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A fluorescent bioprobe for detecting force transmission of paxillin protein in living cells

A fluorescent biological probe, living cell technology, applied in the field of cell biology and molecular biology

Active Publication Date: 2020-08-14
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no effective method for detecting force transmission on Paxillin protein in living cells

Method used

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  • A fluorescent bioprobe for detecting force transmission of paxillin protein in living cells
  • A fluorescent bioprobe for detecting force transmission of paxillin protein in living cells

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Embodiment Construction

[0022] The specific implementation manners of the present invention will be further described below in conjunction with the accompanying drawings and technical solutions.

[0023] After the probe is transfected into living cells with a transfection reagent, the cells can express the remodeled fusion fluorescent protein, and the Ld12(1-1) and Lim1234(1-5) sequences at both ends are respectively associated with the cytoskeleton protein and signal The protein is connected, so that it is automatically integrated into the position of the original Paxillin protein on the local focal adhesion. When an external force is applied, when the force is transmitted to the Paxillin protein site replaced by the probe in the local focal adhesion, the Linker(1-3) contained in the probe shrinks or stretches under the force deformation, causing the fluorescent protein to react to the ECFP(1 -2) and YPet(1-4) spacing changes, so that the FRET efficiency changes. Give the transfected cells excitati...

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Abstract

The invention belongs to the technical fields of cytobiology and molecular biology and particuallry relates to a fluorescent biological probe for detecting the force transmission of Paxillin protein in a living cell. The fluorescent biological probe is a biological probe based on an FRET technique and is capable of dynamically detecting the change of the force transmission of the Paxillin proteinin the living cell. The probe comprises five parts including an FRET fluorescent protein pair ECFP and YPet, an elastic sequence Linker, an Ld12 sequence and a Lim1234 sequence. After the probe and arecombinant plasmid of pcDNA3.1(+) are transfected into the living cell, a fluorescent protein reconstitution fusion probe can be spontaneously expressed in the cell and can be automatically integrated into a local focal adhesion region in which the Paxillin protein is located, the change of force transmission of the Paxillin protein in the living cell can be qualitatively detected, and the probehas no toxic and side effect to the cell.

Description

technical field [0001] The invention belongs to the technical field of cell biology and molecular biology, and specifically relates to a fluorescent bioprobe for detecting the force transfer of Paxillin protein in living cells, which is based on fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET) technology and conventional bioengineering Biological probes designed and prepared by cloning technology. Its probe structure can express itself in living cells, and qualitatively reflect the changes in Paxillin protein force transmission in living cells through changes in fluorescent signals. Background technique [0002] Abnormal responses of cells to external forces are associated with certain diseases. External forces act on the cell membrane of living cells, and then physically transmit within the cell through structures such as cytoskeletal proteins connected to the cell membrane. Paxillin protein is an important structural protein on local ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00G01N33/68
CPCG01N33/6875
Inventor 刘波邓莎
Owner DALIAN UNIV OF TECH
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