Antimicrobial peptide and its application

Abstract

This application discloses an antimicrobial peptide and its application. The antimicrobial peptide is a polypeptide having the sequence shown in Seq ID No. 1, Seq ID No. 2, or Seq ID No. 3. The antimicrobial peptide has preventive and therapeutic effects against tilapia pathogens, providing a new solution and approach for addressing common bacterial diseases in high-density tilapia aquaculture.

Description

Technical Field

[0001] The present application relates to the technical field of antimicrobial peptides, and in particular to an antimicrobial peptide and its application. Background Art

[0002] In recent years, with the continuous development of high-throughput sequencing technology, the research on fish intestinal metagenomics has also made great strides; however, the current reports on tilapia intestinal metagenomic research mainly use the 16S amplicon method to study the composition of intestinal bacteria, observe probiotics such as Streptococcus agalactiae, Lactococcus lactis, and Lactobacillus rhamnosus, and prebiotics such as plant extracts, yeast extracts, and resveratrol, as well as the effects of environmental factors such as temperature and salinity on the distribution of intestinal microbial composition, and compare the microbiota of tilapia, tilapia and grass carp, and other non-fish animals in different breeding environments; there is a lack of analysis of the tilapia intestinal metagenomics, and even more of an in-depth analysis of the functional genes from the tilapia intestinal metagenomic gene set, so as to positively search for relevant endogenous antibacterial genes and potential probiotics.

[0003] GIFT tilapia (Oreochromis niloticus) is an improved tilapia species with strong adaptability and rapid growth. Due to the development of high-density aquaculture, pathogen infection has posed a challenge to the growth performance and immunity of tilapia. In addition, the replacement of antibiotics is also a pressing issue. Studies have shown that the intestinal microbiome of tilapia plays an important role in its nutrient absorption, intestinal immune regulation and intestinal pathogen resistance.

[0004] Currently, the development and quality improvement of the aquaculture industry still face prominent challenges, including a shortage of genetically improved varieties, low rates of improved varieties, severe disease, rapid decline in the production performance of established varieties, and slow progress in aquaculture technology. Solving these problems is no longer feasible with traditional aquaculture techniques alone; instead, it requires continuous technological innovation and widespread application.

[0005] Global tilapia production reached 1.6 billion tons in 2016. However, high-density stocking stress significantly reduces tilapia growth rate, feed utilization, microvilli length, and disease resistance, often leading to reduced production. Furthermore, in response to these stressful conditions, intestinal HSP70 protein expression levels increase. Streptococcal disease, caused by Streptococcus iniae, contributes to significant losses in the tilapia market, estimated at $23 billion annually. However, resistance driven by overuse of antibiotics has compelled scientists to explore alternatives to reduce infection and disease. Effective strategies include supplementation with functional immune enhancers, including probiotics, prebiotics, commensal bacteria, antimicrobial peptides, phage therapy, immunostimulants, and botanical extracts. Studies have shown that the fish gut microbiome plays an integral role in host health by stimulating immune system development through its repertoire of antimicrobial compounds, aiding nutrient acquisition, and outcompeting opportunistic pathogens. For example, antimicrobial peptides (AMPs) produced by Bacillus species isolated from the gastrointestinal (GI) tract of the Indian common carp (Labeo rohita) have attracted attention as alternative tools for controlling pathogen colonization in the fish gut. This could reveal the potential reservoir of antimicrobial compounds in uncultured microorganisms inhabiting the gut microbiota, thereby shedding light on the relationship between the gut microbiota and immune responses or disease resistance.

[0006] The use of intestinal microorganisms as probiotics to regulate fish growth is primarily due to the secretion of physiologically active substances such as vitamins, fatty acids, and digestive enzymes during bacterial metabolism, which benefit animal growth and health. Fish immunity is primarily regulated by the secretion of antimicrobial substances such as antimicrobial peptides, antimicrobial lipopeptides, lysozymes, organic acids, and protein exotoxins, with antimicrobial peptides being a key mechanism. Antimicrobial peptides are peptides (AMPs) synthesized by the ribosomes of many Gram-negative and Gram-positive bacteria. AMP production is a universal phenomenon in all life forms, from multicellular organisms to bacterial cells. In higher organisms, AMPs contribute to innate immunity and serve as the first line of defense against harmful microorganisms. Within bacterial communities, AMP production provides producers with a competitive advantage in certain ecological niches because they mediate the killing of other bacteria. Antimicrobial peptides produced by bacteria are commonly called bacteriocins. Most AMPs have a relatively narrow antimicrobial spectrum, typically targeting species or genera closely related to the producer. Others, such as the lactic acid bacterial antimicrobial peptide nisin, may have a broader spectrum, encompassing important pathogenic or problematic Staphylococci, Listeria, Enterococci, and Streptococci. Furthermore, bacteriocins are typically very potent, acting at picomolar to nanomolar concentrations, whereas eukaryotic AMPs must be active at micromolar concentrations. Furthermore, antimicrobial peptides avoid killing commensal and beneficial cells, exhibit low or no toxicity to eukaryotic cells, and are active against pathogens and their derived antibiotic-resistant strains. Therefore, they hold promise as alternatives to antibiotics for disease prevention and control amidst subtherapeutic antibiotic misuse and the rampant spread of drug resistance.

[0007] The gut microbiome is a dense and diverse microbial ecosystem, making it a promising reservoir for the discovery of novel antimicrobial metabolites. For example, a novel peptide antibiotic produced by human nasal commensals was recently reported to limit Staphylococcus aureus colonization and maintain homeostasis. For example, antimicrobials produced by skin commensals have the potential to treat atopic dermatitis, and antimicrobials derived from human skin commensals protect against Staphylococcus aureus and lack atopic dermatitis. Indeed, given the density and diversity of microbial populations in the gut, in the era of genomics and metagenomics, the rich sequence information is a valuable resource for high-throughput mining of AMPs and AMP variants. Several novel bacteriocins have been identified based on homology searches with known genes involved in biosynthesis, transport, and immunity. More specialized search tools, such as BAGEL2, that use algorithms based on the genetic and physicochemical properties of known bacteriocins can also aid in the identification of novel bacteriocins from published genome sequences.

[0008] The development of molecular tools has eliminated the requirement for functional effects influenced by bacterial culture conditions and induction. This has enabled the identification of putative bacteriocin clusters in bacterial genomes, including those from uncultivable microorganisms. Several public bacteriocin software and databases have been used to align bacteriocin peptide sequences in sequenced bacterial genomes; BAGEL, with 482 sequences, and BACTIBASE, with 345 sequences, are the most commonly used. A more extensive database is available, including the Human Microbiome Project Reference Genome Database. Analysis of this database by Walsh et al. identified 74 putative bacteriocin-encoding gene clusters in various phyla. A more accurate analysis of the distribution and frequency of specific bacteriocin types can be solved using hidden Markov models, enabling the identification of bacteriocin homologs, such as seven new lantibiotics. Drissi et al. developed an extensive bacteriocin database, BUR ("Bacteriocins of the URMITE Database"), which combined BAGEL, BACTIBASE, and NCBI databases for published bacteriocin sequences and searched them. They analyzed 641 available genomes from the gastrointestinal tract to include as much bacteriocin data as possible.

[0009] The rational application of bacteriocins, either directly or as probiotics from bacteria produced by them, is being studied as a method for modulating the structure and function of the intestinal microbiome. Antimicrobial peptides are primarily added to feed in animal production and breeding. Domestic and international research has shown that antimicrobial peptides can improve growth performance, immunity, antioxidant capacity, and pathogen resistance in farmed animals. Adding 5-10 mg / kg of recombinant Chinese shrimp antimicrobial peptide to the GIFT tilapia base feed significantly increased weight gain, while a 5 mg / kg supplement significantly improved survival rate and specific growth rate. The GIFT tilapia group receiving 100 mg / kg of cecropin achieved higher weight gain throughout the entire feeding period than the antibiotics quinocetone and olaquinoxaline. Antimicrobial peptides not only directly protect against pathogens but also affect various parameters, including improving serum antioxidant levels, altering immune organ indices, enhancing immune cell activity and phagocytic capacity, improving immune-related indicators, and enhancing the expression of immune factors. At present, patents in this area are still in the stage of accumulation and rapid development, and new breakthroughs in basic research are urgently needed.

[0010] Therefore, the development of new antimicrobial peptides suitable for tilapia is of great significance for high-density aquaculture of tilapia. Summary of the Invention

[0011] The purpose of this application is to provide a new antimicrobial peptide and its application.

[0012] This application adopts the following technical solutions:

[0013] One aspect of the present application discloses an antimicrobial peptide, which is a polypeptide having a sequence shown in Seq ID No. 1, Seq ID No. 2 or Seq ID No. 3;

[0014] Seq ID No.1: MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKK;

[0015] Seq ID No.2: PHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFK;

[0016] Seq ID No.3:

[0017] TDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARR IRGER.

[0018] Preferably, the antimicrobial peptide is an antimicrobial peptide screened from the intestinal microecology of GIFT tilapia.

[0019] Another aspect of the present application discloses the use of the antimicrobial peptide of the present application in the preparation of tilapia antimicrobial drugs.

[0020] Another aspect of the present application discloses the use of the antimicrobial peptide of the present application in preparing tilapia feed.

[0021] Another aspect of the present application discloses a drug containing the antimicrobial peptide of the present application.

[0022] Preferably, the medicine is used to prevent or treat tilapia pathogens or diseases caused by tilapia pathogens.

[0023] Another aspect of the present application discloses a feed containing the antimicrobial peptide of the present application.

[0024] Another aspect of the present application discloses a method for preparing tilapia intestinal antimicrobial peptides, comprising: obtaining tilapia intestinal contents, extracting metagenomic DNA from the obtained metagenomic DNA, sequencing the extracted metagenomic DNA, filtering the sequencing data, removing host sequences, assembling and removing redundancy, and constructing a non-redundant gene set; starting from the non-redundant gene set, comparing it with a public database to obtain species annotation information, and combining it with a gene abundance table to obtain a species abundance table at different classification levels; simultaneously, performing functional annotation and abundance analysis of metabolic pathways, homologous gene clusters, carbohydrate enzymes, signal peptides, and transmembrane domains; predicting the activity of antimicrobial peptide precursor sequences based on the non-redundant gene set and the results of the comparative analysis thereof, removing inactive predicted antimicrobial peptide precursor sequences to obtain antimicrobial peptides with potential activity, and performing activity verification and detection on the potentially active antimicrobial peptides using an ELISA plate to obtain tilapia intestinal antimicrobial peptides.

[0025] It can be understood that the antimicrobial peptide of the present application is actually prepared by the above method.

[0026] Preferably, the activity of the antimicrobial peptide precursor sequence is predicted, specifically comprising applying an E value ≤ 10 in a non-redundant gene set. -5 The threshold value was set at TBLASTN, and homology searches were performed to predict antimicrobial peptide genes. The queried antimicrobial peptide nucleic acid sequences were then translated into peptide sequences for further comparison and verification on NCBI. At the same time, antimicrobial peptides that only exist in eukaryotes were removed.

[0027] Preferably, after obtaining the antimicrobial peptides with potential activity, the method further includes classifying the sequences according to the annotation information in the APD3 database and performing functional annotation according to the KEGG database; using the pepstats software of EMBOSS-6.6.0 to perform composition analysis of the peptides, calculate the net charge value and isoelectric point at neutral pH; and using HeliQuest to predict the Shiffere-Edmundson helical wheel diagram, hydrophobicity and hydrophobic force; and at the same time, using I-TASSER to predict the tertiary structure of each antimicrobial peptide.

[0028] The beneficial effects of this application are:

[0029] The antimicrobial peptides of the present application have preventive and therapeutic effects on tilapia pathogens, and provide a new solution and approach for solving common bacterial diseases in high-density tilapia farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a flow chart for preparing tilapia intestinal antimicrobial peptides in the examples of this application;

[0031] Figure 2 This is a phylogenetic tree analysis diagram of the probiotics in the examples of this application. DETAILED DESCRIPTION

[0032] Common bacterial diseases in high-density tilapia aquaculture pose a challenge to the industry. This application collects intestinal content samples from GIFT tilapia and uses NGS metagenomic sequencing to construct a non-redundant gene set of the intestinal flora. Combined with an antimicrobial peptide database comparison, antimicrobial peptide genes are mined from the functional gene set. Further combining gene and strain contribution analysis with reported microbial preparations, candidate antimicrobial peptides are predicted, providing new ideas and technical support for the development of GIFT tilapia microbial preparations, which is of great significance for high-density aquaculture and antibiotic substitution.

[0033] The existing technology lacks analysis of the tilapia intestinal metagenome, and even more so, lacks in-depth mining and analysis of functional genes from the tilapia intestinal metagenome gene set, thereby positively searching for relevant endogenous antimicrobial genes and potential probiotics. Therefore, this application constructs a high-quality and complete tilapia intestinal microbial gene set database, and conducts in-depth and high-throughput mining and annotation of functional genes and antimicrobial substance genes. This will help to globally understand which core intestinal microbial flora and their metabolites play an important role in tilapia growth, immunity, disease resistance, etc., and then use microbial functional genes to develop microecological preparations for use in aquaculture.

[0034] This application systematically compares and analyzes the intestinal microbial composition and function of GIFT tilapia under high-density farming, maintaining consistency in seasonal changes, diet, feed, growth and development, etc., which helps to summarize the composition characteristics of tilapia intestinal microorganisms, clarify the metabolic functions of intestinal core flora genes, and high-throughput mining of potential bacteriocin genes, including analysis of other antibacterial metabolites (lysozyme, organic acids, protein exotoxins, etc.), predict potential probiotics that are helpful for antibacterial and growth, and is conducive to the design of microecological preparations and feed additives that regulate intestinal flora in aquaculture, as an alternative to antibiotics, and increase the targeting, globality and effectiveness of aquatic microecological preparation research. This application uses metagenomic data for high-throughput mining and functional identification of antimicrobial peptides, further providing a candidate library for finding valuable candidate strains, and providing new ideas for the development of ecologically efficient growth promoters and disease prevention drugs for tilapia and even aquatic animal farming.

[0035] At present, the development and quality improvement of the aquaculture industry are still faced with prominent problems such as few genetically improved varieties, low rate of good varieties, serious diseases, rapid decline in production performance of bred varieties, and slow progress in aquaculture technology. It is difficult to make breakthroughs by relying solely on traditional aquaculture technology to solve these problems. We must rely on the continuous innovation and promotion of technology, especially molecular-assisted aquaculture technology based on whole genome sequencing. The organic combination of genomics and aquaculture is a necessary condition and inevitable trend for the leapfrog development of the aquaculture field today. The frequent occurrence of diseases in water plant aquaculture such as tilapia has restricted the further development of the aquaculture industry. This application uses the mature microbiology foundation of the Guangdong Institute of Microbiology to promote the improvement of aquaculture and R&D innovation levels from the direction of metagenome, which will be a feasible solution to cope with opportunities and challenges. The antimicrobial peptides of this application have good economic and social benefits.

[0036] This application uses metagenomics technology to construct a non-redundant gene set of tilapia intestinal microorganisms, locate potential endogenous probiotics in the intestinal flora, and thus identify potential endogenous probiotics with development value in host pathogen antagonism and immune promotion, such as Figure 1The implementation of this application will provide a material basis and technical guidance for the research team's exploration of microbial resources and the development of microecological preparations, thereby promoting the development and utilization of probiotics and the transformation of research results.

[0037] The present application is described in detail below through specific embodiments in conjunction with the accompanying drawings. The following embodiments are only used to illustrate the present application and should not be understood as limiting the present application.

[0038] Example 1

[0039] The purpose of this study was to complete metagenomic sequencing analysis of intestinal content samples of GIFT tilapia, and to perform high-throughput prediction of antimicrobial peptide genes and identification of potential probiotics in the metagenomics.

[0040] (1) Tilapia breeding: The GIFT tilapia used in the experiment were the same batch of tilapia that were high-density cultured in an aquaculture base, with an average of 5,000 tilapia in each recirculating aquaculture tank.

[0041] (2) Sample processing: The entire experimental period was 90 days, and the contents of the posterior half of the intestine were collected every 45 days, for a total of two times. Feed was stopped for 24 hours before the intestinal tract was collected. On the day of the experiment, 30 fish were randomly selected from each box, and their weight and length were measured. The collected live fish were dissected under ethical operating standards, and the hindgut contents of the intestine were partially mixed and placed in sterile EP tubes for subsequent experimental operations such as metagenomic DNA extraction, quality measurement, and library construction.

[0042] (3) Metagenomic sequencing analysis: Relying on the laboratory's sequencing platform and metagenomic bioinformatics analysis capabilities, the advanced Illumina NextSeq 500 sequencer is used. The raw data generated by each sample is 12G (Q30>80%). Data that meets the Q30 high-quality sample requirements enter the subsequent analysis. The raw data is filtered through unified quality control standards for removing joints, contamination, and low quality, and then the host sequence is removed before entering the assembly process. The project adopts a single sample assembly strategy. For the assembled scaftigs, MetaGeneMark is used for gene prediction. The genes generated by the assembly prediction of each sample are put together to remove redundancy and construct a non-redundant gene set. Then, species and functional annotation were performed: starting from the non-redundant gene set, it was compared with the NCBI-NR database, and the species annotation information of Unigenes was obtained using Megan software. Combined with the gene abundance table, species abundance tables at different taxonomic levels were obtained; at the same time, the Unigenes were functionally annotated and abundance analyzed for metabolic pathways (KEGG), homologous gene clusters (eggNOG), carbohydrate enzymes (CAZy), signal peptides, and transmembrane domains.

[0043] (4) Prediction and identification of antimicrobial peptides (AMPs): The previously constructed intestinal non-redundant gene set was used as a local tag dataset. Previously verified AMPs were obtained from the online antimicrobial peptide database and used for query. Then, a homology search was performed on TBLASTN with an E value ≤ 10-5 threshold in the above-mentioned metagenome tag dataset to predict antimicrobial peptide genes. The queried AMP nucleic acid sequences were then translated into peptide sequences and submitted to NCBI for further verification using the BLASTP tool. At the same time, those AMPs that only exist in eukaryotes were removed. The activity of AMP precursor sequences was predicted using the GenomicAMPs prediction software V1.2.7 (2021SR0424886), and AMPs predicted to be inactive were removed. Finally, we obtained potentially active AMP polypeptide sequences from the metagenome. These sequences were further classified according to the annotation information in the APD3 database and functionally annotated according to the KEGG database. Peptide composition analysis, net charge at neutral pH, and isoelectric point calculations were performed using EMBOSS 6.6.0's pepstats software. Shiffere-Edmundson helical wheel diagrams, hydrophobicity, and hydrophobic forces were predicted using HeliQuest (http: / / expasy.org / tools). The tertiary structure of each antimicrobial peptide was also predicted using the I-TASSER online website.

[0044] (5) Verification of the activity function of antimicrobial peptides: From the predicted potentially active AMPs, peptides with similar sequences to known sequences, about 30 amino acids, relatively clear and meaningful functions, or obvious differences between species were selected for synthesis. All predicted active peptide fragments were synthesized by solid-phase synthesis using standard amino acid resin Fmoc chemical synthesis to ensure that the purity of the obtained peptide dry powder was above 90%. The peptides were diluted in multiples according to their concentration, added to a clean 96-well ELISA plate, and mixed with pathogenic bacteria; at the same time, negative control wells (peptide solution) and PBS blank control wells were set up, and the absorbance value OD600 was read every half hour. Using statistical software SPSS16.0 and Origin, the growth curves of each test bacteria were drawn according to the OD600 values, and the numerical differences between the control group and the experimental group were calculated to determine whether they were statistically significant, and the minimum inhibitory concentration (MIC) of the peptide was determined.

[0045] According to the above method, three antimicrobial peptides were finally screened in this example. The specific three antimicrobial peptides were polypeptides with sequences shown in Seq ID No. 1, Seq ID No. 2 or Seq ID No. 3;

[0046] Seq ID No.1: MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKK;

[0047] Seq ID No.2: PHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFK;

[0048] Seq ID No.3:

[0049] TDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARR IRGER.

[0050] The antimicrobial peptide with the sequence shown in Seq ID No. 1, when added to feed at a dosage of 4 mg / kg, has a preventive and therapeutic effect on 90% of pathogens; the concentration is 1500 when endotoxin is not tested and 3000 when endotoxin is tested.

[0051] The antimicrobial peptide with the sequence shown in Seq ID No. 2, when added to feed at a dosage of 4 mg / kg, has a preventive and therapeutic effect on 90% of pathogens; the concentration is 1500 when endotoxin is not tested and 3000 when endotoxin is tested.

[0052] The antimicrobial peptide with the sequence shown in Seq ID No. 3, when added to feed at a dosage of 4 mg / kg, has a preventive and therapeutic effect on 90% of pathogens; the endotoxin level is 2500 when not tested and 4200 when tested.

[0053] Feasibility analysis:

[0054] (1) Reliable theoretical basis: The research team has been engaged in the study of fish intestinal metagenomics and probiotics, and has conducted high-density probiotic feeding and aquaculture experiments on tilapia. At the same time, combined with extensive research on tilapia intestinal microbial research literature, it was found that the current research on tilapia intestinal microorganisms, probiotics and antimicrobial metabolites is still mainly based on the feeding of single bacteria and substances. Then, 16S rRNA amplicon analysis was used to study the regulatory effects of probiotics and antimicrobial peptides on intestinal flora. However, there is a lack of research on potential probiotics from the perspective of the entire intestinal microbiome. With the increasing maturity of whole-genome analysis of intestinal microbes, we have discovered potentially exploitable bacteriocins from intestinal microbial sources through high-throughput mining and annotation of functional genes for antimicrobial metabolites, primarily bacteriocins, in the intestinal microbial metagenome. We have also further screened and identified strains expressing these antimicrobial genes based on their characteristics, abundance, and relationships with existing probiotics, resulting in a series of potential probiotics. Metagenomic analysis has been used to characterize the metabolic properties of these strains, which are then subsequently validated through targeted culture, providing a wider range of superior probiotic options for the future. We have already conducted research on the identification of antimicrobial peptides in grass carp intestinal microbes. This project has strong foundations, clear ideas, and is theoretically feasible.

[0055] (2) Feasibility of technical methods: The applicant of this project has a solid theoretical and applied foundation in metagenomic research, has participated in the HMP project for the construction of human intestinal gene sets, and has been engaged in theoretical and applied research on fish metagenomics and probiotics for many years. He has published many articles on fish metagenomics research in Toxins and IJAB. In the early work of the applicant of this project, the intestinal microorganisms of silver carp, grass carp, bighead carp, mudskipper, basket fish, forktail catfish, etc. have been preliminarily studied using 16S rRNA amplicon sequencing and metagenomic sequencing methods. At the same time, the project team also used the complete antimicrobial peptide gene database constructed to mine a large number of antimicrobial peptide genes in the grass carp metagenome, and conducted kinship analysis on probiotics and mature probiotics containing corresponding antimicrobial peptides to further identify the application value of potential probiotics. In the early stage, the research team has used different concentrations of Clostridium butyricum and bacteriocin to feed tilapia to observe the effects of probiotics and bacteriocin on fish growth and immunity under high-density farming conditions. These previous knowledge accumulations and corresponding preliminary research have a strong connection and inheritance with this application project. The experimental methods and analysis methods are basically consistent with this project. Many experimental data and research conditions can be used, which lays a solid technical foundation for the in-depth and systematic research and successful completion of this fund project.

[0056] (3) Feasible foundation and conditions: The unit where this project team is located has NextSeq 500, MiSeq high-throughput sequencing platforms and microbial screening platforms. It has been engaged in animal metagenomic bioinformatics research and probiotic research for a long time and has accumulated rich experience. It has high-density container farming conditions for tilapia. In addition, the microbial research team of this project team has screened probiotics such as Clostridium butyricum and Lactobacillus reuteri. It has the professional foundation and technical conditions to carry out the fish metagenomic research and application of this project.

[0057] Research achievements

[0058] My research team has long been engaged in aquatic metagenomic research and has accumulated extensive experience in fish gut microbiota and probiotics. In 2016, our research team spearheaded a project to study the gut metagenomics of 100 fish species, with the applicant participating in sampling, preservation, and information analysis. We have currently conducted preliminary studies on the gut microbiota of silver carp, grass carp, bighead carp, mudskipper, basketfish, and catfish using 16S amplicon sequencing and metagenomic sequencing (preliminary sampling and analysis results are listed in Table 1). We initially sampled the gut microbiota of eight grass carp individuals, generating 29 gigabytes (Gb) of raw data per sample; four silver carp samples generated 46.7G of raw data; and one bighead carp sample generated 12G of raw data. This data was filtered for host contaminants, resulting in the resulting metagenomic data for further analysis as a whole, and preliminary assembly and annotation analysis was performed. From these annotated metagenomic data, a total of 4,966 species, 1,453 genera, 378 families, 178 orders, 76 classes, and 54 phyla were identified. Five phyla, Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria, were found to dominate the grass carp gastrointestinal microbiome. The results of this comparative metagenomic study of multiple fish species have been submitted to the journal Fish and Shellfish. The experimental techniques and NGS meta-analysis methods used in this project are very similar to those employed in these previous studies, laying a solid foundation for the successful implementation of this project.

[0059] Table 1. Fish metagenomic information analysis work that has been carried out

[0060]

[0061] Our research group, while conducting a fish metagenomics project, has summarized recent research findings on the fish microbiome, encompassing freshwater and marine fish species such as common carp, rainbow trout, Siberian sturgeon, Atlantic salmon, catfish, Atlantic cod, coral reef fish, and Antarctic fish. Interestingly, methods for studying the microbial communities of carp species (such as grass carp, silver carp, Prussian carp, silver crucian carp, and Jian carp) from Asian countries are reviewed. Most previous high-throughput fish microbiome studies employed metagenomic approaches, particularly 16S sequencing (listed in Table 2). Many of these studies were combined with culture-independent techniques such as DDGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). Only a few complete metagenomic studies have been reported, one of which was used to decipher the biosynthetic and metabolic pathways of carbohydrates, amino acids, and lipids in ryegrass-fed grass carp. In this study, microarrays were also used to examine patterns of metabolite changes. Another study revealed the role of the gut microbiome in cellulose metabolism through a metatranscriptomic approach. A comparative metagenomics and 16S analysis study of gibel crucian carp concluded that metagenomics provides more accurate taxonomic classification than pure 16S analysis. However, there are no reports on the use of metagenomics to analyze the gut microbiome of tilapia and comprehensively annotate the tilapia gut microbiome gene set. The results of this review were published in the International Journal of Agriculture and Biology.

[0062] Table 2 Summary of fish metagenomic studies using high-throughput methods

[0063]

[0064] The applicant also conducted antimicrobial peptide and probiotic feeding experiments on GIF tilapia, conducted high-throughput mining of antimicrobial peptide genes in the intestinal metagenome of grass carp (see Table 3), and made further predictions on potential probiotics (see Figure 2 ), the results of which have been published in the journal Toxins; and found that there is a lack of strain-level whole-genome assembly of potential probiotics in tilapia metagenomic studies, and that probiotic research is still in the state of low-throughput, single validation studies.

[0065] Table 3 Summary of grass carp intestinal microbial bacteriocins with the highest similarity to antimicrobial peptides in NCBI

[0066]

[0067]

[0068] Phylogenetic tree analysis of high-abundance bacterial communities, bacterial communities producing predicted antimicrobial peptides, and reported probiotics, such as Figure 2As shown, the highly abundant strains in the grass carp intestine are marked in red fonts, the strains producing predicted antimicrobial peptides are marked in blue fonts, and the reported probiotics are marked in green fonts.

[0069] The experimental techniques and analytical methods used in this study are very similar to those employed in these previous studies, laying a solid foundation for the smooth implementation of this project. Furthermore, this preliminary research will also provide a solid foundation for the successful completion of this project's aquaculture trials and subsequent validation trials of the probiotic antimicrobial peptides.

[0070] Example 2

[0071] 1. Toxicity test

[0072] To confirm the safety and lack of adverse side effects of the three peptides represented by Seq ID No. 1, Seq ID No. 2, or Seq ID No. 3 in this study, two groups of tilapia were intraperitoneally injected with 10- and 20-fold the effective concentration of the peptides, respectively, 48 hours after abstaining from feeding. A control group of 25 tilapia received an equal volume of PBS intraperitoneally. Each group was tested with each of the three peptides. The fish were observed daily for 30 days to determine the presence of adverse reactions and survival rates, which served as safety criteria for the three peptides. The stocking density in this case was 25 tilapia per cubic meter.

[0073] The results showed that after intraperitoneal injection of 10-fold and 20-fold doses of the peptide, the survival rate of all test groups was 100% after 30 days of observation, without any adverse reactions. The situation in the control group was the same, indicating that the three peptides in this experiment are highly safe for fish.

[0074] 3. Peptide protection test

[0075] (1) Feeding test

[0076] 4 mg of polypeptide was added to each kilogram of feed to prepare the feed. No polypeptide was added to the control group. Four types of feed were designed for this experiment: feed supplemented with Seq ID No. 1 polypeptide, feed supplemented with Seq ID No. 2 polypeptide, feed supplemented with Seq ID No. 3 polypeptide, and a conventional feed without any polypeptide added. Therefore, the experiment was divided into four groups, each containing 50 tilapia, with a stocking density of 25 fish / cubic meter. The control group and the corresponding polypeptide-added feed were fed three times a day, 45 g of feed per meal, for 14 consecutive days. After 14 days, a challenge test was performed.

[0077] Toxicity test: A solution containing Streptococcus tilapia was inoculated into the experimental fish via intraperitoneal injection at a dose of 1 mL per fish. The control group was injected with 1 mL of DMEM medium. There were 50 fish in each control and experimental groups, and the survival rate of the fish was observed for 14 consecutive days.

[0078] The results showed that the control group experienced approximately 60% mortality 12 days after challenge, reaching a final mortality rate of 80%. However, the groups receiving peptides maintained a survival rate of at least 70% at the end of the experiment. The survival rate for those fed Seq ID No. 1 peptide was 78%, for those fed Seq ID No. 2 peptide was 70%, and for those fed Seq ID No. 3 peptide was 82%. Furthermore, the survival rate for the group fed Seq ID No. 3 peptide reached a high of 94% on day 10. Although fish mortality occurred over the following days, the survival rate remained at 82%.

[0079] (2) Immersion test

[0080] The experimental soaking solution was prepared by adding 100 mg of the peptide per liter of water. The control group was regular freshwater. Four groups were divided into four groups, each containing 50 tilapia, stocked at a density of 25 per cubic meter. Each group was immersed in the control and peptide-supplemented soaking solutions for one hour daily for 14 days. After 14 days, a challenge test was conducted.

[0081] Challenge test: The test method was the same as in "(1) Feeding test". The test results showed that the mortality rate of the control group reached approximately 70% 12 days after the challenge, and finally stabilized at a high mortality rate of 86%. The survival rate of the group receiving the peptide was still at least 66% at the end of the test. The survival rate of the group fed with Seq ID No. 1 peptide was 74%, the survival rate of the group fed with Seq ID No. 2 peptide was 66%, and the survival rate of the group fed with Seq ID No. 3 peptide was 80%.

[0082] The above experiments demonstrate that feeding or soaking the three peptides tested in this study can enhance the fish's antibacterial capacity, thereby increasing its survival rate. Furthermore, the present application further conducted similar challenge experiments using other pathogenic bacteria of tilapia, including Edwardsiella tarda, Aeromonas hydrophila, and Pseudomonas fluorescens. The results showed that the three peptides represented by the sequences shown in Seq ID No. 1, Seq ID No. 2, or Seq ID No. 3 all had preventive and therapeutic effects against Edwardsiella tarda, Aeromonas hydrophila, and Pseudomonas fluorescens, providing a new solution and approach for common bacterial diseases in high-density tilapia aquaculture.

[0083] The above content is a detailed description of the present application in conjunction with specific implementation methods, and the implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field of the present application, several simple deductions or substitutions can be made without departing from the basic inventive concept of the present application.

Claims

1. An application of an antimicrobial peptide in the preparation of an antimicrobial drug for tilapia, characterized in that: The antimicrobial peptide is a polypeptide having a sequence shown in Seq ID No. 1; Seq ID No.1: MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKK; The antimicrobial peptide is an antimicrobial peptide screened from the intestinal microecology of Gift Tilapia, and the pathogens inhibited by the antimicrobial peptide are Edwardsiella tarda, Aeromonas hydrophila or Pseudomonas fluorescens.

2. Use of the antimicrobial peptide according to claim 1 in preparing tilapia feed.

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