Recombination HEK 293 cell highly expressed by KDR and application thereof

A recombinant cell, high-expression technology, applied in the direction of cells modified by introducing foreign genetic material, determination/inspection of microorganisms, biochemical equipment and methods, etc.

Inactive Publication Date: 2011-06-01
XI AN JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Previous researchers often used vascular endothelial cells with relatively high expression of KDR as research objects, but they had the following defects: 1. Slow cell growth; 2. Cell culture requirements High; 3. The cost of cell culture is high; 4. The abundance of KDR expression on the cell surface does not meet the requirements
Moreover, none of the KDR genes carried by the existing stable and high-expressing KDR cell lines are full-length KDR genes, which affects the complete function of KDR.

Method used

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  • Recombination HEK 293 cell highly expressed by KDR and application thereof
  • Recombination HEK 293 cell highly expressed by KDR and application thereof
  • Recombination HEK 293 cell highly expressed by KDR and application thereof

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Embodiment Construction

[0029] In the present invention, on the basis of constructing eukaryotic expression carrying pcDNA3.1(+)-KDR of the full-length KDR gene, HEK293 cells are transfected to obtain a recombinant HEK293-KDR cell strain with stable and highly expressed KDR molecules. The following is a detailed description of this aspect, which is an explanation of the present invention rather than a limitation.

[0030] The present invention is based on pcDNA3.1 (+) carrier (from the First Affiliated Hospital of Xi'an Jiaotong University donated), its plasmid map is as follows figure 1 As shown, the present invention selects KpnI and XbaI restriction sites as multiple cloning sites for connecting foreign genes.

[0031] 1) Construction of eukaryotic expression vector pcDNA3.1(+)-KDR

[0032] a. Design a pair of primers comprising restriction sites, use the MHS4426-99626176 plasmid (containing the full-length cDNA sequence of the KDR gene, purchased from NCBI, the United States, catalog number BC_...

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Abstract

The invention discloses a recombination HEK 293 cell highly expressed by KDR and an application thereof. The recombination cell takes an HEK 293 cell as a host cell, and an exogenous expression vector transfecting the host cell is an expression vector containing KDR full length genes. Compared with an extracellular region only containing KDR or the recombination cell constructed by partial gene sequence, the recombination HEK 293-KDR cell strain surface containing the KDR full length gene constructed by the invention can stably express KDR molecules, and has complete molecular activity. A cell membrane chromatography has a certain reservation to taspine when detecting the HEK 293-KDR cell and can be applied to drug screen taking VEGFR as a target spot.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the construction of cells with high expression of vascular endothelial growth factor, in particular to a recombinant HEK293 cell with high expression of KDR based on exogenous recombinant plasmid and its application. technical background [0002] Angiogenesis refers to the regeneration of new blood vessels on the basis of the original vascular bed, which is a complex process regulated by multiple factors. Under normal physiological conditions, vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, and is one of the strongest stimulating factors of angiogenesis found so far. VEGF exerts the biological effect of stimulating angiogenesis by combining with its receptor. [0003] The VEGF receptor molecule consists of three parts: an extracellular part formed by seven immunoglobulin-like structures, an intracellular part containing tyrosine kinase, and a transmembrane r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02C12N5/10
Inventor 李旭葛晓雯贺浪冲王嗣岑杜晖张彦民
Owner XI AN JIAOTONG UNIV
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