Two-hybrid yeast and method for detecting thyroid hormone-like/antithyroid hormone compound
A thyroid hormone and anti-thyroid technology, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as large differences in physical and chemical properties, low difficulty in enrichment and purification, etc.
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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, shake culture in a 96-well plate shaker for 2 hours; after the culture, first detect the absorbance value at 600nm; remove 15...
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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