Antibacterial peptide derived from arthropod protein as well as preparation method and application of antibacterial peptide

Abstract

The invention discloses an antibacterial peptide r-WRL derived from arthropod protein and a preparation method and application of the antibacterial peptide r-WRL, and belongs to the field of bioengineering, and the amino acid sequence of the antibacterial peptide r-WRL is as shown in SEQ ID No.1. The invention further discloses application of the antibacterial peptide r-WRL in preparation of a medicine for treating and / or preventing gram-negative bacterium and / or gram-positive bacterium infectious diseases. As the antibacterial peptide r-WRL shows a relatively strong bacteriostatic action, the antibacterial peptide r-WRL can be used for effectively inhibiting gram-negative and gram-positive bacteria, and has the potential of being applied to medicines for treating gram-negative bacteria and gram-positive bacteria infectious diseases. The antibacterial peptide has relatively low hemolytic activity and relatively good physiological salt ion stability. In conclusion, the antibacterial peptide r-WRL 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 derived from arthropod protein and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are non-specific immune defense response products produced by organisms, with a broad spectrum of biological activities such as antiviral, antifungal, antiparasitic, antitumor, and immunomodulatory. The main mechanism by which antimicrobial peptides kill 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 and have extremely broad market application prospects. There are very few natural antimicrobial peptides that can be used as antibiotic substitutes in the medical field. There are two main limiting factors: one is the hemolytic effect of natural antimicrobial peptides on red blood cells; the other is the stability of natural antimicrobial peptides under physiological conditions. Therefore, it is necessary to develop safe and stable antimicrobial peptides through reasonable design. Summary of the invention

[0003] Based on the above needs, the present invention discloses an antimicrobial peptide r-WRL derived from arthropod protein, which can effectively kill Gram-negative and Gram-positive pathogens and has 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] Further, its molecular formula is shown in formula (I):

[0006]

[0007] Another object of the present invention is to provide a method for preparing the antimicrobial peptide r-WRL derived from arthropod protein as described above, which is as follows: using the amino acid sequence fragment of arthropod protein: GAPAQTPSSQ as a template, connecting it with a hydrophilic-hydrophobic alternating antimicrobial fragment, 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 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 physiological salt ion stability of the polypeptide, and finally naming it as antimicrobial peptide r-WRL.

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

[0009] Furthermore, the Gram-negative bacteria are Escherichia coli, Salmonella paratyphi 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 bacteria and / or Gram-negative bacteria infection, wherein the drug contains the arthropod protein-derived antimicrobial peptide r-WRL as described above.

[0012] Advantages and beneficial effects of the present invention: The antimicrobial peptide r-WRL of the present invention has a highly effective inhibitory effect on Gram-negative bacteria such as Escherichia coli and Salmonella paratyphi and Gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis; and has low hemolytic activity (minimum hemolytic concentration>64mM) 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 It is the high performance liquid chromatography-mass spectrum of the antimicrobial peptide r-WRL of the present invention.

[0015] Figure 3 It 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 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.

[0017] Example 1

[0018] Design of antimicrobial peptides

[0019] Arthropod protein is a kind of elastic protein, and it is the most effective elastic protein known so far. Many insects and arthropods contain arthropod protein. Arthropod protein not only has excellent mechanical properties, but also has excellent chemical and cell compatibility, so it has high application potential in biomedical engineering and medicine, including tissue engineering, drug delivery, bioimaging, biosensors, catalysis and bioelectronics. In this example, the fragment sequence of arthropod protein is used as a template to rationally design safe, stable and highly active antimicrobial peptides. The amino acid sequence of the antimicrobial peptide r-WRL is:

[0020] GAPAQTPSSQWWRLWWRLRRR;

[0021] Using the flexible sequence arthropod protein fragment sequence 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 antibacterial 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, 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;

[0030] 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;

[0031] 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, 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, freeze-dry;

[0032] 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%.

[0033] Embodiment 3:

[0034] Determination of antimicrobial activity of antimicrobial peptides

[0035] 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.

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

[0037]

[0038] It can be seen from Table 2 that 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, collect red blood cells; wash with PBS 3 times, and then 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 constant temperature incubator at 37°C 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 PBS was used as a negative control; 50 μL of red blood cells plus 50 μL 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 r-WRL did not show hemolytic activity within the detection range and was significantly different from bee venom in the control group.

[0042] Embodiment 4:

[0043] The salt ion stability of the antimicrobial peptide r-WRL was determined by measuring the antibacterial activity under different physiological salt conditions. The salt ion conditions used were as follows: NaCl 150mM; KCl 4.5mM; CaCl 2 2.5mM; MgCl 2 1mM,FeCl 3 4μM; ZnCl 2 8μM; NH 4 Cl 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[NH 4 + ]]> <![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, K + , NH 4 + , and Zn 2+ Under physiological concentrations of Na + and Mg 2+ In this environment, r-WRL has antibacterial activity. In summary, the antimicrobial peptide r-WRL has excellent stability under physiological salt concentration.

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.

2. The antimicrobial peptide r-WRL derived from arthropod protein according to claim 1, characterized in that: Its molecular formula is shown in formula (I):

3. The method for preparing an antimicrobial peptide r-WRL derived from arthropod protein 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 a hydrophilic-hydrophobic alternating antibacterial fragment, the amino acid sequence of the antibacterial fragment is: WWWRLWWRLRRR, and the designed polypeptide sequence is shown in SEQID No.1; a polypeptide is obtained by solid phase chemical synthesis, and then the polypeptide is prepared after reverse phase high performance liquid chromatography purification and mass spectrometry identification; then the polypeptide is tested for antibacterial activity, hemolytic activity and physiological salt ion stability, and finally named as antimicrobial peptide r-WRL.

4. Use of the arthropod protein-derived antimicrobial peptide r-WRL according to claim 1 in the preparation of a drug for treating and / or preventing Gram-negative and / or Gram-positive bacterial infectious diseases.

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

6. The use according to claim 4, characterized in that: The Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis or Listeria.

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

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