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
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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
[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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