Improved antibacterial peptide Ascapin-5 and application thereof

Abstract

The invention discloses an antibacterial peptide Ascapin-5 modifier and application thereof, and belongs to the field of biological medicine. According to the present invention, the natural antibacterial peptide Ascapin-5 is modified by using the self-developed artificial intelligence model, and the mutation is introduced at the eight positions of the amino acid sequence of the natural antibacterial peptide Ascapin-5, such that the modified antibacterial peptides Perceptide05 (GIKKWIKGWAKKLIKVAKKIAKQ) and Perceptide07 (GIKKWIKGAIKKLIKVAKWIWKQ) are obtained; the modified antibacterial peptides Perceptide 05 and Perceptide 07 can effectively improve the antibacterial activity of Ascapin-5, the minimum inhibitory concentration value can be reduced by 64 times at most (namely, the activity is improved by 64 times), gram-negative bacteria and gram-positive bacteria can be effectively inhibited at the same time, the minimum inhibitory concentrations can reach 0.25 mu M and 0.5 mu M respectively, and the modified antibacterial peptides Perceptide 05 and Perceptide 07 can be used for development of new-generation antibacterial drugs and antibacterial products.

Description

Technical Field

[0001] The present invention relates to an antimicrobial peptide modification and application thereof, in particular to an antimicrobial peptide Ascaphin-5 modification and application thereof in drug preparation, and belongs to the field of biomedicine. Background Art

[0002] Drug-resistant bacterial infections are a serious problem facing the world today. According to statistics from the World Health Organization, drug-resistant bacterial infections currently cause 1.3 million deaths each year, and if no countermeasures are taken, this death toll will increase rapidly by 100,000. The process of developing a new antibiotic is time-consuming and laborious, and the use of small molecule antibiotics may continue to cause bacteria to evolve drug resistance and become ineffective. Antimicrobial peptides, as a class of short peptides, are ideal targets for drug development because they work mainly by destroying bacterial cell membranes rather than targeting specific proteins, thus quickly and effectively inhibiting bacteria and other pathogens in a broad spectrum.

[0003] There are many natural antimicrobial peptides from amphibians such as frogs and toads, whose skin secretions contain these peptide substances that resist microbial invasion. The toad Ascaphus truei is considered to be the most primitive species in the order Anura. 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 relatively limited, and in actual drug development, more active antimicrobial peptides are needed to exert antibacterial effects. Summary of the invention

[0004] In order to obtain highly active antimicrobial peptides, the present invention modified the natural antimicrobial peptide Ascaphin-5 (sequence: GIKDWIKGAAKKLIKTVASNIANQ, SEQ ID No: 1) through an independently developed artificial intelligence model, introduced mutations at 8 positions in its amino acid sequence, and obtained modified antimicrobial peptides Perceptide05 and Perceptide07, whose sequences are shown below:

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

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

[0007] The modified antimicrobial peptides Perceptide05 and Perceptide07 can be synthesized by chemical methods such as peptide solid phase synthesis, and applied to antimicrobial drugs and other antimicrobial products (such as disinfectants, detergents, and feed additives).

[0008] After testing, the modified antimicrobial peptides Perceptide05 and Perceptide07 can effectively enhance the antibacterial activity of Ascaphin-5, and their minimum inhibitory concentration can be reduced by up to 64 times (i.e., the activity is increased by 64 times), and can effectively inhibit both Gram-negative and Gram-positive bacteria, including but not limited to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, with minimum inhibitory concentrations of 0.25μM and 0.5μM, respectively. The modified antimicrobial peptides can be used in the development of a new generation of antimicrobial drugs to improve human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a sequence alignment of the antimicrobial peptide Ascaphin-5 and its modified forms Perceptide05 and Perceptide07.

[0010] Figure 2 This is the sequence wheel diagram of the modified antimicrobial peptides Perceptide05 and Perceptide07. DETAILED DESCRIPTION

[0011] The present invention will be further described below by way of embodiments in conjunction with the accompanying drawings. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0012] Example 1 Source and properties of antimicrobial peptide modifications

[0013] The antimicrobial peptides Perceptide05 and Perceptide07 are modified from the natural antimicrobial peptide Ascaphin-5. The specific method is to input the antimicrobial peptide Ascaphin-5 sequence into the self-developed artificial intelligence model, and the model applies mutations to the sequence, mutating one amino acid at each step. After repeating this process 8 times, the modified sequence is obtained. Figure 1 As shown in the sequence alignment, the artificial intelligence model selected the 8 gray sites for mutation to obtain Perceptide05 and Perceptide07. There are 4 amino acid differences between the two sequences of Perceptide05 and Perceptide07.

[0014] The length of the antimicrobial peptides Perceptide05 and Perceptide07 are both 24 amino acids (consistent with Ascaphin-5), and the hydrophobic amino acids account for 38%. Among them, the molecular weight of Perceptide05 is 2792.55, with 10 positive charges, while the molecular weight of Perceptide07 is 2892.67, with 9 positive charges. The sequence wheel diagrams of the two antimicrobial peptides are shown in Figure 2. Figure 2 As shown, the white background represents polar amino acids, and the black background represents non-polar amino acids, which are roughly distributed on both sides of the antimicrobial peptide helix, showing amphiphilic characteristics.

[0015] Example 2 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0016] The bacterial species used in the minimum inhibitory concentration determination experiment include two Gram-negative strains and two Gram-positive strains, namely Escherichia coli ATCC25922, Pseudomonas aeruginosa PAO1, Staphylococcus aureus ATCC6538, and Bacillus subtilis ATCC6633. The minimum inhibitory concentration of antimicrobial peptides was determined using the Hancock microbroth dilution method. The four standard strains were activated by overnight culture and cultured at 37°C in a shaker at 220 rpm for about 3 hours, and then diluted to OD 625 The values ​​are in the range of 0.08-0.13.

[0017] In order to compare the difference in activity between the modified Perceptide05 and Perceptide07 and the antimicrobial peptide Ascaphin-5 from which they were derived, this example simultaneously tested the minimum inhibitory concentration of these three antimicrobial peptides under the same experimental conditions. The three antimicrobial peptide dry powders were synthesized by Wuhan Dangang Biotechnology Co., Ltd. through solid phase chemical synthesis, and the purity was above 95%. The antimicrobial peptide solution stock was prepared with sterile PBS solution at a concentration of 512 μM. 100 μL of LB culture medium and 100 μL of antimicrobial peptide solution were added to each 96-well plate, and the antimicrobial peptide solution was prepared by diluting the concentration gradient by two times to make the final concentration of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μM. 100 μL of 100-fold diluted bacterial solution was added to each plate, mixed well, and cultured in a 37°C incubator for 16 hours. The lowest antimicrobial peptide solution concentration value at which no obvious bacterial growth was observed by the naked eye is the minimum inhibitory concentration value of the antimicrobial peptide under this condition.

[0018] The test results are shown in Table 1. Compared with the natural antimicrobial peptide Ascaphin-5, its modified forms Perceptide05 and Perceptide07 showed significant improvement in antibacterial activity against four different strains. For example, the antibacterial activity of Perceptide05 against Bacillus subtilis was increased by 64 times compared with Ascaphin-5, and that of Perceptide07 was increased by 32 times. In summary, the modified antimicrobial peptides Perceptide05 and Perceptide07 can effectively inhibit the growth of four strains and have great potential for drug development.

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

[0020]

Claims

1. An antimicrobial peptide, which is a modified form of the natural antimicrobial peptide Ascaphin-5, characterized in that: The antimicrobial peptide is the antimicrobial peptide Perceptide05 or Perceptide07 having the following amino acid sequence: Perceptide05:GIKKWIKGWAKKLIKKVAKKIAKQ; Perceptide07: GIKKWIKGAIKKLIKKVAKWIWKQ.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs.

3. The use according to claim 2, characterized in that The bacteria targeted by the antibacterial drug include Gram-positive bacteria and Gram-negative bacteria.

4. The use according to claim 3, characterized in that The bacteria targeted by the antibacterial drug are one or more of the following bacteria: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis.

5. The use according to claim 2, characterized in that The effective concentration of the antimicrobial peptide Perceptide05 in the antimicrobial drug is greater than or equal to 0.25 μM, or the effective concentration of the antimicrobial peptide Perceptide07 is greater than or equal to 0.5 μM.

6. An antibacterial drug, characterized in that: Contains the antimicrobial peptide Perceptide05 and / or Perceptide07 as described in claim 1.

7. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial products.

8. The use according to claim 7, characterized in that The antimicrobial product is a disinfectant, a detergent or a feed additive.

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