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Branched chain amino acid fluorescent probe and application thereof

A branched-chain amino acid, fluorescent probe technology, applied in the field of fluorescent proteins, can solve problems such as failure to meet monitoring requirements

Pending Publication Date: 2018-08-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method still cannot meet the above monitoring requirements

Method used

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  • Branched chain amino acid fluorescent probe and application thereof
  • Branched chain amino acid fluorescent probe and application thereof
  • Branched chain amino acid fluorescent probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] Construction of pRSETb-LivJ and pRSETb-LivK plasmids

[0185] The LivJ and LivK genes in E. coli genes were amplified by PCR. The PCR product of LivJ was recovered after gel electrophoresis and digested with BamHI and HindIII; the PCR product of LivK was recovered after gel electrophoresis and digested with EcoRI and HindIII. Carry out the corresponding double digestion of the vector. After ligation with T4 DNA ligase, the ligation product was transformed into MachI, and the transformed MachI was spread on LB plates (ampicillin 100ug / mL), and cultured at 37°C overnight. 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.

[0186] Construction and detection of pRSETb-LivJ-cpYFP, ​​pRSETb-LivK-cpYFP fluorescent probes with different insertion sites

[0187] In this example, we used pRSETb-LivJ as the base plasmid to select 11...

Embodiment 2

[0194] Design, Construction and Detection of Branched-chain Amino Acid Fluorescent Probes Based on cpBFP

[0195] According to the method in Example 1, cpYFP was replaced by blue fluorescent protein cpBFP, and fused into branched-chain amino acid binding protein and leucine-binding protein to construct branched-chain amino acid blue fluorescent protein fluorescent probe and leucine blue fluorescence respectively Protein fluorescent probes. The result is as image 3 As shown, the fluorescence detection results showed that 326 / 327 and 327 / 328 of the branched-chain amino acid fluorescent probes responded more than 3 times to branched-chain amino acids; 328 / 329 of the leucine fluorescent probes responded more than 1.5 times to leucine , 329 / 330.

Embodiment 3

[0197] Design, construction and detection of branched-chain amino acid fluorescent probes based on cpmApple

[0198] According to the method in Example 1, cpYFP was replaced by apple red fluorescent protein cpmApple, and fused into branched-chain amino acid binding protein and leucine-binding protein to construct branched-chain amino acid red fluorescent protein fluorescent probe and leucine red fluorescent protein fluorescent probe respectively. probe, the result is as Figure 4 As shown, the fluorescent detection results showed that 326 / 327, 327 / 328 of the branched-chain amino acid fluorescent probes responded more than 3 times to branched-chain amino acids; 328 / 329, 329 of the leucine fluorescent probes responded more than 1.5 times to leucine. / 330.

[0199] The results of Examples 1-3 show that the linker region 325-329 of the branched-chain amino acid binding protein is suitable for fusion of cpFP fluorescent protein to obtain a fusion fluorescent protein branched-chain...

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Abstract

The invention provides a fluorescent probe, which contains a, response polypeptide and b, optical active peptide, wherein the optical active peptide is inserted into the response polypeptide. The invention also provides a nucleotide sequence or a complementary sequence for coding the fluorescent probe of any embodiment. The invention also provides a nucleotide sequence or complementary sequence expression vector in operational connection with an expression control sequence. The invention also provides a cell containing the expression vector. The invention also provides a method for preparing the fluorescent probe. The method comprises the steps of providing the cell containing the vector for expressing the fluorescent probe; culturing the cell under the cell expression condition; separating the fluorescent probe. The invention also provides the fluorescent probe or application of the fluorescent probe prepared by the method to branched chain amino acid detection. In one embodiment, thebranched chain amino acid is selected from aminocarproic acid, isoleucine and valine. The detection can be performed in vivo, in vitro, subcellular fraction, or in-situ level. The invention also provides a kit, which contains the fluorescent probe or the fluorescent probe prepared by the method.

Description

technical field [0001] The present invention relates to fluorescent protein, more specifically to a fluorescent probe for detecting branched-chain amino acids, a preparation method and application thereof. Background technique [0002] Branched-chain amino acids (BCAAs) include leucine, isoleucine, and valine, which serve as potent nutrient signaling molecules for regulating the rate of protein synthesis. These three branched-chain amino acids belong to the nine essential amino acids for humans, accounting for 35% of the essential amino acids in muscle protein and 40% of the amino acids required by mammals. They are important in immunity, brain function, and other physiological aspects (P.C. Calder, 2006; B. Skeie et al., 1990). All three BCAAs are required for lymphocyte growth and proliferation (K. Tajiri and Y. Shimizu, 2013). Branched-chain amino acids may affect brain protein synthesis and energy production, and participate in the synthesis of different neurotransmitt...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N21/64
CPCG01N21/6428C07K14/245C07K2319/60C07K14/195C07K2319/20
Inventor 杨弋赵玉政李写邹叶君张长程
Owner EAST CHINA UNIV OF SCI & TECH
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