Method for gathering and separating endogenous nuclear receptors and deoxyribonucleic acid (DNA) binding sequence special for same

A technology for binding sequences and nuclear receptors, which is applied in the field of enrichment and separation of endogenous nuclear receptors and their special DNA binding sequences, which can solve the problems of limited antibody types, limited isolation and identification, low expression levels of NR and its co-regulatory proteins, etc. , to achieve the effect of solving detection problems and wide application prospects

Inactive Publication Date: 2014-04-02
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of endogenous NR and its co-regulatory proteins still faces great technical difficulties. First, the expression level of NR and its co-regulatory proteins in vivo is very low, which is difficult to detect with conventional technical means; secondly, although antibodies The isolation and identification of endogenous NR and its co-regulatory proteins can also be achieved by immunoprecipitation and immunoprecipitation strategies, but the cost of antibodies is very high, and the types of antibodies currently available are very limited, which greatly limits the identification of endogenous NR and its co-regulatory proteins. clementines

Method used

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  • Method for gathering and separating endogenous nuclear receptors and deoxyribonucleic acid (DNA) binding sequence special for same
  • Method for gathering and separating endogenous nuclear receptors and deoxyribonucleic acid (DNA) binding sequence special for same
  • Method for gathering and separating endogenous nuclear receptors and deoxyribonucleic acid (DNA) binding sequence special for same

Examples

Experimental program
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Embodiment 1

[0029] Example 1. Acquisition of dedicated DNA-binding sequences for enrichment and isolation of endogenous nuclear receptors and their complexes

[0030]According to the specific combination of endogenous nuclear receptor (NR) and hormone response element (hormone response element, HRE), and HRE usually contains two conserved half-sites (5'-AGGTCA-3'), the present invention designs A variety of DNA sequences containing conserved half-site elements in which two conserved half-sites are arranged either as direct repeats (DR) or inverted repeats (IR) and where the two half-sites Spots consist of 0-6bp bases. These DNA sequences can form tandem multi-copy HRE sequences through techniques such as in vitro ligation. A single copy of HRE contains two conserved half-sites, and a 3-5bp linker sequence (Linker) is connected in tandem between each single copy of HRE; Table 1 shows the tandem 3 Copy the DNA binding sequence of HRE, in which the sequence 1-17 in the sequence table is a s...

Embodiment 2

[0057] Example 2. Enrichment and separation of endogenous nuclear receptors in mouse liver cells

[0058] Taking 12×DR1 as an example, the nuclear receptors in mouse liver cells were enriched and isolated, and identified by mass spectrometry. The enrichment separation referred to in the present invention refers to the separation of nuclear receptors and their complexes from complex nucleoproteins. Using the method of the present invention to enrich and isolate endogenous nuclear receptors in mouse liver cells, such as figure 1 As shown, it specifically includes the following steps:

[0059] 1. Obtain a high-purity biotin-labeled DNA bait (about 600bp fragment) containing 12×DR1 by the method of Example 1;

[0060] 2. Immobilize biotin-labeled DNA bait to streptavidin-coated magnetic beads by biotin-streptavidin binding On M-280streptavodin (Invitrogen), the specific operation is as follows:

[0061] 1) Take 60 μl of magnetic beads into a clean EP tube, place them on a mag...

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Abstract

The invention discloses a method for gathering and separating endogenous nuclear receptors and deoxyribonucleic acid (DNA) binding sequence special for the same. The sequence is DNA sequence in series connection with a plurality of single-copy hormonal response elements, each single-copy hormonal response element comprises two conservative half sites which are separated by oligonucleotide chains with different lengths, all the single-copy hormonal response elements are in series connection with each other through 3-5bp linkers and are based on a basic unit design shown by sequence 11 and specifically shown by sequence 1-sequence 17 in a sequence table, and the copy number is 3n (n is a positive integer). The efficient board-spectrum method for gathering and separating the nuclear receptors effectively solves the problem of detection of endogenous natural rubber (NR) and auxiliary regulating proteins and has a wide application prospect in proteomics research, discovery of clinical disease markers, development of new drugs and other aspects.

Description

technical field [0001] The invention belongs to a method for enriching and separating target proteins in the field of biotechnology, in particular to a method for enriching and separating endogenous nuclear receptors and its special DNA binding sequence. Background technique [0002] Nuclear receptors (nuclear receptors, NR) are the largest family of transcription factors in animals. They are widely distributed in organisms and regulate the expression of specific genes by binding to corresponding ligands. important role in physiological processes. According to reports, 48, 49, and 47 NRs are expressed in humans, mice, and rats, respectively. Ligands that bind and activate NRs include lipophilic substrates, such as endogenous hormones, vitamin A, vitamin D and xenobiotics, endocrine blocking agents, etc. Since NR regulates a large number of disease-related genes, including hypertension, cancer, diabetes, cardiovascular disease, cholesterol gallstone disease, and metabolic s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C07K14/705C07K1/14C12Q1/68G01N33/68
Inventor 秦钧刘琼明丁琛刘明伟刘万霖宋雷
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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