Beta-hairpin structure self-assembly capture antibacterial peptide and application thereof

Abstract

The invention discloses a beta-hairpin structure self-assembly capture antibacterial peptide and application thereof, and relates to the technical field of biological medicine. The amino acid sequence of the beta-hairpin structure self-assembly capture antibacterial peptide is as shown in any one of SEQ ID NO. 3-6. A broad-spectrum antibacterial short peptide Jelleine-1 (J-1) from bee royal jelly is selected as a template peptide, design and transformation are performed according to amino acid sequence characteristics of the short peptide J-1, and a hydrophilic and hydrophobic amino acid alternate arrangement structural unit is obtained and serves as a self-assembly peptide fiber skeleton. Beta-hairpin peptide design is carried out by adopting two turning sequences of DPG and RRRF, so that the beta-hairpin peptide has dual effects of resisting bacteria and capturing bacteria. The beta-hairpin structure self-assembly capture antibacterial peptide provided by the invention can form a fiber network structure, has a good antibacterial effect, high stability and excellent biocompatibility, and provides an effective technical support for developing novel antibacterial drugs.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a beta-hairpin structure self-assembly capturing antimicrobial peptide and application thereof. Background Art

[0002] As an important component of the innate immune system, antimicrobial peptides exert their antibacterial effects through physical mechanisms such as changing membrane permeability and destroying cell membrane integrity, and are considered to be a favorable alternative to traditional antibiotics. However, natural AMPs have a series of problems such as weak biological activity, high toxicity, and immature stability, which seriously hinder their application. In recent years, advances in molecular design and optimization strategies, as well as the rapid rise of nanotechnology, have shown the potential to improve the biological and chemical properties of AMPs, making the application of AMPs possible.

[0003] Hydrogen bonding, ionic, hydrophobic and π-π stacking interactions between amino acids are the main driving forces for self-assembly. Supramolecular self-assembly is considered to be one of the methods to improve the antibacterial activity and stability of antimicrobial peptides. The present invention intends to develop nanostructured antimicrobial peptides with self-assembly tendencies, in order to provide technical support for avoiding antibiotic resistance and developing effective clinical anti-infection alternatives. Summary of the invention

[0004] The purpose of the present invention is to provide a β-hairpin structure self-assembly capture antimicrobial peptide and its application to solve the problems existing in the above-mentioned prior art. The β-hairpin structure self-assembly capture antimicrobial peptide provided by the present invention can form a fiber network structure, has good antibacterial effect and high stability, and has excellent biocompatibility, providing effective technical support for the development of new antimicrobial drugs.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a β-hairpin structure self-assembly capture antimicrobial peptide J-1-pG, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide J-1-RF, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide JR-pG, the amino acid sequence of which is shown in SEQ ID NO.5.

[0009] The present invention also provides a β-hairpin structure self-assembly capture antimicrobial peptide JR-RF, the amino acid sequence of which is shown in SEQ ID NO.6.

[0010] The present invention also provides the use of the above-mentioned β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembled capture antimicrobial peptide JR-pG or β-hairpin structure self-assembled capture antimicrobial peptide JR-RF in the preparation of self-assembled capture peptide fibers with antimicrobial function.

[0011] The present invention also provides a self-assembled capture peptide fiber with antibacterial function, wherein the self-assembled capture peptide fiber is obtained by self-assembly of the above-mentioned β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG, β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF, β-hairpin structure self-assembled capture antimicrobial peptide JR-pG or β-hairpin structure self-assembled capture antimicrobial peptide JR-RF.

[0012] The present invention also provides an antimicrobial peptide JR, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The present invention also provides the use of the above-mentioned β-hairpin structure self-assembled captured antimicrobial peptide J-1-pG, β-hairpin structure self-assembled captured antimicrobial peptide J-1-RF, β-hairpin structure self-assembled captured antimicrobial peptide JR-pG, β-hairpin structure self-assembled captured antimicrobial peptide JR-RF, self-assembled captured peptide fiber or antimicrobial peptide JR in the preparation of antimicrobial agents.

[0014] Furthermore, the pathogens targeted by the antibacterial agent include Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

[0015] The present invention also provides an antibacterial agent, the active ingredients of which include the above-mentioned β-hairpin structure self-assembled captured antimicrobial peptide J-1-pG, β-hairpin structure self-assembled captured antimicrobial peptide J-1-RF, β-hairpin structure self-assembled captured antimicrobial peptide JR-pG, β-hairpin structure self-assembled captured antimicrobial peptide JR-RF, self-assembled captured peptide fiber or antimicrobial peptide JR.

[0016] The present invention discloses the following technical effects:

[0017] The present invention selects Jelleine-1 (J-1), a broad-spectrum antibacterial short peptide derived from royal jelly of honey bees, as a template peptide, designs and modifies it according to the amino acid sequence characteristics of the short peptide J-1, obtains a structural unit with alternating hydrophilic and hydrophobic amino acids, and uses it as a self-assembling peptide fiber skeleton. DThe β-hairpin peptide is designed with two turn sequences, PG and RRRF, so that it has the dual effects of antibacterial and bacterial capture. The self-assembled captured antimicrobial peptide in the present invention can form a fiber network structure, has good antibacterial effect and high stability, and has excellent biocompatibility, providing effective technical support for the development of new antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 HPLC chromatograms of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0020] Figure 2 HPLC chromatograms of designed peptides JR, JR-pG, and JR-RF;

[0021] Figure 3 Mass spectra of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0022] Figure 4 Mass spectra of designed peptides JR, JR-pG, and JR-RF;

[0023] Figure 5 Circular dichroism spectra of template peptide J-1 and designed peptides J-1-pG, J-1-RF, JR, JR-pG and JR-RF in three different environments;

[0024] Figure 6 The statistical graphs of the toxicity test results of the template peptide J-1 and the designed peptides J-1-pG, J-1-RF, JR, JR-pG and JR-RF on RAW264.7 macrophages;

[0025] Figure 7 The results of the critical aggregation concentration determination of β-hairpin; A is the fluorescence intensity of hairpin J-1-RF at different concentrations; B is the linear fitting diagram of hairpin J-1-RF concentration and fluorescence intensity; C is the fluorescence intensity of hairpin JR-pG at different concentrations; D is the linear fitting diagram of hairpin JR-pG concentration and fluorescence intensity;

[0026] Figure 8 Transmission electron microscopy images of β-hairpin J-1-RF at different concentrations;

[0027] Fig. 9 Atomic force microscopy for β-hairpin peptide J-1-RF;

[0028] Fig.10 The results of the β-haika peptide bacterial capture ability test are shown in Figure 1. A is a cuvette observation diagram; and B is a statistical diagram of the CFU value of Escherichia coli. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Peptide sequence design and physicochemical parameter analysis

[0035] The invention selects the broad-spectrum antimicrobial peptide Jelleine-1 (i.e., J-1) from royal jelly of honey bees as a template, replaces the first amino acid at its N-terminus with arginine, and constructs a β-sheet structure sequence with alternating hydrophilic and hydrophobic amino acids as a β-hairpin structure self-assembling peptide fiber skeleton;D The sequence design was performed with two turn sequences, PG and RRRF, and a total of four β-hairpin peptides (J-1-pG, J-1-RF, JR-pG, and JR-RF) were obtained. The physicochemical parameters and molecular structures of all tested peptides are shown in Tables 1 and Figure 1 shown.

[0036] Table 1 Amino acid sequence and physicochemical parameters of target peptides

[0037]

[0038]

[0039] Note: a The actual molecular mass is detected by mass spectrometer; b The isoelectric point is calculated by the online tool PepCalc.com-Peptidecalculator; p stands for D P, namely D-proline.

[0040] Hydrophilic cationic residues and hydrophobic residues play an important role in electrostatic interaction with negatively charged bacterial membranes and in promoting the penetration of peptide sequences into the hydrophobic domains of bacterial membranes, respectively. The number of positive charges of the designed peptides in the study increased compared with the template peptides, providing appropriate electrostatic interactions for antibacterial effects.

[0041] Example 2 Synthesis and identification of polypeptides

[0042] 1. Synthesis of antimicrobial peptides

[0043] The peptides shown in Table 1 were synthesized using solid phase synthesis:

[0044] Weigh the resin, add dichloromethane to soak for 5 minutes, wash with N,N-dimethylformamide, then remove the dichloromethane, and then use the prepared deprotecting agent to remove the Fmoc protecting group on the resin (decapping), and the decapping time is 20 minutes; weigh the second amino acid at the C-terminus, add a condensing agent, N,N-dimethylformamide, and put it into the reaction column for reaction (air blowing); use the ninhydrin method for detection, and the detection result is that the solution is bright yellow, the resin is transparent, and there is no variegated color; use the deprotecting solution to remove the Fmoc protecting group on the second amino acid, wash with N,N-dimethylformamide 6 times, and repeat the operation for the remaining amino acids until the last amino acid; use the deprotecting solution to remove the last Fmoc protecting group, and then wash with N,N-dimethylformamide; after the reaction is completed, wash and shrink with dichloromethane and methanol respectively; cut the polypeptide with a cleavage solution, and precipitate the liquid with ether; analyze with a mass spectrometer (MS) and a high performance liquid chromatography (HPLC) for purification.

[0045] 2. Purification of antimicrobial peptides

[0046] (1) Pre-analysis: Take a sample and place it in a 0.5 mL tube, dissolve it in ultrapure water, filter the sample with a 0.45 μm membrane, and then analyze the sample by rapid gradient HPLC (10-100%).

[0047] (2) Sample treatment: Add 200 mg of sample to a 20 mL beaker, then add 15 mL of H2O and 5 mL of methanol. Ultrasonicate the sample until the sample is completely dissolved, and then filter the solution with a 0.45 μm membrane.

[0048] (3) High Performance Liquid Chromatography (HPLC): Samples were collected from 0 to 25 minutes. Pump A was 100% acetonitrile plus 0.1% trifluoroacetic acid, and pump B was 100% water plus 0.1% trifluoroacetic acid. The collected components were analyzed by high performance liquid chromatography (HPLC) to check the purity.

[0049] The chromatograms of the template peptide and the designed peptide after purification are shown in Figure 2. Figure 1-Figure 2 As shown in the figure, each designed peptide has an obvious absorption peak. The purity of the purified peptide reaches more than 95%.

[0050] 3. Identification of antimicrobial peptides

[0051] The purified peptides were collected and the target peaks were identified by LC 6000 reverse phase preparative chromatograph and Waters 2000 mass spectrometer, and the actual peptide molecular weight and purity were finally analyzed. Figure 3-Figure 4 As shown. The results showed that the actual relative molecular mass was basically consistent with the theoretical value, indicating that all peptides were successfully synthesized. The results showed that the actual relative molecular mass was basically consistent with the theoretical value, indicating that all peptides were successfully synthesized.

[0052] Example 3 Determination of the secondary structure of antimicrobial peptides

[0053] 1. Determination of the secondary structure of antimicrobial peptides

[0054] Circular dichroism (CD) was used to determine the secondary structures of the antimicrobial peptides (shown in Table 1).

[0055] The structure in three different simulated solution environments (water environment, prokaryotic cell membrane environment and biomembrane hydrophobic environment). The peptide was dissolved in 10mM PBS buffer solution at pH = 7.4, 30mM sodium dodecyl sulfate (SDS) and 50% trifluoroethanol (TFE) to prepare a peptide solution with a final concentration of 150μM for use. The circular dichroism spectrometer was used to scan in the wavelength range of 190nm to 250nm at room temperature with a scanning rate of 1nm / second. The results are shown in Figure 5 shown.

[0056] In PBS buffer, J-1 has a negative absorption peak at 198nm, showing a random coiled conformation. However, in SDS buffer, JR, J-1-pG, JR-pG, and JR-RF have positive absorption peaks at 203nm and negative absorption peaks around 216nm, indicating a typical β-sheet structure. This suggests that the alternating arrangement of hydrophilic and hydrophobic amino acids contributes to the formation of the β-sheet conformation of the peptide in a simulated hydrophobic environment.

[0057] Example 4 Determination of antibacterial activity

[0058] Determination of minimum inhibitory concentration (MIC): The antibacterial activity was determined by the microdilution method. The strain to be tested was cultured to the logarithmic growth phase, and different concentrations of peptides (as shown in Table 1) and bacterial solution were added to rows 1-11 of the 96-well plate, respectively, and incubated at 37°C for 16 hours. Bacterial solution and culture medium without peptide were used as positive and negative controls, respectively. The MIC value of the peptide was expressed as the critical concentration value when there was no visible turbidity or precipitation. The results are shown in Tables 2 and 3.

[0059] Table 2 MIC values ​​of short peptides against Gram-negative bacteria (μM)

[0060]

[0061]

[0062] Table 3 MIC (μM) of short peptides against Gram-positive bacteria

[0063]

[0064] The five derived peptides (JR, J-1-pG, J-1-RF, JR-pG and JR-RF) all exhibited broad-spectrum antibacterial activity, and the antibacterial activity was significantly enhanced compared with the template peptide. Among them, the designed peptides JR and J-1-RF showed enhanced antibacterial activity, which may be due to the appropriate increase in the number of positive charges.

[0065] Example 5 Cytotoxicity Assay

[0066] The cytotoxicity of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG and JR-RF was tested:

[0067] The cytotoxic effect of peptides on mouse macrophages (RAW 264.7) was evaluated by CCK-8 kit. In summary, cells were seeded into individual wells of a 96-well plate and exposed to different concentrations of peptide solutions for 12 h at 37 °C and 5% CO2. After the incubation time, 10 μL of CCK-8 solution was added to each well, left to stand for 2 h, and the absorbance at 450 nm was measured by a microplate reader. The positive control was cells without peptide treatment, and the negative control was complete culture medium.

[0068] like Figure 6 As shown, the experiment found that with the increase of experimental concentration, the cell survival rate gradually decreased. At a concentration of 4 μM, the cell survival rate of melittin was less than 20%. In contrast, the cell survival rate of the polypeptide designed by the present invention was still greater than 80% at a test concentration of 64 μM. The results show that the series of derivative peptides designed by the present invention have good biocompatibility.

[0069] Example 6 Critical Aggregation Concentration (CAC) Determination

[0070] CAC of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG, and JR-RF was determined using the ANS fluorescence assay:

[0071] ANS was dissolved in dimethylformamide at a concentration of 1 mM, and 1 μL of this solution was then added to 100 μL of peptides at different concentrations. The mixed solution was transferred to a quartz cuvette and tested using an F-4500 fluorescence spectrophotometer (Hitachi, Japan), and the fluorescence spectra were collected from 420 to 550 nm under excitation at 360 nm. The CAC was determined by a matching curve obtained by plotting the ANS fluorescence intensity at 475 nm against different concentrations of peptides. The results are shown in Figure 7 shown.

[0072] The results showed that the critical aggregation concentrations of the two hairpin peptides, J-1-RF and JR-pG, were 12.45 μM and 13.03 μM, respectively. Therefore, above this critical concentration, the hairpin peptides can self-assemble into nanostructures, thus achieving excellent bioactivity and stability.

[0073] Example 7 Characterization of the structure of self-assembled capture peptide fibers

[0074] In order to observe the self-assembly structure formation of β-hairpin peptide, the microstructure of the peptide was observed by negative staining of transmission electron microscopy and atomic force microscopy, and its ability to form fiber structure was evaluated. The steps were as follows:

[0075] 10 μL of different concentrations of hairpin peptide J-1-RF solution were respectively taken and evenly coated on a copper grid (400 square mesh), adsorbed for 5 minutes, stained with 0.1% phosphotungstic acid for 30 seconds, dried at room temperature for 15 minutes, and observed using a transmission electron microscope. Figure 8 shown.

[0076] The results showed that when the concentration of J-1-RF was 12μM, the nanofibers observed by transmission electron microscopy were only short and thin, indicating that they were in the early stages of aggregation. When the concentration increased to 32μM and 128μM, the hairpin peptide J-1-RF further self-assembled into supramolecular nanostructures. The antiparallel β-sheet dominated and drove the formation of a twisted and dense fiber structure, indicating concentration-driven self-assembly. Atomic force microscopy was used to investigate the formation of fiber morphology of J-1-RF hairpin peptide (32μM), and the same results were obtained ( Fig. 9 ).

[0077] Example 8 Bacterial Agglutination Assay

[0078] In order to evaluate the ability of antimicrobial peptides to capture pathogens, peptides J-1, JR, J-1-RF and JR-pG were mixed with Escherichia coli culture solutions in sterile cuvettes, the bacterial agglutination phenomenon was observed, and the supernatant of the culture solution was taken out and the live bacteria were counted after gradient dilution.

[0079] The specific test steps are as follows:

[0080] (1) Preparation of bacteria: Escherichia coli ATCC 25922 was used as a model strain, and a bacterial culture grown to the logarithmic phase was obtained. The culture was centrifuged at 3000 rpm for 5 min and washed three times with HEPES buffer (pH = 7.0).

[0081] (2) Co-culture of peptide and bacteria: OD 600 = 0.25 in an ethanol-sterilized cuvette, add 32 μM nanopeptide solution, and let it stand at room temperature for 8 h. Observe the bacterial agglutination state and take pictures. The group without peptide treatment was used as the control group.

[0082] (3) Result evaluation: 50 μL of bacterial supernatant was taken at 0, 30, 60, and 90 min, and added to 450 μL of high-temperature sterilized PBS for gradient dilution. 100 μL of the dilution was evenly inoculated on the MHA plate medium. The cells were cultured overnight in a 37°C incubator, the number of colonies of each sample was counted, and the CFU value of each sample was calculated. The experiment was repeated three times.

[0083] The results are as follows Fig.10As shown, the results showed that with the increase of time, β-hairpin induced the rapid agglutination and sedimentation of Escherichia coli, and the total number of colonies in the supernatant was significantly reduced. Among them, J-1-RF could basically induce complete sedimentation of bacteria after 90 minutes, and had a strong bacterial capture effect.

[0084] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A β-hairpin structure self-assembly captures antimicrobial peptide J-1-pG, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A β-hairpin structure self-assembly capture antimicrobial peptide J-1-RF, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

3. A β-hairpin structure self-assembly capture antimicrobial peptide JR-pG, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

4. A β-hairpin structure self-assembly capture antimicrobial peptide JR-RF, characterized in that: The amino acid sequence is shown in SEQ ID NO.

6.

5. Use of the β-hairpin self-assembled captured antimicrobial peptide J-1-pG according to claim 1, the β-hairpin self-assembled captured antimicrobial peptide J-1-RF according to claim 2, the β-hairpin self-assembled captured antimicrobial peptide JR-pG according to claim 3, or the β-hairpin self-assembled captured antimicrobial peptide JR-RF according to claim 4 in the preparation of self-assembled captured peptide fibers with antimicrobial function.

6. A self-assembled capture peptide fiber with antibacterial function, characterized in that: The self-assembled capture peptide fiber is obtained by self-assembly of the β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG described in claim 1, the β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF described in claim 2, the β-hairpin structure self-assembled capture antimicrobial peptide JR-pG described in claim 3, or the β-hairpin structure self-assembled capture antimicrobial peptide JR-RF described in claim 4.

7. An antimicrobial peptide JR, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

8. Use of the β-hairpin self-assembled captured antimicrobial peptide J-1-pG according to claim 1, the β-hairpin self-assembled captured antimicrobial peptide J-1-RF according to claim 2, the β-hairpin self-assembled captured antimicrobial peptide JR-pG according to claim 3, the β-hairpin self-assembled captured antimicrobial peptide JR-RF according to claim 4, the self-assembled captured peptide fiber according to claim 6, or the antimicrobial peptide JR according to claim 7 in the preparation of an antimicrobial agent.

9. The use according to claim 8, characterized in that: The pathogenic bacteria targeted by the antibacterial agent include Escherichia coli, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessus or Bacillus cereus.

10. An antibacterial agent, characterized in that The active ingredients include the β-hairpin structure self-assembled capture antimicrobial peptide J-1-pG described in claim 1, the β-hairpin structure self-assembled capture antimicrobial peptide J-1-RF described in claim 2, the β-hairpin structure self-assembled capture antimicrobial peptide JR-pG described in claim 3, the β-hairpin structure self-assembled capture antimicrobial peptide JR-RF described in claim 4, the self-assembled capture peptide fiber described in claim 6, or the antimicrobial peptide JR described in claim 7.

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