Antibacterial peptide PCKR derived from moth source as well as preparation method and application of antibacterial peptide PCKR

Abstract

The invention provides an antibacterial peptide PCKR derived from a moth source as well as a preparation method and application of the antibacterial peptide PCKR, and belongs to the field of bioengineering, and the amino acid sequence of the antibacterial peptide PCKR is as shown in SEQ ID No.1; the preparation method comprises the following steps: by taking an antibacterial peptide Peyn-cec sourced from leaf armyworm as a template, cutting off 15 amino acids at the N end of the antibacterial peptide Peyn-cec, and mutating the amino acid G at the fourteenth site into W; then, amino acid double exchange is carried out on the amino acid IFKR from the fourth site to the seventh site, and the antibacterial peptide derived from the moth source is obtained and named as PCKR. The antibacterial peptide PCKR derived from the moth source has efficient antibacterial activity on various pathogenic microorganisms, and has no toxicity on human red blood cells, so that the antibacterial peptide PCKR has the potential of becoming a feed antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a moth-derived antibacterial peptide PCKR, its preparation method and application. Background Art

[0002] Antibacterial peptides are a class of natural active polypeptides, usually composed of 10 to 30 amino acid residues, and exhibit broad-spectrum antibacterial activity. Different from antibiotics that interfere with the key metabolic pathways of microorganisms, antibacterial peptides usually destroy bacterial cell membranes physically to eliminate pathogenic microorganisms. Therefore, compared with antibiotics, antibacterial peptides are less likely to produce drug resistance and show great potential as alternatives to feed antibiotics.

[0003] Although antibacterial peptides are considered the most promising alternatives to feed antibiotics in the post-antibiotic era, many of the developed antibacterial peptides have disadvantages such as low biological activity, narrow antibacterial spectrum or unsatisfactory toxicity, making it difficult to apply them to livestock production. Peyn-cec is a natural antibacterial peptide derived from Athetis dissimilis, which has antibacterial activity against Gram-negative bacteria but no antibacterial activity against Gram-positive bacteria. Therefore, it is necessary to rationally modify the antibacterial peptide Peyn-cec derived from Athetis dissimilis to obtain a broad-spectrum antibacterial peptide, providing a theoretical basis and technical support for the research and development of antibacterial peptides. Summary of the Invention

[0004] The object of the present invention is to disclose a moth-derived antibacterial peptide PCKR, aiming to create a broad-spectrum antibacterial peptide with the potential to become an alternative to feed antibiotics.

[0005] The object of the present invention is achieved by the following technical solution: a moth-derived antibacterial peptide PCKR, whose amino acid sequence is shown in SEQ ID No.1.

[0006] Further, its molecular formula is shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a preparation method of a derivative antibacterial peptide PCKR derived from a moth-derived antibacterial peptide as described above, as follows: Using the antibacterial peptide Peyn-cec derived from Athetis dissimilis as a template, whose sequence is shown in SEQ ID No.2, truncate its N-terminal 15 amino acids, and mutate the fourteenth amino acid G to W to increase hydrophobicity and reduce the net positive charge; Subsequently, perform an amino acid double exchange on the amino acid sequence from the fourth to the seventh positions: IFKR to obtain the exchanged sequence: KRIF, which is used to further increase its hydrophobicity and reduce the hydrophobic moment, and the sequence of the obtained polypeptide is shown in SEQ ID No.1; Then, synthesize the polypeptide by solid-phase chemical synthesis, purify it by reverse-phase high-performance liquid chromatography and identify it by mass spectrometry, and then measure its bactericidal activity and hemolytic activity, and finally name it antibacterial peptide PCKR.

[0009] Another object of the present invention is to provide an application of a derivative antibacterial peptide PCKR derived from a moth-derived antibacterial peptide as described above in the preparation of a drug for treating Gram-positive bacteria or / and Gram-negative bacteria infectious diseases.

[0010] Further, for the application as described above, the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa or Salmonella choleraesuis.

[0011] Further, for the application as described above, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

[0012] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria or / and Gram-negative bacteria infections, and the drug contains a derivative antibacterial peptide PCKR derived from a moth-derived antibacterial peptide as described above.

[0013] The beneficial effects and advantages of the present invention are as follows: The present invention truncates the N-terminal of the antibacterial peptide Peyn-cec derived from natural Athetis dissimilis, and adopts amino acid substitution and conversion strategies to increase its hydrophobicity, reduce the hydrophobic moment and net positive charge, successfully creates a broad-spectrum antibacterial peptide PCKR, and conducts biological activity detection on it, and finds that it has high antibacterial activity against Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella choleraesuis and Gram-positive bacteria: Staphylococcus aureus, Enterococcus faecalis and Staphylococcus epidermidis. This antibacterial peptide only causes 3.97% hemolysis of red blood cells at a concentration of 128 μM, and it already has the development potential to become a substitute for feed antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a high-performance liquid chromatography diagram of the derivative antibacterial peptide PCKR of the present invention;

[0015] Figure 2 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectrum derived from the moth-derived antimicrobial peptide PCKR of the present invention;

[0016] Figure 3 Comparison chart of hemolytic activities derived from the moth-derived antimicrobial peptide PCKR and melittin ME of the present invention. Detailed implementation manners

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

[0018] Example 1

[0019] Design of the moth-derived antimicrobial peptide PCKR

[0020] Taking the antimicrobial peptide Peyn-cec from Thalassodes proquadraria as a template, whose sequence is shown in SEQ ID No. 2, truncate 15 amino acids at its N-terminus, and mutate the fourteenth amino acid G to W to increase hydrophobicity and reduce the net positive charge; subsequently, perform an amino acid double exchange on the amino acid sequence from the fourth to the seventh positions: IFKR, and the exchanged sequence is KRIF, which is used to further increase its hydrophobicity and reduce the hydrophobic moment, and the obtained mutant moth-derived antimicrobial peptide is named KRIF, as shown in Table 1.

[0021] Table 1 Amino acid sequence of the moth-derived antimicrobial peptide KRIF

[0022]

[0023] The net charge number of the antimicrobial peptide KRIF is +6, the hydrophobic value is 0.541, and the hydrophobic moment is 0.09. This moth-derived antimicrobial peptide not only has high antibacterial activity against a variety of pathogenic microorganisms, but also has no toxicity to human red blood cells, and has the potential to be developed as a substitute for feed antibiotics.

[0024] Example 2

[0025] Synthesis of the moth-derived antimicrobial peptide by solid-phase chemical synthesis method

[0026] 1. The preparation of the moth-derived 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 coupled to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trityl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); following this procedure, the synthesis is carried out from the C-terminus to the N-terminus in turn until the synthesis is completed, and the resin with side-chain protection and Fmoc group removed is obtained;

[0027] 2. In the obtained peptide resin, a cleavage reagent is added, and the reaction is carried out at 20 °C in the dark for 2 h, followed by filtration; 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. Precipitation is carried out 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 a polypeptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) mixed in a mass ratio of 95:2.5:2.5;

[0028] 3. Column equilibration is carried out for 30 min using 0.2 M sodium sulfate (adjusted to pH 7.5 with phosphoric acid). The polypeptide is dissolved in a 90% aqueous acetonitrile solution, filtered, and passed through a C18 reversed-phase normal pressure column. Gradient elution is adopted (the eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), the flow rate is 1 mL / min, the detection wavelength is 220 nm, the main peak is collected and freeze-dried; further purification is carried out using a reversed-phase C18 column. Eluent A is 0.1% TFA / aqueous solution; eluent B is 0.1% TFA / acetonitrile solution, the elution concentration is 25% B - 40% B, the elution time is 12 min, the flow rate is 1 mL / min, and the main peak is collected and freeze-dried as above;

[0029] 4. Identification of the antibacterial peptide: The obtained antibacterial peptide is analyzed by electrospray mass spectrometry. The molecular weight shown in the mass spectrum diagram (as Figure 1 、 2 shown) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antibacterial peptide is greater than 95%.

[0030] Example 3:

[0031] 1. Determination of antibacterial activity: The minimum inhibitory concentration is determined after treating the antibacterial peptide KRIF from moths by the serial dilution method. Using 2 mg / ml BSA (containing 0.01% acetic acid) as the diluent, a series of gradient antibacterial peptide solutions are 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 cells / mL) to each well. Positive control (containing bacterial solution but no antibacterial peptide) and negative control (neither containing bacterial solution nor antibacterial peptide) are set respectively. After incubation at 37 °C for 14 - 18 h, the absorbance is measured at OD 492nm . The minimum antibacterial peptide concentration that inhibits 99.9% of microbial growth is the minimum inhibitory concentration. The test results are shown in Table 2.

[0032] Table 2 Minimum antibacterial activity of the antibacterial peptide KRIF from moths against pathogenic microorganisms

[0033]

[0034] 2. As can be seen from Table 2, the antimicrobial peptide KRIF shows high antibacterial activity against both Gram-negative bacteria (E. coli 25922, E. coli K88, S. typhimurium 14028, Acinetobacter baumannii, K. pneumoniae, P. aeruginosa ATCC27853, and Swine paratyphoid 021493) and Gram-positive bacteria (S. aureus 29213, S. aureus 25923, S. epidermidis ATCC12228, S. aureus 43300, and E. faecalis 29212).

[0035] 3. Determination of hemolytic activity: Collect 1 mL of fresh human blood, anticoagulate with heparin and dissolve it in 2 mL of PBS solution, centrifuge at 3000 rpm for 10 min, and collect red blood cells; wash them 3 times with PBS solution and then resuspend them in 10 mL of PBS solution; take 50 μL of the red blood cell suspension and mix it evenly with 50 μL of antimicrobial peptide solutions at different concentrations, incubate at a constant temperature in a 37 °C incubator for 1 h; then centrifuge at 4 °C and 3000 rpm for 10 min; take out the supernatant and measure the absorbance at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Among them, 50 μL of red blood cells plus 50 μL of PBS solution is used as a negative control, and 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a 10% hemolysis rate. The test results are shown in Figure 3 . Through Figure 3 it can be seen that KRIF does not show hemolytic activity within the detection range, causes 3.97% red blood cell hemolysis at a concentration of 128 μM, and fails to cause 10% red blood cell hemolysis, showing a significant difference from the control group melittin, indicating that the antimicrobial peptide KRIF has the potential to be developed into a feed-type antimicrobial peptide.

Claims

1. A moth-derived antimicrobial peptide PCKR, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The moth-derived antimicrobial peptide PCKR according to claim 1, characterized in that: Its molecular formula is shown in formula (I):

3. The method for preparing the moth-derived antimicrobial peptide PCKR according to claim 1, characterized in that: The method is as follows: using the antimicrobial peptide Peyn-cec from Spodoptera lutea as a template, the sequence of which is shown in SEQ ID No.2, truncating 15 amino acids at the N-terminus, and mutating the fourteenth amino acid G to W to improve hydrophobicity and reduce the net positive charge; subsequently, performing a double amino acid exchange on the fourth to seventh amino acid sequence: IFKR to obtain a replaced sequence: KRIF, which is used to further improve its hydrophobicity and reduce the hydrophobic moment, and the sequence of the obtained polypeptide is shown in SEQ ID No.1; then, the polypeptide is synthesized by a solid phase chemical synthesis method, purified by reversed-phase high performance liquid chromatography and identified by mass spectrometry, and then subjected to bactericidal activity determination and hemolytic activity determination, and finally named as the antimicrobial peptide PCKR.

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

5. The use according to claim 4, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa or Salmonella suis.

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

7. A drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, characterized in that: The drug contains the moth-derived antimicrobial peptide PCKR as claimed in claim 1.

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