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A novel fish-derived antimicrobial peptide mutant and its preparation method and application

A technology for antimicrobial peptides and mutants, applied in the field of new fish-derived antimicrobial peptide moronecidin mutants and its preparation, can solve the problems of low yield of antimicrobial peptides, complicated process, and restrictions on the application of antimicrobial peptides, and achieve broad antibacterial spectrum and antibacterial The effect of high potency, broad market prospect and development value

Inactive Publication Date: 2017-07-11
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction of antimicrobial peptides from animals has low yield, time-consuming, complicated process, and high cost, making it impossible to achieve large-scale production, thus restricting the practical application of antimicrobial peptides

Method used

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  • A novel fish-derived antimicrobial peptide mutant and its preparation method and application
  • A novel fish-derived antimicrobial peptide mutant and its preparation method and application
  • A novel fish-derived antimicrobial peptide mutant and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 The acquisition of novel fish-derived antimicrobial peptide mutants and their genes

[0015] 1. Fish-derived antimicrobial peptide moronecidin (GenBank accession number: AAV65044) is an antibacterial peptide composed of 22 amino acid residues, which has strong antibacterial activity against bacteria. In order to further improve its antibacterial activity, the amino acid sequence of the fish-derived antimicrobial peptide moronecidin was analyzed by biological software, and 2 amino acids (K7R and V20K) were mutated in the 22 amino acids of the fish-derived antimicrobial peptide moronecidin. In order to ensure the amidation of the C-terminus of the antimicrobial peptide, asparagine N was added to the C-terminus to significantly improve the antibacterial efficacy, and a novel fish-derived antimicrobial peptide mutant was obtained, whose amino acid sequence is SEQ ID NO:1.

[0016] 2. According to the obtained amino acid sequence SEQ ID NO:1 of the novel fish-deriv...

Embodiment 2

[0017] Example 2 Construction of genetically engineered fish-derived antimicrobial peptide mutant expression vector and acquisition of engineering bacteria

[0018] 1. Both the vector containing the antimicrobial peptide gene and the yeast expression vector were digested with XhoI and XbaI, and the digested products were recovered and ligated for PCR identification and sequencing.

[0019] 2. After the positive plasmid was linearized by SacI single enzyme digestion, it was added to the competent cell suspension of Pichia pastoris. After electroporation, spread evenly on YPDS selection plate containing 100 μg / mL Zeocin, and incubate at 30°C for 3-5 days. When the positive transformants on the YPDS plate grow larger, each transformant is inoculated onto the YPDS selection plate containing Zeocin 200 μg / mL, 500 μg / mL, and 1000 μg / mL in turn, and the colonies that grow normally on the high-concentration Zeocin plate are Possibly high copy recombinant strains.

[0020] 3. Inocula...

Embodiment 3

[0021] Example 3 Fermentation and purification of novel fish-derived antimicrobial peptide mutants

[0022] 1. Fermentation process

[0023] 1) Inoculate the positive recombinants obtained by screening into Erlenmeyer flasks with 1%-10% inoculum volume after activation, and inoculate 10L with 5%-20% inoculation volume at 28-30°C and 200r / min shaker for 16-24h Fermentation tank (installed medium 7L), temperature 28-30°C, rotation speed 500-1500r / min, medium pH value 5.0-6.0, ventilation volume 0.1-1.0VVM (the amount of oxygen introduced into 1L fermentation broth for 1min), dissolved Fermentation is carried out under the condition of oxygen>20%, and 50% glycerol is fed for 5 hours after culturing for 18-24 hours. When the dissolved oxygen suddenly rises to 100%, methanol is fed until the end of fermentation. The whole fermentation lasts for 48-72 hours.

[0024] 2) After the fermentation, the original tank was steam sterilized at 100°C for 10-20 minutes, discharged, centrifuge...

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Abstract

The invention provides a novel moronecidin mutant, and a preparation method and application thereof. According to the novel moronecidin mutant, and a preparation method and application thereof, spatial structure analysis is performed on amino acid sequences of the moronecidin through biological software; two amino acid sequences (K2R and V20K) of the 22 amino acid sequences of the moronecidin are mutated; meanwhile, asparagine N is added to the tail end C in order to ensure the amidation of the tail end C of an antimicrobial peptide, so that the novel moronecidin mutant is obtained, and the antibacterial efficacy of the novel moronecidin mutant is remarkably improved. On the basis, a novel moronecidin mutant gene is artificially synthesized by selecting and using pichia pastoris preferred codons, and is cloned in pichia pastoris for expressing to obtain a novel antimicrobial peptide recombinant yeast strains; fermentation scale is scaled up to a fermentation tank level; high-density fermentation and high-efficiency expression of an antimicrobial peptide product are realized. Fermentation liquid can be prepared into antimicrobial peptide preparations, such as a power preparation and a liquid preparation, which are used for aquatic animal feed additives or aquatic animal disease prevention and treatment preparations after being further purified.

Description

technical field [0001] The invention belongs to the technical field of functional gene modification, and in particular relates to a novel fish-derived antimicrobial peptide moronecidin mutant and its preparation method and application. Background technique [0002] Antimicrobial peptides are polypeptides with a small molecular weight that exist in organisms. They have broad-spectrum antibacterial activity and have a strong killing effect on bacteria, especially for certain drug-resistant pathogenic bacteria. In addition, some antimicrobial peptides also have a killing effect on some viruses, fungi, protozoa, etc., and even have the effect of improving the immune activity of animals and accelerating the wound healing process. At the same time, it also has the characteristics of good stability and good water solubility. These properties make antimicrobial peptides a research hotspot in related disciplines such as medicine, immunology and molecular biology. As one of the non-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/81A23K20/147A23K50/80A61K38/17A61P31/04
CPCA61K38/00C07K14/461
Inventor 侯竹美丰艳妮
Owner QINGDAO AGRI UNIV
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