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Construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3)

A construction method and a technology for recombining cells, which are applied in the field of recombination cells to achieve the effect of improving specificity and sensitivity

Inactive Publication Date: 2012-07-25
XI AN JIAOTONG UNIV
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Problems solved by technology

However, not all "atopy" individuals will develop an "anaphylaxis" associated with IgE

Method used

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  • Construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3)
  • Construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3)
  • Construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3)

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Embodiment Construction

[0022] The present invention will be described in detail below in conjunction with the accompanying drawings and implementation.

[0023] In the present invention, based on the eukaryotic expression vector pEZ-M02 / H3 that amplifies the full-length H3R gene, HEK293 cells are transfected to obtain a recombinant HEK293 / H3 cell line that is stable and highly expresses H3R. The following is a detailed description of this aspect , the description is to explain rather than limit the present invention.

[0024] A method for constructing recombinant HEK293 cells with high H3R expression, comprising the following steps:

[0025] 1) H3R gene cloning

[0026] Prepare Escherichia coli DH5α competent cells according to the molecular cloning experiment guide, add 10 μL of pEZ-M02 / H3 plasmid to 200 μL competent cells, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, add 10 mL of ampicillin-free LB liquid culture Shake at 37°C for 1h, centrifuge at 3000rpm for 3min, pipette the ce...

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Abstract

The invention relates to a construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3). The recombinant cells are constructed by using HEK 293 cells as host cells, and an exogenous expression vector transfecting the host cells is an expression vector comprising an H3R full length gene. The expression vector comprising the H3R full length gene is pEZ-M02 and is an enkaryotic expression vector. The recombinant HEK293-H3R cells highly expressing H3R are applied in screening antiallergic drugs. The construction method comprises extracting an enkaryotic expression vector, pEZ-M02 / H1R plasmid, transfecting the HEK293 cells with the pEZ-M02 / H1R plasmid, screening by utilizing G418 to obtain a genetic engineering cell line HEK293-H3R highly expressing the full length H1R, and confirming that the cell line can improve the expression of the full length H3R by 12 times.

Description

technical field [0001] The invention belongs to the technical field of recombinant cells and relates to a method for constructing recombinant HEK293 cells with high expression of H3R. Background technique [0002] Histamine is a small molecule amine substance that is a human body's own component. It is synthesized by the action of histidine decarboxylase on L-histidine. Histidine decarboxylase is expressed in the cells of the whole body, including the central nervous system Systemic neurons, gastric parietal cells, mast cells and basophils. [0003] G protein-coupled receptors (GPCRs), also known as seven α-helices transmembrane segment receptors (7TM receptors), is the largest protein superfamily in the body. In terms of structure, it includes seven transmembrane segments. After they bind to the ligand G protein molecular switch, through the mediation of the G protein coupled to the receptor, the second messenger substance is increased or decreased, and in turn changes on ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 张涛贺浪冲王嗣岑黄静韩省力
Owner XI AN JIAOTONG UNIV
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