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Method and system of detecting dioxin-like compounds

一种化合物、复合物的技术，应用在检测类戴奥辛化合物领域

Active Publication Date: 2015-03-04
E BIO ENTERPRISE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method and system of detecting dioxin-like compounds
  • Method and system of detecting dioxin-like compounds
  • Method and system of detecting dioxin-like compounds

Examples

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Embodiment 1

[0043] Embodiment 1: Construction of plasmid

[0044] AHR( Figure 1A build shown), AIP and P23 ( Figure 1B build shown), ARNT ( Figure 1C construction shown), and HSP90 ( Figure 1D Constructs shown) were amplified from HEK293 cDNA and all point mutations were corrected. Renilla luciferase (RL) was derived from pRL-TK (Promega Corporation, Madison, WI, USA) and YFP was derived from pEYFP-N1 (Clontech Laboratories, Inc., Mountain View, CA. USA).

Embodiment 2

[0045] Example 2: Generation of stable transfected cell lines

[0046] Using lipofectamin 2000 TM Transfection agent (Invitrogen, Carlsbad, CA, USA), AHR-RL, AIP-HIS and P23-HIS were transfected into tetracycline-regulated expression cells (T-REx TM cells) (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured in DMEM medium (Gibco, USA), which contained 10% fetal bovine serum (fetal bovine serum, FBS) (Gibco, USA), 5 μg / ml blasticidin (Blasticidin), 250 μg / ml Zeocin and 500 μg / ml G418. Then, the optimized cell line was transfected with ARNT-YFP or HSP90-YFP, and screened with 200 μg / ml hygromycin to obtain a stable cell line transfected with ARNT-YFP (referred to as "AAPA cells") ), and a stable cell line (referred to as "AAPH cells") transfected with HSP90-YFP was obtained.

Embodiment 3

[0047] Example 3: Detection of Dioxin by BRET Analysis in AAPA and AAPH Cells

[0048]The AAPH and AAPA cells prepared in Example 2 were treated with 1 μg / ml tetracycline for 24 hours, and then 10 μM 3MC was added for 3 hours. The induced expression of AHR-RL, AIP-HIS (AIP with His tag), P23-HIS (P23 with His tag), HSP90-YFP, or ARNT-YFP was confirmed by Western blotting. The cytoplasmic and nuclear fragments were detected with specific antibodies, including anti-RL, anti-YFP, anti-LaminA / C and anti-GAPDH. The results of the Western bolt are shown in Figure 2A , which can confirm the localization of AHR and ARNT proteins after AAPH and AAPA cell lines are treated with 3MC.

[0049] In order to induce protein expression and AHR complex formation, according to the method described in Example 2, prepare AAPA cells that were stably co-transfected with AHR-RL, AIP-HIS, P23-HIS and ARNT-YFP, and then 1 μg / ml of tetracycline for 48 hours. Then the treated cells were washed twic...

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Abstract

The invention relates to a method or a cell free system of detecting dioxin-like compounds in a test sample using a whole cell lysate derived from a cell transfected to express a fusion protein comprising an AHR fused to a reporter peptide. The method in combination with bioluminescence resonance energy transfer (BRET) technique is also provided so as to improve the sensitivity.

Description

technical field [0001] The invention relates to a method and system for detecting dioxin-like compounds. Background technique [0002] Dioxins are often formed as a result of incomplete combustion processes and are classified in a group of tenacious and often toxic chemicals, also known as persistent organic pollutants. Among them, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is considered to be the most toxic one and has been The World Health Organization (WHO) classifies it as a carcinogen. The toxicity of other dioxins or mixtures of dioxins has also been described in association with TCDD. [0003] Traditionally, analytical chemistry methods such as chromatography and mass spectrometry are often used to determine the content of dioxin in samples. However, these methods are not cost-effective and time-consuming, and more importantly, the results obtained are not directly related to dioxin toxicity. All these inherent disadvantages ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/567
CPCG01N2430/40G01N21/6428G01N21/763G01N33/5308G01N33/542
Inventor 李心予
Owner E BIO ENTERPRISE LTD
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