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Recombinant yeast for detecting estrogenic compounds and its construction method

A technology of estrogen receptor and yeast, applied in the biological field, can solve the problem of low sensitivity of estrogen compounds

Active Publication Date: 2019-10-25
中国农业科学院研究生院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the currently established recombinant yeast cell detection system is less sensitive to estrogenic compounds

Method used

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  • Recombinant yeast for detecting estrogenic compounds and its construction method
  • Recombinant yeast for detecting estrogenic compounds and its construction method
  • Recombinant yeast for detecting estrogenic compounds and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, construction is used to detect the recombinant yeast of estrogen compound

[0064] The biological effects of estrogen are roughly regulated by three estrogen receptors, namely estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and G protein-coupled estrogen receptor (GPER). Most of the classic estrogen receptors ERα and ERβ exist in the nucleus and act as ligand-activated transcription factors in the cell. The way these two receptors function is mainly through the classical regulatory pathway, that is, estrogen binds to the estrogen receptor (ER) to form a dimer in the nucleus, and then binds to the estrogen response elements (EREs) located on the promoter of the target gene, thus play a role. Among them, the estrogen receptor ERβ is more sensitive to most estrogen compounds than ERα, so ERβ is selected as the estrogen receptor in the estrogen detection system.

[0065] In order to enhance the sensitivity of the classical regulatory pathway of ...

Embodiment 2

[0098] Example 2, the application of recombinant yeast in the detection of estrogenic compounds

[0099] 1. The detection effect of different strains on estrogen compounds

[0100] Cultivate the recombinant yeast p416GDP-ERβ+gDNA (ERE-ADH-GFP) and p426GDP-ERβ+gDNA (ERE-ADH-GFP) obtained in Example 1, add estrogen compounds (17-β estradiol ) to a final concentration of 5 nM. After shaking for 4 hours, the fluorescence intensity was measured with a microplate reader (infiniteM200PRO) (excitation wavelength 485 nm, emission wavelength 525 nm). Each sample was repeated 3 times. Take no estrogen compound (17‐βestradiol) as the control.

[0101] The result is as figure 1 As shown, it can be seen from the figure that compared with recombinant yeast p416GDP-ERβ+gDNA (ERE-ADH-GFP), the fluorescence intensity of recombinant bacteria p426GDP-ERβ+gDNA (ERE-ADH-GFP) is higher, indicating that this bacteria is used as a detection method. Estrogenic compounds work better.

[0102] 2. T...

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Abstract

The invention discloses a recombinant yeast for detecting an estrogenic compound and a construction method of the recombinant yeast. A recombinant bacterium provided by the invention is a recombinant bacterium obtained by expressing an estrogenic receptor beta gene, a resistance screening gene expression cassette and 3 serial ERE (Estrogen Response Element) gene and report gene expression cassettes in saccharomyces cerevisiae. The recombinant yeast constructed by the method can be used for stably and efficiently synthesizing GFP (Green Fluorescent Protein) in the presence of the estrogenic compound; the expression amount of the GFP and the concentration of the estrogenic compound form a direct proportion; EC50 of 17 beta estradiol is 3.25pM and is about 20 times as much as that of an existing document report. The recombinant yeast disclosed by the invention lays a foundation for rapidly and sensitively detecting the estrogenic compound in the environment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant yeast for detecting estrogen compounds and a construction method thereof. Background technique [0002] Estrogenic compounds refer to all compounds with estrogenic activity, which are an important class of environmental endocrine disruptors with various types and structures. Estrogen in the environment enters the biological chain through polluting water, soil, and air, enriches and amplifies in the biological chain, and accumulates and amplifies in livestock and poultry through feed, drinking water, air, etc., endangering the production and reproduction performance of livestock and endangering Animal food safety. These compounds can bind to estrogen receptors in the hypothalamus, pituitary, uterus and other organs, and have adverse effects on the reproductive development process, endocrine system, and cancer mechanisms, and also threaten the survival of biological races....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/65C12Q1/6897C12R1/865
CPCC07K14/721C12N15/65C12N15/81C12N2800/102C12N2830/34C12Q1/6897
Inventor 王海胜赵百锁佟继旭
Owner 中国农业科学院研究生院
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