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Heterologous expression method for plantaricin pln E and pln F

A plant lactobacillus, heterologous expression technology, applied in the field of microbial engineering, can solve the problem of low purification efficiency

Inactive Publication Date: 2016-06-15
ZHEJIANG GONGSHANG UNIVERSITY
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  • Abstract
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Problems solved by technology

However, the biggest challenge faced by natural bacteriocins is the extremely low purification efficiency. In recent years, the method of using heterologous expression to increase the production of bacteriocins has attracted more and more attention. A variety of bacteriocins have achieved their heterologous expression in E. coli. source expression

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  • Heterologous expression method for plantaricin pln E and pln F
  • Heterologous expression method for plantaricin pln E and pln F
  • Heterologous expression method for plantaricin pln E and pln F

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Embodiment Construction

[0030] 1 material

[0031] 1.1 Strains and plasmids

[0032] Escherichia coli E.coliDH5α and Escherichia coli E.coliBL21 (DE3) are preserved by our laboratory. The expression plasmid pET-32a(+) was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. and stored in Escherichia coli E.coliDH5α.

[0033] 1.2 Reagents

[0034]Peptone, Yeast Extract, Tris (Tris), Ampicillin, Ammonium Persulfate (APS), Isopropyl-β-D-thiogalactopyranoside (IPTG), Acrylamide, N, N-methylenebisacrylamide, SDS, bromophenol blue, TEMED, Coomassie brilliant blue R-250, Tricine, agarose, SanPrep column PCR product purification kit, and SDS-PAGE protein standards were purchased from Sangon Bioengineering ( Shanghai) Co., Ltd.; rTaq enzyme, endonuclease restriction enzyme (KpnI, NcoI), T 4 DNA Ligase, dNTPs, DNAMarker, and plasmid extraction kits were purchased from TAKARA; DNA gel recovery kits were purchased from AxyPrep; absolute ethanol, glycerin, glacial acetic acid, sodium hydroxide, and sodiu...

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Abstract

The invention discloses a plant lactobacillus pln E and pln The heterologous expression method of F, found from NCBI pln E and pln The gene sequence of F, the sequence number is, and an enterokinase site is added at the N-terminal of the sequence, and an enzyme cutting site is designed Kpn I and Nco I, synthetic sequence; plasmid pUC57- pln E and pUC57- pln F is the template, and primers are designed for pln E and pln PCR amplification of the F fragment, after double digestion of the pET-32a plasmid and the target fragment, the recombinant plasmid pET-32a- pln E and pET-32a- pln F, transformed into E. coli E. coli DH5α and save. The invention utilizes the heterologous expression system of Escherichia coli to construct an expression plasmid and realize its high-efficiency expression in Escherichia coli, making it possible to prepare a large amount of PlnE / F, thereby providing a basis for the study of its mechanism of action and its future application in the field of food.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a heterologous expression method of plant lactobacillus plnE and plnF. Background technique [0002] Chemical preservatives can effectively inhibit the growth of microorganisms, and the cost is low, but excessive addition will cause harm to the human body. As people's demand for material levels and dietary health continues to increase, chemical preservatives are increasingly showing their own disadvantages. In addition, with the emergence of problems such as the abuse of antibiotics and drug resistance, people began to turn their attention to bacteriocins with the characteristics of safety, high efficiency, no drug resistance, no residue and no pollution. In recent decades, researchers have discovered hundreds of bacteriocins from lactic acid bacteria, but only Nisin is still used in the food industry. The reason is that the wild strains producing bacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C07K14/335
Inventor 郦萍顾青吴双双
Owner ZHEJIANG GONGSHANG UNIVERSITY
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