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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 the introduction of foreign genetic material, the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the high cost of cell culture, slow cell growth, and high cell culture requirements. problems, to achieve the effect of improving sensitivity and specificity, fast growth, and complete molecular activity

Inactive Publication Date: 2010-05-05
XI AN JIAOTONG UNIV
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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

[0028] The present invention is based on constructing the eukaryotic expression vector pcDNA3.1(+)-KDR of the full-length KDR gene and transfecting HEK293 cells to obtain a recombinant HEK293-KDR cell line stably and highly expressing KDR molecules. This aspect is described in detail below, which is an explanation rather than a limitation of the present invention.

[0029] The present invention is based on pcDNA3.1(+) as a basic vector (from the first affiliated hospital of Xi’an Jiaotong University), and 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.

[0030] 1) Construction of eukaryotic expression vector pcDNA3.1(+)-KDR

[0031] a. Design a primer pair containing restriction sites, using the MHS4426-99626176 plasmid (containing the full-length sequence of KDR gene cDNA, purchased from NCBI, USA, catalog number BC_131822) as a template, and PCR amplify the full le...

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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 an 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. It is a complex process controlled by multiple factors. Vascular endothelial growth factor (VEGF) under normal physiological conditions plays a key role in angiogenesis and is one of the strongest angiogenesis-stimulating factors found so far. VEGF exerts the biological effect of stimulating angiogenesis by binding to its receptor. [0003] The VEGF receptor molecule is composed of an extracellular part formed by seven immunoglobulin-like structures, an intracellular part containing tyrosine kinases, and a transmembrane region. A tyrosine inse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
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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