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Pichia pastoris efficient gene knockout method

A Pichia pastoris and gene knockout technology, applied in the field of bioengineering, can solve problems such as excessive glycosylation of fusion proteins, and achieve the effects of improving fermentation performance and high fusion efficiency

Inactive Publication Date: 2018-12-25
惠州卫生职业技术学院
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Problems solved by technology

[0008] In view of this, the present invention provides a high-efficiency gene knockout method for Pichia pastoris, which can effectively improve the glycosylation modification of proteins by Pichia pastoris and solve the problem of excessive glycosylation of fusion proteins. och 1 gene was efficiently knocked out

Method used

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Embodiment

[0028] A high-efficiency gene knockout method for Pichia pastoris, comprising the following steps:

[0029] S1. Construction of knockout plasmid pGE1203-ADE-URA3 through plasmid pYES2;

[0030] S2. Knock the knockout plasmid into the target gene in the Pichia pastoris group och 1 upstream and downstream;

[0031] S3. The protein with knockout plasmid activity recognizes and cuts the nucleotide sequence, and knocks out the target gene och 1. Obtain knockout of the target gene och 1 Pichia pastoris.

[0032] Further, in step S1, the construction method of the knockout plasmid pGE1203-ADE-URA3 is:

[0033] f. Using the plasmid pYES2 as a template, carry out a PCR reaction by amplifying primers P1 and P2 to generate pGE1201;

[0034] g. Design the target gene by using the known Pichia strain as a template och Homology arm sequences P3 and P4 of 1;

[0035] h. Design oligonucleotide primers P5 and P6 through Pichia pastoris URA3 gene;

[0036] i. Design oligonucleoti...

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Abstract

The invention provides a pichia pastoris efficient gene knockout method. The method comprises the following steps: S1, constructing a knockout plasmid pGE1203-ADE-URA3 through a plasmid pYES2; S2, enabling the knockout plasmid to be knocked-in the upstream and the downstream of a target gene och1 in a pichia pastoris group; and S3, identifying a protein with plasmid knockout activity, shearing a nucleotide sequence, and knocking-out the target gene och1, to obtain pichia pastoris in which the target gene och1 is knocked-out. The method is capable of effectively improving galactosylated modification of the pichia pastoris to the protein, solving a problem of fused protein hyperglyeosylation, and efficiently knocking-out the och1 gene of the pichia pastoris.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-efficiency gene knockout method of Pichia pastoris. Background technique [0002] In recent years, with the development of genetic engineering and cell engineering technology, many target genes have been cloned and expressed in prokaryotic and eukaryotic cells to improve the expression ability of foreign proteins. For a long time, Escherichia coli has been used as a host bacterium for expressing exogenous genes, and has successfully expressed a variety of exogenous proteins, but it cannot express proteins with complex structures, and the yield of secreted expression is low. Yeast, as a host bacterium expressing exogenous genes, not only has the characteristics of prokaryotic growth and simple operation, but also has the characteristics of post-translational modification and processing of eukaryotic cells. When expressing certain genetically engineered...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N1/19C12R1/84
CPCC07K14/39C12N15/905
Inventor 李莉玲
Owner 惠州卫生职业技术学院
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