Lysozyme and its derived antibacterial peptides and use thereof in the preparation of antibacterial medicaments

Abstract

The application discloses lysozyme and an antibacterial peptide derived from the lysozyme and application of the antibacterial peptide in preparation of antibacterial drugs. Based on a lysozyme prediction model, 1,640 protein sequences collected in a rumen Haementa ventalina genome database are systematically predicted, and finally it is found that lysozyme with an amino acid sequence shown in SEQ ID No. 1 has relatively strong antibacterial activity and stable antibacterial activity and is less affected by environmental factors. The antibacterial peptide region of the lysozyme is analyzed and determined, and three peptide segments with definite antibacterial activity are identified, and the amino acid sequences of the three peptide segments are shown in SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4. The antibacterial peptide provided by the application has relatively strong membrane damage capacity and broad-spectrum antibacterial activity, is more simple in structure compared with the whole protein, is more flexible in synthesis and modification, and is also easier to break through a bacterial drug resistance mechanism.

Description

Technical Field

[0001] This invention relates to lysozyme, and more particularly to lysozyme and antimicrobial peptides derived therefrom, and their use in the preparation of antimicrobial drugs, belonging to the field of lysozyme and its antimicrobial applications. Background Technology

[0002] Lysozyme is a class of natural defense proteins widely found in plants, animals, and microorganisms, exerting its antibacterial effect by hydrolyzing peptidoglycan in bacterial cell walls. In addition to its overall enzymatic activity, increasing research has revealed that lysozyme molecules also contain short peptide fragments with independent antibacterial functions. These antimicrobial peptides typically exhibit strong membrane-disrupting capabilities and broad-spectrum antimicrobial activity, complementing traditional enzymatic mechanisms. Systematic mining of lysozyme sequences can identify potential antimicrobial peptide regions, thereby expanding the application value of lysozyme in the antimicrobial field. Compared to the whole protein, these peptides are structurally simpler, more flexible in synthesis and modification, and more likely to overcome bacterial resistance mechanisms. Therefore, mining antimicrobial peptides in lysozyme not only helps to understand its multi-layered antimicrobial mechanisms but also provides important resources and ideas for developing novel antimicrobial molecules. Summary of the Invention

[0003] One of the objectives of this invention is to provide a lysozyme with antibacterial activity;

[0004] The second objective of this invention is to systematically mine and screen the lysozyme to obtain antimicrobial peptides with antibacterial activity.

[0005] A third objective of this invention is to provide a method for preparing the aforementioned antimicrobial peptide.

[0006] The fourth objective of this invention is to apply the lysozyme or antimicrobial peptide with antibacterial activity to the preparation of antibacterial drugs.

[0007] The above-mentioned objectives of the present invention are mainly achieved through the following technical solutions:

[0008] One aspect of the present invention is to provide a source from Ophryoscolex caudatus The lysozyme RPL187, which has antibacterial activity, has the amino acid sequence shown in SEQ ID No. 1.

[0009] This invention is based on the LysoMiner predictive model for the presence of caudate caterpillars in the rumen of ruminants. Ophryoscolex caudatusSystematic prediction was performed on 1,640 protein sequences included in the SAGT3 genome database. Oxford cup inhibition zone assays showed that RPL187 exhibited significant antibacterial activity against *Micrococcus luteus*, with inhibition zone diameters of 17.0 ± 0.5 mm and 21.0 ± 1.0 mm at concentrations of 1 mg / mL and 2 mg / mL, respectively. Quantitative detection of RPL187 enzyme activity using turbidimetric assays revealed that the control group showed almost no fluctuation in its OD450, while the RPL187 curve showed a significant decrease in OD450 within 20 min, indicating strong antibacterial activity. Different antibacterial activity detection methods showed that RPL187 consistently demonstrated good efficacy, indicating that the antibacterial activity of this protein is relatively stable and less affected by environmental factors.

[0010] Another aspect of the present invention is to systematically screen and mine lysozyme RPL187 with antibacterial activity to obtain antimicrobial peptides with antibacterial activity, the amino acid sequences of which are shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4 respectively.

[0011] This invention, based on the high specific enzymatic activity of RPL187, further analyzes and determines its antimicrobial peptide regions, identifying three peptides with definite antimicrobial activity. Ultimately, the antimicrobial peptide region RPL187-162 was determined to exhibit the most significant antibacterial effect. This invention utilizes an antimicrobial peptide prediction model to predict antimicrobial peptides in lysozyme RPL187 derived from the genome of the rumen-derived cephalopoda. Based on the antimicrobial peptide prediction model, a sliding window of 13 amino acid lengths was set with a step size of 1 AA, and the fragments in each window were qualitatively predicted as antimicrobial peptides. The distribution of the predicted antimicrobial peptide fragments was statistically analyzed, revealing a concentration in three regions of the RPL187 protein: 24–45, 113–140, and 147–174. The highest prediction score from each region was selected for experimental verification, resulting in the verification of three candidate peptides: RPL187-26, RPL187-118, and RPL187-162. For Gram-positive bacteria, Gamboge microspheres... Micrococcus luteus The MIC of RPL187-162 was 2 μM, while the MIC values ​​of RPL187-26 and RPL187-118 were both 32 μM, approximately 6 times higher than RPL187-162, indicating a significantly weaker antibacterial effect. For Gram-positive bacteria such as Staphylococcus aureus... Staphylococcus aureus The MIC value of RPL187-162 was 64 μM, while RPL187-26 and RPL187-118 showed no inhibitory effect on this strain. For Gram-negative bacteria *Escherichia coli*... Escherichia coliCVCC3367 and RPL187-162 have a MIC value of 128 μM and also exhibit certain antibacterial activity, while RPL187-26 and RPL187-118 did not show significant antibacterial ability. In summary, this invention determines that the antimicrobial peptide RPL187-162 exhibits superior antimicrobial performance compared to other candidate peptides in both the antimicrobial spectrum and MIC level.

[0012] Another aspect of the present invention is to provide the encoding gene of the antimicrobial peptide.

[0013] Another aspect of the present invention is to provide an expression cassette containing the encoding gene of the antimicrobial peptide, a recombinant expression vector, or a recombinant host cell containing the recombinant expression vector; wherein the recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.

[0014] The present invention further provides a method for preparing the antimicrobial peptide, comprising:

[0015] (1) The encoding gene of the antimicrobial peptide is operatively linked to the expression regulatory element to construct a recombinant expression vector;

[0016] (2) Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify the crude protein obtained.

[0017] Another aspect of the present invention is to apply the lysozyme and the antimicrobial peptides with antibacterial activity obtained therefrom to the preparation of drugs that inhibit bacterial infection; wherein the bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, the Gram-negative bacteria are preferably Escherichia coli; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus.

[0018] Another aspect of the present invention provides a pharmaceutical composition for inhibiting bacteria, the pharmaceutical composition comprising a therapeutically effective amount of lysozyme or antimicrobial peptide as described in the present invention and a pharmaceutically acceptable carrier or excipient; wherein the bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, the Gram-negative bacteria preferably being Escherichia coli; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus.

[0019] The antimicrobial peptides provided by this invention possess strong membrane-disrupting capabilities and broad-spectrum antimicrobial activity. Compared to whole proteins, they are structurally simpler, more flexible in synthesis and modification, and easier to overcome bacterial resistance mechanisms. This invention provides new candidate molecules for the development of highly effective antimicrobial peptides with low resistance risk, and shows promise for applications in the preparation of new clinical antibacterial drugs.

[0020] Definitions of terms involved in this invention

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0022] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0023] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may be maintained as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell.

[0024] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.

[0025] The term "transformation" refers to the genetic conversion of polynucleotides or polypeptides into host cells by introducing gene-coding molecules into the host cells.

[0026] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in host cells. Attached Figure Description

[0027] Figure 1 The size of the inhibition zone of lysozyme RPL187 against Micrococcus luteus.

[0028] Figure 2 Results of turbidimetric determination of lysozyme RPL187 activity.

[0029] Figure 3 Different fragments of lysozyme RPL187 were screened for their effects on the Gram-positive bacterium Micrococcus luteus. Micrococcus luteus The antibacterial results.

[0030] Figure 4 Different fragments of lysozyme RPL187 were screened for their effectiveness against Gram-positive bacteria, Staphylococcus aureus. Staphylococcus aureus The antibacterial results of 09057.

[0031] Figure 5 Different fragments of lysozyme RPL187 were screened for their activity against Gram-negative bacteria, Escherichia coli. Escherichia coli Antibacterial results of CVCC3367. Detailed Implementation

[0032] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0033] Experimental Example 1: RPL187 Lysozyme Extraction and Activity Detection Experiment

[0034] 1. Experimental Methods

[0035] Based on the LysoMiner lysozyme prediction model, there are caudal caterpillars in the rumen of ruminants. Ophryoscolex caudatus A systematic prediction was performed on 1,640 protein sequences included in the SAGT3 genome database.

[0036] Oxford cup inhibition zone test: A single colony of *Micrococcus luteus* was picked from the target strain and inoculated onto antibiotic-free LB liquid medium. The culture was incubated overnight at 37 ℃ and 200 rpm with shaking to obtain a seed culture. The bacterial suspension was then diluted to OD600 ≈ 0.4 with antibiotic-free LB liquid medium. A bacterial suspension equivalent to 1% of the total solid medium volume was mixed with melted antibiotic-free LB agar medium (containing 1% agar) and poured into a plate pre-laid with a layer of agar water (bottom layer). The agar was allowed to solidify. The Oxford cups were removed, and the sample solution RPL187 was added to the formed wells. The plates were incubated at 37 ℃ overnight, and the diameter of the inhibition zone was observed and measured the next day.

[0037] Turbidimetric assay for lysozyme activity: At room temperature, 50 μL of lysozyme was added to a 96-well plate, followed by 150 μL of prepared *Micrococcus luteus* suspension. The plate was rapidly pipetted and then quickly placed in a microplate reader to measure the absorbance. The absorbance was recorded every 10 minutes for a total of 1 hour. Negative and positive control groups were included in the experiment. The negative control group consisted of 50 μL phosphate buffer with 150 μL of *Micrococcus luteus* suspension; the positive control group consisted of 50 μL egg white lysozyme (HEWL, 0.02 mg / mL) with 150 μL of *Micrococcus luteus* suspension.

[0038] 2. Experimental Results

[0039] Results of the Oxford Cup Antibacterial Zone Experiment ( Figure 1 The results showed that RPL187 had significant antibacterial activity against Micrococcus luteus, with inhibition zone diameters of 17.0 ± 0.5 mm and 21.0 ± 1.0 mm at concentrations of 1 mg / mL and 2 mg / mL, respectively. The negative control group (CK) was 20 mM Tris-Cl (500 mM NaCl, 10% glycerol).

[0040] The amino acid sequence of the RPL187 protein is shown in SEQ ID No. 1:

[0041] MKNKVTDAQMQKMGWKSYNLNDLNNCIKKFGINTVKRIRHFIAQCSHESALGRYTQELGGPSYCSKYDGRRDLGNTQKGDGCKFKGAGYIQLTGRYNYQQFANYIKDKRVMEGVSYVSKKYPWTSAGFWWNKNGMNKLCDSGASVEVITKRVNGGYNGLADRKKYYQRACGIFK (SEQ ID No. 1).

[0042] The enzyme activity of RPL187 was quantitatively detected by turbidimetric assay, with 20 mM Tris-Cl (500 mM NaCl, 10% glycerol) as a negative control.

[0043] The results showed that the curve of the control group had almost no fluctuation, while the OD450 of the curve of RPL187 decreased significantly within 20 min, indicating that RPL187 has strong antibacterial activity.

[0044] According to different antibacterial activity detection methods, RPL187 showed good results, indicating that the antibacterial activity of this protein is relatively stable and less affected by environmental factors. Finally, the candidate protein RPL187 with potential activity was identified.

[0045] Example 2: Identification and Antimicrobial Peptide Activity Detection Test in RPL187 Protein

[0046] 1. Experimental Methods

[0047] Antimicrobial peptide prediction was performed on lysozyme RPL187 from the genome of rumen-derived caudate caterpillars using an antimicrobial peptide prediction model.

[0048] Based on the antimicrobial peptide prediction model, a sliding window with a length of 1 amino acid was set up, and the fragments in each window were used to make qualitative predictions of antimicrobial peptides.

[0049] Minimum inhibitory concentration (MIC) determination: A single colony of the target strain was picked and inoculated into antibiotic-free LB liquid medium and cultured overnight at 37°C. Subsequently, the bacterial culture was diluted 1:100 and cultured further until the logarithmic growth phase (OD2). 600 =0.4–0.6), adjust the concentration to approximately 5×10 5 CFU / mL. 100 μL of bacterial culture was added to 96-well plates containing different concentrations of antimicrobial peptides (1–128 μM, serially diluted twofold), and incubated at 37°C for 16 h. OD was measured. 600 The changes were assessed to evaluate the minimum inhibitory concentration (MIC) of the peptide and its inhibitory effect on bacterial growth.

[0050] 2. Experimental Results

[0051] The distribution of fragments predicted to be antimicrobial peptides was statistically analyzed, and they were found to be concentrated in three regions of the RPL187 protein: 24–45, 113–140, and 147–174. The fragment with the highest prediction score in each region was selected for experimental verification, and a total of three candidate peptides were verified, namely RPL187-26, RPL187-118, and RPL187-162.

[0052] Table 1. Results of prediction and screening of antimicrobial peptides in lysozyme RPL187 using the AREA model.

[0053]

[0054] For Gram-positive bacteria Micrococcus luteus Micrococcus luteus The MIC of RPL187-162 is 2 μM, while the MIC values ​​of RPL187-26 and RPL187-118 are both 32 μM, which is about 6 times higher than that of RPL187-162, indicating a significantly weaker antibacterial effect. Figure 3 For Gram-positive bacteria Staphylococcus aureus Staphylococcus aureus The MIC value of RPL187-162 was 64 μM, while RPL187-26 and RPL187-118 did not show any inhibitory effect on this strain. Figure 4 For Gram-negative bacteria Escherichia coli Escherichia coli CVCC3367 and RPL187-162 both had a MIC value of 128 μM, exhibiting some antibacterial activity, while RPL187-26 and RPL187-118 showed no significant antibacterial activity. Figure 5 ).

[0055] In summary, the antimicrobial peptide RPL187-162 exhibits superior antimicrobial properties compared to other candidate peptides in both antimicrobial spectrum and MIC level, and has good application potential in the clinical preparation of antimicrobial drugs.

Claims

1. An antimicrobial peptide with antibacterial activity was isolated and identified from lysozyme RPL187, characterized in that, The amino acid sequence of the antimicrobial peptide is selected from the amino acids shown in SEQ ID No. 2 or SEQ ID No.

3.

2. An antimicrobial peptide with antibacterial activity was isolated and identified from lysozyme RPL187, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No.

4.

3. Use of the antimicrobial peptide of claim 1 in the preparation of a medicament for inhibiting bacterial infection; wherein the bacteria is Micrococcus luteus.

4. Use of the antimicrobial peptide of claim 2 in the preparation of a medicament for inhibiting bacterial infection; wherein the bacteria are Micrococcus luteus, Staphylococcus aureus, or Escherichia coli.

5. The gene encoding the antimicrobial peptide according to claim 1 or 2.

6. An expression cassette or recombinant expression vector containing the coding gene of claim 5.

7. A recombinant host cell containing the expression cassette or recombinant expression vector of claim 6.

8. A method for preparing the antimicrobial peptide according to claim 1 or 2, characterized in that, include: (1) The encoding gene of the antimicrobial peptide is operatively linked to the expression regulatory element to construct a recombinant expression vector; (2) Transform the recombinant expression vector into host cells, culture the host cells, induce the expression of recombinant protein, and purify the crude protein obtained.

9. A pharmaceutical composition for inhibiting bacteria, said pharmaceutical composition comprising a therapeutically effective amount of the antimicrobial peptide of claim 1 and a pharmaceutically acceptable carrier or excipient; said bacteria being Micrococcus luteus.

10. A pharmaceutical composition for inhibiting bacteria, said pharmaceutical composition comprising a therapeutically effective amount of the antimicrobial peptide of claim 2 and a pharmaceutically acceptable carrier or excipient; said bacteria being Micrococcus luteus, Staphylococcus aureus, or Escherichia coli.

Images