Real-time fluorescence display method for interaction of proteins in living cells
By fusing fluorescent signal peptides on proteins and observing their localization changes using fluorescence microscopes, the protein interactions are directly judged, and the problems of false positives and detection complexity in the prior art are solved, and high-accuracy protein interactions are achieved in living cells Effect detection.
Patent Information
- Application Number
- CN202411324087.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-27
AI Technical Summary
The existing protein-protein interaction detection methods have high false positives and insufficient accuracy. Especially in the detection of living cells, the existing methods rely on indirect judgment and complex technical means, resulting in cumbersome and exhaustive detection process. Duration.
By establishing a protein fluorescence display sequence, quickly cloning plasmids α, β and γ, fusing specific fluorescent signal peptides to the protein, using fluorescence microscope to observe the localization changes of proteins in cells, directly judge the interaction between proteins, avoid false positives, and improve the Detection accuracy.
This method is simple, fast and reliable, which significantly improves the accuracy of judging protein interactions. It is suitable for all eukaryotic cells. It can observe protein interactions in real time in living cells, reduce equipment requirements, avoid false positives, and improve detection accuracy.
Smart Images
Figure CN119198658A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of protein interaction detection methods, and in particular to a real-time fluorescence display method for protein interaction in living cells. Background Art
[0002] Studying protein-protein interactions can not only reveal the functions of proteins at the molecular level, but is also crucial for studying the laws of life activities such as growth, development, differentiation and apoptosis, and provides an important theoretical basis for exploring the mechanisms of major diseases, disease treatment, disease prevention and new drug development.
[0003] Existing methods for protein-protein interaction are mainly divided into two types: in vitro cell detection methods and in vivo cell detection methods. Among them, in vitro cell detection methods include: immunoprecipitation, tandem affinity purification, proximity labeling, fusion protein precipitation technology, etc. These methods mainly rely on antibodies and mass spectrometry technology, which will produce a large number of false positive results, and the entire detection process is cumbersome and time-consuming. In vivo protein detection methods include: yeast two-hybrid, immunofluorescence co-localization, protein complementation test, fluorescence resonance energy transfer method, etc. Although these methods can detect protein interactions in living cells, these methods are based on the distance between protein molecules to indirectly judge whether proteins interact, and are interfered by protein expression, with high false positives and limitations.
[0004] Due to the above reasons, existing methods for detecting protein-protein interactions have a high false positive rate and are insufficient in accuracy. Summary of the invention
[0005] The purpose of the present invention is to provide a real-time fluorescence display method for protein interactions in living cells, which can avoid false positives when studying protein interactions and improve detection accuracy.
[0006] To achieve the above object, the present invention provides a method for real-time fluorescence display of protein interactions in living cells, comprising:
[0007] Establish protein fluorescence display sequence rapid cloning plasmid α, protein fluorescence display rapid cloning plasmid β and protein fluorescence display rapid cloning plasmid γ;
[0008] The cDNA sequence of protein A was cloned into protein fluorescence display sequence rapid cloning plasmid α and protein fluorescence display sequence rapid cloning plasmid β, and the cDNA sequence of protein B was cloned into protein fluorescence display sequence rapid cloning plasmid γ;
[0009] The protein fluorescence display sequence rapid cloning plasmids α, β, and γ are transduced separately, the protein fluorescence display sequence rapid cloning plasmid α is combined with γ to co-transfect the tool cells, and the protein fluorescence display sequence rapid cloning plasmid β is combined with γ to co-transfect the tool cells;
[0010] After transfection, the changes in the fluorescence signals produced by protein A and protein B in the five groups of cells were observed to determine whether there was a protein-protein interaction between protein A and protein B.
[0011] Wherein, observing the changes in the fluorescence signals generated by protein A and protein B in the five groups of cells after transfection to determine whether there is a protein-protein interaction between protein A and protein B specifically includes:
[0012] When protein A migrates to a specific organelle or structural region, the organelle or structural region shows fluorescence of protein A or the fluorescence intensity is enhanced. At the same time, protein B is also carried into the organelle or structural region, and the fluorescence signal of protein B appears or the fluorescence signal is enhanced at this position, indicating that there is an interaction between protein A and protein B;
[0013] When protein A migrates to a specific organelle or structural region, the fluorescence of protein A appears in the organelle or structural region or the fluorescence intensity is significantly enhanced. At the same time, the position and intensity of the fluorescence signal of protein B do not change, indicating that there is no interaction between protein A and protein B.
[0014] The protein fluorescence display sequence rapid cloning plasmid α expresses a fusion protein with a specific positioning signal peptide and fused with a first fluorescence signal.
[0015] Wherein, the protein fluorescence display rapid cloning plasmid β expresses a fusion protein fused with a first fluorescence signal.
[0016] Wherein, the protein fluorescence display rapid cloning plasmid γ expresses a fusion protein fused with a second fluorescence signal.
[0017] A method for real-time fluorescence display of protein interactions in living cells of the present invention, the protein A that fuses a fluorescent signal peptide (such as green fluorescent protein) is positioned, and then the protein B that fuses another fluorescent signal peptide (such as red fluorescent protein) is detected to see if the positioning changes, thereby identifying whether protein A and protein B have interactions. This method changes the positioning of the protein so that only when there is a real interaction and direct binding between the two proteins can the binding protein be directional displacement with the migrating protein, and the fused fluorescent signal makes it visible in real time in living cells whether the protein has organelle or regional migration, and the phenomenon is obvious, which can significantly improve the accuracy of judging whether there is interaction between proteins. The whole technical method is simple, fast and reliable, avoiding the complexity of the related technology mentioned in the background technology, the false positive caused by indirect judgment and other problems; this method is constructed using signal peptides of different organelles, so it is applicable to all eukaryotic cells; this method is constructed using plasmids, and the plasmids expressing proteins of the same species are the same, so different protein interactions can be detected in different cells of the same species at high throughput, thereby constructing a protein interaction network of eukaryotic organisms; this method is used in living cells, and real-time fluorescence imaging can be performed to observe protein interactions under specific conditions. The method can be observed using a common fluorescence microscope, making the detection method simpler and less demanding on equipment. The invention can avoid false positives when studying interactions between proteins and improve detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.
[0019] Figure 1 The present invention is a flow chart of a method for real-time fluorescence display of protein interactions in living cells.
[0020] Figure 2 It is a schematic diagram of protein migration in a real-time fluorescence display method of protein interaction in living cells of the present invention. DETAILED DESCRIPTION
[0021] See also Figure 1-Figure 2 ,in, Figure 1 is a flow chart of a method for real-time fluorescence display of protein interactions in living cells of the present invention, Figure 2 It is a schematic diagram of protein migration in a real-time fluorescence display method of protein interaction in living cells of the present invention.
[0022] The present invention provides a method for real-time fluorescence display of protein interactions in living cells, comprising:
[0023] S1 established protein fluorescence display sequence rapid cloning plasmid α, protein fluorescence display rapid cloning plasmid β and protein fluorescence display rapid cloning plasmid γ;
[0024] In the embodiment of the present invention, by establishing a universal protein localization sequence, protein fluorescence (such as green fluorescence) displays the sequence for rapid cloning of plasmid α, protein fluorescence (such as green fluorescence) displays the sequence for rapid cloning of plasmid β, and protein fluorescence (such as red fluorescence) displays the sequence for rapid cloning of plasmid γ.
[0025] The protein fluorescence display sequence rapid cloning plasmid α expresses a fusion protein with a specific positioning signal peptide and a first fluorescence signal. The protein fluorescence display rapid cloning plasmid β expresses a fusion protein with a first fluorescence signal. The protein fluorescence display rapid cloning plasmid γ expresses a fusion protein with a second fluorescence signal.
[0026] S2 clones the cDNA sequence of protein A into protein fluorescence display sequence rapid cloning plasmid α and protein fluorescence display sequence rapid cloning plasmid β, and clones the cDNA sequence of protein B into protein fluorescence display sequence rapid cloning plasmid γ;
[0027] In an embodiment of the present invention, the cDNA sequence of protein A is cloned into protein fluorescence display sequence rapid cloning plasmid α and protein fluorescence display sequence rapid cloning plasmid β, and the cDNA sequence of protein B is cloned into protein fluorescence display sequence rapid cloning plasmid γ; protein A expressed by protein fluorescence display sequence rapid cloning plasmid α will carry a specific positioning sequence and a fluorescent signal peptide, protein A expressed by protein fluorescence display sequence rapid cloning plasmid β will only carry one fluorescent signal peptide, and protein B expressed by protein fluorescence display sequence rapid cloning plasmid γ will carry another fluorescent signal peptide.
[0028] S3 transduces protein fluorescence display sequence rapid cloning plasmids α, β, and γ separately, co-transfects the tool cells with protein fluorescence display sequence rapid cloning plasmid α and γ, and co-transfects the tool cells with protein fluorescence display sequence rapid cloning plasmid β and γ;
[0029] After S4 transfection, the changes in the fluorescence signals produced by protein A and protein B in the five groups of cells were observed to determine whether there was a protein-protein interaction between protein A and protein B.
[0030] Specifically include:
[0031] When protein A migrates to a specific organelle or structural region, the organelle or structural region shows fluorescence of protein A or the fluorescence intensity is enhanced. At the same time, protein B is also carried into the organelle or structural region, and the fluorescence signal of protein B appears or the fluorescence signal is enhanced at this position, indicating that there is an interaction between protein A and protein B;
[0032] When protein A migrates to a specific organelle or structural region, the fluorescence of protein A appears in the organelle or structural region or the fluorescence intensity is significantly enhanced. At the same time, the position and intensity of the fluorescence signal of protein B do not change, indicating that there is no interaction between protein A and protein B.
[0033] In the embodiment of the present invention, three protein fluorescence display sequence rapid cloning plasmids of α, β, and γ are constructed, wherein the α plasmid can express a fusion protein with a specific localization signal peptide (such as nuclear entry, mitochondrial entry, or nucleolus, etc.) and a fluorescent signal (such as green fluorescent protein), the β plasmid expresses a fusion protein fused with the same fluorescent signal (such as green fluorescent protein), and the γ plasmid expresses a fusion protein fused with another fluorescent signal (such as red fluorescent protein). The protein A cDNA fragment originally expressed in a certain organelle or a specific region (such as the cytoplasm) is cloned into the α (with a different specific localization signal peptide such as a nuclear entry signal peptide) and β plasmids respectively. Protein B is mainly expressed in a certain organelle or a specific region (such as the cytoplasm) of the cell, and its cDNA is cloned into the plasmid γ. : The α, β, and γ plasmids are divided into 5 groups of 1 (α plasmid), 2 (β plasmid), 3 (plasmid), 4 (α+γ plasmid) and 5 (β+γ plasmid), and are transfected into 293T cells or target cells respectively. After transfection for 48 hours, the localization of the fluorescent protein is observed under a fluorescence microscope. In group 2, green fluorescence was mainly observed in the cytoplasm, in group 3, red fluorescence was mainly observed in the cytoplasm, in group 1, green fluorescence was mainly observed in the nucleus, and in group 5, green and red fluorescence were mainly observed in the cytoplasm. In group 4, two situations may occur: 1. Green fluorescence was observed in the nucleus and red fluorescence was observed in the cytoplasm, which proves that protein A and protein B do not interact with each other; 2. Green fluorescence was observed in the nucleus and red fluorescence was observed in the nucleus at the same time, which proves that protein A and protein B interact with each other.
[0034] The present invention discloses a method for real-time fluorescence display of protein interaction in living cells. Since there are various organelles and structural regions in eukaryotic cells, such as nuclei, mitochondria, endoplasmic reticulum, cell membrane, nucleolus, etc., proteins in these organelles or structural regions in the natural state all have specific positioning sequences. If the specific positioning sequence is artificially connected to a protein that is not in this organelle or structural region (such as the nucleus), the protein (non-nucleus protein) can be directed into this specific organelle or structural region. The fluorescent tracing of proteins is to connect a specific fluorescent display peptide segment to the peptide chain of the target protein, and to observe the generation of specific fluorescence under a fluorescence microscope by laser excitation of a specific wavelength under a fluorescence microscope to determine the expression amount of the protein and the specific organelle or structural region in the cell. Based on the above principle, the present invention locates a protein A fused with a fluorescent signal peptide (such as green fluorescent protein), and then detects whether the location of another protein B fused with another fluorescent signal peptide (such as red fluorescent protein) changes, thereby identifying whether protein A and protein B have an interaction. This method changes the positioning of proteins so that only when there is a real interaction and direct binding between the two proteins can the binding protein be displaced in a directional manner with the migrating protein. The fused fluorescent signal makes it visible in real time in living cells whether the protein undergoes organelle or regional migration, and the phenomenon is obvious, which can significantly improve the accuracy of judging whether there is an interaction between proteins. The entire technical method is simple, fast, and reliable, avoiding the complexity of the related technology mentioned in the background technology, the false positive caused by indirect judgment, and other problems; this method is constructed using signal peptides of different organelles, so it is suitable for all eukaryotic cells; this method is constructed using plasmids, and the plasmids expressing proteins of the same species are the same, so different protein interactions can be detected in different cells of the same species at a high throughput, thereby constructing a protein interaction network of eukaryotic organisms; this method is used in living cells, and real-time fluorescence imaging can be performed to observe protein interactions under specific conditions. This method can be observed using an ordinary fluorescence microscope, making the detection method simpler and requiring less equipment. The present invention can avoid false positives when studying interactions between proteins and improve detection accuracy.
[0035] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.
Claims
1. A method for real-time fluorescence display of protein interactions in living cells, characterized in that: include: Establish protein fluorescence display sequence rapid cloning plasmid α, protein fluorescence display rapid cloning plasmid β and protein fluorescence display rapid cloning plasmid γ; The cDNA sequence of protein A was cloned into protein fluorescence display sequence rapid cloning plasmid α and protein fluorescence display sequence rapid cloning plasmid β, and the cDNA sequence of protein B was cloned into protein fluorescence display sequence rapid cloning plasmid γ; The protein fluorescence display sequence rapid cloning plasmids α, β, and γ are transduced separately, the protein fluorescence display sequence rapid cloning plasmid α is combined with γ to co-transfect the tool cells, and the protein fluorescence display sequence rapid cloning plasmid β is combined with γ to co-transfect the tool cells; After transfection, the changes in the fluorescence signals produced by protein A and protein B in the five groups of cells were observed to determine whether there was protein-protein interaction between protein A and protein B.
2. A method for real-time fluorescence display of protein interactions in living cells as claimed in claim 1, characterized in that: After transfection, observing the changes in the fluorescence signals generated by protein A and protein B in the five groups of cells to determine whether there is a protein-protein interaction between protein A and protein B specifically includes: When protein A migrates to a specific organelle or structural region, the organelle or structural region shows fluorescence of protein A or the fluorescence intensity is enhanced. At the same time, protein B is also carried into the organelle or structural region, and the fluorescence signal of protein B appears or the fluorescence signal is enhanced at this position, indicating that there is an interaction between protein A and protein B; When protein A migrates to a specific organelle or structural region, the fluorescence of protein A appears in the organelle or structural region or the fluorescence intensity is significantly enhanced. At the same time, the position and intensity of the fluorescence signal of protein B do not change, indicating that there is no interaction between protein A and protein B.
3. A method for real-time fluorescence display of protein interactions in living cells as claimed in claim 2, characterized in that: The protein fluorescence display sequence rapid cloning plasmid α expresses a fusion protein with a specific positioning signal peptide and fused with a first fluorescence signal.
4. A method for real-time fluorescence display of protein interactions in living cells as claimed in claim 3, characterized in that: The protein fluorescence display rapid cloning plasmid β expresses a fusion protein fused to a first fluorescence signal.
5. A method for real-time fluorescence display of protein interactions in living cells as claimed in claim 4, characterized in that: The protein fluorescence display rapid cloning plasmid gamma expresses a fusion protein fused to a second fluorescent signal.