ER beta mediated dual luciferase reporter gene detection system and application in drug screening

A dual-luciferase, reporter gene technology, applied in the field of inhibitors or agonists, can solve the problem of lack of ERβ inhibitor or agonist cell screening models and other problems

Pending Publication Date: 2021-02-12
内蒙古自治区中医医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One of the purposes of the present invention is to provide an ERβ-mediated dual-luciferase reporter gene detection system to solve the problem of lack of reporter gene-based estrogen rec...

Method used

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  • ER beta mediated dual luciferase reporter gene detection system and application in drug screening
  • ER beta mediated dual luciferase reporter gene detection system and application in drug screening
  • ER beta mediated dual luciferase reporter gene detection system and application in drug screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] [Example 1] Construction of pcDNA3.1(+)-ERβ expression vector and pGL3-enhancer-5×ERE luciferase reporter vector

[0057] 1. Construction of pcDNA3.1(+)-ERβ expression vector

[0058] (1) Get the target fragment

[0059] The ERβ coding gene ESR2 transcript NM_001291712.2 was retrieved from the NCBI database, and its coding sequence is 1792..3279, with a total of 1488 bp, as shown in sequence 1. A NheI restriction site is added to its 5' end, and a BamHI restriction site is added to its 3' end for chemical synthesis.

[0060] (2) The pcDNA3.1(+) expression plasmid vector and the target gene fragment ERβ were digested with NheI and BamHI respectively.

[0061] The enzyme digestion system is specifically:

[0062]

[0063] After digestion at 37°C for 1.5 h, the digested fragments were separated by 1% agarose gel electrophoresis, and the fragments of the digested products of the pcDNA3.1(+) expression plasmid vector and the fragments of the digested products of ERβ ...

Embodiment 2

[0086] [Example 2] Cell culture and transfection

[0087] (1) Cell recovery

[0088] Take out the 293T cells from the liquid nitrogen tank, thaw them quickly in a 37°C water bath, inoculate the cells into 100mm culture dishes, and place them in 5% CO 2 , Cultivated in a carbon dioxide incubator at 37°C.

[0089] (2) Cell passage and inoculation

[0090] When the cells grow to 80-90% confluence, use trypsin to digest 293T cells, and use 5×10 4 293T cells were seeded in a 24-well plate at a density of cells / well at 37°C in 5% CO 2 incubator overnight.

[0091] (3) Cell transfection

[0092] The next day, remove the medium from the cells, replace with fresh minimal medium without antibiotics and serum, and incubate at 37°C.

[0093] Using the pRL reporter gene plasmid as an internal reference, prepare a co-transfection reagent mixture: take 1.1 μg of the total amount of plasmids in tube A, and the mass ratio of pcDNA3.1-ERβ plasmid:pGL3-enhancer-5×ERE plasmid:pRL plasmid is...

Embodiment 3

[0095] [Example 3] Application of Drug Screening

[0096] The above-mentioned co-transfected cells were treated with the extract of S. Extracting the dried cane of S. sagittarius, concentrating the filtrate under reduced pressure at low temperature, and drying in vacuum to prepare dry powder. Use DMSO to dissolve the extract phases of Sargassum sativa. Since different components have different concentrations, concentrated stock solutions with different concentrations are prepared. See Table 1 for details. The co-transfected cells without component or drug treatment were used as the control group, with 10 -8 The β-estradiol of mol / L concentration is agonist control, 10 -7 The mol / L concentration of tamoxifen was used as the inhibitor control, and the solutions of each extraction phase were respectively applied to the co-transfected cells, and three replicate wells were set up in each group, and the culture was continued for dual luciferase detection.

[0097] Table 1

[009...

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Abstract

The invention discloses an ER beta mediated dual luciferase reporter gene detection system, which comprises a pcDNA3.1(+)-ER beta expression vector constructed by cloning an ER beta coding sequence asshown in sequence 1 to a multiple cloning site of an empty vector pcDNA3.1(+), and a pGL3-enhancer-5*ERE luciferase reporter vector constructed by cloning an ER beta estrogen reaction element 5*ERE base sequence as shown in a sequence 2 to a multiple cloning site of an empty vector pGL3-Enhancer. According to the ER beta-mediated dual luciferase reporter gene detection recombinant expression vector system, the estrogen receptor ER beta-mediated dual luciferase reporter gene detection cell model and the establishment method of the estrogen receptor ER beta-mediated dual luciferase reporter gene detection cell model, a phase combined with ER beta can be screened out from sargentgloryvine stem extract phases, the inhibition effect or the excitation effect is determined, the inhibitor or agonist is identified, and the system is also suitable for screening unknown phases of other Chinese herbal medicines.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to establishing an estrogen receptor ERβ-mediated dual-luciferase reporter gene detection cell model and screening inhibitors or agonists in the extract of Sage vine. Background technique [0002] Estrogen receptor is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, which participates in estrogen response elements or inhibits the transcription of certain genes. There are two subtypes of estrogen receptors, α and β (estrogen receptorαandβ, ERαand ERβ), and these two subtypes have different functions and distributions in tissues. At present, most studies and viewpoints believe that ERα is dominant in the female reproductive system, while ERβ is dominant in cardiovascular, nervous system, and bone tissues. Estrogen has similar affinity to the two receptors, so both can up-regulate the expression of the two receptors. [0003] ERβ is widel...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/12C12N5/10C12Q1/02A61K36/185A61P5/30A61P5/32A61K135/00
CPCC12N15/85C12N15/66C07K14/721C12N5/0686G01N33/5008G01N33/5044A61K36/185A61P5/30A61P5/32C12N2800/107C12N2510/00C12N2503/02G01N2500/10
Inventor 夏瑶宾王潇
Owner 内蒙古自治区中医医院
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