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Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria

A technology of genetically engineered bacteria and Trichoderma reesei, applied in the biological field, can solve the problems of difficulty in separation and purification, low expression of exogenous proteins, etc., and achieve the effects of efficient secretion and expression, saving operation time and improving work efficiency.

Active Publication Date: 2013-03-13
百开盛(上海)生物科技有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] Therefore, the technical problem to be solved in the present invention is to provide a simple and fast method for expressing foreign protein in Trichoderma reesei (Trichoderma reesei) cells, aiming at the low expression level of foreign protein and the difficulty of separation and purification. The equipment and its application and the obtained Trichoderma reesei genetically engineered bacteria can efficiently secrete and express heterologous genes derived from animals, plants and fungi, etc.

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  • Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria
  • Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria
  • Expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria

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preparation example Construction

[0040] The preparation method of Trichoderma reesei expression equipment of the present invention:

[0041] (1) by PCR amplification, respectively obtain Trichoderma reesei exoglucan cellobiohydrolase I promoter (P cbhI ), exoglucan cellobiohydrolase I terminator (T cbhI ), the respective DNA sequences of the hygromycin phosphotransferase gene screening marker cassette.

[0042] (2) the promoter (P cbhI ), secretory peptide, multiple cloning site, terminator (T cbhI ), the hygromycin B selection marker cassette were sequentially connected to the expression vector, and the recombinant expression vector 1, namely pWE32F00, was constructed. The expression vector here mainly plays the role of a large number of copies of the expression equipment and provides the Agrobacterium binding transfer site, so any binary vector of Agrobacterium can be selected, such as pZP100, pPZP201, pPZP201B, pPZP201BK, pBI121, pCAMBIA, pPK2, but It is not limited to these vectors.

[0043] (3) Inse...

Embodiment 1

[0080] Embodiment 1 comprises the construction of the expression vector of Trichoderma reesei expression equipment of the present invention

[0081] 1.1. Extraction and inspection of Trichoderma reesei genome

[0082] Trichoderma reesei RUT C30 was inoculated on PDA medium and incubated at 28°C for 7 days until the spores matured. An appropriate amount of spore suspension was prepared and inoculated in Martin's medium liquid seed medium, and cultured at 30° C. at 180 rpm for 2 days until the mycelium concentration reached 4-5 g / L for genome extraction.

[0083] In this example, the genomic DNA of Trichoderma reesei was extracted by using the rapid genome extraction method of fungi commonly used in the field, and the extracted genomic DNA was tested by agarose gel electrophoresis after the extraction.

[0084]1.2, Trichoderma reesei exoglucan cellobiohydrolase I promoter (P cbhI ) separation

[0085] According to the sequence of the cbhI promoter released on GeneBank, using ...

Embodiment 2

[0103] Example 2 Expression of red fluorescent protein using Trichoderma reesei expression equipment

[0104] 2.1. Acquisition of red fluorescent protein gene Red

[0105] Using the plasmid pDSRed2-N1 (Clontech Company, Cat. No. 632406) as a template, the 697bp red fluorescent protein Red gene was amplified by PCR using primers.

[0106] The upstream primer P7, whose nucleotide sequence is shown in SEQ ID NO: 10, contains a PacI restriction site;

[0107] The downstream primer P8, whose nucleotide sequence is shown in SEQ ID NO: 11, contains an EcoRV restriction site.

[0108] The length of the amplified product was 697bp, and the results of length and sequence analysis were in line with expectations.

[0109] 2.2. The connection between Red and expression equipment

[0110] The above-mentioned amplified product (red gene) was digested with PacI and EcoRV restriction enzymes, and connected to the expression vector PWE32F00 through PacI and EcoRV restriction enzyme digestion...

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Abstract

The invention discloses expression equipment for expressing heterologous protein in Trichoderma reesei cell, and gene engineering bacteria. The expression equipment comprises the following elements from 5' to 3' in turn: (1) a promoter for controlling an exogenous gene to be transcribed in Trichoderma reesei, (2) a coding sequence of signal peptide for controlling the exogenous gene to be secreted and expressed in the Trichoderma reesei, (3) a multi cloning site, and (4) a terminator for controlling the exogenous gene to stop being transcribed in the Trichoderma reesei. The exogenous gene is inserted into the expression equipment, agrobacterium tumefaciens is transformed by a T-DNA binary vector, and agrobacterium tumefaciens transformants are jointed with the Trichoderma reesei to form the Trichoderma reesei gene engineering bacteria; and the obtained Trichoderma reesei gene engineering bacteria can efficiently secrete and express heterologous genes derived from animals, plants, fungi and the like, and the heterologous protein is massively produced. The culture conditions for the Trichoderma reesei are extensive, the Trichoderma reesei is suitable for solid culture and liquid submerged fermentation and cannot generate mycotoxins and antibiotics under the enzyme producing conditions, and the generated extracellular protein is easily separated and purified, and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression device for expressing foreign protein in Trichoderma reesei cells and genetic engineering bacteria thereof. Background technique [0002] Trichoderma reesei is a mesophilic saprophytic filamentous fungus isolated from cotton oilcloth in the Solomon Islands during World War II and has a good ability to degrade cellulosic materials. Because the bacterium was screened and identified by Dr. Reese E.T., it was named Trichoderma reesei. As an industrial strain, T.reesei can produce enzymes that decompose different plant materials, including cellulase, hemicellulase, etc., and the extracellular enzyme protein production of some Trichoderma reesei mutants can reach 60g / L, and the most The main cellulase CBHI (cellobiohydrolase I, cellobiohydrolase I) can reach 50% of the total protein secretion (about 30g / L). Trichoderma reesei has been used as an industrial produ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12R1/885C12R1/01A23K20/147
Inventor 魏东芝王玮吕丹丹
Owner 百开盛(上海)生物科技有限公司
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