Chicken intestinal bacteriophage source antibacterial peptide and application thereof

Abstract

The invention discloses a chicken intestinal bacteriophage source antibacterial peptide, and belongs to the technical field of biology. The antibacterial peptide (CGV-AMP-1) disclosed by the invention shows excellent effects of high efficiency, rapidness and safety on staphylococcus capitis and streptococcus suis type II; the antibacterial peptide (CGV-AMP-1) can inhibit the growth of bacteria at extremely low concentration (MIC 8 g / ml), and has strong bactericidal ability (MBC 32 g / ml); the time sterilization dynamics experiment proves that the bacteria can be completely killed within 120 minutes; meanwhile, the peptide is extremely high in safety, almost has no hemolytic activity in a range of several times of effective concentration, is extremely low in cytotoxicity, and shows huge potential as a novel anti-infection drug.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to chicken intestinal phage-derived antimicrobial peptides and their applications. Background Technology

[0002] In recent years, the widespread use and even abuse of antibiotics in the medical and livestock industries has led to the emergence and spread of multidrug-resistant bacteria (or "superbugs"), which has become a global public health crisis. In particular, the drug resistance of major zoonotic pathogens such as Streptococcus suis type II is becoming increasingly serious, leading to a decline in the effectiveness of traditional antibiotic treatments. Therefore, the search for novel antibacterial agents with entirely new mechanisms of action is urgently needed.

[0003] Antimicrobial peptides are a class of small-molecule polypeptides produced by the innate immune system of organisms. They possess broad-spectrum antimicrobial activity, rapid action, and are less likely to induce bacterial resistance, making them one of the most promising alternatives to traditional antibiotics. Currently known antimicrobial peptides are mostly isolated from animals, plants, insects, or bacteria themselves.

[0004] Despite the promising prospects of antimicrobial peptides, those discovered from traditional sources still face some challenges, such as relatively limited sources, high cytotoxicity or hemolytic activity of some antimicrobial peptides, and easy degradation by proteases in vivo. These limitations restrict their large-scale commercial development and application.

[0005] Currently, mining novel antimicrobial peptides from metagenomic data, especially virome data, is an emerging frontier. Viruses, especially bacteriophages, as the most numerous biological entities on Earth, possess a wealth of genetic resources with unknown functions within their genomes, representing a vast untapped treasure trove of antimicrobial peptides.

[0006] Within the poultry gut microbiota ecosystem, bacteriophages and bacteria, acting as predators and prey, have co-evolved over a long period. Bacteriophages may have evolved numerous functional peptides designed to disrupt or regulate bacterial life processes. These peptides are crucial for bacteriophage survival and reproduction, including potentially highly effective antimicrobial peptides. Therefore, the bacteriophage genome can be considered a preferred resource library of antimicrobial peptides generated through directed evolution. Nevertheless, research specifically targeting chicken gut bacteriophage resources for systematic functional mining and validation of antimicrobial peptides remains to be completed.

[0007] In summary, there is an urgent need in this field to find and develop novel antimicrobial peptides with advantages such as high antimicrobial activity and high safety (low hemolysis and low cytotoxicity) from new and abundant resources in order to address the increasingly serious challenge of bacterial resistance. Chicken intestinal phage resources are one of the best options. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide chicken intestinal phage-derived antimicrobial peptides and their applications. The antimicrobial peptides prepared by this invention have significant antibacterial activity, high safety, and are simple to prepare and suitable for large-scale production.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a chicken intestinal phage-derived antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] This invention provides the application of chicken intestinal phage-derived antimicrobial peptides in the preparation of biological antimicrobial agents and anti-infective drugs.

[0011] It contains at least the following beneficial technical effects: This antimicrobial peptide (CGV-AMP-1) exhibits excellent efficacy, rapid action, and safety against Streptococcus suis type II: it can inhibit bacterial growth at extremely low concentrations (MIC 8 µg / ml) and has strong bactericidal activity (MBC 32 µg / ml); time-dependent bactericidal kinetics experiments confirmed that it can completely kill the bacteria within 120 minutes; at the same time, the peptide has extremely high safety, with almost no hemolytic activity within several times the effective concentration range, and very low cytotoxicity, demonstrating great potential as a novel anti-infective drug. Attached Figure Description

[0012] Figure 1 These are the results of the inhibition zone experiment; Figure 2 This is the sterilization time curve. Detailed Implementation

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0019] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0020] Example 1 The amino acid sequence of the chicken intestinal phage-derived antimicrobial peptide CGV-AMP-1 obtained by screening in this invention is shown in SEQ ID NO:1: CGV-AMP-1RDAANVSRMKKGWLQRIKDFLAGKK.

[0021] The antimicrobial peptide sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. using solid-phase synthesis (90% purity) for subsequent experimental verification.

[0022] 1. The synthesis of peptides using the Fmoc solid-phase synthesis method consists of the following cycles: 1.1 Deprotection: Fmoc-protected columns and monomers must have the amino protecting groups removed using an alkaline solvent (piperidine).

[0023] 1.2 Activation and Cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to cross-link, forming a peptide bond. A large amount of ultra-concentrated reagent is used to drive the reaction in this step. Cycling: These two steps are repeated until the synthesis is complete.

[0024] 1.3 Elution and Deprotection: The peptide is eluted from the column, and its protecting group is eluted and deprotected by a deprotecting agent (TFA).

[0025] Example 2 1. Antibacterial zone experiment Experimental methods: To evaluate the in vitro antimicrobial activity of synthetic antimicrobial peptides against common poultry and livestock pathogens, an inhibition zone experiment was conducted using the agar diffusion method. The experimental strains included *Riemerella anatipestifer*, *Escherichia coli*, *Salmonella*, *Proteus*, *Staphylococcus aureus*, *Manniella*, *Enterococcus faecalis*, *Clostridium perfringens*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Streptococcus suis* type II, totaling 12 pathogens. The specific experimental steps are as follows: (1) Pre-culture of pathogens The original glycerol strains of the pathogen were inoculated into the corresponding solid media and cultured at 37°C for 18-24 hours. After single colonies formed, they were used for liquid culture. The solid media used for each strain were as follows: TSA medium for *Riemerella anatipestifer*, TSC selective medium for *Clostridium perfringens*, MH medium for *Pseudomonas aeruginosa*, THB medium for *Staphylococcus capitella* and *Streptococcus suis* type II, and LB medium for the remaining strains.

[0026] (2) Preparation of logarithmic growth phase bacterial culture Using a sterile inoculation loop, pick a single colony and inoculate it into 5 mL of the corresponding liquid culture medium. *Riemerella anatipestifer* was inoculated on TSB, *C. wiltii* on FT anaerobic medium, *Pseudomonas aeruginosa* on MH liquid medium, *Staphylococcus capitella* and *Streptococcus suis* type II on THB, and the remaining species on LB liquid medium. *C. wiltii* was cultured statically at 37°C under anaerobic conditions, while the other species were cultured with shaking at 37°C and 180 rpm for 9-12 hours, until the culture became noticeably turbid and entered the logarithmic growth phase.

[0027] (3) Standardization of bacterial concentration Centrifuge the cultured bacterial suspension (8000 rpm, 5 min), wash twice with sterile PBS, and resuspend. Adjust the bacterial suspension concentration to approximately 1 × 10⁻⁶ using a turbidimetric method. 5 CFU / mL was used for subsequent antibacterial experiments.

[0028] (4) Preparation of antibacterial plates Mix 1 mL of standardized bacterial suspension with the corresponding agar medium cooled to approximately 50°C, then quickly pour the mixture into a sterile plate. Gently shake to distribute the agar evenly, and allow it to solidify at room temperature. The resulting plate contains bacteria.

[0029] (5) Treatment and incubation of antimicrobial peptides The antimicrobial peptide was prepared at a concentration of 1 mg / mL using sterile ultrapure water and sterilized by aseptic filtration. 3 μL of the antimicrobial peptide solution was spotted onto the surface of a bacterial plate using a micropipette. After each plate was added, it was placed upright at 4°C for 3 hours to allow for sufficient diffusion and absorption of the antimicrobial peptide. The plates were then inverted and incubated at 37°C for 24 hours.

[0030] To verify the effectiveness of the experimental system and rule out non-specific antibacterial effects, a positive control and a negative control were set up. The positive control used meropenem solution, prepared at a concentration of 1 μg / mL using sterile ultrapure water; the negative control used an equal volume of sterile ultrapure water. The sample addition method was the same as for the antimicrobial peptide group: 3 μL was added to a bacterial plate, incubated at 4°C for 3 h to allow for sufficient diffusion, and then the plate was inverted and incubated at 37°C for 24 h. The antimicrobial activity of the antimicrobial peptide was evaluated by observing and measuring the presence and diameter of inhibition zones, and compared with the control group.

[0031] Experimental results: The inhibition zone assay results showed that CGV-AMP-1 exhibited antibacterial effects against Staphylococcus aureus and Streptococcus suis type II. Figure 1 .

[0032] 2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Experimental methods: This experiment used two strains (Staphylococcus capsulatum and Streptococcus suis type II) screened in the previous inhibition zone experiment to determine the minimum inhibitory concentration and minimum bactericidal concentration of CGV-AMP-1 using the micro-broth dilution method.

[0033] The antimicrobial peptide solution was prepared into a stock solution of 1024 μg / mL using sterile ultrapure water. In a 96-well microplate, 50 μL of the stock solution was added to each well in columns 1 and 2; 50 μL of sterile culture medium was added to each well in columns 2 through 12. Subsequently, a 2-fold serial dilution was performed starting from column 2, up to column 12, and excess liquid was discarded. The final concentrations of the antimicrobial peptide (after dilution with bacterial culture) in each well were: 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively.

[0034] Then add 50 μL of bacterial suspension (diluted to a final concentration of 5 × 10⁻⁶) to each well. 5 The total volume was 100 μL (CFU / mL). A positive control was meropenem at the same concentration gradient, and a negative control was sterile ultrapure water. After incubating the 96-well plates at 37°C for 24 hours, the clarity of the liquid was visually observed to determine bacterial growth. The MIC was defined as the lowest concentration of antimicrobial peptide at which no visible bacterial growth (clarity) was observed.

[0035] To further determine MBC, 10 μL of mixed culture medium was taken from wells where no bacterial growth was observed and spread onto antibiotic-free solid culture plates, which were then incubated at 37°C for 18–24 h. If no colony growth was observed on the plate, the concentration of the antimicrobial peptide corresponding to that well was the minimum bactericidal concentration.

[0036] The experimental results are shown in Table 1: CGV-AMP-1 showed good antibacterial effect against Streptococcus suis type II. The MIC for this bacterium was 8 ug / ml, and the MBC was 32 ug / ml.

[0037] Table 1. MIC and MBC determination of CGV-AMP-1 against Staphylococcus aureus and Streptococcus suis type II. 3. Hemolytic test Experimental methods: To evaluate the hemolytic activity of the antimicrobial peptides, a red blood cell suspension was prepared using defibrinated sheep blood. The specific method is as follows: Defibrinated sheep blood was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the red blood cells were repeatedly washed with PBS buffer until the supernatant was clear and transparent. Subsequently, the red blood cells were resuspended in 10 times their volume of PBS to prepare a red blood cell suspension for later use.

[0038] In the experiment, 100 μL of erythrocyte suspension and 100 μL of antimicrobial peptide solutions of different concentrations were mixed in a 1.5 mL centrifuge tube and incubated at 37 °C for 1 hour to induce possible hemolytic reactions. After incubation, the mixture was centrifuged at 1500 r / min for 10 min, and the supernatant was collected.

[0039] Add 100 μL of supernatant to a 96-well plate and measure its absorbance (OD) at 540 nm using a microplate reader. 540 PBS buffer was used as a negative control (0% hemolysis), and 1% Triton X-100 was used as a positive control (100% hemolysis). Each experiment was performed in triplicate, and results are expressed as mean ± standard deviation (Mean ± SD).

[0040] The experimental results are shown in Table 2: CGV-AMP-1 maintained extremely low hemolytic activity (0.31±0.17%) even at a maximum concentration of 128 μg / mL.

[0041] Table 2 Results of hemolytic test 4. Cytotoxicity assay Experimental methods: The CCK-8 assay was used to assess the cytotoxic effects of the peptides. Cells were cultured in DMEM medium (containing 10% fetal bovine serum) and, after reaching the logarithmic growth phase, were collected by trypsin digestion and resuspended in the medium, with the cell density adjusted to 2 × 10⁶ cells / year. 5 Cells were seeded at a rate of 100 μl / well in 96-well plates and incubated overnight to ensure stable cell adhesion. The next day, the culture medium was discarded, and the cells were washed twice with PBS. Different concentrations of peptide (8-128 μg / mL) were added for treatment, with negative controls (cells only) and blank controls (culture medium only) included. Incubation continued overnight. After treatment, the supernatant was discarded, and the cells were washed twice with PBS. Fresh DMEM medium containing 10% CCK-8 reagent was added, and the cells were incubated at 37°C for 2 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [Absorbance of experimental wells - Absorbance of blank wells] / (Absorbance of negative control wells - Absorbance of blank wells)] × 100%, with each group performed in triplicate; Cytotoxicity rate (%) = 100% - Cell viability.

[0042] Table 3: Experimental Results Cytotoxicity assays showed that CGV-AMP-1 maintained more than 70% cell viability at all tested concentrations.

[0043] Table 3 Results of Cytotoxicity Tests 5. Time-based sterilization kinetics experiment Experimental methods: Streptococcus suis type II was cultured under suitable conditions with shaking until the logarithmic growth phase. The turbidity of the bacterial culture was measured and converted to viable cell concentration. The bacterial concentration was adjusted to 2 × 10⁻⁶ using serum-free THB liquid medium. 5 CFU / mL. Subsequently, 500 μL of the treated bacterial culture was mixed with 500 μL of a peptide-containing solution (the final peptide concentration was set to be twice its minimum bactericidal concentration, resulting in a mixed bacterial culture concentration of 1 × 10⁻⁶ CFU / mL). 5 The concentration of the antimicrobial peptide was 1×MBC, and the mixture was thoroughly mixed in a 1.5 mL centrifuge tube.

[0044] The mixture was incubated at a constant temperature of 37°C, and samples were taken at multiple time points (0, 15, 30, 60, 120, 180, and 240 minutes). At each time point, a certain volume of the mixture was taken, serially diluted 10-fold, and 100 μL was evenly spread onto the surface of THB solid medium. The inoculated plates were placed in an incubator at 37°C for 18–24 hours, and the number of colony-forming units (CFU) at each time point was recorded and counted to calculate the relative survival rate of the bacteria.

[0045] A negative control group was prepared by adding an equal volume of sterile water instead of the antimicrobial peptide. Three replicates were set up for each time point, and the entire experiment was independently repeated three times.

[0046] The experimental results are shown in Figure 2 CGV-AMP-1 kills all bacteria within 120 minutes.

[0047] 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 chicken intestinal phage-derived antimicrobial peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the chicken intestinal phage-derived antimicrobial peptide according to claim 1 in the preparation of biological antimicrobial agents and anti-infective drugs.

3. The application according to claim 1, characterized in that, The minimum inhibitory concentration of the chicken intestinal phage-derived antimicrobial peptide is 8 µg / ml, and the minimum bactericidal concentration is 32 µg / ml.

Images