A pearl-capsule antibacterial peptide and application thereof

Abstract

The application discloses a pearl capsule antibacterial peptide and application thereof, and belongs to the technical field of biological medicine. The amino acid sequence of the pearl capsule antibacterial peptide is KRLF. The theoretical molecular weight is 562.76 Da, the isoelectric point is 11.54, the net charge is +2 at pH=7.0, and the antibacterial experiment result shows that the polypeptide has broad-spectrum and high-efficiency antibacterial activity and has obvious inhibiting effect on the proliferation of gram-positive bacteria and gram-negative bacteria. The safety evaluation experiment result shows that the antibacterial peptide is beneficial to the proliferation of keratinocytes and fibroblasts and is safe and non-toxic.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a pearl sac antimicrobial peptide and its application. Background Technology

[0002] Antimicrobial peptides, as a class of polypeptides with potent antibacterial activity, have gradually become a research hotspot in the field of biotechnology in recent years. Antimicrobial peptides possess several significant advantages, such as safety and non-toxicity, broad-spectrum antibacterial activity, good stability, low bactericidal concentration, and weak sensitization, making them ideal alternatives to antibiotics. Through unique antibacterial mechanisms, such as cell membrane damage, intracellular bactericidal activity, and immune regulation, antimicrobial peptides exhibit potent inhibitory effects against Gram-positive bacteria, Gram-negative bacteria, and even drug-resistant strains.

[0003] Pearl sacs, primarily consisting of pearl oyster meat and sac fluid, possess high nutritional and medicinal value. Modern research indicates that pearl sacs are rich in bioactive components, such as amino acids, trace elements, and polypeptides, which exhibit excellent antibacterial, antioxidant, and wound-healing properties. Extracting and purifying the active polypeptides from pearl sacs yields antimicrobial peptides from pearl sacs, preserving the nutritional value of this traditional food while significantly enhancing its antibacterial effects and application potential through modern technology. Summary of the Invention

[0004] The purpose of this invention is to provide a pearl sac antimicrobial peptide and its application, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of this invention is a pearl sac antimicrobial peptide, the amino acid sequence of which is KRLF.

[0007] The second technical solution of the present invention is the application of the pearl sac antimicrobial peptide in the preparation of drugs for diseases caused by bacteria.

[0008] The third technical solution of the present invention is the application of the pearl sac antimicrobial peptide in the preparation of food sterilizing agents.

[0009] The fourth technical solution of the present invention is a bactericide, comprising the pearl sac antimicrobial peptide; the bactericide is used to kill Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] The antimicrobial peptide of this invention is derived from Pinctada martensii, with a theoretical molecular weight of 562.76 Da, an isoelectric point of 11.54, and a net charge of +2. It has a significant inhibitory effect on bacteria causing skin infections, and at high concentrations, it is not cytotoxic to fibroblasts and immortalized human epidermal cells. It can be used as a green antimicrobial agent in the fields of food, skin infection and other related microbial control. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0014] Figure 2 This is a 3D diagram of the interaction between KRLF and GyrB, based on the ligand-protein binding pattern obtained from docking; where A represents the binding mode, B represents the binding site, and the green dashed line represents hydrogen bonding.

[0015] Figure 3 This represents the 2D interaction mode between small molecules and proteins. Green dashed lines represent hydrogen bonding, pink / purple dashed lines represent hydrophobic interactions, and brown dashed lines represent electrostatic interactions.

[0016] Figure 4 The effect of different concentrations of KRLF on the survival rate of human immortalized keratinocytes (HaCaT).

[0017] Figure 5 The effect of different concentrations of KRLF on the survival rate of human fibroblasts (HFF-1). Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0024] This invention provides a pearl sac antimicrobial peptide with the amino acid sequence KRLF.

[0025] This invention also provides the application of the pearl sac antimicrobial peptide in the preparation of drugs for bacterial diseases.

[0026] In some specific implementations, the bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

[0027] This invention also provides the application of the pearl sac antimicrobial peptide in the preparation of food sterilizers.

[0028] In some specific implementations, the bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

[0029] This invention also provides a bactericide, including the pearl sac antimicrobial peptide; the bactericide is used to kill Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

[0030] The pearl sac antimicrobial peptide involved in this invention is isolated from the pearl sac through simple enzymatic hydrolysis and advanced purification technology, and possesses a well-defined molecular structure and biological activity. Experimental verification has shown that this antimicrobial peptide exhibits strong inhibitory and bactericidal effects against a variety of bacteria, providing a new option for clinical antimicrobial therapy.

[0031] In terms of applications, pearl sac antimicrobial peptides have a wide range of potential uses. Firstly, in the medical field, these antimicrobial peptides can be used to develop novel antibiotic drugs, particularly for the treatment of drug-resistant bacteria, showing broad market prospects. Secondly, in the food industry, pearl sac antimicrobial peptides can serve as natural preservatives, extending food shelf life and ensuring food safety. Furthermore, in aquaculture, antimicrobial peptides can also be used as feed additives to improve the growth performance and disease resistance of aquatic animals, reducing antibiotic use and lowering drug residues and environmental pollution.

[0032] Pearl sac antimicrobial peptides, as a novel antimicrobial agent, not only meet the current urgent societal demand for new antimicrobial agents but also demonstrate enormous application potential and economic value in multiple fields. This invention will provide solid technical support for the further research and application of pearl sac antimicrobial peptides, promoting their widespread use in medicine, food, and agriculture.

[0033] Example 1

[0034] Preparation of antimicrobial peptides from pearl sacs

[0035] The pearl sac was washed three times with distilled water to remove impurities. The pearl sac was then enzymatically hydrolyzed with papain at a ratio of 10% pearl sac to water, 0.2% enzyme to substrate, pH 6.5, 50℃, and for 2 hours. After enzyme inactivation at 100℃ for 10 minutes, the mixture was centrifuged at 5500 rpm for 15 minutes. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate containing molecules smaller than 1000 Da was collected to obtain the pearl sac antimicrobial peptides. The enriched pearl sac antimicrobial peptides were analyzed by HPLC-MS / MS, yielding 1805 peptide sequences (including the sequence "KRLF") with molecular weights ranging from 344 to 3182 Da and peptide lengths from 4 to 32.

[0036] The amino acid sequence of the pearl sac antimicrobial peptide of this invention is: KRLF, and its mass spectrometry information is as follows: Figure 1 As shown.

[0037] Pearl sac antimicrobial peptides with a purity of 99% can be obtained using existing solid-phase chemical synthesis methods. In this embodiment, the pearl sac antimicrobial peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using a solid-phase synthesis method, and Shanghai Qiangyao Biotechnology Co., Ltd. provided the polypeptide molecular weight, HPLC, and MS detection information. The physicochemical parameters of the pearl sac antimicrobial peptides are: 4 amino acid residues, relative molecular weight of 562.72 Da, isoelectric point, and net charge of +2.

[0038] Example 2

[0039] Molecular docking screening of pearl capsule antimicrobial peptides

[0040] (1) The protein crystal structure of GyrB was downloaded from the PDB database (PDB ID: 4URO). Other material structures were deleted, and only the GyrB protein structure was retained for docking. The 3D structure of KRLF was constructed using PyMOL 2.5.5, and energy minimization was performed using Chem3D 14.0 under the MMFF94 force field. Molecular docking was performed using AutoDockVina 1.2.3 software. Before docking, all receptor proteins were treated using PyMol 2.5.5, including the removal of water molecules, salt ions, and small molecules. Then, the docking box was constructed. The center of the docking box was defined based on the position of the crystal ligand using the PyMol plugin center_of_mass.py, and the side length of the box was set to 22.5 angstroms. In addition, ADFRsuite 1.0 was used to convert all processed small molecules and receptor proteins into the PDBQT format required for docking with AutoDockVina 1.1.2. During docking, the demi-macro level of the global search was set to 20, and the other parameters were kept at their default settings. The docking conformation with the highest score was the binding conformation. Finally, PyMol 2.5.5 and Discovery Studio Viewer were used to visualize and analyze the docking results.

[0041] (2) Molecular docking screening of monomers: The binding energies of peptide KRLF and GyrB are shown in Table 1. According to the binding energies, the binding of KRLF and GyrB is very stable (the hydrogen bonding between KRLF and GyrB includes GLU58, ASP81, LEU123, HIS124, PRO87, ILE102, VAL105, and VAL119, and the electrostatic interaction with RUNX2 includes LEU123, LEU123, HIS124, and ASP81 (see Table 1)). Figure 2-3 ).

[0042] Table 1. Molecular docking analysis of the pearl sac antimicrobial peptide AutoDock Vina identified by HPLC-MS / MS.

[0043] peptides Quality (Da) length mass-to-charge ratio Docking score (kcal / mol) KRLF 562.72 4 605.3811 -8.463

[0044] (3) Monomer synthesis

[0045] The synthetic peptide KRLF with a purity of over 98% was obtained from Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China).

[0046] Example 3

[0047] Antibacterial activity assay of antimicrobial peptides from pearl sac

[0048] This embodiment of the antibacterial activity assay involves minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Six strains were selected as test subjects: Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Micrococcus luteus (ATCC 4698), Escherichia coli (ATCC 25922), methicillin-resistant Staphylococcus aureus (ATCC 43300), and Aeromonas hydrophila (ATCC 7965). The minimum inhibitory concentration was determined using the microbroth dilution method. The above bacterial strains were inoculated into Muller-Hinton broth and cultured at suitable temperatures for 18-24 hours until the logarithmic growth phase. The bacterial cultures were then diluted to approximately 1×10⁻⁶ using fresh Muller-Hinton broth. 6 CFU / mL, ready for use. Add 100 μL of the above bacterial suspension to a sterile 96-well plate, then add 100 μL of pearl sac antimicrobial peptide dilutions at different concentrations (1, 2, 4, 8, 16, 32, 64, 128 μg / mL) to the 96-well plate sequentially. Incubate the 96-well plate in an incubator for 24 h, and finally measure the absorbance (OD) of each well at 600 nm using a microplate reader. 600 The minimum inhibitory concentration was determined as the absorbance value (OD). 600 The minimum antimicrobial peptide concentration is less than 0.1.

[0049] Minimum bactericidal concentration (MBC) determination: Following the MIC determination method described above, 100 μL of bacterial suspension was taken from each well of the experimental group with an absorbance value <0.1 and added to the corresponding PCA agar plate. The plate was spread evenly and incubated upside down in a constant temperature incubator for 18–24 h. Colony growth was observed. The lowest concentration at which no colony growth occurred was the minimum bactericidal concentration of the pearl capsule antimicrobial peptide against this bacterium.

[0050] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) of the pearl sac antimicrobial peptide against six test bacteria were determined by microdilution, and the results are shown in Table 2. The novel pearl sac antimicrobial peptide provided by this invention exhibits highly efficient antibacterial activity, with MICs of 2-4 μg / mL against *Micrococcus luteus* (ATCC 4698), 2-4 μg / mL against *Staphylococcus aureus* (ATCC 29213), 4-8 μg / mL against methicillin-resistant *Staphylococcus aureus* (ATCC 43300), 4-8 μg / mL against *Escherichia coli* (ATCC 25922), 8-16 μg / mL against *Aeromonas hydrophila* (ATCC 7965), and 16-32 μg / mL against *Pseudomonas aeruginosa* (ATCC 27853).

[0051] Table 2 Results of antibacterial activity assay of pearl pouch antimicrobial peptides

[0052] strains MIC (μg / mL) MBC (μg / mL) Micrococcus luteus (ATCC4698) 2-4 2-4 Staphylococcus aureus (ATCC29213) 2-4 4-8 Methicillin-resistant Staphylococcus aureus (ATCC43300) 4-8 4-8 Escherichia coli (ATCC25922) 4-8 8-16 Aeromonas hydrophila (ATCC 7965) 8-16 16-32 Pseudomonas aeruginosa (ATCC27853) 16-32 32-64

[0053] Example 4

[0054] (1) Effects of pearl cyst peptides on human keratinocytes

[0055] HaCaT cells were cultured in complete medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, at 37°C and 5% CO2) at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. After cell adhesion, the cells were treated with different concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128 μg / mL) of pearl sac peptide and incubated for 24 h and 48 h. The cell proliferation activity of pearl sac peptide was detected by CCK8 assay.

[0056] The results are as follows Figure 4 As shown, a series of concentrations of pearl sac peptides were used to treat cells for 24 hours, and cell viability was detected by CCK8. The results showed that pearl sac antimicrobial peptides could promote the proliferation of HaCaT cells and had no adverse effects on human keratinocytes.

[0057] (2) Effects of pearl cyst peptides on human fibroblasts

[0058] HFF-1 cells were cultured in complete medium (supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, at 37°C and 5% CO2) at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. After cell adhesion, the cells were treated with different concentrations (0, 1, 2, 4, 8, 16, 32, 64, 128 μg / mL) of pearl sac peptide and incubated for 24 h and 48 h. The cell proliferation activity of pearl sac peptide was detected by CCK8 assay.

[0059] The results are as follows Figure 5 As shown, a series of concentrations of pearl sac peptides were used to treat cells for 24 hours, and cell viability was detected by CCK8. The results showed that pearl sac antimicrobial peptides could promote the proliferation of HFF-1 cells and had no adverse effects on human fibroblasts.

[0060] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of a pearl-cocoon antibacterial peptide in the preparation of a medicament for bacterial infection, characterized in that, The amino acid sequence of the pearl sac antimicrobial peptide is KRLF.

2. Use according to claim 1, characterized in that, The bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

3. Use of the pearl-cocoon antibacterial peptide in the preparation of a food sterilizing agent, characterized in that, The amino acid sequence of the pearl sac antimicrobial peptide is KRLF.

4. Use according to claim 3, characterized in that, The bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila.

5. A bactericide characterized by comprising: The product includes pearl pouch antimicrobial peptides; the bactericide is used to kill Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Aeromonas hydrophila; the amino acid sequence of the pearl pouch antimicrobial peptides is KRLF.

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