A new type of pig source antibacterial peptide mutant and its preparation methods and applications

A technology of antimicrobial peptides and mutants, which is applied in the field of novel porcine antimicrobial peptide mutants and their preparation, can solve the problems of narrow antibacterial spectrum, difficulty in clinical application, low antibacterial activity, etc., and achieve broad antibacterial spectrum, antibacterial The effect of high potency, broad market prospect and development value

Inactive Publication Date: 2017-03-01
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most natural antimicrobial peptides have technical problems such as low antibacterial activity, narrow antibacterial spectrum, sensitivity to protease, and hemolytic toxicity, making it difficult to be widely used in clinical practice.

Method used

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  • A new type of pig source antibacterial peptide mutant and its preparation methods and applications
  • A new type of pig source antibacterial peptide mutant and its preparation methods and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Acquisition of novel porcine-derived antimicrobial peptide mutants and their genes

[0015] 1. Porcine antimicrobial peptide PMAP37 (GenBank accession number: L39641.1) is an antibacterial peptide with amphipathic α-helix composed of 37 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 PMAP37 was carried out by biological software, and the hydrophobic amino acid near the position far from the center of the peptide sequence was selected to replace it with the hydrophilic amino acid Lys. It is to reduce the hydrophobicity of both ends. At the same time, Leu was used to replace Lys in the middle of the peptide to compensate for the reduction of the overall hydrophobicity of the peptide caused by the introduction of Lys on the nonpolar side of the peptide. Finally, three amino acid mutations (L...

Embodiment 2

[0017] Example 2 Construction of genetically engineered porcine-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 used xho I and Xba I double enzyme digestion, the enzyme digestion products were recovered and ligated for PCR identification and sequencing.

[0019] 2. Positive plasmid classics Sac After linearization by single enzyme digestion, add Pichia pastoris competent cell suspension. After electrotransformation, spread evenly on the 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 strain...

Embodiment 3

[0021] Example 3 Fermentation and purification of the preparation of novel porcine-derived antimicrobial peptide mutants

[0022] 1. Fermentation process

[0023] 1) Inoculate the screened positive recombinants into 1%-10% inoculum in Erlenmeyer flasks after activation, culture on a shaker at 28-30°C and 200r / min for 16-24 hours, then inoculate 10L with 5%-20% inoculum Fermentation tank (installed medium 6L), 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 in 1min), dissolved Fermentation was carried out under the condition of oxygen>20%, and 50% glycerol was added for 4 hours after culturing for 18-24 hours. When the dissolved oxygen suddenly rose to 100%, methanol was added until the end of fermentation. The whole fermentation lasted for 48-72 hours.

[0024] 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 pig-source antibacterial peptide mutant, a preparation method and an application thereof. According to the invention, the spatial structure analysis is preformed on the amino acid sequence of pig-source antibacterial peptide PMAP37 through biology software and three amino acids (L6K, K20L and L31K) of the thirty-seven amino acids are mutated, so that the novel pig-source antibacterial peptide mutant is acquired and the antibacterial effect is obviously increased. On the basis, a pichia pastoris biased codon is selected and a novel pig-source antibacterial peptide mutant gene is artificially synthesized and is cloned in expression of the pichia pastoris, so that a novel antimicrobial peptide recombined yeast strain is acquired, the fermentation scale is amplified to the fermentation tank level and the high-density fermentation and high-efficient expression of the antibacterial peptide products are realized. After the fermentation liquor is further purified, the fermentation liquor can be prepared into an antibacterial peptide preparation, such as powder and liquid, used for preventing and treating livestock diseases.

Description

technical field [0001] The invention belongs to the technical field of functional gene modification, and in particular relates to a novel porcine-derived antimicrobial peptide mutant and its preparation method and application. Background technique [0002] Antimicrobial peptide is a kind of small molecular protein with antibacterial activity existing in organisms, the number of amino acids is less than 100, often positively charged, and a class of small peptides with broad-spectrum antibacterial properties. The defensive polypeptides of exogenous pathogens are important components of the innate immunity of organisms, and together with interferon and complement, they constitute the host's immune defense system. 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 b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/81C12R1/84
CPCC07K14/47
Inventor 侯竹美
Owner QINGDAO AGRI UNIV
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