High throughput screening and authenticating method for biological effect substance on the basis of recombination nuclear receptor protein

A technology for biological effects and identification methods, applied in the field of high-throughput non-targeted effector identification, can solve the problems of time-consuming and labor-intensive, low selectivity, poor specificity, etc., to avoid blindness, improve specificity and efficiency, The effect of increasing the hit rate

Active Publication Date: 2018-12-25
PEKING UNIV
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Problems solved by technology

However, this method is time-consuming and labor-intensive, and requires gas or liquid chromatography for multi-step fractionation experiments. Chemical analysis and biological activity testing are independent of each other, with poor specificity and low selectivity. Therefore, the identification of trace biological effect substances in complex environmental matrices very ineffective

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  • High throughput screening and authenticating method for biological effect substance on the basis of recombination nuclear receptor protein
  • High throughput screening and authenticating method for biological effect substance on the basis of recombination nuclear receptor protein
  • High throughput screening and authenticating method for biological effect substance on the basis of recombination nuclear receptor protein

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Embodiment Construction

[0035] The technical solution of the present invention will be described in more detail below by taking the identification of biological effect substances in common environmental media as an example.

[0036] 1. Detection of biological effect substances specifically bound to RARα recombinant nuclear receptor protein in indoor dust

[0037] 1. Specific capture chemical method based on the principle of affinity binding

[0038] A. Weigh 0.1g of dust and wash it with 5mL n-hexane, 5mL dichloromethane / n-hexane (1:4, v / v), 5mL dichloromethane / n-hexane (1:1, v / v), 5mL methanol Sonicate for 20 minutes, shake and extract for 30 minutes, then centrifuge at 4000 rpm for 10 minutes, absorb the supernatant, combine the extracts, blow dry with nitrogen, and dilute to volume with 100 μL DMSO.

[0039] B.1ml 10% Ni 2+ For the chelated magnetic bead suspension, wash the magnetic beads 3 times with 1ml S3 buffer, and then add 100 μL S3 buffer.

[0040] C. Take 500 μg of human RARα protein w...

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Abstract

The invention relates to a high throughput screening and authenticating method for a biological effect substance on the basis of recombination nuclear receptor protein. Selected recombination nuclearreceptor protein with a tag is selectively adsorbed to a magnetic bead carrier; according to an affinity binding principle of a nuclear receptor and a micromolecule, the recombination nuclear receptorprotein is used for specifically capturing the effect substance with binding activities from complex environment media; and an ultra-high performance liquid chromatography-quadrupole static electricfield orbitrap high-resolution mass spectrum technology is combined to carry out high throughput and non-targeted effect substance analysis. By use of the method, the specificity and the efficiency ofsubstance authentication are improved, and the method can be applied to the quick screening and the structure authentication of the effect substance combined with the nuclear receptor in the environment, and also can be used for medicine development.

Description

technical field [0001] The invention belongs to the field of biochemical detection, and relates to a method for screening biological effect substances specifically bound to recombinant nuclear receptor proteins, in particular to an affinity binding screening method in which recombinant nuclear receptor proteins are immobilized on nickel magnetic beads And the high-throughput non-target effect substance identification method established on the basis of it. Background technique [0002] Global chemicals are growing at an explosive rate. So far, the American Chemical Abstracts Service has registered more than 108.7 million commercial chemicals. While bringing convenience to people's lives, these substances also enter the environment in various forms, such as house dust, drinking water etc. A large number of studies have shown that some chemicals in the environment can interfere with the endocrine system, thereby affecting ecology and human health, but little is known about the...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/02
CPCG01N30/02G01N33/6848
Inventor 胡建英张虹贾映亭胡文欣张照斌
Owner PEKING UNIV
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