Preparation and application of real-time living cell structural mechanics fluorescent detection probe real-time living cell structural mechanics detection method and application of the method

A technology of fluorescent probes and structural mechanics, which is applied in the field of fluorescent protein pair probe construction, can solve the problems that cannot meet the needs of real-time monitoring and detection of living cells, achieve high-throughput detection of mechanical changes in cell structures, and overcome large structures. , the effect of simple cell influence

Inactive Publication Date: 2015-05-20
郭军
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, this detection technology has certain limitations and cannot meet the needs of real-time monitoring of live cel

Method used

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  • Preparation and application of real-time living cell structural mechanics fluorescent detection probe real-time living cell structural mechanics detection method and application of the method
  • Preparation and application of real-time living cell structural mechanics fluorescent detection probe real-time living cell structural mechanics detection method and application of the method
  • Preparation and application of real-time living cell structural mechanics fluorescent detection probe real-time living cell structural mechanics detection method and application of the method

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Embodiment Construction

[0038] 1. Commercially obtained gene sequences of fluorescent protein pairs and ORF sequence clones of major backbone protein genes, as shown in the following table:

[0039]

[0040] 2. Using gene cloning technology, clone the following framework protein ORFs into eukaryotic expression vectors. Taking the pEGFP-C1 plasmid as the carrier, eBFP and eGFP as the probe fluorescent pair as an example, construct various backbone protein-related fluorescent probes, the main restriction enzymes used are: AgeI, ApaI, BamHI, KpnI, NheI, SpeI , XhoI. The probe DNA clone structure is as follows:

[0041] β-actin

[0042] pCMVie-gctagc(NheI)-βactin - cca accggt(AgeI)ggtggcggaggg-eBFPggtacc(KpnI)gga cca ggtggcggt-eGFP-ggaggtggcggtactagt (SpeI) cca -βactin-ggggccc(ApaI)-

[0043] vimentin

[0044] pCMVie-gctagc(NheI)-vimentin- cca accggt(AgeI)ggtggcggaggg-eBFPggtacc(KpnI)gga cca ggtggcggt-eGFP-ggaggtggcggtactagt (SpeI) cca -vimentin-gggccc(ApaI)-

[0045] laminB1

[0046...

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Abstract

The invention relates to a real-time living cell structural mechanics detection method and application of the method, and belongs to the technical field of bioengineering. The method disclosed by the invention comprises the following steps: selecting fluorescent protein pairs capable of being subjected to fluorescence resonance energy transfer, connecting the protein pairs to angle positions by using short peptide chains, and constructing a mechanical detection probe by utilizing the fluorescence resonance energy transfer caused by the angle change of the protein pairs; and integrating the probe into the cell skeleton proteins by adopting a molecular cloning method and by connecting the probe with a skeleton protein. The fluorescence energy transfer is detected, so that the mechanical change of cell skeleton transfer can be estimated. The lengths of short peptide chains are changed, the initial angles of the protein pairs can be regulated, the sensitivity of the fluorescence probe is improved, and research on a mechanism related to the cell structure mechanics is effectively realized. The improved probe has the detection characteristics of slight influence on a culture medium, rapidness, micro amount, sensitivity, accuracy and high flux and can be used for constructing a cell platform for screening drugs related to the cell structure mechanics.

Description

technical field [0001] The invention relates to the construction and application of "fluorescence protein pair" probes capable of fluorescence resonance energy transfer. The probe is suitable for the detection of indicators of microstructural mechanical changes in living cells, especially for the real-time quantitative or semi-quantitative determination of mechanical changes in vitro and in vivo living cells and tissues induced by dynamic molecules. Background technique [0002] Muscle cells are the earliest cells related to mechanics. In fact, all cells can generate force and feel force, such as nerve, endothelial, bone and stem cells. All life processes involve the action and regulation of mechanics, such as differentiation, retrodifferentiation, division, polarization, movement, invasion and metastasis, and apoptosis. At the same time, the mechanical activities of different tissues and organs exist in the form of "steady state". Once this mechanical homeostasis is brok...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/06
Inventor 不公告发明人
Owner 郭军
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