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Double-cross yeast gER beta 1-GRIP1 for reorganizing estrogen receptor of gobiocypris rarus and application of double-cross yeast gER beta 1-GRIP1

A technology of estrogen receptor and rare gobi carp, which is applied in the biological field and can solve the problems of limited biological detection methods

Inactive Publication Date: 2015-06-17
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also shortcomings in this type of method, and any single biological detection method can only provide limited evidence

Method used

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  • Double-cross yeast gER beta 1-GRIP1 for reorganizing estrogen receptor of gobiocypris rarus and application of double-cross yeast gER beta 1-GRIP1
  • Double-cross yeast gER beta 1-GRIP1 for reorganizing estrogen receptor of gobiocypris rarus and application of double-cross yeast gER beta 1-GRIP1
  • Double-cross yeast gER beta 1-GRIP1 for reorganizing estrogen receptor of gobiocypris rarus and application of double-cross yeast gER beta 1-GRIP1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Construction of recombinant rare gobi estrogen receptor two-hybrid yeast gERβ1-GRIP1

[0099] 1. Construction of the bait plasmid pGBKT7-gERβ1 containing the Ligand Binding Domain (LBD) gene fragment (gERβ1 LBD fragment, ie, SEQ ID No.1) of the rare gobi estrogen receptor β1.

[0100] The sequence between the EcoR I and BamH I recognition sites of pGBKT7 was replaced by SEQ ID No.1, and the rest of the pGBKT7 sequence remained unchanged to obtain the bait plasmid pGBKT7-gERβ1.

[0101] EcoR I and BamH I double digested pGBKT7-gERβ1 or pGBKT7 to obtain the digested products respectively, and the digested products were detected by agarose gel electrophoresis, and the results were as follows figure 1 shown.

[0102] figure 1 It shows that the bait plasmid pGBKT7-gERβ1 can obtain two bands after EcoR I and BamH I double enzyme digestion, which are respectively the backbone fragment (7.3Kb) of the yeast expression plasmid pGBKT7 and the fragment containing the g...

Embodiment 2

[0109] Example 2. Establishment of Natural Estrogen Dose-Effect Curve Using Recombinant Rare Gobi Estrogen Receptor Two-Hybrid Yeast gERβ1-GRIP1

[0110] 1. Yeast cell culture

[0111] Inoculate the recombinant rare gobi estrogen receptor two-hybrid yeast gERβ1-GRIP1 into the auxotrophic liquid medium (SD / -Trp / -Leu / -His) to obtain the initial bacterial liquid, so that the absorbance at 600nm is After culturing at 30°C for 24 hours at 0.1-0.2, the cultured bacterial liquid was obtained, and the absorbance value at 600 nm of the bacterial liquid was adjusted to 0.7-0.8 (the yeast was in the logarithmic growth phase) to obtain the two-hybrid yeast gERβ1-GRIP1 bacterial liquid.

[0112] (The determination of the above OD value is adjusted to zero by auxotrophic liquid medium (SD / -Trp / -Leu / -His))

[0113] 2. Draw a standard curve

[0114] (1) Dissolve 17β-estradiol in dimethylsulfoxide (DMSO) to prepare 17β-estradiol standard concentration reaction solution, the concentration ran...

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Abstract

The invention discloses a double-cross yeast gER beta 1-GRIP1 for reorganizing an estrogen receptor of gobiocypris rarus and application of the double-cross yeast gER beta 1-GRIP1. The invention discloses double-cross yeast. The double-cross yeast is obtained by transferring a coding gene of fusion protein of a ligand binding region and a DNA bonding structure region containing the estrogen receptor beta 1 of the gobiocypris rarus and a coding gene of fusion protein of a common activity factor and a copying activity structure region containing the estrogen receptor into fluorescent yeast of which the gene type is MAT alpha, ura 3-5, ade 2-101 trp 1-901, leu2-3, 112, gal4 delta, met-, gal80 delta, URA:: GAL1UAS-GAL1TATA-Luc. The double-cross yeast gER beta 1-GRIP1 has important application in detecting the toxic equivalent or the size of potential ecological toxicity in natural estrogen and / or similar estrogen compounds in environmental samples, in particular assessing the risk which may exist in the endocrine system of the gobiocypris rarus.

Description

technical field [0001] The invention relates to a recombinant rare gobi estrogen receptor two-hybrid yeast gERβ1-GRIP1 and application thereof, belonging to the field of biotechnology. Background technique [0002] Endocrine disruptors in the environment, also known as environmental hormones, refer to exogenous chemical substances or mixtures that can affect the endocrine system of animals and cause damage to individuals and their offspring or groups (partial subgroups). The U.S. Environmental Protection Agency (EPA) has described the specific process by which it affects the endocrine system: it hinders the normal behavior of organisms and the synthesis, storage, secretion, in vivo transport, combination and clearance of normal hormones related to reproduction and development. The interference effects of endocrine disruptors in the environment include: estrogen interference effects, androgen interference effects, thyroid hormone interference effects, etc., especially estroge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81G01N33/68
Inventor 王子健杨明嘉韦玉梅马梅
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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