Gene engineering antibacterial peptide and preparation method and application thereof

A technology of genetic engineering and antimicrobial peptide, applied in the field of genetic engineering antimicrobial peptide and its preparation, can solve problems such as insufficient antibacterial ability, insufficient antibacterial spectrum, complex structure, etc., achieve strong antibacterial ability, wide antibacterial spectrum, and simple production process Effect

Active Publication Date: 2013-05-01
QINGDAO VLAND BIOTECH INC +1
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a genetically engineered antibacterial peptide and its preparation method and application, which can solve the problems of complex structure, insufficient antibacterial ability and insufficient antibacterial spectrum in the prior art

Method used

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  • Gene engineering antibacterial peptide and preparation method and application thereof
  • Gene engineering antibacterial peptide and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Preparation method of novel antimicrobial peptide

[0035] 1) Design and synthesis of novel antimicrobial peptide genes

[0036] According to the amino acid sequences of Cecropin A and Magainin II in GenBank, the N-terminus of the cecropin A part, that is, the hybrid peptide, has not been changed in any way, and the natural a-helix structure of the original cecropin A has been maintained. Cecropin A was selected in the hinge region. The hinge region AGP serves as the amino acid composition of the variable region. And replace the non-polar amino acid alanine A near the C terminal of Magainin N terminal with positively charged polar amino acid lysine K, so as not to affect its a-helical structure, enhance the number of positive charges at the C terminal, and make the antibacterial Peptides are more easily and more firmly adsorbed on the surface of bacterial cell membranes. At the same time, in order to ensure the amidation of the C-terminus of the antimicrobial peptide,...

Embodiment 2

[0049] 10L-scale fermenter production and formulation preparation of new antimicrobial peptides

[0050] 1) Antimicrobial peptides After activation, the positive recombinants obtained by screening were inoculated in the Erlenmeyer flask at 1%-10% inoculum, cultured at 28-30°C, 200r / min shaker for 16-24h, and then inoculated with 5%-20% inoculum Put into a 10L fermenter (6L loaded medium), temperature 28-30°C, rotation speed 500-1500r / min, medium pH 5.0-6.0, aeration 0.1-1.0VVM (the amount of oxygen introduced into 1L fermentation broth for 1min) Fermentation was carried out under the condition of dissolved oxygen>20%. After 18-24 hours of cultivation, 50% glycerol was added for 4 hours. When the dissolved oxygen suddenly rose to 100%, methanol was added until the end of fermentation. The whole fermentation lasted for 48-72 hours.

[0051] 2) After the fermentation, the original tank was steam sterilized at 100°C for 10-20 minutes, discharged, centrifuged at 5000r / min for 10 mi...

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Abstract

The invention provides gene engineering antibacterial peptide and a preparation method and application thereof, which can solve the problems of complicated structure, low antibacterial capacity and not wide antibacterial spectrum in the prior art. The antibacterial peptide has the amino acid sequence of KWKLFKKIAGPKFLHSKKKFN. According to antibacterial characteristics of cecropinA and magainins, a novel antibacterial peptide amino acid sequence is independently designed. On the basis, a preferred codon of pichia pastoris is selected, a novel antibacterial peptide gene is artificially synthesized and is cloned in the pichia pastoris for expression, a novel antibacterial peptide recombinant yeast strain, the fermentation scale is amplified to the fermentation tank level, and the high-density fermentation and high-efficiency expression of the antibacterial peptide product are realized. Fermentation liquor is further purified and prepared into powder, liquid and other preparations of the antibacterial peptide. The preparations of the gene engineering antibacterial peptide can serve as a livestock feed additive or are used for controlling livestock diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetic engineering antibacterial peptide and its preparation method and application. Background technique [0002] The abuse of antibiotics has not only led to public health problems such as drug residues, but also due to the emergence of drug-resistant strains and new pathogenic subtypes, the prevention and treatment of bacterial diseases has once again become a difficult problem for human beings. Therefore, the development of new antibacterial drugs has become an increasingly important research topic. [0003] Antibacterial peptides (ABP), also known as antimicrobial peptides (Antimicrobial peptides), antibiotic peptides (Antibiotics peptides), are a class of small molecular polypeptides with various biological activities induced by organisms, and are the mainstay of the host immune defense system. An important component, it has the advantages of broad-sp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/81A23K1/17A61K38/16A61P31/00A23K20/195
Inventor 王宏华凌红丽贾德强蒋贻海丛雁方
Owner QINGDAO VLAND BIOTECH INC
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