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

A technology for detecting the environment and estrogen, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problem of lack of receptor co-activation factors, inability to simulate the real situation of eukaryotic cells, and false positives And other issues

Active Publication Date: 2011-03-30
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

Examples

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Effect test

Embodiment 1

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

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

[0037] First, design PCR primers for the estrogen receptor ligand binding domain (hER LBD), and introduce EcoRI and BamHI single restriction sites at the 5'ends of the upstream and downstream primers, namely the upstream primer P1: 5'-TCGCCG GAATTC GCTGGAGACATGAGA-3' (the underlined part is the restriction site of EcoRI); downstream primer P2: 5'-TAACGC GGATCC TCAGACTGTGGCAGG-3' (the underlined part is the restriction site of BamHI); 2.5ng of pASV3 plasmid containing hER LBD (sequence shown in SEQ ID No.1) (gifted by Professor Heery of Nottingham University, UK) 2.5ng was used as a template for PCR amplification The PCR product (0.3pmol) was ligated to the T-vector with T4 DNA ligase (1μL) to construct the T-hER LBD plasmid; pGBKT7 (1μg,...

Embodiment 2

[0044] Example 2: Using two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCC No. 2307) to determine 17β-estradiol (E 2 ) To draw the standard curve.

[0045] 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 that in Example 1), and detect the bacteria at 600nm The absorbance value (using the culture medium as the 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.

[0046] Draw a standard curve: take 0.995 mL of bacterial suspension into the corresponding Eppendorf tube, and add 5 μL of DMSO (blank) or 5 μL of E dissolved in DMSO 2 ; Transfer 200μL of each of the above solutions to a 96-well plate in turn. Then at 130 rpm, 30 ℃ in a 96-well shaker shake culture for 2h; after the end of the culture, first detect the absorbance val...

Embodiment 3

[0050] Example 3: The two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCC No. 2307) biological test method is used to evaluate the toxicity of estrogen-like pollutants in environmental samples.

[0051] The influent and effluent of different water plants in a certain city were collected as samples. Using solid phase extraction method, adding dichloromethane and n-hexane to extract estrogen-like pollutants in the sample, drying the extracted organic solvent in high-purity nitrogen, adding DMSO to dissolve and preparing to be within the linear range of the standard curve Any concentration of test sample solution.

[0052] The two-hybrid Saccharomyces cerevisiae ER-GRIP1 (CGMCC No. 2307) biological test method described in Example 1 was used to evaluate the toxicity of environmental samples of estrogen-like pollutants, and the measurement results were compared with the one-hybrid recombinant estrogen receptor gene yeast method and mass spectrometry The measurement results of the anal...

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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-likecompounds 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 invention relates to a two-hybrid yeast for detecting estrogen-like compounds in the environment and a biological test method, in particular to a two-hybrid yeast containing a recombinant estrogen receptor gene obtained by using yeast two-hybrid technology, and A biological test method that uses 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 chemicals released into the environment during production and life of humans. They have similar chemical structures to hormones in humans and animals, and can Produces a hormone-like effect. They enter the body, disrupt the normal secretion of hormones, disrupt the physiological processes of humans and animals, and cause obstacles to functions such as reproduction and immunity. The interference effects of endocrine disruptors in the environment include: estrogen inter...

Claims

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Application Information

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Patent Type & Authority Patents(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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