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Method for expressing recombinant protein by using trichoderma reesei as host

A technology of Trichoderma reesei and recombinant protein, which is applied in the field of genetic engineering and can solve problems such as expression failure

Active Publication Date: 2018-05-18
深圳中科欣扬生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

For example, if the recombinant gene is inserted into the regulatory region of the silencer, it may directly cause its inability to transcribe and fail to express, and if the foreign gene is inserted into the regulatory region of the enhancer, its expression may also be greatly increased

Method used

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  • Method for expressing recombinant protein by using trichoderma reesei as host
  • Method for expressing recombinant protein by using trichoderma reesei as host
  • Method for expressing recombinant protein by using trichoderma reesei as host

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Embodiment 1

[0040] Embodiment 1, the acquisition and application example of Trichoderma reesei genome insertion site M29 that is beneficial to recombinant gene expression

[0041] In the present invention, a polypeptide 2A sequence derived from Foot and mouth disease virus (FMDV) is used as a connecting peptide to fuse red fluorescent protein gene DsRed and a lipase gene LipA derived from Aspergillus niger in Trichoderma reesei . The 2A sequence can make the DsRed gene and the LipA gene be expressed at a ratio of 1:1, the LipA gene will be secreted outside the cell due to the introduction of the signal peptide sequence, and the red fluorescent protein will remain in the cell as a reporter gene as a marker gene for flow High-throughput screening with cytometers. The fusion expression backbone was randomly integrated into the genome of Trichoderma reesei. Due to the difference in the insertion site and copy number, transformants with different expression levels of recombinant proteins were...

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Abstract

The invention discloses a method for expressing a recombinant protein by using trichoderma reesei as a host. The method for expressing the recombinant protein by using the trichoderma reesei as the host comprises the following step: cloning an expression cassette which contains an encoding gene of a target protein to be expressed in a 5' UTR region of a cel3c gene in a trichoderma reesei genome. Experiments prove that in cbh1 gene deleted strain, an expression skeleton of the cbh1 gene is integrated to an internal ribosome entry site, the expression level of the cbh1 gene is compared with theexpression level of the cbh1 gene of a wild strain, and it discovers that the expression level which is higher than a cbh1 site can be obtained by inserting a recombination gene into the site. The technical scheme of effectively expressing the recombinant protein by using the trichoderma reesei as the host is further enriched and improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing recombinant proteins using Trichoderma reesei as a host. Background technique [0002] The filamentous fungus Trichoderma reesei (Trichoderma reesei) is the main strain producing cellulase and hemicellulase in industry, and it is a food safety grade strain certified by the US FDA. Trichoderma reesei has a strong ability to secrete proteins, and the amount of exocrine proteins of some mutant strains can reach 100g / L, and has a glycosylation system similar to that of higher mammals, so Trichoderma reesei is an ideal recombinant Protein expression hosts have been highly valued by internationally renowned enzyme preparation companies (such as Genencor, Novozymes, etc.), and have been successfully applied to the expression and production of various drugs, chemical reagents, and enzyme preparations. In recent years, with the rapid development of Trichoderma r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/67C12R1/885
CPCC12N15/67C12N15/80
Inventor 秦丽娜董志扬江贤章黄建忠
Owner 深圳中科欣扬生物科技有限公司
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