An antibacterial peptide based on sequence information of the proteome of the family vermetidae and application thereof

Abstract

The application relates to an antibacterial peptide and application thereof, wherein the amino acid sequence of the antibacterial peptide NA is WVKIIKTICRWR, the antibacterial peptide is composed of 12 amino acid residues, has cation amphiphilic characteristics, and can be obtained through chemical synthesis. The antibacterial peptide has stable in-vitro bacteriostatic activity on various serotypes of avian and other animal enteropathogenic Escherichia coli (ExPEC), has good cell compatibility and blood safety in an effective bacteriostatic concentration interval, has low hemolysis rate and small cytotoxicity. The antibacterial peptide can be used for preparing veterinary antibacterial preparations, functional feed additives or animal health management related products, can replace or reduce the use of traditional antibiotics in the breeding field, provides a new technical scheme for the prevention and control of ExPEC related infections, and has important application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and veterinary medicine, specifically to an antimicrobial peptide obtained from the proteome sequence information of Hericidae animals and its application, particularly to the application of the antimicrobial peptide in the prevention and treatment of Escherichia coli-related infections in poultry and other animals. Background Technology

[0002] Extraintestinal pathogenic Escherichia coli (ExPEC) is an important group of pathogens that can cause systemic infections in hosts. It is widely found in poultry, livestock, and the environment, and can cause a variety of diseases such as septicemia, peritonitis, and respiratory infections. ExPEC exhibits significant serotype diversity, with different serotypes showing significant differences in virulence factor composition, host adaptability, and drug sensitivity, making its control increasingly difficult.

[0003] Currently, the prevention and control of ExPEC infection still mainly relies on traditional antibiotics. However, with the long-term, high-dose, or improper use of antibiotics, drug-resistant strains are constantly emerging, and some serotypes of ExPEC have shown resistance to many commonly used antibacterial drugs, leading to unstable treatment effects in clinical and aquaculture production. At the same time, antibiotic residues and the spread of drug-resistant genes also pose risks to food safety and public health.

[0004] To address these issues, antimicrobial peptides, as a class of innate immune molecules, have attracted widespread attention due to their mechanism of action differing from traditional antibiotics and their low likelihood of inducing drug resistance. Existing research indicates that most antimicrobial peptides achieve bactericidal effects through physical interactions with bacterial cell membranes, and their bactericidal process typically does not depend on specific metabolic pathways; therefore, they also have the potential to inhibit drug-resistant strains.

[0005] However, the development of existing antimicrobial peptides still faces several limitations. On the one hand, many reported antimicrobial peptides are mainly derived from insects, amphibians, or microorganisms, and their structural types and action spectra are relatively concentrated, resulting in limited activity stability and applicability when targeting different ExPEC serotypes. On the other hand, some antimicrobial peptides exhibit high cytotoxicity or hemolytic activity at effective inhibitory concentrations, limiting their promotion in the veterinary field and large-scale applications.

[0006] Previous studies have shown that snail-like mollusks contain a variety of natural peptides with antimicrobial activity or antimicrobial peptide candidate fragments. These peptides exhibit amphiphilicity and positive charge distribution in their structure, enabling them to inhibit both Gram-negative and Gram-positive bacteria. Systematic reviews also indicate that mollusc-derived antimicrobial peptides possess rich structural diversity and potential broad-spectrum antimicrobial activity, suggesting that these organisms are an important resource for developing novel antimicrobial peptides. Marine invertebrates, due to their long-term exposure to high microbial load environments, have evolved diverse defense molecular systems, gradually becoming an important potential source of novel antimicrobial peptides. Therefore, selecting marine invertebrate mollusc proteomes for peptide mining is reasonable as a natural antimicrobial peptide candidate library. As typical marine mollusks, the Hericidae family contains a large number of functional segments related to membrane interactions in their proteomes; however, systematic studies on antimicrobial peptides derived from the proteomes of these animals are still relatively limited, especially regarding the prevention and treatment of different ExPEC serotypes, which lacks clear reports.

[0007] Therefore, developing an antimicrobial peptide with novel sources, well-defined structural characteristics, and stable antimicrobial activity against multiple serotypes of ExPEC is of significant research value and practical importance for enriching the sources of antimicrobial peptides and expanding their application in the field of veterinary anti-infectives. Summary of the Invention

[0008] To address the problems of insufficient adaptability to serotype differences, limited sources of effective active substances, and narrow safety windows in the control of Gram-negative pathogenic Escherichia coli (ExPEC) by existing antibacterial agents, the present invention proposes the following technical solution.

[0009] This invention provides an antimicrobial peptide NA obtained from the proteomic sequence information of a Hericidae animal, with the following amino acid sequence: WVKIIKTICRWR. The antimicrobial peptide consists of 12 amino acid residues, containing a reasonable distribution of hydrophobic and positively charged amino acids, exhibiting an overall cationic amphiphilic characteristic. This sequence is independent of any chemical modification or tag structure; its N-terminus and C-terminus are both in the form of natural amino acid terminals.

[0010] Furthermore, the purity of the antimicrobial peptide is not less than 90%, preferably not less than 95%.

[0011] Furthermore, the antimicrobial peptide is prepared by chemical synthesis and purified by reversed-phase high-performance liquid chromatography to obtain a single peptide component.

[0012] The present invention further provides the use of the antimicrobial peptide in the preparation of antimicrobial agents that inhibit or kill pathogenic Escherichia coli.

[0013] Furthermore, the pathogenic Escherichia coli is an extraintestinal pathogenic Escherichia coli (ExPEC) of avian or other animal origin, including but not limited to pathogenic strains of different serotypes.

[0014] Under in vitro conditions, the antimicrobial peptide exhibited stable inhibitory effects against a variety of ExPEC strains within a low concentration range, and its minimum inhibitory concentration (MIC) could be determined by microbroth dilution. Experimental results showed that the antimicrobial peptide exhibited a consistent inhibitory trend among different strains, indicating that its antimicrobial activity is independent of a single serotype.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects and technical advantages: 1. This invention provides a novel antimicrobial peptide with clearly defined properties. The antimicrobial peptide NA provided by this invention is derived from the proteome of a herringbone worm, and its amino acid sequence is well-defined and its composition is stable. The purity, molecular weight, and main physicochemical parameters of this antimicrobial peptide were systematically characterized using reversed-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry, clearly defining its existence as an independent chemical substance, which is beneficial for those skilled in the art to prepare and apply it repeatedly.

[0016] Compared to existing antimicrobial peptides that are mostly derived from bacteria, insects, or artificially modified sequences, the antimicrobial peptides of this invention differ in terms of source type and sequence composition, providing a new technical path for expanding the antimicrobial peptide resource library.

[0017] 2. The antimicrobial peptide NA exhibits reproducible in vitro antibacterial effects against multiple serotypes of ExPEC. In vitro drug susceptibility testing results show that the antimicrobial peptide NA of this invention exhibits clear antimicrobial activity against multiple avian extracorporeal pathogenic Escherichia coli serotypes (including O21, O18, O8, O33, etc.). Within a certain concentration range, the antimicrobial peptide shows a clear antimicrobial trend across different serotypes, indicating that its antimicrobial activity is not dependent on a single serotype.

[0018] The results show that the antimicrobial peptide of the present invention has good applicability consistency in different serotypes of ExPEC, providing experimental basis for its further research and application as a broad-spectrum antimicrobial candidate.

[0019] 3. It exhibits acceptable biocompatibility characteristics within the effective antibacterial concentration range. In vitro evaluations on chicken fibroblast DF-1 cells and avian erythrocytes show that, within the main concentration range corresponding to the antibacterial effect, no significant adverse biological effects conflicting with its antibacterial activity were observed in the antimicrobial peptides of this invention.

[0020] Experimental results show that within a range of usage concentrations below or close to the effective antibacterial concentration, the host cell survival rate remains at a high level, and the erythrocyte hemolysis rate is in a low range, indicating that the antimicrobial peptide has the basic conditions for further research and application as an antimicrobial active ingredient under reasonable dosage control.

[0021] 4. The experimental observations are consistent with the common membrane action characteristics of antimicrobial peptides. Scanning electron microscopy results show that pathogenic E. coli cells treated with the antimicrobial peptides of this invention exhibit changes in membrane structure integrity. This phenomenon is consistent with the general understanding in existing literature that cationic antimicrobial peptides interfere with bacterial cell membrane structure through physical means. Attached Figure Description

[0022] Figure 1 This is a 3D structural simulation diagram of the antimicrobial peptide NA of the present invention.

[0023] Figure 2 This is a helical diagram of the antimicrobial peptide NA of the present invention.

[0024] Figure 3 This is a liquid chromatogram of the antimicrobial peptide NA of the present invention.

[0025] Figure 4 This is the mass spectrum of the antimicrobial peptide NA of the present invention.

[0026] Figure 5 This is a diagram of the ExPEC drug susceptibility test of NA to O21 serotype according to the present invention.

[0027] Figure 6 This is a diagram of the ExPEC drug susceptibility test of NA to O8 serotype according to the present invention.

[0028] Figure 7 This is a diagram of the ExPEC drug susceptibility test of NA to O18 serotype according to the present invention.

[0029] Figure 8 This is a diagram of the ExPEC drug susceptibility test of NA to O33 serotype according to the present invention.

[0030] Figure 9 This is a SEM image of the O21 serum type ExPEC from the normal control group of this invention.

[0031] Figure 10 This is a SEM image of the O21 serotype ExPEC after 1 hour of incubation with the antimicrobial peptide of this invention. Detailed Implementation

[0032] With the continued prevalence of Gram-negative pathogens in livestock and poultry farming and public health, especially the significant differences in drug resistance and pathogenic characteristics among different serotypes of extraintestinal pathogenic Escherichia coli (ExPEC) from poultry and humans, existing anti-infection methods have gradually revealed problems in practical applications, such as limited applicability, insufficient antibacterial stability, and difficulty in simultaneously assessing safety.

[0033] On the one hand, the sources of existing antimicrobial peptides are still mainly bacteria, insects or artificially modified sequences, and their structural types and modes of action are relatively concentrated, which makes it easy to have a narrow antimicrobial spectrum or large fluctuations in activity when dealing with different serotypes of ExPEC. On the other hand, some reported antimicrobial peptides have high hemolytic activity or host cell toxicity near the effective antimicrobial concentration, which limits their practical application in veterinary or cross-species control scenarios.

[0034] Furthermore, although recent studies have attempted to extract antimicrobial active molecules from marine organisms, most of these efforts remain at the level of natural isolation or crude extraction. A technical path has not yet been established that uses the complete proteome as a functional resource library and systematically screens and validates the application of short peptide functional segments. As a result, potentially efficient and safe antimicrobial peptide sequences have not been fully explored and utilized.

[0035] Therefore, the present invention aims to solve the following technical problems: 1. How to provide an antimicrobial peptide with a novel structural source, well-defined sequence characteristics, and stable availability to overcome the problems of existing antimicrobial peptides having a single source and severe structural homogeneity; 2. How to obtain an antimicrobial peptide that can exhibit stable antibacterial activity against multiple serotypes of ExPEC, so as to improve its broad-spectrum applicability in practical prevention and control applications; 3. How to ensure the antibacterial effect while maintaining good cell compatibility and blood safety of the antimicrobial peptide within the effective concentration range, so as to meet the safety requirements of veterinary drug preparations and related application scenarios.

[0036] To address the aforementioned technical problems, this invention has obtained an antimicrobial peptide with a clearly defined amino acid sequence by mining potential functional fragments in the proteome of the Hericidae family. The antimicrobial effect and biosafety of this peptide in different ExPEC serotypes have been systematically verified, thus providing a new solution for the existing antimicrobial peptide technology system.

[0037] (I) Material and technical solutions for antimicrobial peptides This invention provides an antimicrobial peptide NA obtained from the proteomic sequence information of a Hericidae animal, with the following amino acid sequence: WVKIIKTICRWR. The antimicrobial peptide consists of 12 amino acid residues, containing a reasonable distribution of hydrophobic and positively charged amino acids, exhibiting an overall cationic amphiphilic characteristic. This sequence is independent of any chemical modification or tag structure; its N-terminus and C-terminus are both in the form of natural amino acid terminals.

[0038] In a preferred embodiment, the antimicrobial peptide is obtained by chemical synthesis and purified into a single peptide component with a purity of not less than 90%, preferably not less than 95%.

[0039] (II) Technical Scheme for the Application of Antimicrobial Peptides in Inhibiting Pathogenic Escherichia coli The present invention further provides the use of the antimicrobial peptide in the preparation of antimicrobial agents that inhibit or kill pathogenic Escherichia coli.

[0040] In one embodiment, the pathogenic Escherichia coli is an extraintestinal pathogenic Escherichia coli (ExPEC) of avian or other animal origin, including but not limited to pathogenic strains of different serotypes.

[0041] Under in vitro conditions, the antimicrobial peptide exhibited stable inhibitory effects against a variety of ExPEC strains within a low concentration range, and its minimum inhibitory concentration (MIC) could be determined by microbroth dilution. Experimental results showed that the antimicrobial peptide exhibited a consistent inhibitory trend among different strains, indicating that its antimicrobial activity is independent of a single serotype.

[0042] (III) Technical solutions related to the biosafety of antimicrobial peptides In a preferred embodiment, the biosafety of the antimicrobial peptide is verified as follows: Regarding cell compatibility: Different concentrations of antimicrobial peptides were treated using an in vitro cultured animal cell model, and cell viability was assessed using a metabolic activity-based assay. Results showed that within the range corresponding to the effective antibacterial concentration, the antimicrobial peptides did not cause a significant decrease in cell activity, indicating good compatibility with host cells.

[0043] Regarding blood compatibility: The blood safety of the antimicrobial peptide was evaluated using a erythrocyte hemolysis test. Experimental results showed that within the effective antibacterial concentration range, the hemolysis rate induced by the antimicrobial peptide was low, and no significant blood cell destruction effect was observed.

[0044] The above results indicate that the antimicrobial peptides of the present invention, while exerting antibacterial activity, possess the basic conditions for further development and application under reasonable dosage control, and are suitable as active ingredients for the prevention and control of pathogenic Escherichia coli-related infections.

[0045] (iv) Application format description Without limiting the scope of protection, the antimicrobial peptide can be used as an effective active ingredient in antimicrobial preparations, and the specific dosage form, excipient composition and administration method of the preparation can be selected or adjusted according to actual application needs.

[0046] Summary table of main physicochemical parameters of antimicrobial peptide NA Parameter name Numerical / Description amino acid sequence WVKIIKTICRWR Sequence length 12 aa Theoretical molecular weight About 1602 Da Measured molecular weight 1601.85 Da is consistent with the theoretical value (ESI-MS, see...). Figure 4 ) Net charge (pH 7) +3.9 Theoretical isoelectric point (pI) Approximately 11.39 Average hydrophobicity index (GRAVY) 0.075 Instability Index 33.27 (Forecast: Stable) Constructive features It has a structural tendency to form amphiphilic α-helices. Example 1: Preparation and physicochemical characterization of antimicrobial peptide NA This embodiment describes the preparation process and physicochemical properties of the antimicrobial peptide NA (amino acid sequence WVKIIKTICRWR) of the present invention, in order to clarify the composition and basic characteristics of the substance.

[0047] The antimicrobial peptide was prepared by solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography after synthesis. The obtained product was a single peptide component with a purity of not less than 95%, as shown in the corresponding chromatogram. Figure 3 As shown.

[0048] Further molecular weight verification of the antimicrobial peptide was performed using liquid chromatography-mass spectrometry (LC-MS). The results showed that the measured molecular weight corresponding to the main ion peak was 1601.85 Da, which is basically consistent with the theoretical molecular weight of approximately 1602 Da (see [reference]). Figure 4 This indicates that the molecular composition of the prepared antimicrobial peptide matches the target sequence.

[0049] The main physicochemical parameters obtained based on sequence composition calculations are summarized in the table above. The results show that the antimicrobial peptide is positively charged overall under neutral conditions (net charge approximately +3.9, pI approximately 11.39), with an average hydrophobicity index (GRAVY) of 0.075 and an instability index of 33.27, classifying it as a predicted stable peptide. Combined with the three-dimensional structure simulation diagram (…), Figure 1 ) and spiral wheel diagram ( Figure 2 It can be seen that this peptide has a conformational tendency to form an amphiphilic α-helical structure.

[0050] Regarding solubility, the antimicrobial peptide exhibits good solubility in PBS buffer, but slight self-aggregation may occur at higher concentrations. This characteristic has been taken into account in subsequent drug sensitivity tests and does not affect the determination of its antibacterial effect.

[0051] The antimicrobial peptides described in this invention are not obtained by direct isolation of natural peptides from the body of the Hericidae family, but rather by short peptide sequences obtained by calculating, predicting, and screening potentially antimicrobial functional fragments based on the proteome sequence data of the Hericidae family.

[0052] Given that antimicrobial peptides typically correspond to local functional regions in the parent protein, and that such short peptide sequences lack significant conservation across different species, and that the proteome annotation information for the Hericidae family is currently incomplete, it is difficult to use existing public databases to uniquely and inversely locate the parent protein for this short peptide sequence.

[0053] The above situation is a common technical phenomenon in the research of antimicrobial peptides based on proteome sequence mining, and does not affect the certainty, reproducibility and experimental verification results of the antimicrobial peptide sequence of the present invention and its antimicrobial activity.

[0054] Example 2: Evaluation of the in vitro antibacterial activity of antimicrobial peptide NA against different serotypes of ExPEC To evaluate the antimicrobial activity of the antimicrobial peptides of this invention against various avian ExPEC serotypes, the MIC was determined using the microbroth dilution method. ExPEC strains O21, O18, O8, O32, and O33 cultured in logarithmic phase were diluted to 0.5 McFarland solution, and then further diluted 1:1000 to prepare test bacterial solutions. The antimicrobial peptide working solution was serially diluted 2-fold to 2000–15.63 μg / mL, and 50 μL was added to sterile 96-well microplates (50 μL per well), mixed with 100 μL TSB and 50 μL of test bacterial solution, and incubated at 37 °C for 18–24 h. The inhibition rate was calculated using the formula: .

[0055] Table 1. Inhibition rate of NA on ExPEC in O21 serotype Concentration (μg / mL) 2000 1000 500 250 125 62.5 31.25 15.63 Normal control Blank control NA1 0.567 0.274 0.175 0.072 0.033 0.007 0.022 1.024 1.144 0.123 NA2 0.554 0.286 0.148 0.101 0.027 0.016 0.017 1.019 1.153 0.117 NA3 0.467 0.306 0.154 0.127 0.033 0.026 0.948 1.077 1.142 0.115 OD600 average value 0.530 0.288 0.159 0.100 0.031 0.016 0.329 1.040 1.146 0.118 Inhibition rate % 48.48 71.94 84.55 90.24 96.98 98.41 68.00 -1.17 Table 2. Inhibition rate of NA on ExPEC in O8 serotype Concentration (μg / mL) 250 125 62.5 31.25 15.63 7.81 Normal control Blank control NA1 0.167 0.154 0.990 1.070 1.104 1.073 1.048 0.130 NA2 0.172 0.195 1.016 1.077 1.094 1.078 1.074 0.130 NA3 0.182 0.404 1.006 1.089 1.096 1.071 1.083 0.133 OD600 average value 0.174 0.251 1.004 1.079 1.098 1.074 1.068 0.131 Inhibition rate % 95.44 87.22 6.87 -1.09 -3.14 -0.57 Table 3. Inhibition rate of NA on ExPEC in O18 serotype Concentration (μg / mL) 250 125 62.5 31.25 15.63 7.81 Normal control Blank control NA1 0.187 0.134 0.103 0.932 0.971 1.007 0.821 0.072 NA2 0.260 0.142 0.107 0.797 0.953 0.991 0.856 0.075 NA3 0.228 0.140 0.094 0.949 0.973 0.973 0.866 0.079 OD600 average value 0.225 0.139 0.101 0.893 0.966 0.990 0.848 0.075 Inhibition rate % 80.59 91.77 96.65 -5.82 -15.27 -18.43 Table 4. Inhibition rate of NA on ExPEC in O33 serotype Concentration (μg / mL) 250 125 62.5 31.25 15.63 7.81 Normal control Blank control NA1 0.217 0.262 0.160 0.145 0.136 1.081 1.028 0.128 NA2 0.241 0.165 0.163 0.136 0.973 1.049 1.019 0.126 NA3 0.228 0.171 0.144 0.140 1.065 1.029 1.026 0.118 OD600 average value 0.228 0.199 0.156 0.141 0.725 1.053 1.024 0.124 Inhibition rate % 88.41 91.66 96.48 98.16 33.29 -3.18 The experimental results (Tables 1–4) showed that the O21 serotype exhibited ≥ 95% inhibition at concentrations of 31.25–62.5 μg / mL; the O8 serotype exhibited ≥ 95% inhibition at 250 μg / mL; the O18 serotype exhibited ≥ 95% inhibition at 62.5 μg / mL; and the O33 serotype exhibited ≥ 95% inhibition at concentrations of 31.25–62.5 μg / mL.

[0056] Figure 5The orange boxes highlight NA1-NA3, representing three replicates of drug sensitivity testing. The concentrations from left to right are 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL. The blue boxes highlight the normal control group, and the green boxes highlight the blank control group. Figure 6 The orange boxes highlight NA1-NA3, representing three replicates of drug sensitivity testing. The concentrations from left to right are 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL, respectively. The blue boxes highlight the normal control group, and the green boxes highlight the blank control group. Figure 7 The orange boxes highlight NA1-NA3, representing three replicates of drug sensitivity testing. The concentrations from left to right are 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL, respectively. The blue boxes highlight the normal control group, and the green boxes highlight the blank control group. Figure 8 The orange boxes highlight NA1-NA3, representing three replicates of drug sensitivity testing. The concentrations from left to right are 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL, respectively. The blue boxes highlight the normal control group, and the green boxes highlight the blank control group.

[0057] Figure 5-8 It was found that the O33 serotype of ExPEC was most sensitive to NA (MIC 31.25 μg / mL), followed by the O21 and O18 serotypes (MIC 62.5 μg / mL), with the O8 serotype having the highest MIC of 250 μg / mL. The overall trend indicates that this peptide exhibits low MIC values ​​and robust antibacterial activity across most serotypes.

[0058] It is worth noting that a high antibacterial effect was observed in some serotypes O21, O18 and O33 at lower concentrations. This phenomenon has been reported in antimicrobial peptide research and does not affect the overall technical effect of the antimicrobial peptide of the present invention, which has clear antibacterial activity in different serotypes.

[0059] Example 3: Evaluation of the cell compatibility and hemolytic properties of the antimicrobial peptide NA To assess the safety of the antimicrobial peptide NA in biological applications, this embodiment tests its cell compatibility and blood compatibility.

[0060] (1) Evaluation of cell compatibility Chicken fibroblast DF-1 cells (Shanghai Siger Biotechnology) were selected as the model cells. Cells were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic growth phase, then digested, counted, and their density adjusted to 3 × 10⁻⁶ cells / year. 4 Cells (cells / mL) were seeded into 96-well plates and allowed to adhere fully. Different concentrations of antimicrobial peptide NA solution were then added to each well, with corresponding control groups. After a certain treatment time, cell viability was assessed using the MTT assay, and cell viability was calculated. Cell viability was calculated using the following formula:

[0061] Table 5. Cytotoxicity of NA against chicken fibroblast DF-1 cells. Concentration (μg / mL) 312.50 156.25 78.13 39.06 19.53 9.77 4.88 2.44 Normal control Blank control OD490 0.131 0.469 0.648 0.691 0.767 0.861 0.919 0.984 0.966 0.054 0.139 0.364 0.676 0.674 0.767 0.882 0.947 1.001 0.974 0.053 0.134 0.413 0.663 0.793 0.757 0.845 0.934 1.012 0.945 0.052 Cell survival rate % 9.26 32.98 61.76 72.31 76.00 87.88 97.81 102.06 The experimental results are shown in Table 5. At concentrations of 39.06 μg / mL and below, cell viability was generally higher than 70%. At concentrations of 9.77 μg / mL and below, cell viability was maintained above 87%, and no significant inhibition of cell activity was observed.

[0062] (2) Evaluation of hemolysis Fresh poultry blood was collected, and after centrifugation to remove plasma, red blood cells were washed with PBS to prepare a red blood cell suspension of a specific concentration (4% poultry red blood cell suspension). Antimicrobial peptide NA was mixed with the red blood cell suspension at different concentration gradients, incubated at 37 ℃, centrifuged, and the supernatant was collected to measure absorbance and calculate hemolysis rate. The Triton-X-100 treatment group served as a positive control, and the PBS treatment group served as a negative control.

[0063] Hemolysis rate is calculated using the following formula:

[0064] Table 6. Results of the hemolytic effect of NA on duck blood cells. Concentration (μg / mL) 2000 1000 500 250 125 62.5 31.25 15.63 Positive control negative control OD540 0.454 0.299 0.205 0.238 0.170 0.128 0.134 0.127 0.979 0.110 0.927 0.110 0.966 0.110 OD average value 0.957 0.110 hemolysis rate % 40.63 22.25 11.17 15.10 7.11 2.14 2.78 2.00 The results are shown in Table 6. The hemolysis rate was less than 3% in the concentration range of 62.5 μg / mL and below; even at 500 μg / mL, the hemolysis rate remained below 20%, indicating that the antimicrobial peptide has good blood compatibility within the effective antibacterial concentration range.

[0065] Example 4: Observation on the effect of antimicrobial peptide NA on ExPEC cell morphology This embodiment is used to observe the effect of antimicrobial peptide NA on bacterial cell structure from a morphological perspective.

[0066] To clarify the disruptive effect of the antimicrobial peptide of this invention on bacterial membranes, SEM analysis was used to examine the morphological changes of the ExPECO21 serotype after treatment with the antimicrobial peptide. The logarithmic-phase bacterial culture was adjusted to OD600 ≈ 0.1–0.2, co-incubated with a 1×MIC concentration of peptide for 1 h, followed by fixation with 2.5% glutaraldehyde, gradient dehydration with ethanol, and gold sputtering. SEM observations (see Figures 9 and 10) showed that the untreated control group bacteria had intact surface structures and regular morphology; while the bacterial cells treated with the antimicrobial peptide NA exhibited localized collapse and structural discontinuities, suggesting that the antimicrobial peptide can significantly affect bacterial cell structure.

[0067] Based on the sequence conformation features described in Example 1, it can be inferred that the antimicrobial peptide NA may achieve its antibacterial effect by physically interacting with the bacterial cell membrane and disrupting the membrane structure integrity. The above description is only for reasonable interpretation of experimental observations and does not constitute a limitation on the mechanism of action of the antimicrobial peptide of this invention.

[0068] As can be seen from the above embodiments, the antimicrobial peptide NA provided by the present invention has the characteristics of novel origin, well-defined sequence, and clear physicochemical properties, and exhibits stable antibacterial activity against multiple serotypes of ExPEC under in vitro conditions. Furthermore, within the main concentration range corresponding to its antibacterial effect, this antimicrobial peptide exhibits acceptable cell compatibility and blood compatibility characteristics, demonstrating its potential as an antimicrobial active ingredient in the prevention and control of pathogenic Escherichia coli.

Claims

1. An antimicrobial peptide obtained by mining proteomic sequence information of Hericaria species, characterized in that, Its amino acid sequence is: WVKIIKTICRWR.

2. The antimicrobial peptide according to claim 1, characterized in that, The purity of the antimicrobial peptide is not less than 90%.

3. The antimicrobial peptide according to claim 1, characterized in that, The purity of the antimicrobial peptide is not less than 95%.

4. The antimicrobial peptide according to claim 1 or 2, characterized in that, The antimicrobial peptide was prepared by chemical synthesis and purified by reversed-phase high-performance liquid chromatography to obtain a single peptide component.

5. The use of the antimicrobial peptide according to any one of claims 1-4 in the preparation of antimicrobial agents that inhibit or kill pathogenic Escherichia coli.

6. The application according to claim 5, characterized in that, The pathogenic Escherichia coli is extraintestinal pathogenic Escherichia coli, including avian or other animal-derived strains of serotypes O21, O18, O8, and O33.

7. The application according to claim 5, characterized in that, The antibacterial agent is a veterinary antibacterial agent, a functional feed additive, or an animal health management-related product.

8. The application according to any one of claims 4-6, characterized in that, The concentration of the antimicrobial peptide used in the formulation is 31.25 μg / mL to 2000 μg / mL.

Images