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Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain

A recombinant vector, foreign gene technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, fungi, etc., can solve the problems of low growth density, low expression level, unsuitable for edible protein, etc.

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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  • Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain
  • Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain
  • Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0056] Step 1. Amplify the pUC19 plasmid

[0057] Forward primer:

[0058] 5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'

[0059] Reverse primer: 5'-TACAATTTTATGGTGCACTTCTCAGTACAATCTGCT-3'

[0060] 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 as fragment A.

[0061] Step 2, amplify the pcYGW of the Gateway system vector

[0062] Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3'

[0063] Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'

[0064] 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 named Fragment B.

[0065] Step 3, amplifying the PKD1 vec...

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Abstract

The invention provides a signal peptide-free recombinant vector for exogenous gene expression in a Kluyveromyces marxianus nutritional deficient strain as well as a preparation method and application thereof. The recombinant vector sequentially comprises an ampicillin resistance gene, a PKD1 vector, an inulase promoter, multiple cloning sites, an inulase terminator, a nutritional gene promoter and a nutritional gene open reading frame. The signal peptide-free recombinant vector and the preparation method thereof, constructed by the invention, can be used for constructing transformants to realize exogenous gene expression.

Description

technical field [0001] The invention relates to a recombinant expression vector used in auxotrophic strains, in particular to a recombinant expression vector without signal peptide. 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 ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/645
Inventor 吕红余垚袁汉英周峻岗李育阳
Owner FUDAN UNIV
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