A kind of anti-enzymatic nano-capture peptide and its preparation method and application

Abstract

The present invention provides an enzymatically resistant nano-capture peptide, its preparation method, and application, belonging to the field of bioengineering. The sequence of the peptide is shown in SEQ ID No. 1. A preparation method for the antimicrobial peptide is also disclosed. The capture peptide has a strong capture effect on Escherichia coli and Staphylococcus aureus rather than a direct bactericidal effect, effectively reducing bacterial selective pressure. It is virtually non-toxic to mammalian cells and exhibits strong resistance to protease testing, showing potential as an antibiotic alternative.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an enzymatically resistant nano-capture peptide and a preparation method and application thereof. Background Art

[0002] Human α-defensin 6 (HD6) employs a natural supramolecular strategy to capture pathogens by forming nanofiber networks, potentially reducing the tendency for bacteria to acquire drug resistance. This provides a novel strategy for the development of a new generation of antimicrobial agents, termed capture peptides. However, the sensitivity of capture peptides to protease digestion remains a significant constraint on their oral application. Digestive proteases such as pepsin and trypsin can hydrolyze capture peptides into short peptides, such as amino acids or dipeptides, thereby losing their original biological activity. Therefore, molecular improvements in capture peptides focus on enhancing their resistance to protease hydrolysis while retaining their chemical stability and low cytotoxicity to increase their potential for clinical application. Summary of the Invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide an enzymatically resistant nano-capture peptide with a more stable structure, which can avoid the digestive hydrolysis of digestive proteases under physiological conditions and further improve the application potential of the capture peptide.

[0004] The technical solution adopted by the present invention is as follows: an enzymatically resistant nano-capture peptide RF-29, whose amino acid sequence is shown in SEQ ID No. 1.

[0005] Another object of the present invention is to provide a method for preparing an enzymatically resistant nano-capture peptide RF-29, comprising the following steps:

[0006] Step S1: Phenylalanine F is selected to provide hydrophobic interaction; arginine R is selected to provide positive charge, meeting the basic conditions of antimicrobial peptides; the peptide structure adopts a surfactant-like mode to promote overall molecular self-assembly; and proline P is selected to avoid protease hydrolysis;

[0007] Step S2: GSGS was used as a flexible linker to connect to the 123-131 active region of the Mycobacterium tuberculosis natural peptide Rv2626c: LPEHAIVQF to construct a polypeptide with an amino acid sequence as shown in SEQ ID No. 1;

[0008] Step S3: The polypeptide was prepared by solid-phase chemical synthesis and mass spectrometry identification, and then the nanomorphology of the polypeptide, in vitro cytotoxicity, protease stability and bacterial capture ability were measured. Finally, it was named as the enzymatically resistant nano-capture peptide RF-29.

[0009] Furthermore, the self-assembly method of the enzymatically resistant nano-capture peptide RF-29 as described above has the following self-assembly conditions: concentration of 2-512 μM, incubation at 37° C. for 24 hours.

[0010] Another object of the present invention is to provide the use of the enzymatically resistant nano-capture peptide RF-29 described above in the preparation of drugs for treating infectious diseases caused by Escherichia coli and / or Staphylococcus aureus.

[0011] Furthermore, the enzymatically resistant nano-capture peptide RF-29 described above is capable of capturing Escherichia coli and Staphylococcus aureus.

[0012] The present invention has the following advantages and beneficial effects: the enzymatically resistant nano-capture peptide RF-29 of the present invention has strong biological activity and a significant effect of resisting proteases; the bacterial agglutination ability, cytotoxicity and protease stability of the prepared nano-antimicrobial peptide are measured, and it is found that the enzymatically resistant nano-capture peptide RF-29 has a strong capture effect on Escherichia coli and Staphylococcus aureus rather than a direct bactericidal effect, has almost no toxicity to mammalian cells, and has strong resistance under protease conditions, and has the potential to be used as an antibiotic substitute. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the HPLC chromatogram of the enzymatically resistant nano-capture peptide RF-29;

[0014] Figure 2 is the mass spectrum of the enzymatically resistant nanocapture peptide RF-29;

[0015] Figure 3 Scanning electron microscopy nanometer characterization of the enzymatically resistant nanocapture peptide RF-29; (a) 2 μM; (b) 512 μM;

[0016] Figure 4 Figure 2 is a cytotoxicity assay of the enzymatically resistant nanocapture peptide RF-29;

[0017] Figure 5 This is a diagram showing the ability of the enzymatically resistant nano-capture peptide RF-29 to capture Escherichia coli; (a) SYTO9 staining of the control group; (b) SYTO9 staining of the RF-29 group; (c) SYTO9 staining of the bee venom group; (d) PI staining of the control group; (e) PI staining of the RF-29 group; (f) PI staining of the bee venom group; (g) merged picture of the control group; (h) merged picture of the RF-29 group; (i) merged picture of the bee venom group.

[0018] Figure 6Figure 2 shows the ability of the enzymatically resistant nano-capture peptide RF-29 to capture Staphylococcus aureus; (a) SYTO9 staining of the control group; (b) SYTO9 staining of the RF-29 group; (c) SYTO9 staining of the bee toxin group; (d) PI staining of the control group; (e) PI staining of the RF-29 group; (f) PI staining of the bee toxin group; (g) merged picture of the control group; (h) merged picture of the RF-29 group; (i) merged picture of the bee toxin group.

[0019] Figure 7 This is the protease stability diagram of the enzymatically resistant nanocapture peptide RF-29. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0021] Example 1

[0022] Design of enzymatically resistant nanocapture peptides

[0023] Design of the enzymatically resistant nanocapture peptide RF-29: Phenylalanine (F) was selected to provide hydrophobic interaction; arginine (R) was selected to provide positive charge to meet the basic conditions of antimicrobial peptides; the peptide structure adopted a surfactant-like mode to promote overall molecular self-assembly; at the same time, proline (P) was used to protect F and R to prevent them from being hydrolyzed by proteases; GSGS was used as a flexible linker to connect to the 123-131 active region of the natural peptide Rv2626c of Mycobacterium tuberculosis: LPEHAIVQF. The amino acid sequence of the peptide is shown in Table 1.

[0024] Table 1 Amino acid sequence of the enzymatically resistant nanocapture peptide RF-29

[0025]

[0026] Example 2

[0027] Synthesis of capture peptides by solid-phase chemical synthesis

[0028] a. Resin swelling: Peptide synthesis is carried out sequentially from the C-terminus to the N-terminus. Pour Fmoc-Dab-OH-Wang resin into the reaction column and soak for 30 minutes, then drain.

[0029] b. Deprotection: After soaking the resin for 30 minutes, the solution was removed and then deprotected using piperidine for 30 minutes;

[0030] c. Weighing: Within 30 minutes of deprotection, calculate the amount of amino acid, condensing agent, and NMM required for each step based on the amount made, and then weigh the next amino acid;

[0031] d. Deprotection washing: After 30 minutes of deprotection, remove the piperidine and then wash 6 times with DMF. After 6 washes, detect the deprotection color and record it;

[0032] e. Feeding: After washing, deprotection, and testing, add the weighed materials in order, followed by a small amount of reaction solution, and then add alkali and NMM. Adjust the gas evenly, flush the resin stuck to the inner wall of the reaction column with DCM, and then record the reaction time. The reaction time is 30 minutes;

[0033] f. Washing after the reaction: drain the solution in the reaction column, add an appropriate amount of DMF for washing, agitate with nitrogen for 2 minutes, drain, and repeat the operation 3 times;

[0034] g. Detection: After thirty minutes of reaction, remove the reaction solution, wash with DMF three times and then detect whether the reaction is complete;

[0035] h. Post-synthesis Washing and Drying: Drain the peptide, add an appropriate amount of methanol to the reaction column, aerate with nitrogen for 2 minutes, drain, then add an appropriate amount of DCM, aerate with nitrogen for 2 minutes, and repeat the process three times. Finally, add an appropriate amount of methanol to the reactor, aerate with nitrogen for 2 minutes, and drain. Repeat the process two more times. Place the resin in a suitable container and dry it in a vacuum desiccator for 12 hours before cutting.

[0036] Cutting: Place the dried resin into a suitable round-bottom flask, add an appropriate amount of prepared cutting solution (1 g / 10 mL), and place it in a constant temperature shaker at 25°C for 2 hours.

[0037] Filtration: Use a 50mL sand core funnel to filter out the resin particles, then pour the filtrate into a 100mL centrifuge tube, add 6-8 times the volume of anhydrous ether, and stir while adding. The precipitated white solid is the desired crude polypeptide.

[0038] Washing: Seal the centrifuge tube and place it in a centrifuge at 4000 rpm for 3 minutes. Remove the tube, discard the supernatant, add ether, stir evenly with a glass rod, and centrifuge again. Repeat this operation 5 times.

[0039] Drying: After washing five times, place the peptide in a vacuum desiccator and dry it for 24 hours. The resulting white powder is the crude product of the desired peptide, which is weighed and purified.

[0040] Purification: Use 0.2 mol / L sodium sulfate (phosphate pH = 7.4) to equilibrate the column for 30 min, dissolve the polypeptide in 90% acetonitrile aqueous solution, filter, apply to reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), flow rate 1 mL / min, detection wave is 220 nm, collect the main peak, and lyophilize; further purification is performed using reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, flow rate 1 mL / min, collect the main peak as above, and lyophilize;

[0041] Identification: The captured peptides obtained above were analyzed by electrospray mass spectrometry (e.g. Figure 1 The purity of the captured peptides was greater than 95% (as shown in Figure 2 shown).

[0042] Example 3

[0043] Scanning electron microscopy nanometer characterization of the enzymatically resistant nanocapture peptide RF-29:

[0044] Nanomorphology analysis: To further analyze the nanomorphology of the enzymatically resistant nanocapture peptide RF-29, the peptide (1.28 mM) was diluted to 512 μM and 2 μM concentrations in deionized water and incubated in a 37°C incubator for 24 hours. The sample was smeared on a glass slide and coated with metal using a Polaron SC7640 sputter coater after natural drying. The sample was observed at 5 kV by a Hitachi S-4800 SEM (Hitachi, Japan). The test results are shown in Figure 3 .

[0045] from Figure 3 (a) It can be seen that 2 μM enzymatically resistant nanocapture peptide RF-29 forms nanoparticles with larger size and loose structure; Figure 3 (b) It can be seen that 512 μM enzymatically resistant nanocapture peptide RF-29 forms nanoparticles with smaller size and tighter structure.

[0046] Example 4

[0047] In vitro cytotoxicity assay of the anti-enzymatic nanocapture peptide RF-29: The cells frozen in liquid nitrogen were revived and inoculated into a culture medium containing 10% fetal bovine serum and 1% double-antibody, and subcultured at 37°C and 5% CO2. The cultured cells were digested with 0.25% trypsin and the cell density was adjusted to 2-4×10 5cells / mL. 50 μL of cell suspension was mixed with 50 μL of peptides of different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 24 h. Subsequently, 25 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 h. After incubation, the supernatant was discarded, and the crystals at the bottom of the well were dissolved with 100 μL of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader. The culture medium wells served as blank controls. The test results are shown in Figure 4 .

[0048] from Figure 4 It can be seen that even after treatment with high concentrations of the enzymatically resistant nano-capture peptide RF-29, the cell survival rate of piglet jejunal epithelial cells (IPEC-J2) and mouse peritoneal macrophages (RAW 264.7) still remained at 80%, indicating that the enzymatically resistant nano-capture peptide RF-29 has good biocompatibility and has the potential to become an antibiotic alternative.

[0049] Example 5

[0050] Assay for bacterial capture by the enzyme-resistant nanocapture peptide RF-29: To evaluate the ability of the enzyme-resistant nanocapture peptide RF-29 to capture pathogens, ultrahigh-resolution fluorescence microscopy was used to observe bacterial aggregation mediated by the enzyme-resistant nanocapture peptide RF-29. Escherichia coli particles containing green fluorescent protein were prepared, centrifuged at 3000 rpm for 5 minutes, and resuspended in PBS buffer. The peptide solution and melittin were added to a final concentration of 32 μM and incubated with the bacterial suspension at 37°C for 2 hours. Propidium iodide (PI) dye was then added to a final concentration of 10 μg / mL and incubated at 4°C for 15 minutes. Free PI was then removed by centrifugation. The sample was resuspended, applied to a glass slide, air-dried, covered with a coverslip, and mounted with nail polish. The sample was observed using ultrahigh-resolution fluorescence microscopy at excitation wavelengths of 488 nm and 535 nm. Untreated cells served as a negative control.

[0051] Depend on Figure 5 and Figure 6 (ai) As can be seen, unlike the direct bactericidal effect of melittin, 32 μM enzymatically resistant nanocapture peptide RF-29 exhibited a significant capture effect on E. coli 25922 and S. aureus 29213, without directly killing the bacteria. This antibacterial mechanism can minimize the selective pressure exerted on pathogens and reduce the likelihood of them developing drug resistance.

[0052] Example 6

[0053] Determination of the protease stability of the enzymatically resistant nano-capture peptide RF-29: In order to detect the anti-protease ability of the peptide, artificial simulated gastric / intestinal fluid, 10 mg / mL gastric, pancreatic, and chymotrypsin were mixed with equal volumes of peptide (2.56 mM), incubated at 37°C for different times, and the peptide that had not been treated with protease was used as a control. The bands were then reflected using Tricine-SDS-PAGE protein gel.

[0054] pass Figure 7 It can be seen that after protease treatment, the enzymatically resistant nano-capture peptide RF-29 has protein bands similar to those in the control group, further proving that the enzymatically resistant nano-capture peptide RF-29 has strong resistance to multiple proteases.

Claims

1. An enzymatically resistant nanopeptide capture peptide RF-29, characterized by: Its amino acid sequence is shown in SEQ ID No.

1.

2. The method for preparing an enzymatically resistant nano-capture peptide RF-29 according to claim 1, characterized in that: The steps are as follows: Step S1: Phenylalanine F is selected to provide hydrophobic interaction, arginine R is selected to provide positive charge, and proline P is selected to avoid protease hydrolysis; Step S2: GSGS was used as a flexible linker to connect to the 123-131 active region of the Mycobacterium tuberculosis natural peptide Rv2626c: LPEHAIVQF to construct a polypeptide with an amino acid sequence as shown in SEQ ID No. 1; Step S3: The polypeptide was prepared by solid-phase chemical synthesis and mass spectrometry identification, and then the nanomorphology of the polypeptide, in vitro cytotoxicity, protease stability and bacterial capture ability were measured. Finally, it was named as the enzymatically resistant nano-capture peptide RF-29.

3. The self-assembly method of the enzymatically resistant nano-capture peptide RF-29 according to claim 1, characterized in that: The self-assembly conditions are as follows: the concentration is 2-512 μM, and the nanostructure can be self-assembled after incubation at 37° C. for 24 hours.

4. Use of the enzymatically resistant nano-capture peptide RF-29 according to claim 1 in preparing nanoparticles for capturing Escherichia coli and / or Staphylococcus aureus.

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