An antimicrobial peptide derived from arthropod protein and its preparation method and application

Abstract

The present invention discloses an antimicrobial peptide r-WRL derived from arthropod protein, its preparation method and application, and belongs to the field of bioengineering. The amino acid sequence of the antimicrobial peptide r-WRL is shown in SEQ ID No. 1. The present invention also discloses the application of the antimicrobial peptide r-WRL in the preparation of drugs for treating and / or preventing Gram-negative and / or Gram-positive bacterial infectious diseases. Since the antimicrobial peptide r-WRL exhibits a strong antibacterial effect, it can effectively inhibit Gram-negative and Gram-positive bacteria and has the potential for application in drugs for treating Gram-negative and Gram-positive bacterial infectious diseases. The antimicrobial peptide has low hemolytic activity and good physiological salt ion stability. In summary, the antimicrobial peptide r-WRL of the present invention has the development potential to become an antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to an antimicrobial peptide derived from arthropod protein, a preparation method and an application thereof. Background Art

[0002] Antimicrobial peptides are nonspecific immune defense response products produced by organisms, exhibiting broad-spectrum biological activities such as antiviral, antifungal, antiparasitic, antitumor, and immunomodulatory properties. The primary mechanism by which antimicrobial peptides kill bacteria is through physical permeation, disrupting the bacterial cell membrane. However, bacteria and other microorganisms find it difficult to alter the structure of their own phospholipid bilayer membranes. Therefore, antimicrobial peptides have become attractive candidates for addressing drug resistance in pathogens, offering promising market applications. However, few natural antimicrobial peptides are suitable for use in medical applications as antibiotic alternatives. These are primarily limited by two factors: their hemolytic effect on red blood cells and their low stability under physiological conditions. Therefore, rational design is crucial for the development of safe and stable antimicrobial peptides. Summary of the Invention

[0003] Based on the above needs, the present invention discloses an antimicrobial peptide r-WRL derived from arthropod protein, which effectively kills Gram-negative and Gram-positive pathogens while having very low hemolytic activity and good physiological salt stability.

[0004] The technical solution adopted by the present invention is as follows: the amino acid sequence of an antimicrobial peptide r-WRL derived from arthropod protein is shown in SEQ ID No.1.

[0005] Furthermore, its molecular formula is shown in formula (I):

[0006]

[0007] Another object of the present invention is to provide a method for preparing the arthropod-derived antimicrobial peptide r-WRL, as described above. The method comprises: using an arthropod protein amino acid sequence fragment: GAPAQTPSSQ as a template, linking it to a hydrophilic-hydrophobic alternating antimicrobial fragment, wherein the amino acid sequence of the antimicrobial fragment is WWWRLWWRLRRR, and the designed polypeptide sequence is shown in SEQ ID No. 1; obtaining the polypeptide by solid-phase chemical synthesis, and then purifying it by reverse-phase high-performance liquid chromatography and identifying it by mass spectrometry to complete the preparation of the polypeptide; then testing the antimicrobial activity, hemolytic activity, and physiological salt ion stability of the polypeptide, and finally naming it the antimicrobial peptide r-WRL.

[0008] Another object of the present invention is to provide use of the above-mentioned arthropod-derived antimicrobial peptide r-WRL in the preparation of a drug for treating and / or preventing Gram-negative and / or Gram-positive bacterial infectious diseases.

[0009] Furthermore, the Gram-negative bacteria are Escherichia coli, Salmonella paratyphi suis or Pseudomonas aeruginosa.

[0010] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis or Listeria monocytogenes.

[0011] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, wherein the drug contains the arthropod protein-derived antimicrobial peptide r-WRL as described above.

[0012] Advantages and benefits of the present invention: The antimicrobial peptide r-WRL exhibits highly effective inhibitory effects against Gram-negative bacteria such as Escherichia coli and Salmonella paratyphi, as well as Gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis. It also exhibits low hemolytic activity (minimum hemolytic concentration >64 mM) and good physiological salt stability. In summary, the antimicrobial peptide r-WRL is an antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the high performance liquid chromatogram of the antimicrobial peptide r-WRL of the present invention.

[0014] Figure 2 This is a high performance liquid chromatography-mass spectrometry diagram of the antimicrobial peptide r-WRL of the present invention.

[0015] Figure 3 This is a comparison chart of the hemolytic activities of the antimicrobial peptide r-WRL of the present invention and melittin ME. DETAILED DESCRIPTION

[0016] The present invention will be 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 and reagents used are all commercially available unless otherwise specified.

[0017] Example 1

[0018] Design of antimicrobial peptides

[0019] Arthropodin is a type of elastic protein, and it is the most effective elastic protein known. Many insects and arthropods contain arthropodin. Arthropodin not only has excellent mechanical properties, but also has excellent chemical and cytocompatibility, and therefore has high application potential in biomedical engineering and medicine, including tissue engineering, drug delivery, bioimaging, biosensors, catalysis, and bioelectronics. This example uses arthropodin fragment sequences as templates to rationally design safe, stable, and highly active antimicrobial peptides. The amino acid sequence of the antimicrobial peptide r-WRL is:

[0020] GAPAQTPSSQWWWRLWWRLRRR;

[0021] Using a flexible sequence arthropod protein fragment as a template, a stimulus-responsive antimicrobial peptide was designed and named r-WRL. The sequence of the antimicrobial peptide is shown in Table 1.

[0022] Table 1 Amino acid sequence

[0023]

[0024] Its molecular formula is shown in formula (I):

[0025]

[0026] The charge number of the antimicrobial peptide r-WRL is +5. By connecting the hydrophilic and hydrophobic alternating antimicrobial fragments with the fragment sequence derived from arthropod protein, it has the high biocompatibility of arthropod protein and the antimicrobial activity of antimicrobial peptides.

[0027] Example 2

[0028] Synthesis of antimicrobial peptide r-WRL by solid-phase chemical synthesis

[0029] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using 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); follow this procedure from the C-terminus to the N-terminus until the synthesis is complete, and obtain the resin with the side chain protection of the Fmoc group removed;

[0030] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with TFA (trifluoroacetic acid) to precipitate, mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether. Precipitate at -20°C for 3 hours to precipitate a white powder. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0031] 3. Use 0.2 mol / L sodium sulfate (phosphoric acid adjusted to pH = 7.5) to equilibrate the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and use a 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), the flow rate is 1 mL / min, the detection wave is 220 nm, collect the main peak, and freeze-dry; further purification is performed using a reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12 minutes, the flow rate is 1 mL / min,, and the main peak is collected as above and freeze-dried;

[0032] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray 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%.

[0033] Example 3:

[0034] Determination of antimicrobial activity of antimicrobial peptides

[0035] 1. Determination of antimicrobial activity: The minimum inhibitory concentration (MIC) of several antimicrobial peptides was determined using the broth microdilution method. 0.01% acetic acid (containing 0.2% BSA) was used as the diluent, and a series of gradient antimicrobial peptide solutions were prepared 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 100mg / mL) were added to each well. Positive control (containing bacterial solution but not antimicrobial peptide) and negative control (containing neither bacterial solution nor peptide) were set up respectively. Incubate at 37℃ for 14-18h and analyze the samples at 492nm (OD 492nm ) to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0036] Table 2 Antibacterial activity of antimicrobial peptide r-WRL

[0037]

[0038] As can be seen from Table 2, the antimicrobial peptide r-WRL exhibits high antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0039] Table 3 MHC (μM), GM (μM) and SI values ​​of antimicrobial peptides

[0040] peptides <![CDATA[MHC a ]]> <![CDATA[GM b ]]> <![CDATA[Selection Index (SI) c > r-WRL >64 7.5 17.0 ME 0.25 2.31 0.11

[0041] 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, and collect red blood cells; wash it with PBS 3 times, and then resuspend it with 10 mL of PBS; take 50 μL of red blood cell suspension and mix it evenly with 50 μL of antimicrobial peptide solution of different concentrations dissolved in PBS, and incubate it in a constant temperature incubator at 37°C for 1 hour; take it out after 1 hour, centrifuge it 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 concentration of antimicrobial peptide 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 r-WRL did not show hemolytic activity within the detection range, and showed significant difference from the bee venom in the control group.

[0042] Example 4:

[0043] The salt ion stability of the antimicrobial peptide r-WRL was determined by measuring its antibacterial activity under different physiological salt conditions. The salt ion conditions used were as follows: NaCl 150mM; KCl 4.5mM; CaCl2 2.5mM; MgCl2 1mM, FeCl3 4μM; ZnCl2 8μM; NH4Cl 6μM. The test results are shown in Table 4.

[0044] Table 4 Salt ion stability of antimicrobial peptide r-WRL (μM)

[0045] comparison <![CDATA[Na + ]]> <![CDATA[K + ]]> <![CDATA[Ca 2+ ]]> <![CDATA[Mg 2+ ]]> <![CDATA[NH4 + ]]> <![CDATA[Zn 2+ ]]> S. aureus 29213 2 16 4 >64 32 4 4 P.aeruginosa PAO1 2 16 4 >64 16 4 2

[0046] It can be seen from Table 4 that at physiological concentrations of K + , NH4 + , and Zn 2+ Under physiological concentrations of Na + and Mg 2+ In the presence of saline, r-WRL exhibits antimicrobial activity. In summary, the antimicrobial peptide r-WRL has excellent stability at physiological salt concentrations.

Claims

1. An antimicrobial peptide r-WRL derived from arthropod protein, characterized in that Its amino acid sequence is shown in SEQ ID No. 1, and its molecular formula is shown in formula (I):

2. The method for preparing an arthropod protein-derived antimicrobial peptide r-WRL according to claim 1, characterized in that: The method is as follows: an amino acid sequence fragment of arthropod protein: GAPAQTPSSQ is used as a template, and it is connected with an alternating hydrophilic and hydrophobic antibacterial fragment, the amino acid sequence of the antibacterial fragment is: WWWRLWWRLRRR, and the designed polypeptide sequence is shown in SEQ ID No. 1; a polypeptide is obtained by solid-phase chemical synthesis, and then purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry to complete the preparation of the polypeptide; then, the polypeptide is tested for antibacterial activity, hemolytic activity and physiological salt ion stability, and finally named as the antimicrobial peptide r-WRL.

3. Use of the arthropod protein-derived antimicrobial peptide r-WRL according to claim 1 in the preparation of a medicament for treating and / or preventing infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria, wherein the Gram-negative bacteria are Escherichia coli, Salmonella Paratyphi suis, or Pseudomonas aeruginosa, and the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, or Listeria monocytogenes.

4. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains the arthropod-derived antimicrobial peptide r-WRL as claimed in claim 1.

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