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A kind of yeast engineering bacteria, its preparation method and application

A yeast engineering and yeast technology, applied in the field of genetic engineering, can solve the problem that the Pa-AMP05 gene cannot be expressed safely and efficiently, and achieve the effect of high-efficiency expression

Inactive Publication Date: 2011-11-30
SHENZHEN SUNSMILE BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the embodiment of the present invention provides a yeast engineering bacterium to solve the technical problem that the Pokeweed antimicrobial peptide Pa-AMP05 gene cannot be expressed safely and efficiently in the prior art

Method used

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  • A kind of yeast engineering bacteria, its preparation method and application
  • A kind of yeast engineering bacteria, its preparation method and application
  • A kind of yeast engineering bacteria, its preparation method and application

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preparation example Construction

[0027] The embodiment of the present invention also provides a preparation method of yeast engineered bacteria, comprising the following steps:

[0028] a) amplifying the Pokeweed antimicrobial peptide Pa-AMP05 gene;

[0029] b) recombining pokeweed antimicrobial peptide Pa-AMP05 gene and yeast shuttle plasmid to obtain a recombinant plasmid;

[0030] c) Transforming the recombinant plasmid into yeast cells and restoring the culture to obtain a yeast engineered bacterium containing the pokeweed antimicrobial peptide Pa-AMP05 gene. The sequence of the pokeweed antimicrobial peptide Pa-AMP05 gene is shown in SEQ ID NO:1.

[0031] The yeast engineering bacteria obtained by the preparation method of the embodiment of the present invention need to be expanded and cultivated, and then separated and purified to obtain pokeweed antimicrobial peptide Pa-AMP05, pokeweed antimicrobial peptide Pa-AMP05 gene expressed pokeweed antimicrobial peptide Pa-AMP05 The amino acid sequence of the ...

Embodiment 1

[0069] An engineering yeast bacterium, the yeast is Saccharomyces cerevisiae S78, the yeast engineering bacterium comprises a recombinant plasmid composed of a yeast shuttle plasmid pVT102U / α and pokeweed antimicrobial peptide Pa-AMP05 gene, and pokeweed antimicrobial peptide Pa-AMP05 gene sequence As shown in SEQ ID NO: 1, the preservation number of the engineered Saccharomyces cerevisiae is CGMCC 3681.

[0070] see figure 1 , figure 1 It shows the structural diagram of the recombinant plasmid in the engineering bacteria of the embodiment of the present invention. The recombinant plasmid uses the yeast shuttle plasmid pVT102U / α as the carrier and recombines the Pokeweed antimicrobial peptide Pa-AMP05 gene. The expression cassette of the recombinant plasmid is ADH1-α- MFL-Pa-AMP05-TT, where ADH1 is the promoter, α-MFL is the base sequence of the α factor signal peptide, TT is the terminator, and the antimicrobial peptide Pa-AMP05 refers to the base of pokeweed antimicrobial p...

Embodiment 2

[0072] The preparation method of the yeast engineering bacterium of the embodiment of the present invention, concrete steps are as follows:

[0073] Step S201 amplifies pokeweed antimicrobial peptide Pa-AMP05 gene.

[0074] The upstream primer and downstream primer of pokeweed antimicrobial peptide Pa-AMP05 gene were designed, the sequence of the upstream primer is shown in SEQ ID NO:2, and the sequence of the downstream primer is shown in SEQ ID NO:3. An enzyme cutting site for restriction endonuclease XbaI was introduced into the upstream primer sequence, and an enzyme cutting site for restriction endonuclease HindIII was introduced into the downstream primer sequence. details as follows:

[0075] Upstream primer: 5'-CTAG AGATCT GCTGGTTGTATT-3', the underline is the enzyme cutting site of restriction endonuclease XbaI;

[0076] Downstream primer: 5'-CCGGG TTCGAA TTATCTA-3', the underline is the enzyme cutting site of restriction endonuclease HindIII,

[0077] Use the...

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Abstract

The invention is applicable to the technical field of genetic engineering and provides a yeast engineering bacterium, its preparation method and application. The yeast engineering bacterium of the present invention comprises a recombinant plasmid composed of a yeast shuttle plasmid and pokeweed antimicrobial peptide Pa-AMP05 gene. The sequence of the pokeweed antimicrobial peptide Pa-AMP05 gene is shown in SEQ ID NO:1. The yeast engineering bacteria of the embodiment of the present invention uses yeast as the expression system of pokeweed antimicrobial peptide Pa-AMP05 gene, which realizes safe and efficient expression of the gene and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a yeast engineering bacterium, its preparation method and application. Background technique [0002] Antimicrobial peptides are small molecular polypeptides produced in organisms to defend against the invasion of foreign pathogens. They are widely found in insects, plants, animals and humans. It has broad application prospects in the fields of medicine and health, agricultural production, and food industry. Antimicrobial peptides have a unique mechanism of action and are not easy to produce drug-resistant bacteria, so they are considered as substitutes for traditional antibiotics. Pokeweed antimicrobial peptide Pa-AMP05 is a new type of antimicrobial peptide with great application prospects. [0003] However, the content of antimicrobial peptides in the body is low, and separation and purification are difficult, which is a difficult problem restricting t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/29C12N15/81A23L1/305A23L3/3526A23K1/17A61K38/16A61P31/00C12R1/865A23K20/195
Inventor 彭永鹤李永新黄毓茂张捷周洪波
Owner SHENZHEN SUNSMILE BIOTECH
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