Application of antibacterial peptide in preparation of medicine for preventing or treating tilapia mossambica lake virus disease

Abstract

The invention discloses an application of an antibacterial peptide in preparation of a medicine for preventing or treating tilapia lake virus diseases. The antibacterial peptide is a polypeptide with a sequence as shown in Seq ID No.1. The research finds that the polypeptide with the sequence shown as Seq IDNo.1 can effectively inhibit the growth of the tilapia lake virus in the tilapia body, so that the effect of preventing and treating the tilapia lake virus is achieved, and a new scheme and way are provided for preventing and treating the tilapia lake virus.

Description

Technical Field

[0001] The present application relates to the technical field of tilapia farming, and in particular to the use of an antimicrobial peptide in the preparation of a drug for preventing or treating tilapia lake virus disease. Background Art

[0002] Tilapia Lake Virus Disease (TiLVD) is a disease caused by Tilapia Lake Virus (TiLV) infection, with a mortality rate of over 90%, and is an important disease affecting tilapia farming. Although studies have pointed out that early detection of Tilapia Lake Virus can promote early treatment, thereby effectively reducing the economic losses of the farming industry, and corresponding Tilapia Lake Virus detection technology has been developed; however, the lack of treatment options for Tilapia Lake Virus Disease has greatly limited the prevention and control of Tilapia Lake Virus.

[0003] Therefore, developing new agents or solutions for preventing or treating tilapia lake virus disease remains a research focus and difficulty in the field of tilapia aquaculture technology. Summary of the invention

[0004] The purpose of the present application is to provide an application of an antimicrobial peptide in the preparation of a drug for preventing or treating tilapia lake virus disease.

[0005] This application adopts the following technical solutions:

[0006] The present application discloses an application of an antimicrobial peptide in the preparation of a drug for preventing or treating tilapia lake virus disease, wherein the antimicrobial peptide is a polypeptide having a sequence shown in Seq ID No.1;

[0007] Seq ID No. 1: MALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQ.

[0008] The present application also discloses a nucleic acid encoding the antimicrobial peptide of the present application, namely, a nucleic acid encoding the polypeptide of the sequence shown in Seq ID No.1.

[0009] The present application also discloses a recombinant plasmid containing the nucleic acid of the present application, that is, a recombinant plasmid containing a nucleic acid encoding a polypeptide of the sequence shown in Seq ID No. 1.

[0010] The present application also discloses a microbial host containing the recombinant plasmid of the present application, such as a competent cell transformed with the recombinant plasmid.

[0011] The present application also discloses the use of the nucleic acid of the present application, the recombinant plasmid of the present application or the microbial host of the present application in the preparation of a drug for preventing or treating tilapia lake virus disease.

[0012] The present application also discloses a drug for preventing or treating tilapia lake virus disease, wherein the drug contains a polypeptide having a sequence shown in Seq ID No. 1;

[0013] Seq ID No. 1: MALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQ.

[0014] The beneficial effects of this application are:

[0015] The antimicrobial peptide of the present application has preventive and therapeutic effects on tilapia pathogenic bacteria, and provides a new solution and approach for solving common bacterial diseases in high-density tilapia farming. DETAILED DESCRIPTION

[0016] Tilapia lake virus disease is a common bacterial disease in high-density tilapia farming. In the present application, intestinal microorganisms of Nile tilapia are collected as research objects, and genome sequencing and analysis of the intestinal microorganisms are performed using high-throughput sequencing technology, and antimicrobial peptides are mined from the antimicrobial peptides in combination with comparison to an antimicrobial peptide database. Then, peptides with good preventive and therapeutic effects on tilapia lake virus disease are screened from the antimicrobial peptides for optimization and improvement, thereby obtaining a polypeptide with a sequence shown in Seq ID No. 1 of the present application.

[0017] The polypeptide of the sequence shown in Seq ID No. 1 of the present application can effectively inhibit the growth of tilapia lake virus in tilapia, thereby playing a role in preventing and treating tilapia lake virus disease, and is particularly suitable for high-density tilapia farming, providing a new solution and approach for the prevention and treatment of tilapia lake virus disease.

[0018] The present application is described in detail below through specific examples. The following examples are only used to illustrate the present application and should not be construed as limiting the present application.

[0019] Embodiment 1

[0020] Nile tilapia intestinal microbial sequencing:

[0021] (1) Tilapia breeding: The Nile tilapia used in the experiment were high-density farmed tilapia from the same batch, with a stocking density of 30 tilapia / m3. A total of 1,000 tilapia were farmed in the same batch for the experiment.

[0022] (2) Sample processing: Tilapia intestinal microorganisms were extracted on the 45th and 90th days of culture for testing. Specifically, 20 tilapia were randomly selected for each sampling, and their weight and length were measured. Then, they were dissected and the intestinal hindgut contents were taken for metagenomic DNA extraction. Genomic DNA was extracted using Stool DNA Kit.

[0023] (3) Sequencing analysis: All DNA samples were sequenced by Shenzhen Qingke Biotechnology Co., Ltd. The raw data were filtered through unified quality control standards such as removal of adapters, contamination, and low quality, and then the host sequence was removed before assembly to obtain intestinal microbial genome information. MetaGeneMark was used for gene prediction, and the genes generated by the assembly prediction of each sample were put together to remove redundancy; then species and functional annotation were performed: the species annotation information of Unigenes was obtained by comparing with the NCBI-NR database using Megan software, and combined with the gene abundance table to obtain species abundance tables at different classification levels; at the same time, the Unigenes were functionally annotated and analyzed for abundance of metabolic pathways (KEGG), homologous gene clusters (eggNOG), carbohydrate enzymes (CAZy), signal peptides, and transmembrane domains.

[0024] (4) Prediction and identification of antimicrobial peptides (AMPs): In the intestinal microbial gene dataset obtained in “(3) Sequencing analysis”, AMPs obtained from the online antimicrobial peptide database were used for comparison and query, and a threshold of E value ≤ 10-5 was used to perform homology search in TBLASTN to predict antimicrobial peptide genes.

[0025] The queried AMP nucleic acid sequence was then translated into a polypeptide sequence and verified using BLASTP. At the same time, AMPs that only exist in eukaryotes were removed. The activity was predicted using the Genomic AMPs prediction software V1.2.7 (2021SR0424886), inactive AMPs were removed, and potentially active AMPs were obtained. The annotation information in the APD3 database was used for classification, functional annotation was performed according to the KEGG database, peptide analysis was performed using the pepstats software of EMBOSS-6.6.0, Shiffere-Edmundson spiral wheel diagram prediction was performed using HeliQuest (http: / / expasy.org / tools), and the tertiary structure of each AMP was predicted using the I-TASSER online website.

[0026] (5) Verification of the activity and 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 significant differences between species were selected for synthesis. The peptides screened in this experiment were synthesized by Notai Biotechnology.

[0027] According to the above method, a polypeptide with better effect was finally screened out in this example, namely the polypeptide with the sequence shown in Seq ID No.1;

[0028] Seq ID No. 1: MALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQ.

[0029] Embodiment 2

[0030] 1. Toxicity test

[0031] In order to confirm whether the polypeptide of the sequence shown in Seq ID No.1 of this experiment is highly safe and has no adverse side effects on tilapia, this experiment stopped feeding for 72 hours, and injected the polypeptide intraperitoneally into two groups of tilapia at a dose of 200 mg / tail and 600 mg / tail, respectively. At the same time, a control group was set up for intraperitoneal injection of the same amount of PBS, with 100 tilapia in each group, and the conditions of the two groups of tilapia were observed every day for 30 days. The breeding density of tilapia is 30 tails / cubic meter.

[0032] The results showed that the survival rates of the two test groups were 100% at the doses of 200 mg / tail and 600 mg / tail for intraperitoneal injection, and no adverse reactions occurred, which was the same as the control group, indicating that the polypeptide sequence shown in Seq ID No.1 is highly safe for fish.

[0033] 3. Peptide protection test

[0034] (1) Feeding test

[0035] The antimicrobial peptide of the sequence shown in Seq ID No.1 was added to the feed at a dosage of 10 mg / kg, and no antimicrobial peptide was added to the control group. Therefore, the experiment was divided into two groups, namely the experimental group and the control group, each group containing 100 tilapia, with a stocking density of 30 tails / cubic meter, the experimental group was fed with feed added with antimicrobial peptide, and the control group was fed with control feed without antimicrobial peptide added, three times a day, 100 g of feed each time, for 10 consecutive days, and then the challenge test was carried out.

[0036] In the challenge test, a solution containing Tilapia Lake Virus Disease was inoculated into the experimental fish by intraperitoneal injection at a dose of 1 mL per fish, and a control test of injecting 1 mL of water was designed, that is, the 100 fish in the experimental group were divided into two parts, one part with 50 fish inoculated with Tilapia Lake Virus Disease, and the other part with 50 fish injected with water as a control test; similarly, the control group was also inoculated with Tilapia Lake Virus Disease in one part with 50 fish, and the other part with 50 fish injected with water as a control test. The survival rate of the fish was observed for 30 consecutive days.

[0037] The test results showed that the mortality rate of the control group injected with tilapia lake virus disease for 10 days was 82%, and the final mortality rate of 30 days of continuous observation was as high as 88%, and the final mortality rate of the control group injected with water was only 4%; the final mortality rate of the experimental group injected with tilapia lake virus disease for 30 days of continuous observation was 6%, and the final mortality rate of the control group injected with water was 2%. It can be seen that the antimicrobial peptide sequence shown in Seq ID No.1 can effectively inhibit the growth of tilapia lake virus in tilapia, thereby playing a role in preventing and treating tilapia lake virus disease.

[0038] (2) Immersion test

[0039] The antimicrobial peptide of the sequence shown in Seq ID No.1 was added to water to prepare an antimicrobial peptide aqueous solution with a concentration of 200 mg / L as an immersion solution, and the control group was pure water without adding antimicrobial peptides. The experimental group and the control group each contained 100 tilapia, and the breeding density was 30 fish / cubic meter; the experimental group was immersed in the immersion solution for 1 hour, once a day, for 10 consecutive days; the control group was immersed in pure water without adding antimicrobial peptides for 1 hour, once a day, for 10 consecutive days; and then the toxicity test was carried out.

[0040] Poison attack test: The test method is the same as “(1) Feeding test”.

[0041] The test results showed that the mortality rate of the control group reached about 80% 10 days after the virus attack, and the final mortality rate after continuous observation for 30 days was as high as 84%, while the final mortality rate of the control group injected with water was only 2%. The test group using antimicrobial peptides injected with tilapia lake virus disease and observed continuously for 30 days had a final mortality rate of 4%, while the final mortality rate of the control group injected with water was 2%.

[0042] The above experiments show that feeding or soaking the antimicrobial peptides of the sequence shown in Seq ID No.1 of this experiment can enhance the antibacterial ability of fish, thereby improving their survival rate, providing a new solution and approach for the prevention and treatment of tilapia lake virus disease in high-density tilapia farming.

[0043] The above content is a detailed description of the present application in combination with specific implementation methods, and it cannot be determined that the implementation of the present application is 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. Use of an antimicrobial peptide in the preparation of a drug for preventing or treating tilapia lake virus disease, characterized in that: The antimicrobial peptide is a polypeptide with a sequence shown in Seq ID No.1; Seq ID No. 1: MALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQ.

2. A nucleic acid encoding the antimicrobial peptide for use according to claim 1.

3. A recombinant plasmid containing the nucleic acid according to claim 2.

4. A microbial host containing the recombinant plasmid according to claim 3.

5. Use of the nucleic acid according to claim 2, the recombinant plasmid according to claim 3 or the microbial host according to claim 4 in the preparation of a drug for preventing or treating tilapia lake virus disease.

6. A drug for preventing or treating tilapia lake virus disease, characterized in that: The drug contains a polypeptide having a sequence shown in Seq ID No. 1; Seq ID No. 1: MALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQ.