Anti-enzymolysis antibacterial peptide FRL as well as preparation method and application thereof

Abstract

The invention discloses an anti-enzymolysis antibacterial peptide FRL as well as a preparation method and application thereof, and belongs to the field of bioengineering, and the sequence of the antibacterial peptide FRL is as shown in SEQ ID No.1. The antibacterial peptide FRL shows a narrow-spectrum antibacterial effect, can effectively inhibit gram-negative bacteria, and has the potential of being applied to drugs for treating gram-negative bacteria infectious diseases. The antibacterial peptide has very low hemolytic activity and eukaryotic cell toxicity. The antibacterial peptide cannot cause 10% red blood cell hemolysis under the concentration of 128 [mu] mol / L, and the survival rate of mouse macrophages RAW264.7 reaches 80% or above. The antibacterial peptide can be used for treating trypsin, pepsin, chymotrypsin and protease K (0.4 mg / mL) under the conditions that the pH is equal to 5 and the temperature is 37 DEG C; 0.5 h), good stability is shown. In conclusion, the antibacterial peptide FRL has the development potential of becoming an antibiotic substitute.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to an antimicrobial peptide FRL resistant to enzymatic hydrolysis and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are active polypeptides with antibacterial effects that are widely present in organisms and are the response products of biological nonspecific immune defense. Antimicrobial peptides have a broad spectrum of biological activities such as antiviral, antifungal, antiparasitic, antitumor, and immunomodulatory. The main mechanism of antimicrobial peptides in killing bacteria is to destroy the bacterial cell membrane through physical penetration; and it is difficult for bacteria and other microorganisms to change the cell membrane structure of their own phospholipid bilayer; therefore, antimicrobial peptides have become an attractive candidate for solving the drug resistance of pathogenic microorganisms. At present, antimicrobial peptides have become a research hotspot in the fields of drug development and have extremely broad market application prospects.

[0003] Although thousands of antimicrobial peptides have been extracted from various plants and animals, there are very few antimicrobial peptides that can be used as antibiotic substitutes in the medical field. There are two main reasons that limit natural antimicrobial peptides from becoming antibiotic substitutes: one is the hemolytic effect of natural antimicrobial peptides on red blood cells; the other is the low stability of natural antimicrobial peptides in the body. Therefore, it is imperative to find a method to develop stable and safe antimicrobial peptides. Summary of the invention

[0004] Based on the above requirements, the present invention discloses an antimicrobial peptide FRL resistant to enzymatic hydrolysis, which has high stability in the gastrointestinal tract and very low hemolytic activity.

[0005] The technical solution adopted by the present invention is as follows: the amino acid sequence of an antimicrobial peptide FRL resistant to enzymatic hydrolysis is shown as SEQ ID No. 1.

[0006] Another object of the present invention is to provide a method for preparing the antimicrobial peptide FRL resistant to enzymatic hydrolysis as described above, as follows: using the flexible sequence Resilin as a template, connecting two hydrophilic and hydrophobic alternating antimicrobial fragments, the sequence of the antimicrobial fragment is: FFRLFFRLRRR, and the designed polypeptide sequence is shown in SEQ ID No. 1; obtaining the polypeptide by solid phase chemical synthesis, and then completing the preparation of the polypeptide after reverse phase high performance liquid chromatography purification and mass spectrometry identification; then testing the antimicrobial activity, hemolytic activity and protease stability of the polypeptide, and finally naming it as antimicrobial peptide FRL.

[0007] Another object of the present invention is to provide the use of the above-mentioned antimicrobial peptide FRL resistant to enzymolysis in the preparation of drugs for treating Gram-negative bacterial infectious diseases.

[0008] In a further application as described above, the Gram-negative bacteria are Escherichia coli, Salmonella pullorum, Pseudomonas aeruginosa or Salmonella typhimurium.

[0009] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-negative bacterial infection, wherein the drug contains the antimicrobial peptide FRL resistant to enzymatic degradation as described above.

[0010] Advantages and beneficial effects of the present invention: The antimicrobial peptide FRL of the present invention has a highly effective inhibitory effect on Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium, but has a weaker inhibitory effect on Staphylococcus aureus and Staphylococcus epidermidis; it has a narrow-spectrum antibacterial effect. And it has very low hemolytic activity (minimum hemolytic concentration>128mM). Absorbance detection found that the antimicrobial peptide FRL showed temperature response characteristics at pH=5; it had a tendency to aggregate as the temperature increased, indicating that it has the characteristic of stably passing through the front part of the gastrointestinal tract. In the case of oral administration, it has the potential to effectively retain antibacterial activity to the end of the gastrointestinal tract. In summary, the antimicrobial peptide FRL is an antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the high performance liquid chromatogram of the antimicrobial peptide FRL of the present invention.

[0012] Figure 2 It is the high performance liquid chromatography-mass spectrum of the antimicrobial peptide FRL of the present invention.

[0013] Figure 3 It is a comparison chart of the hemolytic activity of the antimicrobial peptide FRL of the present invention and melittin ME.

[0014] Figure 4 This is a comparison chart of the cytotoxic effects of the antimicrobial peptide FRL melittin ME of the present invention on mouse macrophages.

[0015] Figure 5 The OD of the antimicrobial peptide FRL of the present invention under different environments 350nm Absorbance.

[0016] Figure 6 The high performance liquid chromatograms of the antimicrobial peptide FRL of the present invention after incubation with different proteases for 0.5 hours. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0018] Example 1

[0019] Design of antimicrobial peptides

[0020] The amino acid sequence of the antimicrobial peptide FRL is:

[0021] GGRPSDSWGAPGGGNFFRLFFRLRRR;

[0022] Using the flexible sequence Resilin as a template, a stimulus-responsive antimicrobial peptide was designed and named FRL. The sequence of the antimicrobial peptide is shown in Table 1.

[0023] Table 1 Amino acid sequence

[0024]

[0025] Its molecular formula is shown in formula (I):

[0026]

[0027] The charge number of the antimicrobial peptide FRL is +5. By connecting the hydrophilic-hydrophobic alternating antimicrobial fragments with the sequence module derived from Resilin, it has the characteristics of high biocompatibility, low immunogenicity and stimulation response of Resilin.

[0028] Example 2

[0029] Synthesis of antimicrobial peptide FRL by solid phase chemical synthesis

[0030] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and a resin with side chain protection removed from the Fmoc group is obtained;

[0031] 2. Add a cleavage agent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0032] 3. Use 0.2 mol / L sodium sulfate (phosphoric acid adjusted to pH = 7.5) to balance the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, C18 reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1 mL / min, detection wave is 220 nm, collect the main peak, and freeze-dry; further purify using reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25% B to 40% B, elution time is 12 min, flow rate is 1 mL / min, collect the main peak as above, and freeze-dry;

[0033] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weights (e.g. Figure 1 , 2 The molecular weight of the antimicrobial peptide was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide was greater than 95%.

[0034] Embodiment 3:

[0035] Determination of antimicrobial activity of antimicrobial peptides

[0036] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the microbroth dilution method. 0.01% acetic acid (containing 0.2% BSA) was used as the diluent, and a series of gradient antimicrobial peptide solutions were prepared in sequence using the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5 pc / mL) in each well. Set up positive control (containing bacterial solution but not antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) respectively. Incubate at 37℃ for 14-18h and read the sample at 492nm (OD 492nm ) to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0037] Table 2 Antibacterial activity of antimicrobial peptide FRL

[0038]

[0039] It can be seen from Table 2 that the antimicrobial peptide FRL exhibits high antibacterial activity against Gram-negative bacteria.

[0040] Table 3 MHC (μM), GM (μM) and SI values ​​of short peptides

[0041]

[0042] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, centrifuge at 1000g for 5 minutes, collect red blood cells; wash with PBS three times, and resuspend with 10 mL PBS; take 50 μL of red blood cell suspension and 50 μL of antimicrobial peptide solution of different concentrations dissolved in PBS, mix them evenly, and incubate them in a 37°C incubator for 1 hour; take out after 1 hour, centrifuge at 4°C and 1000g for 5 minutes; take out the supernatant and measure the absorbance value at 570nm with an enzyme marker; take the average value of each group and compare and analyze. 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 was used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a hemolysis rate of 10%. See the test results. Figure 3 .pass Figure 3 It can be seen that the antimicrobial peptide FRL did not show hemolytic activity within the detection range and was significantly different from bee venom in the control group.

[0043] Eukaryotic cytotoxicity assay: MTT was used to detect cytotoxicity using mouse macrophage RAW264.7 cells.

[0044] (1) Preparation of culture medium and cell culture: Complete culture medium was prepared by mixing DMEM (culture medium) and fetal bovine serum at a ratio of 9:1, and mouse macrophage RAW264.7 cells were resuscitated in liquid nitrogen until the cells covered 80%-90% of the bottom of the flask.

[0045] (2) Experimental treatment of cells to be used: Wash and resuspend the cells three times with sterile PBS, digest the cells with 0.25% trypsin solution to make them fall off the bottom of the bottle, rinse with complete culture medium to obtain a single cell suspension, and fill a 96-well plate with a final concentration of about 2×10 4 50 μL of cell suspension.

[0046] (3) Antimicrobial peptide treatment: Add 10 μL of antimicrobial peptide to the first well of a separate 96-well plate and dilute it in multiples. Then take out 50 μL of the peptide solution and add it to wells 1-10 of the original 96-well plate. Add 50 μL of complete medium to well 11 and 100 μL of complete medium to well 12. Incubate at constant temperature for 4 h.

[0047] (4) Toxicity test: 50 μL of 5 mg / mL MTT solution was added to a 96-well plate. After culturing for 3-4 h, 150 μL of DMSO (dimethyl sulfoxide) was added and the OD value was measured by a microplate reader. 570nm Measure the absorbance. The higher the absorbance value, the weaker the toxicity, and vice versa. See the test results for Figure 4 .

[0048] pass Figure 4It can be seen that the antimicrobial peptide FRL did not show toxicity to mouse macrophages within the detection range, and was significantly different from bee venom in the control group.

[0049] Embodiment 4:

[0050] Environmental response characteristics of antimicrobial peptide FRL were detected by OD 350nm The absorbance at 400 μM was used to measure the absorbance. The antimicrobial peptide FRL was dissolved in deionized water to obtain a peptide solution with a final concentration of 64 μM. Solutions of pH = 9 and pH = 5 were prepared with NaOH and HCl. Mix pH = 9, pH = 5, and deionized water with an equal volume of 64 μM peptide solution to obtain antimicrobial peptide FRL solutions of pH = 9, pH = 7, and pH = 5 with a final concentration of 32 μM. In a transparent 96-well plate, the above solutions were added, three parallels of each solution, 100 μL for each parallel, and the absorbance at different temperatures was read three times at each temperature; the reading method was to increase, decrease, and then increase from the lowest temperature in sequence. See the test results in the following table. Figure 5 .

[0051] pass Figure 5 It can be seen that at pH = 5, the antimicrobial peptide FRL exhibits temperature-responsive characteristics and shows a tendency to aggregate as the temperature rises. This feature enables the antimicrobial peptide FRL to have the potential for stable antibacterial activity in the gastrointestinal tract.

[0052] The stability of the polypeptide in the gastrointestinal tract was determined by testing the stability of the polypeptide to proteases present in the gastrointestinal tract under a pH = 5 environment. As described above, a 0.2 mg / mL peptide solution at pH = 5 was prepared, mixed with an equal volume of a 0.8 mg / mL protease solution, and incubated at 37°C for 0.5 hours. The degradation of the polypeptide by multiple proteases was detected by HPLC. The HPLC parameters were the same as those in Example 2 ( Figure 1 ) are the same. The test results are shown in Figure 6 .

[0053] pass Figure 6 It can be seen that at pH = 5, the structure of the antimicrobial peptide FRL remained relatively intact after incubation at 37°C for 0.5 hours in the presence of 0.4 mg / mL chymotrypsin, pepsin, proteinase K, and trypsin, indicating good stability.

[0054] The pH of the human stomach is 1.5-3.5, the pH of the duodenum is 6, and the pH from the jejunum to the colon is about 6.8-9. The pH of the stomach of monogastric animals, such as pigs, is 3-5, and the pH from the duodenum to the colon increases from 6 to 7-8. Proteases that have a greater impact on peptide activity, such as pepsin, trypsin, and chymotrypsin, are mostly secreted in the front end of the gastrointestinal tract (lower pH segment). Therefore, the antimicrobial peptide FRL has a tendency to aggregate under low pH conditions, indicating that it has the characteristic of stably passing through the front part of the gastrointestinal tract. In the case of oral administration, it has the potential to effectively retain antibacterial activity to the end of the gastrointestinal tract. In summary, the antimicrobial peptide FRL is an antimicrobial peptide with high application value.

Claims

1. An antimicrobial peptide FRL resistant to enzymatic hydrolysis, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The method for preparing an antimicrobial peptide FRL resistant to enzymatic hydrolysis according to claim 1, characterized in that: The method is as follows: using the flexible sequence Resilin as a template, connecting the two hydrophilic and hydrophobic alternating antibacterial fragments, the sequence of the antibacterial fragment is: FFRLFFRLRRR, and the designed polypeptide sequence is shown in SEQ ID No. 1; using solid phase chemical synthesis to obtain the polypeptide, and then after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed; then the antibacterial activity, hemolytic activity and protease stability of the polypeptide are tested, and finally named as the antimicrobial peptide FRL.

3. Use of the antimicrobial peptide FRL resistant to enzymolysis according to claim 1 in the preparation of a drug for treating Gram-negative bacterial infectious diseases.

4. The use according to claim 3, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella pullorum, Pseudomonas aeruginosa or Salmonella typhimurium.

5. A drug suitable for treating and / or preventing Gram-negative bacterial infection, characterized in that: The medicine contains the antimicrobial peptide FRL resistant to enzymatic degradation as claimed in claim 1.

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