Recombinant bacterium for preparing bacteriocin durancin GL and preparation method and application of recombinant bacterium

A technology of recombinant bacteria and bacteriocin, applied in the field of microbial biology, can solve the problems of difficult to guarantee purity, high cost, complex fermentation liquid composition, etc., and achieve the effect of simple composition

Active Publication Date: 2015-05-06
NANJING UNIV OF FINANCE & ECONOMICS
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  • Abstract
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AI Technical Summary

Problems solved by technology

The bacteriocin durancin GL is prepared by fermentation of Enterococcus durancinus, the concentration of the target product in the fermentation broth is low, and the composition of the fermentation broth is complex, so the process of isolating the active bacteriocin is time-consuming, laborious, costly, and the purity is difficult to guarantee

Method used

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  • Recombinant bacterium for preparing bacteriocin durancin GL and preparation method and application of recombinant bacterium
  • Recombinant bacterium for preparing bacteriocin durancin GL and preparation method and application of recombinant bacterium
  • Recombinant bacterium for preparing bacteriocin durancin GL and preparation method and application of recombinant bacterium

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Embodiment 1

[0028] 1. Cloning of bacteriocin durancin GL gene

[0029] According to the sequence of the bacteriocin durancin GL gene in the GenBank database (HQ696461.1), the primer dur1 (SEQ ID NO: 5) was designed: 5′-ggg agg caa tta tat gaa gaa aaa att tgt tag tat t-3′ and dur2 ( SEQ ID NO: 6): 5′-CCA gTg CCC CCA TAA ACT ATA CAC CC-3′, a gene fragment of bacteriocin durancin GL was amplified from Enterococcus durable 41D (a bacterium that has been published), and the fragment is composed of The composition of genes and immune genes, the sequence is shown in SEQ ID NO:1.

[0030] The gene of the bacteriocin durancin GL can also be prepared by chemical synthesis.

[0031] 2. Sequence verification of bacteriocin durancin GL gene fragment

[0032] The amplified durancin GL gene fragment was recovered, and then ligated with pEASY-Blunt Simple Cloning Vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the recombinant plasmid pEASY / durAB. During the ligation pro...

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Abstract

The invention provides a recombinant bacterium for preparing bacteriocin durancin GL, a preparation method and application of the recombinant bacterium and relates to the microbial biotechnical field. The recombinant bacterium carries a coding gene of a fusion protein formed by connecting the bacteriocin durancin GL, a purification tag and a restriction enzyme cutting site. The method for preparing the bacteriocin durancin GL comprises the following steps: inducing the recombinant bacterium to express the fusion protein; crushing to obtain lysate; centrifuging and taking a supernate; carrying out HisTrap HP column and GSTrap 4B column separation or GSTrap 4B column separation and collecting an eluting peak; and carrying out enzymolysis by enterokinase and removing the purification tag to obtain the bacteriocin durancin GL. The recombinant bacterium provided by the invention is convenient to construct and can efficiently express the recombinant bacteriocin durancin GL. The bacteriocin durancin GL is efficiently separated by virtue of affinity chromatography, and the restriction enzyme cutting site is hydrolyzed to obtain the bacteriocin durancin GL with activity.

Description

Technical field [0001] The present invention relates to the field of microbial biotechnology, in particular to a recombinant bacteria for preparing bacteriocin durancin GL, a preparation method and application. Background technique [0002] Bacteriocins are a class of proteins or polypeptides with antibacterial activity produced by ribosomes. They are widely found in Gram-positive and Gram-negative bacteria. They have low concentration and high efficiency in inhibiting target bacteria. Today, when more and more attention is paid to the potential safety hazards of chemical preservatives, the application of bacteriocins derived from food-grade microorganisms as biological preservatives meets consumers' requirements for safety, health and naturalness. [0003] The bacteriocin durancin GL, which is derived from durable Enterococcus and can specifically inhibit Listeria, has important potential application value in the food industry. The bacteriocin durancin GL is prepared by fermentat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/31C12P21/02
Inventor 鞠兴荣陈信全都立辉袁建何荣和肖营吴学友刘凌平施荣华
Owner NANJING UNIV OF FINANCE & ECONOMICS
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