Preparation method and application of animal-derived cationic antimicrobial peptides

A technology of antimicrobial peptides and cations, which is applied in the field of preparation of animal-derived cationic antimicrobial peptides, can solve the problems that the safety and effectiveness of protein poultry and animals have not been proven, and achieve the improvement of feed remuneration and breeding profits, high antibacterial activity, The effect of broad-spectrum antibacterial action

Active Publication Date: 2013-04-17
山东迅达康兽药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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
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Embodiment 1

[0032] The antimicrobial peptide gallinacin 14 N-terminal 1-24 amino acid residue fragment (MGIFLLFLVLLAVPQAAPESDTVT (SEQ ID NO.3)) and the antimicrobial peptide Spheniscin-2 C-terminal 5-38 amino acid residue fragment (CRLRRGFCARGRCRRFPSIPIGRCSRFVQCCRRVW (SEQ ID NO.4)) hybrid ( The two fragments are linked together) to obtain the following amino acid sequence: MGIFLLFLVLLAVPQAAPESDTVTCRLRRGFCARGRRFPSIPIGRCSRFVQCCRRVW.

[0033] According to the above amino acid sequence, the preferred codons of genetically engineered bacteria, that is, the common codes of highly expressed genes, are selected for amino acid encoding, and then the unsuitable part of the codon tandem organization is appropriately modified, adjusted and optimized by means of DNA analysis software (that is, the expression of the protein is selected). The amino acid codons preferred by the host bacteria to improve expression efficiency), and finally we obtained the nucleotide sequence and amino acid sequence of the o...

Embodiment 2

[0043]Cloning of the antimicrobial peptide gene: the obtained antimicrobial peptide gene fragment (gal-sphe) was cloned into the selected vector IMPACT-CN, and then the plasmid (IMPACT-CN) was transformed into the genetically engineered bacteria (ER2566), containing 100 μg / ml ampicillin and medium coated with 20mg / ml X-gal, 1mM / ml IPTG (Tryptone (tryptone): 10g / L, Yeast Extract (yeast extract): 5g / L, NaCl (sodium chloride ): 10g / L, Agar agar 15g / L) culture (37 ℃, 12-16 hours), screening out the bacterium colony that contains recombinant plasmid (IMPACT-CN-gal-sphe), and bacterium colony is preserved in 4 ℃.

Embodiment 3

[0045] Expression and purification of antimicrobial peptides:

[0046] 1. Bacterial culture: Take a single colony directly, add it to 1L LB liquid medium (containing 100 μg / ml ampicillin), and culture with shaking at 37°C until the OD600 reaches 0.5-0.7. Note: A single colony newly transformed with a recombinant expression plasmid can be picked, first inoculated in 10ml liquid medium (containing ampicillin 100μg / ml), shaken at 37°C for 3-4 hours, and then inoculated in 1L liquid medium (containing ampicillin 100 μg / ml), cultured at 37°C until OD600 reached 0.5.

[0047] 2. Induction of protein expression: Add IPTG at a final concentration of 0.3 mM, shake and culture at 37° C. for 3 h, and induce protein expression. Set up a negative control. Take a sample of 10-20 μl for SDS-PAGE electrophoresis, and take a sample of 1-2 μl for Western blot identification.

[0048] 3. Cell collection: centrifuge at 5000×g at 4°C for 10 minutes, discard the supernatant. Store at -20 / -80°C....

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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 in particular 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 on antimicrobial peptides, mainly related to antimicrobial pathogen infection and antimicrobial peptides used in fresh-keeping technology; Abroad, only the French company Entomcd has obtained a number of patent applications, and uses these new peptides for the treatment of human diseases worldwide. Now many antimicrobial peptides have 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 and effectiveness o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47A23K20/195
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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