Broad-spectrum antibacterial peptides and uses thereof

Abstract

The present application belongs to the technical field of biology, and specifically relates to a broad-spectrum antibacterial peptide and application thereof. The present application screens two broad-spectrum antibacterial peptides HB-141 and antibacterial peptide SC-165 through bioinformatics and artificial intelligence big models, and the two broad-spectrum antibacterial peptides have good inhibitory effect on pathogenic bacteria such as enterotoxigenic Escherichia coli and Staphylococcus aureus. The safety of the two broad-spectrum antibacterial peptides is good through hemolysis experiment test, the two broad-spectrum antibacterial peptides can improve infection and inflammatory injury of the body, replace the use of antibiotics, and have good application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to broad-spectrum antimicrobial peptides and their applications. Background Technology

[0002] The overuse and improper use of antibiotics in livestock and poultry farming have led to a series of problems, including continuously increasing bacterial resistance and excessive veterinary drug residues in animal-derived foods. These problems not only seriously endanger food safety but also pose a potential threat to human health, and have become critical issues that urgently need to be addressed in the field of veterinary public health.

[0003] Antimicrobial peptides (AMPs) are broadly classified into two categories based on their origin: natural and artificially designed and synthesized. Natural antimicrobial peptides have a wide range of sources, including mammals, plants, amphibians, insects, microorganisms, birds, and marine organisms. Artificially synthesized peptides, on the other hand, are prepared through chemical synthesis or genetic engineering after structural optimization using strategies such as chemical modification and molecular coupling. Their antibacterial effects manifest in two ways: firstly, they can directly induce the death of pathogens, targeting key structural and functional components of microorganisms (such as cell membranes, cell walls, biofilms, and important intracellular substances); secondly, they can regulate the host's innate and adaptive immunity, thereby enhancing the body's overall immune defense. In short, as an important component of the innate immune system, antimicrobial peptides, with their broad-spectrum antibacterial activity and low likelihood of inducing drug resistance, are considered ideal alternatives to antibiotics. However, traditional antimicrobial peptide development heavily relies on experimental screening and functional verification, and is severely constrained by factors such as research efficiency, cost, and time constraints, significantly hindering its industrialization. In recent years, artificial intelligence technology has rapidly developed in bioinformatics fields such as protein sequence analysis and functional prediction, providing new insights for overcoming traditional research bottlenecks.

[0004] To overcome the shortcomings of existing technologies, this invention, through extensive and in-depth research and by combining artificial intelligence technology for screening and verification, obtained two novel broad-spectrum antimicrobial peptides, thus completing this invention. Summary of the Invention

[0005] In one aspect, the present invention discloses a broad-spectrum antimicrobial peptide, wherein the antimicrobial peptide is antimicrobial peptide HB-141 or antimicrobial peptide SC-165, wherein the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No. 4.

[0006] In one aspect, the present invention discloses a recombinant expression vector containing a sequence as shown in SEQ ID No. 1 or a sequence as shown in SEQ ID No. 2.

[0007] In some embodiments, the expression vector is selected from eukaryotic expression vectors, prokaryotic expression vectors, and insect expression vectors; preferably, the expression vector is a prokaryotic expression vector; more preferably, the prokaryotic expression vector is a pET expression vector.

[0008] In one embodiment, the prokaryotic expression vector is pET-32a. The sequences of antimicrobial peptide genes (HB-141, SC-165) are linked to the pET-32a vector using genetic engineering techniques to construct recombinant expression vectors pET-32a-HB-141 and pET-32a-SC-165. The nucleotide sequence of the antimicrobial peptide HB-141 is shown in SEQ ID No. 1; the nucleotide sequence of the antimicrobial peptide SC-165 is shown in SEQ ID No. 2.

[0009] In one aspect, the present invention discloses a recombinant expression system obtained by transfecting a recombinant expression vector into a host, wherein the recombinant expression vector expresses the antimicrobial peptide HB-141 or the antimicrobial peptide SC-165.

[0010] In some embodiments, the host is selected from animal cells, insect cells, Escherichia coli, and yeast. Preferably, the host is Escherichia coli.

[0011] In one aspect, the present invention discloses the use of the antimicrobial peptide HB-141 or antimicrobial peptide SC-165 described herein in the preparation of a drug.

[0012] In some embodiments, the drug is used for antibacterial and anti-inflammatory purposes, and for regulating the intestinal microbiota.

[0013] In one aspect, the present invention discloses the use of the antimicrobial peptide HB-141 or antimicrobial peptide SC-165 described herein in the preparation of additives.

[0014] In some embodiments, the additive is used in food, cosmetics, and animal feed.

[0015] In one aspect, the present invention discloses an antimicrobial reagent or kit containing antimicrobial peptide HB-141 and / or antimicrobial peptide SC-165, wherein the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No. 4.

[0016] In one aspect, the present invention discloses a method for preparing antimicrobial peptide HB-141 or antimicrobial peptide SC-165, wherein the antimicrobial peptide is prepared by chemical methods or biosynthesis.

[0017] In this invention, the antimicrobial peptide HB-141 or antimicrobial peptide SC-165 can be synthesized by chemical methods or prepared by biosynthesis. Chemical synthesis methods include Fmoc solid-phase peptide synthesis, liquid-phase peptide synthesis, and microwave-assisted solid-phase peptide synthesis. Biosynthesis methods can utilize the expression vectors and recombinant strains / cells described above, and the specific operations, separation, and purification methods are well known to those skilled in the art.

[0018] In one aspect, the present invention discloses a method for preparing antimicrobial peptide HB-141 or antimicrobial peptide SC-165, the method comprising the following steps: (1) Cloning the antimicrobial peptide gene sequence using SEQ ID No. 1 or SEQ ID No. 2 as a template; (2) Construct recombinant expression vectors pET-32a-HB-141 and pET-32a-SC-165; (3) Preparation of Escherichia coli BL21(DE3)pLysS competent cells; (4) The recombinant expression vector was transformed into BL21(DE3)pLysS competent cells, and the positive transformants of recombinant Escherichia coli pET-32a-HB-141 and pET-32a-SC-165 were screened by LB medium and PCR identification. (5) IPTG was used to induce recombinant Escherichia coli strains pET-32a-HB-141 and pET-32a-SC-165 to express the antimicrobial peptides HB-141 or SC-165; (6) Separate and purify the expression product.

[0019] Beneficial effects

[0020] This invention uses bioinformatics and artificial intelligence models to screen and obtain two broad-spectrum antimicrobial peptides, which have good inhibitory effects on enterotoxigenic Escherichia coli and Staphylococcus aureus. The two antimicrobial peptides exert their antibacterial effect by disrupting the bacterial cell membrane structure, and hemolysis experiments show that they have good biosafety.

[0021] The antimicrobial peptides of this invention can improve infection and inflammatory damage in the body, and can be used in pharmaceuticals or additives to replace antibiotics, thus having good application value. Attached Figure Description

[0022] Figure 1 The simulated structural predictions of two antimicrobial peptides are shown. In the figure, A is SC-165, whose structure is mainly composed of two relatively long α-helices connected by a short, irregular coil, resulting in a compact overall structure. The two α-helices are arranged in approximately parallel lines. Figure 1In the case of HB-141, the α-helical region is relatively short and is separated by multiple irregularly coiled segments, making the overall structure more flexible.

[0023] Figure 2 Western blot identification of recombinant proteins.

[0024] Figure 3 Figure 1 shows the antibacterial curves of proteins HB-141 and SC-165 against ATCC35401, where A is the antibacterial curve of protein HB-141 against ATCC35401; and B is the antibacterial curve of protein SC-165 against ATCC35401.

[0025] Figure 4 Diluted ATCC35401 protein at different concentrations.

[0026] Figure 5 Diluted ATCC35401 protein SC-165 at different concentrations.

[0027] Figure 6 Fluorescence microscopy observation of live and dead bacteria of the two recombinant proteins.

[0028] Figure 7 Electron micrographs of proteins HB-141 and SC-165 co-cultured with ETEC.

[0029] Figure 8 The hemolytic activities of different recombinant proteins, where (A) is the hemolytic activity of protein HB-141 and (B) is the hemolytic activity of protein SC-165.

[0030] Figure 9 Blood routine examinations of mice in different treatment groups. Among them, ns, P >0.05; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001; A: Changes in white blood cell count in each experimental group; B: Changes in neutrophil ratio in each experimental group; C: Changes in lymphocyte ratio in each experimental group; D: Changes in red blood cell count in each experimental group; E: Changes in platelet count in each experimental group.

[0031] Figure 10 Immune organ indices of mice in different treatment groups.

[0032] Figure 11 The effects of different treatment groups on the intestines of mice, where A is a jejunal section of mice in different treatment groups; and B is an ileal section of mice in different treatment groups.

[0033] Figure 12 Results of intestinal bacterial load measurement in mice of each group, where A is the bacterial load result of ileum tissue and B is the bacterial load result of jejunum tissue.

[0034] Figure 13 Intestinal gene expression levels in mice from different experimental groups. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0036] Example 1: Computer-aided artificial intelligence screening of antimicrobial peptides (1) By integrating the annotated sequences of four internationally recognized antimicrobial peptide public databases, namely CAMP, APD, DRAMP and dbAMP, a rigorous screening was conducted to ensure the validity of the samples. Samples with amino acid lengths <10 or >100 were removed because peptides with amino acid lengths that are too short (<10) are difficult to form a stable antimicrobial conformation and cannot exert antimicrobial effects, while peptides with lengths that are too long (>100) are prone to spatial folding abnormalities and do not conform to the conventional length range of natural antimicrobial peptides. Finally, 2518 valid positive samples were obtained, and a positive sample data dataset was constructed. (2) The peptide sequences labeled with "antibacterial" related keywords were first removed one by one to avoid confusion with the positive samples. Then, the amino acid lengths of the samples that did not meet the above range were removed. Finally, 1748 sequences were randomly selected from the qualified sequences as the final negative sample dataset.

[0037] (3) Five traditional machine learning models integrating BERT pre-training strategies were systematically evaluated, including Random Forest (RF), Support Vector Machine (SVM), Decision Tree (DT), K-Nearest Neighbors (KNN), and XG Boost. The focus was on comparing the feature extraction ability, expression accuracy, and prediction performance of each model under BERT word vector embedding. The comprehensive dataset used in the study contained 2518 positive samples and 1748 negative samples. After five-fold cross-validation and comprehensive evaluation using multiple indicators such as accuracy and precision, the results showed that XG Boost was the best performing single model (accuracy 0.9653). Based on this, composite models were constructed using hard voting, weighted soft voting, and stacking ensembles, respectively, and their performance was compared. The results showed that the model obtained by ensemble based on the stacking generalization strategy had the best prediction effect, with an accuracy of 96.91%. The accuracy and F1 score were also the best, significantly outperforming each single model and other ensemble methods. Subsequently, this strategy model was used to virtually screen the previous porcine gut microbiome sequences, ultimately identifying two candidate antimicrobial peptide sequences with the highest scores, and their structures were simulated (see [link to relevant documentation]). Figure 1 The two antimicrobial peptides were named HB-141 (amino acid sequence: GAGRIRVTHQCAGRRQAYCYALAAPRLACVKPVASVHPEPGSNSSLL, SEQ ID No. 3) and SC-165 (amino acid sequence: CRMRCAYPAYKVSIVGRIRRLRRIRQIPGPDLPGFTHIWKAKCLVT, SEQ ID No. 4) for subsequent functional verification.

[0038] Example 2 Prokaryotic expression and purification of antimicrobial peptides (1) Based on the predicted antimicrobial peptide sequence and Escherichia coli codon preference, the gene sequence of the candidate antimicrobial peptide was optimized (see Table 1). After being synthesized by Beijing Qingke Biotechnology Co., Ltd., it was cloned into the pET-32a vector to construct the pET-32a-HB-141 and pET-32a-SC-165 recombinant plasmids.

[0039] Table 1: Optimization of antimicrobial peptide gene sequences ; (2) Transform the recombinant plasmids pET-32a-HB-141 and pET-32a-SC-165 into Escherichia coli BL21(DE3) pLyss competent cells.

[0040] (3) Randomly select several positive strains with correct sequencing results and inoculate them into LB liquid medium containing ampicillin and chloramphenicol at an inoculation rate of 1:100. Incubate at 37°C and 180 r / min with shaking until OD600nm=1.0. Then, transfer them into LB liquid medium containing ampicillin and chloramphenicol at an inoculation rate of 1:100. Incubate at 37°C and 180 r / min with shaking until OD600nm=0.4-0.8. Add IPTG to make the final concentration 0.6mmol / L and incubate at 37°C and 180 r / min with shaking for 10 h. Take 2 mL of bacterial culture, centrifuge at 10000 r / min for 10 min, discard the supernatant, wash three times with PBS, resuspend the bacterial cells in 100 μL of PBS, take 20 μL of the resuspended solution, add 5 μL of 5×SDS loading buffer, mix well, boil for 10 min, centrifuge at 10000 r / min for 4 min, collect the supernatant, and perform SDS-PAGE electrophoresis to detect its expression. Simultaneously, pET-32a bacterial protein was induced using the same method as a negative control. Strains with high protein expression levels were selected for subsequent experiments.

[0041] (4) The strain was induced to express by IPTG. After expression, the supernatant was collected by centrifugation and disruption. The supernatant was purified by magnetic beads. The purified fusion protein was concentrated in an ultrafiltration tube with a molecular weight cutoff of 3 kDa. After repeated centrifugation, PBS was added for desalting and imidazole removal to obtain recombinant proteins HB-141 and SC-165. The protein concentration was measured by a BCA protein kit and the protein was verified by Western blot.

[0042] See Figure 2 Western blot analysis showed clear and specific bands at approximately 6.1 kDa and 6.6 kDa, confirming the successful expression of both recombinant proteins HB-141 and SC-165.

[0043] Example 3: Validation of the in vitro antibacterial activity of recombinant proteins HB-141 and SC-165 3.1 Detection of in vitro antibacterial activity of recombinant proteins HB-141 and SC-165 against five pathogens Five pathogenic bacteria—enterotoxigenic Escherichia coli ATCC35401, Staphylococcus aureus strain ATCC6538, Staphylococcus aureus strain ATCC25923, Escherichia coli strain ATCC25922, and Pseudomonas aeruginosa strain ATCC9027—were activated on LB solid medium and incubated overnight at 37°C. Single colonies were picked and transferred to LB liquid medium, cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase. The cultures were then transferred to LB liquid medium at a 1:100 inoculum and cultured until a concentration of 1×10⁻⁶ was achieved. 5 -1×10 6 CFU / mL. The antimicrobial peptides were diluted with PBS to a protein solution concentration of 800 μg / mL. In a sterile 96-well plate, 100 μL of LB liquid medium was added to each well. In the first column, 100 μL of protein solution was added to each well and mixed. Then, 100 μL of this protein solution was added to the second column and mixed thoroughly. This serial dilution was repeated until the fifth column. In the sixth column, 100 μL of LB liquid solution was added as a negative control. In the seventh column, 100 μL of pathogenic bacterial suspension was added as a positive control. The plates were then incubated at 37°C, and absorbance was measured every 2 hours.

[0044] See results Figure 3 When the concentrations of both proteins were ≥200 μg / mL, both HB-141 and SC-165 showed inhibitory effects on ATCC35401. Referring to Table 3, recombinant protein SC-165 showed inhibitory effects on enterotoxigenic Escherichia coli (ETEC) ATCC35401, Staphylococcus aureus ATCC6538 / ATCC25923, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC9027, exhibiting broad-spectrum antibacterial activity; while protein HB-141 showed no significant inhibitory effect on Pseudomonas aeruginosa, but exhibited antibacterial activity against the other tested strains.

[0045] Table 2: Inhibition of pathogens by two antimicrobial peptides ; 3.2 Dilution spot test 100 μL of a concentration of 1×10 6 CFU / mL ETEC ATCC35401 bacterial culture was mixed thoroughly with 100 μL of protein HB-141 and protein SC-165 at different concentrations (to achieve final concentrations of 0, 50, 100, 150, 200, and 250 μg / mL), and then diluted separately at different ratios (0, 10...).-2 10 -3 10 -4 10 -5 10 -6 Take 5 μL of the mixture corresponding to each concentration and dilution factor and drop it onto a square LB solid medium. Let it stand overnight for incubation.

[0046] The results are as follows Figure 4 , Figure 5 As shown, near the minimum inhibitory concentration (MIC), the antibacterial effect of the compound formulation on ATCC 35401 changes significantly, and the number of colonies decreases significantly with increasing concentration of the compound formulation.

[0047] 3.3 ETEC DMAO-PI double staining and fluorescence microscopy detection The operation steps are as follows: (1) Bacterial culture.

[0048] Culture the bacteria in a suitable liquid culture medium until the logarithmic growth phase.

[0049] (2) Bacterial preparation.

[0050] Take an appropriate amount of bacterial culture, centrifuge at 10000xg for 5 minutes at room temperature, discard the supernatant, wash once with physiological saline (ST341), and then adjust the bacterial concentration to approximately 10 with physiological saline. 8 bacteria / ml

[0051] (3) Staining.

[0052] Add 1 μL of staining working solution (100X) to every 100 μL of bacterial culture and mix well. Incubate at 37°C in the dark for 15 minutes. The optimal incubation time may vary for different bacterial species. Use 15 minutes as the initial incubation time. The staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining result.

[0053] (4) Detection.

[0054] After incubation, 10 μl of bacterial culture was dropped onto a glass slide (FSL051) and covered with a 24 mm square coverslip (FCGF24). The staining effect was observed under a fluorescence microscope. DMAO was green fluorescence, Ex / Em=503 / 530nm; PI was red fluorescence, Ex / Em=535 / 617nm.

[0055] Results: Observation by live-dead bacteria fluorescence staining (see [link]). Figure 6When proteins HB-141 and SC-165 were applied to ETEC, the amount of red and green fluorescence in the fluorescence field was basically the same, and the two signals highly overlapped. After superposition, the main fluorescence was yellow, which was highly consistent with the fluorescence characteristics of the dead bacteria control group. No characteristic features of live bacteria with predominantly green fluorescence were observed in the field of view after MERGE.

[0056] 3.4 Co-culture of HB-141, SC-165 with ETEC and electron microscopy analysis ETEC cultured to the logarithmic growth phase. Equal volumes of bacterial suspension were mixed with 150 μg / mL proteins HB-141 and SC-165, respectively, and co-cultured overnight at 37°C. The mixture was then washed three times with sterile PBS buffer. The bacterial suspension was transferred to 6-well plates containing crawling slides. The precipitated samples were then transferred to 6-well plates containing crawling slides. The slides were fixed in 2.5% glutaraldehyde at 4°C for 2 hours, washed with sterile PBS buffer, and then fixed again in 3% glutaraldehyde at 4°C for 6 hours. The slides were washed with sterile PBS buffer for 20 minutes. The plates were then sequentially placed in 30%, 50%, 70%, 80%, and 100% ethanol for 20 minutes each, followed by 100% acetone and freeze-dried under vacuum. The control group was treated with PBS instead of protein and then subjected to the same treatment. The completed samples were observed and photographed using an electron microscope.

[0057] See results Figure 7 , Figure 7 Medium A (Normal Control Group ETEC): Under normal conditions, ETEC exhibits a typical short rod-shaped morphology with an intact bacterial structure and clear, sharp edges. The bacterial surface is relatively smooth, with a few visible pili or flagella-like appendages. The bacterial cells are arranged relatively dispersedly or in a locally ordered distribution, without obvious signs of aggregation or rupture. The overall morphology is regular, consistent with the ultrastructural characteristics of typical enterotoxigenic Escherichia coli. Figure 7 ETEC co-cultured with protein HB-141 (Category B): After co-culturing with protein HB-141, the morphology of ETEC cells changed significantly, showing obvious shrinkage, swelling, or severe deformation (such as bending and breakage). The cell surface was rough and uneven, with obvious depressions, pores, or membrane structure damage visible in some areas. The number of accessory structures such as pili and flagella decreased, and abnormal aggregation or adhesion between cells was observed, indicating increased extracellular matrix secretion and severe damage to cell integrity. Figure 7In the middle category (ETEC co-cultured with protein SC-165): After co-culturing with protein SC-165, the ETEC cells remained rod-shaped overall, but some cells showed swelling, deformation, and blurred edges. The cell surface was relatively rough, with some membrane structure damage visible. Some cell surfaces had flocculent or granular material attached, and the number of accessory structures such as pili and flagella was significantly reduced. Obvious aggregation and adhesion were observed between cells, with some cells intertwined, suggesting increased extracellular matrix secretion and enhanced intercellular interactions.

[0058] 3.5 In vitro hemolytic activity assay of recombinant proteins HB-141 and SC-165 First, collect 10 mL of healthy rabbit blood using the cardiac blood sampling method. Centrifuge the rabbit blood at 5000 r / min for 5 min and discard the supernatant. Add 10 mL of physiological saline, mix well, centrifuge at 5000 r / min for 5 min, discard the supernatant, and repeat three times, with the last centrifugation for 10 min. Prepare a 4% rabbit red blood cell suspension with sterile physiological saline and store at 4℃ for later use. Adjust the concentration of proteins HB-141 and SC-165 to 900 µg / mL, and dilute from 900 µg / mL to well 10. Add an equal volume of PBS solution to well 11 and an equal volume of 1% Triton X-100 solution to well 12. Then add an equal volume of 4% rabbit red blood cell suspension. Incubate the 96-well plate at 37℃ for 1 h, centrifuge at 5000 r / min for 5 min, and measure the absorbance of the supernatant at a wavelength of 492 nm using a microplate reader. Hemolysis rate (%) = [OD 492nm (peptide)-OD 492nm (PBS)] / [OD 492nm (TritonX-100)-OD 492nm (PBS)]×100%.

[0059] Result: As Figure 8 As shown, proteins HB-141 and SC-165 exhibit very low hemolytic activity. The hemolysis rate of erythrocytes in all experimental groups did not exceed 5%, which fully demonstrates that proteins HB-141 and SC-165 demonstrate excellent performance in terms of hemolytic safety, and their low hemolytic activity can effectively reduce the potential risks caused by erythrocyte damage.

[0060] Example 4: Test of intervention of recombinant proteins HB-141 and SC-165 in ETEC-infected mice An ETEC infection model was constructed based on previously reported methods. Furthermore, based on previous in vitro experimental results, the optimal dosage for proteins HB-141 and SC-165 was determined to be 5.2 mg / mouse, and the optimal dosage for ampicillin (working concentration: 60 mg / kg) was set at 3.6 mg / mouse. SPF-grade mice were randomly divided into 5 groups of 8 mice each: blank control group, model group, protein HB-141 group, protein SC-165 group, and positive control group (ampicillin). All mice were administered medication via gavage. Forty-eight hours prior to challenge, streptomycin at a concentration of 5 mg / mL was added to the mice's drinking water to inhibit some intestinal symbiotic flora, creating conditions conducive to subsequent pathogen colonization. Twelve hours prior to challenge, all mice were fasted but allowed water intake to improve the success rate of pathogen infection. Thirty minutes before challenge, each mouse was administered 0.2 mL of 3% sodium bicarbonate solution by gavage to neutralize the acidic environment in the stomach and reduce the killing effect of gastric acid on the subsequently challenged bacterial strain. Except for the blank control group, all other groups of mice were administered ETEC bacterial suspension (concentration 1×10⁻⁶) by gavage at a dose of 0.1 mL / mouse. 11 Mice were challenged with a concentration of CFU / mL for 3 consecutive days. After challenge, mice in the blank control group and model group were administered an equal volume of physiological saline (0.1 mL / mouse) by gavage once; mice in the protein HB-141 group were administered 0.3 mL of protein HB-141 solution by gavage once, mice in the protein SC-165 group were administered 0.3 mL of protein SC-165 solution by gavage once, and mice in the positive control group were administered 0.3 mL of ampicillin solution by gavage once. All the above intervention drugs were administered by gavage twice a day for 3 consecutive days.

[0061] 4.1. Clinical observation and status assessment Mice in each treatment group were observed daily for their general condition, including mental state, fur color, responsiveness, and activity level, as well as anal and fecal conditions. The weight of each mouse was measured and recorded on time. Based on the visual and anatomical changes in the spleen and intestines of mice in different treatment groups, scores were assigned according to the clinical symptom scoring scale (Table 3), and objective quantitative assessments were performed.

[0062] Table 3: Clinical Symptom Scoring Table for Mice ; ; Results: After being challenged with ETEC bacterial suspension in groups, mice in the model group, positive control group, HB-141 group, and SC-165 group all developed typical clinical symptoms 2 hours after challenge: reduced activity, small amounts of loose stool, accompanied by severe depression, significantly decreased activity, huddling together and lying still, and fluffy, rough, and dull fur. In contrast, mice in the blank control group maintained good mental state throughout the process, were agile, had smooth and clean fur, normal food and water intake, and did not exhibit diarrhea, mental abnormalities, or other adverse symptoms; their overall general condition remained unchanged. The efficacy scores are shown in Table 4. Regarding body weight, the weight of mice in the negative control group remained stable throughout the observation period, consistently above 100% and showing a slow upward trend. The body weight of mice in the model group decreased significantly after challenge, reaching approximately 82% by day 3. The weight changes in the positive control group, HB-141 group, and SC-165 group were similar to those in the negative control group, remaining around 100% or slightly increasing, without significant weight loss. The HB-141 group, however, saw its body weight rise to approximately 112% by day 3. Overall, ETEC challenge resulted in a significant decrease in body weight in the model group mice, while the positive control, HB-141, and SC-165 treatments inhibited the weight loss induced by the challenge.

[0063] Table 4. Therapeutic efficacy scoring table for mice in different treatment groups ; 4.2 Complete blood count Blood was collected from all mice using blood collection tubes containing anticoagulants, and complete blood counts (white blood cell count, neutrophil ratio, lymphocyte ratio, red blood cell count, and platelet count) were measured.

[0064] Results: See Figure 9 The white blood cell count and neutrophil ratio in the challenge group were significantly higher than those in the model control group, HB-141 group, SC-165 group and positive control group; the decrease in lymphocyte ratio in the challenge group was greater than that in the model control group, HB-141 group, SC-165 group and positive control group.

[0065] 4.3 Immune organ index measurement The spleen and thymus were weighed and recorded to calculate the immune organ index.

[0066] See results Figure 10The spleen immune index results showed that compared with the negative control group, the spleen immune index of mice in the model control group was significantly decreased (P<0.01). After intervention with HB-141, SC-165, and positive control drugs, the spleen immune index of mice was significantly restored compared with the model control group (P<0.01), and there was no significant difference between the intervention groups and the positive control group, suggesting that HB-141 and SC-165 can effectively reverse the decrease in spleen immune index caused by ETEC challenge. The thymus immune index results showed that the thymus immune index of mice in the model control group was significantly decreased compared with the negative control group (P<0.01). The thymus immune index of the HB-141 group, SC-165 group, and positive control group was significantly higher than that of the model control group, with the recovery effect of the SC-165 group being similar to that of the positive control group. In conclusion, ETEC challenge can significantly reduce the spleen and thymus immune indices of mice, while intervention with HB-141 and SC-165 can effectively restore immune organ indices, reduce the damage to immune organs caused by ETEC infection, and exert an immunoprotective effect.

[0067] 4.4 Preparation of small intestinal pathological sections and HE staining examination As a result, Figure 11 Figure A shows pathological sections (HE staining) of jejunal tissue from different treatment groups in an ETEC-infected mouse model, reflecting the intestinal pathological changes in each group in terms of structural integrity and degree of damage: Group (A) (negative control group): The jejunal villi were intact and neatly arranged, with continuous epithelial cells and no obvious inflammatory cell infiltration in the lamina propria, indicating a normal intestinal barrier structure. Group (B) (model control group): The jejunal villi showed obvious breakage, fusion, and atrophy, with a large number of epithelial cells detached and obvious inflammatory cell infiltration in the lamina propria, indicating severe damage to the intestinal barrier, a typical pathological manifestation of ETEC infection. Group (C) (HB-141 treatment group): The jejunal villi were basically intact and relatively neatly arranged, with only a small amount of epithelial cell detachment and mild inflammatory infiltration, indicating significantly less intestinal damage than the model control group. Group (D) (SC-165 treatment group): The jejunal villi were intact, with good epithelial cell continuity and less inflammatory cell infiltration in the lamina propria, indicating effective protection of the intestinal barrier function. (E) Group (Positive Control Group): The jejunal villus structure remained largely intact, with only minor damage and inflammation in localized areas, similar to the protective effects of the HB-141 and SC-165 treatment groups. In summary, ETEC challenge can cause severe pathological damage to the jejunal tissue of mice, while HB-141 and SC-165 treatments effectively reduced intestinal tissue damage and maintained the integrity of the intestinal barrier structure, with protective effects comparable to the positive control. This suggests that these two treatments have a significant protective effect against jejunal damage caused by ETEC infection. Figure 11Figure B shows pathological sections of ileal tissue from different treatment groups in an ETEC-infected mouse model, reflecting the intestinal pathological changes in each group in terms of structural integrity and degree of damage: (A) Group (negative control group): Ileal villi were intact and neatly arranged, epithelial cells were continuous, and there was no obvious inflammatory cell infiltration in the lamina propria; the intestinal barrier structure was normal. (B) Group (model control group): Ileal villi showed obvious breakage, fusion, and atrophy; a large number of epithelial cells were shed; obvious inflammatory cell infiltration was visible in the lamina propria; the intestinal barrier was severely damaged, which is a typical pathological manifestation of ETEC infection. (C) Group (HB-141 treatment group): Ileal villi were basically intact and neatly arranged; only a small amount of epithelial cell shed and mild inflammatory infiltration were observed; the intestinal damage was significantly milder than in the model control group. (D) Group (SC-165 treatment group): Ileal villi were intact; epithelial cell continuity was good; there was less inflammatory cell infiltration in the lamina propria; the intestinal barrier function was effectively protected. (E) Group (Positive Control Group): The ileal villi structure remained largely intact, with only minor damage and inflammation in localized areas, similar to the protective effects of the HB-141 and SC-165 treatment groups. In summary, ETEC challenge can cause severe pathological damage to the ileal tissue of mice, while HB-141 and SC-165 treatments effectively alleviated intestinal tissue damage and maintained the integrity of the intestinal barrier structure, with protective effects comparable to the positive control. This suggests that these two treatments have a significant protective effect against ileal damage caused by ETEC infection.

[0068] 4.5 Determination of bacterial load in tissues under different treatments After weighing a portion of jejunum and ileum, add sterile PBS for grinding, record the volume of the grinding solution, and then serially dilute the tissue grinding solution (10⁻⁶ ppm). -2 ~10 -7 Afterwards, the tissue was spread onto LB solid medium and incubated in a 37°C oven for 24 hours before the bacterial load was calculated.

[0069] Results: See Figure 12 ETEC challenge can significantly increase bacterial load in the ileum and jejunum of mice, while HB-141 and SC-165 treatment can significantly reduce bacterial load in the intestine and effectively inhibit ETEC colonization and proliferation in the intestine. Their protective effect is comparable to that of the positive control, providing important evidence for their selection as a candidate drug against ETEC infection.

[0070] 4.6 Measurement of changes in gene expression levels in intestinal tissue RNA was extracted from small intestinal tissue, followed by RT-PCR and qPCR to detect the genes β-actin, TNFα, IL-1β, IL-6, and IL-8. Results are as follows: Figure 13As shown, compared with the negative control group, the mRNA expression levels of IL-6, IL-8, IL-1β and TNF-α in the ileum tissue of mice in the model control group were significantly increased (P<0.01 or P<0.001). After intervention with HB-141 and SC-165 and treatment with the positive control, the mRNA expression levels of the above four pro-inflammatory factors were significantly decreased compared with the model control group (P<0.05, P<0.01 or P<0.001). Among them, the downregulation effect of SC-165 intervention group on IL-6 and IL-1β was similar to that of the positive control group, and the inhibitory effect of HB-141 intervention group on IL-8 and TNF-α was significant.

[0071] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A broad-spectrum antimicrobial peptide, characterized in that, The antimicrobial peptide is either antimicrobial peptide HB-141 or antimicrobial peptide SC-165, wherein the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No.

4.

2. A recombinant expression system, characterized in that, The recombinant expression system is obtained by introducing a recombinant expression vector into a host, wherein the recombinant expression vector expresses antimicrobial peptide HB-141 or antimicrobial peptide SC-165, wherein the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No.

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3. The recombinant expression system according to claim 2, characterized in that, The host is Escherichia coli.

4. The application of antimicrobial peptide HB-141 or antimicrobial peptide SC-165 in the preparation of antimicrobial drugs, characterized in that, The amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3, and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No. 4; the bacteria are Staphylococcus aureus or enterotoxin-producing Escherichia coli.

5. The application of antimicrobial peptide HB-141 or antimicrobial peptide SC-165 in the preparation of additives, characterized in that, The additive is used in feed; the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No.

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6. An antibacterial reagent or kit, characterized in that, The reagent or kit contains antimicrobial peptide HB-141 and / or antimicrobial peptide SC-165, wherein the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; and the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No.

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7. A method for preparing antimicrobial peptide HB-141 or antimicrobial peptide SC-165, characterized in that, The antimicrobial peptides are prepared by chemical or biosynthetic methods; the amino acid sequence of antimicrobial peptide HB-141 is shown in SEQ ID No. 3; the amino acid sequence of antimicrobial peptide SC-165 is shown in SEQ ID No.

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8. A method for preparing antimicrobial peptide HB-141 or antimicrobial peptide SC-165, characterized in that, The method includes the following steps: (1) Using SEQ ID No. 1 or SEQ ID No. 2 as a template, clone the antimicrobial peptide gene sequence, wherein the antimicrobial peptide HB-141 gene sequence is shown in SEQ ID No. 1; and the antimicrobial peptide SC-165 gene sequence is shown in SEQ ID No. 2; (2) Construct recombinant expression vectors pET-32a-HB-141 and pET-32a-SC-165; (3) Preparation of Escherichia coli BL21(DE3)pLysS competent cells; (4) The recombinant expression vector was transformed into BL21(DE3)pLysS competent cells, and the positive transformants of recombinant Escherichia coli pET-32a-HB-141 and pET-32a-SC-165 were screened by LB medium and PCR identification. (5) IPTG was used to induce recombinant Escherichia coli strains pET-32a-HB-141 and pET-32a-SC-165 to express the antimicrobial peptides HB-141 or SC-165. (6) Separate and purify the expression product.

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