Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus

The technology of lfcinb15-mag12 and pet-32a-lfcinb15-mag12 is applied in the technical field of LfcinB15-Mag12 encoding gene, and can solve the problems of unsatisfactory activity, difficulty in extraction, limited sources of natural antimicrobial peptides and the like

Inactive Publication Date: 2009-08-19
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, the sources of natural antimicrobial peptides are limited, the extraction is difficult, and most of them have the defects of unsatisfactory activity or hemolytic toxicity. Therefore, using naturally occurring antimicrobial peptides as templates to design synthetic derivatives is more important for the study of antimicrobial peptide mechanism of action and synthetic activity. Antimicrobial peptides with lower toxicity, more stability and broader spectrum are of great significance. The method of constructing bioreactors to obtain antimicrobial peptides through genetic engineering technology shows broad prospects and great potential

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  • Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus
  • Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus
  • Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus

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Embodiment Construction

[0027] The present invention will be further described below in conjunction with accompanying drawing and specific embodiment:

[0028] to combine figure 2 , the technical route taken in this embodiment is:

[0029] 1. Directly introduce cohesive ends at both ends of the synthetic LfcinB15-Mag12 coding gene fragment, connect it into the expression vector pET-32a, and construct the expression vector pET-32a-LfcinB15-Mag12;

[0030] 2. Transform the expression vector pET-32a-LfcinB15-Mag12 into the expression host E.coli BL21(DE3), induce with 0.1mmol / LIPTG at 30°C for 3h, and produce a specifically induced expression band with the expected molecular weight of 23.9kD;

[0031] 3. Optimize the optimal induction conditions. Under the determined optimal induction conditions, the induction starts when the cell density reaches OD600 = 0.6, 0.3mmol / L IPTG, 30°C induction for 4h, the expression of the fusion protein accounted for 22% of total body protein;

[0032] 4. The fusion pr...

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Abstract

The invention provides a method for synthesizing an LfcinB15-Mag12 encoding gene and expressing the same in colon bacillus. Active gene fragments enconding LfcinB 1-15 amino acid and active gene fragments of Magainin from 1 to 12 are designed according to E. coli codon preference, a recombinant expression vector pET-32a-Lfcin B15-Mag12 is constructed by an expression vector pET-32a, codon encoding 6 histidine labels exists before multiple cloning sites of pET-32a, and stop codon TAG and TAA are added. The method successfully expresses the LfcinB15-Mag12 in the colon bacillus by a gene engineering method, establishes basis of theory and practice for scale production of hybrid peptide and other noble peptides by the gene engineering method, and is significant for establishing peptide gene engineering strains and commonness technology of antibacterial peptide research.

Description

(1) Technical field [0001] The invention belongs to the application field of agriculture, animal husbandry and veterinary medicine, and specifically relates to an LfcinB15-Mag12 coding gene technology. (2) Background technology [0002] The development of antibiotics has a history of more than 50 years. Because it can prevent the occurrence of livestock and poultry diseases, improve animal production capacity, and improve the efficiency of breeding industry, it has always been an important part of the additive premix. However, the long-term use of antibiotic additives can cause many pathogenic bacteria to develop drug resistance, and at the same time, serious drug residues will be produced in animal products, which will have adverse effects on the further prevention and treatment of livestock and poultry diseases and human health. Therefore, the use of antibiotic additives is increasingly strictly restricted in countries all over the world. Since January 1, 2006, the Europe...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00
Inventor 单安山高鹏毕重鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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