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Hybrid antibacterial peptide CE-PR and application thereof

A CE-PR, hybrid antimicrobial peptide technology, applied in the field of peptide screening, can solve the problem of eukaryotic cell toxicity

Inactive Publication Date: 2015-03-25
青岛宏昊生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Natural antimicrobial peptides have various disadvantages in clinical application, and some are often accompanied by hemolysis while having high killing activity against pathogenic microorganisms, and some are toxic to eukaryotic cells (such as natural antibacterial peptide bombesin, defense toxin and melittin, etc.)

Method used

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  • Hybrid antibacterial peptide CE-PR and application thereof
  • Hybrid antibacterial peptide CE-PR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 The acquisition of hybrid antimicrobial peptide CE-PR and its gene

[0014] 1. Use biological software to analyze the spatial structure of the amino acid sequences of porcine antimicrobial peptide cecropin P1 (GenBank accession number: AB186032) and antimicrobial peptide PR-39 (GenBank accession number: NM_214450.1), and select the N-terminal of antibacterial peptide cecropin P1 Based on 19 amino acids with an α-helical structure, the 12th hydrophobic amino acid was replaced with a hydrophilic amino acid lysine (K12N), and then combined with the N-terminal 26 with strong antibacterial activity of the antibacterial peptide PR-39 amino acids were spliced, and protein Lingker (GPG) was added in the middle to obtain a novel hybrid antimicrobial peptide CE-PR, whose amino acid sequence is SEQ ID NO:1.

[0015] 2. According to the amino acid sequence SEQ ID NO: 1 of the obtained novel hybrid antimicrobial peptide CE-PR, redesign according to the codon preference ...

Embodiment 2

[0016] Example 2 Construction of Genetic Engineering Hybrid Antimicrobial Peptide CE-PR Expression Vector and Acquisition of Engineering Bacteria

[0017] 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.

[0018] 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.

[0019] 3. Inoculate a singl...

Embodiment 3

[0020] Embodiment 3 Fermentation, the preparation of purification novel hybrid antimicrobial peptide CE-PR

[0021] 1. Fermentation process

[0022] 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.

[0023] 2) After the fermentation, the original tank was steam sterilized at 100°C for 10-20 minutes, discha...

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Abstract

The invention provides a novel hybrid antibacterial peptide CE-PR as well as a preparation method and application of the novel hybrid antibacterial peptide CE-PR. The preparation method comprises the steps of splicing 19 amino acids at the N end of an antibacterial peptide cecropin P1 and 26 amino acids at the N end of an antibacterial peptide PR-39 on the basis of spatial structural analysis on amino acid sequences of swine-derived antibacterial peptides PR-39 and cecropin P1; adding protein Lingker (GPG) in the intermediate; and carrying out mutation (K12N) on an amino acid at the position 1 in the N end of the antibacterial peptide cecropin P1 to obtain the novel hybrid antibacterial peptide CE-PR with a remarkably-improved antibacterial effect. On the basis, a pichia pastoris optimal codon is selected to artificially synthesize a novel hybrid antibacterial peptide CE-PR gene which is cloned into pichia pastoris to express to obtain a novel antibacterial peptide recombinant yeast strain, and the fermentation scale is amplified to the level of a fermentation tank, so that the high-density fermentation and efficient expression of an antibacterial peptide product are realized. After being further purified, a fermentation solution can be prepared into powder, liquid and other antibacterial peptide preparations for preventing and treating livestock and poultry diseases.

Description

technical field [0001] The invention belongs to the technical field of polypeptide screening, and in particular relates to a hybrid antibacterial peptide and its application. Background technique [0002] Antibacterial peptides (antibacterial peptides) is a kind of polypeptide with strong antibacterial effect produced by organisms, and is an important component of biological innate immunity. So far, more than 600 endogenous antibacterial active peptides have been found in many organisms including animals, plants and prokaryotes. Porcine antimicrobial peptide PR-39 (GenBank accession number: NM_214450.1) consists of 39 amino acids and was first isolated from pig intestine by Agerberth B et al. in 1991. It is rich in two amino acid residues, arginine (Arg) and proline (Pro), forming a Pro-Arg-Pro structure, which may be related to the interaction of bacterial phospholipid membranes. Compared with the antimicrobial peptide PR-39 itself, the synthetic peptide with 26 amino aci...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/81A23K1/17A61P31/04A61P37/04A23K20/195
CPCY02A40/70
Inventor 王宏华
Owner 青岛宏昊生物科技有限公司
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