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Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound

A thyroid hormone and anti-thyroid technology, applied in the direction of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of large differences in physical and chemical properties, low and difficult enrichment, purification, etc.

Active Publication Date: 2011-04-06
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The physical and chemical properties of these substances are quite different, and the concentration in water is usually extremely low, so it is difficult to enrich and purify; at present, there is no report on the sample pretreatment method for detecting the interference effect of thyroid hormone in samples. Therefore, it is urgent to establish the interference effect of thyroid hormone. Enrichment and purification methods for detection, especially for the detection of analogues / antithyroid hormones in environmental samples

Method used

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  • Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound
  • Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound
  • Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Preparation of two-hybrid yeast cell TR-GRIP1

[0063] 1. Purify the pGBT9-TR yeast expression plasmid comprising the thyroid hormone receptor gene fragment (ie, the sequence shown in SEQ ID No.1).

[0064] 100 μL of competent Escherichia coli DH5α was transformed with 10 μL of bait plasmid pGBT9-TR (gifted by Professor Stallcup, University of California, USA), positive clones were screened in a medium containing ampicillin, and the plasmid was extracted and purified. The size of the plasmid DNA was identified by 0.8% agarose gel electrophoresis, and the plasmid DNA was sequenced at the same time. The sequencing primer was the pGBT9 universal primer (the sequencing work was completed by Beijing Nuosai Genome Research Center Co., Ltd.). The medium containing ampicillin comprises 100 mg / L ampicillin, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agarose.

[0065] 2. Purifying the pGAD424-GRIP1 yeast expression plasmid containing the thyroid hormone recept...

Embodiment 2

[0072] Saccharomyces cerevisiae CGMCC No.2306 was used to determine the induction effect of triiodothyronine (T3) to draw a standard induction curve.

[0073] Yeast cell culture: inoculate the prepared Saccharomyces cerevisiae CGMCC No.2306 into SD / -Trp / -Leu liquid medium (composition is the same as in Example 1), and detect the absorbance value at 600nm of the bacterial liquid (taking 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.

[0074] Draw a standard curve: Take 0.995 mL of the bacterial suspension into the corresponding Eppendorf tube, add 5 μL of DMSO (blank) or 5 μL of T3 dissolved in DMSO; 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; rem...

Embodiment 3

[0079] Saccharomyces cerevisiae CGMCC No.2306 was used to measure the inhibitory effect of amiodarone hydrochloride to draw a standard inhibition curve.

[0080] Yeast cell culture: inoculate the prepared Saccharomyces cerevisiae CGMCC No.2306 into SD / -Trp / -Leu liquid medium (composition is the same as in Example 2), and detect the absorbance value at 600nm of the bacterial liquid (taking 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.

[0081] Draw a standard curve: Take 0.995 mL of the bacterial suspension into the corresponding Eppendorf tube, add 5 μL of LT3 and 5 μL of amiodarone hydrochloride dissolved in DMSO; 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 va...

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Abstract

The invention provides a two-hybrid yeast cell for detecting thyrotropic hormone-like / antithyrotropic hormone compounds in a sample and a preparation method thereof, wherein a yeast contains pGBT9-TR yeast expression plasmids and pGAD424-GRIP1 yeast expression plasmids, wherein the pGBT9-TR yeast expression plasmids contain thyroid hormone receptor genes, and the pGAD424-GRIP1 yeast expression plasmids contain thyroid hormone receptor coactivated factor genetic fragments with the sequence of SEQ ID No.2. The invention also provides a bioassay method for detecting the thyrotropic hormone-like / antithyrotropic hormone compounds in the sample. The yeast and the bioassay method greatly improve the sensitivity of the thyrotropic hormone-like / antithyrotropic hormone compounds. The constructed yeast has stable characteristics and is easy to culture and screen; the screening process of the whole thyrotropic hormone-like / antithyrotropic hormone 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 used for detection of compounds / anti-thyroid hormones and the construction method of the yeast and its detection method, in particular to a yeast two-hybrid technology containing recombinant thyroid hormone receptor Two-hybrid yeast with somatic genes, and a bioassay method using the yeast to detect thyroid hormone-like / anti-thyroid hormone compounds. 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 disrupto...

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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