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Cell model stably expressing human-derived CaSR gene and construction method of cell model

A technology of cell model and construction method, applied in the field of genetic engineering, to achieve the effect of simple and controllable preparation method, high accuracy, good stability and reliability

Inactive Publication Date: 2020-01-07
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is no cell model capable of high-throughput screening of active substances targeting CaSR

Method used

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  • Cell model stably expressing human-derived CaSR gene and construction method of cell model
  • Cell model stably expressing human-derived CaSR gene and construction method of cell model
  • Cell model stably expressing human-derived CaSR gene and construction method of cell model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The construction of embodiment 1 cell model

[0043] The cell model was established using Chinese hamster ovary cells (CHO) as recipient cells. CHO cells were cultured in F12 medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Incubator cultivation.

[0044] (1) Recombinant plasmid construction

[0045] Use the human source CaSR cDNA sequence to amplify to obtain the target gene CaSR, fuse the amplified CaSR gene with SNAP through the connection sequence cacgcgt and then connect it with the pcDNA5 / FRT expression vector by ligase catalysis, and transform Escherichia coli; amplify, extract Plasmid DNA was extracted and sequenced to identify positive clones, and the positive clones identified correctly were recombinant plasmids, named pcDNA5 / FRT Flag-Snap-CaSR (human).

[0046] (2) Transfection of recombinant plasmids into CHO cells

[0047] Preparation of DNA and liposome (Lipofectamine2000) mixture (10cM petri dish)

[0048] A solution:

[0049]

[0050] ...

Embodiment 2

[0058] Embodiment 2 Cell model of the present invention is used for the specificity of drug screening

[0059] CaSR is mainly associated with G q Protein-coupled, CaSR is activated mainly by activating PLC β Signaling pathway, promotes the generation of DAG and IP3, and IP3 promotes the release of intracellular calcium ions, so it can be detected by detecting Ca 2+ The release determines the activation of CaSR; in addition, the generated IP3 will be gradually degraded into IP1 in the cell and accumulate in the cell. The LiCl added to the medium can make IP1 exist stably, and the accumulation of IP1 can be used to reflect the activation of CaSR. In this example, in order to identify cell lines that can stably express functional CaSR, the first method is mainly used.

[0060] To test the specificity of this cell model for drug screening, it was ensured that the response of the response element was regulated by changes in the activity of the CaSR. Multiple agonists of CaSR, CaCl...

Embodiment 3

[0073] Example 3 Evaluation of drug screening methods based on cell models

[0074] The purpose of establishing a screening model is to screen active substances and reflect the biological activity of the substance. Therefore, it is necessary to objectively evaluate whether the screening model can accurately reflect the biological activity of the test substance. There are many indicators to evaluate the quality of a screening model, such as model stability, sensitivity, specificity, etc. In addition, in order to make the model meet the requirements of screening, quantitative evaluation of the screening model is also required, mainly to analyze whether the model can effectively reflect the activity of the sample. Commonly used quantitative technical parameters for evaluating drug screening models are as follows:

[0075] (1) Signal to background ratio (signal to background, S / B)

[0076] The signal-to-background ratio reflects the data obtained from the drug screening model (M...

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Abstract

The invention provides a cell model stably expressing a human-derived CaSR gene. The cell model is preserved in China Center for Type Culture Collection and named as Chinese hamster ovary cell CHO-CaSR, with a preservation number being CCTCC NO: C201893. A construction method of the cell model is as follows: a SNAP tag and a CaSR gene are linked by a linking sequence cacgcgt to obtain the CaSR gene fused with the SNAP tag, the CaSR gene is linked with an expression vector to construct a recombinant plasmid containing the CaSR gene, the recombinant plasmid is transfected into mammalian cells, and a cell strain capable of stably expressing the CaSR gene is cultured by drug screening. The cell model can be used for screening CaSR-targeted active substances, and can also be used in conjunctionwith homogeneous time resolved fluorescence (HTRF) through SNAP-tag to accurately and quickly study interaction of CaSR with other proteins and oligomerization of the CaSR. The cell model can screenhigh-throughput and low-cost active substances targeting the CaSR, and the screening method is simple and controllable in steps, low in cost, high in throughput, and very good in stability and reliability.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a method for constructing a cell model, in particular to a cell model stably expressing a human CaSR (calcium sensitive receptor) gene and a method for constructing the cell model. Background technique [0002] Calcium-sensing receptor (CaSR), as a member of the family C G protein-coupled receptors (GPCRs) superfamily, has been studied in many tissues and organs since it was first discovered in 1993, and it has been found that its function changes are related to certain associated with the development of certain diseases. [0003] The human CaSR gene is located on the long arm of chromosome 3 (3q13.3-21), and is mainly composed of a polypeptide chain of 1078 amino acid residues, including 3 structural domains: ① The amino extracellular region composed of 612 amino acids ( NH2-terminalextracellular domain, ECD), forming a dimer with Ca 2+ 、Gd 3+ , Mg 2+ , spermine, gentamicin a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02
CPCC12N5/0603C12N5/0682C12N5/0686C12N15/85C12N2503/00C12N2510/00C12N2800/107G01N33/5008
Inventor 刘剑峰殷学梁汤恒敏王素云
Owner HUAZHONG UNIV OF SCI & TECH
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