Antibacterial peptide AMP1 and application thereof

Abstract

The application relates to the technical field of biological pharmacy, in particular to an antibacterial peptide AMP1 and application thereof. The application provides the antibacterial peptide AMP1, the amino acid sequence of the antibacterial peptide AMP1 is shown as SEQ ID NO. 1, the antibacterial peptide can effectively inhibit escherichia coli; and can also effectively treat septicemia caused by escherichia coli and infection.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to an antimicrobial peptide AMP1 and its applications. Background Technology

[0002] Faced with the increasingly severe crisis of antibiotic resistance and the global policy trend of "banning or reducing antibiotic use" in animal husbandry, the discovery of safe and effective natural antimicrobial alternatives has become a cutting-edge research hotspot in life sciences and medicine. Pigs, as important economic animals and ideal models for human disease research, possess an extremely rich array of small open reading frames (smORFs) in their gut microbiome. These smORFs are typically short (encoding less than 100 amino acids) and are easily overlooked or classified as non-coding sequences in conventional genome annotation, resulting in the undeveloped potential of many functional small peptides.

[0003] smORFs can be effectively translated into functional small peptides with various biological activities, including antibacterial, antifungal, and pheromone communication functions. Among them, antimicrobial peptides (AMPs) exhibit significant advantages over traditional antibiotics due to their unique physical membrane-breaking mechanism, making them less likely to induce bacterial resistance. In human medicine, antimicrobial peptides are considered highly promising new antimicrobial agents that can be used to combat infections caused by drug-resistant pathogens. In livestock production, as novel green feed additives, they can not only enhance animal immunity and maintain intestinal health but also improve muscle quality and growth performance, significantly increasing the economic benefits of animal husbandry while reducing medication costs, thus possessing broad prospects for industrial transformation.

[0004] Based on this, this study fully utilizes the high-quality porcine gut microbiota smORF resource library independently constructed by our research group in the past, and innovatively introduces machine deep learning algorithms to efficiently extract features and predict activity of small peptide sequences, thus overcoming the technical bottlenecks of low throughput and long cycle time in traditional wet experimental screening. Simultaneously, to further verify the actual biological efficacy of candidate molecules, the study will use mouse models to conduct in vitro and in vivo functional experiments, systematically evaluating their antibacterial activity, safety, and immunomodulatory effects. This study aims to establish a complete research pathway from "big data resources" to "functionally active molecules" and then to "in vivo application verification," in order to provide theoretical basis and antimicrobial peptide resource reserves for human antimicrobial drug development and "One Health." Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide AMP1; this antimicrobial peptide can effectively inhibit Escherichia coli; and can also effectively treat sepsis caused by Escherichia coli infection.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides an antimicrobial peptide AMP1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides the application of the aforementioned antimicrobial peptide AMP1 in the preparation of antimicrobial preparations.

[0008] Preferably, the inhibitory target of the formulation includes Escherichia coli.

[0009] Preferably, the concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

[0010] The present invention also provides the use of the aforementioned antimicrobial peptide AMP1 in formulations for the treatment of sepsis.

[0011] Preferably, the sepsis is caused by Escherichia coli infection.

[0012] Preferably, the concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

[0013] The present invention also provides an antimicrobial preparation comprising the aforementioned antimicrobial peptide AMP1 and a pharmaceutically acceptable carrier.

[0014] Preferably, the concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

[0015] Beneficial effects: This invention uses machine learning to screen and identify broad-spectrum antimicrobial peptides in porcine intestinal microorganisms smORF, resulting in an antimicrobial peptide AMP1. This antimicrobial peptide can effectively inhibit Escherichia coli and can also effectively treat sepsis caused by Escherichia coli infection. Attached Figure Description

[0016] Figure 1 The results validate the therapeutic effect of AMP1 on a mouse model of sepsis caused by Escherichia coli infection. In this model, A represents mouse survival rate, B represents TNF-α expression level, C represents IL-6 expression level, D represents IL-1β expression level, and F represents pathological results of the liver, spleen, and kidneys. Detailed Implementation

[0017] This invention provides an antimicrobial peptide AMP1, the amino acid sequence of which is shown in SEQ ID NO.1; The SEQ ID NO.1 is: ILALGYPWGYTKTVPIRYFYNIPPCPHPV.

[0018] The present invention also provides the application of the aforementioned antimicrobial peptide AMP1 in the preparation of antimicrobial preparations.

[0019] In this invention, the inhibitory target of the formulation includes Escherichia coli.

[0020] In this invention, the concentration of antimicrobial peptide AMP1 in the preparation is 5~20 mg / mL, preferably 10~15 mg / mL, and more preferably 12.5 mg / mL.

[0021] The present invention also provides the use of the aforementioned antimicrobial peptide AMP1 in formulations for the treatment of sepsis.

[0022] In this invention, the sepsis is caused by Escherichia coli infection.

[0023] In this invention, the concentration of antimicrobial peptide AMP1 in the preparation is 5~20 mg / mL, preferably 10~15 mg / mL, and more preferably 12.5 mg / mL.

[0024] The present invention also provides the use of the aforementioned antimicrobial peptide AMP1 in a formulation for treating human vaginal epithelial cell damage.

[0025] The present invention also provides an antimicrobial preparation comprising the aforementioned antimicrobial peptide AMP1 and a pharmaceutically acceptable carrier.

[0026] Preferably, the concentration of the antimicrobial peptide AMP1 in the formulation is 5-20 mg / mL, more preferably 10-15 mg / mL, and even more preferably 12.5 mg / mL.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] In this invention, all statistical analyses and visualizations were performed using R v4.0.2, data processing was done using the dplyr package, and graphing was done using the ggplot2 package. Alpha diversity indices for single samples and Bray-Curtis distances (beta diversity distances between samples) were calculated using the vegan package. Alpha diversity indices for species, resistance genes, and AMPs, the number of detected target genes, total abundance, and inter-group differences in abundance at different sub-levels were determined using Turkey's HSD test (multiple groups, multcomp package). Heatmaps were generated using the pheatmap package, and Venn plots were drawn using the VennDiagram package. NMDS, ANOSIM, Mantel test, and Procrustes analyses were performed using the vegan package. PCoA analysis was performed using the ape package. Male C57BL / 6 mice, 8 weeks old, weighing 20-22g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The E. coli originated from Jilin University; TUNEL staining was performed using the One-step TUNEL in Situ ApoptosisiKit (Green, FITC) (E-CK-320) kit from Elabscience; mounting medium (containing DAPI) was purchased from Beyotime Biotechnology Co., Ltd.; and the Roche cytotoxicity assay kit was purchased from Roche Biotechnology Co., Ltd.

[0029] Example 1: Obtaining Antimicrobial Peptides

[0030] (1) Screening and identification of antimicrobial peptides based on machine learning

[0031] Based on smORF data, and following the method described in the Cell paper (doi:10.1016 / j.cell.2024.05.013), five mainstream software programs—amPEP, ampir, amplify, ampscan, and APIN—were used to identify AMPs. For AMPs targeting pathogenic bacteria, the default models and parameters of these software programs were used. For AMPs targeting pathogenic fungi, all models were reconstructed when using these software programs. The training data used were 1,813 known antifungal peptides from the APD6 antimicrobial peptide database (https: / / aps.unmc.edu / AP / ). For screening positive candidate antimicrobial peptides (bacteria + fungi), the selection criteria for positive AMPs in different software programs are as follows: an AMP is considered a positive candidate antimicrobial peptide only if it is identified as positive by at least two software programs. The parameter settings are as follows: AmPEP score > 0.5; Ampir score > 0.8; Amplify score > 0.5; amp-scanner (v2) score > 0.5 and APIN score = 1. AMPs were predicted simultaneously for MAG sequences and non-MAG contig sequences. Only one read from each positive AMP with completely identical amino acid sequences was retained, resulting in the final non-redundant AMPs. Considering the inclusion of public data and the significant differences in data volume from different sources, all data were flattened to approximately 20 million read pairs (about 6 Gb) to avoid bias in relative abundance calculations. The ARGs_OAP v2.0 tool (https: / / github.com / xinehc / args_oap / ) was then used to calculate the TPM abundance of the AMPs. A total of 1,534,716 positive candidate antibacterial peptides were obtained from this database, and AMP1 (ILALGYPWGYTKTVPIRYFYNIPPCPHPV) was selected for validation.

[0032] (2) Validation of antimicrobial peptides

[0033] To verify the antibacterial activity of antimicrobial peptides, six antimicrobial peptides were selected for validation. The inhibition zone method (disk diffusion method) was used to evaluate the antibacterial activity of the six antimicrobial peptides against Escherichia coli (LB medium). The final concentration of the antimicrobial peptides was 20 mg / mL. Ampicillin (10 μg / tablet) was used as a positive control for bacteria, and distilled water was used as a negative control. Each sample was tested in triplicate. After incubation at 37°C for 12 h, the diameter of the inhibition zone was observed to evaluate the antimicrobial spectrum and efficacy of each antimicrobial peptide. The results showed that, based on the inhibition zone, AMP1 (ILALGYPWGYTKTVPIRYFYNIPPCPHPV) had a strong inhibitory effect on Escherichia coli, so AMP1 was selected for animal experiments to verify this effect.

[0034] Example 2: Effects of AMP1 on mice with septicemia infected with Escherichia coli

[0035] Forty-eight male C57BL / 6 mice were randomly divided into a control group, an E. coli infection group, an AMP1 intervention group, and an AMP1 treatment group, with eight mice in each group. Injections were administered daily at 10:00 AM according to the following regimen: Control group (NT): Each mouse was injected intraperitoneally with 0.2 mL of sterile PBS; Escherichia coli infection group (E. coli): Each mouse was intraperitoneally injected with 0.2 mL of E. coli bacterial solution at a concentration of 10. 4 CFU; AMP1 intervention group (AMP1): Each mouse was injected intraperitoneally with 0.2 mL of AMP1 solution with a concentration of 20 mg / mL for 4 consecutive days; AMP1 treatment group (E.coil+AMP1): Each mouse was injected intraperitoneally with 0.2 mL of E. coli bacterial solution for 1 hour, followed by intraperitoneal injection of 0.2 mL of AMP1 at a concentration of 20 mg / mL. AMP1 was then injected intraperitoneally at the same time point every day for 4 consecutive days. After the experiment, the mortality rate of mice was recorded; the expression levels of inflammatory factors in serum were measured using a kit; and hematoxylin-eosin (HE) staining was used to detect pathological changes in liver, spleen, and kidney tissues. The results are as follows: Figure 1 As shown; The results showed that E. coli began to die on the second day, with the survival rate dropping below 50%, and all mice died within about 4 days (survival rate of 0%), indicating that mice could not resist septicemia caused by E. coli infection, and the mortality process was the fastest after infection. Figure 1 A); Simultaneously, 24 hours after infection, the expression levels of TNF-α, IL-6, and IL-1β in mouse serum were significantly increased, indicating that E. coli infection can rapidly trigger a strong systemic inflammatory response in the body, and the excessive release of inflammatory factors may be an important reason for septic shock and death in mice. Figure 1 B, C, and D); Pathological examination of liver, spleen, and kidney sections revealed: in the Enterobacter infection group, liver cells showed edema, necrosis, and nuclear fragmentation, accompanied by extensive infiltration of lymphocytes and granulocytes; renal tubular epithelial cells showed vacuolar degeneration; in the spleen, the white pulp showed lymphocyte necrosis, nuclear pyknosis, fragmentation, and slight hemorrhage, with a small amount of surrounding fibrous connective tissue hyperplasia; the red pulp was congested, and a large amount of basophilic granules were visible. Figure 1 E).

[0036] In contrast, the survival rate of mice treated with AMP1 fluctuated slightly after infection, but remained consistently above 90%, significantly higher than that of the infection-only group. This indicates that AMP1 has a significant protective effect against E. coli infection and can effectively reduce mortality. Figure 1 A); The expression levels of TNF-α, IL-6, and IL-1β in the serum of mice in the AMP1-treated group were significantly lower than those in the infection group, indicating that AMP1 can alleviate the cytokine storm induced by infection by inhibiting the excessive secretion of inflammatory factors. Figure 1 B, C, and D); meanwhile, pathological results of the liver, spleen, and kidneys showed that AMP1 could effectively alleviate the above-mentioned pathological symptoms (B, C, and D). Figure 1 E).

[0037] As can be seen from the above embodiments, the present invention provides an antimicrobial peptide AMP1 and its applications. This antimicrobial peptide can effectively inhibit Escherichia coli; it can also effectively treat sepsis caused by Escherichia coli infection.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide AMP1, characterized in that, The amino acid sequence of the antimicrobial peptide AMP1 is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide AMP1 according to claim 1 in the preparation of antimicrobial preparations.

3. The application according to claim 2, characterized in that, The inhibitory targets of the formulation include Escherichia coli.

4. The application according to claim 2, characterized in that, The concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

5. The use of the antimicrobial peptide AMP1 according to claim 1 in a formulation for treating sepsis.

6. The application according to claim 5, characterized in that, The sepsis was caused by Escherichia coli infection.

7. The application according to claim 5, characterized in that, The concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

8. An antibacterial preparation, characterized in that, It includes the antimicrobial peptide AMP1 as described in claim 1 and a pharmaceutically acceptable carrier.

9. The antibacterial agent according to claim 8, characterized in that, The concentration of the antimicrobial peptide AMP1 in the formulation is 5~20 mg / mL.

Images