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Recombinant vector used in Kluyveromyces marxianus auxotrophic strain

A Kluyveromyces and auxotrophic technology, applied in the field of recombinant vectors, can solve problems such as low expression level, unsuitable for edible protein, and low growth density

Inactive Publication Date: 2015-11-18
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the protein expression of Pichia pastoris needs to be induced by methanol, so it is not suitable for the production of edible protein
Saccharomyces cerevisiae is prone to ethanol production, with low growth density and low expression level

Method used

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  • Recombinant vector used in Kluyveromyces marxianus auxotrophic strain
  • Recombinant vector used in Kluyveromyces marxianus auxotrophic strain
  • Recombinant vector used in Kluyveromyces marxianus auxotrophic strain

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0080] Step 1, construct the first recombinant vector

[0081] 1.1. Amplification of pUC19 plasmid

[0082] Forward primer:

[0083] 5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'

[0084] Reverse primer: 5'-TACAATTTTATGGTGCACTTCTCAGTACAATCTGCT-3'

[0085] The pUC19 plasmid was amplified using the primers. The PCR amplification reaction was carried out according to the instruction manual of PhantaSuperFidelityDNA Polymerase of Vazyme Company, the annealing temperature was 58°C, the extension time was 3 minutes, and 30 cycles. The PCR product was named A1 fragment.

[0086] 1.2, Amplify the pcYGW of the Gateway system vector

[0087] Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3'

[0088] Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'

[0089] The pcYGW of the Gateway system vector was amplified using the primers. PCR amplification conditions were the same as step 1, except that the extension time was 1 min. The PCR product was nam...

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Abstract

The invention provides a recombinant vector used in a Kluyveromyces marxianus auxotrophic strain, and a preparation method and application thereof. The recombinant vector sequentially comprises a PKD1 vector, an inulase promoter, an inulase signal peptide, a polyclone site, an inulase terminator, a nutrient gene promoter and a nutrient gene open reading frame. The constructed recombinant vector and preparation method thereof can be used for constructing a transformant, thereby implementing expression of the exogenous gene.

Description

technical field [0001] The invention relates to a recombinant vector, in particular to a recombinant expression vector used in auxotrophic strains of Kluyveromyces marxe. Background technique [0002] Yeast is a single-celled eukaryote, which has both the characteristics of microorganisms and the protein synthesis and processing system of eukaryotes. Therefore, it is widely used to express a variety of foreign eukaryotic proteins. The current mainstream yeast expression systems include Pichia pastoris and Saccharomyces cerevisiae expression systems. However, the protein expression of Pichia pastoris needs to be induced by methanol, which is not suitable for the production of edible protein. Saccharomyces cerevisiae is prone to ethanol production, with low growth density and low expression level. Aiming at these series of problems, it is of great industrial value to develop a new yeast expression system with high safety and high yield. [0003] Kluyveromyces marxianus has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19
Inventor 吕红余垚周峻岗马骁骁
Owner FUDAN UNIV
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