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Antibacterial peptide coexpression vector, construction and expression method thereof

A technology of co-expression vectors and construction methods, applied in the field of co-expression vectors, can solve the problems of changing protein structure, affecting the activity and production of antimicrobial peptides, and achieving the effect of overcoming the lack of expression

Inactive Publication Date: 2014-11-05
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

[0010] Aiming at the defects of the prior art, the present invention discloses a co-expression vector, which can simultaneously stably and efficiently express up to four antibacterial peptides in the same host bacterium. The co-expressed polypeptides have high-efficiency broad-spectrum antibacterial and antiviral activities. Consistent with natural antimicrobial peptides, it solves the problem of changing the protein structure during fusion expression and affecting the activity and production of antimicrobial peptides

Method used

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  • Antibacterial peptide coexpression vector, construction and expression method thereof
  • Antibacterial peptide coexpression vector, construction and expression method thereof
  • Antibacterial peptide coexpression vector, construction and expression method thereof

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Embodiment 1

[0049] Embodiment 1: Construction of antimicrobial peptide co-expression vector

[0050] 1. Amplification of genes encoding LEAP-2, TAP, SBD-1, and Lysozyme

[0051] The primers are used to amplify the genes encoding antimicrobial peptides LEAP-2, TAP, SBD-1 and Lysozyme to obtain the respective antimicrobial peptide genes.

[0052] PCR reaction system: 50 μL MasterMix, 2 μL each of upstream and downstream primers, 16 μL template, 30 μL deionized water; DNA gel recovery kit recovered four antimicrobial peptide coding genes;

[0053] 2. Construction of pETDuet-1-L2 and pRSFDuet-1-S1 vectors

[0054] The two vectors pETDuet-1 and pRSFDuet-1 were digested with EcoRI and NotI.

[0055] 40 μL digestion system: 4 μL of 10×H buffer, 1 μL of EcoRI and NotI, 24 μL of carrier, 10 μL of deionized water.

[0056] After 1% agarose electrophoresis, the digested products were recovered by the gel recovery kit;

[0057] Then the LEAP-2 and SBD-1 genes were double-digested with EcoRI and N...

Embodiment 2

[0071] Example 2: Co-expression of LEAP-2, TAP, SBD-1, Lysozyme four antimicrobial peptide co-expression vectors

[0072] 0.5 μL plasmids of pETDuet-1-L2-T and pRSFDuet-1-S1-Ly were transformed into BL21(DE3) host bacteria at the same time, and single-clonal colonies were picked and inoculated in fresh 10ml LB medium, and the bacteria were shaken overnight at a ratio of 1:100. Proportional transfer to fresh 10ml LB medium, when the OD value reaches 0.6-0.8, take 1ml of the bacterial liquid as an uninduced sample, then add 10 μL IPTG with a final concentration of 1.0mM to induce expression for four hours and collect 1ml of the bacterial liquid sample;

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Abstract

The invention discloses an antibacterial peptide coexpression vector, a construction and expression method thereof. The antibacterial peptides fall into four types, and coding genes of the four antibacterial peptides are pairwise connected to double expression vectors pETDuet-1 and pRSFDuet-1 undergoing expression simultaneously in one host bacterium respectively. The antibacterial peptide coexpression vector provided by the invention can efficiently and stably express four antibacterial peptides at the same time, thus having efficient broad-spectrum antibacterial and antiviral activity. The expressed antibacterial peptide protein structure keeps consistent with natural antibacterial peptide, thus solving the problem that change of the protein structure during fusion expression can influence the activity and yield of antimicrobial peptides.

Description

technical field [0001] The invention relates to a co-expression carrier capable of simultaneously expressing multiple antimicrobial peptides, belonging to the technical field of bioengineering. Background technique [0002] Due to the abuse of antibiotics and chemically synthesized drugs in animal husbandry for many years, many pathogenic bacteria have developed drug resistance and drug residues. Therefore, researchers have begun to use modern biological technology to develop green, high-efficiency, beneficial to the health of organisms, and beneficial to the environment. A new biotherapeutic agent that can replace antibiotics to protect and improve farming efficiency. [0003] Among the existing biological agents, antimicrobial peptides are small molecular active substances widely present in natural biological organisms, usually composed of 15-45 amino acid residues, which can be induced to act on microbial cell membranes, making cell membranes Perforation destroys the int...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66
Inventor 周继章陈启伟李兆才曹小安宫晓炜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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