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A fish-derived antimicrobial peptide hepcidin mutant and its application

A peptide mutant and antibacterial peptide technology, applied in the field of novel fish-derived antibacterial peptide hepcidin mutant and preparation thereof, can solve the problems of high price of chemically synthesized peptides, narrow antibacterial scope, low yield of natural antibacterial peptides, etc., and achieve broad market prospects and development value, broad antibacterial spectrum and high antibacterial titer

Inactive Publication Date: 2017-04-05
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although antimicrobial peptides have remarkable functions and attractive application prospects in aquaculture, the yield of natural antimicrobial peptides is low, the antibacterial range is narrow, and they cannot be extracted from animals in large quantities, and chemically synthesized peptides are expensive. The artificial modification of peptides to obtain better antimicrobial peptide mutants is a hot spot in the current research on antimicrobial peptides

Method used

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  • A fish-derived antimicrobial peptide hepcidin mutant and its application
  • A fish-derived antimicrobial peptide hepcidin mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 The acquisition of novel fish-derived antimicrobial peptide hepcidin mutant and its gene

[0015] 1. Fish-derived antimicrobial peptide hepcidin (GenBank accession number: AAT01563) is an antimicrobial peptide composed of 25 amino acid residues. In order to further improve its antibacterial activity, the spatial structure analysis of the amino acid sequence of the fish-derived antimicrobial peptide hepcidin was carried out by biological software. 13 C 14 The formation of C) further improves the bacteriostatic activity of the antimicrobial peptide, and replaces the lysine (K) at the 15th position of the antimicrobial peptide hepcidin with arginine (R), which is more polar. At the same time, in order to balance the structure of the antimicrobial peptide, the methionine (M) at the 6th position was replaced with lysine (L), and the tryptophan (W) at the 9th position was replaced with glycine (G). Three amino acid mutations (M6L, W9G and K15R) in the peptide hepc...

Embodiment 2

[0017] Example 2 Construction of fish-derived antimicrobial peptide hepcidin 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. Inoculate a single co...

Embodiment 3

[0021] Example 3 Fermentation and purification of novel fish-derived antimicrobial peptide hepcidin mutant preparation

[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 culture medium 6L), temperature 28-30 ℃, 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 4 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 min...

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Abstract

The invention provides a fish-derived antibacterial peptide hepcidin mutant and an application thereof. The novel fish-derived antibacterial peptide hepcidin mutant is obtained by carrying out spatial structure analysis on an amino acid sequence of the fish-derived antibacterial peptide hepcidin and mutating three amino acids in 25 amino acids of the fish-derived antibacterial peptide hepcidin, and the antibacterial efficacy of the fish-derived antibacterial peptide hepcidin is obviously improved. On the basis, a novel fish-derived antibacterial peptide hepcidin mutant gene is artificially synthesized by selecting a pichia pastoris biased codon and is cloned in pichia pastoris to be expressed, thus obtaining a novel antibacterial peptide recombinant yeast strain, and the fermentation scale is enlarged to the fermentation tank level, thus achieving high-density fermentation and high-efficiency expression of antibacterial peptide products. The fermentation liquor can be prepared into antibacterial peptide preparations, such as powder and liquid, after being further purified, and the antibacterial peptide preparations are used as aquatic animal feed additives or preparations for preventing and treating diseases of aquatic animals.

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 hepcidin mutant and its preparation method and application. Background technique [0002] Antimicrobial peptides (AMPs) are persistent in organisms, encoded by genes, and synthesized by ribosomes with broad-spectrum anti-bacterial, anti-fungal, anti-protozoan or anti-viral active polypeptides. They are widely present in animals and plants and are biological immune systems. important component of nonspecific immunity. Most antimicrobial peptides are small in molecular weight (less than 100 amino acids), positively charged and amphipathic. Antimicrobial peptides have the characteristics of unique antibacterial mechanism, broad-spectrum antibacterial activity, rapid bactericidal ability, not easy to produce drug resistance, stability, and good water solubility. As an ideal substitute for antibiotics, people...

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 李超侯竹美
Owner QINGDAO AGRI UNIV
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