Antimicrobial peptide ascaphin-5 modifier and use thereof

Abstract

The application discloses an antibacterial peptide Ascaphin-5 modified body and application thereof, and belongs to the field of biological medicine. An artificial intelligent model independently developed by the application is used for modifying a natural antibacterial peptide Ascaphin-5, mutations are introduced into the amino acid sequence of the natural antibacterial peptide Ascaphin-5 at 8 positions, and modified bodies, antibacterial peptides Perceptide05 (GIKKWIKGWAKKLIKKVAKKIAKQ) and Perceptide07 (GIKKWIKGAIKKLIKKVAKWIWKQ) are obtained. The modified bodies, antibacterial peptides Perceptide05 and Perceptide07, can effectively improve the antibacterial activity of Ascaphin-5, the minimum inhibitory concentration value can be reduced by 64 times at most (i.e. the activity is improved by 64 times), and can simultaneously effectively inhibit gram-negative bacteria and gram-positive bacteria, the minimum inhibitory concentration can reach 0.25 micromoles and 0.5 micromoles respectively, and can be used for development of a new generation of antibacterial drugs and antibacterial products.

Description

TECHNICAL FIELD

[0001] The present application relates to an antimicrobial peptide and its application, in particular to an antimicrobial peptide Ascaphin-5 and its application in the preparation of a medicament, and belongs to the field of biological medicine. BACKGROUND

[0002] Drug-resistant bacterial infection is a serious problem in the world today. According to the statistics of WHO, drug-resistant bacterial infection currently causes 1.3 million deaths per year, and if no measures are taken, this number will rapidly increase by 1 million per year. The development of a new antibiotic is time-consuming and laborious, and the use of small molecule antibiotics can still lead to the evolution of bacteria with drug resistance and thus lose their effect. Antimicrobial peptides, as a class of short peptides, are ideal drug development objects, because antimicrobial peptides mainly act by destroying bacterial cell membranes, and do not target specific proteins, so they can quickly and effectively inhibit bacteria and other pathogens.

[0003] There are many natural antimicrobial peptides from amphibians such as frogs and toads, whose skin secretions contain these polypeptide substances that resist microbial invasion. Ascaphium Ascaphus truei Ascaphium is considered to be the most primitive species in the order of Anura, and researchers have isolated a variety of antimicrobial peptides from its skin secretions, including Ascaphin-1, Ascaphin-5, Ascaphin-8, etc., which have certain antimicrobial activity (Biochemical and Biophysical Research Communications, 320(1), 170-175, 2004). However, the antimicrobial activity of these natural antimicrobial peptides is still limited, and higher activity antimicrobial peptides are needed to exert the bacteriostatic effect in actual drug development. SUMMARY

[0004] In order to obtain antimicrobial peptides with high activity, the natural antimicrobial peptide Ascaphin-5 (sequence: GIKDWIKGAAKKLIKTVASNIANQ, SEQ ID No: 1) was modified by an artificial intelligence model independently developed by the present application, and mutations were introduced at 8 positions of the amino acid sequence, obtaining modified antimicrobial peptides Perceptide05 and Perceptide07, whose sequences are as follows:

[0005] Perceptide05: GIKKWIKGWAKKLIKKVAKKIAKQ (SEQ ID No: 2)

[0006] Perceptide07: GIKKWIKGAIKKLIKKVAKWIWKQ (SEQ ID No: 3)

[0007] The modified antibacterial peptides Perceptide05 and Perceptide07 can be synthesized by polypeptide solid-phase synthesis and other chemical methods, and are applied in antibacterial drugs and other antibacterial products (such as disinfectants and detergents).

[0008] Tests show that the modified antibacterial peptides Perceptide05 and Perceptide07 can effectively improve the antibacterial activity of Ascaphin-5, and the minimum inhibitory concentration value can be reduced by 64 times (i.e. the activity is improved by 64 times), and can effectively inhibit gram-negative bacteria and gram-positive bacteria at the same time, including but not limited to Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis, and the minimum inhibitory concentration can reach 0.25 μM and 0.5 μM, respectively. The modified antibacterial peptides can be used for the development of a new generation of antibacterial drugs to improve human health and well-being. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Sequence alignment of antibacterial peptide Ascaphin-5 and its modified peptides Perceptide05 and Perceptide07.

[0010] Figure 2 Sequence wheel diagram of the modified antibacterial peptides Perceptide05 and Perceptide07. DETAILED DESCRIPTION

[0011] The present application will be further described below with reference to the accompanying drawings and examples. However, it should be understood that these descriptions are only to further illustrate the features and advantages of the present application, and are not limiting on the claims of the present application.

[0012] Example 1 Source and properties of antibacterial peptide modified bodies

[0013] The antibacterial peptides Perceptide05 and Perceptide07 are based on the modification of the natural antibacterial peptide Ascaphin-5. The specific method is to input the sequence of the antibacterial peptide Ascaphin-5 into the self-developed artificial intelligence model, and the model applies mutations to the sequence, with 1 amino acid per step, and repeats the process 8 times to obtain the modified sequence. As shown in the sequence alignment of Figure 1 As shown in the sequence alignment of

[0014] The lengths of the antibacterial peptides Perceptide 05 and Perceptide 07 are both 24 amino acids (consistent with Ascaphin-5), and the proportion of hydrophobic amino acids is 38%. Among them, the molecular weight of Perceptide 05 is 2792.55, with 10 positive charges, while the molecular weight of Perceptide 07 is 2892.67, with 9 positive charges. The sequence wheel diagram of the two antibacterial peptides is shown in Figure 2 , wherein the white background is a polar amino acid and the black background is a non-polar amino acid, which are roughly distributed on both sides of the antibacterial peptide helix, showing the characteristics of amphiphilicity.

[0015] Example 2 Determination of the minimum inhibitory concentration of antibacterial peptides

[0016] The bacterial strains used in the minimum inhibitory concentration determination experiment include two gram-negative bacterial strains and two gram-positive bacterial strains, which are Escherichia coli Escherichia coli ATCC 25922, Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1, Staphylococcus aureus Staphylococcus aureus ATCC 6538, Bacillus subtilis Bacillus subtilis ATCC 6633. The minimum inhibitory concentration of the antibacterial peptide was determined using the Hancock broth dilution method. Four standard strains were activated by overnight culture, diluted to an OD 625 value of 0.08-0.13 after being cultured at 37°C in a shaking incubator at 220 rpm for about 3 hours.

[0017] To compare the activity differences between the modified Perceptide 05, Perceptide 07 and the source antibacterial peptide Ascaphin-5, this example simultaneously tests the minimum inhibitory concentrations of the three antibacterial peptides under the same experimental conditions. The three antibacterial peptide dry powders were synthesized by Wuhan Dangang Biotechnology Co., Ltd. through solid-phase chemical synthesis method, and the purity was above 95%. The antibacterial peptide solution stock was prepared using sterile PBS solution, with a concentration of 512 μM. In a 96-well plate, 100 μL of LB culture medium and 100 μL of antibacterial peptide solution were added, and the antibacterial peptide solution was diluted to a final concentration of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μM in a two-fold concentration gradient. Each added 100 μL of diluted 100 times bacterial solution, mixed well and cultured in a 37°C incubator for 16 hours. The lowest antibacterial peptide solution concentration value observed by naked eye without obvious bacterial growth was the minimum inhibitory concentration value of the antibacterial peptide under the condition.

[0018] The test results are shown in Table 1. Compared with the natural antibacterial peptide Ascaphin-5, the modified bodies Perceptide 05 and Perceptide 07 show significant improvement in antibacterial activity against four different strains, for example, the antibacterial activity of Perceptide 05 against Bacillus subtilis B.subtilis is 64 times that of Ascaphin-5, and Perceptide 07 is 32 times that of Ascaphin-5. In summary, the modified antibacterial peptides Perceptide 05 and Perceptide 07 can effectively inhibit the growth of four strains, and have great potential for drug development.

[0019] Table 1. Minimum inhibitory concentration test results of antibacterial peptides (unit: μM)

[0020]

Claims

1. An antimicrobial peptide, which is a modification of the natural antimicrobial peptide Ascaphin-5, characterized in that, The antibacterial peptide is an antibacterial peptide Perceptide05 or Perceptide07 as shown in the following amino acid sequence: Perceptide05: GIKKWIKGWAKKLIKKVAKKIAKQ; Perceptide07: GIKKWIKGAIKKLIKKVAKWIWKQ.

2. Use of the antibacterial peptide of claim 1 in the preparation of an antibacterial medicament against one or more of the following bacteria: Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis.

3. Use according to claim 2, wherein the compound is ###0002### The effective concentration of the antibacterial peptide Perceptide05 in the antibacterial drug is greater than or equal to 0.25 μM, or the effective concentration of the antibacterial peptide Perceptide07 is greater than or equal to 0.5 μM.

4. An antibacterial agent, characterized by, The antibacterial product comprises the antibacterial peptide Perceptide05 and / or Perceptide07 as claimed in claim 1.

5. Use of the antibacterial peptide of claim 1 in the manufacture of an antibacterial product directed against one or more of the following bacteria: Escherichia coli 、 Pseudomonas aeruginosa 、 Staphylococcus aureus 、 Bacillus subtilis.

6. The use according to claim 5, wherein the compound is ###0002### The antibacterial product is a disinfectant or a detergent.

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