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Recombinant estrogen receptor fluorescent two-hybrid yeast as well as preparation method and application

An estrogen receptor, two-hybrid technology, applied in biochemical equipment and methods, chemical instruments and methods, antibody mimics/scaffolds, etc. time-consuming issues

Inactive Publication Date: 2015-04-08
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a detection method, the detection of LacZ usually requires steps such as breaking cells and developing color in a warm bath, which is cumbersome, time-consuming and labor-intensive.
These shortcomings have become obstacles to the high-throughput and rapid detection of estrogen-like environmental endocrine disruptors in recombinant estrogen receptor two-hybrid yeast

Method used

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  • Recombinant estrogen receptor fluorescent two-hybrid yeast as well as preparation method and application
  • Recombinant estrogen receptor fluorescent two-hybrid yeast as well as preparation method and application
  • Recombinant estrogen receptor fluorescent two-hybrid yeast as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Construction of recombinant estrogen receptor fluorescent two-hybrid yeast (hER-GRIP1)

[0083] 1. Co-transformation

[0084] (1) Inoculate the fluorescent yeast Yluc in 10ml of YPD medium, and culture it with shaking at 30°C overnight to obtain a culture solution;

[0085] (2) Take the culture solution to 50ml of fresh YPD medium to make OD600=0.2-0.3;

[0086] (3) Shake culture at 30°C until OD600=0.6-0.8, about 5-7 hours, depending on the selection culture;

[0087] (4) Centrifuge at 1000 g for 5 min at room temperature. Discard the supernatant, take 10ml of sterile water to resuspend the yeast cells;

[0088] (5) Centrifuge at 1000g for 5min at room temperature; discard the supernatant, and take 1.0ml of solution A (competent solution) to resuspend.

[0089] (6) Shake culture at 30°C for 30 minutes to obtain fluorescent yeast Yluc competent cells;

[0090] (7) Transform fluorescent yeast Yluc competent cells with pGBKT7-hER plasmid and pGAD424-GRIP1 ...

Embodiment 2

[0095] Example 2. Establishment of Natural Estrogen Dose-Effect Curve Using Recombinant Estrogen Receptor Fluorescent Two-hybrid Yeast hER-GRIP1

[0096] 1. Yeast cell culture

[0097] Inoculate the recombinant estrogen receptor fluorescent two-hybrid yeast hER-GRIP1 into SD / -Trp / -Leu liquid medium, use the medium as a blank, detect the OD600 of the bacterial liquid, adjust the OD600 to 0.1-0.2, and culture at 30°C with shaking After 24 hours, adjust the OD600 of the bacterial solution to 0.7-0.8.

[0098] Second, the drawing of the standard curve

[0099] Natural estrogens mainly include 17β-estradiol (E2), estrone (E1) and estriol (E3). The concentration gradients of E1, E2, and E3 were selected for the research on the recombinant recombinant estrogen receptor fluorescent two-hybrid yeast hER- GRIP1 is induced. The following takes E2 as an example to describe in detail, and all operation steps of E1 and E3 are the same.

[0100] (1) Dissolve 17β-estradiol (E2) in dimethy...

Embodiment 3

[0126] Example 3. Using recombinant estrogen receptor fluorescent two-hybrid yeast (hER-GRIP1) biological assay method to evaluate the estrogen-like pollutant effect of environmental samples

[0127] 1. Collect surface runoff water samples from e-waste demolition sites in a certain city as samples. The estrogen-like compounds were extracted by solid-phase extraction, and the specific method was: the sample was loaded into the Oasis C18 column solid-phase extraction column (Waters, USA), and methanol:ethyl acetate (volume ratio 1:1) was added. Mixed solvents were used for elution, and the eluate was collected as the crude fraction; then the crude fraction was loaded onto a silica gel column (3g silica gel, column i.d.0.9cm), using n-hexane: dichloromethane (volume ratio 1:1 ) mixed solvent for elution, remove the non-polar components, then add ethyl acetate for elution, collect the eluent, and obtain the purified components; dry the organic solvent of the purified components in...

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Abstract

The invention discloses recombinant estrogen receptor fluorescent two-hybrid yeast as well as a preparation method and an application thereof. The invention discloses recombinant estrogen receptor fluorescent two-hybrid yeast, which is obtained by introducing the encoding gene of fusion protein containing an estrogen receptor and a DNA combination domain and the encoding gene of fusion protein containing an estrogen receptor nuclear receptor coactivator and a transcription activating domain into fluorescent yeast Yluc with genotype of MATalpha, ura 3-5, ade 2-101 trp 1-901, leu 2-3, 112, gal4detla, met-, gal80 delta, URA::GALiUAS-GAL1TATA-Luc. The yeast disclosed by the invention is stable in gene character, is easy to culture and screen and can be used for detecting xenoestrogen compounds or estrogenic compounds in environment, and the detection method is simple, convenient and quick and can be suitable for the requirements of high-throughput quick detection.

Description

technical field [0001] The invention relates to a recombinant estrogen receptor fluorescent double-hybrid yeast and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] Environmental endocrine disruptors (Endocrine disruptors, environmental endocrine disrupting chemicals, EDCs) refers to the general designation of a large class of exogenous compounds that affect the endocrine system in the body and have harmful effects on the development, reproduction, nervous and immune systems of humans and wild animals (NIH, 2013). Many natural or man-made compounds are endocrine disruptors, including drugs, dioxins, dioxin-like substances, PCBs, pesticides (such as DDT), plasticizers (such as bisphenol A). Many everyday products may contain endocrine disruptors, such as: plastic bottles, metal cans, detergents, flame retardants, food, toys, cosmetics, pesticides. [0003] The interrelationship between chemical exposure and healt...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12Q1/02
CPCC07K14/721C07K2319/71C07K2319/80C12Q1/02
Inventor 王子健杨明嘉马梅
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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