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Method for transient expression of target protein by 293T cell and application thereof

A target protein, transient expression technology, applied in cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of low transient expression level and high production cost, and achieve the goal of improving target protein level, reduce production cost, and increase protein expression yield

Pending Publication Date: 2022-04-29
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention solves the current technical problem of low transient expression level of recombinant protein in 293T cells, resulting in high production cost, and realizes the technical effect of improving the transient expression level of recombinant protein in 293T cells and reducing production cost

Method used

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  • Method for transient expression of target protein by 293T cell and application thereof

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Experimental program
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Effect test

Embodiment 1

[0062] Zinc transporter 8 (ZnT8), a member of slc30 family (znts proteins), mainly transports intracellular zinc ions to extracellular matrix or intracellular vesicles. It is mainly located in pancreatic islets β The overexpression of ZnT8 in cells can affect the synthesis, storage and secretion of insulin. It is an important marker of type I diabetes and has become an important supplement to current diagnostic reagents. Therefore, increasing the expression of ZnT8 protein plays an important role as the raw material of in vitro diagnostic reagent.

[0063] Taking the transient expression of ZnT8 protein by 293T cells as an example, this embodiment includes the following steps:

[0064] 1、 Obtain target DNA

[0065] According to the use method of Plasmid Extraction Kit, the recombinant plasmid pcDNA3 with high concentration after endotoxin removal was obtained 3-znt8, after measuring the concentration with a micro spectrophotometer, freeze it at - 20 ℃ for standby, and its concentr...

Embodiment 2

[0085] Taking the transient expression of ZnT8 protein by 293T cells as an example, this embodiment includes the following steps:

[0086] 1、 Obtain target DNA

[0087] According to the use method of Plasmid Extraction Kit, the recombinant plasmid pcDNA3 with high concentration after endotoxin removal was obtained 3-znt8, after measuring the concentration with a micro spectrophotometer, freeze it at - 20 ℃ for standby, and its concentration is 2mg / ml.

[0088] 2、 Obtain host cells to be transfected

[0089] With 1 × ten 6 293T suspension cells were subcultured into trans293 medium at the density of cells / ml; After cell counting the next day, adjust the cell density to 4.0 with fresh trans293 medium × ten 6 Pieces / ml, 100ml medium in total.

[0090] 3、 Transfection

[0091]Dilute 1ug plasmid DNA with 100ul cloning medium, dilute Pei with equal volume cloning medium at the same time, and the mass ratio of plasmid DNA to Pei is 1:4. Add 10ml cloning medium into two 50ml centrif...

Embodiment 3

[0101] Taking the transient expression of ZnT8 protein by 293T cells as an example, this embodiment includes the following steps:

[0102] 1、 Obtain target DNA

[0103] According to the use method of Plasmid Extraction Kit, the recombinant plasmid pcDNA3 with high concentration after endotoxin removal was obtained 3-znt8, after measuring the concentration with a micro spectrophotometer, freeze it at - 20 ℃ for standby, and its concentration is 2mg / ml.

[0104] 2、 Obtain host cells to be transfected

[0105] With 1 × ten 6 293T suspension cells were subcultured into trans293 medium at the density of cells / ml; After cell counting the next day, adjust the cell density to 4.0 with fresh trans293 medium × ten 6 Pieces / ml, 100ml medium in total.

[0106] 3、 Transfection

[0107] Dilute 1ug plasmid DNA with 100ul cloning medium, dilute Pei with equal volume cloning medium at the same time, and the mass ratio of plasmid DNA to Pei is 1:4. Add 10ml cloning medium into two 50ml centri...

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Abstract

The invention provides a method for transient expression of target protein by 293T cells and application thereof, and the method comprises the following steps: S10: obtaining target DNA and 293T cells, and transfecting to obtain transfected 293T cells; s20, culturing the transfected 293T cells, and adding a culture medium and a histone deacetylase inhibitor and / or copper ions; s30, maintaining the culture temperature to be lower than 37 DEG C, and supplementing the material to obtain a 293T cell supernatant after material supplementation; and S40, purifying the 293T cell supernatant after material supplementation to obtain the target protein. The technical problem of high production cost caused by low transient expression level of the current recombinant protein in 293T cells is solved, and the technical effects of improving the transient expression level of the recombinant protein in the 293T cells and reducing the production cost are achieved.

Description

technical field [0001] The invention relates to the technical field of gene expression, in particular to a method for transient expression of target protein by 293T cells and its application. Background technology [0002] Transient expression refers to the method that the foreign gene exists on the free vector and is not integrated into the chromosome after entering the receptor cell, and the expression product of the target gene can be obtained in a short time. Compared with stable expression, transient expression greatly shortens the production process of micro recombinant proteins. It is suitable for proteins that are toxic to host cells, or proteins that need to provide a small number but a wide variety of proteins. [0003] 293T cell is a cell line derived from 293 cell through gene technology. It is transfected with adenovirus E1A gene and can express SV40 large T antigen, including SV40 replication starting point and promoter region. Many eukaryotic expression vectors suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K14/47C12N5/10
CPCC12N15/85C07K14/47C12N5/0686C12N2510/00C12N2800/107C12N2501/065C12N2500/20
Inventor 邹炳德邹继华宋丹丽唐云霞丁建静章玉胜
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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