Antibacterial peptide based on shellfish and application thereof

Abstract

The invention relates to an antibacterial peptide based on shellfish and application of the antibacterial peptide. The antibacterial peptide has an amino acid sequence as follows: Lys-Val-Ala-Phe-Val-Lys-Val-Val-Lys-Val-Leu-Gly-Cys-Phe-Lys-Lys-Arg, wherein the amino acid sequence of the antibacterial peptide is shown in the description; meanwhile, the invention discloses application of the antibacterial peptide in preparation of an antibacterial agent and / or an antibacterial drug, and the antibacterial drug adopting the antibacterial peptide as an active component. The antibacterial peptide disclosed by the invention has a definite bactericidal effect and relatively low cytotoxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibacterial peptides, and particularly relates to a kind of antibacterial peptides based on shellfish and application thereof. BACKGROUND

[0002] Antibacterial peptides (Antimicrobial Peptides) are also called host defense peptides, which are a kind of natural small molecule polypeptide substances with antibacterial activity produced in vivo, generally composed of less than 100 amino acids, and have broad-spectrum antibacterial properties, and are important immune defense molecules in the innate immune defense system of multicellular organisms. The source includes human, animal, plant, insect and microorganism, and the antibacterial peptide can also be called "peptide antibiotic", which has the function of antibiotic, and has good inhibition or killing effect on various bacteria, fungi, viruses and parasites. Antibacterial peptides have the characteristics of broad-spectrum antibacterial activity, no drug resistance, no residue, etc., and are a potential high-quality antibiotic substitute, which has great development prospect. The plasma of marine shellfish contains a variety of pure natural active peptides, which have potential antibacterial properties, and provides valuable resources for the development of new antibacterial drugs, which requires further comprehensive research to find the technology for treating multiple drug-resistant bacteria (CR-AB). SUMMARY

[0003] In view of the above deficiencies, the present application provides an antibacterial peptide based on shellfish and application thereof.

[0004] The technical scheme adopted by the present application to solve the technical problem is: an antibacterial peptide based on shellfish, having the following amino acid sequence: Lys-Val-Ala-Phe-Val-Lys-Val-Val-Lys-Val-Leu-Gly-Cys-Phe-Lys-Lys-Arg.

[0005] According to the application of the aforementioned antibacterial peptide in the preparation of antibacterial agents and / or antibacterial drugs, the bacteria are gram-negative bacteria.

[0006] As a preferred embodiment, the gram-negative bacteria are carbapenem-resistant Acinetobacter baumannii.

[0007] An antibacterial drug contains the antibacterial peptide as described above as an active ingredient.

[0008] The present application has the advantages that: the antibacterial peptide has low cytotoxicity to mammalian cells in a lower effective concentration range, has clear bactericidal effect and low cytotoxicity, and can be used as a drug against CR-AB. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1This is a structural and helical diagram of the antimicrobial peptide (OAMP) of the present invention; Figure 2 This is a sequence alignment diagram of the antimicrobial peptide of this invention with other antimicrobial peptides; Figure 3 This is a comparison diagram of the hydrophobicity and positive charge of the antimicrobial peptide of this invention and the natural antimicrobial peptide (AMP12); Figure 4 This is a diagram illustrating the pharmacodynamic effects of the antimicrobial peptide of this invention in combination with different antibiotics against CR-AB strains. Figure 5 This is a cytotoxicity evaluation diagram of the antimicrobial peptide of this invention. Detailed Implementation

[0010] To more clearly illustrate the objectives, technical solutions, and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It is clear and complete that the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0011] The various databases, analysis tools, and software mentioned in this invention, as well as their usage methods, are all prior art in this application and will not be described in detail here.

[0012] Examples of embodiments of the present invention Figures 1 to 3 As shown, a shellfish-based antimicrobial peptide (OAMP) has the following amino acid sequence: Lys-Val-Ala-Phe-Val-Lys-Val-Val-Lys-Val-Leu-Gly-Cys-Phe-Lys-Lys-Arg, e.g. Figure 1 As shown, the left side is the structural diagram of the antimicrobial peptide, and the right side is its helical diagram. In the helical diagram, yellow and gray represent nonpolar hydrophobic amino acids, blue represents basic and charged amino acids, red represents acidic amino acids, and the arrows indicate the hydrophobic direction. Figure 2The image shows the sequence alignment of the antimicrobial peptide (OAMP) with AMP12 and Arenicin-1. Arenicin is an antimicrobial peptide sequence extracted from the marine algae Arenicola marina. In the alignment diagram, conserved (*) or partially conserved (:&.) residues are colored according to their biochemical characteristics. AMP12 is a predicted antimicrobial peptide sequence extracted from existing oyster hemolymph peptide data in the dbAMP database. These extracted peptide sequences were then input into the CAMP and Antimicrobial Peptide Scanner vr.2 databases, and peptides with scores higher than 0.5 were selected as candidate peptides. After bactericidal experiments with pathogenic microorganisms, AMP12 with the strongest antimicrobial activity was obtained. The antimicrobial peptide (OAMP) is an antimicrobial peptide designed by evaluating and optimizing the antimicrobial peptide sequence of AMP12 using the Silico tool (https: / / aps.unmc.edu / design / improve, Antimicrobial Peptide Designer), and named OAMP. The application of OAMP in the preparation of antimicrobial agents and / or antimicrobial drugs targets Gram-negative bacteria, specifically carbapenem-resistant Acinetobacter baumannii.

[0013] An antimicrobial drug containing, as described above, an antimicrobial peptide as an active ingredient.

[0014] Example 1: Synthesis and structural performance verification of the antimicrobial peptide OAMP: 1) Prediction of antimicrobial activity of oyster plasma antimicrobial peptides: First, dbAMP (Database of Antimicrobial Peptides) (https: / / awi.cuhk.edu.cn / ~AMPActiPred / ) was used to predict raw oyster plasma peptide data and construct a dataset of peptide sequences with antimicrobial activity. Then, these predicted peptide sequences were applied to the CAMP (https: / / camp.bicnirrh.res.in / ) platform, and their antimicrobial activity was further validated using a prediction model based on the Random Forest (RF) algorithm. Simultaneously, the antimicrobial peptide scanner vr.2 (https: / / www.dveltri.com / ascan / ) was used to score and analyze the predicted peptide sequences, selecting those sequences with scores greater than 0.5 across multiple databases as our final dataset. Finally, the Silico tool (https: / / aps.unmc.edu / design / improve, Antimicrobial Peptide) was used. The Designer evaluated and optimized the antimicrobial peptide sequences in the final dataset to select peptides with strong antimicrobial activity. These selected peptide sequences were then chemically synthesized for subsequent antimicrobial activity verification experiments. The selected peptides were then subjected to bactericidal experiments with pathogenic microorganisms to select AMP12, which had the strongest antimicrobial activity.

[0015] 2) Design, synthesis, and structural performance verification of the antimicrobial peptide OAMP: Based on the amino acid composition of peptide AMP12, an antimicrobial peptide OAMP was designed, and OAMP samples were obtained through chemical artificial synthesis, such as... Figures 1 to 3 As shown, the antimicrobial activity of OAMP was evaluated and compared using entries in the APD3 (Antimicrobial Peptide Database); the clusterW software was used to align the sequences of OAMP with similar antimicrobial peptides to assess the similarity and differences in their structures; the three-dimensional structure of OAMP was analyzed using the online peptide structure prediction software PEP-FOLD; and the hydrophobicity and net charge number of OAMP were analyzed using the HeliQuest tool (https: / / heliquest.ipmc.cnrs.fr / ).

[0016] Example 2, Synergistic Experiment of OAMP and Antibiotics: like Figure 4As shown, synergistic experiments were conducted on OAMP and antibiotics such as meropenem, tigecycline, and polymyxin. The drug interactions of OAMP with each of the three antibiotics in combination with five CR-AB strains were analyzed using a micro-broth checkerboard dilution method. In the experiments, CAMHB broth was used. The experimental concentrations of meropenem, tigecycline, and polymyxin ranged from 0 mg / L to 32 mg / L, and different concentrations were prepared using CAMHB broth. The experimental concentrations of OAMP ranged from 0 µM to 64 µM, and different concentrations were prepared using DMSO as the solvent. Columns 1-11 contain serially 2-fold dilutions of the antibiotics (meropenem, tigecycline, and polymyxin, respectively), rows AG contain serially 2-fold dilutions of OAMP, column 12 contains only serially diluted OAMP, and row H contains only serially diluted antibiotics. The five CR-AB strains were strains with five different drug resistance gene types, obtained from different patients and screened using existing gene detection methods. - 1 belongs to the OXA-23 type, without an external discharge pump; CR-AB - 2 belongs to the OXA-58 type; CR-AB - 3 belongs to the NDM type of metallo-β-lactamase; CR-AB - 4 belongs to the OXA-23 type and is accompanied by a PmrB gene mutation; CR-AB - 5 belongs to the OXA-23 type and is equipped with an AdeABC effluent pump. As can be seen from the figure, OAMP exhibits a low MIC value, indicating that it has strong antibacterial activity; the synergistic effect of OAMP in combination with commonly used clinical antibiotics reduces antibiotic dosage, decreases side effects, and has the potential to effectively combat drug-resistant bacteria.

[0017] Example 3, Cytotoxicity analysis of the antimicrobial peptide OAMP: like Figure 5 As shown in the figure, the cytotoxicity of OAMP was analyzed using the MTT assay. The MTT assay is an existing technology, and its methods and steps will not be described in detail here. The cells selected in this example are RAW264.7 cells; the concentrations of OAMP are 0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM respectively; the results in the figure show that within the concentration range effective against CR-AB, its cytotoxicity is relatively low, which meets the requirements.

[0018] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A shellfish-based antimicrobial peptide, characterized in that, It has the following amino acid sequence: Lys-Val-Ala-Phe-Val-Lys-Val-Val-Lys-Val-Leu-Gly-Cys-Phe-Lys-Lys-Arg.

2. The use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial agents and / or antimicrobial drugs, wherein the bacteria are Gram-negative bacteria.

3. The application according to claim 2, characterized in that, The Gram-negative bacterium is carbapenem-resistant Acinetobacter baumannii.

4. An antibacterial drug, characterized in that, It contains the antimicrobial peptide as described in claim 1 as an active ingredient.

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