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Construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli

A technology of genetically engineered bacteria and Escherichia coli, which is applied in the fields of genetic engineering and biology, can solve the problems of small proportion of target fragments and reduced actual expression yield of target fragments, etc., and achieves the effects of increased expression, convenient separation and purification, and inhibition of toxicity.

Active Publication Date: 2011-10-12
广东希普生物科技股份有限公司
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  • Abstract
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Problems solved by technology

However, if the sequence of the fusion head is too long, the proportion of the target fragment in the fusion protein will be too small, which will reduce the actual expression yield of the target fragment

Method used

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  • Construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli
  • Construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli
  • Construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli

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Experimental program
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Embodiment Construction

[0038] figure 1 , 2 The principle and production process of constructing cationic antimicrobial peptide G13 Escherichia coli genetically engineered bacteria are shown, and the steps are as follows:

[0039] 1. Synthetic antimicrobial peptide gene:

[0040] G13 was synthesized by Shanghai Sangon Biotechnology Co., Ltd. using a solid-phase phosphoramidite triester method with a DNA synthesizer;

[0041] 2. Gene amplification:

[0042] Using the synthesized G13 sequence as a template, an EcoR I restriction site and a Met codon are added to the upstream primer, and a Sal I restriction site and a stop codon are added to the downstream primer; the primers are as follows: G13F: 5'-AGAATTCATGCAGCGTTCTGTGTCTAAC-3', G13R : 5'-ATTGTCGACTTACCAACGAGAACGACCAG-3', reaction conditions: 94°C for 30s, 62°C for 15s, 72°C for 15s, 30 cycles, 72°C for 10min, the PCR product was detected by 1% agarose electrophoresis, and the G13 fragment was recovered by the kit;

[0043] 3. Cloning and transf...

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Abstract

The invention discloses construction of gene engineering bacteria for expressing recombinant cationic antimicrobial peptides (AMPs) G13 escherichia coli, and belongs to the technical field of gene engineering and biology. A fusion head gene is connected with an AMPs gene to construct recombinant plasmid and convert escherichia coli BL21 competent cells, so that the expression yield is improved, and more effective treatment is obtained. The invention aims to design a shorter fusion head which has higher hydrophobic property, and the C final fragment of the fusion head has more negative charges, so that the fusion head can protect small molecular target AMPs; the toxicity of the AMPs to host cells is neutralized; and the cationic AMPs comprise alpha spiral amphipathic AMPs with net positivecharges and contain nucleotide sequences SEQ ID: NO 1 and SEQ ID: NO 2. According to the construction method, DNA recombination and gene engineering technology such as restriction endonuclease cutting, ligase connection and the like are used.

Description

technical field [0001] The invention proposes the construction of a recombinant cationic antibacterial peptide G13 Escherichia coli genetically engineered bacterium, which belongs to the field of genetic engineering and biotechnology, and involves using an optimized fusion head to connect the fusion head gene with the antibacterial peptide gene, construct a recombinant plasmid, and transform Escherichia coli BL21 competent cells can increase the expression yield. Background technique [0002] The overuse of antibiotics has caused serious health, health and environmental problems, and finding natural alternatives to traditional antibiotics is a current research hotspot. Cationic antimicrobial peptides (AMPs) are amphiphilic small molecule active peptides with net positive charges produced by the immune system of organisms under inducing conditions. Cationic antimicrobial peptides interact with anions on the cell membrane, destroy the membrane structure, form ion channels to ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12R1/19
Inventor 查向东卢颖虎刘杨梁琳恽辉余忠丽彭霞
Owner 广东希普生物科技股份有限公司
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