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Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide

A technology of genetically engineered bacteria and construction methods, which is applied in the field of construction of genetically engineered bacteria secreting and expressing Metchnikowin Escherichia coli, can solve the problems of unreported Metchnikowin recombinant expression and few reports of proline-containing antimicrobial peptides, and achieve cost low effect

Inactive Publication Date: 2013-05-08
安徽希普生物科技有限公司
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Problems solved by technology

At present, there are not many reports on the recombinant expression of proline-containing antimicrobial peptides, and there is no report on the recombinant expression of Metchnikowin

Method used

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  • Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide
  • Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide
  • Construction method of colon bacillus genetic engineering strain for secretory expression of Metchnikowin antibacterial peptide

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Embodiment Construction

[0021] The present invention will be described in further detail below according to the drawings and embodiments.

[0022] 1. Gene amplification:

[0023] (1) Amplification of the Metchnikowin gene: Using the plasmid containing the Metchnikowin gene sequence as a template, an EcoRI restriction site was designed downstream of it, primer MF: 5'-CATCGTCATCAGGGTCCG-3'; MR: 5'-GGAATTCTTAATAAATCGG -3', Reaction conditions: 94°C for 45s, 50°C for 45s, 72°C for 30s, 30 cycles, 72°C for 10min. The reaction products were detected by 1.5% agarose gel electrophoresis and recovered by gel recovery kit.

[0024] (2) Amplification of the fusion protein gene: using the plasmid containing the fusion protein gene as a template, design an Nco I restriction site upstream of the sequence, primers: CF: 5'-CATGCCATGGATCACCATCATCATCAT-3', CR:: 5'-AAGGGTTTCCGAAGGCTTGG -3'.

[0025] Reaction conditions: 94°C for 45s, 50°C for 45s, 72°C for 30s, 30 cycles, 72°C for 10min. The reaction products were ...

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Abstract

The invention discloses a construction method of a colon bacillus genetic engineering strain for secretory expression of antibacterial peptide Metchnikowin. The construction method comprises the steps of: genetically connecting a Metchnikowin antibacterial peptide gene, constructing a recombinant pET-22b plasmid, and transforming a colon bacillus BL21 competent cell. The construction method disclosed by the invention has the beneficial effects that the colon bacillus genetic engineering strain has efficient and stable secretory expression, and the protein cleavage is low in cost, and safe due to a downstream DP hydrolysis locus.

Description

technical field [0001] The invention relates to a construction method for secreting and expressing antimicrobial peptide Metchnikowin Escherichia coli genetic engineering bacteria. Background technique [0002] Antimicrobial peptides are small molecular active peptides produced by the immune defense system of organisms under inducing conditions, and are widely distributed in plants, animals and humans. Antimicrobial peptides are encoded by genes and synthesized by ribosomes. At present, more than one thousand antimicrobial peptides have been discovered and identified, most of which have a net positive charge. From the structural point of view, it can be divided into α-helical type, β-sheet type, rich in disulfide bonds, rich in proline and other types. Antimicrobial peptides have a broad antibacterial spectrum, and some antimicrobial peptides can also specifically inhibit the growth of certain tumor cells. Antimicrobial peptides can be used in medicine and health, food ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C12R1/19
Inventor 查向东恽辉余忠丽程林春赵大伟耿玉静
Owner 安徽希普生物科技有限公司
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