Detection method for realizing visual protein-protein interaction through co-transfer of outer nuclear membrane and application

Abstract

The invention provides a detection method for realizing visual protein-protein interaction by co-transferring an outer nuclear membrane. The method comprises the following steps: constructing an expression vector of an outer nuclear membrane localization protein and a fluorescent protein; recombining the expression fragment of the bait protein with an expression vector to construct a bait protein expression vector; transforming and extracting plasmids; constructing a prey protein expression vector; transforming and extracting plasmids; transfecting bait protein expression vector plasmids and prey protein expression vector plasmids into cells in proportion, and culturing; and performing fluorescence imaging. And the co-localization condition of the protein on the outer nuclear membrane is directly observed by utilizing fluorescence labeling, so that the interaction of the protein can be judged. Positioning is clear and visual; and the method has high specificity, living cell compatibility and high throughput potential, so that the method has a wide application prospect in protein function research and drug screening.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and in particular relates to a detection method and application for visualizing protein-protein interactions by co-transferring the outer nuclear membrane. Background Art

[0002] Protein interactions are crucial for life functions and the development of disease. Verifying protein interactions is a fundamental requirement in modern life sciences and medicine. Currently, biochemical techniques commonly used in laboratories include immunoprecipitation, yeast hybridization, GST-pulldown, bimolecular fluorescence complementation, and proximity labeling. However, these techniques often require long experimental cycles, complex procedures, or expensive instrumentation or kits. Furthermore, most experimental techniques rely on cell disruption, which can lead to false-positive or false-negative results. Additionally, some methods rely on protein translocation, such as nuclear cotranslocation of cytoplasmic proteins. However, these methods are limited in their use with large proteins, as they do not translocate as efficiently as smaller proteins. In summary, methods for detecting protein interactions should meet the following requirements: 1. They should be simple to operate and easy to establish; 2. They should be simple to use, requiring no specialized equipment and applicable to most laboratories; 3. They should be reliable, with research conditions more closely resembling physiological conditions; and 4. They should provide a distinct signal that is easily observed. Summary of the Invention

[0003] The present invention provides a method for detecting protein-protein interactions by co-transferring the outer nuclear membrane to solve the problems of the related art. The technical solution is as follows:

[0004] In a first aspect, the present invention provides a method for visualizing protein-protein interactions by co-transferring the outer nuclear membrane.

[0005] Construct outer nuclear membrane localization and fluorescent protein expression vector;

[0006] Recombining the expression fragment of the bait protein with the expression vector to construct a bait protein expression vector; transforming and extracting the plasmid;

[0007] Construct prey protein expression vector; transform and extract plasmid;

[0008] The bait protein expression vector plasmid and the prey protein expression vector plasmid are transfected into cells in a certain proportion, and the cells are cultured; and fluorescence imaging is performed.

[0009] In one embodiment, the outer nuclear membrane localization protein is the C-terminal domain mDTK of mouse SYNE1 protein, the nucleotide sequence of which is shown in SEQ ID NO.1

[0010] SEQ ID NO.1:

[0011] Tcatggtcttctgcagatgagctggacacctcaggatctgtgagtcccacatccggaagaagtaccccaaacagacaga

[0012] aatcgccacgaggcaaatgtagtctctcacagcctggaccctctgtcagcagcccaaagagcaggtccacaagagatg

[0013] gctccgattcctcccgttctgaccccaggccagaacgggtgggtcgagccttcctgttccggatcctccgggcagctcttc

[0014] cctttcagctgctcctgctgctgcttattggactcacctgccttgtacccatgtcagagaaagactacagctgtgccctctccaacaactttgcccgatccttccatccgatgctcagatataccaacggtcctcctccactctga.

[0015] The corresponding amino acid sequence is shown in SEQ ID NO.10;

[0016] SEQ ID NO.10:

[0017] SWSSADELDTSGSVSPTSGRSTPNRQKSPRGKCSLSQPGPSVSSPKSRST RDGSDSSRSDPRPERVGRAFLFRILRAALPFQLLLLLLIGLTCLVPMSEKDYSC ALSNNFARSFHPMLRYTNGPPPL.

[0018] In one embodiment, the mDTK and fluorescent protein expression sequences are amplified using amplification primers to obtain amplified products;

[0019] The amplified product is recombined with a plasmid vector to obtain an expression vector.

[0020] In one embodiment, primers are designed and the expression fragment of the bait protein is amplified. After amplification, the expression fragment of the bait protein is recombined into the expression vector digested with Kpn1 and BamH1 to obtain the bait protein expression vector.

[0021] In one embodiment, the fluorescent protein is red fluorescent protein mCherry.

[0022] In one embodiment, primers are designed and the expression fragment of the prey protein is amplified, and the prey protein expression fragment is recombined with a eukaryotic cell fluorescent expression plasmid to obtain a prey protein expression vector;

[0023] The eukaryotic cell fluorescence expression plasmid is pEGFP-C1 expressing green fluorescent protein GFP, which is digested with endonucleases EcoRV and Xho1 and then recombined with the expression fragment of the amplified prey protein.

[0024] The primers used to amplify mDTK include a forward primer and a reverse primer, wherein the forward primer is shown in SEQ ID NO.2; the reverse primer is shown in SEQ ID NO.3;

[0025] mDTK-F (SEQ ID NO. 2):

[0026] GCATGGACGAGCTGTACAAGTCATGGTCTTCTGCAGATGA;

[0027] mDTK-R (SEQ ID NO. 3):

[0028] TTCGAATGGGTGACCTCGAGTCAGAGTGGAGGAGGACCG.

[0029] The primers used to amplify the expression sequence of mCherry include a forward primer and a reverse primer, the forward primer is shown in SEQ ID NO.4; the reverse primer is shown in SEQ ID NO.5;

[0030] mCherry-F (SEQ ID NO. 4):

[0031] TGTGGTGGAATTCTGCAGATATGGTGAGCAAGGGCGAG;

[0032] mCherry-R (SEQ ID NO. 5):

[0033] TCATCTGCAGAAGACCATGACTTGTACAGTCGTCCATGC

[0034] The plasmid vector is pcDNA TM 3.1 (-) ; The pcDNA TM 3.1 (-) The plasmid was recombined after digestion with EcoRV and Xho1.

[0035] In one embodiment, the bait protein is TLE6, and the prey protein is OOEP; or the bait protein is NLRP7, and the prey protein is TLE6.

[0036] In one embodiment, the primers used to amplify the expression sequence of TLE6 include a forward primer and a reverse primer, wherein the forward primer is shown as SEQ ID NO.6; the reverse primer is shown as SEQ ID NO.7;

[0037] TLE6-F (SEQ ID NO.6):

[0038] GGCTAGTTAAGCTTGGTACCATGACCTCTAGGGACCAGCC;

[0039] TLE6-R (SEQ ID NO.7):

[0040] CACACTGGACTAGTGGATCCGTAGGGTGATCTGGTACACGG

[0041] The primers used to amplify the expression sequence of OOEP include a forward primer and a reverse primer, wherein the forward primer is shown as SEQ ID NO.8; the reverse primer is shown as SEQ ID NO.9;

[0042] OOEP-F (SEQ ID NO. 8):

[0043] CCGGACTCAGATCTCGAGCTATGGTCGATGATGCTGGTG;

[0044] OOEP-R (SEQ ID NO. 9):

[0045] TACCGTCGACTGCAGAATTCTTAAGCAACAGGATCCTGGGGAG

[0046] In one embodiment, the transfection and plasmid extraction process is:

[0047] The plasmid was transformed into E. coli Top10 competent cells and plated on solid LB medium containing ampicillin.

[0048] A single clone was picked and inoculated into LB culture medium containing ampicillin for culture, and the endotoxin-free plasmid was extracted.

[0049] In one embodiment, if the sequence of the expression fragment contains repetitive sequences, the TOP10 competent vector is replaced with, for example, the stabl3 competent vector.

[0050] In one embodiment, the bait protein expression vector plasmid and the prey protein expression vector plasmid are transfected into cells in a proportional manner, and during the culture process,

[0051] The cells are HEK293T cells; the ratio of the bait protein expression vector plasmid to the prey protein expression vector plasmid is 5-8:2.

[0052] In a second aspect, the embodiments of the present application provide a kit for visualizing protein-protein interactions, comprising the expression vector prepared as described above.

[0053] In a third aspect, an embodiment of the present application provides an application of mDTK, a C-terminal domain based on mouse SYNE1 protein, in visualizing protein-protein interactions by co-transferring the outer nuclear membrane.

[0054] The advantages or beneficial effects of the above technical solution include at least:

[0055] The present application discloses a method for visualizing protein-protein interactions by co-transferring the outer nuclear membrane, which utilizes the specific binding of the KASH domain to the SUN protein to ensure that the bait protein is accurately positioned to the outer nuclear membrane, and then uses fluorescent markers (mCherry and GFP) to directly observe the co-localization of proteins on the outer nuclear membrane, thereby determining the interactivity of the proteins. Since the interaction is confined to the outer nuclear membrane region, the interference of other signals in the cytoplasm or nucleus is reduced, thereby improving the signal-to-noise ratio. Moreover, this method does not require fixing cells or destroying cell structures, and protein interactions can be observed in real time in living cells, which is closer to physiological conditions. It provides clear spatial positioning information and avoids interference from complex backgrounds in the cytoplasm or nucleus. Direct observation of interactions through fluorescent signals eliminates the need for complex subsequent processing or data analysis, and the results are intuitive and easy to analyze.

[0056] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0058] Figure 1 A schematic diagram of the method of the present application for detecting protein-protein interactions;

[0059] Figure 2 This is the fluorescence staining image after cells were transfected with the negative control plasmid pCDNA3.1-mCherry-mDTK, LAMINB was used to mark the nuclear membrane, and α-Tubulin was used to mark the outline of the entire cell;

[0060] Figure 3 This is the fluorescence staining image of the sample treated with a mixture of the negative control vector pCDNA3.1-mCherry-mDTK and the prey protein expression vector GFP-OOEP;

[0061] Figure 4 This is the fluorescence staining image of the sample treated with a mixture of the bait protein expression vector TLE6-mCherry-mDTK and the prey protein expression vector GFP-OOEP;

[0062] Figure 5 This is the fluorescence staining image of the sample treated with a mixture of the negative control vector pCDNA3.1-mCherry-mDTK and the prey protein expression vector TLE6-GFP;

[0063] Figure 6 This is the fluorescence staining image of the sample treated with a mixture of the bait protein expression vector NLRP7-mCherry-mDTK and the prey protein expression vector TLE6-GFP. DETAILED DESCRIPTION

[0064] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0065] Among existing protein interaction monitoring methods, nuclear translocation detection of protein interactions is susceptible to interference from other fluorescent signals within the cell, requiring careful control analysis. Immunoprecipitation (Co-IP) requires subsequent detection such as immunoblotting, which has multiple steps, limited sensitivity, and is susceptible to interference from nonspecific binding. Yeast two-hybrid methods rely on reporter gene expression for judgment, requiring additional detection steps (such as X-gal staining), and relatively indirect visualization. Fluorescence complementation methods can be difficult to observe when the fluorescent signal is weak, requiring optimized experimental conditions. PLA (proximity ligation analysis) signal detection relies on specific ligation reactions and amplification, and background signals may affect observation. Therefore, all of these methods have various shortcomings.

[0066] The bait protein is fused with a fluorescent protein and the C-terminal domain of the mouse SYNE1 protein; SYNE1 is an outer nuclear membrane localized protein that connects the nuclear membrane to the perinuclear cytoskeleton. Its C-terminal domain (called mouse DTK, mDTK) contains disordered sequences outside the outer nuclear membrane, transmembrane sequences that penetrate the nuclear membrane, and a KASH domain that binds to the SUN protein in the nuclear membrane cavity. The mDTK structure can transfer the bait protein expressed in the cytoplasm to the outer nuclear membrane for localization; if the bait protein interacts with the prey protein, the two will co-localize outside the outer nuclear membrane. Figure 1 As shown, when the bait-mCherry-mDTK was successfully translocated to the outer nuclear membrane, the interaction between the bait protein and the prey protein (fused with GFP fluorescent protein) was directly observed by fluorescence microscopy, showing the co-localization of the two on the outer nuclear membrane.

[0067] Therefore, the method of this application simulates the in vivo nuclear membrane environment, closely resembling physiological conditions. Using a common fluorescence microscope or confocal microscope, it can visualize the two-color signal surrounding the cell nucleus, making it easy to distinguish and interpret the image. It is an efficient, intuitive, and reliable method for detecting protein-protein interactions, particularly suitable for interactions between cytoplasmic proteins. Its high spatial resolution, specificity, compatibility with living cells, and high-throughput potential give it broad application prospects in protein function research and drug screening.

[0068] Example 1

[0069] 1. Required reagents

[0070] 1) Transfection reagent PEI (1 mg / mL, pH 7.0), stored in aliquots at -20°C.

[0071] 2) Cell culture medium: high-glucose DMEM supplemented with 5% fetal bovine serum and 0.1% penicillin-streptomycin;

[0072] 3) Endotoxin-free plasmid extraction kit (DP118, Tiangen);

[0073] 4) Optional reagents: If fixation is required, use 4% PFA (paraformaldehyde);

[0074] 5) Vector construction system: One Step Cloning Kit (C112-01, Vazyme);

[0075] 2. Required instruments

[0076] Fluorescence microscopy or confocal laser scanning microscopy.

[0077] 1. Construct vectors containing the target gene to be tested and negative control vectors

[0078] 1. Construction of mCherry-mDTK expression vector:

[0079] Amplify the mCherry expression fragment using amplification primers and purify and recover it;

[0080] mCherry-F (SEQ ID NO. 4):

[0081] TGTGGTGGAATTCTGCAGATATGGTGAGCAAGGGCGAG;

[0082] mCherry-R (SEQ ID NO. 5):

[0083] TCATCTGCAGAAGACCATGACTTGTACAGTCGTCCATGC

[0084] Amplify the mTDK expression fragment using amplification primers and purify and recover it;

[0085] mDTK-F (SEQ ID NO. 2):

[0086] GCATGGACGAGCTGTACAAGTCATGGTCTTCTGCAGATGA;

[0087] mDTK-R (SEQ ID NO. 3):

[0088] TTCGAATGGGTGACCTCGAGTCAGAGTGGAGGAGGACCG.

[0089] pcDNA was digested with EcoRV and Xho1 TM 3.1 (-) (V855-20, Thermofisher), purified and recovered; according to the instructions of One Step Cloning Kit (C112-01, Vazyme), the amplified mCherry expression fragment and the amplified mDTK-R expression fragment were cleaved with enzyme-digested pcDNA TM 3.1 (-) Plasmid recombination construction was performed; this plasmid was named pCDNA3.1-mCherry-mDTK, which is the basic plasmid and negative control vector of this application.

[0090] 2. Use TLE6 as bait protein to construct bait protein expression vector:

[0091] Design primers to amplify TLE6 expression fragments:

[0092] TLE6-F (SEQ ID NO. 6):

[0093] GGCTAGTTAAGCTTGGTACCATGACCTCTAGGGACCAGCC;

[0094] TLE6-R (SEQ ID NO. 7):

[0095] CACACTGGACTAGTGGATCCGTAGGGTGATCTGGTACACGG.

[0096] The expression fragment of TLE6 was amplified and purified, and then the amplified fragment was recombined into pCDNA3.1-mCherry-mDTK digested with Kpn1 and BamH1 using a One Step Cloning Kit to obtain the bait protein expression vector TLE6-mCherry-mDTK;

[0097] 3. Using OOEP as prey protein, construct prey protein expression vector:

[0098] Design primers to amplify the OOEP expression fragment:

[0099] OOEP-F (SEQ ID NO. 8):

[0100] CCGGACTCAGATCTCGAGCTATGGTCGATGATGCTGGTG;

[0101] OOEP-R (SEQ ID NO. 9):

[0102] TACCGTCGACTGCAGAATTCTTAAGCAACAGGATCCTGGGGAG.

[0103] The expression fragment of OOEP was amplified and purified, and then the amplified fragment was recombined into pEGFP-C1 (Clontech) digested with Xho1 and EcoR1 using a One Step Cloning Kit to obtain a prey protein expression vector;

[0104] Each plasmid was sequenced by Sanger sequencing to ensure the correct insert sequence; since mDTK binds to SUN via its C-terminus, this fragment needs to be at the C-terminus of the fusion protein.

[0105] 2. Extraction of plasmid

[0106] 1) The negative control vector, bait protein expression vector, and prey protein expression vector plasmids were transformed into competent E. coli Top10 cells, plated on LB solid medium containing 100 μg / ml ampicillin, and cultured in a 37°C incubator overnight.

[0107] 2) Pick a single clone and inoculate it into 50 ml of LB culture medium containing 100 μg / ml ampicillin for 14 hours. Extract the endotoxin-free plasmid according to the instructions and adjust the concentration to 100 ng / ml.

[0108] If the expression fragment contains repetitive sequences, use, for example, stabl3 competent cells instead of TOP10 competent cells.

[0109] III. Cell Culture, Inoculation, and Transfection

[0110] 1) 24 hours before transfection, HEK293T cells were digested into single cells and seeded into 96-well glass-bottom plates, with 10 cells seeded per well. 4 The culture medium is DMEM + 5% fetal bovine serum + 0.1% double-antibody. When the cells grow to 80% confluency, transfection is performed.

[0111] 2) Mix and dilute the bait protein expression vector / negative control vector (ng): prey protein expression vector (ng) in a ratio of 8:2 in 20 ml of OPTI-DMEM, with the total amount of the two not exceeding 100 ng; add 300 ng of PEI to the above plasmid mixture, mix thoroughly, and incubate at room temperature for 15 minutes; add 100 ml of cell culture medium to the incubated plasmid mixture and mix thoroughly;

[0112] 3) The culture medium in the 96-well plate was removed and the culture medium containing the plasmid mixture was added again; the cells were cultured at 37° C. and 5% CO 2 for 24 hours.

[0113] If the cell culture system is expanded, the corresponding plasmids and transfection system can be increased proportionally. The plasmid ratio can be adjusted using a gradient to optimize the conditions during the actual operation. In principle, the expression level of the bait protein should be higher than that of the prey protein to prevent excessive background of the prey protein. After transfection, the culture medium can be changed every six hours.

[0114] 4. Fluorescence Imaging

[0115] 1) Use a laser confocal microscope with a 40x water objective to observe the subcellular localization of green fluorescent GFP (488 nm) and red fluorescent mCherry (594 nm);

[0116] 2) Taking the largest surface of the cell nucleus as the image acquisition surface, randomly capture 5 fields of view;

[0117] 3) The fluorescence distribution (measured in the direction across the cell nuclear diameter) and fluorescence colocalization analysis of the expressed cells were performed using Image J software (NIH); the proportion of cells with similar fluorescence patterns was counted.

[0118] 4) The transfected cells can be fixed (with 4% paraformaldehyde at 37° C. for 10 minutes) and then subjected to immunofluorescence staining.

[0119] After the negative control plasmid pCDNA3.1-mCherry-mDTK was transfected into the cells, LAMINB was used to mark the nuclear membrane and α-Tubulin was used to mark the outline of the entire cell. The fluorescence staining images are shown in the figure below. Figure 2 The fluorescence staining of the sample treated with the negative control vector pCDNA3.1-mCherry-mDTK mixed with the prey protein expression vector GFP-OOEP is shown in Figure 2. Figure 3 As shown; wherein the bait protein expression vector TLE6-mCherry-mDTK and the prey protein expression vector GFP-OOEP were mixed and treated with the fluorescent staining diagram of the sample. Figure 4 As shown;

[0120] from Figure 2 As can be seen in the figure, α-Tubulin marks the entire cell outline, LAMINB marks the nuclear membrane, and pCDNA3.1-mCherry-mDTK is localized to the outer nuclear membrane and also observed in perinuclear endoplasmic reticulum structures. The right panel shows the distribution of mCherry and LAMINB signals, indicating the proportion of cells with positive interactions.

[0121] Figure 4 In the figure, pCDNA3.1-mCherry-mDTK was used as a negative control. It can be seen that pCDNA3.1-mCherry-mDTK was concentrated at the outer nuclear membrane and localized on the outer nuclear membrane. Since OOEP did not interact with it, GFP-OOEP showed a diffuse distribution instead of a clear distribution outside the nuclear membrane. Figure 3 In the case of pCDNA3.1-mCherry-mDTK, due to the attachment of the bait protein TLE6 to the outer nuclear membrane, when mCherry-mDTK in TLE6-mCherry-mDTK is localized to the outer nuclear membrane, the interaction between the attached bait protein TLE6 and OOEP causes GFP-OOEP to also be distributed at the nuclear membrane, rather than being diffusely distributed. TLE6-mCherry-mDTK and GFP-OOEP clearly colocalize at the outer nuclear membrane, demonstrating the interaction between the bait protein TLE6 and OOEP.

[0122] Example 2

[0123] Interaction between TLE6 and NLRP7.

[0124] The experiment was conducted according to the method of Example 1, using TLE6 as the prey protein and NLRP7 as the bait protein. TLE6 was fused with GFP, NLRP7 was fused with mCherry-mDTK, and pCDNA3.1-mCherry-mDTK was used as the negative control vector. Figure 5 and Figure 6 As shown, Figure 5 The fluorescence staining images of the sample treated with negative control vector pCDNA3.1-mCherry-mDTK mixed with prey protein expression vector TLE6-GFP are shown in Figure 2. Figure 5 As shown; the fluorescence staining diagram of the sample treated with the bait protein expression vector NLRP7-mCherry-mDTK and the prey protein expression vector TLE6-GFP is shown Figure 6 As shown;

[0125] from Figure 6 As can be seen in the figure, pCDNA3.1-mCherry-mDTK is used as a negative control. It can be seen that pCDNA3.1-mCherry-mDTK is concentrated at the outer nuclear membrane and localized on the outer nuclear membrane. Since TLE6 does not interact with it, GFP-TLE6 is diffusely distributed instead of being clearly distributed outside the nuclear membrane. Figure 5 In the case of pCDNA3.1-mCherry-mDTK, the NLRP7 bait protein is attached to the outer nuclear membrane. When the mCherry-mDTK in the NLRP7-mCherry-mDTK is localized to the outer nuclear membrane, the interaction between the attached bait protein NLRP7 and TLE6 causes GFP-TLE6 to be localized to the nuclear membrane, rather than being diffusely distributed. The clear colocalization of NLRP7-mCherry-mDTK and GFP-TLE6 at the outer nuclear membrane demonstrates the interaction between the bait proteins NLRP7 and TLE6.

[0126] The bait protein and prey protein are not limited to TLE6 / OOEP and NLRP7 / TLE6 in the examples. In actual use, the scope of use can be extended to all soluble proteins localized in the cytoplasm that can be detected by this method.

[0127] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0128] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0129] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A method for visualizing protein-protein interactions by co-transferring the outer nuclear membrane, characterized in that Construct expression vectors for outer nuclear membrane localization proteins and fluorescent proteins; Recombining the expression fragment of the bait protein with the expression vector to construct a bait protein expression vector; transforming and extracting the plasmid; Construct prey protein expression vector; transform and extract plasmid; The bait protein expression vector plasmid and the prey protein expression vector plasmid are transfected into cells in a certain proportion, and the cells are cultured; and fluorescence imaging is performed.

2. The method according to claim 1, characterized in that The outer nuclear membrane localization protein is the C-terminal domain mDTK of mouse SYNE1 protein, and the nucleotide sequence is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that Amplification primers were used to amplify the mDTK and fluorescent protein expression sequences to obtain amplified products; The amplified product is recombined with a plasmid vector to obtain an expression vector.

4. The method according to claim 1, wherein Primers are designed and the expression fragment of the bait protein is amplified. After amplification, the expression fragment of the bait protein is recombined into the expression vector after being digested by Kpn1 and BamH1 to obtain the bait protein expression vector.

5. The method according to claim 1, characterized in that Design primers and amplify the expression fragment of the prey protein, and perform plasmid recombination with the eukaryotic cell fluorescence expression plasmid to obtain the prey protein expression vector; The eukaryotic cell fluorescent expression plasmid is pEGFP-C1 expressing green fluorescent protein GFP, which is digested by endonucleases EcoRV and Xho1 and then recombined with the expression fragment of the amplified prey protein.

6. The method according to claim 3, characterized in that The fluorescent protein is red fluorescent protein mCherry; The primers used to amplify mDTK include a forward primer and a reverse primer, wherein the forward primer is shown in SEQ ID NO.2; the reverse primer is shown in SEQ ID NO.3; The primers used to amplify the expression sequence of mCherry include a forward primer and a reverse primer, the forward primer is shown as SEQ ID NO.4; the reverse primer is shown as SEQ ID NO.5; The plasmid vector is pcDNA TM 3.1 (-) ; The pcDNA TM 3.1 (-) The plasmid was recombined after digestion with EcoRV and Xho1.

7. The method according to any one of claims 1 to 6, characterized in that The bait protein is TLE6, and the prey protein is OOEP; or the bait protein is NLRP7, and the prey protein is TLE6.

8. The method according to claim 7, characterized in that The primers used to amplify the expression sequence of TLE6 include a forward primer and a reverse primer, the forward primer is shown in SEQ ID NO.6; the reverse primer is shown in SEQ ID NO.7; The primers used to amplify the expression sequence of OOEP include a forward primer and a reverse primer. The forward primer is shown as SEQ ID NO.8; the reverse primer is shown as SEQ ID NO.

9.

9. A kit for visualizing protein-protein interactions, characterized in that: The invention also comprises the expression vector prepared according to any one of claims 1 to 3.

10. Application of the C-terminal domain mDTK of mouse SYNE1 protein to visualize protein-protein interactions by co-translocation to the outer nuclear membrane.

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