Antibacterial peptide NyAmp-a and application thereof

Abstract

The invention discloses an antibacterial peptide NyAmp-a and application thereof, and relates to the technical field of biological medicines. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO. 1. The invention also provides an application of the antibacterial peptide NyAmp-a in preparation of an antibacterial product for inhibiting staphylococcus aureus, bacillus subtilis, escherichia coli and aeromonas hydrophila, and a minimum inhibitory concentration of the antibacterial peptide NyAmp-a to each bacterium. The invention provides a novel efficient antibacterial solution for four common pathogenic bacteria, namely staphylococcus aureus, bacillus subtilis, escherichia coli and aeromonas hydrophila. The antibacterial peptide NyAmp-a has the characteristic that drug resistance is not easy to generate, a related antibacterial composition can be applied to the fields of medicine, agriculture, food and the like, has important practical application value, and effectively solves the problems that traditional antibiotics are easy to generate drug resistance, and existing antibacterial peptides are high in cytotoxicity and lack of selectivity for specific strains.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an antimicrobial peptide NyAmp-a and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) belongs to the genus Staphylococcus and is a Gram-positive bacterium. Under a microscope, S. aureus bacteria are arranged in grape-like clusters. They lack spores, flagella, and most lack capsules. This bacterium commonly parasitizes the skin, nasal cavity, throat, gastrointestinal tract, carbuncles, and purulent sores of humans and animals, and is ubiquitous in the environment, including air and sewage. Reports indicate that after colonizing human skin, S. aureus can invade subcutaneous tissue, directly affecting nerve cells and causing itching. Drug resistance in S. aureus develops frequently, and its exotoxins can cause toxic diseases in humans, as well as scalded skin syndrome and toxic shock syndrome. (Bacillus subtilis...) Bacillus subtilis *Escherichia coli* belongs to the genus *Bacillus* and is a Gram-positive bacillus. The bacteria are rod-shaped, capable of forming spores, and can survive under adverse conditions. They are widely distributed in soil, water, air, and on the surface and intestinal tracts of animals and plants. A few strains may cause infection in immunocompromised individuals or cause food spoilage. Escherichia coli *Escherichia coli* belongs to the genus *Escherichia* and is a Gram-negative short rod-shaped bacterium. The bacteria are short rods with blunt ends, non-spore-forming, and most possess flagella, enabling motility. It is an opportunistic pathogen, normally coexisting symbiotically with humans, but may cause infection under special circumstances such as weakened immunity. Symptoms of *E. coli* infection vary depending on the site of infection. Intestinal infections may present with diarrhea, abdominal pain, nausea, and vomiting; acute infections may also include fever, chills, and limb pain. Extraintestinal infections, such as urinary tract infections, may present with urinary urgency, frequency, and painful urination. Neonatal infections may cause persistent high fever, and in severe cases, even coma. *Aeromonas hydrophila* (… Aeromonas hydrophila Aeromonas belongs to the genus Aeromonas and is a Gram-negative short rod-shaped bacillus. The bacteria are rod-shaped, non-spore-forming, and most possess flagella, enabling motility. Widely distributed in freshwater environments, silt, and aquatic animals, it is an opportunistic pathogen. When the body's immunity is weakened or the skin and mucous membranes are damaged, it can cause intestinal infections in humans, resulting in symptoms such as abdominal pain, diarrhea, and watery stools; it can also cause skin and soft tissue infections, sepsis, etc., and can induce various diseases in fish and other aquatic animals.

[0003] Antimicrobial peptides, also known as antimicrobial peptides, are widely distributed in organisms and are part of the innate immune defense system. Unlike conventional antibiotics, antimicrobial peptides are protein products encoded by specific genes, thus possessing unique antimicrobial mechanisms. Nevertheless, the clinical application of antimicrobial peptides still faces challenges, such as strong cytotoxicity and lack of selectivity for specific bacterial strains. Summary of the Invention

[0004] To address the aforementioned shortcomings in the prior art, this invention provides an antimicrobial peptide NyAmp-a and its applications, offering a new active ingredient for preparing antibacterial products targeting Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Aeromonas hydrophila. This solves the problems of traditional antibiotics easily developing resistance and existing antimicrobial peptides exhibiting strong cytotoxicity and lack of selectivity for specific strains.

[0005] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide an antimicrobial peptide NyAmp-a, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The above-mentioned antimicrobial peptide NyAmp-a is used in the preparation of products that inhibit Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Aeromonas hydrophila.

[0007] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Staphylococcus aureus is 25 μg / mL.

[0008] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Bacillus subtilis is 25 μg / mL.

[0009] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Escherichia coli is 25 μg / mL.

[0010] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Aeromonas hydrophila is 50 μg / mL.

[0011] Furthermore, the product is a bacteriostatic agent or antibacterial drug.

[0012] Furthermore, the only effective ingredient in antibacterial agents or antimicrobial drugs is the antimicrobial peptide NyAmp-a.

[0013] Furthermore, the preparation method of the antibacterial agent includes the following steps: dissolving the antimicrobial peptide NyAmp-a in sterile water to obtain the antibacterial agent.

[0014] Furthermore, the concentration of the antimicrobial peptide NyAmp-a in the antibacterial agent is 25-50 μg / mL.

[0015] Furthermore, antimicrobial agents include the antimicrobial peptide NyAmp-a and pharmaceutically acceptable excipients.

[0016] Furthermore, the excipients are those required for the preparation of water extracts, powders, detergents, oils, emulsions, ointments, plasters, or aerosols.

[0017] In summary, the present invention has the following beneficial effects: 1. The antimicrobial peptide NyAmp-a provided by this invention has a minimum inhibitory concentration (MIC) of 25 μg / mL against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli, and a MIC of 50 μg / mL against Aeromonas hydrophila. The antimicrobial peptide NyAmp-a can effectively inhibit the growth and reproduction of these four pathogens and has a significant antimicrobial effect. This invention provides a new active ingredient for the preparation of antimicrobial products targeting Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Aeromonas hydrophila.

[0018] 2. As part of the innate immune defense system, antimicrobial peptides possess unique antimicrobial mechanisms. The antimicrobial peptide NyAmp-a exhibits a low tendency to induce drug resistance, overcoming the problem of resistance development associated with traditional antibiotics and solving the technical challenges of existing antimicrobial peptides' high cytotoxicity and lack of selectivity for specific bacterial strains.

[0019] 3. The antimicrobial peptide NyAmp-a can be prepared into various dosage forms such as water extract, powder, lotion, oil, emulsion, ointment, plaster or aerosol. Antimicrobial products use antimicrobial peptide NyAmp-a as the only active ingredient, which simplifies the formulation process. Related antimicrobial compositions can be applied in many fields such as medicine, agriculture, and food. Attached Figure Description

[0020] Figure 1 The growth of the antimicrobial peptide NyAmp-a against Staphylococcus aureus is shown in the figure. Figure 2 The growth of the antimicrobial peptide NyAmp-a against Staphylococcus aureus is shown in the figure. Figure 3 The growth of the antimicrobial peptide NyAmp-a against the minimum inhibitory concentration of Bacillus subtilis is shown in the figure. Figure 4 The growth of the antimicrobial peptide NyAmp-a against the minimum inhibitory concentration of Bacillus subtilis is shown in the figure. Figure 5 The growth pattern shows the minimum inhibitory concentration (MIC) of the antimicrobial peptide NyAmp-a against Escherichia coli. Figure 6 The growth pattern shows the minimum inhibitory concentration (MIC) of the antimicrobial peptide NyAmp-a against Escherichia coli. Figure 7 The growth of the antimicrobial peptide NyAmp-a against the minimum inhibitory concentration of Aeromonas hydrophila is shown in the figure. Figure 8The growth of the antimicrobial peptide NyAmp-a against the minimum inhibitory concentration of Aeromonas hydrophila is shown in the figure. Figure 9 The graph shows the minimum inhibitory concentration (MIC) of the antimicrobial peptide NyAmp-a against four types of bacteria. Detailed Implementation

[0021] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1: Preparation of the antimicrobial peptide NyAmp-a The antimicrobial peptide NyAmp-a was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity greater than 95%. The amino acid sequence of the antimicrobial peptide NyAmp-a is N-MHLLIPKIGTKQKKNMAMRM. YIQKVSLQIKQNEQLQRVLTLTFLLLWLT-C.

[0023] Example 2: Determination of minimum inhibitory concentration The four bacteria used in the experiment—Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Aeromonas hydrophila—were all existing, commonly used strains. The minimum inhibitory concentration (MIC) of the antimicrobial peptide NyAmp-a was determined using the serial dilution method.

[0024] Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were inoculated onto LB solid medium, while Aeromonas hydrophila was inoculated onto TSB solid medium for activation. After overnight incubation, single colonies were collected and transferred to LB liquid medium, while Aeromonas hydrophila was inoculated onto TSB liquid medium and incubated at 37°C with shaking (27°C for Aeromonas hydrophila) until the logarithmic growth phase. The cultures were centrifuged, the supernatant was discarded, and the bacterial cells were collected. The cells were washed with sterile water three times, and then resuspended in sterile water to adjust the bacterial concentration to 10⁻⁶. 5The concentration of NyAmp-a was used for subsequent experiments. The antimicrobial peptide NyAmp-a was diluted with sterile PBS to a stock solution of 5 mg / mL. This diluted peptide was then mixed with suspensions of four different bacteria, resulting in a final volume of 200 μL. This yielded final concentrations of NyAmp-a of 150 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 0 μg / mL, respectively. This 200 μL mixture was incubated in a shaker at 37 °C for 2 h. Two hours later, the mixture was removed from the incubator. 25 μL of the mixture was taken from the laminar flow hood and inoculated into the corresponding solid culture medium, with four replicates for each peptide concentration. The plates were placed in a 37 °C incubator and incubated for 12 hours. After 12 hours, the plates were removed for observation, and the colony counts at each concentration were counted. The final concentrations of NyAmp-a were 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively, which achieved 100% inhibition of bacteria. The inhibition rates of other concentrations are shown in Table 1.

[0025] Table 1. Results of Antibacterial Rate

[0026] The growth of the antimicrobial peptide NyAmp-a against Staphylococcus aureus is shown in the figure below. Figure 1 and Figure 2 As shown, Figure 1 In the bacterial cultures from column 1 to column 4, the concentrations of antimicrobial peptides were 0 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. Figure 2 In the figure, the concentrations of the antimicrobial peptide in the bacterial cultures from column 5 to column 8 were 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL, respectively. The growth pattern of the antimicrobial peptide NyAmp-a against Bacillus subtilis is shown in the figure below. Figure 3 and Figure 4 It can be seen that, Figure 3 In the bacterial cultures of columns 1 to 4, the concentrations of antimicrobial peptides were 0 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. Figure 4 In the figure, the concentrations of the antimicrobial peptide in the bacterial cultures in columns 5 to 8 were 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL, respectively. The growth pattern of the antimicrobial peptide NyAmp-a against Escherichia coli is shown in the figure below. Figure 5 and Figure 6 As shown, Figure 5In the bacterial cultures of columns 1 to 4, the concentrations of antimicrobial peptides were 0 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. Figure 6 In the figure, the concentrations of the antimicrobial peptide in the bacterial suspensions from column 5 to column 8 were 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL, respectively. The growth pattern of the antimicrobial peptide NyAmp-a against Aeromonas hydrophila is shown in the figure below. Figure 7 and Figure 8 As shown. Figure 7 In the bacterial cultures from column 1 to column 4, the concentrations of antimicrobial peptides were 0 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. Figure 8 In the bacterial cultures from column 5 to column 8, the concentrations of antimicrobial peptides were 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL, respectively.

[0027] Depend on Figures 1-8 It was found that when the concentration of the antimicrobial peptide NyAmp-a was 25 μg / mL, *Staphylococcus aureus*, *Bacillus subtilis*, and *Escherichia coli* showed no growth; however, when the concentration was below 25 μg / mL, all bacteria showed varying degrees of growth. When the concentration of the antimicrobial peptide NyAmp-a was 50 μg / mL, *Aeromonas hydrophila* showed no growth; however, when the concentration was below 50 μg / mL, all bacteria showed varying degrees of growth. Therefore, the minimum inhibitory concentration (MIC) of the antimicrobial peptide NyAmp-a against *Staphylococcus aureus*, *Bacillus subtilis*, and *Escherichia coli* was 25 μg / mL, and against *Aeromonas hydrophila* it was 50 μg / mL, indicating that it has a good antibacterial effect against these four bacteria. The results of the MIC of the antimicrobial peptide NyAmp-a against the four bacteria are shown in the figure below. Figure 9 As shown.

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antimicrobial peptide NyAmp-a, characterized in that, The amino acid sequence of the antimicrobial peptide NyAmp-a is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide NyAmp-a according to claim 1 in the preparation of products that inhibit Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Aeromonas hydrophila.

3. The application as described in claim 2, characterized in that, The minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Staphylococcus aureus is 25 μg / mL. The minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Bacillus subtilis is 25 μg / mL; The minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Escherichia coli is 25 μg / mL; The minimum inhibitory concentration of the antimicrobial peptide NyAmp-a against Aeromonas hydrophila is 50 μg / mL.

4. The application as described in claim 2, characterized in that, The product is an antibacterial agent or antimicrobial drug.

5. The application as described in claim 4, characterized in that, The sole active ingredient of the antibacterial agent or antimicrobial drug is the antimicrobial peptide NyAmp-a.

6. The application as described in claim 4 or 5, characterized in that, The method for preparing the antibacterial agent includes the following steps: dissolving the antimicrobial peptide NyAmp-a in sterile water to obtain the antibacterial agent.

7. The application as described in claim 6, characterized in that, The concentration of the antimicrobial peptide NyAmp-a in the antibacterial agent is 25-50 μg / mL.

8. The application as described in claim 4 or 5, characterized in that, The antimicrobial agent includes the antimicrobial peptide NyAmp-a and pharmaceutically acceptable excipients.

9. The application as described in claim 8, characterized in that, The excipients are those required for preparing water extracts, powders, detergents, oils, emulsions, ointments, plasters, or aerosols.

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