A cell-based high-throughput screening assay for Farnesoid X receptor agonists / 生物医学与环境科学(英文)
Biomedical and Environmental Sciences
;
(12): 465-469, 2007.
Article
in English
| WPRIM
| ID: wpr-249824
ABSTRACT
<p><b>OBJECTIVE</b>To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.</p><p><b>METHODS</b>cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.</p><p><b>RESULTS</b>After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.</p><p><b>CONCLUSION</b>A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Transcription Factors
/
Base Sequence
/
Transfection
/
Cell Line
/
Chemistry
/
Reproducibility of Results
/
Receptors, Cytoplasmic and Nuclear
/
DNA, Complementary
/
DNA Primers
Type of study:
Diagnostic study
/
Prognostic study
/
Screening study
Limits:
Humans
Language:
English
Journal:
Biomedical and Environmental Sciences
Year:
2007
Type:
Article
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