Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method

A detection environment, two-hybrid technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of false positive, lack of receptor coactivator, false negative, etc.

Active Publication Date: 2009-07-01
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the lack of receptor coactivator when using recombinant estrogen receptor gene single hybrid yeast to measure estrogen-like pollutants in the prior art, so it cannot simulate the real situation of eukaryotic cells, and it is easy to produce false negatives or is the defect of false positive results, so as to construct a two-hybrid yeast capable of expressing estrogen receptor and receptor coactivator at the same time, and on this basis, establish a cost-effective biological method for evaluating the interference activity of environmental samples on estrogen receptor testing method

Method used

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  • Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method
  • Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method
  • Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method

Examples

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Embodiment 1

[0035] Example 1: Preparation of Saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) cells

[0036] 1. Construct the pGBKT7-hER yeast expression plasmid comprising the estrogen receptor gene fragment (ie, the sequence shown in SEQ ID No.1).

[0037] Firstly, PCR primers for the estrogen receptor ligand binding domain (hER LBD) were designed, and EcoR I and BamH I single restriction sites were introduced at the 5′ ends of the upstream and downstream primers respectively, that is, the upstream primer P1: 5′-TCGCCGGAATTCGCTGGAGACATGAGA -3' (the underlined part is the restriction site of EcoRI); downstream primer P2: 5'-TAACGCGGATCC

[0038]TCAGACTGTGGCAGG-3' (the underlined part is the restriction site of BamH I); carry out PCR amplification with 2.5 ng of the pASV3 plasmid (gifted by Professor Heery, University of Nottingham, UK) containing hER LBD (sequence shown in SEQ ID No.1) as template The PCR product (0.3pmol) was connected to the T-vector with T4 DNA ligase (1μL) to const...

Embodiment 2

[0045] Embodiment 2: Determination of 17β-estradiol (E 2 ) to draw a standard curve.

[0046] Yeast cell culture: inoculate the prepared two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCCNo.2307) cells into SD / -Trp / -Leu liquid medium (the composition is the same as in Example 1), and detect the bacterium liquid at 600nm Absorbance value (take the culture medium as blank), adjust the absorbance value to 0.1-0.2, and adjust the absorbance value at 600nm of the bacterial solution to 0.7-0.8 after culturing in an air-bath shaker at 30°C for 24 hours.

[0047] Draw the standard curve: take 0.995mL of the bacterial suspension into the corresponding Eppendorf tube, add 5μL of DMSO (blank) or 5μL of E 2 ; Transfer 200 μL of each of the above solutions to a 96-well plate in turn. Then at 130rpm, 30°C, vibrate and culture on a 96-well plate shaker for 2 hours; after the culture, first detect the absorbance value at 600nm; remove 150μL of yeast liquid; add 120μL of test buffer (3.33mL...

Embodiment 3

[0051] Example 3: Evaluation of the toxicity of estrogen-like pollutants in environmental samples using the two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCC No. 2307) biological assay method.

[0052] The influent and effluent water from different water plants in a certain city were collected as samples. Using solid phase extraction method, add dichloromethane and n-hexane to extract the estrogen-like pollutants in the sample, dry the extracted organic solvent in high-purity nitrogen, add DMSO to dissolve and prepare it within the linear range of the standard curve Test sample solution of any concentration.

[0053] Utilize the two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCCNo.2307) bioassay method described in Example 1 to evaluate the toxicity of estrogen-like pollutants in environmental samples, and the measurement results are compared with the single-hybrid recombinant estrogen receptor gene yeast method, mass spectrometry The results of the analytical methods were...

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Abstract

The invention provides a two-hybrid yeast for detecting estrogen-like compounds in environmental samples and a preparation method thereof, wherein the yeast contains pGBKT7-ER yeast expression plasmids and pGAD424-GRIP1 yeast expression plasmids, wherein the pGBKT7-ER yeast expression plasmids contain estrogen receptor genes, and the pGAD424-GRIP1 yeast expression plasmids contain estrogen receptor coactivated factor genetic fragments with the sequence of SEQ ID No.2. The invention also provides a bioassay method for detecting the estrogen-like compounds in the environment, which comprises: co-culturing two-hybrid yeast cells and a sample to be detected, adding a reaction liquid of o-nitrobenzene-beta-D-galactopyranoside for reaction, and calculating the concentration of the estrogen-like compounds according to the detected absorbance value of supernatant at 420 nanometers after the reaction stops. The invention adopts the two-hybrid yeast of recombinant estrogen receptor genes for test, and is more close to the actual action conditions of an endocrine system of a mammal; constructed yeast cell genes have stable character and are easy to culture and screen; the screening process of the whole estrogen-like effect is simple to operate; and the required quantity of the sample is small, and the cost is low.

Description

technical field [0001] The present invention relates to a two-hybrid yeast for detecting estrogen-like compounds in the environment and a biological testing method, in particular to a two-hybrid yeast containing a recombinant estrogen receptor gene obtained by using yeast two-hybrid technology, and A bioassay method using the yeast to detect estrogen-like compounds in the environment. Background technique [0002] Endocrine disruptors in the environment, also known as environmental hormones, refer to certain toxic chemical substances released into the environment by humans during production and life. They have similar chemical structures to hormones in humans and animals, and can Produce hormone-like effects. When they enter the body, they disrupt the normal secretion of hormones, disrupt the physiological processes of humans and animals, and lead to reproductive and immune dysfunction. The interference effects of endocrine disruptors in the environment include: estrogen i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/12C12N15/81C12Q1/06C12R1/865
Inventor 王子健李剑马梅饶凯锋
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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