistant bacterial infections. However, most chemically modified self-assembled antimicrobial peptides have a non-negligible toxic effect, and self-assembled antimicrobial peptides driven by intermolecular forces of amino acids tend to have weak activity, making them impossible as antimicrobial agents against drug-resistant bacteria. Thus, there is an additional need to develop antimicrobial peptides that are non-toxic and have high efficacy against drug-resistant bacteria.

Disclosure of Invention

Based on the defects, the invention aims to provide the self-assembled antibacterial peptide 3RF for resisting the drug-resistant bacteria, and the preparation method and the application thereof, solve the problems of high toxicity and poor activity on the drug-resistant bacteria of the existing self-assembled antibacterial peptide, and have the capabilities of low toxicity and high efficiency for inhibiting the drug-resistant bacteria.

The self-assembled antibacterial peptide 3RF for resisting drug-resistant bacteria comprises a branched chain A and a branched chain B, wherein the amino acid sequence of the branched chain A is RRRCFFF, the amino acid sequence of the branched chain B is FFFCRRR, and the branched chain A and the branched chain B are connected through disulfide bonds formed between thiol groups in two cysteine residues.

Further, the molecular formula of the antibacterial peptide 3RF is shown as the formula (I):

Further, the antibacterial peptide 3RF is dissolved in PB buffer solution with the concentration of 10mM and the pH of 7.4, the concentration of the antibacterial peptide 3RF is 32-256 mu M, and the antibacterial peptide 3RF is incubated for 12 hours at room temperature to self-assemble into a nano structure.

The invention further aims to provide a preparation method of the self-assembled antibacterial peptide 3RF for resisting drug-resistant bacteria, which comprises the steps of designing two branched chains, namely RRRCFFF a sequence of the branched chain A, FFFCRRR a sequence of the branched chain B, connecting the branched chain A and the branched chain B through disulfide bonds formed between thiol groups in two cysteine residues, driving the branched chain A and the branched chain B to self-assemble through cation-pi action, respectively synthesizing the branched chain A and the branched chain B by adopting a solid-phase chemical synthesis method, connecting the branched chain A and the branched chain B through disulfide bonds formed between thiol groups in two cysteine residues, purifying the polypeptide through mass spectrum identification and reverse-phase high performance liquid chromatography, thus obtaining the polypeptide, and finally obtaining the antibacterial peptide 3RF through observation of the self-assembled form of the polypeptide, antibacterial activity determination and hemolytic toxicity determination.

The invention also aims to provide an application of the self-assembled antibacterial peptide 3RF for resisting the drug-resistant bacteria in preparing a drug for treating infectious diseases caused by the drug-resistant bacteria, wherein the drug-resistant bacteria are methicillin-resistant staphylococcus aureus, kanamycin-resistant escherichia coli or spectinomycin-resistant pseudomonas aeruginosa.

The self-assembled antibacterial peptide 3RF has the advantages that the self-assembled antibacterial peptide 3RF can form a stable fiber-containing structure, has excellent inhibition effect on drug-resistant bacteria such as methicillin-resistant staphylococcus aureus, kanamycin-resistant escherichia coli and spectinomycin-resistant pseudomonas aeruginosa, and has almost no toxicity on human erythrocytes.

Drawings

FIG. 1 is a mass spectrum of antimicrobial peptide 3 RF;

FIG. 2 is a high performance liquid chromatogram of antimicrobial peptide 3 RF;

FIG. 3 is a graph of antimicrobial peptide 3RF minimum aggregation concentration (CAC);

FIG. 4 is a graph showing the hemolytic activity of antibacterial peptide 3 RF.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

Design of antibacterial peptides

The design principle is that two branched chains are designed, a branched chain A, a sequence RRRCFFF, a branched chain B and a sequence FFFCRRR, wherein the branched chain A and the branched chain B are connected through disulfide bonds formed between thiol groups in two cysteine residues, a polypeptide sequence drives self-assembly by utilizing a cation-pi action (R-F), then the branched chain A and the branched chain B are respectively synthesized by adopting a solid-phase chemical synthesis method, and the branched chain A and the branched chain B are connected through the disulfide bonds formed between the thiol groups in the two cysteine residues.

The amino acid sequence of the antimicrobial peptide 3RF is as follows:

TABLE 1 amino acid sequence of antibacterial peptides

The molecular formula of the self-assembled antibacterial peptide 3RF is shown as a formula (I),

Example 2

1. Synthesis of antibacterial peptide by solid phase chemical synthesis method

1. Fmoc-Arg (pbf) -OH was first attached to Rink resin, then after a piperidine deprotection reaction for 30min, piperidine was removed and washed with Dimethylformamide (DMF) and the deprotection color was detected with ninhydrin. And sequentially connecting the subsequent straight-chain amino acids until Fmoc-Trp (boc) -OH at the N end is connected, removing Fmoc at the N end to obtain FFFC (Trt) R (pbf) R (pbf) R (pbf), and cleaving the monomer peptide from the resin by using 95% TFA through the resin, and simultaneously cutting off all side chain protecting groups of the sequence. Obtaining FFFC (Trt) R (pbf) R (pbf) R (pbf) crude product, purifying liquid phase and freeze-drying.

2. Preparation of R (pbf) R (pbf) R (pbf) C (Trt) FFF in the same manner as in step 1 cleaves the monomeric peptide from the resin while cleaving all side chain protecting groups of the sequence. Crude products of R (pbf) R (pbf) R (pbf) C (Trt) FFF monomers are obtained, purified in liquid phase and freeze-dried.

3. And (3) oxidizing and synthesizing, namely dissolving two monomer peptides, regulating Ph to 7.5-8 by using an ammonium bicarbonate solution after mixing, stirring for 1 hour, monitoring the oxidation condition by using mass spectrum and liquid phase, purifying by HPLC after the oxidation is completed, and freeze-drying to obtain the target substance.

2. Purifying and identifying:

1. Detecting crude MS, namely taking a small amount of crude, dissolving, determining that the molecular weight (shown in figure 1) is basically consistent with the theoretical molecular weight in table 1 by using LC-MS, and purifying.

2. Purifying the polypeptide by high performance liquid chromatography to obtain polypeptide with purity >95%, and purifying the antibacterial peptide 3RF by high performance liquid chromatography as shown in figure 2.

Example 3

The Critical Aggregation Concentration (CAC) of the nanopeptides was determined by a sodium 1-anilino-8-naphthalene sulfonate (ANS) fluorescent probe. Dye preparation ANS powder was dissolved in N, N-Dimethylformamide (DMF) to a concentration of 40mM and stored in a dark place at low temperature for use. Different concentrations of the antimicrobial peptide 3RF (1-256. Mu.M) were dissolved in PB (10 mM, pH 7.4)) buffer and incubated for 12 hours at room temperature. Different concentrations of antimicrobial peptide 3RF were then mixed with ANS. The change of fluorescence intensity is monitored at the excitation wavelength of 360nm and the emission wavelength of 420-670nm by using an enzyme-labeled instrument, the slit width is 2nm, and as shown in figure 3, obvious fluctuation of fluorescence intensity can be observed, which shows that the antibacterial peptide 3RF has a certain aggregation self-assembly capability.

Example 4

Antibacterial activity assay for antibacterial peptide 3RF

The Minimum Inhibitory Concentration (MIC) of the peptides was determined using standard micro broth dilution. The bacteria in log phase were diluted to 2X 10 ⁵ CFU/mL. 50 μl of different concentrations of antimicrobial peptide 3RF (final concentration 1-128 μM) and equal volumes of bacterial suspension were added to each well of a 96-well plate, negative controls (medium only) and positive controls (bacteria and medium) were set simultaneously, and then the 96-well plate was placed in a 37 ℃ incubator for 18-20 hours. The absorbance at 492nm was measured using a microplate reader, two replicates were set for each test, and these tests were repeated at least three times. The results are shown in Table 2.

TABLE 2 minimum inhibitory concentration (μM) of antimicrobial peptide 3RF

As can be seen from Table 2, the antibacterial peptide 3RF has excellent activity against methicillin-resistant Staphylococcus aureus MRSA, kanamycin-resistant Escherichia coli M15 and spectinomycin-resistant Pseudomonas aeruginosa 109004, and the minimum inhibitory concentration is 2-4. Mu.M.

Example 5

Determination of haemolytic Activity of antibacterial peptide 3RF

Blood preparation by drawing fresh human blood, centrifuging at 3000-3500 rpm for 10 min at 4 ℃, sucking out the supernatant, filtering the phosphate buffer (PBS ph=7.4) with a 0.22 μm aqueous filter, adding the filtered solution to erythrocytes, centrifuging the erythrocytes three times, and finally re-suspending the erythrocytes in 10 volumes of PBS. Dilution of antimicrobial peptide 3RF PBS was added to 1-12 columns in a 96-well plate, with 90. Mu.L added to the first column and 50. Mu.L added to the other columns. mu.L of the dissolved nano peptide solution was pipetted into column 1 of all 96 well plates and diluted to column 10. Red blood cell addition red blood cells after resuspension were added to columns 1-12 of 96-well plates, 50 μl per well. 0.1% Triton X-100 was added as positive control (100% hemolysis) to column 12 and column 11 as negative control. The 96-well plate was placed in a 37 ℃ incubator for 1 hour, and then the 96-well plate was centrifuged at 1000×g for 5 minutes at 4 ℃, and 50 μl of the supernatant of each well was extracted and transferred to a new 96-well plate. The absorbance at 570nm was measured by using a microplate reader. Each test was run in duplicate and these tests were repeated at least three times.

As shown in fig. 4, the antimicrobial peptide 3RF showed negligible hemolysis at all concentrations tested, indicating that the antimicrobial peptide 3RF has good biocompatibility.

In conclusion, the antibacterial peptide 3RF has excellent antibacterial activity on methicillin-resistant staphylococcus aureus MRSA, kanamycin-resistant escherichia coli M15 and spectinomycin-resistant pseudomonas aeruginosa 109004, has little toxicity on human erythrocytes and has extremely high application potential.

Claims

1. A self-assembled antibacterial peptide 3RF for resisting drug-resistant bacteria is characterized by having a molecular formula shown in a formula (I) and comprising a branched chain A and a branched chain B, wherein the amino acid sequence of the branched chain A is RRRCFFF, the amino acid sequence of the branched chain B is FFFCRRR, the branched chain A and the branched chain B are connected through disulfide bonds formed between thiol groups in two cysteine residues,

2. The self-assembly method of self-assembled antibacterial peptide 3RF for resisting drug-resistant bacteria according to claim 1, wherein the nano self-assembly condition is that the antibacterial peptide 3RF is dissolved in PB buffer solution with the concentration of 10mM and the pH of 7.4, the concentration of the antibacterial peptide 3RF is 32-256 mu M, and the self-assembly is performed at room temperature for 12 hours to form a nano structure.

3. The preparation method of the self-assembled antibacterial peptide 3RF for resisting drug-resistant bacteria, which is characterized by comprising the steps of designing two branches, namely RRRCFFF a sequence of a branch A, FFFCRRR a sequence of a branch B, connecting the branch A and the branch B through disulfide bonds formed between thiol groups in two cysteine residues, driving the self-assembly of the branch A and the branch B through cation-pi action, respectively synthesizing the branch A and the branch B by adopting a solid-phase chemical synthesis method, connecting the branch A and the branch B through disulfide bonds formed between thiol groups in two cysteine residues, carrying out mass spectrum identification and reversed-phase high performance liquid chromatography purification on the polypeptide, and finally obtaining the polypeptide 3RF through observation of self-assembled forms of the polypeptide, antibacterial activity measurement and hemolytic toxicity measurement.

4. The use of a self-assembled antimicrobial peptide 3RF against drug-resistant bacteria according to claim 1 for the preparation of a medicament for the treatment of infectious diseases caused by drug-resistant bacteria, said drug-resistant bacteria being methicillin-resistant staphylococcus aureus, kanamycin-resistant escherichia coli or spectinomycin-resistant pseudomonas aeruginosa.