Fusion proteome based on m6A methyltransferase and application thereof
By constructing a fusion protein group based on proximity labeling technology, the fusion of the cleavage APEX2 enzyme with METTL3 and METTL14 is achieved, and the labeling of the m6A methyltransferase dimer binding protein with high specificity and high sensitivity is solved, which solves the problem of identification in the prior art and improves the reliability and depth of the research.
Patent Information
- Application Number
- CN202510462311.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art lacks high specificity, stable and reliable methods for identifying binding proteins of m6A methyltransferase dimers, making it difficult to accurately distinguish direct and indirect interaction proteins.
A fusion protein group based on proximity labeling technology was constructed, and the cleavage APEX2 enzyme was fused with METTL3 and METTL14, respectively, and reconstituted into a complete APEX2 enzyme for biotin labeling in cells, capturing potential m6A methyltransferase dimer binding proteins, and co-expression was achieved through stable expression of the cell lines isolated from the APEX2 system.
It significantly improves the comprehensiveness and authenticity of protein interaction research, provides more reliable research methods, deepens the understanding of the biological functions of m6A modified, and lays the foundation for the treatment strategies of related diseases.
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Figure CN120290512A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fusion proteins and protein interaction detection, and particularly relates to a fusion proteome based on m 6 A methyltransferase and its application in constructing a cell model for stably expressing the METTL3-METTL14-APEX2 proximity labeling system. Background Art
[0002] m 6 A modification, as one of the most abundant RNA modifications inside eukaryotic mRNA, specifically refers to the methylation process occurring at the 6th nitrogen atom (N 6 ) of the adenosine in the RNA molecule. This modification is dynamically reversible and is mainly catalyzed by a heterodimeric complex composed of METTL3 and METTL14 proteins. In these dimers, METTL3 acts as the catalytic active subunit responsible for performing the methylation reaction; while METTL14 serves as the RNA-binding active subunit to assist METTL3 in accurately recognizing the RNA substrate. Existing studies have shown that multiple proteins can bind to the METTL3-METTL14 dimer, thereby finely regulating its catalytic activity. For example, WTAP promotes the occurrence of m 6 A modification by stabilizing the core subunits and guiding the dimer to nuclear speckles; while proteins such as HAKAI and VIRMA bind to the METTL3-METTL14 dimer through different mechanisms to jointly regulate the level of m 6 A methylation modification. It is worth noting that although METTL3 plays a key role in m 6 A modification, it can only bind to approximately 22% of the m 6 A modification sites and has other biological functions independent of m 6 A modification. This increases the complexity of identifying the regulatory subunits of the methyltransferase dimer. Traditionally, researchers mainly rely on co-immunoprecipitation based on METTL3 or METTL14 combined with mass spectrometry techniques to identify these regulatory subunits. However, since METTL3 and METTL14 must bind to each other to exert catalytic functions, these methods have limitations in terms of specificity and are difficult to accurately distinguish direct and indirect interacting proteins.
[0003] Therefore, the existing technology currently lacks a highly specific, stable and reliable method to identify the binding proteins of the m 6 A methyltransferase dimer. Given the importance of the interaction between the m 6 A methyltransferase dimer binding protein and other proteins in cell life activities, it is necessary to seek new research tools to reveal these interactions. Summary of the Invention
[0004] To overcome the limitations in the background art, the present application constructs a fusion proteome based on the proximity labeling technology (PL) for labeling the interacting proteins of the METTL3-METTL14 dimer. The proximity labeling technology refers to a class of technologies that can capture protein interaction relationships with different strengths, including transient or weak interactions. In the peroxidase system, enzymes such as APEX / APEX2 have extensive activities in living cells and can catalyze biotin phenol to generate highly reactive free radicals. These free radicals can covalently bind to specific amino acids (such as Tyr, Trp, Cys, and His) in neighboring proteins within an extremely short time (<1 ms half-life), thereby achieving biotin labeling. Due to the membrane impermeability and short-range action range (about 20 nm) of free radicals, therefore, using enzymes such as APEX / APEX2 can efficiently and specifically label the interacting proteins around the target protein;
[0005] The fusion proteome provided by the present application is obtained by respectively fusing the split APEX2 enzyme with METTL3 and METTL14, so that it is reconstructed into a complete APEX2 enzyme in the cell. APEX2 catalyzes the biotin labeling of neighboring proteins, thereby capturing potential m 6 A methyltransferase dimer-binding protein; and based on the fusion proteome provided by the present application, the present invention has also gradually constructed a cell line stably expressing the separated APEX2 system to achieve the co-expression of the APEX2 enzyme and the m 6 A methyltransferase dimer-binding protein without active interference between the two.
[0006] In the first aspect, the primary object of the present invention is to provide a fusion proteome based on m 6 A methyltransferase, the fusion proteome comprising a first fusion protein and a second fusion protein;
[0007] The first fusion protein is obtained by binding the amino acid fragment nAEPX2 of the 1st to 201st positions of the APEX2 enzyme to the METTL3 protein through a first linker peptide and then binding a Myc tag protein; for example, the first fusion protein obtained by binding the C-terminus of the nAPEX2 to the METTL3 protein through the first linker peptide and then binding the N-terminus to the Myc tag protein is Myc-METTL3-nAPEX2;
[0008] The second fusion protein is obtained by binding the amino acid fragment cAPEX2 of the 201st to 250th positions of the APEX2 enzyme to the METTL14 protein through a second linker peptide and then binding a Flag tag protein; for example, the second fusion protein obtained by binding the C-terminus of the cAPEX2 to the METTL14 protein through the second linker peptide and then binding the N-terminus to the Flag tag protein is Flag-METTL14-cAPEX2;
[0009] Among them, the amino acid sequence of the nAPEX2 from the N-terminus to the C-terminus is as shown in SEQ ID NO: 1 in the sequence listing; the amino acid sequence of the cAPEX2 from the N-terminus to the C-terminus is as shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence of the first linker peptide from the N-terminus to the C-terminus is as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the second linker peptide from the N-terminus to the C-terminus is as shown in SEQ ID NO: 4 in the sequence listing.
[0010] As a preferred embodiment of the fusion protein group of the present invention, in the fusion protein group, the first fusion protein is Myc-METTL3-nAPEX2, and its amino acid sequence from the N-terminus to the C-terminus is as shown in SEQ ID NO: 5 in the sequence listing; the second fusion protein is Flag-METTL14-cAPEX2, and its amino acid sequence from the N-terminus to the C-terminus is as shown in SEQ ID NO: 6 in the sequence listing.
[0011] In a second aspect, based on the fusion protein group provided in the first aspect, the present invention further provides a recombinant plasmid group, which is composed of two recombinant plasmids that respectively express the first fusion protein and the second fusion protein. The construction steps of the recombinant plasmid group include: first, construct fusion gene fragments that express the first fusion protein and the second fusion protein respectively, and recombine these two gene fragments onto the FKBP-V5-AP-nes_pLX304 and HA-Halotag-FRB-EX-nes_pLX304 expression vectors respectively.
[0012] In a third aspect, based on the recombinant plasmid group provided in the second aspect, the present invention further provides a cell model of a proximity labeling system capable of stably expressing METTL3-METTL14-APEX2 and a construction method thereof. The construction method includes the steps of: co-transfecting the successfully constructed recombinant plasmids into HEK293 cells, labeling with Biotin-phenol, catalyzing with H2O2, and detecting the labeling effect by Western Blotting; constructing a stable cell line that stably expresses the first fusion protein and the second fusion protein by lentiviral packaging technology with the above-mentioned constructed recombinant plasmids, performing resistance screening with Blasticidin (BSD), and combining immunofluorescence to detect the labeling efficiency.
[0013] Technical principle: The APEX2 enzyme is split into two inactive fragments. Through genetic engineering techniques, these two fragments are fused and expressed with the target protein. When the target proteins interact with each other, these two originally inactive APEX2 fragments can recombine to form APEX2 with complete enzyme activity, thereby achieving highly specific proximity labeling in a specific time and space. This method significantly reduces the non-specific labeling background and improves the accuracy and sensitivity of labeling.
[0014] Specifically, as Figure 1 shown in the experimental procedure of the split APEX2 enzyme proximity labeling system. In this system, the APEX2 enzyme is split into two functionally inactivated fragments: the N-terminal fragment, i.e., nAPEX2, corresponds to Figure 1 nAP in Figure 1 and the C-terminal fragment, i.e., cAPEX2, corresponds to
[0015] cEX2 in The nAPEX2 fragment is fused to the C-terminus of the METTL3 protein by genetic engineering means to construct the Myc-METTL3-nAPEX2 recombinant protein; the cAPEX2 fragment is fused to the C-terminus of the METTL14 protein to construct the Flag-METTL14-cAPEX2 recombinant protein; when METTL3 and METTL14 have a specific interaction, the nAPEX2 and cAPEX2 fragments approach and recombine spatially to form the METTL3-METTL14-APEX2 protein composition with complete APEX2 enzyme activity; subsequently, biotinylated phenol is used to biotinylate the interacting proteins, and then catalyzed by H2O2 to covalently label biotin to the neighboring proteins. Subsequently, streptavidin is used to enrich the biotin-labeled proteins, and the biotin signal of the interacting proteins can be detected by Western Blotting experiment.
[0015] Beneficial effects: Based on the principle of split APEX2 enzyme labeling, the present invention constructs recombinant plasmids by connecting the split APEX2 enzyme to the C-terminus of Myc-METTL3 and Flag-METTL14 respectively, namely Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 recombinant plasmids, and co-transfects HEK293 cells to confirm their normal expression. After biotinylated phenol labeling and H2O2 catalysis, Western Blotting experiments prove that the split APEX2 enzyme resumes its labeling function with the binding of METTL3 and METTL14. A stable cell line model stably expressing the MEETL3-METTL14-APEX2 recombinant protein is obtained by lentiviral packaging technology, and immunofluorescence shows good labeling effects. Finally, a method for labeling m 6A cell model of a methyltransferase dimer-binding protein. The constructed cell model can ensure the normal expression and function of the APEX2 enzyme, maintain its high spatio-temporal resolution, applicability to living cells, and high sensitivity in the proximity labeling system, significantly improving the comprehensiveness and authenticity of protein interaction research, and providing a more reliable research method for related fields. The above cell model belongs to a cell line stably expressing the separated APEX2 system, and provides a powerful experimental platform for studying the regulatory subunits and molecular mechanisms of m 6 A methyltransferase dimer, laying a solid foundation for developing treatment strategies for related diseases. This innovation not only is expected to deepen the understanding of the biological functions of m 6 A modification, but also lays a solid foundation for developing treatment strategies for related diseases. Brief Description of the Drawings
[0016] Figure 1 is the experimental procedure of the split APEX2 enzyme proximity labeling system;
[0017] Figure 2a is the gene map of the recombinant plasmid Myc-METTL3-nAPEX2 in Example 1;
[0018] Figure 2b is the gene map of the recombinant plasmid Flag-METTL14-cAPEX2 in Example 1;
[0019] Figure 3 is a schematic diagram of the expression of the fusion protein in 293 cells and the result of biotin-labeled Western Blotting;
[0020] Figure 4 is the co-localization of Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 in cells, and the scale bar is 5 μM;
[0021] Figure 5 is the result of Western Blotting experiment to verify the specificity of the proximity labeling system designed in Example 1. Detailed Embodiments
[0022] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.
[0023] In the examples where specific technologies or conditions are not specified, they shall be in accordance with the technologies or conditions described in the literature in this field (for example, refer to "Molecular Cloning: A Laboratory Manual" written by J. Sambrook et al. and translated by Huang Peitang et al., the third edition, Science Press) or in accordance with the product instructions. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.
[0024] The gene fragments and primer synthesis in the examples were all synthesized by Tsingke Biological Co., Ltd.
[0025] The vector plasmid FKBP-V5-AP-nes_pLX304 was purchased from addgene.
[0026] The vector plasmid HA-Halotag-FRB-EX-nes_pLX304 was purchased from addgene.
[0027] The cells HEK293(293) and 293T used were taken from this laboratory.
[0028] Literature [1]: Han Y, Branon T C, Martell J D, et al. Directed Evolution of Split APEX2 Peroxidase[J]. ACS Chemical Biology, 2019,
[0029] 14(4). DOI: 10.1021 / acschembio.8b00919.
[0030] Example 1 Design of Recombinant Plasmids and Construction of Overexpression Vectors
[0031] According to Figure 1 the labeling principle shown, the present invention designed the following experiment:
[0032] First, the inventors of the present application queried and downloaded the genomic DNA sequences of METTL3 and METTL14 according to the NCBI database. Among them, the gene ID of METTL3 is 56339, and the species is human; the gene ID of METTL14 is 57721, and the species is human. The genomic sequence of APEX2 is the sequence corresponding to APEX2 in APEX2-NLS of Plasmid #124617 publicly available on the addgene website. The 1-200 amino acid fragment of the lytic APEX2 enzyme disclosed in reference [1] was named nAEPX2, and the 201-250 amino acid fragment was named cAPEX2. nAPEX2 and cAPEX2 were respectively constructed at the C-terminus of METTL3 and METTL14. The Primer Primer online design tool was used to design primer sequences, and EcoR I and Xho I restriction sites were added to the 5' and 3' ends of the primers. The sticky ends formed after digestion are shown in Table 1. The designed recombinant plasmid gene map is as Figure 2a and Figure 2b shown.
[0033] Table 1 PCR primer sequences
[0034]
[0035]
[0036] According to the above design of the recombinant plasmid, an overexpression vector was constructed. The specific operations are as follows:
[0037] Step S1, perform overlapping PCR on the METTL3 and METTL14 gene fragments and the nAPEX2 and cAPEX2 gene fragments;
[0038] Step S2, use EcoRI and XhoI restriction endonucleases to digest 1 μg of two vector plasmids, FKBP-V5-AP-nes_pLX304 and HA-Halotag-FRB-EX-nes_pLX304, and two fusion genes, Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2. Use the Novoprotein gel recovery kit (DC301-01) to purify the digested PCR products; ligate the digested and recovered fragments and vectors at room temperature for 4 h according to the ligation system shown in Table 2;
[0039] Step S3, transform the ligated plasmid into competent cells DH5α, evenly spread it on an LB solid medium plate containing ampicillin resistance, and invert it in a 37°C incubator for 12-16 hours; the next day, pick a single colony for culture, extract the plasmid and send it to Tsingke Biotechnology Company for sequencing;
[0040] Step S4: Compare and verify the sequencing results to obtain the recombinant plasmids Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2.
[0041] Table 2 PCR Ligation System
[0042] reagent system 10×T4 DNA ligase buffer 10 μL vector 50 ng fragment 150 ng T4 DNA ligase 1 μL enzyme-free water make up the volume to 20 μL
[0043] Example 2: Transfect cells with recombinant plasmids to verify the effectiveness of the APEX2 enzyme labeling system
[0044] Since this fusion protein is overexpressed in the eukaryotic system and may not necessarily have labeling activity, it is necessary to verify the activity of the protein before starting the labeling experiment. Specifically, in this Example 3, first, the recombinant plasmids constructed in Example 1 were transfected into cells according to the following experimental design groups. After 48 hours of transfection, the cells were treated with biotin-phenol, and then hydrogen peroxide (H2O2) was added to catalyze the reaction. Then, the construction of the APEX2 proximity labeling system was verified by Western Blotting experiment.
[0045] Experimental group: Add Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 and treat with both biotin-phenol and H2O2;
[0046] Negative group: Add Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 and treat only with biotin-phenol, without adding H2O2 treatment;
[0047] Blank control group 1: Add Myc-METTL3-nAPEX2 without adding Flag-METTL14-cAPEX2 and treat with biotin-phenol and H2O2;
[0048] Blank control group 2: Add Flag-METTL14-cAPEX2 without adding Myc-METTL3-nAPEX2 and treat with biotin-phenol and H2O2.
[0049] Process of biotin phenol labeling experiment: First, treat the above experimental groups with biotin-phenol and H2O2, and then all experimental groups were terminated with the termination solution. Each plate of 293 cells was scraped with 1 mL of PBS, and the cell pellet was obtained by centrifugation at 2000 rpm for 5 min. After collecting the cell supernatant, it was collected with 30 μL of 2×SDS loading buffer, and finally, the labeling effect was verified by Western blotting.
[0050] The experimental process of verifying protein expression by Western Blotting: Equal amounts of biotin-labeled proteins were loaded onto a 15 μL SDS-PAGE gel. After blocking with 5% skim milk for 1 h, the streptavidin antibody labeled with horseradish peroxidase (streptavidin-HRP) was diluted at 1:5000 and used. Incubation was carried out overnight at 4 °C, and then washed three times with TBST (0.1% Tween 20), and then the exposure solution could be added for exposure.
[0051] Figure 3 It is a schematic diagram of the expression of the fusion protein in 293 cells and the results of biotin-labeled Western Blotting. Figure 3 In the experimental results, from left to right, they are the experimental group, the negative group, blank control group 1, and blank control group 2.
[0052] The experimental results showed that both Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 fusion proteins were highly expressed in cells. After using streptavidin-horseradish peroxidase (streptavidin-HRP) to specifically recognize and bind biotinylated proteins, signals of a large number of biotinylated proteins were detected, indicating that the APEX2 proximity labeling system successfully achieved proximity labeling of proteins, confirming the feasibility and effectiveness of this system.
[0053] Example 3 Lentivirus packaging and infection
[0054] 1) Lentivirus packaging
[0055] One day before transfection, 293T cells were seeded into a 60 mm cell culture dish and allowed to grow to a confluence of 60%-70%. The next day, two 1.5 ml EP tubes were prepared respectively, and 500 μl of DMEM medium was added. One tube was added with 0.625 μg VSVG, 2.5 μg PSPAX, and 5 μg Myc-METTL3-nAPEX2 plasmid, and the other tube was added with 0.625 μg VSVG, 2.5 μg PSPAX, and 5 μg Flag-METTL14-cAPEX2 plasmid, and gently flicked with fingers to mix evenly. Another 1.5 ml EP tube was prepared, added with 500 μl of DMEM medium and 35 μl of transfection reagent, and gently flicked with fingers to mix evenly. After incubation at room temperature for 5 min, the DNA-containing solution was added dropwise to the EP tube containing the transfection reagent, gently flicked to mix evenly, and incubated at room temperature for 15 min. The cells were taken out of the incubator, and the DNA mixture was carefully added dropwise into the cell culture medium, mixed evenly, and then the cells were placed back into the incubator for continued culture. After 4 h of transfection, the medium was replaced with complete medium for continued culture. After 48 h of transfection, the virus products were collected and filtered through a 0.45 μm pvdf filter membrane, and the virus supernatant was stored at -80 °C.
[0056] 2) Infected target cells
[0057] The target cells were HEK 293 cells. On the day of virus infection, the frozen lentivirus was taken out from the -80 °C refrigerator and thawed on ice. The original culture medium of the target cells was discarded, and the virus supernatant was added. At the same time, a polybrene solution with a final concentration of 8 μg / ml was added. The culture dish was gently shaken to mix and cover the cells. The culture dish was placed in an incubator and cultured overnight at 37 °C and 5% CO2. After 24 h, the medium containing the virus was discarded and the required fresh DMEM medium was replaced. The culture dish was placed in the incubator and cultured overnight at 37 °C and 5% CO2. After 48 h of infection, the culture solution was discarded, and fresh DMEM cell culture solution containing 1 μg / ml blasticidin solution was added and screened and cultured at 37 °C and 5% CO2. The cells were observed within the next week, and 1 μg / mL blasticidin was ensured to be contained in the medium during passage. One week after transfection, a small amount of cells were taken for Western blotting detection to observe whether there was a stable overexpression effect.
[0058] Example 4 Identification of stable transfected cell line
[0059] 1) Immunofluorescence
[0060] The stable transfected cell line screened in Example 4 was treated with biotin and catalyzed by H2O2, and then washed 3 times with PBS; subsequently, cell fixation was carried out, and 4% paraformaldehyde (diluted with PBS) was added and fixed at room temperature for 20 min, and then washed three times with PBS; cell perforation was carried out, and 0.1% TritonX-100 (diluted with PBS) was added for 10 min, and then washed three times with PBS; blocking was carried out, and 1% BSA (diluted with PBS) was added and blocked at room temperature for 45 min, and then washed three times with PBS; primary antibody incubation was carried out, with a ratio of 1:5000 (prepared with 1% BSA), and incubated overnight at 4 °C. The next day, the primary antibody was discarded, and Strep-Cy3 fluorescent secondary antibody (prepared with 1% BSA) was incubated at room temperature for 1 h, and then washed three times with PBS, paying attention to light avoidance; nuclear staining was carried out, and Hoechst dye was added, with a ratio of 1:2000 (diluted with PBS), and incubated in the dark for 5 min, and then washed twice with PBS; mounting was carried out. 17 μL of anti-fluorescence quencher was added dropwise onto the glass slide. The coverslip was carefully taken out with a syringe needle, with one side first against the bottom, and carefully placed downwards. The anti-quencher was evenly contacted with the cells, and nail polish was dropped around and placed on the glass slide, and left at room temperature for 10 min - 20 min; photographing was carried out.
[0061] As Figure 4 shown, it is the co-localization of Myc-METTL3-nAPEX2 and Flag-METTL14-cAPEX2 in the cells. It can be seen from the immunofluorescence detection results that a stable transfected cell line expressing METTL3-METTL14-APEX2 was constructed, and it had a good labeling efficiency.
[0062] 2) Verification of the specificity of the APEX2 proximity labeling system by IP-Western Blotting
[0063] Treat the cell line stably expressing the recombinant protein with biotin-phenol. The experiment is divided into a control group (-H2O2) and an experimental group (+H2O2). Add H2O2 to catalyze the reaction for 1 minute in the experimental group, and then immediately terminate the reaction with the termination solution. Scrape the cells in each plate with 1 mL of pre-cooled PBS buffer. After collecting the cell suspension, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and retain the cell pellet; resuspend the cell pellet with RIPA-buffer, place it on ice for lysis for 20 min, and then perform sonication; after sonication, filter the cell debris, add streptavidin magnetic beads and incubate overnight at 4°C; take the magnetic beads, wash the magnetic beads 5 times with high-salt washbuffer, resuspend the magnetic beads in 30 μL of 2×SDS loading buffer, fully lyse the cells and denature the proteins, incubate with METTL3, METTL14, WTAP, HAKAI antibodies and incubate the whole lane with streptavidin antibody. The experimental results are as Figure 5 shown. The experimental results show that compared with the -H2O2 group, the WTAP signal and HAKAI signal in the +H2O2 group are significantly enriched, indicating that under the catalysis of hydrogen peroxide, the APEX2 enzyme can efficiently mediate the biotinylation reaction and specifically enrich the known m 6 A methyltransferase complex METTL3, METTL14, WTAP and HAKAI proteins. This result proves that the split APEX2 proximity labeling system has high specificity.
Claims
1. A fusion proteome based on m 6 A methyltransferase, characterized in that, The fusion protein group includes a first fusion protein and a second fusion protein; the first fusion protein is obtained by binding the amino acid fragment nAPEX2 at positions 1 to 201 of the APEX2 enzyme to the METTL3 protein through a first linker peptide and then binding the Myc tag protein; the second fusion protein is obtained by binding the amino acid fragment cAPEX2 at positions 201 to 250 of the APEX2 enzyme to the METTL14 protein through a second linker peptide and then binding the Flag tag protein; wherein, the amino acid sequence of the nAPEX2 from the N-terminus to the C-terminus is as shown in SEQ ID NO: 1 in the sequence listing; the amino acid sequence of the cAPEX2 from the N-terminus to the C-terminus is as shown in SEQ ID NO: 2 in the sequence listing.
2. The fusion proteome according to claim 1, characterized in that The amino acid sequence of the first linker peptide from the N-terminus to the C-terminus is as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the second linker peptide from the N-terminus to the C-terminus is as shown in SEQ ID NO: 4 in the sequence listing.
3. The fusion proteome according to claim 1, wherein The amino acid sequence of the first fusion protein from the N-terminus to the C-terminus is as shown in SEQ ID NO: 5 in the sequence listing; the amino acid sequence of the second fusion protein from the N-terminus to the C-terminus is as shown in SEQ ID NO: 6 in the sequence listing.
4. A recombinant plasmid group comprising the fusion protein group according to claim 1, wherein the recombinant plasmid group is composed of two recombinant plasmids that respectively express the first fusion protein and the second fusion protein.
5. The construction method of the recombinant plasmid group according to claim 4, characterized in that, The construction method includes the steps of: first constructing fusion gene fragments that express the first fusion protein and the second fusion protein respectively, and recombining these two gene fragments onto the FKBP-V5-AP-nes_pLX304 and HA-Halotag-FRB-EX-nes_pLX304 expression vectors respectively.
6. Use of the recombinant plasmid according to claim 4 in constructing a cell model of a proximity labeling system that stably expresses METTL3-METTL14-APEX2.
7. The application according to claim 6, wherein The use includes the steps of: co-transfecting the successfully constructed recombinant plasmid into HEK293 cells, labeling with biotin phenol, catalyzing with H2O2, and detecting the labeling effect by Western Blotting; constructing a stable cell line that expresses the first fusion protein and the second fusion protein by lentiviral packaging technology for the above-mentioned constructed recombinant plasmid, performing resistance screening with blasticidin, and combining immunofluorescence to detect the labeling efficiency.