A kind of porcine antimicrobial peptide cecropin P1 mutant and its preparation method and application

An antibacterial peptide and mutant technology, applied in the field of functional peptide screening, can solve the problems of unclear systemic or local toxicity, instability and drug resistance, and achieve broad market prospects and development value, antibacterial spectrum. The effect of broad and high antibacterial titer

Inactive Publication Date: 2017-10-10
ANHUI HAOXIANG AGRI & ANIMAL HUSBANDRY +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with traditional antibiotics, antimicrobial peptides have more advantages and may become a substitute for antibiotics, but only a few antimicrobial peptides have entered clinical trials, and others have stopped in various phases of clinical trials due to various reasons, and the reasons for failure Unknown Presumably due to systemic or local toxicity, instability, and drug resistance

Method used

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  • A kind of porcine antimicrobial peptide cecropin P1 mutant and its preparation method and application
  • A kind of porcine antimicrobial peptide cecropin P1 mutant and its preparation method and application
  • A kind of porcine antimicrobial peptide cecropin P1 mutant and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Acquisition of novel porcine-derived antimicrobial peptide cecropin P1 mutant and its gene

[0015] 1. Porcine antibacterial peptide cecropin P1 (GenBank accession number: AB186032) is an antibacterial peptide with amphipathic α-helix composed of 31 amino acids, which has strong antibacterial activity against bacteria. In order to further improve its antibacterial activity, the spatial structure analysis of the amino acid sequence of the porcine antimicrobial peptide AB186032 was carried out by biological software, and the two amino acids (10L, 12N) of the alpha helix at the hydrophilic end were selected to use the more hydrophilic arginine Acid (R) and lysine (K) were replaced in order to further improve the hydrophilicity and its ability to bind to the cell membrane, while adding asparagine at the C-terminus of the antimicrobial peptide to promote the amidation of the C-terminus, and finally A novel porcine antimicrobial peptide cecropin P1 mutant is obtaine...

Embodiment 2

[0017] Example 2 Construction of genetically engineered pig-derived antimicrobial peptide cecropin P1 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]...

Embodiment 3

[0021] Example 3 Fermentation and purification of novel porcine-derived antimicrobial peptide cecropin P1 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-...

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Abstract

The invention provides a swine-derived antibacterial peptide cecropin P1 mutant as well as a preparation method and application of the swine-derived antibacterial peptide cecropin P1 mutant. The preparation method comprises the steps of carrying out spatial structural analysis on an amino acid sequence of a swine-derived antibacterial peptide cecropin P1 through biological software; carrying out mutation (R10L, K12N) on 2 amino acids selected from 31 amino acids; and adding asparagine at the C tail end so as to be beneficial to the amidation of the C tail end to obtain a novel swine-derived antibacterial peptide mutant with a remarkably-improved antibacterial effect. On the basis, a pichia pastoris optimal codon is selected to artificially synthesize a novel swine-derived antibacterial peptide cecropin P1 mutant 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 functional polypeptide screening, and in particular relates to a novel porcine-derived antimicrobial peptide cecropin P1 mutant and a preparation method and application thereof. Background technique [0002] The abuse of antibiotics has not only led to public health problems such as drug residues, but also due to the emergence of drug-resistant strains and new pathogenic subtypes, the prevention and treatment of bacterial diseases has once again become a difficult problem for human beings. Therefore, the development of new antibacterial drugs has become an increasingly important research topic. Antibacterial peptide (ABP), also known as antimicrobial peptide (Antimicrobial peptide), antibiotic peptide (Antibiotics peptide), is a class of small molecular polypeptides with various biological activities induced by organisms, and is a key component of the host immune defense system. An important component, i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/81A23K20/147A23K20/195A61K38/17A61P31/04C12R1/84
CPCA23K20/195A61K38/00C07K14/47Y02A40/70
Inventor 高亚飞顾有方殷宗俊高亚成李升和孙裴高静李郁蒋广志
Owner ANHUI HAOXIANG AGRI & ANIMAL HUSBANDRY
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