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Pyruvate optical probe as well as preparation method and application thereof

An optical probe, pyruvate technology, applied in the field of optical probes, can solve the problem of in situ, real-time, dynamic, high-throughput and high-space-time resolution detection, cell fragmentation, separation, extraction and purification, not suitable for living cell research, etc. problems, to achieve the effect of large dynamic changes in fluorescence, easy maturation, and elimination of sample processing steps

Pending Publication Date: 2021-09-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are not suitable for living cell research, and there are many defects: time-consuming sample processing is required, such as cell disruption, separation, extraction and purification, etc.; in situ, real-time, dynamic, high Detection of throughput and high spatiotemporal resolution

Method used

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  • Pyruvate optical probe as well as preparation method and application thereof
  • Pyruvate optical probe as well as preparation method and application thereof
  • Pyruvate optical probe as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment approach

[0062]In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention can be selected from 117 / 121, 141 / 143, 191 / 192, 191 / 193, 191 / 194, 191 of PdhR (96-254) One or more sites of / 195, 192 / 193, 192 / 194, 192 / 195, 193 / 194, 193 / 195, 194 / 195, 210 / 214 are inserted with cpYFP and have one or more selected from the following Mutant probes: Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q, E193L, M194D, M194V, M194H, M194W, M194V and S190E and R191N and R192D (M194V / S190E / R191N / R190D and M1902D), R191Y and R192T (M194V / S190D / R191Y / R192T), S190P and R191H and R192P (S190P / R191H / R192P), S190R and R191S and R192P (S190R / R191S / R192P), S190L and R191V / R190L) (S190L) (S190L) R191Q and R192E (S190T / R191Q / R192E) or R191S and R192T (R191S / R192T). Preferably, the optical probe is a probe with cpYFP inserted at the 191 / 193 site of PdhR(96-254) and having Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q or E193L mutations, or a probe of PdhR(96-254) -254) with cpYFP inserted at...

Embodiment 1

[0121] Example 1: Pyruvate-binding protein particles

[0122] The PdhR (96-254) gene in the Escherichia coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII, and the pCDFDuet1 vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was transformed into MachI, and the transformed MachI was spread on LB plates (streptomycin sulfate 50ug / mL) and incubated overnight at 37°C. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

Embodiment 2

[0123] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0124] In this example, based on pCDFDuet-PdhR(96-254), the following site was selected to insert cpYFP according to the crystal structure of pyruvate-binding protein, and the corresponding pCDFDuet-PdhR(96-254)-cpYFP plasmid was obtained: 117 / 118, 117 / 119, 117 / 120, 117 / 121, 118 / 119, 118 / 120, 118 / 121, 119 / 120, 119 / 121, 120 / 121, 140 / 141, 140 / 142, 140 / 143, 141 / 142, 141 / 143, 142 / 143, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 161 / 162, 161 / 163, 161 / 164, 162 / 163, 162 / 164, 163 / 164, 174 / 175, 174 / 176, 175 / 176, 191 / 192, 191 / 193, 191 / 194, 191 / 195, 192 / 193, 192 / 194, 192 / 195, 193 / 194, 193 / 195, 194 / 195, 210 / 211, 210 / 212, 210 / 213, 210 / 214, 211 / 212, 211 / 213, 211 / 214, 212 / 213, 212 / 214 or 213 / 214. Exemplary sequences are shown in Table 1.

[0125] Table 1. Sequences of Optical Probes

[0126] sequence insertion site SEQ ID NO:6 117 / 121 SEQ ID NO:7 141 / 143 SEQ ID NO:8 1...

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Abstract

The invention relates to a pyruvate optical probe as well as a preparation method and application thereof. In one aspect, the invention relates to an optical probe. The optical probe comprises a pyruvate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the pyruvate-sensitive polypeptide. The invention also relates to a preparation method of the probe and application of the probe in pyruvate detection.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to a pyruvate optical probe and its preparation method and application. Background technique [0002] Pyruvate is an organic acid with two functional groups, carbonyl and carboxyl, and is also the simplest α-keto acid, which widely exists in various animals and plants. Pyruvate is inseparable from energy metabolism. During glycolysis, one molecule of glucose or glycogen and two molecules of ATP are consumed to produce two molecules of NADH, four molecules of ATP and two molecules of pyruvate. In the cytoplasm, pyruvate can be reduced to provide energy, and it can also be transported to the mitochondria to become the main fuel of the tricarboxylic acid cycle. Pyruvate enters the mitochondria under aerobic conditions, and is oxidatively decarboxylated by the pyruvate dehydrogenase complex to produce NADH, CO2, and acetyl-CoA. Acetyl-CoA enters the tricarboxylic acid cycle and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C09K11/06G01N33/68G01N33/58
CPCC07K14/245C09K11/06G01N33/68G01N33/582C07K2319/60C09K2211/10G01N33/58C12N15/62C07K19/00C12N15/09
Inventor 杨弋赵玉政李写张则一张秀泽陈念黄立
Owner EAST CHINA UNIV OF SCI & TECH
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