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Proline optical probe and preparation 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., and achieve the effects of easy maturation, good specificity, and large dynamic changes in fluorescence

Active Publication Date: 2022-07-29
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 preparation method and application thereof
  • Proline optical probe and preparation method and application thereof
  • Proline optical probe and preparation method and application thereof

Examples

Experimental program
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Embodiment approach

[0071] In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be Atu2422 fused to cpYFP at site 121 / 122 and having a compound selected from the group consisting of F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Probes formed upon mutation of Y275F are shown in SEQ ID NOs: 16-24.

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

Embodiment 1

[0163] Example 1: Proline-Binding Protein Particles

[0164] The proline-binding protein (here Atu2422) gene in Agrobacterium tumefaciens was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and EcoRI, while the pRSETb vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, MachI was transformed with the product, and the transformed MachI was spread on an LB plate (100 ug / mL of ampicillin) and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmids were sequenced correctly, the subsequent plasmid construction was 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 to fuse cpYFP based on pRSETb-Atu2422 to obtain the corresponding pRSETb-Atu2422-cpYFP plasmids: 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, 328 / 3...

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Abstract

The present invention relates to a proline optical probe and its preparation method and application. In one aspect, the present invention relates to an optical probe comprising a proline-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located in the proline-sensitive polypeptide or its functional variant within the sequence of functional variants. The present invention also relates to the preparation method of the above probe and its application 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 a preparation method and application thereof. Background technique [0002] As one of 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 proline content in plants will increase dramatically. The mechanism of this phenomenon and its physiological role in plants have not been clearly studied. 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 metabolism pathway on intracellular ROS, people have gradually refocused on the study of proline and its metabolic pathway in mammalian cells. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(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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