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Method for detecting interaction of proteins

A detection method and protein technology, which can be used in measurement devices, biological tests, material inspection products, etc., can solve the problems of inability to synchronously detect multiple pairs of protein interactions in parallel, unable to detect protein interactions, and unable to solve multiple pairs of proteins. Prospects for clinical application, good bleaching resistance, and the effect of strong tissue penetration

Inactive Publication Date: 2010-01-06
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
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  • Claims
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Problems solved by technology

However, using the BiFC method to detect protein interactions, green fluorescent protein is often used, which has poor penetration and cannot solve the detection of multiple pairs of protein interactions; and the known red fluorescent protein (mRFP-Q66T) can also be used for BiFC research, but this Fluorescent proteins not only have relatively low brightness, but are also unstable to pH (pKa=7.5). It is difficult to study protein interactions in an acidic environment, cannot work at a physiological temperature of 37°C, cannot simultaneously detect multiple pairs of protein interactions in parallel, and cannot Imaging to detect protein interactions

Method used

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Embodiment 1

[0124] This implementation example uses the BiFC phenomenon of TagRFP to study the interaction between transcription factors bFos and bJun. Based on this purpose, it is necessary to connect bFos to the N-terminal of the TagRFP protein or sequence (hereinafter referred to as TN), and bJun to the C-terminal of the TagRFP protein or sequence (hereinafter referred to as TC), and to quantitatively characterize each fusion construct in the study ( bFos-TN or bJun-TC), the commonly used yellow fluorescent protein mCerulean and the commonly used cyan fluorescent protein mCitrine are respectively connected to the above fusion constructs as fluorescent markers.

[0125] refer to Figure 8 with Figure 9 , Figure 8 It is a BiFC molecular probe structure based on the TagRFP155 site, which is used to study the BiFC system for the interaction between bJun and bFos. The probe consists of fluorescent protein mCerulean / mCitrine, target protein bJun / bFos / ΔbFos and TagRFP-C segment / N segment ...

Embodiment 2

[0130] Dimerization of BACE enzyme, BACE enzyme and γ-secretase enzymatically digest and hydrolyze amyloid precursor protein (amyloid precursor protein, APP) in turn to produce 4kD β-amyloid peptide (amyloid β-peptide, Aβ). The accumulation of Aβ leads to the occurrence of Alzheimer's disease (AD). Recently, Haass C group used non-denaturing gel electrophoresis and co-immunoprecipitation methods to find that BACE exists in the form of homodimers. Therefore, BACE dimerization serves as a good model for protein interactions in living cells.

[0131] Figure 13 It is a schematic diagram of the principle of the BiFC system based on the TagRFP155 site to study the dimerization of BACE enzyme cells, Figure 14 It is a single-cell image of HeLa cells BACE(FL)-TagRFP155N and BACE(FL)-TagRFP155C cultured at 26°C for 24 hours. The two fragments (TN155 and TC155) at position 155 were cloned and inserted into the BACE enzyme vector to construct BACE(FL)-TN155 and BACE(FL)-TC155, and th...

Embodiment 3

[0138] Research on protein interaction in nematodes. Nematodes grow fast, can be cultured in large quantities, and are prone to mutations. In addition, it has a simple structure and a transparent body, which is easy to track and image. The first to apply BiFC to nematodes was Martin Chalfie, the 2008 Nobel Prize winner in chemistry. He fused the fluorescent fragments of GFP mutants (cleaved at position 157) with antiparallel leucine short peptides (NZ and CZ) respectively. , to study the regulation of gene expression and protein localization by different promoters. In addition, in 2008, Professor Hu used the Venus-based system to study the interaction between Jun and Fos proteins in nematodes. However, there is no report on the application of the red BiFC system to nematodes.

[0139] The fusion fragments C-bJ-TC151, Y-bF-TN151 and Y-ΔbF-TN151 were cloned downstream of the heat shock promoter hsp16-2 to construct pHsp-C-bJ-TC151, pHsp-Y-bF-TN151 and pHsp- YΔbF-TN151. The h...

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Abstract

The invention relates to a detection method which belongs to the field of biomolecules. The method can be used for detecting the interaction among proteins by a dimolecular fluorescent complement technology on the basis of orange red fluorescent protein and infrared fluorescent protein. The invention can be applied to detect the interaction of the proteins by living body imaging and can synchronously detect the interaction of multiple pairs of proteins in parallel.

Description

technical field [0001] The invention relates to a detection method, which is used for detecting the protein function and belongs to the field of biomolecules. Background technique [0002] Protein-protein interaction (hereinafter referred to as "PPI") is a physiological process that plays an important role in maintaining the normal physiological functions of organisms. PPI involves almost all important life activities, including DNA replication and transcription, protein Synthesis and secretion, signal transduction and metabolism, etc. For example, the signal pathway that precisely regulates and realizes the physiological functions of cells realizes signal transduction and amplification through the interaction between signal molecules and corresponding receptors, so as to realize the stress response of cells to external environmental stimuli. In addition, a variety of protein molecules with important functions function in the form of dimers, trimers or oligomers, and the in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64G01N21/84
Inventor 骆清铭张智红储军秦岭松
Owner HUAZHONG UNIV OF SCI & TECH
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