Antibacterial peptide and application thereof as medicine and feed additive

Abstract

The invention discloses an antibacterial peptide and application thereof as a medicine and a feed additive. The antibacterial peptide is obtained by modifying an amino acid sequence as shown in SEQ ID No: 1. A potential antibacterial peptide sequence is excavated from channel catfish proteome, and a novel antibacterial peptide which has broad-spectrum antibacterial activity and particularly has a remarkable inhibition effect on APEC is obtained through structural modification and optimization. The antibacterial peptide can effectively inhibit the growth of various drug-resistant APEC strains in vitro, shows good safety in cytotoxicity and red blood cell hemolysis experiments, and can be used as a powerful supplement for traditional antibiotic and vaccine prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of antimicrobial peptide bioengineering and biological control of poultry diseases, specifically to antimicrobial peptides and their applications as drugs and feed additives. Background Technology

[0002] In the poultry farming sector, avian pathogenic Escherichia coli (Avian Pathogenic Escherichia coli) Escherichia coli Infectious diseases such as septicemia and peritonitis caused by APEC (Animal Crossings and Infectious Diseases) are widespread and are exacerbated by antibiotic resistance, becoming a significant factor affecting the healthy development of the industry. National animal-derived bacterial resistance monitoring results show that common pathogens such as Escherichia coli exhibit high resistance rates and multidrug resistance (MDR) in livestock and poultry. Studies generally agree that antibiotic resistance is a prominent issue in APEC, particularly high resistance to fluoroquinolones and β-lactam antibiotics. This indicates an urgent need to explore new and more efficient antibiotic alternatives in waterfowl farming.

[0003] Currently, commonly used antibacterial alternatives mainly include probiotics, organic acids, and plant extracts, but their overall antibacterial activity is limited and they are difficult to produce ideal inhibitory effects against specific pathogens such as APEC.

[0004] Furthermore, in the current situation of the poultry industry, the following problems are common: First, APEC's resistance to traditional antibiotics is increasing, and multidrug-resistant (MDR) strains are widespread, leading to a significant decline in the effectiveness of drug treatment; Second, the prevalent serotypes in APEC are complex and variable, and existing inactivated or recombinant vaccines often only target a limited number of serotypes, making it difficult to cover new prevalent strains in a timely manner, resulting in unstable immune protection and unsatisfactory clinical prevention and control effects.

[0005] Therefore, providing a novel, highly efficient, safe, and targeted antimicrobial peptide against major drug-resistant pathogens such as APEC is a problem that this invention urgently needs to solve. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to overcome the technical difficulties in the prevention and control of APEC in the prior art, and to provide a novel antimicrobial peptide with broad-spectrum antibacterial activity, especially with a significant inhibitory effect on APEC, and its application as a drug and feed additive.

[0007] To achieve the above objectives, the present invention provides an antimicrobial peptide obtained by modifying the amino acid sequence shown in SEQ ID No:1.

[0008] In this invention, the antimicrobial peptide is derived from the spotted catfish.

[0009] The modification methods described here can be implemented in a manner that is understandable and conventionally used by those skilled in the art. For example, in the specific operation of this invention, in order to improve antibacterial activity and stability, the amino acid sequence shown in SEQ ID No:1 (specifically, its sequence is KGSMRAVAKNKRKRNMKAKKGRSKSCKCRKKGC) is modified to obtain an antimicrobial peptide with an amino acid sequence shown in SEQ ID No:2 (specifically, its sequence is AKWKVKLSLKAKKGRSFWAK).

[0010] Preferably, the optimized antimicrobial peptide has a net charge of +8, a hydrophobicity index of 0.35–0.40, and exhibits a typical amphiphilic α-helical conformation.

[0011] The present invention also provides a drug for preventing and controlling pathogenic Escherichia coli in birds, the drug comprising the antimicrobial peptides described above.

[0012] Preferably, the drug is used for serotypes O8, O18, O21, O32, and O33 of pathogenic Escherichia coli in birds.

[0013] The present invention also provides a feed additive for the prevention and control of pathogenic Escherichia coli in poultry, the feed additive comprising the antimicrobial peptides described above.

[0014] Through the above technical solution, this invention mines potential antimicrobial peptide sequences from the proteome of the channel catfish and, through structural modification and optimization, obtains a novel antimicrobial peptide with broad-spectrum antimicrobial activity, particularly showing significant inhibitory effects against APEC. This antimicrobial peptide can effectively inhibit the growth of various drug-resistant APEC strains in vitro and exhibits good safety in cytotoxicity and erythrocyte hemolysis experiments, making it a powerful supplement to traditional antibiotics and vaccines for prevention and control. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a helical diagram of the antimicrobial peptide provided by the present invention; Figure 2 This is a three-dimensional spatial structure simulation diagram of the antimicrobial peptide provided by the present invention; Figure 3 This is a liquid chromatogram of the antimicrobial peptide provided by the present invention; Figure 4 This is the mass spectrum of the antimicrobial peptide provided by the present invention; Figure 5 This is a graph showing the detection results of the antimicrobial effect of the antimicrobial peptide against APEC O21 serotype in Verification Example 1 of this invention; Figure 6 This is a graph showing the detection results of the antimicrobial effect of the antimicrobial peptide on APEC O8 serotype in Verification Example 2 of this invention; Figure 7 This is a graph showing the detection results of the antimicrobial effect of the antimicrobial peptide on APEC O18 serotype in Example 3 of the present invention; Figure 8 This is a graph showing the detection results of the antimicrobial effect of the antimicrobial peptide on APEC O32 serotype in Example 4 of this invention; Figure 9 This is a graph showing the detection results of the antimicrobial effect of the antimicrobial peptide on APEC O33 serotype in Example 5 of this invention; Figure 10 This is a SEM image of the control APEC cells in Example 3 of this invention; Figure 11 This is a SEM image of APEC after incubation with the antimicrobial peptide for 1 hour in Example 3 of this invention. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0017] The antimicrobial peptides used in the following examples are antimicrobial peptides with the amino acid sequence shown in SEQ ID No:2, prepared by Sangon Biotech (Shanghai) Co., Ltd. using a solid-phase peptide synthesis method (Fmoc strategy), and purified by desalting and lyophilized. The purity was ≥95% as determined by HPLC, and the molecular weight was consistent with the theoretical value as verified by mass spectrometry.

[0018] Chicken fibroblast cell line (DF-1) or chicken embryo fibroblasts were commercially available products from Siger Biosciences; the test strains were provided by the Jiangsu Provincial Key Laboratory of Veterinary Biopharmaceutical High Technology Research. The aforementioned test strains have been publicly used in existing literature, for example, [1] Sun Fan, Su Yuhang, Dong Hongyan, et al. Distribution, drug resistance and transmission characteristics of rmtB gene, 16S rRNA methyltransferase in duck-derived Escherichia coli [J]. Animal Husbandry and Veterinary Medicine, 2025, 57 (10): 76-83. [2] Guo Changming, Yuan Cheng, Feng Qi, et al. Isolation, identification and biological characteristics analysis of pathogenic Escherichia coli from geese in some areas of Jiangsu Province from 2021 to 2023 [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2025, 52(10):4989-4999. DOI:10.16431 / j.cnki.1671-7236.2025.10.043. [3] Gu Chenyi, Dong Hongyan, Guo Changming, et al. Investigation and analysis of the prevalence of major bacterial diseases in waterfowl in Jiangsu Province [J]. Modern Animal Husbandry and Veterinary Medicine, 2023, (09): 61-64. DOI: 10.20154 / j.cnki.issn1672-9692.2023.09.014. [4] Guo Changming, Chen Huaijun, Yuan Cheng, et al. Isolation, identification and biological characteristics of extraintestinal pathogenic Escherichia coli in waterfowl in some areas of Jiangsu Province [J]. Chinese Journal of Animal Infectious Diseases, 2023, 31(03):25-33. DOI:10.19958 / j.cnki.cn31-2031 / s.2023.03.024. [5] Guo Changming, Wu Zhi, Zhu Shanyuan, et al. Study on serotypes, virulence factors and drug resistance of pathogenic Escherichia coli in ducks in Jiangsu and surrounding areas [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2020, 47(12):4076-4084. DOI:10.16431 / j.cnki.1671-7236.2020.12.032.

[0019] The strains used in the aforementioned published documents are the same as the test strains used in this invention. Unless otherwise specified, the reagents used are conventional commercially available products that are understood and used in the art.

[0020] In specific embodiments, the various verification or testing tests of the present invention are performed as follows.

[0021] The validation experiment for the antimicrobial activity against multiple APEC serotypes was conducted as follows: 2 mg of the synthesized antimicrobial peptide was centrifuged at 12000 rpm for 1 min and dissolved in 250 μL of 1×PBS (pH 7.4) that had been sterilized by 0.25 μm filtration as the test stock solution. The bacterial culture in the logarithmic growth phase was first adjusted to a concentration of 0.5 Mc units using sterile TSB medium, and then diluted 1000 times to obtain the test bacterial solution. A sterile, disposable 96-well cell culture plate was placed in a biosafety cabinet. 50 μL of test solution (i.e., the test stock solution or the solution obtained after sterilization) was prepared in each well using a multi-channel pipette. The test stock solution was serially diluted (the highest concentration of the test solution was 8 mg / mL, and subsequent concentrations were diluted 2-fold with PBS). Then, 100 μL of sterile TSB medium was added to each well using a multi-channel pipette, and finally 50 μL of test bacterial solution was added to each well. The normal control well contained 50 μL of PBS + 50 μL of test bacterial solution + 100 μL of TSB, and the blank control contained 50 μL of PBS + 150 μL of TSB. Each well was replicated in triplicate. The prepared 96-well plate was placed in a microplate reader and incubated at 37°C with low-speed shaking for 24 h. The OD600 was measured, and the antimicrobial peptide antibacterial effect and MIC were calculated according to the following formula.

[0022]

[0023] The cytotoxicity of antimicrobial peptides to avian cells was detected using the MTT assay. The specific steps were as follows: Chicken fibroblast cell line (DF-1) or chicken embryo fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator until the logarithmic growth phase was reached. After the cells reached a suitable density, they were digested with trypsin and resuspended in fresh medium to prepare a homogeneous cell suspension. The concentration of the cell suspension was adjusted to approximately 3 × 10⁻⁶ cells / mL. 4 Cells / mL: Take a disposable sterile 96-well cell culture plate and add 180 μL of cell suspension to each well in a biosafety cabinet to achieve a cell density of approximately 3000–10000 cells / well. Incubate at 37°C and 5% CO2 for 6–12 hours to allow cells to adhere fully. Take the antimicrobial peptide stock solution (i.e., the previously prepared test stock solution, concentration 8 mg / mL) and perform serial dilutions of 2-fold with PBS as the solvent to prepare antimicrobial peptide working solutions of different concentrations. Aspirate the original culture medium from the wells and add 180 μL of fresh culture medium and 20 μL of the corresponding concentration of antimicrobial peptide working solution to each well. Add the same volume of PBS to the control wells. Blank: Add culture medium only to each well, with 3 parallel wells per group; incubate the 96-well plate at 37℃ in a 5% CO2 incubator for 24 hours; after incubation, carefully discard the supernatant, add 90 μL of fresh culture medium to each well, followed by 10 μL of 5 mg / mL MTT solution (yellow), and continue incubation for 4 hours in the dark; after incubation, carefully aspirate the supernatant, add 110 μL of Formazan crystal dissolving solution (DMSO or special dissolving solution) to each well, and shake on a shaker at low speed for 10 minutes to fully dissolve the purple crystals; measure the absorbance (OD) of each well at 490 nm using a microplate reader. 490 Cell viability is calculated using the following formula, where OD... treatment OD measured for experimental wells (cells + antimicrobial peptides) 490 Value, OD blank OD measured in blank wells (culture medium + MTT, cell-free) 490 Value, OD control OD measured for control wells (cells + culture medium) 490 value.

[0024]

[0025] The hemolytic activity of duck erythrocytes was assessed using the following method: 5 mL of fresh duck blood was collected using a disposable sterile anticoagulant blood collection tube and transferred to a sterile conical flask. The blood was then stirred for 10 minutes with a sterile spreader to remove fibrin. 3 mL of blood was transferred to a sterile 50 mL centrifuge tube, and 30 mL of sterile physiological saline was added. After mixing, the mixture was centrifuged at 1500 rpm for 15 minutes, and the supernatant was discarded. Washing was repeated 2-3 times until the supernatant was colorless. The resulting erythrocytes were prepared into a 4% suspension using 0.9% sodium oxide solution for testing. 100 μL of the antimicrobial peptide test stock solution (the aforementioned prepared test stock solution) was added to a 96-well plate pre-filled with 100 μL of sterile PBS. The solution was serially diluted 2-fold using a multi-stage pipette to prepare the test solution. A positive control was prepared using 4% sterile PBS. Triton-X-100 (100 μL / well), negative control was sterile PBS (100 μL / well); add 100 μL of red blood cell suspension (i.e., the 4% suspension prepared above) to each well, and incubate at 37°C with low-speed constant temperature shaking for 1 h in an ELISA reader; after incubation, centrifuge at 1000g for 3 min; carefully aspirate 100 μL of supernatant into a new 96-well plate and detect OD540nm.

[0026]

[0027] The effects of antimicrobial peptides on bacterial morphology (in this invention, the focus is on observing the membrane-penetrating effect of APEC) were observed using scanning electron microscopy. The specific procedure was as follows: the test strain (specifically APEC O21 serotype) was taken and cultured at 37°C to the logarithmic growth phase. The bacterial culture was centrifuged at 1000 r / min for 10 min, the supernatant was discarded, and the culture was washed twice with sterile PBS buffer and resuspended at OD200. 600 =0.1–0.2; Take the antimicrobial peptide stock solution (i.e., the test stock solution prepared above) and adjust it to a concentration of 1×MIC. Mix it with an equal volume of the bacterial solution and incubate at 37℃ for 1 h. Use the untreated bacterial solution as a control. After incubation, centrifuge each group of samples at 5000 r / min for 5 min, discard the supernatant, collect the bacterial precipitate, and wash it three times with PBS to remove residual antimicrobial peptides. Add the washed bacterial cells to 2.5% glutaraldehyde fixative and fix overnight at 4℃. Then, dehydrate them sequentially with 50%, 70%, 90%, and 100% ethanol gradients, treating each concentration for 10 min. After dehydration, place the sample in a mixture of 100% ethanol and tert-butanol (1:1, v / v) for 30 min, and then further dry the sample with pure tert-butanol. Place the dried bacterial cells on the sample stage, sputter-coated with gold, observe the changes in bacterial surface morphology under a scanning electron microscope, and record the micrographs.

[0028] Furthermore, the helical diagram of the antimicrobial peptide of the present invention (hereinafter referred to as antimicrobial peptide LP or LP) is as follows: Figure 1 As shown, the three-dimensional spatial structure simulation diagram is as follows: Figure 2 As shown, its liquid chromatogram is as follows: Figure 3 As shown, the mass spectrum is as follows Figure 4 As shown.

[0029] in, Figure 1 The spatial distribution of amino acid residues in the peptide chain under the α-helical conformation is shown. Hydrophobic residues (yellow) are mainly distributed on one side of the helix, while positively charged basic residues (red) are concentrated on the other side, forming a typical amphipathic α-helix structure. This structure facilitates the effective insertion and binding of peptide molecules into the hydrophobic and hydrophilic regions of the cell membrane, thereby enhancing their ability to penetrate and disrupt bacterial membranes, explaining the basis of the peptide's excellent antibacterial activity.

[0030] Figure 2 The left side of the image shows the spatial structure model of the peptide chain, revealing that the peptide has a regular single α-helix as its main secondary structure. The color intensity on the right side represents the model confidence level (pLDDT value), with an overall blue color (>90), indicating high prediction accuracy and a stable and reliable spatial conformation. This result further validates that the designed peptide has a typical amphipathic helical configuration, consistent with the structural characteristics of natural antimicrobial peptides, providing a structural basis for its transmembrane disruption of bacterial cell membranes. Verification Example 1: Antibacterial effect against APEC O21 serotype

[0031] Test results as follows Figure 5 As shown in Table 1, the MIC of LP was 62.5 μg / mL; within the concentration range of 62.5–1000 μg / mL, the color of the bacterial solution in the wells became significantly lighter, and the OD... 600 The value was significantly lower than that of the normal control well, indicating that bacterial growth was significantly inhibited; when the concentration dropped to below 62.5 μg / mL, the antibacterial effect gradually weakened, and the turbidity in the well was close to the level of the normal control.

[0032] Figure 5 In the diagram, the blue boxes highlight LP1-LP3, representing three replicate drug sensitivity tests. The concentrations from left to right are 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.63 μg / mL. The orange boxes highlight the normal control group, and the light green boxes highlight the blank control group. In other words, the three rows from top to bottom represent three replicate tests, with each row representing one experiment. The blue boxes highlight the antimicrobial peptides used in this invention, the orange boxes highlight the normal control group, and the green boxes highlight the blank control group. This labeling method is also used in the following verification examples.

[0033] Table 1 Concentration (μg / mL) LP1 LP2 LP3 <![CDATA[OD 600 Average value Inhibition rate % 1000 0.1496 0.1481 0.155 0.1509 99.36 500 0.1358 0.146 0.1447 0.1421 100.24 250 0.1381 0.1417 0.1379 0.1392 100.54 125 0.1375 0.1412 0.141 0.1399 100.47 62.5 0.1436 0.1405 0.1408 0.1416 100.3 31.25 1.1589 1.1721 1.0963 1.1424 -0.91 15.63 1.1846 1.1666 1.1713 1.1741 -4.11 Normal control 1.1192 1.1487 1.1324 1.1334 Blank control 0.1429 0.146 0.1449 0.1446 Verification Example 2: Antibacterial effect against APEC O8 serotype

[0034] Test results as follows Figure 6 As shown in Table 2, the MIC of LP was <7.82 μg / mL; within the concentration range of 7.82–250 μg / mL, the color of the bacterial solution in the wells became significantly lighter, and the OD... 600 The value was significantly lower than that of the normal control well, indicating that bacterial growth was significantly inhibited.

[0035] Similarly, Figure 6 In the diagram, the blue boxes indicate the drug sensitivity replicates for three groups, with concentrations from left to right of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL. The orange boxes indicate the normal control group, and the light green boxes indicate the blank control group.

[0036] Table 2 Concentration (μg / mL) LP1 LP2 LP3 <![CDATA[OD 600 Average value Inhibition rate % 250 0.1135 0.1214 0.1234 0.1194 101.24 125 0.1173 0.1229 0.1218 0.1206 101.10 62.5 0.1156 0.1253 0.1234 0.1214 101.02 31.25 0.1166 0.1204 0.1181 0.1183 101.35 15.63 0.1246 0.1279 0.1199 0.1241 100.73 7.81 0.1211 0.1224 0.1191 0.1208 101.08 Normal control 1.0483 1.0736 1.0833 1.0684 Blank control 0.1297 0.1304 0.1331 0.1310 Verification Example 3: Antibacterial effect against APEC O18 serotype

[0037] Test results as follows Figure 7 As shown in Table 3, the MIC of LP was 15.63 μg / mL; within the concentration range of 15.63–250 μg / mL, the color of the bacterial solution in the wells became significantly lighter, and the OD... 600 The value was significantly lower than that of the normal control well, indicating that bacterial growth was significantly inhibited.

[0038] Similarly, Figure 7 In the diagram, the blue boxes indicate the drug sensitivity replicates for three groups, with concentrations from left to right of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL. The orange boxes indicate the normal control group, and the light green boxes indicate the blank control group.

[0039] Table 3 Concentration (μg / mL) LP1 LP2 LP3 <![CDATA[OD 600 Average value Inhibition rate % 250 0.0734 0.0836 0.0813 0.0794 99.44 125 0.0746 0.0809 0.08 0.0785 99.56 62.5 0.0739 0.0793 0.0785 0.0772 99.72 31.25 0.0729 0.0788 0.0778 0.0765 99.82 15.63 0.074 0.0762 0.0762 0.0755 99.95 7.81 0.7753 0.7965 0.825 0.7989 6.31 Normal control 0.8206 0.8563 0.8662 0.8477 Blank control 0.0723 0.0745 0.0785 0.0751 Verification Example 4: Antibacterial effect against APEC O32 serotype

[0040] Test results as follows Figure 8 As shown, the MIC of LP was 15.63 μg / mL; within the concentration range of 15.63–250 μg / mL, the color of the bacterial solution in the wells became significantly lighter, and the OD... 600 The value was significantly lower than that of the normal control well, indicating that bacterial growth was significantly inhibited.

[0041] Similarly, Figure 8In the diagram, the blue boxes indicate the drug sensitivity replicates for three groups, with concentrations from left to right of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL. The orange boxes indicate the normal control group, and the light green boxes indicate the blank control group.

[0042] Verification Example 5: Antibacterial effect against APEC O33 serotype Test results as follows Figure 9 As shown in Table 4, the MIC of LP was 15.63 μg / mL; within the concentration range of 15.63–250 μg / mL, the color of the bacterial solution in the wells became significantly lighter, and the OD... 600 The value was significantly lower than that of the normal control well, indicating that bacterial growth was significantly inhibited.

[0043] Similarly, Figure 9 In the diagram, the blue boxes indicate the drug sensitivity replicates for three groups, with concentrations from left to right of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, and 7.81 μg / mL. The orange boxes indicate the normal control group, and the light green boxes indicate the blank control group.

[0044] Table 4 Concentration (μg / mL) LP1 LP2 LP3 OD600 average value Inhibition rate % 250 0.1417 0.1235 0.1263 0.1305 99.28 125 0.122 0.1253 0.1286 0.1253 99.86 62.5 0.1272 0.1249 0.1259 0.126 99.78 31.25 0.1183 0.1267 0.1277 0.1242 99.98 15.63 0.1167 0.1219 0.1249 0.1211 100.32 7.81 0.9575 1.0027 0.9969 0.9857 4.28 Normal control 1.0278 1.0194 1.0256 1.0242 Blank control 0.1281 0.1263 0.1178 0.1240 Example 1: Cytotoxicity against DF-1 in chicken fibroblasts

[0045] The test results are shown in Table 5. It can be seen that the cell viability of the antimicrobial peptide of the present invention remained above 90% in the range of 4.88–625 μg / mL, and reached 103.83% at the highest concentration (625 μg / mL). No obvious dose-dependent decreasing trend was observed, indicating that the antimicrobial peptide has good biocompatibility and low cytotoxicity to avian cells within the effective antimicrobial concentration range.

[0046] Among them, OD 490 -1、OD 490 -2 and OD 490 -3 corresponds to three sets of parallel experiments.

[0047] Table 5 Concentration (μg / mL) <![CDATA[OD 490 -1]]> <![CDATA[OD 490 -2]]> <![CDATA[OD 490 -3]]> Cell survival rate % 625.00 2.785 2.4542 2.7154 103.83 312.50 2.7282 2.5063 2.3946 96.97 156.25 2.8396 2.8961 2.1787 102.67 78.13 2.2797 2.705 2.4187 90.05 39.06 2.6583 2.1938 2.9756 96.80 19.53 2.0405 2.2973 2.5504 92.85 9.77 2.1485 2.6075 2.6351 97.24 4.88 2.2458 2.6176 2.2335 96.37 Normal control 2.7282 2.5063 2.3946 Blank control 0.0556 0.0556 0.0556 Test Case 2, Hemolytic Safety Assessment

[0048] The hemolytic safety test of the antimicrobial peptide of this invention was conducted in only one group, with three parallel tests each for the negative and positive controls. The results are shown in Table 6. It can be seen that the hemolysis rate was 18.95% at the highest concentration of 500 μg / mL, and the hemolysis rate decreased significantly as the concentration decreased. Below 62.5 μg / mL, the hemolysis rate was consistently below 5%, specifically 2.01% at 31.25 μg / mL, 1.32% at 15.63 μg / mL, and only 0.39% at 7.81 μg / mL. These results indicate that the antimicrobial peptide of this invention has almost no hemolytic effect on avian erythrocytes within the effective antibacterial concentration range (16–32 μg / mL), demonstrating good biocompatibility and safety.

[0049] Table 6 Concentration (μg / mL) <![CDATA[OD 540 ]]> OD average value hemolysis rate % 500 0.2661 18.95 250 0.2071 11.74 125 0.1732 7.59 62.5 0.1464 4.31 31.25 0.1276 2.01 15.63 0.122 1.32 7.81 0.1144 0.39 Positive control 0.9002 0.9395 0.9458 0.9285 negative control 0.1138 0.1092 0.1104 0.1111 Example 3: Observation of the membrane penetration effect on APEC

[0050] Observation results as follows Figure 10 and Figure 11 As shown, where, Figure 10 SEM images of the control group. Figure 11 SEM image of APEC after 1 hour of incubation with antimicrobial peptide.

[0051] like Figure 10 As shown, the untreated normal control bacteria are intact, smooth, and dense in structure, without obvious breakage or collapse, indicating that the bacteria are in a normal physiological state. Figure 11 As shown, the bacterial cells treated with the antimicrobial peptide exhibit significant shrinkage, depression, and rupture on the cell surface. Some cells show severe deformation or even dissolution, with blurred cell outlines, indicating that the antimicrobial peptide caused significant damage to the cell membrane. These results demonstrate that the antimicrobial peptide can directly act on the APEC cell membrane, causing loss of membrane structural integrity, leading to intracellular leakage and cell dissolution, thereby achieving its bactericidal mechanism. These results verify that the antimicrobial peptide possesses typical membrane-penetrating antimicrobial characteristics, providing morphological evidence for its application as a novel anti-infective agent.

[0052] As shown in Examples 1-5, this invention selected serotypes of avian pathogenic Escherichia coli O8, O18, O21, O32, and O33, and determined the minimum inhibitory concentration (MIC) using the broth dilution method. The results showed that the MICs of the antimicrobial peptides of this invention against most APEC strains ranged from 15.63 to 62.5 μg / mL, with O8 being particularly sensitive (MIC < 7.81 μg / mL), superior to most natural antimicrobial peptide control groups.

[0053] As shown in Example 1, this invention uses chicken fibroblasts (DF-1) as a model and employs the MTT cell proliferation and cytotoxicity assay kit to detect cytotoxicity; the results show that the cell survival rate is >90% at the effective concentration and there is no obvious toxicity.

[0054] As shown in Example 2, the present invention uses duck erythrocyte suspension as a model, and the test results show that the hemolysis rate is less than 5% in the range of 0–512 μg / mL, indicating good safety.

[0055] As shown in Example 3, the present invention further examined avian pathogenic Escherichia coli treated with the antimicrobial peptide using scanning electron microscopy. In the experiment, the bacterial cells were incubated for 1 hour with the antimicrobial peptide of the present invention (1 times the MIC concentration) and a control buffer, respectively, followed by glutaraldehyde fixation, gradient ethanol dehydration, critical point drying, and metal sputtering treatment. The surface morphology of the bacterial cells was then observed using scanning electron microscopy. The results showed that the control group cells had smooth, intact surfaces and plump morphologies; while the cells treated with the antimicrobial peptide of the present invention exhibited obvious surface collapse, membrane rupture, and leakage of contents, with blurred cell outlines and some cells completely dissolved.

[0056] In summary, the results indicate that the antimicrobial peptides of this invention exert their antibacterial effect by binding to the bacterial cell membrane with positively charged amphiphilic peptide segments, thereby disrupting the bacterial cell membrane structure. This mechanism of action conforms to the membrane disruption pattern of typical amphiphilic cationic peptides. Therefore, the antimicrobial peptides of this invention can be widely applied in: 1. the development of drugs for the prevention and treatment of pathogenic Escherichia coli infections in poultry; 2. animal feed additives and antibiotic-free alternatives.

[0057] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0059] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide was obtained by modifying the amino acid sequence shown in SEQ ID No:

1.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide comprises the amino acid sequence shown in SEQ ID No:

2.

3. The antimicrobial peptide according to claim 2, characterized in that, The antimicrobial peptide has a net charge of +8, a hydrophobicity index of 0.35-0.40, and exhibits a typical amphiphilic α-helical conformation.

4. A drug for preventing and treating pathogenic Escherichia coli in poultry, characterized in that, The drug includes an antimicrobial peptide as described in any one of claims 1-3.

5. The drug according to claim 4, characterized in that, The drug is used for serotypes O8, O18, O21, O32, and O33 of pathogenic Escherichia coli in birds.

6. A feed additive for the prevention and control of pathogenic Escherichia coli in poultry, characterized in that, The feed additive includes the antimicrobial peptide as described in any one of claims 1-3.

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