A self-assembled antimicrobial peptide 2D2W with low toxicity and high antibacterial activity

Abstract

The present invention discloses a self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity, belonging to the field of biotechnology. It comprises a branch A and a branch B, the amino acid sequence of the branch A is shown in SEQ ID No.1, the amino acid sequence of the branch B is shown in SEQ ID No.2, and the branch A and the branch B are connected by a disulfide bond formed between the thiol groups in the two cysteine ​​residues. The antimicrobial peptide 2D2W of the present invention self-assembles into a nanofiber structure in an aqueous solution, and has strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria, and has almost no cytotoxicity and hemolytic toxicity, and has extremely high application potential in the treatment of bacterial infections.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity. Background Art

[0002] Bacterial infection is an important clinical disease with a high mortality rate, which poses a great threat to the patient's life and imposes a huge economic burden on the country and society. The most traditional treatment is antibiotics, which can quickly and effectively kill bacteria and save the patient's life. However, the long-term and widespread use of antibiotics has led to the evolution of bacterial resistance. Infectious diseases caused by resistant bacteria are increasing. Studies predict that by 2050, 10 million people will die each year from resistant bacterial infections.

[0003] As an important component of the host immune system, antimicrobial peptides have multiple biological activities such as antibacterial, antifungal, and immunomodulatory. At the same time, due to their multiple antibacterial mechanisms based on membrane destruction, they are not easy to make bacteria resistant, and have great potential to replace antibiotics. Self-assembly is an important process in organisms. It usually builds polymer structures from repeated "building blocks" through non-covalent interactions. Non-covalent interactions such as peptide side chain hydrogen bonds, hydrophobicity, and hydrophilicity can drive antimicrobial peptides to assemble into stable structures, and can also obtain regular geometric appearances through chemical modification. The construction of self-assembled antimicrobial peptides helps to improve the original biological activity of antimicrobial peptides, thereby reducing the dosage and frequency of administration. In addition, self-assembled 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.

[0004] However, most chemically modified self-assembling antimicrobial peptides have toxic effects that cannot be ignored, and self-assembling antimicrobial peptides driven by the intermolecular forces of peptide side chain amino acids often have poor activity, making them unable to become highly effective antimicrobial agents against bacteria. Therefore, it is particularly necessary to develop self-assembling antimicrobial peptides that are non-toxic and have highly effective antibacterial activity. Summary of the invention

[0005] Based on the above shortcomings, the purpose of the present invention is to provide a self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity, which solves the disadvantages of high toxicity and poor activity of self-assembling antimicrobial peptides.

[0006] The technical solution adopted by the present invention is as follows: a self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity, comprising a branch A and a branch B, the amino acid sequence of the branch A is shown in SEQ ID No.1, the amino acid sequence of the branch B is shown in SEQ ID No.2, and the branch A and the branch B are connected by a disulfide bond formed between the thiol groups in the two cysteine ​​residues.

[0007] Furthermore, the molecular formula of the antimicrobial peptide 2D2W as described above is shown in formula (I):

[0008]

[0009] Furthermore, the nano self-assembly conditions of the antimicrobial peptide 2D2W as described above are as follows: dissolved in water, at a concentration of 64 μM, at room temperature, for 12 hours, and self-assembled into a nanofiber structure.

[0010] Another object of the present invention is to provide a method for preparing the self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity as described above, as follows: Design principle: Two branches are designed, the amino acid sequence of branch A is: DDCWW, the amino acid sequence of branch B is: WWCRRAAARRRR, branch A and branch B are connected by a disulfide bond formed between the thiol groups in the two cysteine ​​residues, and branch A and branch B are connected by electrostatic attraction of oppositely charged amino acids: aspartic acid and arginine and π-π stacking of aromatic amino acids: tryptophan The effect drives its self-assembly. At the same time, in order to improve its ability to bind to the bacterial membrane, the four arginines on the branch B are used to provide the positive charge required for antibacterial activity. Then, the solid phase chemical synthesis method is used to synthesize the branch A and the branch B respectively, and then the branch A and the branch B are connected through the disulfide bond formed between the thiol groups in the two cysteine ​​residues. The polypeptide is identified by mass spectrometry and purified by reversed-phase high-performance liquid chromatography to obtain the polypeptide. After the self-assembly morphology of the polypeptide is observed, the antibacterial activity is determined, and the cell and hemolytic toxicity are determined, it is finally named antimicrobial peptide 2D2W.

[0011] Another object of the present invention is to provide a use of the self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity as described above in the preparation of a drug for treating Gram-positive and / or Gram-negative bacterial infectious diseases.

[0012] Furthermore, in the application described above, the Gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa.

[0013] Furthermore, in the application described above, the Gram-positive bacteria is Staphylococcus epidermidis or Staphylococcus aureus.

[0014] The present invention has the following advantages and beneficial effects: the self-assembling antimicrobial peptide 2D2W of the present invention can form a stable fiber-assembled structure, and at the same time has strong antibacterial activity against common Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, etc.; and has almost no toxicity to human red blood cells, mouse macrophages, and human renal epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the mass spectrum of the antimicrobial peptide 2D2W;

[0016] Figure 2 It is the HPLC chromatogram of the antimicrobial peptide 2D2W;

[0017] Figure 3 The assembly structure of the self-assembled antimicrobial peptide 2D2W under a scanning electron microscope;

[0018] Figure 4 It is the cytotoxicity graph of the antimicrobial peptide 2D2W;

[0019] Figure 5 This is a graph showing the hemolytic activity of the antimicrobial peptide 2D2W. DETAILED DESCRIPTION

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

[0021] Example 1

[0022] Design of antimicrobial peptides

[0023] Design principle: Two branches are designed, the amino acid sequence of branch A is: DDCWW, and the amino acid sequence of branch B is: WWCRRAAARRRR. Branch A and branch B are connected by a disulfide bond formed between the thiol groups in two cysteine ​​residues. The self-assembly of branch A and branch B is driven by the electrostatic attraction of oppositely charged amino acids: aspartic acid and arginine and the π-π stacking effect of aromatic amino acid: tryptophan. At the same time, in order to improve its binding ability with bacterial membranes, the four arginines on branch B are used to provide the positive charge required for antibacterial activity; then, solid-phase chemical synthesis is used to synthesize branch A and branch B respectively, and then branch A and branch B are connected by a disulfide bond formed between the 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, antibacterial activity is determined, and cell and hemolytic toxicity are determined, and it is finally named antimicrobial peptide 2D2W.

[0024] The amino acid sequence of antimicrobial peptide 2D2W is as follows:

[0025] Disulfide bonds

[0026]

[0027] Table 1 Amino acid sequences of antimicrobial peptides

[0028]

[0029] The molecular formula of the self-assembling antimicrobial peptide 2D2W is shown in formula (I),

[0030]

[0031] Example 2

[0032] 1. Synthesis of antimicrobial peptides by solid phase chemical synthesis

[0033] 1. First, connect Fmoc-Arg(pbf)-OH to Rink resin, then remove piperidine after 30 minutes of piperidine deprotection reaction, and wash with dimethylformamide (DMF), and detect the deprotection color with ninhydrin. Then connect the subsequent straight-chain amino acids in sequence until it is connected to Fmoc-Trp(boc)-OH at the N-terminus, and remove the Fmoc at the N-terminus to obtain NH 2 -W(boc)W(boc)C(npys)R(pbf)R(pbf)AAAR(pbf)R(pbf)R(pbf)R(pbf)-Rink Resin.

[0034] The above resin was cleaved with 95% TFA to remove the monomer peptide from the resin, and all the side chain protecting groups of the sequence were cut off to obtain the crude product WWC(npys)RRAAARRRR-NH2, which was purified by liquid phase and freeze-dried.

[0035] 2. Prepare NH using the same method as step 1 2 -D(otbu)D(otbu)C(trt)W(boc)W(boc)-CTCResin, 95% TFA cleaves the monomer peptide from the resin and simultaneously cuts off all the side chain protecting groups of the sequence to obtain the crude product of DDCWW monomer, which is liquid phase purified and freeze-dried.

[0036] 3. Oxidative synthesis: dissolve two monomer peptides, mix them, adjust the pH to 7.5-8 with ammonium bicarbonate solution, stir for 1 hour, monitor the oxidation status by mass spectrometry and liquid chromatography, and after complete oxidation, purify by HPLC, freeze-dry, and you can get the target product.

[0037] 2. Purification and Identification:

[0038] 1. Detection of crude product MS: Take a small amount of crude product, dissolve it and use LC-MS to determine the molecular weight (such as Figure 1 The molecular weight of the product was basically consistent with the theoretical molecular weight in Table 1 before purification.

[0039] 2. Purification: Use high performance liquid chromatography to purify the peptide to obtain a peptide with a purity of >95%; Figure 2 Shown is the HPLC chromatogram of the antimicrobial peptide 2D2W.

[0040] Example 3

[0041] Scanning electron microscopy observation of antimicrobial peptide nanostructures

[0042] The antimicrobial peptide 2D2W was dissolved in water at a concentration of 64 μM and placed at room temperature for 12 hours to self-assemble into a nanofiber structure. Sample staining: The sample was applied to a 300 mesh copper-coated carbon grid and the nanoparticles were negatively stained with 1% phosphotungstic acid for 30 seconds. Then it was placed at room temperature for air drying for 18 hours. Result observation: The dyed nanoparticles were observed using a Hitachi h-7800TEM (Hitachi, Japan). The accelerating voltage was 100 kV.

[0043] like Figure 3 As shown, the antimicrobial peptide 2D2W forms a stable nanofibrous structure in aqueous solution.

[0044] Example 4

[0045] Determination of antibacterial activity of antimicrobial peptides

[0046] The minimum inhibitory concentration (MIC) of the peptides was determined by standard microbroth dilution method. Bacteria in logarithmic phase were diluted to ~2×10 5 CFU / mL. 50 μl of peptides of different concentrations (final concentration of peptides was 1-128 μM) and an equal volume of bacterial suspension were added to each well of a 96-well plate, and a negative control (medium only) and a positive control (bacteria and medium) were set up at the same time. The 96-well plate was then placed in a 37°C constant temperature incubator for 18 to 20 hours. The absorbance at 492 nm was measured using an ELISA reader. Two parallels were set up for each test, and these tests were repeated at least three times. The results are shown in Table 2.

[0047] Table 2 Minimum inhibitory concentration of antimicrobial peptide 2D2W (μM)

[0048]

[0049] It can be seen from Table 2 that the antimicrobial peptide 2D2W exhibits strong antibacterial activity against Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and other bacteria, with the minimum inhibitory concentration being 8-16 μM.

[0050] Example 5

[0051] Determination of cytotoxicity of antimicrobial peptides

[0052] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) colorimetric method was used to determine the effects of nanopeptides on human embryonic kidney epithelial cells (HEK293) and mouse macrophages (RAW 264.7).

[0053] Preparation of cell samples: When the cells cover about 80% of the bottom of the cell culture bottle, the culture medium is extracted, and the cells are removed from the bottom of the culture bottle using 0.25% trypsin digestion solution. The cells are counted using a cell counter, and the cells are diluted to about 2×104 (cells / well) using the corresponding culture medium. Then the cells are added to columns 1-11 of the 96-well plate, and complete culture medium without cells is added to column 12, 50 μL per well. The 96-well plate is placed in an environment of 37°C and humidified air containing 5% CO2 and cultured for 24 hours. Unprepared cell culture medium is added to columns 1-12 of the 96-well plate, and continuously diluted to the tenth column. The diluted nanopeptide solution is extracted and added to columns 1-10 of the above-mentioned 96-well plate containing cells, and unprepared cell culture medium is added to columns 11-12. The 96-well plate is placed in an environment of 37°C and humidified air containing 5% CO2 and cultured for 24 hours, wherein column 11 contains cells as a positive control and column 12 as a negative control. Addition of MTT: Dissolve MTT into a 5 mg / mL solution with sterile PBS and add it to the 96-well plate incubated with the above nanopeptide solution and cells, 25 μL per well. After 4 hours of incubation in the dark, carefully aspirate all the liquid in the wells, add 150 μL of dimethyl sulfoxide (DMSO) to each well, and dissolve the formazan crystals in DMSO. Use an ELISA reader to measure its absorbance at 570 nm. Set up two parallels for each test, and repeat these tests at least three times. Cell viability (%) = [(sample OD 570 - Negative control OD 570 ) / (positive control OD 570 - Negative control OD 570 )]×100%.

[0054] like Figure 4 As shown, the antimicrobial peptide 2D2W has cytotoxic effects on mouse macrophages RAW 264.7 and renal epithelial cells HEK 293T. In the concentration range of 8-128 μM, the antimicrobial peptide 2D2W has no effect on the survival rate of mouse macrophages and still remains above 90%, indicating that the antimicrobial peptide 2D2W has good biocompatibility.

[0055] Example 6

[0056] Determination of hemolytic activity of antimicrobial peptides

[0057] Preparation of blood: fresh human blood was drawn, centrifuged at 3000-3500rpm for 10 minutes at 4°C, the supernatant was aspirated, phosphate buffer (PBS PH=7.4) was filtered with a 0.22μM water filter, then added to the red blood cells, the red blood cells were centrifuged and washed three times, and finally the red blood cells were resuspended in 10 times the volume of PBS. Dilution of nanopeptides: PBS was added to columns 1-12 of a 96-well plate, 90μL was added to the first column, and 50μL was added to the other columns. 10μL of the dissolved nanopeptide solution was drawn and added to the first column of the 96-well plate, and diluted to the 10th column. Addition of red blood cells: the resuspended red blood cells were added to columns 1-12 of the 96-well plate, 50μL per well. 0.1% Triton X-100 was added to column 12 as a positive control (100% hemolysis), and column 11 as a negative control. The 96-well plate was placed in a 37°C incubator for 1 hour, and then the 96-well plate was centrifuged at 1000×g for 5 minutes at 4°C, and 50 μL of the supernatant from each well was transferred to a new 96-well plate. Result determination: The absorbance at 570 nm was determined using an ELISA reader. Two parallels were set for each test, and these tests were repeated at least three times.

[0058] like Figure 5 As shown, the antimicrobial peptide 2D2W exhibited negligible hemolytic effect at all tested concentrations, indicating that the antimicrobial peptide 2D2W has good biocompatibility.

[0059] In summary, the antimicrobial peptide 2D2W has strong antibacterial activity against common bacteria such as Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. It has almost no toxicity to macrophages, red blood cells, and human renal epithelial cells, and has extremely high application potential.

Claims

1. A self-assembled antimicrobial peptide 2D2W with low toxicity and high antibacterial activity, Features: Its molecular formula is shown in formula (I), including branch A and branch B, the amino acid sequence of branch A is shown in SEQ ID No.1, the amino acid sequence of branch B is shown in SEQ ID No.2, and the branch A and branch B are connected by a disulfide bond formed between the thiol groups in two cysteine ​​residues.

2. A self-assembly method for a self-assembling antimicrobial peptide 2D2W with low toxicity and high antibacterial activity according to claim 1, Features ,The nano self-assembly conditions are as follows: dissolved in water, at a concentration of 64μM, at room temperature, for 12 hours, and self-assembled into nanostructures.

3. A method for preparing a self-assembled antimicrobial peptide 2D2W with low toxicity and high antibacterial activity according to claim 1 or 2, It is characterized in that The method is as follows: Design principle: Two branches are designed, the amino acid sequence of branch A is: DDCWW, and the amino acid sequence of branch B is: WWCRRAAARRRR. Branch A and branch B are connected by a disulfide bond formed between the thiol groups in two cysteine ​​residues. The self-assembly of branch A and branch B is driven by the electrostatic attraction of oppositely charged amino acids: aspartic acid and arginine and the π-π stacking effect of aromatic amino acid: tryptophan. At the same time, in order to improve its binding ability with bacterial membranes, the four arginines on branch B are used to provide the positive charge required for antibacterial activity; then, solid-phase chemical synthesis is used to synthesize branch A and branch B respectively, and then branch A and branch B are connected by a disulfide bond formed between the 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, antibacterial activity is determined, and cell and hemolytic toxicity are determined, and it is finally named antimicrobial peptide 2D2W.

4. Use of the self-assembled antimicrobial peptide 2D2W with low toxicity and high antibacterial activity according to claim 1 in the preparation of a drug for treating Gram-positive and Gram-negative bacterial infectious diseases, wherein the Gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa, and the Gram-positive bacteria are Staphylococcus epidermidis or Staphylococcus aureus.

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