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Proline optical probe and preparing method and application thereof

An optical probe, proline technology, applied in the field of optical probes, can solve the problems of time-consuming, cell fragmentation, etc.

Active Publication Date: 2019-04-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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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  • Proline optical probe and preparing method and application thereof
  • Proline optical probe and preparing method and application thereof
  • Proline optical probe and preparing method and application thereof

Examples

Experimental program
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Effect test

Embodiment approach

[0071] 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 the group consisting of F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and The probes formed when Y275F is mutated are shown in SEQ ID NO: 16-24.

[0072] The optical probe provided by the present invention comprises any one of the amino acid sequences of SEQ ID NO: 10-24 or variants thereof. In one embodiment, the optical probe provided by the invention comprises 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of any of the amino acid sequences of SEQ ID NO: 10-24 %, 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: 10-24. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO:23...

Embodiment 1

[0163] Example 1: Proline-binding protein particles

[0164] The proline-binding protein (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

[0165] Example 2: Expression and detection of cpYFP optical probes at different fusion sites

[0166]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 / 329...

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Abstract

The invention relates to a proline optical probe and a preparing method and application thereof. On one hand, the optical probe comprises a proline sensitive polypeptide or a functional variant thereof and an optical activity polypeptide or a functional variant thereof, wherein the optical activity polypeptide or the functional variant thereof is located in a sequence of the proline sensitive polypeptide or the functional variant thereof. The invention further relates to the preparing method of the probe and application of the probe in detecting proline.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to a proline optical probe and its preparation method and application. Background technique [0002] As one of the 20 natural amino acids, proline is widely distributed in various animals and plants. In plants, proline accumulation is a very common and important phenomenon. When plants are harmed by the external environment, the content of proline in plants will increase dramatically. The mechanism of this phenomenon and its physiological role in plants are still not clear. In mammalian cells, the physiological role of proline as a non-essential amino acid was not widely recognized at the beginning. maintenance plays an important role. However, in recent years, with the discovery of the regulatory role of proline metabolic pathways on intracellular ROS, people have gradually refocused their attention on the study of proline and its metabolic pathways in mammalian cells. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C07K19/00C12N15/62G01N21/64
CPCG01N21/6428C07K14/195C07K2319/60G01N2021/6439
Inventor 杨弋赵玉政李写徐磊王巧慧
Owner EAST CHINA UNIV OF SCI & TECH
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