Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof

A technology of PR-39 and antimicrobial peptides, which is applied in the field of new pig-derived antimicrobial peptide PR-39 mutants and its preparation, can solve the problems of low potency of antimicrobial peptides and difficulty in development and use, and achieve broad market prospects and development value , The effect of improving antibacterial efficacy

Active Publication Date: 2015-02-18
安阳中农大生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual use, the potency of the antimicrobial peptide PR-39 is not high, making it difficult to carry out actual development and use

Method used

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  • Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof
  • Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof
  • Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 The acquisition of novel porcine-derived antimicrobial peptide PR-39 mutant and its gene

[0017] 1. Porcine antimicrobial peptide PR-39 (GenBank accession number: NM_214450.1) is an antimicrobial peptide composed of 39 amino acids. In order to further improve its antibacterial activity, three amino acid mutations (K9L, H29G and K34F) in the porcine antimicrobial peptide PR-39 were finally completed by analyzing the spatial structure of the amino acid sequence of the porcine antimicrobial peptide PR-39, and a The novel porcine antimicrobial peptide PR-39 mutant has an amino acid sequence of SEQ ID NO:1.

[0018] 2. According to the obtained amino acid sequence SEQ ID NO: 1 of the new porcine antimicrobial peptide PR-39 mutant, redesign according to the codon preference of the Pichia pastoris gene, and obtain the coding sequence of the new porcine antimicrobial peptide PR-39 mutant Nucleotide sequence SEQ ID NO:2. And a Kex2 restriction site was introduced at...

Embodiment 2

[0019] Example 2 Construction of genetically engineered porcine-derived antimicrobial peptide mutant expression vector and acquisition of engineering bacteria

[0020] 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.

[0021] 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.

[0022] 3. Inoc...

Embodiment 3

[0023] Example 3 Fermentation and purification of the preparation of novel pig-derived antimicrobial peptide PR-39 mutant

[0024] 1. Fermentation process

[0025] 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.

[0026] 2) After the fermentation, the original tank was steam sterilized at 100°C for 10-20 ...

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Abstract

The invention provides a new swine antibacterial peptide PR-39 mutant as well as a preparation method and application thereof. The new swine antibacterial peptide PR-39 mutant is obtained by carrying out space structure analysis on the amino acid sequence of swine antibacterial peptide PR-39 through biosoftware and mutating three amino acids of 39 amino acids, so that the antibacterial effect of the new swine antibacterial peptide PR-39 mutant is obviously improved. On the basis, a novel swine antibacterial peptide PR-39 mutant gene is artificially synthesized by adopting a pichia pastoris preferred codon, a new antibacterial peptide recombination yeast strain is obtained by expressing the novel swine antibacterial peptide PR-39 mutant gene by cloning into pichia pastoris, and a fermentation scale is expanded to a fermentation tank level, so that the high-density fermentation and high-efficiency expression of an antibacterial peptide product are realized. After fermentation liquor is further purified, antibacterial peptide preparations, namely powder, liquid and the like, are prepared for preventing and treating livestock and poultry diseases.

Description

technical field [0001] The invention belongs to the technical field of functional gene modification, and in particular relates to a novel pig-derived antimicrobial peptide PR-39 mutant and its preparation method and application. Background technique [0002] The world's first antimicrobial peptide, cecropin, was induced and isolated from Cecropin chrysalis by Swedish scientist G.Boman et al. in 1980, and then many antimicrobial peptides were isolated and purified. So far, more than a thousand antimicrobial peptides have been isolated, and they are widely distributed in organisms such as mammals, crustaceans, fish, insects, amphibians, birds, plants, bacteria and viruses. [0003] Natural antibacterial peptides generally have low antibacterial activity, narrow antibacterial spectrum, and technical problems such as hemolytic toxicity, which greatly limit their clinical application. In recent years, with the in-depth research on antimicrobial peptides, researchers have tried t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12A23K1/165A23K1/18
CPCA23K20/147C07K14/47
Inventor 王宏华
Owner 安阳中农大生物技术有限公司
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