Transcriptional activation system for detecting RXR/ PPAR dimer interferent, and detection method

A technology of transcription activation and dimerization, applied in the detection of potential endocrine disruptors in the environment, the field of yeast transcription activation system, can solve the problem that the influence and interference of the reporter gene on the yeast two-hybrid system cannot be ruled out.

Active Publication Date: 2021-09-03
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The yeast two-hybrid system widely used in protein interaction often takes several days to complete the substrate colorimetry, and the reporter gene used to detect the expression of the yeast two-hybrid system needs to be connected with the target gene and then transferred into the yeast strain to activate the reporter gene However, the influence of the reporter gene on the yeast two-hybrid system cannot be ruled out. Some researchers have constructed an in vitro detection system based on the principle of the yeast two-hybrid GAL4 factor to evaluate the binding ability of organic tin and other pollutants to RXR. Research on aggregates is blank

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  • Transcriptional activation system for detecting RXR/ PPAR dimer interferent, and detection method
  • Transcriptional activation system for detecting RXR/ PPAR dimer interferent, and detection method
  • Transcriptional activation system for detecting RXR/ PPAR dimer interferent, and detection method

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Embodiment 1

[0039] Construction of embodiment 1 transcriptional activation system

[0040] The construction of yeast transcription activation system is characterized in that it comprises the following steps:

[0041] (1) Connect the retinoid X receptor gene (RXRa) of Chlamys farreri to the cloning vector pGBT9 to construct the recombinant plasmid pGBT9-RXRa:

[0042] In this embodiment, the cDNA sequence of the retinoic acid X receptor gene of Chlamys farreri is selected as the target sequence (GenBank accession number is JQ778315); firstly, primers of RXRa are designed by Primer5.0 software, and SmaI and PstΙ enzyme cutting site, the primer sequence is shown in SEQ ID NO.3-4, the underlined part is the restriction endonuclease sequence, and the italic part is the vector linker sequence;

[0043]

[0044]

[0045] Using the above primers to amplify the conserved region of the RXRa target gene, using the cDNA template of the gonad tissue of Chlamys farreri and high-fidelity enzymes ...

Embodiment 2

[0055] Example 2 Activation method and activity detection of yeast transcription activation system

[0056] (1) Cultivation of yeast strains

[0057] Pick a monoclonal recombinant yeast strain (Y187) and add it to SD / -Leu / -Trp liquid medium for shaking culture until OD 600 The reading is 0.1-0.4; then add a series of samples to be tested (DMSO solutions containing different concentrations of natural ligands or endocrine disruptors, such as 9-cis retinoic acid 9cRA, tributyltin chloride TBT, triphenyl chloride Tin TPT, tributyl tin oxide TBTO, tri-n-propyl tin chloride TPrT), in a constant temperature shaker at 30 ° C for 18 h, measure and record the OD of the culture solution at this time 600 value.

[0058] (2) Induction and wall-breaking treatment of yeast strains

[0059] A. Take 1mL of the culture medium to be tested in a 1.5mLEP tube, centrifuge at 13,300rpm for 1min, discard the supernatant; add 1mL Zbuffer buffer, centrifuge again and discard the supernatant; add 100...

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Abstract

The invention discloses a transcriptional activation system for detecting a RXR/ PPAR dimer interferent, and a detection method. The detection method comprises the following steps of: connecting a Chlamys farreri retinoic acid receptor gene RXRa to a carrier pGBT9, and constructing a yeast expression plasmid pGBT9-RXRa; connecting a Chlamys farreri peroxisome proliferator-activated receptor PPAR[alpha] to a carrier pGADT7 to construct a yeast expression plasmid pGADT7-PPAR[alpha]; and jointly converting the pGBT9-RXRa and the pGADT7-PPAR[alpha] into a Y187 yeast cell to construct a RXR/ PPAR dimer transcriptional activation system, and carrying out screening, culturing and positive clone. The saccharomyces cerevisiae Y187 is taken as a host bacterial strain, construction of a reporter plasmid is simplified, the sensitivity and the accuracy of the detection method are improved, and a false positive generation probability is lowered. The transcriptional activation system greatly improves specificity for authenticating or detecting a chemical capable of interfering with the internal secretion interference activity of the RXR/ PPAR dimer.

Description

technical field [0001] The present invention relates to a transcriptional activation system and method for detecting RXR / PPAR dimer interferers, specifically obtaining a yeast transcriptional activation system comprising RXR and PPAR by constructing a recombinant plasmid, and using this system to detect potential endocrine disruptors in the environment method. Background technique [0002] As a ligand-dependent transcription factor, retinoic acid X receptor (RXR) can not only form a homodimer with itself, but also interact with other nuclear receptors such as peroxisome proliferator-activated receptor (PPAR), vitamin The formate receptor (RAR) and thyroid hormone receptor (TR) form heterodimers and are therefore key players in the signaling pathway of the nuclear receptor superfamily. The transcription mechanism of RXR is the same as that of classical nuclear receptors, that is, the RXR dimer binds to the response element in the promoter region of the target gene, and regul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/65C12N15/12C12Q1/02C12R1/865
CPCC12N15/81C12N15/65C07K14/70567C07K14/705C12Q1/025G01N2333/395
Inventor 苗晶晶赵安冉潘鲁青
Owner OCEAN UNIV OF CHINA
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