Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application

A synthesis method and antibacterial peptide technology are applied in the field of genetic recombinant pig-derived antibacterial peptides and their biosynthesis, which can solve the problems of high price of antibacterial peptides and low natural yield of antibacterial peptides, and achieve easy amplification, easy operation and high expression efficiency. Effect

Inactive Publication Date: 2008-09-17
ZHEJIANG SCI-TECH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many antimicrobial peptides are being developed into medicines, but the natural yield of antimicrobial peptides is very low, and the price of chemically synthesized antimicrobial p...

Method used

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  • Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application
  • Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application
  • Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application

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Effect test

Embodiment 1

[0036] 1. Cloning of a recombinant pig-derived antimicrobial peptide PG4 gene

[0037] (1) Design and synthesize two oligonucleotides: PG4-1 and PG4-2, and then obtain a double-helix DNA fragment through annealing and renaturation, and its nucleotide sequences are respectively:

[0038] PG4-1:

[0039] 5'-ATTCAGGGATCCATGCGCGGTGGCCGTCTGTGCTATTGTCGCGGTTGGATCTGCTTCTGTGTGGGTCGTTAAAAGCTTAATTCG-3';

[0040] PG4-2:

[0041] 5'-CGAATTAAGCTTTTAACGACCCACACAGAAGCAGATCCAACCGCGACAATAGCACAGACGGCCACCGCGCATGGATCCCTGAAT-3'.

[0042] Two oligonucleotide fragments, PG4-1 and PG4-2, were chemically synthesized using Escherichia coli preferred codons. The double-helix DNA obtained by annealing PG4-1 and PG4-2 corresponds to the amino acid sequence of SEQ ID NO4:Ile- Gln-Gly-Ser-Met-Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Gly-Trp-Ile-Cys-Phe-Cys-Val-Gly-Arg-Terminator-Lys-Leu -Asn-Ser.

[0043]At both ends of the two oligonucleotide fragments, BamH I and HindIII restriction endonuclease sites were...

Embodiment 2

[0066] Antibacterial Test of Recombinant Antimicrobial Peptide PG4

[0067] 1. Recombinant antimicrobial peptide PG4 anti-Escherichia coli E.coli test:

[0068] (1) Two pieces of filter paper with a diameter of 1 cm were taken, sterilized by ultraviolet irradiation (irradiation time was 30 minutes), and infiltrated with sterile water and recombinant antimicrobial peptide PG4 solution (0.1 mg / mL) respectively. exist Figure 4 Among them, sample 1 and sample 2 in turn;

[0069] (2) Put the above two samples into sterile cell culture dishes respectively, and then add 50 μL E. coli culture solution (10 6 CFU / mL), and cultivated in a 37°C incubator for 2 hours;

[0070] (3) Add 1 mL of phosphate buffer solution to each petri dish, and clean it with ultrasonic waves (64kHz, 2 minutes), then take 50 μL of gradient dilution, add 50 μL of bacterial buffer solution of different dilutions to the culture dish, and use a coating Spread the bacterial solution evenly on the solid agar me...

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Abstract

The invention discloses a recombinant swine antibacterial peptide PG4, the amino acid sequence of which has amino acid sequence as shown in SEQ ID NO:1. The invention also discloses a method for synthesizing the recombinant swine antibacterial peptide PG4, which includes: (1) constructing antibacterial peptide PG4 prokaryotic expression vector, (2) transforming into Escherichia coli host cell, and constructing expression engineering bacteria; (3) fermenting and culturing the engineering bacteria, and performing inducible expression of inducer; and (4) purifying. The PG4 has the advantages of high expression efficiency, simple separation and purification, convenient operation, easy magnification, good stability, and suitability for large scale industrialized production. The PG4 is of important value for the research of novel high-efficiency antibacterial drug, and has wide application prospect in the fields of pharmacy industry and biological antibacterial material.

Description

technical field [0001] The invention relates to the expression and purification of recombinant proteins in the field of genetic engineering, in particular to a gene recombinant pig-derived antimicrobial peptide and a biosynthetic method thereof. Background technique [0002] Mammalian antimicrobial peptides are the product of a series of non-specific immune responses in mammals to the infection of external pathogenic substances, and are an important part of the host's innate defense system. It has the characteristics of broad-spectrum antibacterial, thermal stability, strong alkalinity, soluble in water, positive charge, low molecular weight, almost no toxic side effects on eukaryotic cells, and only kills prokaryotic cells and eukaryotic cells with pathological changes. Because it kills prokaryotic cells and diseased eukaryotic cells by changing the permeability of bacterial lipid membranes, bacteria are not easy to develop drug resistance, while traditional antibiotics wor...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/12C12N15/70A61K38/10A61P31/00
Inventor 姚菊明刘琳马廷方
Owner ZHEJIANG SCI-TECH UNIV
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