Preparation and expression method of chicken beta-defensin 9 yeast engineered bacteria

An expression method and technology of engineering bacteria, which are applied in the fields of biotechnology and genetic engineering pharmaceuticals, can solve the problems of inconvenient operation and long production cycle of foreign proteins, and achieve the effects of simple operation, avoiding waste of host expression potential, and good stability

Inactive Publication Date: 2014-03-05
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the fermentation process, toxic and volatile methanol is used as an inducer, and carbon source conversion is required, so the operation is inconvenient, and the production cycle of exogenous proteins is longer

Method used

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  • Preparation and expression method of chicken beta-defensin 9 yeast engineered bacteria
  • Preparation and expression method of chicken beta-defensin 9 yeast engineered bacteria
  • Preparation and expression method of chicken beta-defensin 9 yeast engineered bacteria

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Embodiment 1

[0048] In the present invention, the recipient bacterium is Pichia pastoris GS115, and the carrier plasmid is pGHKα; the target gene is a codon-optimized nucleotide fragment corresponding to 41 amino acids; The chicken AvBD9 target gene fragment was inserted between them; the recombinant plasmid was digested and linearized by SacⅠ and BglⅡ sequentially, and then electrotransformed into highly efficient Pichia pastoris GS115 competent cells; positive recombinants were screened out by MD plate primary screening and PCR identification, and G418 antibody High-copy recombinants were screened, and high-expression strains were selected by protein expression level, and then fermented and cultured, the expression product was directly secreted outside the cell, and the supernatant obtained by centrifugation contained the expressed target protein product - AvBD9.

Embodiment 2

[0050] The specific operation steps of the chicken β-defensin 9 yeast engineering bacteria preparation and expression method are as follows:

[0051] (1) Codon optimization and synthesis of the AvBD9 gene sequence, the optimized gene sequence is as follows figure 1 Shown, upper row: original sequence. Lower row: codon-optimized sequences. Bold font: modified bases. Underline: restriction endonuclease cleavage site. Italics: coding sequence of protease Kex2 restriction site. Inside the box: stop codon;

[0052] According to the AvBD9 gene sequence published by GenBank (accession number: AY534894.1), referring to the preferred codons of Pichia pastoris, the DNASTAR software was used to optimize the gene sequence without changing the amino acid sequence, and EcoRI restriction enzyme was added to the 5′ end of the gene site and Kex2 restriction site coding sequence AAAAGA, add TAA site and NotⅠ restriction site at the 3′ end of the gene, the optimized gene is 146bp in length,...

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Abstract

The invention discloses preparation and an expression method of a chicken beta-defensin 9 yeast engineered bacteria, and belongs to the fields of biological technology and gene engineering pharmaceutical technology. The preparation process comprises: utilizing a pichia yeast consistently-used codon to optimize chicken beta-defensin 9 gene, adding a Kex2 restriction enzyme cutting site coding sequence at the front terminal of the gene, and artificially synthesizing the gene; then inserting the synthetic beta-defensin 9 gene sequence into a yeast constitutive expression vector, integrating into a the chromosome of a methylotrophy type pichia yeast by homologous recombination; and screening for multiple times to obtain the yeast engineered bacteria for producing chicken beta-defensin 9. The yeast engineered bacteria is capable of continuously performing high-level protein expression after fermentation and culturing, the expressed product is high in yield, is relatively close to the natural structure, is secreted in the broth, and also has relatively strong anti-foodborne pathogenic activity. The broth is subjected to preliminary separation and can be directly used as a forage additive after concentrated or dried, and the forage additive is applied to animal breeding industry, so that the production cost of defensin is reduced and the economic benefit of a production enterprise is improved. The technical scheme provided by the invention helps to establish a technological base for industrial large-scale production and application of beta-defensin 9 and development of poultry forage antibiotic additive substitutes.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and genetic engineering pharmacy, and in particular relates to a preparation and expression method of chicken β-defensin 9 yeast engineering bacteria. Background technique [0002] Since the 1950s, subtherapeutic doses of antibiotics have been added to feed to promote animal growth. The abuse of a large number of antibiotics in the breeding industry has led to the continuous emergence of drug-resistant bacteria, and some infectious pathogens have even seriously threatened human health and the development of animal husbandry. With growing concern and the spread of drug-resistant pathogens, Europe has moved to ban the use of antibiotics in animal feed to promote animal growth. Therefore, it is imminent to find a natural, pollution-free new feed additive that can replace antibiotics. It not only has antibacterial function, but also can promote animal growth to reduce feeding costs. [0003] D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/465C12P21/02C12R1/84
Inventor 祁克宗涂健张永正彭开松周秀红汪雪雁刘红梅张明陈玎玎付瑞燕薛秀恒
Owner ANHUI AGRICULTURAL UNIVERSITY
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