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Preparation method and application of animal-derived cationic antimicrobial peptides

An antibacterial peptide and cation technology, which is applied in the field of preparation of animal-derived cationic antibacterial peptides, can solve problems such as unproven safety and effectiveness of protein poultry and animals, and achieve improved feed returns and breeding profits, and broad-spectrum antibacterial effects. , the effect of high antibacterial activity

Active Publication Date: 2011-11-02
山东迅达康兽药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The above patents and inventions are all based on plant, insect or human-derived antimicrobial peptides. Due to the differences between species, the safety and effectiveness of these proteins for poultry and animals have not been confirmed.

Method used

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  • Preparation method and application of animal-derived cationic antimicrobial peptides
  • Preparation method and application of animal-derived cationic antimicrobial peptides
  • Preparation method and application of animal-derived cationic antimicrobial peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The antimicrobial peptide gallinacin 14 N-terminal 1-24 amino acid residue fragment (MGIFLLFLVLLAVPQAAPESDTVT (SEQ ID NO.3)) and antimicrobial peptide Spheniscin-2 C-terminal 5-38 amino acid residue fragment (CRLRRGFCARGRCRFPSIPIGRCSRFVQCCRRVW (SEQ ID NO.4)) were hybridized ( That is to connect the two fragments) together, the amino acid sequence obtained is as follows: MGIFLLFLVLLAVPQAAPESDTVTCRLRRGFCARGRCRFPSIPIGRCSRFVQCCRRVW.

[0032] According to the above-mentioned amino acid sequence, the preferred codons of genetically engineered bacteria, that is, the commonly used codes of highly expressed genes, are used for amino acid encoding, and then with the help of DNA analysis software, the unsuitable part of the codon tandem organization is appropriately modified, adjusted and optimized (that is, the protein expressing the protein is selected. The host bacteria prefer to use amino acid codons to improve expression efficiency), and finally we get the optimized nucleotide se...

Embodiment 2

[0042] Cloning of antimicrobial peptide gene: clone the obtained antimicrobial peptide gene fragment (gal-sphe) into the selected vector IMPACT-CN, and then transform the plasmid (IMPACT-CN) into genetic engineering bacteria (ER2566), containing 100μg / ml ampicillin and medium coated with 20mg / ml X-gal, 1mM / ml IPTG (Tryptone: 10g / L, Yeast Extract: 5g / L, NaCl (sodium chloride) ): 10g / L, Agar agar 15g / L) culture (37°C, 12-16 hours), screen out colonies containing recombinant plasmid (IMPACT-CN-gal-sphe), and store the colonies at 4°C.

Embodiment 3

[0044] Expression and purification of antimicrobial peptides:

[0045] 1. Bacterial culture: directly take a single colony and add it to 1L LB liquid medium (containing 100μg / ml of ampicillin), and shake culture at 37°C until OD600 reaches 0.5-0.7. Note: You can pick a single colony newly transformed with recombinant expression plasmid, first inoculate it in 10ml liquid medium (containing 100μg / ml of ampicillin), culture with shaking at 37°C for 3-4 hours, and then inoculate it in 1L liquid medium (containing ampicillin) 100μg / ml), incubate at 37°C until OD600 reaches 0.5.

[0046] 2. Induce protein expression: add IPTG with a final concentration of 0.3 mM, culture with shaking at 37°C for 3 hours to induce protein expression. Set up a negative control. Sampling 10-20μl for SDS-PAGE electrophoresis, sampling 1-2μl for Western blot identification.

[0047] 3. Cell collection: Centrifuge at 5000×g for 10 minutes at 4°C and discard the supernatant. Store at -20 / -80℃. Freeze and tha...

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Abstract

The invention discloses a preparation method and an application of animal-derived cationic antimicrobial peptides. The preparation method comprises the following steps: cloning cationic antimicrobial peptide genes shown as SEQ ID NO.1 onto a carrier; transforming plasmids into genetic engineering strains; culturing for 12-16 hours on a culture medium; screening out bacterial colonies containing recombinant plasmids; selecting a single bacterial colony and placing in an LB (Load Balance) liquid culture medium; shaking and culturing till OD600 reaches 0.5-0.7; adding IPTG (Isopropyl Thiogalactoside), shaking and culturing for 3 hours and inducing protein expression; centrifuging for 10 minutes at 4 DEG C and collecting thallus; utilizing a lysis buffer solution to re-suspend the thallus, performing ice-bath, and ultrasonically breaking cells, thereby releasing protein; and utilizing a Chitin column to purify the protein. A test proves that the animal-derived cationic antimicrobial peptides have a broad spectrum antimicrobial function and an ultrahigh antimicrobial activity and can be used for replacing antibiotics for preventing and treating poultry and livestock bacterial infections. A new method for developing a novel clinic antimicrobial drug is demonstrated.

Description

Technical field [0001] The invention belongs to the field of animal health product production and application research, and specifically relates to a preparation method and application of animal-derived cationic antibacterial peptides. Background technique [0002] With the deepening of research on antimicrobial peptides, its unique antibacterial effect has attracted people's attention. There are 3 domestic patents related to antimicrobial peptides, which mainly involve antimicrobial peptides against microbial pathogen infection and antimicrobial peptides for preservation technology; Only the French company Entomcd has obtained multiple patent applications abroad and uses these new peptides for the treatment of human diseases worldwide. Many antimicrobial peptides have now entered Phase II or Phase III clinical trials. [0003] The above patents and inventions are all based on plant, insect or human-derived antimicrobial peptides. Due to the differences between species, the safety...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63A61K38/17A61P31/04A23K1/17A23K20/195
Inventor 汪安国王尚明刘思当高蕾
Owner 山东迅达康兽药有限公司
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