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Antibacterial peptide as well as preparation thereof and application thereof in animal feed additive

An antibacterial peptide and bacteriostatic agent technology, applied in the field of antibacterial peptide and its preparation and application in animal feed additives, can solve the problems of antibiotic residues, health and environmental hazards, human physical hazards, etc. Application prospect, effect of inhibiting antiviral activity

Active Publication Date: 2019-01-25
INNER MONGOLIA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibiotics will produce residues in animals and animal products, causing potential harm to the human body; at the same time, the problem of drug resistance produced by pathogenic bacteria has huge potential harm to human health and the environment

Method used

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  • Antibacterial peptide as well as preparation thereof and application thereof in animal feed additive
  • Antibacterial peptide as well as preparation thereof and application thereof in animal feed additive
  • Antibacterial peptide as well as preparation thereof and application thereof in animal feed additive

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Freshwater Crayfish Recombinant Antimicrobial Peptide rPc-Crustin 6 Protein

[0024] (1) The total RNA of the mixed tissue samples of freshwater crayfish hepatopancreas, gills, and intestines was extracted using the animal total RNA extraction kit, and the total RNA samples of freshwater crayfish mixed tissues were obtained.

[0025] (2) Use the freshwater crayfish total RNA sample obtained in step (1) as a template, and use the specific primer 5'-cagtaatatattttattaatacaatg-3' as the post-primer of the reverse transcription synthesis reaction, and use a one-step RT-PCR amplification kit to carry out cDNA was synthesized by reverse transcription, and cDNA samples were obtained.

[0026] (3) Design the front and rear primers Pc-crustin 6-F and Pc-crustin 6-R for amplifying the expression fragment (mature peptide) of the freshwater crayfish antimicrobial peptide gene Pc-crustin 6 coding region:

[0027] Pc-crustin 6-F: 5′-GCC GAATTC CACCTGGGCTAC...

Embodiment 2

[0036] Example 2 Liquid antibacterial experiment of recombinant antimicrobial peptide rPc-Crustin 6 and four common pathogenic bacteria

[0037] The recombinant antimicrobial peptide rPc-Crustin 6 protein preparation obtained in Example 1 was subjected to a liquid antibacterial experiment with common pathogenic bacteria in animal feed. First, Staphylococcus aureus, Escherichia coli, Penicillium, and Trichoderma were selected as pathogenic bacteria for routine overnight liquid culture. On the next day, take 100 μL of the recombinant antibacterial peptide rPc-Crustin 6 protein preparation and mix with 100 μL of the target pathogenic bacteria solution, and place them in the spotting wells of a clean disposable 96-well microplate plate for incubation. At the same time, each spotting 100 μL of liquid poor (PB) medium (1% tryptone, 0.5% NaCl by mass and volume ratio, pH 7.0) was added to the well. Afterwards, under the condition of 28°C, cultured statically for 24 hours; during this...

Embodiment 3

[0039] Example 3 Recombinant antimicrobial peptide rPc-Crustin 6 on the antibacterial and mold removal experiments of rabbit granular feed during storage

[0040] The recombinant antimicrobial peptide rPc-Crustin 6 protein liquid preparation obtained in Example 1 and the rabbit pelleted feed sold in the market were subjected to antibacterial and mildew removal experiments during storage. In the first step, the granular feed is sprayed and mixed with the recombinant antimicrobial peptide rPc-Crustin 6 protein preparation. Get one bag (5kg) of rabbit granular feed bought in the market, utilize the recombinant antimicrobial peptide rPc-Crustin 6 protein liquid preparation that 50mL embodiment 1 obtains to spray and mix, seal the bag mouth afterwards, under normal temperature condition, clean plastics indoors Store in buckets. The second step is the preparation of granular feed extracts at different time points. Sampling experiments were carried out at intervals of 15 days, with...

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Abstract

The invention discloses antibacterial peptide as well as preparation thereof and application thereof in an animal feed additive, belonging to the technical fields of biotechnology and anti-bacteria. The antibacterial peptide provided by the invention is derived from freshwater crayfish, and the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO 1. The preparation of the antimicrobial peptide comprises the following steps: extracting total ribonucleic acid (RNA) of the freshwater crayfish as a template, performing reverse transcription to obtain complementary deoxyribonucleic acid (cDNA); using the cDNA as the template for carrying out polymerase chain reaction (PCR) amplification to obtain a nucleotide fragment containing an antibacterial peptide coding sequence, enabling the amplified product and an expression vector to be digested, ligated and transformed to construct an escherichia coli engineering strain which can express the recombinant antibacterial peptide, and carrying out induced culture on the escherichia coli engineering strain to express the recombinant antibacterial peptide. The antibacterial peptide provided by the invention has functions of bacteriostasis, mold removal and antivirus, thus being used for preparing a bacteriostatic agent, a mold removing agent or an antiviral agent; the antibacterial peptide can be used as the animal feed additive for storing feed, and is free from chemical residue and environmental pollution risk, thus having a broad application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and antibacterial technology, in particular to an antibacterial peptide derived from freshwater crayfish (Procambarus clarkii), its preparation and its application in animal feed additives. Background technique [0002] In the process of animal breeding, feed is an important material basis that affects its development, and it accounts for about 70% of the breeding cost of a family or enterprise. In the general breeding process, granular feed, powdered feed, and silage feed are the types of feed often used by farmers. Animal feed is an important food source for livestock, poultry and other animals. These substances are generally required to be rich in protein to meet the protein needs of animals during growth. In the breeding process, granular feed is often produced and stored in large quantities by production enterprises, and will be purchased and stored in large quantities by breeding enterprises. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A23K20/147
CPCA23K20/147C07K14/43509C12N15/70
Inventor 杜志强沈秀丽王凯
Owner INNER MONGOLIA UNIV OF SCI & TECH
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