Novel antibacterial peptide wrGVC-AMP-S7 derived from wild rodent enteric bacteriophage and application of novel antibacterial peptide wrGVC-AMP-S7

Abstract

The invention discloses a novel antibacterial peptide wrGVC-AMP-S7 derived from intestinal bacteriophage of a wild rodent and application of the novel antibacterial peptide wrGVC-AMP-S7, and belongs to the technical field of biology. According to the present invention, the MIC determination result shows that the antibacterial peptide wrGVC-AMPS7 provides different degrees of antibacterial activity on a variety of gram-positive bacteria and gram-negative bacteria; under the condition that the highest test concentration is 128 mu g / mL, the hemolysis rate is still lower than 10%, and low hemolysis activity and good biological safety are shown. A cytotoxicity test result shows that the antibacterial peptide AMPS7 does not show obvious cytotoxicity to IEC-6 cells. Even under the condition of a high concentration of 128 [mu] g / mL, the cell survival rate is still kept at 95% or above, which indicates that the antibacterial peptide has good cytocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel antimicrobial peptide wrGVC-AMP-S7 derived from wild rodent intestinal bacteriophages and its uses. Background Technology

[0002] In modern livestock production, antibiotics have long been widely used for animal disease prevention and control, as well as for regulating production performance. However, while the continued use of antibiotics effectively controls bacterial infections, it also accelerates the emergence and spread of drug-resistant pathogens, posing potential risks to animal health, food safety, and public health. As the problem of antibiotic resistance becomes increasingly prominent, many countries and regions have successively introduced relevant policies to strictly limit the use of antibiotics in livestock farming. Against this backdrop, exploring safe and effective antibiotic alternatives has become an important research direction for promoting the sustainable development of livestock farming.

[0003] Antimicrobial peptides are a class of small-molecule bioactive peptides widely found in nature. They typically exhibit rapid action and diverse mechanisms of action, demonstrating promising application potential in inhibiting or killing pathogens. In recent years, research on antimicrobial peptides as novel anti-infective molecules has received sustained attention, and they are considered to have significant value in livestock disease control and antibiotic alternatives. Further expanding the types and sources of antimicrobial peptides and continuously exploring new antimicrobial peptide resources has become an important research focus in this field.

[0004] With the rapid development of metagenomic sequencing technology, researchers can systematically analyze the genetic information of complex microbial communities without isolating bacteria, providing new technical means for high-throughput mining of functional molecules. Meanwhile, artificial intelligence methods such as deep learning have shown significant advantages in biological sequence pattern recognition and functional prediction, and can be used to efficiently screen potential candidate peptide sequences with antimicrobial activity from massive sequence data. Mining novel antimicrobial peptides based on metagenomic data combined with deep learning models has become an important development direction and research hotspot in the field of antimicrobial peptide research.

[0005] Wild rodents are widely distributed in diverse natural ecosystems, and their gut microbiota, formed through long-term evolution and the influence of complex environmental factors, exhibits significant diversity and uniqueness. Among these, gut bacteriophages, as an important component of the microbial community, play a crucial role in regulating bacterial community structure and microecological balance, and their genomes may contain antimicrobial-related functional molecules with potential applications. However, current research on the systematic screening, function, and application of antimicrobial peptides derived from wild rodent gut bacteriophages remains relatively limited, and related resources require further development.

[0006] Therefore, the discovery and identification of antimicrobial peptides with broad-spectrum antimicrobial activity from wild rodent gut bacteriophage resources is of great significance for enriching the source of antimicrobial peptides and promoting the development of antibiotic alternative technologies. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a novel antimicrobial peptide wrGVC-AMP-S7 derived from wild rodent intestinal bacteriophages and its uses. The novel antimicrobial peptide wrGVC-AMP-S7 of this invention has broad-spectrum antimicrobial activity and high safety.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a novel antimicrobial peptide wrGVC-AMP-S7 derived from intestinal bacteriophages of wild rodents, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] This invention provides the application of the novel antimicrobial peptide wrGVC-AMP-S7 in the preparation of broad-spectrum antimicrobial agents.

[0010] Furthermore, the broad-spectrum antibacterial agent has an inhibitory effect on the following bacteria: Anatidae, Mansonia, Proteus mirabilis, hemolytic Staphylococcus, Pasteurella, Clostridium perfringens, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus suis type II, Enterococcus faecalis, Klebsiella pneumoniae.

[0011] Furthermore, the concentration of the novel antimicrobial peptide wrGVC-AMP-S7 is 16-128 μg / mL.

[0012] It contains at least the following beneficial technical effects: The MIC determination results of this invention show that the antimicrobial peptide wrGVC-AMP_S7 exhibits varying degrees of antibacterial activity against a variety of Gram-positive and Gram-negative bacteria. Specifically, it shows strong inhibitory effects against hemolytic Staphylococcus aureus, Clostridium perfringens, Salmonella (ATCC 14028), and Staphylococcus aureus (ATCC 29213), with MIC values ​​of 16 μg / mL for each. Simultaneously, it also shows varying degrees of inhibitory effects against other tested strains. These results indicate that wrGVC-AMP_S7 possesses antibacterial activity against a variety of livestock-related pathogens, demonstrating its potential application value as a novel antimicrobial agent in the control of livestock pathogens.

[0013] At the highest tested concentration of 128 μg / mL, its hemolysis rate remained below 10%, demonstrating low hemolytic activity and good biocompatibility. Cytotoxicity assays showed that the antimicrobial peptide AMP_S7 did not exhibit significant cytotoxicity against IEC-6 cells. Even at the high concentration of 128 μg / mL, cell viability remained above 95%, indicating good cell compatibility of this antimicrobial peptide. Attached Figure Description

[0014] Figure 1 The time-dependent bactericidal curve of the novel antimicrobial peptide wrGVC-AMP-S7 against Staphylococcus aureus is shown. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0021] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0022] Example 1 The sequence of the antimicrobial peptide wrGVC-AMP-S7 is as follows: KFRRHGRSRRGSRRRSRPRSVFAKRVGNRL; Solid-phase synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The purity of all synthesized peptides exceeded 90%.

[0023] Example 2 1. Experimental method for determining the minimum inhibitory concentration (MIC): The antimicrobial activity of the screened antimicrobial peptides was evaluated against 13 common poultry and livestock pathogens, including 7 clinical isolates (Rimeria anatidae, Mansonia solani, Proteus mirabilis, Staphylococcus aureus, Pasteurella multocida, Clostridium perfringens, and Pseudomonas aeruginosa) and 6 standard reference strains: Salmonella (ATCC 14028), Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Streptococcus suis type II (ATCC 43765), Enterococcus faecalis (ATCC 29212), and Klebsiella pneumoniae (ATCC 46117).

[0024] MIC was determined using the broth microdilution method. The specific procedure was as follows: The test strains were streaked onto their respective solid culture media. *Rimersella anatipestifer* was inoculated onto TSA medium, *Clostridium perfringens* onto TSC medium, *Streptococcus suis* type II onto THB medium, and the remaining strains onto MH medium. The inoculated plates were then incubated overnight at 37°C until clearly identifiable single colonies formed.

[0025] The obtained single colonies were inoculated into 5 mL of the corresponding liquid culture medium for amplification culture. *Riemerella anatipestifer* was cultured on TSB medium, *Clostridium perfringens* on FT medium, *Streptococcus suis* type II on THB medium, and the remaining strains on MH medium. *Clostridium perfringens* was cultured statically at 37°C, while the other strains were cultured with shaking at 37°C and 220 rpm for 9–12 h, until the bacterial culture reached the logarithmic growth phase and showed obvious turbidity.

[0026] The antimicrobial peptide wrGVC-AMP_S7 was prepared into gradient solutions with the required concentrations using sterile ultrapure water. In sterile 96-well plates, 100 μL of the corresponding liquid culture medium was added to wells 1–8, followed by 100 μL of the prepared antimicrobial peptide solution to well 1. This process was repeated sequentially up to well 10. After dilution, the final concentrations of the antimicrobial peptide in wells 1–9 were 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively.

[0027] Following the order of increasing antimicrobial peptide concentration, 100 μL of diluted bacterial suspension was added to each well sequentially, until the final bacterial concentration in each well reached 1 × 10⁻⁶. 6 CFU / mL. Only the corresponding culture medium was added to the 10th well as a blank control. After adding the samples, the plate was covered and sealed with sealing film, and incubated at 37°C for 14–16 h. Results were observed after incubation, where MIC was defined as the lowest concentration of antimicrobial peptide at which no visible bacterial growth was observed.

[0028] The MIC test results are shown in Table 1: Table 1. MIC Results of wrGVC-AMP_S7 MIC assay results showed that the antimicrobial peptide wrGVC-AMP_S7 exhibited varying degrees of antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria. It showed strong inhibitory effects against hemolytic Staphylococcus aureus, Clostridium perfringens, Salmonella (ATCC 14028), and Staphylococcus aureus (ATCC 29213), with MIC values ​​of 16 μg / mL for each. It also showed varying degrees of inhibitory effects against other tested strains. These results indicate that wrGVC-AMP_S7 possesses antimicrobial activity against a variety of livestock-related pathogens, demonstrating its potential application value as a novel antimicrobial agent in the control of livestock pathogens.

[0029] 2. Hemolytic test The hemolytic activity of defibrinated sheep erythrocytes against antimicrobial peptides was evaluated. First, sheep erythrocytes were repeatedly washed with phosphate-buffered saline (PBS) until the supernatant was colorless and transparent. Then, the washed erythrocyte pellet was resuspended in 19 volumes of PBS to prepare a 5% (v / v) erythrocyte suspension.

[0030] Take 50 μL of erythrocyte suspension and mix it with an equal volume of antimicrobial peptide solutions of different concentrations, wherein the final concentration of antimicrobial peptides ranges from 1 to 128 μg / mL. Incubate the mixture at 37°C for 1 h, then centrifuge at 8,000 g for 10 min. After centrifugation, transfer the supernatant to a 96-well plate and measure the absorbance at 540 nm using a microplate reader.

[0031] The PBS treatment group was used as a negative control, and the 1% Triton X-100 treatment group was used as a positive control. The hemolytic activity of the antimicrobial peptides was evaluated by comparing the changes in absorbance between the treatment groups and the control group.

[0032] All experiments were conducted in triplicate, and the results are expressed as mean plus or minus standard deviation.

[0033] The experimental results are shown in Table 2: Table 2 Results of hemolytic test Experimental results: The hemolytic activity assay results showed that the antimicrobial peptide wrGVC-AMP_S7 did not produce significant hemolytic activity against erythrocytes within the tested concentration range. Even at the highest tested concentration of 128 μg / mL, its hemolysis rate remained below 10%, demonstrating low hemolytic activity and good biocompatibility.

[0034] 3. Cytotoxicity assay The cytotoxic effects of the antimicrobial peptide wrGVC-AMP_S7 on cells were evaluated using the CCK-8 assay. Rat small intestinal crypt epithelial cells IEC-6 were used as a model cell, and the cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS).

[0035] IEC-6 cells in logarithmic growth phase were divided into groups of 2 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates, and incubated overnight at 37°C and 5% CO2 to allow for full cell adhesion. Subsequently, different concentrations of antimicrobial peptide solution were added to each well to achieve a final concentration range of 8–128 μg / mL. Cells without antimicrobial peptide served as a negative control, and wells containing only culture medium served as a blank control.

[0036] After culturing overnight at 37°C, the original culture medium was discarded, and fresh DMEM medium containing 10% CCK-8 reagent was added to each well. The cells were then incubated at 37°C for 2 h. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated based on the changes in absorbance between the treatment and control groups to evaluate the cytotoxicity of the antimicrobial peptides to IEC-6 cells.

[0037] All experiments were conducted in triplicate, and the results are expressed as mean plus or minus standard deviation.

[0038] The experimental results are shown in Table 3: Table 3 Results of Cell Viability Assay Experimental results: Cytotoxicity assays showed that the antimicrobial peptide AMP_S7 did not exhibit significant cytotoxicity against IEC-6 cells. Even at a high concentration of 128 μg / mL, cell viability remained above 95%, indicating that the antimicrobial peptide has good cell compatibility.

[0039] 4. Time-sterilization curve The dynamic bactericidal efficiency of the antimicrobial peptide wrGVC-AMP_S7 was evaluated using a time-based bactericidal curve assay. Staphylococcus aureus (ATCC 29213) was used as the test strain, cultured under suitable conditions to the logarithmic growth phase, and the bacterial concentration was adjusted to 2 × 10⁻⁶. 6 CFU / mL.

[0040] Subsequently, an equal volume of bacterial suspension was mixed with the antimicrobial peptide solution and placed in a 1.5 mL sterile centrifuge tube to achieve a final bacterial concentration of 1 × 10⁻⁶. 6 CFU / mL. The antimicrobial peptides were set at three treatment concentrations: 0.5 × MIC, 1 × MIC, and 2 × MIC.

[0041] The above mixture was incubated at 37°C, and samples were taken at 0, 30, 60, 90, and 120 min. The samples were serially diluted 10-fold, and 100 μL of each dilution was evenly spread onto MH agar plates and incubated at 37°C for 16–18 h. Colonies were then counted to evaluate the bactericidal effect of the antimicrobial peptide at different time points. Each time point was set up in triplicate, and the entire experiment was independently repeated three times.

[0042] The test results are shown in Figure 1 : Experimental results: The experimental results showed that the antimicrobial peptide wrGVC-AMP_S7 could kill 100% of Staphylococcus aureus ATCC29213 within 30 min at a concentration of 2×MIC.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A novel antimicrobial peptide wrGVC-AMP-S7 derived from intestinal bacteriophages of wild rodents, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the novel antimicrobial peptide wrGVC-AMP-S7 according to claim 1 in the preparation of broad-spectrum antimicrobial agents.

3. The application according to claim 2, characterized in that, The broad-spectrum antibacterial agent has an inhibitory effect on the following bacteria: Anatidae, Mansonia, Proteus mirabilis, hemolytic Staphylococcus, Pasteurella, Clostridium perfringens, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Escherichia coli, Streptococcus suis type II, Enterococcus faecalis, Klebsiella pneumoniae.

4. The application according to claim 2 or 3, characterized in that, The concentration of the novel antimicrobial peptide wrGVC-AMP-S7 is 16-128 μg / mL.

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