Method for expressing cecropin AD by virtue of bacillus subtilis and preparation method of cecropin AD

A technology of Bacillus subtilis and cecropin, applied in the field of preparation of biological preparations, can solve the problems of low content of natural antibacterial peptides, high cost of chemical synthesis, difficulty in large-scale production, etc., and achieves reduced enzyme activity, safe, efficient, and easy price. Effects of Competence Conversion

Inactive Publication Date: 2017-10-10
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the low content of natural antimicrobial peptides, direct extraction is not only difficult but als

Method used

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  • Method for expressing cecropin AD by virtue of bacillus subtilis and preparation method of cecropin AD
  • Method for expressing cecropin AD by virtue of bacillus subtilis and preparation method of cecropin AD
  • Method for expressing cecropin AD by virtue of bacillus subtilis and preparation method of cecropin AD

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Embodiment 1

[0027] A method utilizing Bacillus subtilis to express cecropin AD, as follows:

[0028] (1) Gene synthesis: artificially design the coding target gene fragment, fuse the signal peptide SPsacB gene, six histidine genes, SUMO tag upstream of the cecropin AD gene, and add an EcoRI restriction site at the 5' end. Add stop codon and BamHI restriction site at the 3' end, the entire fusion gene SPsacB-6×His-SUMO-Cecropin AD, its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID As shown in NO.2;

[0029] (2), construction and transformation of the expression plasmid: the expression vector uses the Escherichia coli-Bacillus subtilis shuttle vector pGJ148 as the backbone, the promoter is the maltose promoter Pglv, and the gene-deficient strain Bacillus subtilis WB800N is used as the expression host bacterium. Transformation method The recombinant vector is directly transformed into the expression host Bacillus subtilis WB800N, and the express...

Embodiment 2

[0032] A method utilizing Bacillus subtilis to prepare cecropin AD, as follows:

[0033] 1. Gene synthesis

[0034] The amino acid sequence of cecropin AD is: KWKLFKKIEK VGQRVRDAVI SAGPAVATVA QATALAK, the coding gene fragment is artificially designed, and an EcoRI restriction site and signal peptide SP are added at the 5' end sacB , 6×His tag, SUMO, plus a stop codon and a BamHI restriction site at the 3’ end, the entire fusion gene SPsacB-6×His-SUMO-Cecropin AD, its nucleotide sequence is as shown in SEQ ID NO.1 As shown, its amino acid sequence is shown in SEQ ID NO.2, and the gene synthesis was completed by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0035] 2. Construction and transformation of expression plasmids

[0036] 1) Cloning the artificially synthesized coding gene into the Bacillus subtilis expression vector to construct a recombinant expression vector, and the constructed recombinant expression vector is positively screened by single and double enzyme diges...

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Abstract

The invention relates to a method for expressing cecropin AD by virtue of bacillus subtilis and a preparation method of cecropin AD. A fusion protein coding gene (SPSacB)SUMO-CAD is designed and synthesized by taking bacillus subtilis WB800N as a host strain and escherichia coli-bacillus subtilis shuttle vector as an expression vector, the nucleotide sequence of the fusion protein coding gene is represented by SEQ ID NO.1, the amino acid sequence is represented by SEQ ID NO.2, and a recombinant expression vector pGJ148-(SPSacB)SUMO-CAD is successfully constructed. The recombinant expression vector is converted into a host bacteria competent cell in a chemical conversion manner, induced fermentation is carried out by virtue of maltose so as to successfully express foreign proteins, a fusion protein with relatively high purity is obtained through purification through an NI-NTA column and a protein purification analysis meter and is subjected to SUMO digestion so as to obtain recombinant antibacterial peptide, and then fermentation conditions are optimized. The method is low in cost and beneficial to separation and purification.

Description

technical field [0001] The present invention relates to a preparation method of a biological preparation, in particular to a method for expressing cecropin AD by using Bacillus subtilis and a preparation method thereof. Background technique [0002] The extensive use of broad-spectrum antibiotics has not only led to the frequent emergence of drug-resistant bacteria around the world, but also killed normal bacteria while eliminating pathogenic bacteria, causing serious damage to the micro-ecological balance of the body, and secondary Negative consequences such as infection. Antimicrobial peptides are favored for their excellent antibacterial activity and unique mechanism of action. Antimicrobial Peptide (AMP), also known as Host Defense Peptide (HDP), is an important part of the body's innate immune system and a barrier to protect the host against the invasion of foreign pathogens. Antimicrobial peptides come from a wide range of sources, and have biological functions such ...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/62C12N1/21C12R1/125
CPCC07K14/43586C07K2319/02C07K2319/21C07K2319/35C12N2830/34
Inventor 单安山李建平李晓丹
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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