High-efficiency antibacterial peptide of pig intestinal microorganism source as well as preparation method and application of high-efficiency antibacterial peptide

Abstract

The invention discloses a pig intestinal microorganism source efficient antibacterial peptide P13 as well as a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence of the antibacterial peptide is shown as SEQ NO.1, and the antibacterial peptide is prepared through pig intestinal metagenome data mining, bioinformatics prediction screening and solid-phase chemical synthesis. The antibacterial peptide P13 has broad-spectrum antibacterial activity, and is effective on various gram negative and / or positive bacteria such as escherichia coli, staphylococcus aureus, methicillin-resistant staphylococcus aureus and the like; no obvious hemolytic activity exists below 64 mu M, the safety is high, and the stability in a physiological salt environment is high. The action mechanism of the antibacterial peptide P13 is to reduce the flowability of a bacterial membrane, increase the permeability of an outer membrane and destroy the potential of the plasma membrane. The invention breaks through the drug resistance and safety bottlenecks of the traditional antibacterial agent, can be used for preparing a gram negative and / or positive bacterium infection treatment drug, and provides a solution for antibiotic substitution and green animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly efficient antimicrobial peptide derived from porcine intestinal microorganisms, its preparation method, and its application. Background Technology

[0002] The deterioration of pig health caused by intensive farming is becoming increasingly prominent, and the spread of drug resistance due to the overuse of traditional antibiotics is exacerbating the industry crisis. Exploring new disease control strategies is now urgently needed. Antimicrobial peptides, as naturally occurring immunomodulatory molecules in organisms, have become one of the most promising candidates for antibiotic alternatives due to their unique mechanism of action (primarily acting on bacterial cell membranes, less likely to induce drug resistance), rapid metabolism with no residue, and low toxicity to host cells. The gut microbiota, as a key regulator of host health, not only participates in nutrient metabolism and immune regulation but also produces a large number of antimicrobial metabolites through interspecies competition or host interactions. Among these, gut microbial-derived antimicrobial peptides, due to their adaptation to the host's physiological environment and strong specificity, have become a core resource for the research and development of novel antimicrobial peptides.

[0003] In recent years, with the deep integration of metagenomics, bioinformatics, and artificial intelligence, the development of antimicrobial peptides has broken through the limitations of traditional isolation and screening, forming a complete technical system encompassing "metagenomic data mining—bioinformatics prediction—artificial intelligence sequence optimization—in vitro and in vivo experimental verification." Existing research has successfully mined hundreds of novel antimicrobial peptides from the gut microbiota of model animals such as mice and rabbits using deep learning algorithms. However, systematic research on antimicrobial peptides derived from livestock (especially pigs) gut microbiota is still in its early stages. On the one hand, reported antimicrobial peptides from livestock mainly focus on host-secreted defensins and cathelicidin families, with insufficient exploration of antimicrobial peptides from gut microbiota. On the other hand, some discovered microbial-derived antimicrobial peptides suffer from narrow antimicrobial spectra, high hemolytic activity, and poor stability under physiological saline conditions, making it difficult to meet practical application needs. Therefore, screening and optimizing novel antimicrobial peptides from the pig gut microbiota that possess broad-spectrum antimicrobial activity, low toxicity, and high physiological stability has significant theoretical and applied value for promoting green animal husbandry and solving the problem of antibiotic resistance. Summary of the Invention

[0004] Based on the above shortcomings, the purpose of this invention is to obtain a novel, highly efficient antimicrobial peptide P13 derived from porcine intestinal microorganisms through screening and verification. P13 has high bactericidal activity, low hemolytic activity and high physiological salt stability.

[0005] The technical solution adopted in this invention is as follows: The amino acid sequence of a highly efficient antimicrobial peptide P13 derived from porcine intestinal microorganisms is shown in SEQ NO.1.

[0006] Furthermore, its molecular formula is shown in formula (Ⅰ):

[0007]

[0008] Another object of the present invention is to provide the use of P13, a highly effective antimicrobial peptide derived from porcine intestinal microorganisms, in the preparation of medicaments for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0009] Furthermore, the Gram-negative bacteria mentioned are Escherichia coli, Salmonella typhimurium, Salmonella pullorum, or Acinetobacter baumannii.

[0010] Furthermore, the Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus suis, methicillin-resistant Staphylococcus aureus, or Listeria monocytogenes.

[0011] Another object of the present invention is to provide a medicament suitable for treating and / or preventing infections caused by Gram-negative bacteria and / or Gram-positive bacteria, said medicament containing a highly effective antimicrobial peptide P13 of porcine intestinal microorganism origin as described above.

[0012] The present invention has the following advantages and beneficial effects: The present invention obtains highly efficient intestinal microbial-derived antimicrobial peptide P13 from the pig intestinal metagenomics through screening and verification. It has broad-spectrum antimicrobial activity, low hemolytic activity and strong physiological salt stability, which breaks through the drug resistance and safety bottleneck of traditional antimicrobial agents and provides an innovative solution for antibiotic alternative therapy and green animal husbandry. Attached Figure Description

[0013] Figure 1 This is the mass spectrum of the antimicrobial peptide P13.

[0014] Figure 2 This is a high-performance liquid chromatogram of the antimicrobial peptide P13.

[0015] Figure 3 This is a graph showing the hemolytic activity of the antimicrobial peptide P13.

[0016] Figure 4 The figure shows the effect of antimicrobial peptide P13 on bacterial membrane fluidity.

[0017] Figure 5 This figure shows the effect of antimicrobial peptide P13 on bacterial outer membrane permeability.

[0018] Figure 6 The figure shows the effect of antimicrobial peptide P13 on bacterial plasma membrane depolarization. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0020] Example 1

[0021] In this embodiment, a highly efficient intestinal microbial antimicrobial peptide P13 was obtained from a porcine intestinal metagenomic dataset through screening and validation. Its amino acid sequence, actual molecular weight, average hydrophobicity, and charge are shown in Table 1. A porcine intestinal microbial metagenomic dataset (ID: CNP0000824) was downloaded from the CNGBdb database. This dataset covers 787 samples from feces, cecum, ileum, and jejunum. Macrel software was used to predict cationic antimicrobial peptides, yielding candidate cationic peptide sequences. CD-Hit software was used to remove redundant sequences, and a sequence similarity threshold of 70% was set to obtain non-redundant candidate sequences. Amplify and amPEPpy software were used on the non-redundant sequences to obtain high-confidence antimicrobial peptide sequences. The high-confidence sequences were screened according to the following criteria to determine candidate peptides: ≤30 amino acids, no cysteine ​​residues, no four or more consecutive hydrophobic amino acids (meeting the synthetic feasibility rule), and meeting water solubility requirements (meeting the solubility standard). The minimum inhibitory concentration (MIC) of candidate peptides against common pathogenic bacteria was determined using the microbroth dilution method. Among them, the antimicrobial peptide P13 showed the best overall antimicrobial activity.

[0022] Table 1. Antimicrobial peptide P13, actual molecular weight, average hydrophobicity, and charge.

[0023]

[0024] The molecular formula is shown in formula (Ⅰ):

[0025]

[0026] Example 2

[0027] Solid-phase chemical synthesis of antimicrobial peptides: The peptide backbone was prepared sequentially from the C-terminus to the N-terminus using a peptide synthesizer. 1 eq of the first protecting amino acid, 1.5 eq of DIEA, and DMF were reacted with the resin for 2 h. The resin was dried, washed three times with DMF, and then capped with methanol and DIEA for 1 h. The resin was washed again, and Fmoc was removed with a 20% piperidine DMF solution, reacting for 10 min, repeated twice. After washing the resin, 3 eq of the second amino acid, 3 eq of HOBT, and 3 eq of DIC were reacted with DMF for 1.5 h. These two steps were repeated until the last N-terminal amino acid residue was attached and the N-terminal Fmoc was removed. The resin was washed and dried. The resin and peptide side chain protecting groups were cleaved with 95% TFA + 2% Tis + 2% EDT + 1% H2O, reacting for 2 h. The resin was filtered, the filtrate was washed with ice-cold diethyl ether, centrifuged, and the precipitate was collected as the crude product. The crude product was purified by liquid chromatography and lyophilized. The obtained antimicrobial peptides were analyzed by electrospray mass spectrometry (see appendix). Figure 2 The purity of the antimicrobial peptides is greater than 95% (see appendix). Figure 1 ).

[0028] Example 3

[0029] In vitro antibacterial activity, hemolytic activity, and physiological salt stability of antimicrobial peptides were tested.

[0030] (1) In vitro antibacterial activity assay: Single bacterial colonies were picked and cultured overnight in MHB medium, then transferred to fresh MHB medium and grown to mid-log phase. The bacterial suspension was centrifuged and resuspended to a final concentration of 1-5 × 10⁻⁵. 6 CFU / mL. 50 μL of antimicrobial peptides at different concentrations (final concentration 1-64 μM) were added to each well of a 96-well plate, along with an equal volume of bacterial culture. Negative controls (culture medium only) and positive controls (bacteria and culture medium) were also included. The plates were incubated at 37℃ for 18-20 h. The absorbance was measured at 492 nm using a microplate reader to determine the minimum inhibitory concentration (MIC). Results are shown in Table 1.

[0031] Table 1. In vitro antibacterial activity of antimicrobial peptide P13

[0032]

[0033] Note: "-" indicates that no antibacterial activity was detected within the test range, and the same applies below.

[0034] As shown in Table 1, the antimicrobial peptide P13 exhibits high antibacterial activity against common pathogens.

[0035] (2) Hemolytic activity assay: Fresh human red blood cell suspension was collected and diluted 10-fold with PBS (pH=7.4). 50 μL of the solution was then added to the solution.

[0036] Human erythrocyte suspension and equal volumes of antimicrobial peptides at different concentrations (final concentration 1-64 μM) were added to 96-well plates. A negative control (untreated human erythrocyte suspension) and a positive control (human erythrocyte suspension treated with 0.1% Triton X-100) were also included. The plates were incubated at 37°C for 1 hour. After centrifugation (1000g, 5 min, 4°C), 50 μL of supernatant was collected from the mixture and transferred to a new 96-well plate. The absorbance was measured at OD 570 nm using a microplate reader, and the hemolysis rate was calculated. The minimum hemolysis concentration was defined as the antimicrobial peptide concentration that caused a 10% hemolysis rate. Formula: Hemolysis rate (%) = [(sample OD 570 nm) / (sample OD 570 nm)] 570 nm - Negative control OD 570 nm ) / (Positive control OD 570 nm - Negative control OD 570

[0037] nm )]×100%. See attached results. Figure 3 The lower the hemolysis rate, the safer the peptide. Antimicrobial peptide P13 showed no significant hemolytic activity at concentrations below 64 μM, indicating its extremely high potential for clinical application.

[0038] (3) Salt ion stability determination: Escherichia coli (ATCC 25922) was selected as a Gram-negative bacterial model, and Staphylococcus aureus (ATCC 43300) was selected as a Gram-positive bacterial model to detect the stability of antimicrobial peptides under physiological conditions. Salt powder was dissolved in 0.2% BSA solution, and the minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined, following the steps described in Example 2(1). Final salt concentrations: NaCl 150mM, KCl 4.5mM, MgCl2 1mM, NH4Cl

[0039] 6mM, CaCl2 2mM, ZnCl2 8mM, FeCl3 4mM.

[0040] Table 2. Minimum inhibitory concentration (μM) of antimicrobial peptide P13 against E. coli 25922 after salt ion treatment.

[0041]

[0042] As shown in Table 2, under physiological concentration conditions, the monovalent ion Na+... + K + and NH4 + divalent Zn 2+ and trivalent ions Fe 3+ No significant effect on the activity of antimicrobial peptide P13, Ca 2+ and Mg 2+ This resulted in a significant reduction in its antibacterial activity against E. coli ATCC 25922.

[0043] Example 4

[0044] Detection of the mechanism of action of antimicrobial peptides:

[0045] (1) Bacterial membrane fluidity assay: Escherichia coli (E. coli ATCC 25922) was grown to mid-log phase. The bacterial culture was centrifuged and resuspended until the absorbance at 600 nm (OD value) was 0.4. The bacteria were incubated with Laurdan probe (final concentration 20 μM) at 37°C in the dark for 1 h. 50 μL of antimicrobial peptides at different concentrations (final concentrations 0-64 μM) and an equal volume of bacteria and Laurdan mixture were added to a 96-well plate and incubated at 37°C in the dark for 1 h. Fluorescence intensity was measured using a microplate reader at emission wavelengths of 435 nm, 490 nm, and excitation wavelength of 350 nm, and GP was calculated. Formula: GP = (I 435 nm -I 490nm ) / (I 435 nm +I 490 nm (Results are attached.) Figure 4 Compared with the control, as the concentration of antimicrobial peptide P13 increased, the fluidity of bacterial cell membranes decreased and their rigidity increased, and a dose-dependent effect was observed.

[0046] (2) Detection of bacterial outer membrane permeability: Escherichia coli (E. coli ATCC 25922) was grown to mid-log phase. The bacterial culture was centrifuged and resuspended until the absorbance at 600 nm (OD value) was 0.2. The bacteria were incubated with NPN dye (final concentration 10 μM) at 37 °C in the dark for 30 min. 50 μL of antimicrobial peptides at different concentrations (final concentrations 0-64 μM) and an equal volume of the bacterial-NPN mixture were added to a 96-well plate. Fluorescence intensity was measured using a microplate reader at an emission wavelength of 420 nm and an excitation wavelength of 350 nm. Results are attached. Figure 5 The antimicrobial peptide P13 can increase the permeability of bacterial outer membranes in a dose-dependent manner, indicating that it can penetrate bacterial outer membranes.

[0047] (3) Detection of bacterial plasma membrane depolarization: Escherichia coli (E. coli ATCC 25922) was grown to mid-log phase. The bacterial culture was centrifuged and resuspended until the absorbance at 600 nm (OD value) was 0.05. The bacteria were incubated with DiSC3-5 dye (final concentration 0.4 μM) at 37°C in the dark for 1 h, followed by the addition of saturated KCl solution (final concentration 100 mM) and incubation at 37°C in the dark for 30 min. 2 mL of the bacterial culture was added to a 24-well plate, and the fluorescence intensity was measured until the values ​​stabilized. Then, different concentrations of antimicrobial peptides (final concentrations 0-8 μM) were added to the wells, and the fluorescence intensity was measured using a microplate reader at an emission wavelength of 670 nm and an excitation wavelength of 620 nm. Results are attached. Figure 6 The antimicrobial peptide P13 can rapidly increase fluorescence intensity, and this effect is dose- and time-dependent, indicating that it forms ion channels in the bacterial cell membrane and disrupts the membrane potential.

Claims

1. A highly effective antimicrobial peptide P13 derived from porcine intestinal microorganisms, characterized in that: Its amino acid sequence is shown in SEQ NO.

1.

2. The highly effective antimicrobial peptide P13 derived from porcine intestinal microorganisms according to claim 1, characterized in that, Its molecular formula is shown in formula (Ⅰ):

3. The use of the highly effective antimicrobial peptide P13 derived from porcine intestinal microorganisms according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

4. The application according to claim 3, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli, Salmonella typhimurium, Salmonella pullorum, or Acinetobacter baumannii.

5. The application according to claim 3, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, Streptococcus suis, methicillin-resistant Staphylococcus aureus, or Listeria monocytogenes.

6. A drug suitable for treating and / or preventing infections caused by Gram-negative bacteria and / or Gram-positive bacteria, characterized in that, The drug contains P13, a highly effective antimicrobial peptide derived from porcine intestinal microorganisms as described in claim 1.

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