An anti-enzymatic antibacterial peptide FRL and its preparation method and application

Abstract

The present invention discloses an anti - enzymolysis antibacterial peptide FRL, its preparation method and application, belonging to the field of bioengineering. The sequence of the antibacterial peptide FRL is shown as SEQ ID No.1. This antibacterial peptide FRL exhibits narrow - spectrum antibacterial activity, can effectively inhibit Gram - negative bacteria, and has the potential to be applied in drugs for treating Gram - negative bacterial infectious diseases. This antibacterial peptide has very low hemolytic activity and eukaryotic cell toxicity. This antibacterial peptide fails to cause 10% erythrocyte hemolysis at a concentration of 128 μmol / L, and the survival rate of mouse macrophage RAW264.7 reaches more than 80%. This antibacterial peptide shows good stability against trypsin, pepsin, chymotrypsin, and proteinase K (0.4 mg / mL; 0.5 h) at pH = 5 and 37°C. In summary, the antibacterial peptide FRL has the development potential to become an alternative to antibiotics.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to an enzyme-resistant antibacterial peptide FRL, its preparation method and application. Background Art

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

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

[0004] Based on the above needs, the present invention discloses an enzyme-resistant antibacterial peptide FRL, which has high stability in the gastrointestinal tract and low hemolytic activity.

[0005] The technical solution adopted by the present invention is as follows: The amino acid sequence of an enzyme-resistant antibacterial peptide FRL is shown in SEQ ID No.1.

[0006] Another object of the present invention is to provide a preparation method of an enzyme-resistant antibacterial peptide FRL as described above, as follows: Using the flexible sequence Resilin as a template, by connecting the hydrophilic-hydrophobic alternating antibacterial fragments, the sequence of the antibacterial fragment is: FFRLFFRLRRR, and the designed polypeptide sequence is shown in SEQ ID No.1; The polypeptide is obtained by solid-phase chemical synthesis, 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 detected, and finally it is named antibacterial peptide FRL.

[0007] Another object of the present invention is to provide the application of an enzyme-resistant antibacterial peptide FRL as described above in the preparation of drugs for treating Gram-negative bacterial infectious diseases.

[0008] For further applications 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 infections, said drug containing an anti-hydrolytic antibacterial peptide FRL as described above.

[0010] Advantages and beneficial effects of the present invention: The antibacterial peptide FRL of the present invention has a high inhibitory effect on Gram-negative bacterial species such as Escherichia coli and Salmonella typhimurium, but has a weak inhibitory effect on Staphylococcus aureus and Staphylococcus epidermidis; it has a narrow-spectrum antibacterial effect. Moreover, it has very low hemolytic activity (minimum hemolytic concentration > 128 mM). Absorbance detection found that the antibacterial peptide FRL exhibited a temperature-responsive characteristic at pH = 5; it tended to aggregate with increasing temperature, indicating its characteristic of stably passing through the anterior segment of the gastrointestinal tract. In the case of oral administration, it has the potential to effectively retain antibacterial activity until the end of the gastrointestinal tract. In summary, the antibacterial peptide FRL is an antibacterial peptide with high application value. Description of the Drawings

[0011] Figure 1 It is the high-performance liquid chromatography chart of the antibacterial peptide FRL of the present invention.

[0012] Figure 2 It is the high-performance liquid mass spectrometry chart of the antibacterial peptide FRL of the present invention.

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

[0014] Figure 4 It is the comparison chart of the cytotoxic effects of the antibacterial peptide FRL and melittin ME of the present invention on mouse macrophages.

[0015] Figure 5 It is the OD of the antibacterial peptide FRL of the present invention under different environments 350nm Absorbance.

[0016] Figure 6 It is the high-performance liquid chromatography chart of the antibacterial peptide FRL of the present invention after incubation with different proteases for 0.5 hours. Detailed Embodiments

[0017] The present invention will be further described in detail below with reference to the accompanying drawings of the specification. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0018] Example 1

[0019] Design of the antibacterial peptide

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

[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 antimicrobial peptide FRL is +5. By connecting the hydrophilic-hydrophobic alternating antimicrobial fragment with the sequence module derived from Resilin, it has the characteristics of high biocompatibility, low immunogenicity and stimulus response of Resilin.

[0028] Example 2

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

[0030] 1. The preparation of the antimicrobial peptide is carried out one by one from the C-terminus to the N-terminus and completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is attached to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-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 turn until the synthesis is completed, and the resin with side-chain protection of the Fmoc group removed is obtained;

[0031] 2. In the peptide resin obtained above, a cleavage reagent is added, and the reaction is carried out at 20 °C in the dark for 2 h, and then filtered; the precipitate is washed with TFA (trifluoroacetic acid), the washing solution is mixed with the above filtrate, concentrated by a rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether is added, and precipitated at -20 °C for 3 h to precipitate a white powder, centrifuged at 2500 g for 10 min, the precipitate is collected, and the precipitate is washed with anhydrous ether and dried in vacuo to obtain the polypeptide, wherein the cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) mixed in a mass ratio of 95:2.5:2.5;

[0032] 3. Column equilibration was carried out for 30 min using 0.2 mol / L sodium sulfate (adjusted to pH = 7.5 with phosphoric acid). The polypeptide was dissolved in 90% aqueous acetonitrile solution, filtered, and passed through a C18 reversed-phase normal pressure column. Gradient elution was employed (the eluent was a mixture of methanol and aqueous sodium sulfate solution in a volume ratio of 30:70 to 70:30), the flow rate was 1 mL / min, the detection wavelength was 220 nm, the main peak was collected, and freeze-dried; further purification was carried out using a reversed-phase C18 column. Eluent A was 0.1% TFA / aqueous solution; eluent B was 0.1% TFA / acetonitrile solution. The elution concentration was 25% B - 40% B, the elution time was 12 min, the flow rate was 1 mL / min, and the main peak was collected and freeze-dried as above;

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

[0034] Example 3:

[0035] Determination of antibacterial activity of antibacterial peptides

[0036] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antibacterial peptides was determined by the microbroth dilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antibacterial peptide solutions were prepared successively by the two-fold dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, and then add an equal volume of the test bacterial solution (~10 5 CFU / mL) to each well. Positive control (containing bacterial solution but no antibacterial peptide) and negative control (neither containing bacterial solution nor peptide) were set respectively. Incubate at 37 °C for 14 - 18 h, and measure the optical absorbance at 492 nm (OD 492nm ) with an enzyme-linked immunosorbent assay reader to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0037] Table 2 Antibacterial activity of antibacterial peptide FRL

[0038]

[0039] As can be seen from Table 2, antibacterial peptide FRL showed 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 antibacterial peptide FRL did not show toxicity to mouse macrophages within the detection range, and there was a significant difference compared with melittin in the control group.

[0049] Example 4:

[0050] The environmental response characteristics of antibacterial peptide FRL were determined by detecting the absorbance at OD 350nm . The antibacterial peptide FRL was dissolved in deionized water to obtain a peptide solution with a final concentration of 64 μM. Solutions with pH = 9 and pH = 5 were prepared using NaOH and HCl. The solutions with pH = 9, pH = 5, deionized water were mixed with an equal volume of 64 μM peptide solution to obtain antibacterial peptide FRL solutions with pH = 9, pH = 7, pH = 5 and a final concentration of 32 μM. In a transparent 96-well plate, the above solutions were added, with three parallels for each solution and 100 μL for each parallel, and the absorbance at different temperatures was read. It was read three times at each temperature; the reading method was to increase, decrease, and then increase in sequence starting from the lowest temperature. The detection results are shown in Figure 5 .

[0051] It can be seen through Figure 5 that at pH = 5, antibacterial peptide FRL showed the characteristics of temperature response and exhibited a tendency to aggregate as the temperature increased. This characteristic endows antibacterial peptide FRL with the potential for stable antibacterial activity in the gastrointestinal tract.

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

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

[0054] Since 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 around 6.8 - 9. For monogastric animals, such as pigs, the pH of the stomach 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 secreted mostly in the front end of the gastrointestinal tract (the lower pH segment). Therefore, the antimicrobial peptide FRL has an aggregation tendency under low pH conditions, indicating its characteristic of stably passing through the front segment of the gastrointestinal tract. In the case of oral administration, it has the potential to effectively retain its antibacterial activity until 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 enzymolysis-resistant antimicrobial peptide FRL according to claim 1 in the preparation of a drug for treating Gram-negative bacterial infectious diseases, wherein the Gram-negative bacteria are Escherichia coli, Salmonella pullorum, Pseudomonas aeruginosa or Salmonella typhimurium.

4. 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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