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Cell model and constructing method thereof, and applications of cell model in screening active substances targeting serotonin receptor

A cell model, cell technology, applied in the fields of genetic engineering and molecular pharmacology

Inactive Publication Date: 2018-09-25
EZHOU INST OF IND TECH 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 5-HT1BR

Method used

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  • Cell model and constructing method thereof, and applications of cell model in screening active substances targeting serotonin receptor
  • Cell model and constructing method thereof, and applications of cell model in screening active substances targeting serotonin receptor
  • Cell model and constructing method thereof, and applications of cell model in screening active substances targeting serotonin receptor

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Experimental program
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Embodiment 1

[0031] The construction of embodiment 1 cell model

[0032] 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 cultured in an incubator.

[0033] (1) Recombinant plasmid construction

[0034] Use the human 5-HT1BR cDNA sequence to amplify to obtain the target gene 5-HT1BR, connect the amplified 5-HT1B gene and the pcDNA5 / FRT expression vector by ligase, and transform Escherichia coli; amplify, extract plasmid DNA, Extraction and sequencing were performed to identify positive clones, and the positive clones identified correctly were recombinant plasmids, named pcDNA5 / FRT-5-HT1BR.

[0035] (2) Transfection of recombinant plasmids into CHO cells

[0036] Preparation of DNA and liposome (Lipofectin2000) mixture (60mM petri dish)

[0037] A solution:

[0038] Recombinant plasmid pcDNA5 / FRT-5-HT1BR 0.8μg

[0039] Plasmid pOG44 expressing recombinase...

Embodiment 2

[0051] Embodiment 2 Cell model of the present invention is used for the specificity of drug screening

[0052] 5-HT1BR is a Gi / o-coupled G protein-coupled receptor. After activation, it inhibits the production of cAMP by inhibiting adenylyl cyclase (AC), so the activation of 5-HT1BR can be detected by detecting the production of cAMP ; In addition, the Gqi9 chimeric G protein can be transfected in this cell line, and the 5-HT1 B The signal activated by R is coupled to the PLC signaling pathway, which in turn promotes the release of intracellular calcium ions, and thus can be detected by detecting Ca 2+ Release to determine the activation of 5-HT1BR. In this example, in order to identify cell lines that can stably express functional 5-HT1BR, the second method is used, that is, to detect Ca 2+ release.

[0053] 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 5-HT...

Embodiment 3

[0073] Embodiment 3 Evaluation of the drug screening method based on the cell model of the present invention

[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. The 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 obt...

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Abstract

The invention discloses a cell model for stably expressing a 5-HT1B (serotonin) gene, and a constructing method thereof, and applications of the cell model in screening of active substances targeting5-HT1BR (serotonin receptor), particularly in screening of anti-depression drugs and anti-migraine drugs, wherein the cell model is mammalian cells capable of carrying and stably expressing the 5-HT1Bgene. The constructing method comprises: linking a 5-HT1B gene to an expression vector to construct recombinant plasmid, introducing the recombinant plasmid into mammalian cells, and carrying out screening culture to obtain the cell line capable of stably expressing the 5-HT1B gene, wherein the cell line can be used for screening the active substance targeting 5-HT1BR. According to the present invention, whether the to-be-screened substance is screened is judged by determining the yield or the activation degree of the response element after the to-be-screened substance is added to the cell model culture medium, such that the high-throughput, low-cost and accurate screening of the active substance targeting 5-HT1BR can be achieved, and the good stability and the good reliability can be achieved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and molecular pharmacology, relates to a gene expression cell model and its construction method and application, in particular to a cell model stably expressing 5-HT1B (serotonin) gene and its construction method, and the Application of cell model in screening active substances targeting 5-HT1BR (serotonin receptor). Background technique [0002] With the progress of society, the increasing environmental pollution and life pressure, neurological diseases are increasingly plagued by human beings. The creation of new drugs for neurological diseases has been included in the national key development strategy and has become a major drug research and development company in the world. focus of attention. Most human serotonin receptors are abundant in the central nervous system, participate in the regulation of various mental activities, and are closely related to mental diseases, because the...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/02
CPCC07K14/47C12N5/0603C12N5/0682C12N5/0686C12N15/85C12N2503/02C12N2510/00C12N2800/107G01N33/5008G01N2500/10
Inventor 刘剑峰许婵娟郑玲玉马淑敏
Owner EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH
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