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Yeast recombination system for detecting endocrine disrupting chemicals and method thereof

A technology for recombination system and detection environment, which is applied in the field of yeast recombination system for detection of environmental estrogens, can solve problems such as the inability to rule out the influence of reporter genes, laborious immunological detection methods, and the influence of enzyme activity detection, etc., and achieves strong biological toxicity accumulation ability Effect

Pending Publication Date: 2019-12-13
OCEAN UNIV OF CHINA
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AI Technical Summary

Problems solved by technology

While the assays described above assess the ability of a chemical to bind to a hormone receptor, detection of ligand binding requires the use of rather laborious immunological assays, such as mammalian cell lysis steps
On the other hand, the yeast two-hybrid system, which is widely used in the detection of human estrogen receptor, usually takes several days to complete the substrate colorimetry; and the reporter gene used to detect the expression of the yeast two-hybrid system needs to be connected with the target gene and then transferred Yeast strains, thereby starting the expression of the reporter gene, cannot rule out the influence of the reporter gene on the yeast two-hybrid system; in addition, the yeast two-hybrid system uses chemical reagents to lyse yeast cells, which is easy to affect the subsequent enzyme activity detection

Method used

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  • Yeast recombination system for detecting endocrine disrupting chemicals and method thereof
  • Yeast recombination system for detecting endocrine disrupting chemicals and method thereof
  • Yeast recombination system for detecting endocrine disrupting chemicals and method thereof

Examples

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Embodiment 1

[0038] The construction of embodiment 1 yeast recombination system

[0039] (1) Connect the long oyster estrogen receptor gene CgER to the cloning vector pGADT7 to construct a recombinant expression plasmid:

[0040]In this embodiment, the cDNA sequence CgER of the estrogen receptor gene of the long oyster (Crassostrea gigas) is selected as the target sequence (GenBank accession number is AB259818.1); firstly, the primer of CgER is designed by Primer5.0 software, and the primer is added at both ends of the primer. EcoRI / XhoI restriction site, the primer sequence is shown in SEQ ID NO.1-2, the underlined part is the restriction endonuclease sequence, and the italicized part is the vector linker sequence;

[0041]

[0042] Use the above primer pair CgER to amplify the conserved region fragment of the target gene, and use the cDNA template of oyster gonad tissue and high-fidelity enzyme Max DNA Polymerase for PCR reaction, gel recovery and purification to obtain the inserted...

Embodiment 2

[0055] Example 2 Activation method and activity detection of yeast recombination system

[0056] (1) Cultivation of recombinant strains

[0057] Pick a monoclonal recombinant yeast strain (Y187) and add it to the YPDA full nutrient liquid medium for shaking culture until the OD 600 The reading is 0.1-0.4; the non-transfected bacterial strain (represented by the symbol Y187) is used as the blank control group, only the transfected expression plasmid strain (represented by the symbol Cg-ER) is used as the negative control group, and the recombinant strain (represented by the symbol Cg-ER / ERE) as the experimental group.

[0058] Add a series of samples to be tested (DMSO solutions containing different concentrations of steroids or endocrine disruptors, such as 17β-estradiol E2, estrone E1, progesterone P, testosterone T, nonylphenol 4-NP) Shake culture in a constant temperature shaker for 18 hours, measure and record the OD of the culture solution at this time 600 value.

[...

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Abstract

The invention relates to a yeast recombination system for detecting endocrine disrupting chemicals and a method thereof, and the system is constructed by the following method: connecting a crassostreagigas estrogen receptor gene CgER to a vector pGADT7 to construct a recombinant expression plasmid; connecting a xenopus vitellogenin gene ERE to a vector pGBKT7 to construct a recombinant bait plasmid; and co-transforming the recombinant expression plasmid and the bait plasmid into a Y187 yeast cell, and screening and culturing a positive clone strain. The crassostrea gigas living in a benthic fixation manner is high in biotoxicity accumulation capacity, and potential hazards of marine pollutants to organisms can be revealed, so that the crassostrea gigas is regarded as an ideal marine environment monitoring organism. The Y187 strain is used as a yeast host and contains a reporter gene Lac Z, so that the construction of reporter plasmids is simplified, and the probability of false positive of recombinant yeast is greatly reduced. According to the yeast recombination system, the specificity of identifying or detecting chemicals with endocrine disruption activity is greatly improved.

Description

technical field [0001] The invention relates to a yeast recombination system and method for detecting environmental estrogens, belonging to the technical field of biological detection. Background technique [0002] Environmental estrogen disruptors (EDCs) are important environmental issues that affect human health and ecological safety. The difficulty in the study of endocrine disruption in ecotoxicology is that species of different phyla have different endocrine systems. By analyzing the molecular mechanism of EDCs, sensitive molecular markers can be identified, which can be used to indicate the environmental concentration of pollutants and assess exposure risks, and provide a basis for extrapolating cross-species interference effects. [0003] Nonylphenol (NP) is an important class of environmental estrogen disruptors with persistence and bioaccumulation. Because the structure of NP, especially 4-NP, has the characteristic of estrogen, it can activate the target gene dow...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/81C12Q1/40
CPCC12N15/66C12N15/81C12Q1/40G01N2333/938
Inventor 苗晶晶刘力铷潘鲁青刘佩佩
Owner OCEAN UNIV OF CHINA
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