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A transcription regulator specifically responsive to d-2-hydroxyglutarate and its application

A technology of transcriptional regulatory factor and hydroxyglutarate, which is applied in application, genetic engineering, plant gene improvement, etc., to achieve the effects of easy preparation, simple composition, and convenient operation

Active Publication Date: 2022-06-14
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the search found that the transcriptional regulators that specifically respond to D-2-hydroxyglutarate and the methods for the biological detection of D-2-hydroxyglutarate based on transcriptional regulators have not been reported

Method used

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  • A transcription regulator specifically responsive to d-2-hydroxyglutarate and its application
  • A transcription regulator specifically responsive to d-2-hydroxyglutarate and its application
  • A transcription regulator specifically responsive to d-2-hydroxyglutarate and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1: Acquisition and identification of the transcriptional regulator DhdR specifically responsive to D-2-hydroxyglutarate

[0066] The culture medium and reagent used in this embodiment are as follows:

[0067] LB medium: 0.5% yeast powder, 1% peptone, 1% NaCl

[0068] Loading buffer: 20mM Na 2 HPO 4 , 20mM imidazole, 500mM NaCl, pH 7.4.

[0069] Elution buffer: 20mM Na 2 HPO 4 , 500 mM imidazole, 500 mM NaCl, pH 7.4.

[0070] Binding buffer: 10 mM Tris-HCl, 50 mM KCl, 0.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, pH 7.4.

[0071] Gel retardation electrophoresis buffer: 89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3.

[0072] (1) Expression and purification of DhdR

[0073] The DhdR used in the present invention is derived from the repressor protein of Achromobacter denitrificans NBRC15125, using the genome of Achromobacter denitrificans NBRC15125 as a template, the dhdR nucleotide fragment is obtained by PCR amplification, using SacI / HindIII restriction...

Embodiment 2

[0087] Example 2: Constructing a biodetection sensor B using the transcriptional regulator DhdR that specifically responds to D-2-hydroxyglutarate D2HG -0

[0088] The reagents used in this embodiment are as follows:

[0089] HBS-P buffer: 10 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.005% Tween-20, pH 7.4.

[0090] (1) Amplification and purification of biotin-labeled dhdO fragments

[0091] Biotinylated dhdO fragments were obtained by two rounds of PCR:

[0092] The first round uses the Bio-dhdO upstream primer and the Bio-dhdO downstream primer to amplify the unlabeled dhdO fragment by overlapping PCR; the second round uses the unlabeled dhdO fragment of the product of the first round of PCR as a template, and uses the primer Bio upstream primer Amplify with Bio-dhdO downstream primers to obtain biotin-labeled dhdO fragments, use a gel recovery kit to purify and recover the amplified biotin-labeled dhdO fragments, and measure the DNA concentration by NanoDrop ND-1000;

[0093] The...

Embodiment 3

[0103] Example 3: Constructing a biodetection sensor B using the transcriptional regulator DhdR that specifically responds to D-2-hydroxyglutarate D2HG -1

[0104] The reagents used in this embodiment are as follows:

[0105] HBS-P buffer: 10 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.005% Tween-20, pH 7.4.

[0106] (1) Amplification and purification of biotin-labeled dhdO-1 fragment

[0107] Biotinylated dhdO-1 fragments were obtained by two rounds of PCR:

[0108] The first round uses the Bio-dhdO upstream primer and the Bio-dhdO-1 downstream primer to amplify the unlabeled dhdO-1 fragment by overlapping PCR; the second round uses the unlabeled dhdO-1 fragment of the first round of PCR as a template , using the primers Bio upstream primer and Bio-dhdO-1 downstream primer to amplify the biotin-labeled dhdO-1 fragment, use the gel extraction kit to purify and recover the amplified biotin-labeled dhdO-1 fragment, and use NanoDrop ND DNA concentration was measured at -1000; the prim...

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Abstract

The invention discloses a transcription regulation factor specifically responding to D-2-hydroxyglutarate and its application in the biological detection of D-2-hydroxyglutarate. Wherein said transcription regulator is named as DhdR, and its nucleotide sequence is as shown in SEQ ID NO.1, and the D-2-hydroxyglutarate biosensor B constructed by the transcription regulator is D2HG ‑0 and D‑2‑Hydroxyglutarate Biosensor B D2HG ‑1 is capable of detecting biological samples containing D‑2‑hydroxyglutarate. Experiments show that the biosensor can achieve high sensitivity, specificity and accuracy detection of D‑2‑hydroxyglutarate, which provides a new method and approach for the detection of D‑2‑hydroxyglutarate, and also provides a basis for D‑2‑hydroxyglutarate detection. The diagnosis and treatment of 2-hydroxyglutarate-related diseases and the development of targeted drugs provide new means.

Description

technical field [0001] The present invention relates to a transcriptional regulatory factor and its application, in particular to a transcriptional regulatory factor specifically responsive to D-2-hydroxyglutarate and its application in the biological detection of D-2-hydroxyglutaric acid, belonging to field of biological detection. Background technique [0002] D-2-Hydroxyglutaric Acid Is Considered an Abnormal Metabolite Associated with D-2-Hydroxyglutaric Acidemia of Neurometabolic Disorder [1] . In glioma, acute leukemia, chondroma, cholangiocarcinoma and other tumor cells, the mutation of isocitrate dehydrogenase can also cause the accumulation of D-2-hydroxyglutarate [2] . In addition, D-2-hydroxyglutarate is also an important intermediate metabolite in L-serine synthesis, lysine catabolism, and 4-hydroxybutyrate catabolism [3-5] , can be catabolized to 2-ketoglutarate by D-2-hydroxyglutarate dehydrogenase, however, the regulatory mechanism of D-2-hydroxyglutarate ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C12N15/31G01N21/76
CPCC07K14/195G01N21/763
Inventor 高超肖丹张文马翠卿许平
Owner SHANDONG UNIV
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