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A CHO cell secretion capacity evaluating system

A cell secretion and evaluation system technology, applied to cells modified by the introduction of foreign genetic material, introduction of foreign genetic material using a vector, recombinant DNA technology, etc. improved effect

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Problems solved by technology

[0005] At present, most of the protein content secreted by cells is detected by western blot, which is complicated, time-consuming and labor-intensive.

Method used

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  • A CHO cell secretion capacity evaluating system
  • A CHO cell secretion capacity evaluating system
  • A CHO cell secretion capacity evaluating system

Examples

Experimental program
Comparison scheme
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Embodiment Construction

[0028] The present invention will be illustrated by specific examples below, but these specific examples should not be construed as limiting the present invention, and modifications to some details will still fall within the protection scope of the present invention.

[0029] 1. Construction of recombinant plasmids

[0030] 1. Construction of GS expression unit:

[0031] 1) First obtain the pMV fragment, the promoter PGK fragment, the intron sequence EEF1a intron, and the SV40polyA fragment respectively by PCR, and connect the above four fragments to obtain the pMV-PGK-EEF1a intron-SV40polyA vector.

[0032] 2) Whole gene synthesis of the plasmid pUC57-mGS plasmid with glutamine synthetase gene, enzyme digestion to obtain the mGS fragment, and insert it into the enzyme-digested vector pMV-PGK-EEF1aintron-SV40polyA to obtain the complete expression unit of the GS gene pPGK-mGS plasmid.

[0033] 2. Construction of luciferase reporter gene expression unit

[0034] 1) Amplify t...

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Abstract

The invention relates to the field of genetic engineering, and particularly discloses a set of system for evaluating whether CHO expression and secretion capacities are influenced after CHO cells are subjected to gene modification. The expression system includes a eukaryotic expression vector and a CHO expression evaluating cell strain. The eukaryotic expression vector and the stable cell strain include a piggy transposon regulating and control unit, a screened gene expression unit and an antibody-LucGFP fused reporter gene expression unit. The expression system can achieve site-specific integration and efficient expression of an exogenous gene in the CHO cell genome. The system can be used for evaluating influences of any exogenous DNA segment including genes, RNAs and the like on the secretion function of the CHO cells, and has a wide application prospect in the field of modifying the CHO cells to increase the protein yield.

Description

technical field [0001] The invention relates to a eukaryotic expression vector and its application field, in particular to an expression vector and system capable of evaluating whether exogenous DNA fragments will affect the secretion ability of CHO cells, and relates to the field of genetic engineering and pharmaceuticals. Background technique [0002] The CHO expression system is currently one of the most important expression systems for protein or antibody drugs, and has been widely used in biopharmaceuticals. In addition to having the correct folding of guiding proteins that other mammalian cells have, it provides complex N-type glycosylation and accurate O-type glycosylation and other post-translational processing of proteins, and produces protein drugs that are closest to natural activity In addition to other advantages, compared with other cells, CHO cells can obtain higher product yields and resist relatively strong metabolic pressure. In addition, CHO cells are fibr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 庄峰锋李春梅
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