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Preparation method of artificial antibacterial peptide PR39-R1T and application thereof

A PR39-R1T, antibacterial peptide technology, applied in the field of genetic engineering, can solve the problems of low biological activity and hemolytic activity of natural antibacterial peptides, and achieve the effects of strong broad-spectrum antibacterial activity, good stability and low production cost

Inactive Publication Date: 2015-10-21
刘诚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of low biological activity and hemolytic activity of existing natural antimicrobial peptides, to provide an artificial antimicrobial peptide PR39-R1T with high biological activity and no hemolytic activity, and a large-scale production-based High-efficiency preparation method of bacillus expression system, while providing artificial antimicrobial peptide PR39-R1T for the application of various bacteria

Method used

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  • Preparation method of artificial antibacterial peptide PR39-R1T and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of prokaryotic expression vectors and genetically engineered bacteria.

[0019] According to the amino acid sequences of the mature peptides of the antibacterial peptide PR-39 (No. AAB20869.1) and Ranatuerin-1T (No. P82740.1) in GenBank, the 22nd position of the antibacterial peptide Ranatuerin-1T was replaced with methionine (Met) Tryptophan (Trp), artificially designed the amino acid sequence SEQ ID No.1 of the artificial antimicrobial peptide PR39-R1T of the present invention, wherein DDDDK is the cleavage site of enterokinase. Then the nucleotide sequence encoding the antimicrobial peptide was designed using E. coli preferred codons, and nucleic acid restriction endonucleases were added at both ends xho and Bam H Identify the site to form the target gene, the sequence of which is shown in SEQ ID No.2.

[0020] The designed target gene sequence is 5′-cac ctcgag gatgatgatgataaacatcatcgtcgtcgtccgcgtccgccgtatctgccgcgtccgcgtccgccgccgtttt...

Embodiment 2

[0024] Example 2: Obtaining and purifying the artificial antimicrobial peptide PR39-R1T.

[0025] Use enterokinase to digest the expression product according to the following enzyme digestion system: 10×Buffer 10.0 μL, ddH 2O 38.0 μL, target protein 50.0 μL, enterokinase 2.0 μL, placed at 23°C for 20h.

[0026] The artificial antimicrobial peptide PR39-R1T of the present invention was obtained by purifying the HisPur Ni-NTA Spin Purification Kit from Thermo Scientific through nickel ion affinity chromatography. After pretreating the resin column according to the instructions, add the enzyme-cut supernatant equal to the amount of High-capacity nickel-IMAC resin, place it in a 4°C environment for 1 hour, elute the protein, and collect the eluate for SDS- PAGE detection results showed that there was an obvious protein band between 4.6-10.0kD, which was in line with the experimental design size, that is, the artificial antimicrobial peptide PR39-R1T. Its concentration was determ...

Embodiment 3

[0027] Example 3: Detection of biological activity of artificial antimicrobial peptide PR39-R1T.

[0028] Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of artificial antimicrobial peptide PR39-R1T against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus suis type 2 and Bacillus anthracis by microdilution method ). The specific experimental steps are as follows: the experimental strains were prepared into 10 6 Cfu / mL bacterial solution was added to 96-well cell culture plates, 90 μL per well. The artificial antimicrobial peptide PR39-R1T solution was diluted with PBS to 40.0, 20.0, 10.0, 8.0, 6.0, 4.0, 2.0, 1.0, 0.5 μg / mL, and added to a 96-well cell culture plate, 10 μL per well. The blank control group was set to contain only LB liquid medium, the conditional control group only contained the bacterial solution of the experimental strain, and the enzyme digestion control g...

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Abstract

The invention relates to a preparation method of an artificial antibacterial peptide PR39-R1T and application thereof. Amino acid sequences of the artificial antibacterial peptide PR39-R1T are designed artificially, and escherichia coli preference codons are adopted for designing and coding the amino acid sequences of the artificial antibacterial peptide; an overlap region amplification gene splicing method is adopted for synthesizing target genes to construct a prokaryotic expression vector, and the prokaryotic expression vector is converted into escherichia coli BL21 or Rosetta for induction expression; enzyme digestion and nickel ion affinity chromatographic purification are performed on an expression product through enterokinase, and then the artificial antibacterial peptide PR39-R1T can be obtained. The antibacterial peptide has the high broad-spectrum antibacterial activity, can inhibit gram-positive bacteria and gram-negative bacteria and does not have the hemolytic activity. Therefore, the artificial antibacterial peptide can be used as a novel antibacterial agent, has the good application prospect on the aspects of food corrosion prevention, disease treatment and the like, and has the developing potential. The preparation method is high in expression efficiency, simple in separation and purification, low in production cost, good in stability and capable of being applied to large scale production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of artificial antibacterial peptide PR39-R1T. Background technique [0002] The long-term inappropriate application of antibiotics in clinical treatment has resulted in the obvious drug resistance of many pathogenic bacteria, which has become a major problem threatening human and animal health. What is more serious is the emergence of multi-drug resistant strains clinically. The superbug NDM-1 discovered in 2010 is even insensitive to most antibiotics. At present, it generally takes about 10 years to develop a new antibiotic, but it only takes 2 years to produce a generation of drug-resistant bacteria. The situation of anti-infection is very severe, and it is imminent to develop and design new antibacterial drugs. [0003] Antimicrobial peptides are a class of small molecular polypeptides produced by the host that can...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/70C12N1/21A61K38/16A61P31/04
Inventor 刘诚
Owner 刘诚
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