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Alpha-ketoglutaric acid optical probe and preparation method and application thereof

A technology of ketoglutaric acid and optical probes, applied in screening compounds or drugs, optical probes for detecting α-ketoglutarate, quantifying α-ketoglutarate, detection, the above-mentioned detection probes In the field of preparation, it can solve problems such as cumbersome operation steps, inability to use α-KG detection, and long time for sample processing

Active Publication Date: 2019-05-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] The concentration of α-KG in mammalian cells is about 0.2mM (Fan J., Molecular Systems Biology, 2013,9:712), and the traditional method for measuring the concentration of α-KG mainly includes chromatography-mass spectrometry analysis (Rocchiccioli F. , Biomed Mass Spectrom, 1984,11:24), isotope ratio analysis method (Bennett B.D., Nature Protocol, 2008,3:1299), nuclear magnetic resonance analysis method (Teng R., NMR in Biomedicine, 2009,22:292), although The above method can achieve more accurate measurement, but the operation steps are cumbersome, the sample processing time is long, and the sample cannot be used for high-throughput detection. In addition, it cannot be used for the detection of α-KG at the level of living cells, subcellular cells, and living animals.
The above shortcomings limit their application in clinical disease diagnosis and drug precursor research, and to a certain extent restrict the development of α-KG related research fields

Method used

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  • Alpha-ketoglutaric acid optical probe and preparation method and application thereof
  • Alpha-ketoglutaric acid optical probe and preparation method and application thereof
  • Alpha-ketoglutaric acid optical probe and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0258] pRSETb-Glnk1 plasmid construction

[0259] The Glnk1 gene was obtained through biosynthesis, and the gene was amplified by PCR. The PCR product of Glnk1 was recovered after gel electrophoresis, and digested with BamHI and HindIII; at the same time, corresponding double digestion was performed on the pRSETb vector. After ligation with T4 DNA ligase, the ligation product was transformed into Mach1, and the transformed Mach1 was spread on LB plates (ampicillin 100 μg / mL) and cultured at 37°C overnight. The growing Mach1 transformants were subjected to plasmid extraction and PCR identification. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

Embodiment 2

[0261] Plasmid construction and detection of different insertion sites of pRSETb-Glnk1-cpYFP, ​​pRSETb-Glnk1-cpmVenus, pRSETb-Glnk1-cpBFP, pRSETb-Glnk1-cpmApple optical probe

[0262] In this example, we used pRSETb-Glnk1 as the base plasmid to select 48 / 49, 48 / 50, 48 / 51, 48 / 52, 48 / 53, 48 / 54, 49 / 50, 49 / 51 according to the Glnk1 crystal structure , 49 / 52, 49 / 53, 49 / 54, 50 / 51, 50 / 52, 50 / 53, 50 / 54, 51 / 52, 51 / 53, 51 / 54, 52 / 53, 52 / 54, 53 The / 54 site was inserted into cpYFP, ​​cpmVenus, cpBFP, cpmApple.

[0263] The DNA fragments of cpYFP, ​​cpmVenus, cpBPF, cpmApple were generated by PCR, and the DNA fragments were inactivated by adding phosphorus at the 5' end, and the pRSETb-Glnk1 linearized vector containing different breakage sites was amplified by reverse PCR , Ligate the linearized pRSETb-Glnk1 and the phosphorylated cpYFP, ​​cpmVenus, cpBPF, cpmApple fragments at the 5' end under the action of PEG 4000 and T4 DNA ligase to generate recombinant plasmids, transform the ligat...

Embodiment 3

[0268] Plasmid construction and detection of pRSETb-Glnk1-cpYFP tandem, pRSETb-Glnk1-cpmVenus tandem, pRSETb-Glnk1-cpBFP tandem, pRSETb-Glnk1-cpmApple tandem optical probe insertion sites

[0269] In this example, we used the plasmids of pRSETb-Glnk1-cpYFP, ​​pRSETb-Glnk1-cpmVenus, pRSETb-Glnk1-cpBFP, and pRSETb-Glnk1-cpmApple fluorescent protein in Example 2 as the basic plasmids, and then connected two plasmids A Glnk1 protein truncation mutant that deletes the T-loop region.

[0270] Using the pRSET-Glnk1 plasmid as the base plasmid, carry out truncation mutations (SEQ ID NO: 11 and SEQ ID NO: 7) on Glnk1 by reverse PCR and self-circularization ligation reaction. After the mutation is completed, two truncation mutations are generated by PCR After the Glnk1 DNA fragment. At the same time, pRSETb-Glnk1-cpYFP, ​​pRSETb-Glnk1-cpmVenus, pRSETb-Glnk1-cpBFP, pRSETb-Glnk1-cpmApple linearized vectors containing different insertion sites were generated by inverse PCR amplification. ...

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Abstract

The invention provides an optical probe, which comprises a) a response polypeptide, and b) an optically active polypeptide, wherein the optically active polypeptide is inserted into the response polypeptide. The invention provides a nucleic acid sequence encoding the optical probe according to any embodiment of the invention or a complementary sequence thereof, provides an expression vector comprising the nucleic acid sequence operably linked to an expression control sequence or the complementary sequence thereof, provides cells comprising the expression vectors, and provides a method of preparing the optical probe. The method of preparing the optical probe comprises the following steps of: providing the cell comprising a vector expressing the optical probe, culturing the cell under conditions of expression of the cell, and isolating the optical probe. The invention provides the optical probe and the application of the optical probe prepared by the method disclosed by the invention inthe detection of alpha-ketoglutaric acid. The invention also provides a kit, which comprises the optical probe of the invention or the optical probe prepared by the method of the invention.

Description

technical field [0001] The present invention relates to detection probes for alpha-ketoglutarate, more particularly to optical probes for detection of alpha-ketoglutarate. The present invention also relates to the preparation method of the detection probe and its application in detection, quantification of α-ketoglutarate, and screening of compounds or medicines. The present invention also relates to a kit comprising the above-mentioned detection probe. Background technique [0002] α-Ketoglutarate (α-KG) is an important intermediate substance in the tricarboxylic acid cycle. It is an important node connecting energy metabolism, glutamine decomposition and lipid synthesis, and cell proliferation, differentiation, aging and death and other life activities are closely related. α-KG plays an important role in the regulation of epigenetics (Carey B.W., Nature, 2015, 518:413; Hwang I.Y., Cell Metab, 2016, 24:494; TeSlaa T., Cell Metab, 2016, 24:485 ), it can also promote prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N21/64
Inventor 杨弋赵玉政魏玉凤王傲雪邹叶君
Owner EAST CHINA UNIV OF SCI & TECH
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