Recombinant vector used for carrying out foreign gene secretory expression in auxotrophic kluyveromyces marxianus strain

A Kluyveromyces and recombinant vector technology, applied in the field of secretion and expression of recombinant vectors, can solve the problems of low expression level, unsuitable for edible protein, low growth density, 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 produc...

Method used

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  • Recombinant vector used for carrying out foreign gene secretory expression in auxotrophic kluyveromyces marxianus strain
  • Recombinant vector used for carrying out foreign gene secretory expression in auxotrophic kluyveromyces marxianus strain
  • Recombinant vector used for carrying out foreign gene secretory expression in auxotrophic kluyveromyces marxianus strain

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Experimental program
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Embodiment 1

[0062] Embodiment 1, construction of recombinant vector PUKD112

[0063] The construction method of the recombinant vector PUKD112 in this embodiment is as follows:

[0064] Step 1. Amplify the pUC19 plasmid

[0065] Forward primer:

[0066] 5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'

[0067] Reverse primer: 5'-TACAATTTTATGGTGCACTTCTCAGTACAATCTGCT-3'

[0068] 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 recovered according to the instructions of Simgen's Gel Recovery Kit, which was named Fragment A.

[0069] Step 2, amplify the pcYGW of the Gateway system vector

[0070] Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3'

[0071] Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'

[0072] T...

Embodiment 2

[0116] Embodiment 2, construction of recombinant vector PUKD117

[0117] Step 1, amplify the PPIC9K vector

[0118] Forward primer:

[0119] 5'-CCCATAAGTGACACATTTAATTTTTTTTTTTGTTAGATATGAGATTTCCTTCAATTTTTACTGC-3'

[0120] Reverse primer:

[0121] 5'-CTAGTCCCGGGGTCACCGTCGTAAGCTTCAGCCTCTTTTTCTCG-3'

[0122]The PPIC9K vector was amplified with the primers described. The conditions for PCR amplification were the same as those in Step 1 of Example 1 when constructing the PUKD112 plasmid, except that the extension time was 1 minute. The PCR product was recovered according to the instructions of Simgen's Gel Recovery Kit, which was named Fragment A. Fragment A contains the Saccharomyces cerevisiae alpha factor signal peptide.

[0123] Step 2, mutant PUKD112 vector plasmid

[0124] The PUKDN112 plasmid was mutated according to the instructions of the QuikChangeII kit of Agilent. The primer used for mutation is A segment. The mutated plasmid is PUKDN117. PUKDN117 was sequenc...

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Abstract

The invention provides a recombinant vector used for carrying out foreign gene secretory expression in an auxotrophic kluyveromyces marxianus strain as well as a preparation method and application of the recombinant vector. The recombinant vector comprises ampicillin resistance genes, a PKD1 vector, inulase promoters, signal peptides, multiple cloning sites, inulase terminators, nutritional gene promoters and a nutritional gene open reading flame according to the sequence. The constructed recombinant vector used for carrying out foreign gene secretory expression and the preparation method of the recombinant vector can be used for constructing a transformant to achieve secretory expression of foreign genes.

Description

technical field [0001] The invention relates to a secretory expression recombinant vector, in particular to a recombinant expression vector applied to exogenous gene expression in Kluyveromyces marx auxotrophic strains. 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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19
Inventor 吕红余垚袁汉英周峻岗李育阳
Owner FUDAN UNIV
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