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Antibacterial peptide and application thereof in aquaculture

A technology of antimicrobial peptides and bacteria, applied in the field of antimicrobial peptides and their application in aquaculture, can solve the problems of limited natural extraction resources of antimicrobial peptides, high cost of chemically synthesized peptides, complicated separation and purification process, etc. The effect of drug properties, chemical synthesis difficulty, and broad-spectrum antibacterial activity

Active Publication Date: 2021-12-07
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are still some problems in the current research on the application of antimicrobial peptides: for example, the natural extraction resources of antimicrobial peptides are limited, the process is complicated, and the cost is high; chemically synthesized peptides are too expensive and difficult to industrialize; , Antiviral ability makes it difficult to use commonly used bacteria and viruses as expression systems, antimicrobial peptides have small molecular weights and are easily hydrolyzed by proteases, the separation and purification process is complicated, and the yield of gene expression is not high, etc.

Method used

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  • Antibacterial peptide and application thereof in aquaculture
  • Antibacterial peptide and application thereof in aquaculture
  • Antibacterial peptide and application thereof in aquaculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 antimicrobial peptide caNKL2 102-119 selection and synthesis

[0031] 1. Acquisition of NK-lysins sequence of crucian carp

[0032] In order to find out the possible AMP gene in crucian carp, using the AMP gene sequences of zebrafish, grass carp, rainbow trout, carp, etc. (http: / / www.ncbi.nlm.nih.gov / blast) BLASTP search program analyzed the similarity between the homologous sequences of crucian carp AMP and other reported fish AMPs. ClustalX 2.0 was used to perform multiple sequence alignments on the searched AMP amino acid sequences, MEGA 5.0 software was used to construct a phylogenetic tree, and combined with PCR amplification and tissue quantitative data to confirm, two segments of crucian carp NK-lysins sequences were named caNK-lysins1 and caNK respectively. -lysins2, as a result of multiple sequence alignment, it was found that NK-lysins sequences all contain signal peptide, saposin B domain and 6 conserved cysteines (such as figure 1 and figur...

Embodiment 2

[0035] Embodiment 2 antimicrobial peptide caNKL2 102-119 Antibacterial activity verification

[0036] Testing the Synthetic Peptide caNKL2 Using the Oxford Cup Method 102-119 For the antibacterial activity against Escherichia coli, Staphylococcus aureus and Aeromonas hydrophila, kanamycin was used as a positive control, and the dissolution carrier was formic acid.

[0037] First, Escherichia coli, Staphylococcus aureus and Aeromonas hydrophila were streaked and inoculated on LB solid medium (Luria-Bertani medium), placed in a constant temperature incubator at 37°C for 18 hours, and single colonies of each strain were picked Placed in LB liquid medium, cultured at 37°C with constant temperature and shaking for 12h. Measure the OD of the bacterial solution 630 value (the absorbance value of the solution at a wavelength of 630nm), and dilute it to 1×10 6 CFU / mL. Then the bacteria were co-incubated with a series of antimicrobial peptide solutions with different final concentr...

Embodiment 3

[0040] Example 3 Antimicrobial Peptide caNKL2 102-119 cytotoxicity assay

[0041] In this example, the activity of the cells is detected by the MTT assay, and then the antimicrobial peptide caNKL2 is tested 102-119 Whether it is toxic to fish cells. The animal cells used in the experiment are carp epithelioma cell line (Epithelioma papulosum cyprini cell line, EPC).

[0042] Experimental principle: MTT entering living cells can be acted upon by intracellular enzymes to produce blue-purple crystals, which are deposited in the cells. Dead cells cannot form blue-purple crystals. The organic solvent DMSO can dissolve the blue-purple precipitate formed. Use a spectrophotometer to detect the absorbance of the blue-purple crystals in the sample, which can reflect the activity of the cells. The absorbance value of blue-purple crystals in the sample is positively correlated with the number of living cells.

[0043] Specific steps are as follows:

[0044] (1) Cell recovery, recov...

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Abstract

The invention discloses an antibacterial peptide and application thereof in aquaculture. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO.1. A section of micromolecule polypeptide caNKL2102-119 with the length of 18aa is selected and artificially synthesized through bioinformatics analysis and fitting of a three-dimensional space structure of a crucian carp NK-lysin protein, and research finds that the micromolecule polypeptide has excellent antibacterial and antiviral activity, does not easily generate drug resistance, is short in synthesis sequence, small in molecular weight, small in chemical synthesis difficulty, and high in bioactivity, can greatly save the cost of large-scale production on the basis of killing pathogenic bacteria in organisms, is expected to become an effective substitute of antibiotics in aquaculture, has high economic value and production and application value, and can inhibit replication of spring viremia virus (SVCV) of carp, a new thought is provided for preparation of aquatic antiviral drugs, and prevention and control of related aquatic viral diseases are facilitated.

Description

technical field [0001] The invention belongs to the technical field of antimicrobial peptides, and in particular relates to an antimicrobial peptide and its application in aquaculture. Background technique [0002] The discovery and clinical application of antibiotics provide a powerful means for human to control infectious diseases. However, the problem of antibiotic resistance caused by the misuse of antibiotics has become one of the biggest hidden dangers threatening global health. Studies have shown that it takes about ten years or more to develop a new type of antibiotic, but it takes less than two years for bacteria to develop drug resistance. The speed of new drug development is far behind the speed of bacterial resistance. And once there are "super bacteria" with multiple resistance genes, people will have no medicine available for them. In fact, the problem of antibiotic resistance is related to the overuse of antibiotics in human agriculture and medicine and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46A61K38/17A61P31/04A61P31/14G16B30/00
CPCC07K14/461A61P31/04A61P31/14G16B30/00A61K38/00Y02A50/30
Inventor 袁军法戴彩姣张玉军李莉娟
Owner HUAZHONG AGRI UNIV
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