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Amino acid optical probe, and preparation method and application thereof

An optical probe and amino acid technology, applied in the field of optical probes, can solve the problems of high instrument requirements, inability to live cells and subcellular organelles in situ, real-time, dynamic, high-throughput and high spatio-temporal resolution detection, time-consuming and other problems

Active Publication Date: 2019-07-12
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods either require complicated sample extraction and refinement, or have high requirements for instruments, and the equipment is expensive
[0014] In summary, these amino acid detection methods in the prior art are not suitable for living cell research, and there are many defects: they need to go through time-consuming sample processing processes, such as cell disruption, separation, extraction and purification, etc.; they cannot be used in living cells and subcellular organelles Perform in situ, real-time, dynamic, high-throughput and high spatio-temporal resolution detection

Method used

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  • Amino acid optical probe, and preparation method and application thereof
  • Amino acid optical probe, and preparation method and application thereof
  • Amino acid optical probe, and preparation method and application thereof

Examples

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Embodiment approach

[0096] In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention can be fused with cpYFP at the 121 / 122 site of Atu2422 and has a protein selected from F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, The probes formed when G227S and Y275F are mutated are shown in SEQ ID NO: 16-25.

[0097] The optical probe provided by the invention comprises any one of the amino acid sequences of SEQ ID NO: 10-25 or variants thereof. In one embodiment, the optical probe provided by the present invention comprises 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of any of the amino acid sequences of SEQ ID NO: 10-25 %, 95%, 99% sequence identity. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any of the amino acid sequences of SEQ ID NO: 10-25. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO:24.

[0098] In tw...

Embodiment 1

[0187] Example 1: Amino Acid Binding Protein Particles

[0188]The AABP (here Atu2422) gene in the Agrobacterium agrobacterium gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and EcoRI, and the pRSETb vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was used to transform MachI, and the transformed MachI was spread on LB plate (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.

Embodiment 2

[0189] Example 2: Expression and detection of cpYFP optical probes at different fusion sites

[0190] In this example, the following sites were selected for fusion with cpYFP based on pRSETb-Atu2422 to obtain the corresponding pRSETb-Atu2422-cpYFP plasmid: 117 / 118, 117 / 119, 117 / 120, 117 / 121, 118 / 119, 118 / 120, 118 / 121, 119 / 120, 119 / 121, 120 / 121, 120 / 122, 120 / 123, 121 / 122, 121 / 123, 122 / 123, 249 / 250, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 249 / 259, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 250 / 259, 251 / 252, 251 / 253, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 251 / 259, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 252 / 259, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 253 / 259, 254 / 255, 254 / 256, 254 / 257, 254 / 258, 254 / 259, 255 / 256, 255 / 257, 255 / 258, 255 / 259, 256 / 257, 256 / 258, 256 / 259, 257 / 258, 257 / 259, 258 / 259, 323 / 330, 324 / 330, 325 / 330, 326 / 327, 326 / 328, 326 / 329, 326 / 330, 327 / 328, 327 / 329, 327 / 330, 328 / 32...

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Abstract

The invention relates to an amino acid optical probe, and a preparation method and an application thereof. On one hand, the invention relates to an optical probe comprising an amino acid sensitive polypeptide or a functional variant thereof and an optical active polypeptide or a functional variant thereof, wherein the optical active polypeptide or the functional variant thereof is located in a sequence of the amino acid sensitive polypeptide or the functional variant thereof. The invention also relates to the preparation method of the probe and an application of the probe in detection of aminoacids. The amino acid optical probe provided by the invention has relatively small molecular weight, has the advantages of easy maturation, large fluorescence dynamic change and good specificity, canbe expressed in different subcellular organelles of cells, and can detect amino acids inside and outside cells in high flux and quantitatively.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to an amino acid optical probe and its preparation method and application. Background technique [0002] Amino acid is a compound in which the hydrogen atom on the carbon atom of a carboxylic acid is replaced by an amino group, and is an organic compound containing a basic amino group and an acidic carboxyl group. The amino acids obtained after protein hydrolysis are all α-amino acids with amino groups connected to α-carbons, and there are only more than 20 kinds of them. They are the basic units of proteins. Amino acids play a role in the human body through metabolism, such as synthesizing tissue proteins; synthesizing ammonia-containing substances such as acids, hormones, antibodies, and creatine; converting them into carbohydrates and fats; or oxidizing them into carbon dioxide, water, and urea to generate energy. [0003] Alanine, as one of the 20 natural amino acids, is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N21/64
CPCC07K14/195G01N21/6428C07K2319/60G01N2021/6439
Inventor 杨弋赵玉政李写徐磊黄立
Owner EAST CHINA UNIV OF SCI & TECH
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