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Method for high-throughput screening recombinant trichoderma reesei for efficiently expressing foreign protein

An exogenous protein, Trichoderma reesei technology, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc.

Inactive Publication Date: 2013-01-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on high-throughput screening of Trichoderma reesei engineering bacteria that express recombinant proteins efficiently by using flow cytometry sorting technology

Method used

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  • Method for high-throughput screening recombinant trichoderma reesei for efficiently expressing foreign protein
  • Method for high-throughput screening recombinant trichoderma reesei for efficiently expressing foreign protein
  • Method for high-throughput screening recombinant trichoderma reesei for efficiently expressing foreign protein

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Embodiment 1

[0086] Embodiment 1, the screening of the recombinant Trichoderma reesei strain of high production heterologous lipase

[0087] In this embodiment, two transformants were screened, namely pSKLR transformants and pSKRL transformants.

[0088] 1. Construction of fusion expression vectors pSKLR and pSKRL of red fluorescent protein and heterologous lipase

[0089] 1) PCR amplification of cellobiohydrolase I (CBH1) gene promoter and signal peptide fragment PScbh1:

[0090] Using Trichoderma reesei (Trichoderma reesei) strain QM9414 (ATCC 26921) genomic DNA as a template, Pcbh1-F, PScbh1-R as primers for PCR amplification, the amplification conditions are: 95 ° C pre-denaturation for 5 min, 94 ° C denaturation for 30 s, Annealing at 61.1°C for 30s, extension at 72°C for 2min, 30 cycles; finally extension at 72°C for 10min, the amplified promoter fragment PScbh1 containing the signal peptide of cbh1 gene was obtained. The target band was recovered by the agarose gel electrophoresis...

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Abstract

The invention discloses a method for high-throughput screening recombinant trichoderma reesei for efficiently expressing a foreign protein. The method comprises the steps as follows: comparing the red fluorescence intensity of recombinant trichoderma reesei for expressing the foreign protein identified by a flow cytometry meter(FCM); identifying the expressing ability of the foreign protein according to the red fluorescence intensity; and obtaining a result that the expressing ability of the foreign protein of the recombinant trichoderma reesei for expressing the foreign protein having higher red fluorescence intensity is strong. The gene of the foreign protein is led into the trichoderma reesei in a mode of a fusion gene, the fusion gene is formed by connecting a red fluorescence protein gene with the foreign protein gene through a connecting peptide 2A sequence; the foreign protein gene is positioned in the upstream or the downstream of the red fluorescence protein gene; a signal peptide sequence is formed at the 5' end of the foreign protein gene; and connecting peptides 2A are shown in 290th to 306th positions of the 8th sequence in the coding sequence list of the connecting peptide 2A sequence.

Description

technical field [0001] The invention relates to a method for high-throughput screening of recombinant Trichoderma reesei highly expressing foreign proteins. Background technique [0002] Filamentous fungus Trichoderma reesei (Trichoderma reesei) is the main industrial production strain of cellulase and hemicellulase. The amount of exocrine protein of the mutant strain can reach 100g / L, and it has a glycosylation system similar to that of higher mammals. Therefore, Trichoderma reesei is a very ideal host for recombinant protein expression and has been obtained by internationally renowned enzyme preparation companies (such as Genencor , Novozymes, etc.) are highly valued and successfully applied to the expression and production of various drugs, chemical reagents and enzyme reagents. At the same time, the fermentation process of Trichoderma reesei is mature, and the fermentation cost is low, which has laid a good foundation for its industrial production. In recent years, wit...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/64G01N15/14G01N21/64C12N15/62C12N15/63C12N5/10C12N1/15C12N9/20C12R1/885
Inventor 董志扬秦丽娜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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