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Method for preparing food grade Saccharomyces cerevisiae recombinant plasmid

A technology of Saccharomyces cerevisiae and recombinant plasmids, which is applied in the field of biological genetic engineering and can solve problems such as hidden dangers of fermentation system pollution

Inactive Publication Date: 2011-05-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, the exogenous gene expression plasmid existing in the yeast cell or the exogenous gene expression unit integrated on the chromosome contains the Escherichia coli plasmid sequence and the drug resistance gene sequence, resulting in a certain potential contamination of the entire fermentation system.

Method used

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  • Method for preparing food grade Saccharomyces cerevisiae recombinant plasmid
  • Method for preparing food grade Saccharomyces cerevisiae recombinant plasmid
  • Method for preparing food grade Saccharomyces cerevisiae recombinant plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Cloning vector P inu -RH build

[0055] (1) pAG61 plasmid Not1 digestion, electrophoresis recovery Escherichia coli plasmid replicon (Ori)-ampicillin resistance gene (Ap r ) part, alkaline phosphatase treatment to remove phosphorus.

[0056] (2) Using Kluyveromyces cicerisporus CBS4857 genomic DNA as a template, PCR amplification of the inulinase gene with P1 and P2 as primers (GenBank accession number AF178979, sequence such as SEQ.ID.NO 13, Wen T.Q., Cloning and analysis of the inulinase gene from Kluyveromyces cicerisporus CBS4857, World Journal of Microbiology & Biotechnology 19:423-426, 2003), GenBank accession number is AF17897. ) promoter Pinu fragment. Saccharomyces cerevisiae S288c genomic DNA was used as a template, and the terminator Tcyc fragment of CYC1 gene (SGD accession number YJR048W) was amplified by PCR with primers P3 and P4. Carry out corresponding restriction endonuclease digestion treatment according to the enzyme cutting sites desi...

Embodiment 2

[0070] Example 2: Shuttle carrier pHR-P inu Construct

[0071] (1) The pHC11 vector was digested with Bgl I and Sal double enzymes, and a large fragment of 8.7kb (including the complete 2μ plasmid sequence and Leu2 gene sequence) was recovered, and treated with Klenow enzyme to make up.

[0072] (2) Primers P7 and P8 PCR amplified inulinase gene promoter Pinu, and primers P9 and P10 PCR amplified CYC1 gene terminator Tcyc. The Pinu PCR product was digested with EcoR1 and BamH1, and the Tcyc PCR product was digested with BamH1 and HindIII. The Pinu and Tcyc fragments were ligated with the plasmid pSP72 that had been digested with HindIII and EcoR1, and transformed into Escherichia coli DH5α. And sequence identification, get pSP72-Pinu-Tcyc plasmid.

[0073] (3) The pSP72-Pinu-Tcyc plasmid was linearized with BamH1 single-enzyme digestion, filled in with Klenow enzyme, ligated with the pHC11 enzyme-digested fragment (8.7kb) in (1), transformed into Escherichia coli DH5α, posit...

Embodiment 3

[0085] Embodiment 3: construction of inulinase gene expression cassette

[0086] Kluyveromyces cicerisporus CBS4857 genomic DNA was used as a template, primers P11 and P12 were used to PCR amplify the open reading frame ORF of the inulinase gene, and the PCR product was digested with HindIII and Sal1, and combined with Pinu - RH vector ligated to transform Escherichia coli DH5α, identify the transformant by PCR, extract the recombinant plasmid of the positive transformant, sequence and identify, obtain the recombinant plasmid in which the inulinase gene is cloned in the MCS region of the Pinu-RH vector, and the recombinant plasmid has a Pinu promoter- The inulinase gene expression cassette (Pin-Inu-Tcyc) composed of the inulinase gene-Tcyc terminator, the recombinant plasmid was digested with Not1, separated by electrophoresis, and the inulinase gene expression cassette (Pin-Inu-Tcyc) was recovered.

[0087] Inulinase gene open reading frame ORF cloning primers:

[0088] P11:...

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Abstract

The invention belongs to the technical field of biological gene engineering, and provides a method for preparing a food grade Saccharomyces cerevisiae recombinant plasmid. The method comprises: firstly, constructing shuttle plasmid yeast carrier part which having a yeast 2mu plasmid complete sequence, a yeast nutritional deficiency screening gene, a yeast promoter and a yeast terminator; constructing an exogenous gene expression box which has a yeast promoter, a yeast terminator and a target exogenous gene; using the shuttle plasmid yeast carrier part and exogenous gene expression box in combination to convert a corresponding auxotrophic Saccharomyces cerevisiae strain, and screening a culture convertor in an auxotrophic selective culture medium; and finally, separating and purifying to obtain target Saccharomyces cerevisiae recombinant plasmid. The expression plasmid can survive only in the cells of the Saccharomyces cerevisiae and does not transfer, and can be widely used for expressing various exogenous genes safely and efficiently.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a preparation method and application of a food-grade Saccharomyces cerevisiae recombinant plasmid. Background technique [0002] Saccharomyces cerevisiae (Saccharomyces cerevisiae) expression system is the earliest established yeast expression system. Since 1981, Hitzeman et al. first expressed human alpha interferon in Saccharomyces cerevisiae (Hitzeman, R.A et al., Expression of a human gene for interferon in yeast .Nature293:717-722, 1981), hundreds of foreign genes have been successfully expressed in Saccharomyces cerevisiae, and dozens of them have been listed as genetic engineering drugs. Although the overall expression level is lower than that of Pichia pastoris, Saccharomyces cerevisiae expresses some exogenous genes can also reach the level of gram / liter (as Saccharomyces cerevisiae expresses Aspergillus niger glucose oxidase reaches 9g / L, Park...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N9/24C12R1/865
Inventor 刘建平李育阳江佳稀
Owner FUDAN UNIV
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