A barchatus Nat10 antibacterial peptide, its coding sequence and use and preparation method

Abstract

The application discloses a bocachito Nat10 antibacterial peptide, a coding sequence and use and a preparation method thereof, and relates to a method based on multiomics, a biological big data based on multiomics data of bocachito, screening and identifying an antibacterial peptide precursor gene sequence, and obtaining an amino acid sequence of the bocachito Nat10 antibacterial peptide through translation design, as shown in SEQ ID NO: 3. Through activity simulation prediction and in-vitro experiment verification, the bocachito Nat10 antibacterial peptide shows inhibitory effects on the growth activities of Streptococcus agalactiae, Salmonella enteritidis, Staphylococcus aureus, Vibrio anguillarum and Escherichia coli, especially on Staphylococcus aureus, and shows the best antibacterial effect at a concentration of 2.5 micromoles per liter. The bocachito Nat10 antibacterial peptide can be used for preparing various bactericidal preparations, including bacteriostatic agents, medicines or additives, and has good practical value and social ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a basa fish Nat10 antimicrobial peptide, its coding sequence, uses, and preparation method. Background Technology

[0002] The Bocourti catfish (Pangasius bocourti), commonly known as the basa fish, belongs to the family Panthidae and the genus Panthidae. Basa fish are characterized by rapid growth, large size, high yield, broad diet, ease of rearing, lack of intramuscular bones, and ease of processing, thus possessing high economic value and being an important economic fish species in Southeast Asia. Simultaneously, basa fish exhibit extremely strong tolerance to low oxygen and disease resistance, indicating a powerful innate immune system. Further research into its defense system may have potential value in enriching the green resource of antimicrobial peptides. In 2021, BGI Marine Science published the world's first basa fish chromosome-level genome in the international journal *Genomics*. The research team obtained a 731.7 Mb chromosome-level reference genome through whole-genome sequencing and assembly of the basa fish (Pangasianodon hypophthalmus), with contigs and scaffold N50 of 3.5 Mb and 29.5 Mb, respectively. This achievement lays the foundation for genomic research on basa fish, not only elucidating the genetic basis of basa fish but also providing rich data resources for the study of functional proteins in basa fish.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides with antimicrobial activity, widely found in animals, plants, and microorganisms. They are an important component of the innate immune system in organisms, capable of resisting the invasion of external pathogens. The antibacterial mechanism of antimicrobial peptides is mainly through the positively charged antimicrobial peptides acting on the negatively charged phospholipid molecules of the bacterial plasma membrane, causing membrane depolarization or the formation of "pores," allowing them to enter the cell and act on DNA or RNA, directly killing the bacteria. Because of this unique mechanism of action, bacteria do not have time to undergo genetic mutations to compensate for this damage, making it less likely for superbugs to develop. Therefore, with the increasing severity of the global antibiotic resistance problem, antimicrobial peptides are considered a green biological resource with the greatest potential to replace antibiotics.

[0004] In recent years, with the continuous development of high-throughput sequencing (HTS) technology, it has demonstrated enormous application potential and market value in multiple fields. Furthermore, the increasingly close integration of HTS with bioinformatics, artificial intelligence, and other fields has boosted the efficiency of data processing and analysis, and has also shown its role in the prediction and synthesis of antimicrobial peptides. For example, Chinese patent applications CN116024223A and CN115850427A disclose the preparation method and application of conopod double α-helix (β-sheet) antimicrobial peptides. Based on a multi-omics approach, the precursor gene sequences of antimicrobial peptides are identified from conopod multi-omics datasets, and then, through translational design, multiple active antimicrobial peptide amino acid sequences are artificially synthesized, providing a new approach to obtaining antimicrobial peptides. However, current identification of antimicrobial peptides from animals mostly focuses on those that produce venom. For example, 15 short venom peptide sequences from 5 ant species have been reported to have antimicrobial activity; 13 short venom peptide sequences from 7 bee species have been reported to have antibody activity; 10 short venom peptide sequences from 9 snake species have been reported to have antibody activity; and 55 active antimicrobial peptides have been found from more than 30 centipede species. However, the number of antimicrobial peptides identified from fish is not large.

[0005] To date, over 3,000 antimicrobial peptides have been identified from animals, fungi, plants, and bacteria. Most of these peptides consist of 10–100 amino acid residues, and the majority are cationic (positively charged) or amphiphilic (hydrophilic or hydrophobic) α-helix / β-sheet peptide molecules. However, the naturally occurring amounts of these identified antimicrobial peptides in organisms are extremely rare, making their isolation and purification very difficult and hindering their development and utilization. Furthermore, many of these antimicrobial peptides suffer from limited antimicrobial function, narrow antimicrobial spectrum, and insignificant antibacterial effects. Additionally, some antimicrobial peptides exhibit toxicity; for example, some possess high hemolytic activity, causing red blood cell rupture and harm to living organisms when used for in vivo disinfection.

[0006] Nat10 (N-Acetyltransferase 10) is a multifunctional enzyme with dual activities as both a protein acetyltransferase and an RNA acetyltransferase, and it is widely involved in cellular physiological and pathological processes. Current functional studies on Nat10 mainly focus on its role in immune regulation, cell division and aging, DNA damage repair, and the treatment of tumors, autoimmune diseases, and inflammation; however, research on its antibacterial activity has not yet been reported. Summary of the Invention

[0007] To address the aforementioned issues of insufficient existing bio-derived antimicrobial peptide resources, particularly the scarcity of research on antimicrobial peptides from basa fish, the lack of reported antimicrobial peptide sequences, and the absence of research on the antibacterial function of Nat10, this invention provides a basa fish Nat10 antimicrobial peptide, its encoding sequence, uses, and preparation method. Based on a multi-omics approach, antimicrobial peptide precursor gene sequences are identified from basa fish multi-omics datasets through biological big data screening. Then, through translational design, the antimicrobial-active basa fish Nat10 antimicrobial peptide is artificially synthesized. Activity simulation prediction and in vitro experimental verification show that this basa fish Nat10 antimicrobial peptide exhibits inhibitory effects on the growth of *Streptococcus agalactiae*, *Salmonella enteritidis*, *Staphylococcus aureus*, *Vibrio anguillarum*, and *Escherichia coli*. The specific technical solution is as follows:

[0008] One objective of this invention is to provide a basa fish Nat10 antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO:3.

[0009] The aforementioned basa fish Nat10 antimicrobial peptide contains an α-double helix structure.

[0010] The second objective of this invention is to provide a basa fish Nat10 antimicrobial peptide precursor, the amino acid sequence of which is shown in SEQ ID NO:2.

[0011] The third objective of this invention is to provide a basa fish Nat10 antimicrobial peptide precursor gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0012] The fourth objective of this invention is to provide an application of basa fish Nat10 antimicrobial peptide in the preparation of antibacterial products, wherein the bacteria include Streptococcus agalactiae, Salmonella enteritidis, Staphylococcus aureus, Vibrio anguillarum, and Gram-negative Escherichia coli.

[0013] The aforementioned application of basa fish Nat10 antimicrobial peptide in the preparation of antibacterial products includes at least one of the following: preparation of antibacterial agents, preparation of drugs, or preparation of additives.

[0014] The aforementioned application of basa fish Nat10 antimicrobial peptide in the preparation of antibacterial products is as follows: for products against Staphylococcus aureus, the concentration of basa fish Nat10 antimicrobial peptide is 2.5 μmol / L or higher; for products against Gram-positive Streptococcus agalactiae or Gram-negative Escherichia coli, the concentration of basa fish Nat10 antimicrobial peptide is 5 μmol / L or higher; for products against Vibrio anguillarum, the concentration of basa fish Nat10 antimicrobial peptide is 10 μmol / L or higher; and for products against Salmonella enteritidis, the concentration of basa fish Nat10 antimicrobial peptide is 20 μmol / L or higher.

[0015] As a preferred technical solution, the aforementioned application of basa fish Nat10 antimicrobial peptide in the preparation of antibacterial products is as follows: In products used against Staphylococcus aureus, the concentration of basa fish Nat10 antimicrobial peptide is 2.5 μmol / L to 40 μmol / L; in products used against Gram-positive Streptococcus agalactiae or Gram-negative Escherichia coli, the concentration is 5 μmol / L to 40 μmol / L; in products used against Vibrio anguillarum, the concentration is 10 μmol / L to 40 μmol / L; and in products used against Salmonella enteritidis, the concentration is 20 μmol / L to 40 μmol / L.

[0016] As a preferred technical solution, the aforementioned application of basa fish Nat10 antimicrobial peptide in the preparation of antibacterial products, when used for live disinfection, involves a concentration of basa fish Nat10 antimicrobial peptide of 2.5 μmol / L to 5 μmol / L in the antimicrobial product.

[0017] The fifth objective of this invention is to provide a method for preparing basa fish Nat10 antimicrobial peptide. This method involves identifying antimicrobial peptide precursor gene sequences from a basa fish multi-omics dataset using homology alignment, translating the identified precursor genes into antimicrobial peptide precursor proteins, and then designing the potential mature active peptide regions of the translated precursor proteins using known antimicrobial peptides as reference sequences to obtain the amino acid sequence of the basa fish Nat10 antimicrobial peptide. The basa fish multi-omics dataset includes the basa fish genome, transcriptome dataset, and protein dataset. The basa fish genome is an assembled basa fish T2T genome. The transcriptome and protein datasets are derived from eight tissues of the basa fish: eyes, gills, gonads, kidneys, liver, muscle, skin, and spleen.

[0018] The beneficial effects of this invention are:

[0019] 1) This invention utilizes a multi-omics approach, screening and identifying antimicrobial peptide precursor gene sequences from a basa fish multi-omics dataset at the biological big data level. Through translational design, the amino acid sequence of the basa fish Nat10 antimicrobial peptide is obtained. Activity simulation prediction and in vitro experimental verification demonstrate that the basa fish Nat10 antimicrobial peptide exhibits inhibitory effects on the growth of *Streptococcus agalactiae*, *Salmonella enteritidis*, *Staphylococcus aureus*, *Vibrio anguillarum*, and *Escherichia coli*, particularly against *Staphylococcus aureus* at a concentration of 2.5 μmol / L. It exhibits the best antibacterial effect at a concentration of 2.5 μmol / L against Gram-positive Streptococcus agalactiae and Gram-negative Escherichia coli, with the best effect observed at 5 μmol / L. Against Vibrio anguillarum, it shows good antibacterial effect at 5 μmol / L, with the best effect observed at 10 μmol / L. Even against Salmonella enteritidis, it exhibits good antibacterial effect at a concentration of 20 μmol / L. It can be used to prepare various bactericidal preparations, including bacteriostatic agents, drugs, or additives.

[0020] 2) This invention presents a novel antimicrobial peptide from basa fish, namely the Nat10 antimicrobial peptide. It is the first time that Nat10 has been shown to possess antimicrobial activity, and it is also the first time that the basa fish genome has been thoroughly explored, providing a new source for antimicrobial peptides. This valuable biological genetic resource provides a solid theoretical foundation for the in-depth development of novel marine antimicrobial drugs and the research on the clinical therapeutic applications of antimicrobial peptides. It also further enriches the existing antimicrobial peptide database, providing more options for biologically derived antibiotics to replace traditional antibiotics.

[0021] 3) The basa fish Nat10 antimicrobial peptide of this invention has a low hemolysis rate. The hemolytic activity is 5.53% at 5 μmol / L and almost 0 at 2.5 μM. When the concentration is controlled at 5 μmol / L or below, it can be used to disinfect live bacteria, providing a better guarantee for green and healthy aquaculture and other aquaculture industries. It has good application value and social and ecological value. Attached Figure Description

[0022] Figure 1 The three-dimensional structure diagram of the Nat10 antimicrobial peptide from basa fish was constructed.

[0023] Figure 2 The results of liquid chromatography analysis of synthesized basa fish Nat10 antimicrobial peptide;

[0024] Figure 3 Mass spectrometry results of synthesized basa fish Nat10 antimicrobial peptides;

[0025] Figure 4 The inhibitory effects of 2.5μM, 5μM, 10μM, 20μM, and 40μM basa fish Nat10 antimicrobial peptide on Streptococcus agalactiae were evaluated.

[0026] Figure 5 The inhibitory effects of 2.5μM, 5μM, 10μM, 20μM, and 40μM basa fish Nat10 antimicrobial peptide on Salmonella enteritidis were evaluated.

[0027] Figure 6 The inhibitory effects of 2.5μM, 5μM, 10μM, 20μM, and 40μM basa fish Nat10 antimicrobial peptide on Staphylococcus aureus were evaluated.

[0028] Figure 7 The inhibitory effects of 2.5μM, 5μM, 10μM, 20μM, and 40μM basa fish Nat10 antimicrobial peptide on Escherichia coli were evaluated.

[0029] Figure 8 The inhibitory effects of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM basa fish Nat10 antimicrobial peptide on Vibrio anguillarum were evaluated.

[0030] Figure 9 The graph shows the hemolysis rate test results of 2.5μM, 5μM, 10μM, 20μM, and 40μM basa fish Nat10 antimicrobial peptides. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described in detail below with reference to examples. Reagents not provided in the following experiments are all commercially available reagents, and methods not described in detail are all conventional and well-known experimental methods. The abbreviations used in the full-length amino acid sequences are abbreviations commonly used by those skilled in the art. Specifically, R represents arginine (Arg), L represents leucine (Leu), G represents glycine (Gly), N represents asparagine (Asn), C represents cysteine ​​(Cys), T represents threonine (Thr), V represents valine (Val), M represents methionine (Met), A represents alanine (Ala), K represents lysine (Lys), F represents phenylalanine (Phe), S represents serine (Ser), P represents proline (Pro), E represents glutamic acid (Glu), I represents isoleucine (Ile), Q represents glutamine (Gln), and W represents tryptophan (Trp).

[0032] The specific implementation method is as follows:

[0033] Example 1: Obtaining the precursor gene DNA and encoding precursor protein sequence of the Nat10 antimicrobial peptide from basa fish

[0034] 1) Preparation of multi-omics datasets

[0035] The multi-omics dataset includes a basa fish genome, transcriptome dataset, and protein dataset. The basa fish genome is an assembled T2T genome. The transcriptome and protein datasets were obtained from samples of eight basa fish tissues: eyes, gills, gonads, kidneys, liver, muscle, skin, and spleen. The T2T gene is a complete genome without any deletions, offering higher integrity. Since genes in different organs are specifically expressed, using these eight tissues—eyes, gills, gonads, kidneys, liver, muscle, skin, and spleen—is to collect samples from as many organs as possible and obtain gene sequences expressed in all organs for comprehensive analysis and extraction.

[0036] The protein-coding regions of each transcript were predicted using TransDecoder software, and the transcriptional level of genes was calculated using FPKM values. All protein-coding sequences from the genome and transcriptome were translated into amino acids to construct a comprehensive protein set for basa fish.

[0037] 2) Screening antimicrobial peptide precursor gene DNA sequences from multi-omics datasets

[0038] First, an index library for sequence alignment was established using the gene set annotated from the basa fish genome. Potential AMP precursor genes were predicted from the genome using the TBLASTN algorithm in the Blast software (e-value less than 1e-5), retaining sequences with an alignment rate greater than 60%. The AMPml software (2021, China, 2021SR0424886) was used to predict the activity of segments containing potential antimicrobial peptide protein sequences, retaining alignment sequences with a value greater than 0.5 in both the CTDD and PAAC models.

[0039] Then, using the same alignment method, antimicrobial peptide precursor genes were predicted from transcriptome datasets of eight transcriptomes. Subsequently, the common antimicrobial peptide precursor gene sequences in the genomic and transcriptome datasets were aligned with the proteome data to build an index library. Blastp software was used for sequence similarity alignment to obtain the final common antimicrobial peptide sequence set of the three omics datasets, where sequences with an alignment length greater than 60% and an alignment rate greater than 80% were considered reliable sequences.

[0040] Finally, we obtained the DNA sequence of a potential antimicrobial peptide precursor gene from genomic, transcriptomic, and proteomic datasets, as shown in SEQ ID NO:1. After alignment analysis, we named it the basa fish Nat10 antimicrobial peptide precursor gene. Using transcription rules, we translated the above antimicrobial peptide precursor gene into the corresponding precursor protein sequence, namely the basa fish Nat10 antimicrobial peptide precursor, whose amino acid sequence is shown in SEQ ID NO:2.

[0041] SEQ ID NO:1: Nucleic acid coding sequence of basa fish nat10 antimicrobial peptide precursor gene

[0042]

[0043] SEQ ID NO:2: Amino acid sequence of basa fish nat10 antimicrobial peptide precursor gene

[0044]

[0045] Example 2: Prediction and Synthesis of the Active Structure of Nat10 Antimicrobial Peptide from Basa Fish

[0046] The 2927 validated antimicrobial peptides in the public antimicrobial peptide database APD3 were used as reference sequences.

[0047] Based on the antimicrobial peptide precursor protein sequence SEQ ID NO:2 identified from the multi-omics dataset in Example 1, and referring to the known antimicrobial peptide sequences in the public antimicrobial peptide database APD3, the potential mature active peptide region was designed to obtain the amino acid sequence of the basa fish Nat10 antimicrobial peptide, as shown in SEQ ID NO:3.

[0048] SEQ ID NO:3: Amino acid sequence of nat10, an antimicrobial peptide from basa fish.

[0049] LMGLFNRLMRKVVQLFN

[0050] The physicochemical properties of basa fish antimicrobial peptides were predicted using the ProtParam tool on the Expasy website (https: / / web.expasy.org / protparam / ), including molecular weight, isoelectric point, net charge value, and average hydrophilicity, as shown in Table 1.

[0051] Table 1. Predicted physicochemical properties of Nat10 antimicrobial peptides from basa fish

[0052]

[0053] The three-dimensional structure of the antimicrobial peptide was constructed using PEP-FOLD3 software, which is suitable for short peptides. Figure 1 As shown.

[0054] To ensure a high probability of the peptides possessing antibacterial activity, the obtained peptides all met the following conditions:

[0055] (1) Polypeptides are positively charged cations;

[0056] (2) Isoelectric point is greater than 8.0;

[0057] (3) CTDD and PAAC values ​​are greater than 0.5;

[0058] (4) It has an α-helix, β-fold, ring, or extended structure;

[0059] (5) The sequence contains fewer than 5 cysteine ​​residues;

[0060] (6) The sequence length does not exceed 30 amino acids.

[0061] Based on the designed amino acid sequence and constructed three-dimensional model of the basa fish Nat10 antimicrobial peptide, a basa fish Nat10 antimicrobial peptide containing an α-helix structure was synthesized chemically. In this embodiment, the full sequence of the basa fish Nat10 antimicrobial peptide was synthesized using a standard amino acid resin chemical synthesis method, which was customized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized peptide product was purified using a high-performance liquid chromatography (HPLC) system, followed by elution with an acetonitrile gradient of 1 mL / min. The parameters were as follows: column type: SHIMADZU shim-pack GIST C18 (4.6*250MM*5UM), phase A: 0.1% trifluoroacetic acid acetic acid solution, phase B: 0.1% trifluoroacetic acid aqueous solution; detection method: 0.5 mg of sample was dissolved in 0.5 mL of 30% acetic acid aqueous solution (v / v) for elution; volume: 30 μL, detection wavelength: 214 nm, flow rate: 1.0 mL / min. The detection results are as follows. Figure 2 As shown, the purity of the Nat10 polypeptide powder determined by HPLC-MS / MS is above 95%.

[0062] The synthesized peptide product was analyzed by mass spectrometry with the following parameters: 0.1 mg sample dissolved in 0.5 mL of 50% ACN aqueous solution; injection volume: 1 μL; interface: ESI; bias voltage: +4.5 kV; pointer: -0.2 kV; nebulizer gas flow rate: 1.50 L / min; CDL temperature: 250 °C; CDL volume: 0 v; block temperature: 200 °C; T. flow: 0.2 mL / min; B. conc: 50% methanol aqueous solution (v / v). The detection results are as follows. Figure 3 As shown in the figure, the molecular weight of the synthesized linear peptide is 2079.60 Daltons (Da), which is the basa fish Nat10 antimicrobial peptide. Finally, it was freeze-dried into powder and packaged (1 mg each) and stored at -80℃.

[0063] Example 3: Activity test of Nat10 antimicrobial peptides from basa fish

[0064] The basa fish Nat10 antimicrobial peptides synthesized in Example 2 (1 mg each) were diluted in phosphate buffer (PBS, NaCl 136.89 mM; KCl 2.67 mM; Na2HPO4 8.1 mM; KH2PO4 1.76 mM) for later use.

[0065] In this embodiment, the inhibitory effects of basa fish fry Nat10 antimicrobial peptide on *Streptococcus agalactiae*, *Salmonella enterica*, *Staphylococcus aureus*, *Vibrio anguillarum*, *Escherichia coli*, and *Aeromonas hydrophila* are verified in detail. This does not mean that basa fish fry Nat10 antimicrobial peptide has an inhibitory effect only on these specific bacteria. The purpose of this embodiment is to demonstrate that basa fish fry Nat10 antimicrobial peptide has an inhibitory effect on bacteria including those mentioned above, and can be used in the preparation of antibacterial products, including the preparation of antibacterial agents, drugs, or additives. Specific verification is as follows:

[0066] Streptococcus agalactiae, Salmonella enteritidis, Staphylococcus aureus, Vibrio anguillarum, Escherichia coli, and Aeromonas hydrophila were cultured separately. Each strain was cultured to the logarithmic growth phase and then diluted with medium (10⁻⁶). 4 CFU / mL). 100 μL of the diluted bacterial culture was added to each well of a 96-well plate containing 100 μL of the antimicrobial peptide, resulting in concentrations of basa fish Nat10 antimicrobial peptide of 2.5 μM (μmol / L), 5 μM, 10 μM, 20 μM, and 40 μM, respectively. An equal volume of PBS (previously prepared) was added for negative control. All 96-well plates were incubated at 37°C in a microbial incubator. OD600 values ​​were measured at 0h, 2h, 4h, 8h, and 20h, for a total of 20 hours, to plot the growth curve of the detected microorganisms. Each peptide was used in triplicate, and all experiments were repeated twice to obtain reliable results. All data were analyzed using the Social Science Statistical Package and GraphPadPrism software, and are expressed as mean ± standard deviation (n = 3). Paired-samples t-tests and multiple comparisons were used to test the significance of differences between groups. Results are shown in Table 2. Figures 4 to 8 As shown.

[0067] Table 2: Antimicrobial activity of Nat10 antimicrobial peptides from basa fish

[0068]

[0069] From Table 2 and Figures 4 to 8The results showed that the Nat10 antimicrobial peptide from basa fish exhibited inhibitory activity against the growth of *Streptococcus agalactiae*, *Salmonella enteritidis*, *Staphylococcus aureus*, *Vibrio anguillarum*, and *Escherichia coli*. Particularly against *Staphylococcus aureus*, the best antibacterial effect was observed at a concentration of 2.5 μmol / L. Against Gram-positive *Streptococcus agalactiae* and Gram-negative *Escherichia coli*, good antibacterial effects were also observed at a concentration of 2.5 μmol / L, with the best effect observed at 5 μmol / L. Against *Vibrio anguillarum*, good antibacterial effects were observed at a concentration of 5 μmol / L, with the best effect observed at 10 μmol / L. Even against *Salmonella enteritidis*, good antibacterial effects were observed at a concentration of 20 μmol / L. Therefore, this study verifies that the Nat10 antimicrobial peptide identified in basa fish possesses different antibacterial capabilities, particularly showing significant efficacy against *Staphylococcus aureus* at low effective concentrations.

[0070] Example 4: Detection of hemolytic activity of Nat10 antimicrobial peptides

[0071] To further verify the cytotoxicity of the Nat10 antimicrobial peptide, its hemolytic activity was tested in this embodiment. The procedure is as follows:

[0072] (1) The basa fish Nat10 antimicrobial peptide synthesized in Example 2 was dissolved and diluted with PBS (NaCl 136.89 mM; KCl 2.67 mM; Na2HPO4 8.1 mM; KH2PO4 1.76 mM) to 80 μM, 40 μM, 20 μM, 10 μM and 5 μM for later use;

[0073] (2) Take 100 μL of diluted antimicrobial peptide + 100 μL of 4% red blood cell suspension;

[0074] Negative: 100μL PBS + 100μL 4% red blood cell suspension;

[0075] Positive result: 100 μL 0.2% Tritonx-100 + 100 μL red blood cells;

[0076] (3) As for 37℃ for 1 hour;

[0077] (4) Centrifuge at 2500 rpm for 5 min, transfer the supernatant to a new 96-well plate and measure the OD value at λ=576 nm;

[0078] (5) Calculation: Hemolysis rate (%) = [(sample - negative) / (positive - negative)] * 100%;

[0079] Test results as follows Figure 9 As shown. By Figure 9The identification results show that the hemolytic activity of the basa fish Nat10 antimicrobial peptide is 5.53% at 5 μM and almost 0 at 2.5 μM, indicating that it has low hemolytic activity. When applying it, the concentration should be controlled below 5 μM. It can be used for sterilization of live organisms, which can provide better guarantee for green and healthy aquaculture and other aquaculture industries. It has good practical value and social and environmental benefits.

[0080] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A basa fish Nat10 antimicrobial peptide, characterized in that: The amino acid sequence of the basa fish Nat10 antimicrobial peptide is shown in SEQ ID NO:

3.

2. The basa fish Nat10 antimicrobial peptide according to claim 1, characterized in that: The basa fish Nat10 antimicrobial peptide contains an α-double helix structure.

3. The application of the basa fish Nat10 antimicrobial peptide according to claim 1 or 2 in the preparation of antibacterial products, characterized in that: The bacteria are any one of agalactiae, Salmonella enteritidis, Staphylococcus aureus, Vibrio anguillarum, and Gram-negative Escherichia coli.

4. The application of the basa fish Nat10 antimicrobial peptide according to claim 3 in the preparation of antibacterial products, characterized in that: The application includes at least one of the following: preparing antibacterial agents, preparing drugs, or preparing additives.

5. The application of the basa fish Nat10 antimicrobial peptide according to claim 4 in the preparation of antibacterial products, characterized in that: In products used to combat Staphylococcus aureus, the concentration of basa fish Nat10 antimicrobial peptide is 2.5 μmol / L or higher; In products used against Gram-positive Streptococcus agalactiae or Gram-negative Escherichia coli, the concentration of basa fish Nat10 antimicrobial peptide is 5 μmol / L or higher. In products used to combat Vibrio anguillarum, the concentration of basa fish Nat10 antimicrobial peptide is 10 μmol / L or higher; In products used to combat Salmonella enteritidis, the concentration of basa fish Nat10 antimicrobial peptide is 20 μmol / L or higher.

6. The application of the basa fish Nat10 antimicrobial peptide according to claim 5 in the preparation of antibacterial products, characterized in that: In products used to combat Staphylococcus aureus, the concentration of basa fish Nat10 antimicrobial peptide is 2.5 μmol / L to 40 μmol / L; In products used against Gram-positive Streptococcus agalactiae or Gram-negative Escherichia coli, the concentration of basa fish Nat10 antimicrobial peptide is 5 μmol / L to 40 μmol / L. In products used to combat Vibrio anguillarum, the concentration of basa fish Nat10 antimicrobial peptide is 10 μmol / L to 40 μmol / L; In products used to combat Salmonella enteritidis, the concentration of basa fish Nat10 antimicrobial peptide is 20 μmol / L to 40 μmol / L.

7. The application of the basa fish Nat10 antimicrobial peptide according to claim 4 in the preparation of antibacterial products, characterized in that: When used for live animal disinfection, the concentration of basa fish Nat10 antimicrobial peptide in antibacterial products is 2.5 μmol / L to 5 μmol / L.

Images