Antibacterial peptide chicken nk lysin mutants and uses thereof

Abstract

This invention relates to the field of agricultural biotechnology, specifically to the antimicrobial peptide chicken NK lysin mutant and its applications. The chicken NK lysin mutant of this invention is effective against various bacteria, such as Salmonella typhimurium (…). Salmonella typhimurium 541, Gram-negative bacteria), Escherichia coli ( Escherichia coli O157:H7 1491, Gram-negative bacteria), Gram-negative bacteria Salmonella pullorum ( salmonella pullorum ) and Staphylococcus aureus ( Staphylococcus aureus The antibacterial activity of ) is enhanced.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to the antimicrobial peptide chicken NK lysin mutant and its applications. Background Technology

[0002] Antimicrobial peptides, with their advantages of low resistance, broad-spectrum antimicrobial activity, and strong thermal stability, have shown broad application prospects in medicine, agriculture, food, and animal husbandry, and are expected to become an important solution to replace traditional antibiotics. NK-lysin (NKL), an antimicrobial peptide, is an important immunologically active protein derived from natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). It possesses broad-spectrum antibacterial and tumor cell-killing activities, as well as immunomodulatory functions.

[0003] Chicken NK-lysin contains 75 amino acids, rich in basic amino acids, and contains 6 cysteine ​​residues forming 3 pairs of disulfide bonds. It also contains 5 α-helices, each with different antibacterial activity. Generally, the second and third α-helices have higher levels of basic amino acids and the strongest antibacterial activity.

[0004] Although chicken NK-lysin possesses broad-spectrum antibacterial activity, its antibacterial activity needs to be enhanced compared to antibiotics. Therefore, screening for mutants with enhanced antibacterial activity is essential. However, the synthesis and exogenous expression of full-length chicken NK-lysin are both challenging, making mutant screening time-consuming and labor-intensive. Since chicken NK-lysin contains five α-helices, and α-helices are the most common secondary structures among natural antimicrobial peptides, each α-helix is ​​a potential antimicrobial peptide. Each α-helix contains varying degrees of basic and hydrophobic amino acids, and the position and number of these basic and hydrophobic amino acids affect antibacterial function, playing a crucial role in antibacterial activity. Summary of the Invention

[0005] The purpose of this invention is to provide a chicken NK lysin mutant with increased antibacterial activity of the antimicrobial peptide.

[0006] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptide chicken NK lysin mutant.

[0007] The chicken NK lysinα helical mutant according to the present invention includes:

[0008] The mutant of cNKL-α1 chicken NK lysin, the first α-helical peptide (SEQ ID No:1, cNKL-α1: IKCRFCVSLVKKVQKIVGDDPD), has the amino acid sequence shown in SEQ ID No:2.

[0009] cNKL-α1M (SEQ ID No:2): RKCRFCVSLWKKVQKICGDGPD;

[0010] The mutant peptides cNKL-α3M1 and cNKL-α3M2 of the third α-helical peptide of chicken NK lysin (SEQ ID No:3), wherein the amino acid sequence of the mutant peptide cNKL-α3M1 is shown in SEQ ID No:4 and the amino acid sequence of the mutant peptide cNKL-α3M2 is shown in SEQ ID No:5.

[0011] SEQ ID No: 3, cNKL-α3: GRRQRSICKQLLKKLRQ,

[0012] SEQ ID No: 4, cNKL-α3M1: GRRFWSICKQLLKKLRQ,

[0013] SEQ ID No: 5, cNKL-α3M2: GRRQRSICIQLLKKLRQ.

[0014] Beneficial technical effects:

[0015] This invention synthesizes the five α-helices of chicken NK lysin and performs combined mutations on the composition and position of amino acids, particularly basic and hydrophobic amino acids, in each α-helix, aiming to screen for antimicrobial peptides with enhanced antimicrobial function. The animal-derived NK lysin mutants of this invention are effective against various bacteria, such as Salmonella typhimurium (…). Salmonella typhimurium 541, Gram-negative bacteria), Escherichia coli ( Escherichia coli O157:H7 1491, Gram-negative bacteria), Gram-negative bacteria Salmonella pullorum ( Salmonella chickens ) or Staphylococcus aureus ( Staphylococcus golden The antibacterial activity of ) is enhanced. Attached Figure Description

[0016] Figure 1 The comparison shows the antibacterial activity of the first α-helical peptide cNKL-α1 and the mutant peptide cNKL-α1M of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (CNKL-α1). Salmonella typhimurium CVCC Figure 541), B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), Figure C shows Salmonella pullorum (…). Salmonella chickens CVCC 1789);

[0017] Figure 2The comparison shows the antibacterial activity of the third α-helical peptide cNKL-α3 and the mutant peptide cNKL-α3M1 of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (CNKL-α3). Salmonella typhimurium CVCC Figure 541), B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), Figure C shows Salmonella pullorum (…). Salmonella chickens CVCC 1789), Figure D shows Staphylococcus aureus ( Staphylococcus aureus );

[0018] Figure 3 The comparison shows the antibacterial activity of the third α-helical peptide cNKL-α3 and the mutant peptide cNKL-α3M2 of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (CNKL-α3). Salmonella typhimurium CVCC Figure 541), B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), Figure C shows Salmonella pullorum (…). Salmonella chickens CVCC 1789). Detailed Implementation Example 1

[0019] The following peptides were obtained through chemical synthesis: HPLC purity ≥ 95%

[0020] cNKL-α1 is a mutant of the first α-helical peptide of chicken NK lysin, the amino acid sequence of which is shown in SEQ ID No:2. cNKL-α3 are mutant peptides of the third α-helical peptide of chicken NK lysin, cNKL-α3M1 and cNKL-α3M2, the amino acid sequence of which is shown in SEQ ID No:4. The amino acid sequence of the mutant peptide cNKL-α3M2 is shown in SEQ ID No:5.

[0021] Staphylococcus aureus ( Staphylococcus aureus ), belonging to the genus Staphylococcus, is a representative of Gram-positive bacteria and a common foodborne pathogen; Salmonella typhimurium ( Salmonella typhimurium Salmonella (E. coli) is a Gram-negative, long, slender bacillus, an important zoonotic pathogen that can infect humans, poultry, livestock, rodents, and other wild animals. Its infection rate ranks first among Salmonella infections. Escherichia coli O157:H7 Escherichia coli is a Gram-negative bacterium that can cause hemorrhagic diarrhea and enteritis in humans and animals; Salmonella pullorum (… Salmonella chickens This bacterium is the pathogen of pullorum disease in chickens, mainly affecting chicks and causing acute septicemia. This bacterium is currently resistant to many antibiotics.

[0022] The MIC (minimal inhibitory concentration) of antimicrobial peptides was determined using a fully automated growth curve analyzer, as follows:

[0023] 500 μL of the bacterial culture was inoculated into 50 mL of antibiotic-free CaMHB liquid medium and cultured overnight at 37°C with shaking. The next day, 1% of the inoculum was transferred to 50 mL of CaMHB medium and cultured at 37°C with shaking until OD (outlet capacity) was reached. 600 =0.4, the cultured bacterial solution was diluted 1000 times with CaMHB medium.

[0024] The peptide samples used were first dissolved in sterile water to obtain a stock solution with a concentration of 1 mM, and then diluted with sterile water to the required concentrations, corresponding to final concentrations of 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, and 4 μM.

[0025] After sample and bacterial culture dilution, 200 μL of sterile water was added around the perimeter of a 100-well honeycomb culture plate in a clean bench. In the center wells, 100 μL of samples at different concentrations, 100 μL of sterile water (negative control), and 100 μL of 50 μg / mL kanamycin (positive control) were added. Finally, 100 μL of diluted bacterial culture was added to each of these wells. The honeycomb culture plate was then capped and placed in an automated growth curve analyzer, incubated at 37°C, and OD was measured every 30 minutes. 600 The culture lasted for 16 hours.

[0026] Figure 1 The comparison shows the antibacterial activity of the first α-helical peptide cNKL-α1 and the mutant peptide cNKL-α1M of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (CNKL-α1). Salmonella typhimurium CVCC 541), the results showed that the minimum inhibitory concentration (MIC) of cNKL-α1 was 128 µM, while that of cNKL-α1M was 32 µM, representing a 4-fold increase in antibacterial activity; Figure B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), the results showed that the minimum inhibitory concentration (MIC) of cNKL-α1 was greater than 128 µM, while the MIC of cNKL-α1M was 16 µM, indicating an antibacterial activity increase of more than 8 times; Figure C shows Salmonella pullorum (…). salmonella chickens (CVCC 1789) The results showed that the minimum inhibitory concentration (MIC) of cNKL-α1 was greater than 128 µM, while the MIC of cNKL-α1M was 128 µM, indicating that the antibacterial activity was more than doubled.

[0027] Figure 2The comparison shows the antibacterial activity of the third α-helical peptide cNKL-α3 and the mutant peptide cNKL-α3M1 of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (CNKL-α3). Salmonella typhimurium CVCC 541) The results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was greater than 128 µM, while the MIC of cNKL-α3M1 was 8 µM, indicating a more than 16-fold increase in antibacterial activity; Figure B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), the results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was 64 µM, while that of cNKL-α3M1 was 8 µM, representing an 8-fold increase in antibacterial activity; Figure C shows Salmonella pullorum (…). salmonella chickens (CVCC 1789) The results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was 128 µM, while that of cNKL-α3M1 was 16 µM, representing an increase in antibacterial activity of more than 8 times; Figure D shows Staphylococcus aureus ( Staphylococcus aureus The results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was 128 µM, while that of cNKL-α3M1 was 16 µM, representing an 8-fold increase in antibacterial activity.

[0028] Figure 3 Comparison of the antibacterial activities of the third α-helical peptide cNKL-α3 and the mutant peptide cNKL-α3M2 of chicken NK lysin. Figure A shows the antibacterial activity of Salmonella typhimurium (Salmonella typhimurium). Salmonella typhimurium CVCC 541), the results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was greater than 128 µM, while the MIC of cNKL-α3M2 was 16 µM, indicating an antibacterial activity that was more than 8 times higher; Figure B shows Escherichia coli ( Escherichia coli O157:H7 CVCC 1491), the results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was 64 µM, while that of cNKL-α3M2 was 16 µM, representing a 4-fold increase in antibacterial activity; Figure C shows Salmonella pullorum (…). salmonella chickens (CVCC 1789) The results showed that the minimum inhibitory concentration (MIC) of cNKL-α3 was greater than 128 µM, while the MIC of cNKL-α3M2 was 64 µM, which increased the antibacterial activity by more than 2 times.

[0029] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. An alpha-helix mutant of the antibacterial peptide chicken NK lysin with improved antibacterial activity, characterized in that, The alpha helix mutant of the antibacterial peptide chicken NK lysin with improved antibacterial activity includes: a mutant of the first alpha helix peptide of the antibacterial peptide chicken NK lysin, the amino acid sequence of which is shown as SEQ ID No: 2, or, a mutant of the third alpha helix peptide of the antibacterial peptide chicken NK lysin, the amino acid sequence of which is shown as SEQ ID No: 4 or SEQ ID No:

5.

2. The use of the alpha helix mutant of the antibacterial peptide chicken NK lysin with improved antibacterial activity according to claim 1 for the non-therapeutic purpose of inhibiting pathogenic bacteria, wherein, Mutants of the first alpha-helix peptide of the antibacterial peptide chicken NK lysin having the amino acid sequence of SEQ ID No: 2 for use in inhibiting Salmonella typhimurium ( Salmonella typhimurium ), Escherichia coli ( Escherichia coli ) or Salmonella pullorum ( salmonella pullorum ); The mutant of the antibacterial peptide chicken NK lysin 3rd alpha-helix peptide with the amino acid sequence shown as SEQ ID No: 4 is used for inhibiting Salmonella typhimurium ( Salmonella typhimurium ), Escherichia coli ( Escherichia coli ), Salmonella pullorum ( salmonella pullorum ) or Staphylococcus aureus ( Staphylococcus aureus ); Mutants of the anti-bacterial peptide chicken NK lysin 3rd alpha-helix peptide having the amino acid sequence of SEQ ID No: 5 for inhibiting Salmonella typhimurium ( Salmonella typhimurium ), Escherichia coli ( Escherichia coli ) or Salmonella pullorum ( salmonella pullorum ).

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