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Cell strain for expressing metabotropic glutamate receptor 2 and G protein chimera

A glutamate receptor, metabotropic technology, applied in the field of biomedicine, can solve the problems of increased drug development and difficulty, and achieve the effects of stable receptor expression and reduced time and cost

Active Publication Date: 2022-07-19
BIOISLAND LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]Metabotropic glutamate receptor 2 is an important drug target. At present, there is no stable expression cell line suitable for drug screening, which virtually hinders drug development. increased the difficulty

Method used

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  • Cell strain for expressing metabotropic glutamate receptor 2 and G protein chimera
  • Cell strain for expressing metabotropic glutamate receptor 2 and G protein chimera
  • Cell strain for expressing metabotropic glutamate receptor 2 and G protein chimera

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Target, host and expression plasmid construction

[0055] (1) Construction of Metabotropic Glutamate Receptor 2 Expression Plasmid

[0056] 1. Target:

[0057] Gene ID gene name species 24415 GRM2 rat

[0058] 2. Host cell: HEK293

[0059] 3. Resistance: Puromycin

[0060] 4. Expression plasmid construction:

[0061] The target gene (as shown in Table 1) was constructed on the expression vector using restriction endonuclease digestion and T4 DNA ligase ligation method to obtain a plasmid: pAAVS1-HA-SNAP-rmGlu2-Puro-DNR (recombinant expression vector map like figure 1 shown). The vector is a specific gene knock-in vector, and co-transfection with the donor pCas-Guide-AAVS1 plasmid can stably knock the target gene into the AAVS1 site of the cell genome.

[0062] Table 1

[0063]

[0064]

[0065]

[0066] (2) Construction of Gqi9 expression plasmid

[0067] Gqi9 replaces 9 amino acids at the C-terminus of Gq protein w...

Embodiment 2

[0073] Example 2: Cell Culture

[0074] 1. Cell recovery

[0075] Remove the cells stored in liquid nitrogen or -80°C freezer, and quickly move the cells to a 37°C water bath to thaw. Prepare a 15ml centrifuge tube in advance and add 3ml of fresh medium. Add 1 ml of cell suspension in the cryovial to the pre-prepared medium, and place it in a centrifuge at 1000 rpm / min for 3 minutes. During this time, prepare a 100mm cell culture dish and add 10ml of fresh medium. After centrifugation, discard the supernatant, resuspend the cells with 1 ml of fresh culture medium, add the cell suspension evenly to the pre-prepared culture dish, and place it at 37°C, 5% CO. 2 Cultivated in an incubator.

[0076] 2. Cell passage

[0077] 1) First observe the cell state under a microscope to determine whether to proceed with cell passage. After confirming the passage, first discard the old medium, and then use PBS (5mL PBS / 100mm culture dish) to wash away dead cells and cell debris.

[007...

Embodiment 3

[0084] Example 3: Cell Line Construction

[0085] 1. Construction of Gqi9 polyclonal cell line

[0086]1) Construction method: construct the target gene Gqi9 into the neomycin-G418-resistant pcDNA3.1 vector, and transfect the constructed plasmid into the eukaryotic cell HEK293 through liposome, and the target gene will be randomly integrated into the cell in the genome. As the cells grow and proliferate, the target gene can be stably expressed; in the process of cell growth, G418 antibiotics are continuously added for screening, and the cells that have not integrated the target gene into the cell genome will be gradually killed by the antibiotic, thus obtaining a stable expression of the target gene. polyclonal cell lines;

[0087] 2) Plasmid construction: construct the Gqi9 target sequence into the pcDNA3.1-G418 vector, and verify that it has been correctly inserted into the vector after sequencing;

[0088] 3) Cell transfection: 6-well plate cells, transfect 1μg plasmid p...

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Abstract

The invention relates to a recombinant expression vector for expressing a metabotropic glutamate receptor 2, an expression system and a cell with the recombinant expression vector or the expression system. The invention also relates to cells expressing the metabotropic glutamate receptor 2 and the G protein chimera Gqi9. The cell provided by the invention can be used for function detection and drug development by taking the metabotropic glutamate receptor 2 as a target spot.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a cell line expressing a metabotropic glutamate receptor 2 receptor and a G protein chimera. Background technique [0002] Glutamate is the most abundant and most widely used excitatory neurotransmitter in the central nervous system. It is involved in a variety of important physiological activities in the brain, such as regulating cell excitability, mediating the proliferation and development of neurons and glial cells. , survival and death, participation in synaptic transmission, etc. Glutamate receptors can be divided into two categories: metabotropic receptors and ionotropic receptors. Metabotropic glutamate receptors (mGluRs) belong to the C family of G protein-coupled receptors and are widely distributed in the central nervous system. It is associated with a variety of psychiatric and neurological diseases, such as depression, anxiety, migraine, spasticity and neurodegenerative d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/12C12Q1/02G01N33/68
CPCC12N15/85C07K14/705G01N33/5008G01N33/6893C12N2800/107G01N2800/28
Inventor 刘剑峰范治然白晓宇李文征
Owner BIOISLAND LAB
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