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Expression equipment for expressing exogenous protein by secretion in trichoderma reesei and application of expression equipment

A technology of Trichoderma reesei and exogenous protein, applied in the biological field, can solve the problems of difficulty in separation and purification, low expression of exogenous protein, etc., and achieves the effects of saving operation time, improving transformation efficiency and low cost

Active Publication Date: 2012-01-04
百开盛(上海)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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.

Method used

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  • Expression equipment for expressing exogenous protein by secretion in trichoderma reesei and application of expression equipment
  • Expression equipment for expressing exogenous protein by secretion in trichoderma reesei and application of expression equipment
  • Expression equipment for expressing exogenous protein by secretion in trichoderma reesei and application of expression equipment

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preparation example Construction

[0038] The preparation method of Trichoderma reesei expression equipment of the present invention:

[0039] (1) By PCR amplification, the Trichoderma reesei exoglucan cellobiohydrolase II promoter (P cbhII ), exoglucan cellobiohydrolase II terminator (T cbhII ), the respective DNA sequences of the hygromycin phosphotransferase gene screening marker cassette.

[0040] (2) the promoter (P cbhII ), multiple cloning sites, terminators (T cbhII ), the hygromycin B selection marker cassette were sequentially connected to the expression vector, and the recombinant expression vector 1, namely pWEIIF00, 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.

[0041] (3) Insert the exogenous gene of in...

Embodiment 1

[0078] Embodiment 1 comprises the construction of the expression vector of Trichoderma reesei expression equipment of the present invention

[0079] 1.1. Extraction and inspection of Trichoderma reesei genome

[0080] 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.

[0081] 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.

[0082] 1.2, Trichoderma reesei exoglucan cellobiohydrolase II promoter (P cbhII ) isolation According to the sequence of the cbhII promoter released on GeneBank, the Trichoder...

Embodiment 2

[0100] Example 2 Expression of red fluorescent protein using Trichoderma reesei expression equipment

[0101] 2.1. Acquisition of red fluorescent protein gene Red

[0102] Using the plasmid pDSRed2-N1 (Clontech Company, Cat. No. 632406) as a template, the red fluorescent protein Red gene of 695 bp (including restriction sites) was amplified by PCR using primers P7 and P8.

[0103] The upstream primer P7, whose nucleotide sequence is shown in SEQ ID NO: 9, contains an XbaI restriction site;

[0104] The downstream primer P8, whose nucleotide sequence is shown in SEQ ID NO: 10, contains an EcoRV restriction site.

[0105] The length of the amplified product and the results of sequence analysis were in line with expectations.

[0106] 2.2. The connection between Red and expression equipment

[0107] Both EcoRV and StuI restriction sites are blunt ends, so the two ends can be connected to each other. The above-mentioned amplified product (red gene) was subjected to XbaI and Ec...

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Abstract

The invention discloses expression equipment for expressing exogenous protein by secretion in trichoderma reesei cells. The expression equipment comprises the following elements from 5' to 3': (1) an exo-glucan cellobiose hydrolase II promoter of trichoderma reesei; (2) a secretively-expressed signal peptide; (3) a polyclonal locus sequence; and (4) an exo-glucan cellobiose hydrolase II terminator of the trichoderma reesei. Exogenous genes are inserted into the expression equipment, agrobacterium tumefaciens is converted by a T-deoxyribonucleic acid (DNA) binary vector and is jointed with thetrichoderma reesei to obtain trichoderma reesei genetic engineering bacteria which can express heterologous genes from animals, plants, fungi and the like efficiently by secretion, and a large amountof exogenous protein is obtained from the trichoderma reesei genetic engineering bacteria. The trichoderma reesei has an extensive culture condition, and is suitable for solid culture and liquid submerged fermentation; and mycotoxin and antibiotics cannot be generated under the zymogenic condition, and the generated extracellular protein is easy to separate and purify 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 secreting and expressing foreign protein in Trichoderma reesei and its application. 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 bacteria 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), CBHII (cellobiohydrolase II, cellobiohydrolase II), can respectively reach 50% (about 30g / L) and 40% of the total protein secreti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/21C12N1/15C12P21/02C12N9/00A23K1/16A61K38/00C12R1/885C12R1/01A23K20/147
Inventor 王玮魏东芝吕丹丹
Owner 百开盛(上海)生物科技有限公司
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