Antibacterial peptide PG3L derived from ant source as well as preparation method and application of antibacterial peptide PG3L

Abstract

The invention provides an antibacterial peptide PG3L derived from an ant source as well as a preparation method and application of the antibacterial peptide PG3L, and belongs to the field of bioengineering, and the sequence of the antibacterial peptide PG3L derived from the ant source is as shown in SEQ ID No.1; the preparation method comprises the following steps: by taking ant-derived antibacterial peptide Ponericin G3 as a template, cutting off 17 amino acids at the N end of the antibacterial peptide Ponericin G3; then, D, N and E at the fourth site, the seventh site and the eleventh site are mutated into hydrophobic amino acids L, V and L respectively, so that the antibacterial peptide PG3L derived from the ant source is obtained. The antibacterial peptide PG3L derived from the ant source has efficient antibacterial activity on various pathogenic microorganisms, and has no toxicity on human red blood cells, so that the antibacterial peptide PG3L 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 derivative of ant-derived antibacterial peptide PG3L, its preparation method and application. Background Art

[0002] Different from antibiotics that interfere with the key metabolic pathways of microorganisms, antibacterial peptides usually kill pathogenic microorganisms by physically disrupting the bacterial cell membrane. Therefore, compared with antibiotics, antibacterial peptides are less likely to develop drug resistance and show great potential as alternatives to feed antibiotics.

[0003] In recent years, the technology for creating antibacterial peptides has become increasingly mature. However, many natural antibacterial peptides have low biological activities due to their overly long peptide chains, unable to meet the needs in livestock production. Peptide chain truncation and amino acid mutation techniques may be beneficial for finding the active centers of antibacterial peptides. By truncating the N-terminal / C-terminal of the peptide chain, it may effectively improve the antibacterial activity of natural antibacterial peptides against some pathogenic microorganisms. In addition, mutating some amino acids may also improve the biological potency of antibacterial peptides. Polypeptide Ponericin G3 is a natural antibacterial peptide derived from ants and has varying degrees of antibacterial activity against a variety of Gram-negative and Gram-positive bacteria. However, the antibacterial activity of natural Ponericin G3 is low, with the MIC in the range of 400 - 500 mM, making it difficult to be applied as a highly efficient antibacterial peptide in feed additives. Therefore, a highly efficient and low-toxic antibacterial peptide is needed to provide a theoretical basis and technical support for the research and development of feed-type antibacterial peptides. Summary of the Invention

[0004] Based on the above deficiencies, the purpose of the present invention is to provide a derivative of ant-derived antibacterial peptide PG3L to solve the problem that existing natural antibacterial peptides have low biological activities due to their overly long peptide chains and cannot meet the needs in livestock production.

[0005] The purpose of the present invention is achieved through the following technical solution: A derivative of ant-derived antibacterial peptide PG3L, whose amino acid sequence is as shown in SEQ ID No.1.

[0006] Further, for a derivative of ant-derived antibacterial peptide PG3L as described above, its molecular formula is as shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a preparation method of an antibacterial peptide PG3L derived from the ant-derived antibacterial peptide as described above, as follows: Using the antibacterial peptide Ponericin G3 from ants as a template, whose sequence is shown in SEQ ID No.2, truncate 17 amino acids at its N-terminus, and end with the flexible amino acid G at the C-terminus; Subsequently, mutate D, N, and E at the fourth, seventh, and eleventh positions into hydrophobic amino acids L, V, and L respectively to improve the overall hydrophobicity of the antibacterial peptide and reduce the hydrophobic moment, so the sequence of the obtained polypeptide is shown in SEQ ID No.1; Then synthesize the polypeptide by solid-phase chemical synthesis method, purify it by reverse-phase high-performance liquid chromatography and identify it by mass spectrometry, and then measure the bactericidal activity and hemolytic activity, and finally name it antibacterial peptide PG3L.

[0009] Another object of the present invention is to provide the use of an antibacterial peptide PG3L derived from the ant-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 use as described above, the Gram-negative bacteria are Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella choleraesuis or Salmonella typhimurium.

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

[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 an antibacterial peptide PG3L derived from the ant-derived antibacterial peptide as described above.

[0013] The beneficial effects and advantages of the present invention are as follows: The present invention reasonably improves the hydrophobicity of the natural ant-derived antibacterial peptide Ponericin G3 and reduces the hydrophobic moment through N-terminal truncation and amino acid mutation techniques, successfully creates a highly efficient and low-toxic antibacterial peptide PG3L, and conducts biological activity detection on it, and finds that it has high antibacterial activity against Gram-negative bacteria: Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella choleraesuis and Salmonella typhimurium and Gram-positive bacteria: Enterococcus faecalis, Staphylococcus epidermidis and Staphylococcus aureus, the MIC is reduced to 1 - 8 μM, and the biological activity is increased by about 400 times compared with the original peptide. This antibacterial peptide only causes 4.25% 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 the high-performance liquid chromatography chart of the antibacterial peptide PG3L derived from the present invention.

[0015] Figure 2 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry diagram derived from the ant-derived antimicrobial peptide PG3L of the present invention;

[0016] Figure 3 Hemolytic activity diagram derived from the ant-derived antimicrobial peptide PG3L 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 ant-derived antimicrobial peptide PG3L

[0020] Taking the ant-derived antimicrobial peptide Ponericin G3 as a template, truncating 17 amino acids at the N-terminus of the natural ant-derived antimicrobial peptide, and mutating D, N, and E at the fourth, seventh, and eleventh positions into hydrophobic amino acids L, V, and L, to obtain the mutant ant-derived antimicrobial peptide PG3L, and the sequence is shown in Table 1.

[0021] Table 1 Amino acid sequence of the ant-derived antimicrobial peptide PG3L

[0022]

[0023] The net charge number of the antimicrobial peptide PG3L is +6, the hydrophobicity value is 0.519, and the hydrophobic moment is 0.266. This ant-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 ant-derived antimicrobial peptide by solid-phase chemical synthesis method

[0026] 1. The preparation of the ant-derived antimicrobial peptide is carried out one by one from the C-terminus to the N-terminus and completed by a polypeptide 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-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 with the Fmoc group removed is obtained;

[0027] 2. In the obtained peptide resin, a cleavage reagent is added, and the reaction is carried out for 2 h at 20 °C in the dark, 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, which is 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 antibacterial peptide: The obtained antibacterial peptide is analyzed by electrospray mass spectrometry. The molecular weight shown in the mass spectrum (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 bacteriostatic activity: The minimum inhibitory concentration is determined after treating the ant-derived antibacterial peptide PG3L 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 CFU / 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 bacteriostatic activity of ant-derived antibacterial peptide PG3L against pathogenic microorganisms

[0033]

[0034] As can be seen from Table 2, the antibacterial peptide PG3L exhibits high antibacterial activity against 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] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after heparin anticoagulation, 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 antibacterial peptide solutions at different concentrations, and incubate them at a constant temperature in an incubator at 37 °C for 1 h; then centrifuge at 3000 rpm at 4 °C for 10 min; take out the supernatant and measure the absorbance at 570 nm with an enzyme-linked immunosorbent assay 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 concentration of the antibacterial peptide when it causes a 10% hemolysis rate. The detection results are shown in Figure 3 . Through Figure 3 it can be seen that PG3L does not exhibit hemolytic activity within the detection range, causes 4.25% red blood cell hemolysis at a concentration of 128 μM, fails to cause 10% red blood cell hemolysis, and shows a significant difference from the control group melittin, indicating that the ant-derived antibacterial peptide PG3L has the potential to be developed into a feed-type antibacterial peptide.

Claims

1. An antimicrobial peptide PG3L derived from ants, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The antimicrobial peptide PG3L derived from ants according to claim 1, wherein the molecular formula is as shown in formula (I):

3. The method for preparing the ant-derived antimicrobial peptide PG3L according to claim 1, characterized in that: The method is as follows: using the antimicrobial peptide Ponericin G3 from ants as a template, the sequence of which is shown in SEQ ID No. 2, truncating the 17 amino acids at the N-terminus, and ending with a flexible amino acid G at the C-terminus; subsequently, mutating the D, N and E at the fourth, seventh and eleventh positions to hydrophobic amino acids L, V and L respectively to increase the overall hydrophobicity of the antimicrobial peptide and reduce the hydrophobic moment, so that the sequence of the obtained polypeptide is shown in SEQ ID No. 1; then synthesizing the polypeptide by solid phase chemical synthesis, and subjecting it to reverse phase high performance liquid chromatography purification and mass spectrometry identification, and then subjecting it to bactericidal activity determination and hemolytic activity determination, it is finally named as antimicrobial peptide PG3L.

4. Use of the ant-derived antimicrobial peptide PG3L according to claim 1 in the preparation of a medicament 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, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium or Salmonella typhimurium.

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

7. 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 ant-derived antimicrobial peptide PG3L as described above.

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