Design and construction of mTORC1 fluorescent probe SEaTOR and application of mTORC1 fluorescent probe SEaTOR

Abstract

The invention relates to design and construction and application of an mTORC1 fluorescent probe, and belongs to the technical field of biomedicine and cell imaging. The invention discloses an mTORC1 fluorescent probe, the structure of the mTORC1 fluorescent probe comprises an induction structural domain and two fluorescent modules at the two ends of the induction structural domain, and the induction structural domain is p70S6K1 protein or a fragment thereof; and the mTORC1 fluorescent probe can also be connected with a TOS Motif (Total Operating System Motif). The mTORC1 fluorescent probe is high in detection sensitivity, the activity state of mTORC1 can be represented only by observing the intensity of a fluorescence signal, and the mTORC1 fluorescent probe is simple, visual and easy to popularize and apply.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and cell imaging, and particularly relates to the design, construction and application of an mTORC1 fluorescent probe based on p70S6K1. Background Art

[0002] Mammalian target of rapamycin (mTOR) is a serine / threonine protein kinase that is highly conserved in evolution and exists in different species such as rats and humans. It belongs to the phosphatidylinositol kinase-related kinase (PIKK) protein family. Intracellular mTOR is involved in the formation of two different complexes, mTORC1 and mTORC2. Although the protein components of the two complexes are partially the same, they can phosphorylate different substrates. Among them, mTORC1 regulates key biological processes including cell growth, metabolism and protein synthesis, largely by phosphorylating two key effectors, 70KD ribosomal protein S6 kinase 1 (p70S6K1) and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 4EBP (Ahmed, A.R., et al., Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells. Sci Rep, 2019. 9(1): p. 3408.). They respectively promote cap-dependent translation and EJC-related translation through their downstream effectors.

[0003] Studies have shown that mTOR plays an important regulatory role in biological processes such as cell proliferation, survival, metabolism, autophagy, apoptosis, and migration. Abnormalities in the signaling pathways mediated by it (such as the PI3K / Akt / mTOR signaling pathway, mTOR / p70S6K signaling pathway, AMPK / mTOR signaling pathway, etc.) are involved in the occurrence and development of many human diseases. Studies on model organisms by scientists have shown that inhibiting mTORC1 or the downstream signaling pathway of mTORC1 can extend the lifespan of yeast, nematodes, and fruit flies. It has also been found that the activity level of mTORC1 signal is relatively lower in the long-lived Ames dwarf mouse model compared to normal mice, and this signal plays an important role in the longevity of mice. And the follow-up study on the primate marmoset has shown that mTORC1 is also related to its healthy lifespan. So far, researchers have not found evidence that mTORC2 plays a role by phosphorylating S6K1. Therefore, more studies focus on the recruitment and localization of mTORC1 complex in living cells and the phosphorylation of S6K1. Monitoring the activity of mTORC1 at the cellular and in vivo levels is an important technical means for basic research on the mTOR signaling pathway and drug development.

[0004] To achieve this goal, the methods mainly used by current researchers can be classified into two categories. One is the biosensor based on bioluminescence resonance energy transfer (BRET), and the other is the biosensor based on fluorescence resonance energy transfer (FRET). For example, the BRET-based biosensor (AIMTOR) designed by Nathalie Bouquier et al. (Bouquier, N., et al., AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions. BMC Biol, 2020. 18(1): p. 81.) contains a phosphorylatable T757 peptide derived from the mTOR substrate, ULK1, a flexible linker, and a WW domain that can recognize phosphorylated serine or threonine residues. These components are located between a nanoluciferase donor that emits light at 460 nm after binding furacilin and a YPET acceptor protein that emits light at 530 nm. After mTOR phosphorylation, the WW domain binds to the phosphorylated peptide, shortening the distance between the nanoluciferase and YPET, which results in an increase in BRET detected using a photometer or microscope device in living cells. Xin Zhou et al. (Zhou, X., et al., Dynamic Visualization of mTORC1 Activity in Living Cells. Cell Rep, 2015. 10(10): p. 1767-1777.) designed and characterized a genetically encoded mTORC1 activity reporter (TORCAR) based on FRET, enabling the characterization of mTORC1 signaling dynamics in single living cells. The principles of these two types of biosensors are similar. Both have a kinase activity-dependent molecular switch flanked by a pair of fluorescent proteins / nanoluciferases or a pair of FRET fluorescent proteins. The engineered molecular switch is constructed by coupling a sensing domain (usually a peptide substrate that can be recognized and phosphorylated by the kinase of interest) to a domain that specifically binds phosphoamino acids (phosphoamino acid binding domain [PAABD]). When phosphorylated, the substrate falls into the binding pocket of the PAABD, and this engineered conformational change of the molecular switch can be interpreted as a change in BRET or FRET.

[0005] However, both BRET- and FRET-based mTOR biosensors have obvious limitations. An ideal BRET or FRET interaction system requires a suitable pair of fluorescent substances, that is, there is a significant overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor; and the excitation wavelength of the donor should not affect the acceptor, and the emission spectra of the donor and the acceptor should be completely separated, otherwise it is easy to cause spectral interference and make the reaction system unstable. For BRET-based biosensors, imaging requires a dedicated inverted fluorescence microscope for bioluminescence, and an EMCCD detector and an evolved camera with back-illuminated on-chip multiplication gain are required to collect imaging data. In addition, a specific luciferase substrate needs to be applied a few minutes before imaging, and the action time is strictly required, making it difficult to apply to experiments with a large time span. For FRET-based fluorescence lifetime imaging, the fluorescence lifetime needs to be measured at each pixel point, so the imaging speed is relatively slow, resulting in certain limitations in imaging applications that require rapid detection of protein activity; and this imaging method also requires special and costly imaging equipment, restricting its popularization and application in some laboratories and research institutions.

[0006] With the in-depth study of the structure-function of fluorescent proteins, researchers have found that by restructuring the structure of fluorescent proteins through methods such as circular permutation to rearrange and recombine their C- and N-terminal positions, inserting foreign fragments at specific sites, or splitting and then recombining them, fluorescent proteins can fold correctly and emit fluorescence, and their spectral properties remain unaffected. These specific sites have gradually evolved into sites for splitting fluorescent proteins during structural restructuring. Among them, a representative technique in the structural restructuring of fluorescent proteins is bimolecular fluorescence complementation (BiFC). Currently reported fluorescent proteins used for BiFC detection include GFP, BFP, CFP, YFP, Venus, citrine, cerulean, mCherry, etc. Oksana M. Subach et al. (Subach, O.M., et al., YTnC2, an improved genetically encoded green calcium indicator based on toadfish troponin C. FEBS Open Bio, 2023. 13(11): p. 2047-2060.) introduced four internal mutations from the bright fluorescent protein mClover3 into the fluorescent domain of EYFP in the gene-encoded calcium probe YTnC based on truncated troponin C, and developed an enhanced version of YTnC2 (with 20 amino acid mutations compared to YTnC) using directed molecular evolution in the bacterial system, increasing the brightness and dynamic range of the YTnC indicator. Based on BiFC detection, it is possible to directly judge whether proteins interact by observing the presence or absence of fluorescent signals using a fluorescence microscope, which has the characteristics of simplicity and intuitiveness, and has been widely used in the visualization detection of interactions between biological macromolecules. BiFC can also be used in drug development. First, important proteins related to diseases are selected, and BiFC stable cell lines are developed based on BiFC technology. After treatment with different drugs, fluorescent signals are detected to screen for potential therapeutic drugs. Therefore, it has good application prospects in high-throughput screening technologies for finding potential disease treatment targets in pathways. For example, DiscoverX Corporation has developed a series of BiFC cell lines related to GPCRs for screening drugs related to the GPCR family.

[0007] Therefore, it is of great significance to develop a high-performance and popular mTOR-specific probe based on BiFC technology to improve its convenience in practical applications for research related to the mTOR pathway and high-throughput screening technologies for finding potential disease treatment targets in the pathway. Summary of the Invention

[0008] Inspired by previous research, the inventors introduced four internal mutations from the bright fluorescent protein mClover3 into the fluorescent domain of EYFP to increase the brightness and dynamic range of the fluorescence index. Meanwhile, a BiFC-based detection method was adopted, and the activity state of mTORC1 could be characterized simply and intuitively by observing the intensity of the fluorescence signal, which was easy to popularize and apply.

[0009] The object of the present invention is to overcome the defects existing in the above-mentioned background technology, and to provide a design, construction and application of an mTORC1 fluorescent probe SEaTOR (S6K1-EYFP activity reporter of mTOR) based on the complementary technology of p70S6K1 and EYFP fluorescent protein, which has a large dynamic range, high detection sensitivity, is easy to popularize and apply, and can detect the change of the activity state of mTORC1 after cells are stimulated.

[0010] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] One aspect of the present invention provides an mTORC1 fluorescent probe SEaTOR.

[0012] In some embodiments, the present invention provides the application of the fluorescent probe in fluorescence imaging.

[0013] In some embodiments, the amino acid sequence of SEaTOR is as shown in SEQ ID NO. 1, and preferably the nucleotide sequence encoding the protein is as shown in SEQ ID NO. 2.

[0014] Furthermore, the peak value of the single-photon absorption wavelength of the fluorescent probe SEaTOR is 514 nm.

[0015] Furthermore, the single-photon excitation wavelength of the fluorescent probe SEaTOR is 480 - 520 nm.

[0016] On the other hand, the present invention provides a preparation method of an mTORC1 fluorescent probe SEaTOR. The method is as follows: using the substrate p70S6K1 downstream of mTORC1 as the sensing domain, and connecting the two ends thereof with the sequences of the amino-terminal part and the carboxyl-terminal part of the yellow fluorescent protein EYFP (the two parts constitute the fluorescent module of the probe), so that it contains a mutated amino acid sequence, and thus the fluorescent probe SEaTOR is obtained.

[0017] Furthermore, the sensing domain p70S6K1 of the fluorescent probe SEaTOR is the sequence of rat-derived S6K1 after removing the nuclear localization sequence (amino acids 25 - 525), with a mutation of R367P. Its amino acid sequence is shown in SEQ ID NO. 3, and the nucleotide sequence encoding the protein is shown in SEQ ID NO. 4.

[0018] Furthermore, the fluorescent module EYFP of the fluorescent probe SEaTOR is cleaved at the 145th amino acid to form two fluorescent modules, an amino-terminal (amino acids 1 - 145) and a carboxyl-terminal (amino acids 146 - 238), and four internal mutations from the mClover3 bright fluorescent protein are introduced. The complete amino acid sequence of EYFP is shown in SEQ ID NO. 5, and the nucleotide sequence encoding the protein is shown in SEQ ID NO. 6.

[0019] Specifically, to construct the fluorescent probe SEaTOR, the upstream primer containing the NheI restriction site (N-EYFPNheI up (SEQ ID NO. 7)) and the downstream primer containing the KpnI restriction site and the FDL linker sequence (between N-EYFP and p70S6K1) (N-EYFP KpnI down (SEQ ID NO. 8)) were used to amplify the amino-terminal part of the mutant EYFP. The upstream primer containing the MluI restriction site and the GI linker sequence (between C-EYFP and p70S6K1) (C-EYFP MluI up (SEQ ID NO. 9)) and the downstream primer containing the NotI restriction site (C-EYFP NotI down (SEQ ID NO. 10)) were used to amplify the carboxyl-terminal part of the mutant EYFP. The EYFP plasmid template (SEQ ID NO. 11) for amplification was synthesized by Sangon Biotech. The amplified N-EYFP and C-EYFP fragments were loaded onto the pRK5 plasmid vector (addgene 96895) through homologous recombination (Vazyme C113-02) to obtain the pRK5-EYFP (SEQ ID NO. 25) plasmid. Using the sequence shown in SEQ ID NO. 12 as the template for amplifying p70S6K1, the upstream primer used was p70S6K1 KpnI up containing the KpnI restriction site (SEQ ID NO. 13), and the downstream primer was p70S6K1 MluI down containing the MluI restriction site (SEQ ID NO. 14). The amplified p70S6K1 fragment was loaded onto the pRK5-EYFP plasmid by restriction enzyme digestion and ligation methods, and thus the fluorescent probe SEaTOR we needed was obtained. All recombinant vectors were identified by sequencing.

[0020] On the other hand, the present invention provides an application of the mTORC1 fluorescent probe SEaTOR in the detection of mTORC1 signals, wherein the excitation wavelength of the single photon used in the signal detection is 488 nm.

[0021] On the other hand, the present invention provides an application of the mTORC1 fluorescent probe SEaTOR in the detection of mTORC1 signals in living cells, wherein the excitation wavelength of the single photon used in the signal detection is 488 nm.

[0022] On the other hand, the present invention provides a fluorescence imaging detection method capable of characterizing the active state of mTORC1 in living cells, comprising the following steps:

[0023] (1) Transfect the mTORC1 fluorescent probe SEaTOR into mammalian cells;

[0024] (2) Perform imaging analysis using a fluorescence microscope to detect the intensity change of the probe fluorescence, wherein the excitation wavelength of the single photon used in the imaging analysis is 488 nm;

[0025] Preferably, it comprises the following steps:

[0026] (1) Transfect the mTORC1 fluorescent probe SEaTOR into mammalian cells;

[0027] (2) Stimulate mammalian cells to increase or decrease their intracellular mTORC1 activity;

[0028] (3) Perform imaging analysis using a fluorescence microscope to detect the intensity change of the probe fluorescence before and after the above stimulation, wherein the excitation wavelength of the single photon used in the imaging analysis is 488 nm.

[0029] On the other hand, the fluorescent probe SEaTOR provided by the present invention uses the substrate p70S6K1 downstream of mTORC1 as the sensing domain, and its two ends are respectively connected with a sequence of the EYFP fluorescent protein in the Figure 1 shown manner to make it contain a mutant amino acid sequence, thus obtaining the fluorescent probe SEaTOR.

[0030] On the other hand, the fluorescent probe SEaTOR provided by the present invention is a yellow mTORC1 probe, with an absorption wavelength peak of 514 nm and an emission wavelength peak of about 525 nm. In cells cultured at a physiological temperature of 37 °C, under 488 nm single photon excitation, SEaTOR has a range (ΔF / F0) of about 0.28 - 3.52, and the average value of ΔF / F0 reaches 1.33, with a relatively large range and high detection sensitivity in cell imaging, and can simply and intuitively characterize the activity of mTORC1.

[0031] On the other hand, the fluorescent probe SEaTOR provided by the present invention is most different from the prior art in that the bimolecular fluorescence complementation (BiFC) technology is introduced into the detection of mTORC1 activity. A single fluorescent protein is divided into two non-fluorescent fragments, which are fused to both ends of p70S6K1. After mTORC1 is activated, p70S6K1 is phosphorylated and undergoes a conformational change, so that the two fragments of the fluorescent protein can complement each other to form a complete fluorescent protein and emit fluorescence.

[0032] Compared with the existing mTORC1 sensors based on BRET or FRET, the probe provided by the present invention only requires a single fluorescent protein instead of a pair of demanding fluorescent substances. Transfecting the fluorescent probe SEaTOR provided by the present invention into mammalian cells, the change of the mTORC1 activity state after the cells are stimulated by a specific stimulus can be detected by using an ordinary fluorescence microscope.

[0033] A fluorescence imaging detection method for characterizing the mTORC1 activity state in living cells, the method comprising the following steps:

[0034] (1) Cell culture, the complete medium is DMEM (Gibco 11965-092) containing 10% fetal bovine serum (Gibco 10099-141), 1% Glutamax (Gibco 35050-061), 1% sodium pyruvate (Gibco 11360-070), 1% MEM-NEAA (Gibco 11140-050) and 1% penicillin-streptomycin (Gibco 15140-122), the culture temperature is 37 °C, and the CO2 content is 5%;

[0035] When the cell density is 50±5%, transfect the SEaTOR plasmid with the CalPhos Mammalian Transfection Kit (Takara 631312) kit;

[0036] (2) The transfected cells are cultured overnight, and the cells are starved for 8 h with a serum-free medium (the serum-free medium is DMEM containing 1% Glutamax, 1% sodium pyruvate, and 1% penicillin-streptomycin), the culture temperature is 37 °C, and the CO2 content is 5%;

[0037] (3) Fluorescence microscope imaging analysis, wherein the excitation wavelength of single photons is 488 nm.

[0038] The present invention also provides another fluorescence imaging detection method for characterizing the mTORC1 activity in living cells.

[0039] A fluorescence imaging detection method for characterizing mTORC1 activity in mammalian cells, the method comprising the following steps:

[0040] (1) Cell culture, the complete medium is DMEM containing 10% fetal bovine serum, 1% Glutamax, 1% sodium pyruvate, 1% MEM-NEAA and 1% penicillin-streptomycin, the culture temperature is 37 °C, and the CO2 content is 5%;

[0041] When the cell density is 50 ± 5%, transfect the SEaTOR plasmid using the CalPhos Mammalian Transfection Kit (Takara 631312) kit;

[0042] (2) Incubate the transfected mammalian cells overnight, starve the cells for 8 hours with serum-free medium (the serum-free medium is DMEM containing 1% Glutamax, 1% sodium pyruvate, and 1% penicillin-streptomycin), and perfuse with the complete medium containing serum for 50 minutes to promote cell growth and division;

[0043] (3) Fluorescence microscopy imaging analysis, wherein the excitation wavelength of single photon is 488 nm.

[0044] The present invention also provides another fluorescence imaging detection method for characterizing mTORC1 activity in living cells.

[0045] A fluorescence imaging detection method for characterizing mTORC1 activity in living cells, the method comprising the following steps:

[0046] (1) Cell culture, the medium is DMEM containing 10% fetal bovine serum, 1% Glutamax, 1% sodium pyruvate, 1% MEM-NEAA and 1% penicillin-streptomycin, the culture temperature is 37 °C, and the CO2 content is 5%;

[0047] When the cell density is 50 ± 5%, transfect the SEaTOR plasmid using the CalPhos Mammalian Transfection Kit (Takara 631312) kit;

[0048] (2) Incubate the transfected mammalian cells overnight, add 200 nM Rapamycin (mTORC1 inhibitor, MCE HY-10219) for 5 minutes to fully inhibit mTORC1;

[0049] (3) Fluorescence microscopy imaging analysis, wherein the excitation wavelength of single photon is 488 nm.

[0050] The fluorescent probe SEaTOR provided by the present invention uses the substrate p70S6K1 downstream of mTORC1 as the sensing domain, and both ends thereof are connected to a sequence of EYFP fluorescent protein in the Figure 1 manner shown, so that it contains a mutated amino acid sequence, and thus the fluorescent probe SEaTOR is obtained.

[0051] The present invention provides the following technical solutions:

[0052] 1. An mTORC1 fluorescent probe, characterized in that the structure of the probe includes a sensing domain of p70S6K1 protein or a fragment thereof and two fluorescent modules at both ends of the sensing domain.

[0053] 2. The mTORC1 fluorescent probe according to item 1, characterized in that the amino acid sequence of the sensing domain is as shown in SEQ ID NO. 3 or SEQ ID NO. 16.

[0054] 3. The mTORC1 fluorescent probe according to item 1 or 2, characterized in that the nucleotide sequence encoding the sensing domain of the probe is as shown in SEQ ID NO. 4 or SEQ ID NO. 17.

[0055] 4. The mTORC1 fluorescent probe according to any one of items 1-3, characterized in that the probe further includes a TOS Motif, and the sequence of the TOS Motif is as shown in SEQ ID NO.19, and preferably the TOS Motif is located at the N-terminus.

[0056] 5. A method for preparing an mTORC1 fluorescent probe, characterized in that the substrate p70S6K1 protein or a fragment thereof downstream of mTORC1 is used as the sensing domain, and both ends thereof are respectively connected to the amino-terminal part and the carboxyl-terminal part of a fluorescent protein to prepare an mTORC1 fluorescent probe;

[0057] Preferably, the mTORC1 fluorescent probe is further connected with a TOS Motif, more preferably the TOS Motif is located at the N-terminus, and the sequence of the TOS Motif is as shown in SEQ ID NO.19.

[0058] 6. The method according to item 5, characterized in that the arginine (R) at the 367th position of the amino acid sequence of the p70S6K1 protein is replaced by proline (P);

[0059] Preferably, the amino acid sequence of the p70S6K1 protein is as shown in SEQ ID NO. 3; preferably, the fragment of the p70S6K1 protein is Linker-Hydrophobic Motif, and its amino acid sequence is as shown in SEQ ID NO. 16.

[0060] 7. The method according to item 5 or 6, wherein the fluorescent protein is yellow fluorescent protein EYFP.

[0061] 8. The method according to any one of items 5-7, wherein the sequence of the p70S6K1 protein is the sequence of S6K1 after removing the nuclear localization sequence, that is, amino acids 25-525.

[0062] 9. Application of the mTORC1 fluorescent probe defined in any one of items 1-4 in the detection of mTORC1 signal, wherein the mTORC1 fluorescent probe is used to detect the activity of mTORC1 in living cells, preferably to detect the change of the activity state of mTORC1.

[0063] 10. A fluorescent imaging detection method for characterizing the activity state of mTORC1 in living cells, comprising the following steps:

[0064] (1) Transfect the mTORC1 fluorescent probe defined in any one of items 1-4 into mammalian cells;

[0065] (2) Perform imaging analysis using a fluorescence microscope to detect the change in the fluorescence intensity of the mTORC1 fluorescent probe, and the excitation wavelength of the single photon used in the imaging analysis is 488 nm;

[0066] Preferably, it includes the following steps:

[0067] (1) Transfect the mTORC1 fluorescent probe defined in any one of items 1-4 into mammalian cells;

[0068] (2) Stimulate mammalian cells to increase or decrease the intracellular mTORC1 activity;

[0069] (3) Perform imaging analysis using a fluorescence microscope to detect the change in the fluorescence intensity of the mTORC1 fluorescent probe before and after the above stimulation, and the excitation wavelength of the single photon used in the imaging analysis is 488 nm.

[0070] 11. Application of an mTORC1 fluorescent probe in the preparation of a kit for disease detection or diagnosis, wherein the kit includes the mTORC1 fluorescent probe defined in any one of items 1-4.

[0071] 12. The application according to item 11, wherein the disease is a disease affected by mTORC1 expression, including cancer, neurodegenerative diseases, and metabolic diseases.

[0072] Compared with the prior art, the advantages of an mTORC1 fluorescent probe SEaTOR, its design and construction, application, and kit provided by the present invention are as follows:

[0073] (1) It is easy to popularize and apply, has low requirements for fluorescent substances and imaging equipment, and only requires a common confocal microscope to complete the acquisition of data, and can simply and intuitively characterize the activity of mTORC1.

[0074] (2) It has high detection sensitivity.

[0075] (3) It has a large range.

[0076] (4) It can be used in the detection of the active state of mTORC1 in living cells.

[0077] (5) It can complete detection over a longer time span.

[0078] On the other hand, the present invention provides an application of an mTORC1 fluorescent probe SEaTOR in the preparation of a disease detection or diagnosis kit, wherein the kit includes the fluorescent probe SEaTOR.

[0079] On the other hand, the present invention provides an application of an mTORC1 fluorescent probe SEaTOR in the preparation of a disease detection or diagnosis kit, wherein the disease is a disease affected by mTORC1 expression, preferably cancer, neurodegenerative diseases, and metabolic diseases.

[0080] The term "sensing domain" refers to a peptide substrate that can be recognized and phosphorylated by a kinase of interest. For example, the p70S6K1 protein or its fragment that can be used in the SEaTOR probe of the present invention.

[0081] In some embodiments, the sensing domains involved in the present invention include: the p70S6K1 protein module, the fragment AGC-kinase C-terminal Domain (S6K1 353-419AA, SEQ ID NO.15) or its fragment Linker-Hydrophobic Motif (S6K1 381-417AA, SEQ ID NO. 16) that changes the conformation of p70S6K1 after phosphorylation, and Turn-Hydrophobic Motif (S6K1 392-417AA, SEQ ID NO. 18).

[0082] In some embodiments, the sensing domain may additionally include a TOS Motif (S6K128 - 32AA, SEQ ID NO. 19) at the N - terminus or C - terminus to assist in the localization of the probe and its phosphorylation by mTORC1.

[0083] The term "fluorescent module" refers to two or more non - fluorescent fragments into which a complete fluorescent protein is divided. When they complement each other to form a complete fluorescent protein, they can fold correctly and emit fluorescence without affecting their own spectral properties. For example, in the SEaTOR probe of the present invention, the EYFP protein can be divided into two fluorescent protein fragments, N - EYFP and C - EYFP, through a cleavage site in the EYFP protein.

[0084] In some embodiments, the cleavage sites between the fluorescent module N - EYFP and C - EYFP of the present invention may include, for example, the cleavage sites between amino acids 145 and 146, and between amino acids 166 and 167.

[0085] In some embodiments, the fluorescence change range of the probe can be adjusted and the applicable range of the probe can be increased by mutating amino acids at different sites (for example, adding mutations I48L, S175G, S208F, V224L, H231F, and D234N (SEQ ID NO. 20) of the YPet fluorescent protein to the existing EYFP; the positions of the above - mentioned mutation sites correspond to the EYFP sequence); or changing the protein serving as the fluorescent module to fragments of fluorescent proteins such as mNeonGreen (SEQ ID NO. 21, whose cleavage site includes, for example, the cleavage site between amino acids 145 and 146, and inserting a linker sequence EAQ before the cleavage site and a linker sequence IYF after the cleavage site), EGFP (SEQ ID NO. 22, whose cleavage site includes, for example, the cleavage site between amino acids 145 and 146, and inserting a linker sequence LE before the cleavage site and a linker sequence TR after the cleavage site). BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1, is a schematic diagram of the principle for detecting the activity state of mTORC1 using a p70S6K1-based mTORC1 probe provided by the present invention. After adding serum, mTORC1 is activated and phosphorylates its downstream substrate p70S6K1, resulting in a conformational change of p70S6K1, causing EYFP-N and EYFP-C at both ends of p70S6K1 to approach and form a complete EYFP, thus emitting fluorescence. After adding Rapamycin (an mTORC1 inhibitor), the downstream substrate p70S6K1 of mTORC1 is dephosphorylated, and the conformation of p70S6K1 returns to that before phosphorylation, causing EYFP-N and EYFP-C at both ends to separate, leading to a decrease or even disappearance of most of the fluorescence.

[0087] Figure 2 , is a confocal microscopy image showing the activity state of mTORC1 in HEK 293T cells under different conditions, DIV 3. From left to right: a, the activity of mTORC1 when 293T cells are in a serum-free starvation state for a long time (8 hours) (the brightness of the flame false color represents the average fluorescence intensity of the mTORC1 probe in HEK 293T cells within 30 seconds, and the fluorescence intensity values represented by the colors are as shown in the color scale below Figure c. The bright areas represent where mTORC1 is activated); b, the activity of mTORC1 50 minutes after adding serum to 293T cells; c, the activity of mTORC1 5 minutes after adding 200 nM Rapamycin (an mTORC1 inhibitor) to HEK 293T cells; d, the fluorescence of mCherry-βActin shows the morphology of HEK 293T cells, and the scale bar in the figure is 10 μm.

[0088] Figure 3 , is a waveform diagram of the fluorescence change (ΔF / F0) of the mTORC1 probe in HEK 293T cells after adding serum following 8 hours of serum-free starvation.

[0089] Figure 4 , is a waveform diagram of the fluorescence change (ΔF / F0) of the mTORC1 probe after adding 200 nM Rapamycin to 293T cells.

[0090] Figure 5 , is a statistical chart of the fluorescence intensity of the mTORC1 probe after 8 hours of starvation, after adding serum (>50 minutes), and after adding 200 nM Rapamycin (>5 minutes). Each black dot on the graph represents the data of a 293T cell, ***, p < 0.001.

[0091] Figure 6, is a statistical graph of the FRET ratio of the mTORCAR probe after 8 hours of starvation, after adding serum (>50 minutes), and after adding 200 nM Rapamycin (>5 minutes). Each black dot on the graph represents the data of a 293T cell, n.s., no significant, *, p < 0.05, ***, p < 0.001.

[0092] Figure 7 , is a schematic diagram of the principle for detecting the active state of mTORC1 by the mTORC1 probe based on the p70S6K1 fragment Linker-Hydrophobic Motif (LH Motif) provided by the present invention. After adding serum, mTORC1 is activated and phosphorylates its downstream substrate LH Motif, resulting in a conformational change of LH Motif, causing EYFP-N and EYFP-C at both ends of LH Motif to approach and form a complete EYFP, thus emitting fluorescence. After adding Rapamycin (an mTORC1 inhibitor), the downstream substrate LH Motif of mTORC1 is dephosphorylated, and the conformation of LH Motif returns to that before phosphorylation, causing EYFP-N and EYFP-C at both ends of it to separate, resulting in a decrease or even disappearance of most of the fluorescence.

[0093] Figure 8 , is a statistical graph of the fluorescence intensities of the mTORC1 probes SEaTOR and SEaTOR-LH and the cytoskeleton mCherry-βActin after 8 hours of starvation and after adding serum (>50 minutes). Each black dot on the graph represents the data of a 293T cell, ***, p < 0.001, n.s., no significant.

[0094] Figure 9, is a confocal microscopy image showing the fluorescence intensity of HEK 293T cells under different conditions, DIV3. From top to bottom are: a, the fluorescence intensity of the mTORC1 probe SEaTOR-LH in 293T cells after adding 50 ng / ml PDGF growth factor (5 minutes, 10 minutes, 20 minutes, >25 minutes) and after adding 200 nM Rapamycin (the brightness of the flame false color represents the fluorescence intensity of the mTORC1 probe in HEK 293T cells during shooting, and the fluorescence intensity values represented by the colors are shown in the color scale below Figure a. The bright areas represent where mTORC1 is activated); b, the fluorescence intensity of mCherry-βActin expressed in 293T cells under different conditions; c, the fluorescence intensity of pRK5-EYFP in 293T cells after adding 50 ng / ml PDGF growth factor (5 minutes, 10 minutes, 20 minutes, >25 minutes) and after adding 200 nM Rapamycin (the fluorescence intensity values represented by the flame false color are shown in the color scale below Figure c). The scale bar in the figure is 10 μm.

[0095] Figure 10 , is a statistical chart of the fluorescence intensity changes of the mTORC1 probe SEaTOR-LH, pRK5-EYFP and the cytoskeleton mCherry-βActin after adding 50 ng / ml PDGF growth factor (5 minutes, 10 minutes, 20 minutes, >25 minutes). ***, p < 0.001, n.s., no significant; pRK5-EYFP is the original plasmid (without the sensing domain) used to construct the probe. Detailed implementation method

[0096] To enable those skilled in the art to better understand the technical solution of the present invention, the following examples further describe the present invention in detail. The following examples are only for more detailed illustration and do not limit the scope of the present invention.

[0097] Example 1: Construction of SEaTOR

[0098] Combined with Figure 1 shown, using p70S6K1 that can bind to mTORC1 as the sensing domain, it is connected to the EYFP fluorescent protein in the manner shown in Figure 1 shown, so that it contains a mutated amino acid sequence, and thus the fluorescent probe SEaTOR is obtained. The amino acid sequence of SEaTOR is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0099] The sensing domain p70S6K1 is derived from rat, with the nuclear localization sequence removed and having an R367P mutation. EYFP-N and EYFP-C are the amino-terminal part and carboxyl-terminal part of the yellow fluorescent protein EYFP (the two parts constitute the fluorescent module of the probe), p70S6K1 (S6K1_rat 25 - 525AA) is the domain that undergoes conformational changes after being phosphorylated by mTORC1 (sensing module), and linker peptide 1 (amino acid sequence FDL) and linker peptide 2 (amino acid sequence GI) are the connecting parts between the sensing module and different parts of the fluorescent protein.

[0100] Example 2: Response of the fluorescent probe SEaTOR in HEK293T cells

[0101] HEK293T cells were cultured in a glass-bottom dish, and the culture medium was DMEM containing 10% fetal bovine serum, 1% Glutamax, 1% sodium pyruvate, 1% MEM-NEAA, and 1% penicillin-streptomycin. The culture temperature was 37 °C, and the CO2 content was 5%.

[0102] When the cell density reached 50 ± 5%, the SEaTOR plasmid was transfected using the CalPhos Mammalian Transfection Kit (Takara 631312).

[0103] The transfected mammalian cells were cultured overnight, starved with serum-free medium (serum-free medium was DMEM containing 1% Glutamax, 1% sodium pyruvate, and 1% penicillin-streptomycin) for 8 hours, and then imaged and analyzed using an Olympus IX-81-ZDC laser confocal microscope (objective lens was Olympus Plan Apo 100× NA 1.45, imaging CCD was ANDOR iXon DU-897D-500 camera, excitation light wavelength 488 nm, emission wavelength filter 515 / 30nm). The results are as Figure 2 shown in a.

[0104] The transfected mammalian cells were cultured overnight, starved with serum-free medium for 8 hours, then serum was added for 50 minutes to promote cell growth and division, and then the medium was replaced with a colorless and transparent buffer for fluorescence imaging. Imaging and analysis were performed using an Olympus IX-81-ZDC laser confocal microscope (excitation wavelength 488 nm, emission wavelength filter 515 / 30 nm), and it was found that the fluorescence intensity of SEaTOR increased significantly. The results are as Figure 2 shown in b.

[0105] The transfected mammalian cells were cultured overnight. After starving the cells for 8 hours with serum-free medium, 50 minutes after perfusion with complete medium containing serum, 200 nM Rapamycin (an mTORC1 inhibitor) was added for 5 minutes to fully inhibit mTORC1. It was found that the fluorescence intensity of SEaTOR was significantly reduced compared to before the addition of Rapamycin. Live cell imaging was performed throughout the process, and imaging analysis was carried out using an Olympus IX-81-ZDC laser confocal microscope (excitation wavelength 488 nm, emission wavelength filter 515 / 30 nm). The results are as Figure 2 shown in

[0106] Figure Figure 2 a. After starving the cells for 8 hours ( Figure 2 a), 50 minutes after serum stimulation ( Figure 2 b), and 5 minutes after inhibition with 200 nM Rapamycin ( Figure 2 c), confocal microscopy images of the average fluorescence intensity of SEaTOR in the cells. The fluorescence of mCherry-βActin in

[0107] Figure 3 Figure Figure 4 d showed the morphology of HEK 293T cells. The scale bar in the figure is 10 μm. Figure 5 Figure

[0108] is the waveform diagram of the fluorescence change (ΔF / F0) of the mTORC1 probe in 293T cells after perfusion with complete medium containing serum after 8 hours of serum-free starvation. The fluorescence change curve indicates that the fluorescence intensity of SEaTOR increased significantly after serum stimulation; Figure 3 Figure Figure 4 is the waveform diagram of the fluorescence change (ΔF / F0) of the mTORC1 probe in 293T cells after the addition of 200 nM Rapamycin. The fluorescence change curve indicates that the fluorescence intensity of SEaTOR decreased rapidly after the addition of Rapamycin to inhibit mTORC1. Figure 5 Figure

[0109] Example 3: Response of the mTORCAR probe in HEK293T cells

[0110] HEK293T cells were cultured in glass-bottomed dishes with DMEM medium containing 10% fetal bovine serum, 1% Glutamax, 1% sodium pyruvate, 1% MEM-NEAA, and 1% penicillin-streptomycin at a culture temperature of 37 °C and a CO2 content of 5%.

[0111] When the cell density reached 50 ± 5%, the mTORCAR plasmid was transfected using the CalPhos Mammalian Transfection Kit (Takara 631312).

[0112] The transfected mammalian cells were cultured overnight, starved for 8 hours with serum-free medium (serum-free medium was DMEM containing 1% Glutamax, 1% sodium pyruvate, and 1% penicillin-streptomycin), and then imaged and analyzed using a Zeiss LSM710 laser confocal microscope (objective: W Plan-Apochromat 20x / 1.0 DIC M27 70 mm; excitation wavelength of mCerulean was 458 nm, emission wavelength was 459 - 510 nm; excitation wavelength of YPet was 514 nm, emission wavelength was 522 - 614 nm; FRET Raw excitation wavelength was 458 nm, emission wavelength was 522 - 614 nm).

[0113] The transfected mammalian cells were cultured overnight, starved for 8 hours with serum-free medium, then serum was added for 50 minutes to promote cell growth and division, and then the medium was replaced with a colorless and transparent buffer for fluorescence imaging. Imaging and analysis were performed using a Zeiss LSM710 laser confocal microscope (objective: W Plan-Apochromat 20x / 1.0 DIC M27 70 mm; excitation wavelength of mCerulean was 458 nm, emission wavelength was 459 - 510 nm; excitation wavelength of YPet was 514 nm, emission wavelength was 522 - 614 nm; FRET Raw excitation wavelength was 458 nm, emission wavelength was 522 - 614 nm).

[0114] The transfected mammalian cells were cultured overnight. After starving the cells for 8 hours with serum-free medium, complete medium containing serum was perfused and added for 50 minutes, and then 200 nM Rapamycin (mTORC1 inhibitor) was added for 5 minutes to fully inhibit mTORC1. Live cell imaging was performed throughout the process using a Zeiss LSM710 laser confocal microscope (objective: W Plan-Apochromat 20x / 1.0 DIC M27 70 mm; excitation wavelength of mCerulean: 458 nm, emission wavelength: 459 - 510 nm; excitation wavelength of YPet: 514 nm, emission wavelength: 522 - 614 nm; FRET Raw excitation wavelength: 458 nm, emission wavelength: 522 - 614 nm) for imaging analysis.

[0115] Data processing of the fluorescence imaging of the mTORCAR probe refers to the article by Broussard et al. (Broussard, Joshua A et al. “Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine / threonine kinase Akt.” Nature protocols vol. 8,2 (2013): 265 - 81. doi:10.1038 / nprot.2012.147). Before any calculations are applied, all images (FRET Raw , YPet and mCerulean) need to be corrected for background and field inhomogeneity. Background correction is done by measuring the average intensity of the region of interest (ROI) in the image without cells and subtracting this intensity from the gray value of each pixel in the image. FRET Raw is the original FRET image acquired on the microscope. A is the percentage of YPet excitation crosstalk in the FRET image calculated from the control YPet sample. YPet is the image of YPet expression in the sample after direct excitation by the 514 nm laser line. B is the percentage of mCerulean emission crosstalk in the FRET channel calculated from the control mCerulean sample. mCerulean is the image of the sample acquired in the mCerulean channel. The correction factor for YPet direct excitation crosstalk (A) obtained by spectral calculation is A ≈ 0.557, and the correction factor for mCerulean emission crosstalk (B) is B ≈ 0.108. Thereafter, the corrected FRET image (FRET ) can be calculated based on the equation Corr .

[0116] Then, according to the equation calculate the ratio image; the numerator is essentially the normalized FRET Corr image, and the denominator is the normalized mCerulean image.

[0117] The results are shown in the following table. Table 1 is a statistical table of mTORCAR probe FRET-related data after 8 hours of starvation, after adding serum (>50 minutes), and after adding 200 nM Rapamycin (>5 minutes).

[0118] Table 1 Fluorescence intensity of mTORCAR probe

[0119]

[0120] Table 2 Comparison of fluorescence intensity of SEaTOR probe with the prior art

[0121]

[0122] From the comparative analysis of the above statistical data, it can be seen that the probe SEaTOR constructed by us has higher sensitivity than the mTORCAR probe published in previous studies in detecting the active state of mTORC1, and can more significantly reflect the change of the active state of mTORC1 after being stimulated. In addition, our probe has at least two advantages over the previous FRET-based mTORCAR probe: First, the data processing is simpler and more convenient, without the need for multiple complex calibration calculations; Second, the fluorescent proteins used in the FRET-based mTORCAR probe need to be excited by lasers at 458 nm and 514 nm, and most of the microscopes commonly used in the laboratory do not have these two lasers, so the imaging effect of the mTORCAR probe will be poor, while our fluorescent probe SEaTOR only needs to be excited by a 488 nm laser, has higher sensitivity, and has a wider range of applications, which is convenient for popularization and application.

[0123] Example 4: Construction of SEaTOR-LH

[0124] The construction method of SEaTOR-LH is similar to that of SEaTOR in Example 1. As shown in combination Figure 7 taking the Linker-Hydrophobic Motif of p70S6K1 that can bind to mTORC1 (S6K1 381-417AA, whose amino acid sequence is shown in SEQ ID NO. 16 and nucleotide sequence is shown in SEQ ID NO. 17) fragment as the sensing domain, and connecting with the EYFP fluorescent protein through as Figure 7It is connected in the manner shown, and a TOS Motif (S6K1 28-32AA, SEQ ID NO. 19) is added to the N-terminus to assist in the positioning of the probe and the phosphorylation of it by mTORC1, thereby obtaining the fluorescent probe SEaTOR-LH. The amino acid sequence of SEaTOR-LH is shown in SEQ ID NO. 23, and the nucleotide sequence is shown in SEQ ID NO. 24.

[0125] The Linker-Hydrophobic Motif of the sensing domain p70S6K1 is from rat. EYFP-N and EYFP-C are respectively the amino-terminal part and the carboxyl-terminal part of the yellow fluorescent protein EYFP (the two parts constitute the fluorescent module of the probe). The Linker-Hydrophobic Motif (S6K1 381-417AA) fragment is the domain whose conformation changes after being phosphorylated by mTORC1 (sensing module). Linker 1 (amino acid sequence FDL) and Linker 2 (amino acid sequence GI) are the connecting parts between the sensing module and different parts of the fluorescent protein. The N-terminus contains a TOS Motif (S6K1 28-32AA) to assist in the positioning of the probe and the phosphorylation of it by mTORC1.

[0126] Example 5: Response of the fluorescent probe SEaTOR-LH in HEK293T cells

[0127] HEK293T cells were cultured in a glass-bottom dish, and the culture medium was DMEM containing 10% fetal bovine serum, 1% Glutamax, 1% sodium pyruvate, 1% MEM-NEAA, and 1% penicillin-streptomycin. The culture temperature was 37 °C, and the CO2 content was 5%.

[0128] When the cell density reached 50 ± 5%, the SEaTOR-LH plasmid and the mCherry-βActin plasmid for labeling cell morphology were transfected using the CalPhos Mammalian Transfection Kit (Takara 631312). As a control group, the pRK5-EYFP plasmid (SEQ ID NO. 25) without the Linker-Hydrophobic Motif was transfected. pRK5-EYFP is the original plasmid for constructing the probe (without the sensing domain) and was used as the experimental control group;

[0129] The transfected mammalian cells were cultured overnight, starved with serum-free medium (the serum-free medium was DMEM containing 1% Glutamax, 1% sodium pyruvate and 1% penicillin-streptomycin) for 8 hours, and then imaged and analyzed using an Olympus IX-81-ZDC laser confocal microscope (the objective lens was Olympus Plan Apo 100× NA 1.45, the imaging CCD was ANDOR iXon DU-897D-500 camera, the excitation light wavelength was 488 nm, and the emission wavelength filter was 515 / 30 nm). The results are as Figure 8 shown.

[0130] The transfected mammalian cells were cultured overnight, starved with serum-free medium for 8 hours, then serum was added for 50 minutes to promote cell growth and division, and imaged and analyzed using an Olympus IX-81-ZDC laser confocal microscope (excitation wavelength 488 nm, emission wavelength filter 515 / 30 nm). The results are as Figure 8 shown. After adding serum, the fluorescence intensities of SEaTOR and SEaTOR-LH increased significantly, while the fluorescence intensity of co-expressed mCherry-βActin, which labeled the neuron morphology, did not change significantly.

[0131] In addition, the transfected mammalian cells were cultured overnight, imaged using an Olympus IX-81-ZDC laser confocal microscope (the objective lens was Olympus Plan Apo 100× NA 1.45, the imaging CCD was ANDOR iXon DU-897D-500 camera, the excitation light wavelength was 488 nm, and the emission wavelength filter was 515 / 30 nm), then 50 ng / ml of PDGF growth factor was added to stimulate the cells to promote the activity and protein expression of mTORC1, and the change in the fluorescence intensity of the mTORC1 probe was analyzed. The results are as Figure 9 and Figure 10As shown, the fluorescence intensity of SEaTOR-LH increased significantly, while the fluorescence intensity of co-expressed mCherry-βActin only increased significantly 20 minutes after the addition of PDGF and did not change significantly under other conditions. In addition, the fluorescence intensity of pRK5-EYFP only decreased significantly 20 minutes after the addition of PDGF and then returned to the original fluorescence intensity level, without significant changes under other conditions. The transfected mammalian cells were cultured overnight, and after perfusion with complete medium containing 50 ng / ml PDGF for 30 minutes, 200 nM Rapamycin (an mTORC1 inhibitor) was added for 5 minutes to fully inhibit mTORC1. Live cell imaging was performed throughout the process, and imaging statistical analysis was carried out using an Olympus IX-81-ZDC laser confocal microscope (excitation wavelength 488 nm, emission wavelength filter 515 / 30 nm). The results are as Figure 10 shown. It was found that the fluorescence intensity of SEaTOR-LH decreased significantly compared to before the addition of the drug and did not change significantly compared to before the PDGF stimulation, while the fluorescence intensities of mCherry-βActin and pRK5-EYFP did not change significantly after the addition of Rapamycin. Therefore, the fluorescent probe SEaTOR-LH can better characterize the change in the activity state of mTORC1.

[0132] It should be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0133] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various transformations can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0134] In addition, the above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the idea of the present invention, several simple deductions, substitutions, and combinations can be made, which should all be regarded as belonging to the protection scope of the present invention.

[0135] Sequence Listing:

[0136] SEQ ID NO. 1: Amino acid sequence of SEaTOR

[0137] MVSKGEELFTGVVPILVEMVGDVNGHRFSVSGEGEGIATYGMLTLKLICTTGELPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTIFFKGDGYYKTRAEVKFEGDTLVNRIELKGFDFREDGNILGHKLGYNFDLGTAGVFDIDLDQPEDAGSEDELEEGGQLNESMDHGGVGPYELGMEHCEKFEISETSVNRGPEKIRPECFELLRVLGKGGYGKVFQVRKVTGANTGKIFAMKVLKKAMIVRNAKDTAHTKAERNILEEVKHPFIVDLIYAFQTGGKLYLILEYLSGGELFMQLEREGIFMEDTACFYLAEISMALGHLHQKGIIYRDLKPENIMLNHQGHVKLTDFGLCKESIHDGTVTHTFCGTIEYMAPEILMRSGHNRAVDWWSLGALMYDMLTGAPPFTGENRKKTIDKILKCKLNLPPYLTQEARDLLKKLLKRNAASRLGAGPGDAGEVQAHPFFRHINWEELLARKVEPPFKPLLQSEEDVSQFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPSVLESVKEKFSFEPKIRSPRRFIGSPRTPVSPVKFSPGDFWGRGASASTANPQTPVEYPMETSGIEQMDVTTSGEASAPLPIRQPNSGPYKKQAFPMISKRPEHLRMNLTRGINSHNVYITADKQKNGVKAHFEIRHNLEDGSVQLADHYQLNTPIGDGPVLLPDNHYLRHQSALSKDPNEKRDHMVLQEFVTAAGITHGMDELYK

[0138] SEQ ID NO. 2: SEaTOR nucleotide sequence

[0139]

[0140] SEQ ID NO. 3: Amino acid sequence of p70S6K1

[0141] AGVFDIDLDQPEDAGSEDELEEGGQLNESMDHGGVGPYELGMEHCEKFEISETSVNRGPEKIRPECFELLRVLGKGGYGKVFQVRKVTGANTGKIFAMKVLKKAMIVRNAKDTAHTKAERNILEEVKHPFIVDLIYAFQTGGKLYLILEYLSGGELFMQLEREGIFMEDTACFYLAEISMALGHLHQKGIIYRDLKPENIMLNHQGHVKLTDFGLCKESIHDGTVTHTFCGTIEYMAPEILMRSGHNRAVDWWSLGALMYDMLTGAPPFTGENRKKTIDKILKCKLNLPPYLTQEARDLLKKLLKRNAASRLGAGPGDAGEVQAHPFFRHINWEELLARKVEPPFKPLLQSEEDVSQFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPSVLESVKEKFSFEPKIRSPRRFIGSPRTPVSPVKFSPGDFWGRGASASTANPQTPVEYPMETSGIEQMDVTTSGEASAPLPIRQPNSGPYKKQAFPMISKRPEHLRMNL

[0142] SEQ ID NO. 4: Nucleotide sequence of p70S6K1

[0143]

[0144] SEQ ID NO. 5: Amino acid sequence of EYFP

[0145] MVSKGEELFTGVVPILVEMVGDVNGHRFSVSGEGEGIATYGMLTLKLICTTGELPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTIFFKGDGYYKTRAEVKFEGDTLVNRIELKGFDFREDGNILGHKLGYNNSHNVYITADKQKNGVKAHFEIRHNLEDGSVQLADHYQLNTPIGDGPVLLPDNHYLRHQSALSKDPNEKRDHMVLQEFVTAAGITHGMDELYK

[0146] SEQ ID NO. 6: Nucleotide sequence of EYFP

[0147] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGATGGTCGGCGACGTAAACGGCCATAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCATTGCCACCTACGGCATGCTGACCCTGAAGCTCATCTGCACCACCGGCGAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTCGGCTACGGCGTGGCGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAGGGCTACGTCCAGGAACGCACCATCTTCTTCAAAGGCGACGGCTACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCTTCGACTTCAGGGAGGACGGCAACATCCTGGGTCACAAGCTGGGGTACAACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCGTCAAGGCCCACTTCGAGATCCGCCACAACCTCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCTGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGCGCCACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCAGGAGTTCGTGACTGCCGCCGGGAtCACTCACGGCATGGACGAGCTGTACAAG

[0148] SEQ ID NO. 7: N-EYFP NheI up

[0149] TACGACTCACTATAGGCTAGCATGGTGAGCAAGGGCGAG

[0150] SEQ ID NO. 8: N-EYFP kpnI down:

[0151] CCACGCGTCCCCCCGGTACCCAAGTCGAAGTTGTACCC

[0152] SEQ ID NO. 9: C-EYFP MluI upstream:

[0153] GGTACCGGGGGGACGCGTGGCATCAACAGCCACAAC

[0154] SEQ ID NO. 10: C-EYFP NotI downstream:

[0155] CATGTCTGCTCGAAGCGGCCGCTCACTTGTACAGCTCGTCCAT

[0156] SEQ ID NO. 11: Nucleotide sequence of plasmid template for amplifying EYFP

[0157]

[0158] SEQ ID NO. 12: Nucleotide sequence of the plasmid template for amplifying p70S6K1

[0159]

[0160] SEQ ID NO. 13: p70S6K1 KpnI upstream:

[0161] CTTGGGTACCGCAGGAGTGTTTGACATAG

[0162] SEQ ID NO. 14: p70S6K1 MluI downstream:

[0163] TGCCACGCGTTAGATTCATACGCAGGTGC

[0164] SEQ ID NO. 15: AGC-kinase C-terminal Domain amino acid sequence

[0165] RHINWEELLARKVEPPFKPLLQSEEDVSQFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPSVL

[0166] SEQ ID NO.16: Linker-Hydrophobic Motif amino acid sequence

[0167] QFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPS

[0168] SEQ ID NO.17: Linker-Hydrophobic Motif nucleotide sequence

[0169] CAGTTTGATTCAAAGTTTACTCGTCAGACACCTGTTGACAGCCCCGATGACTCAACTCTCAGTGAAAGTGCCAACCAGGTCTTTCTGGGTTTTACATATGTGGCTCCATCT

[0170] SEQ ID NO.18: Turn-Hydrophobic Motif amino acid sequence

[0171] VDSPDDSTLSESANQVFLGFTYVAPS

[0172] SEQ ID NO.19: TOS Motif amino acid sequence

[0173] FDIDL

[0174] SEQ ID NO.20: Amino acid sequence of EYFP after adding YPet mutation

[0175] MVSKGEELFTGVVPILVEMVGDVNGHRFSVSGEGEGIATYGMLTLKLLCTTGELPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTIFFKGDGYYKTRAEVKFEGDTLVNRIELKGFDFREDGNILGHKLGYNNSHNVYITADKQKNGVKAHFEIRHNLEDGGVQLADHYQLNTPIGDGPVLLPDNHYLRHQSALFKDPNEKRDHMVLQEFLTAAGITFGMNELYK

[0176] SEQ ID NO.21: Amino acid sequence of mNeonGreen

[0177] MVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

[0178] SEQ ID NO.22: Amino acid sequence of EGFP

[0179] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0180] SEQ ID NO. 23: Amino acid sequence of SEaTOR-LH

[0181] MFDIDLGGTGGSTGMVSKGEELFTGVVPILVEMVGDVNGHRFSVSGEGEGIATYGMLTLKLLCTTGELPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTIFFKGDGYYKTRAEVKFEGDTLVNRIELKGFDFREDGNILGHKLGYNFDLGTQFDSKFTRQTPVDSPDDSTLSESANQVFLGFTYVAPSTRGINSHNVYITADKQKNGVKAHFEIRHNLEDGGVQLADHYQLNTPIGDGPVLLPDNHYLRHQSALFKDPNEKRDHMVLQEFLTAAGITHGMNELYK

[0182] SEQ ID NO. 24: SEaTOR-LH nucleotide sequence

[0183] ATGTTTGACATAGACCTGGGAGGAACAGGTGGTTCTACCGGTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGATGGTCGGCGACGTAAACGGCCATAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCATTGCCACCTACGGCATGCTGACCCTGAAGCTCTTATGCACCACCGGCGAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTCGGCTACGGCGTGGCGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAGGGCTACGTCCAGGAACGCACCATCTTCTTCAAAGGCGACGGCTACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCTTCGACTTCAGGGAGGACGGCAACATCCTGGGTCACAAGCTGGGGTACAACTTCGACTTGGGTACCCAGTTTGATTCAAAGTTTACTCGTCAGACACCTGTTGACAGCCCCGATGACTCAACTCTCAGTGAAAGTGCCAACCAGGTCTTTCTGGGTTTTACATATGTGGCTCCATCTACGCGTGGCATCAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCGTCAAGGCCCACTTCGAGATCCGCCACAACCTCGAGGACGGCGGTGTGCAGCTCGCCGACCACTACCAGCTGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGCGCCACCAGTCCGCCCTGTTCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCAGGAGTTCTTGACTGCCGCCGGGATCACTCACGGCATGAATGAGCTGTACAAG

[0184] SEQ ID NO. 25: Nucleotide sequence of pRK5-EYFP

[0185] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGATGGTCGGCGACGTAAACGGCCATAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCATTGCCACCTACGGCATGCTGACCCTGAAGCTCATCTGCACCACCGGCGAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTCGGCTACGGCGTGGCGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAGGGCTACGTCCAGGAACGCACCATCTTCTTCAAAGGCGACGGCTACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCTTCGACTTCAGGGAGGACGGCAACATCCTGGGTCACAAGCTGGGGTACAACTTCGACTTGGGTACCGGGGGGACGCGTGGCATCAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCGTCAAGGCCCACTTCGAGATCCGCCACAACCTCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCTGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGCGCCACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCAGGAGTTCGTGACTGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAG。

Claims

1. An mTORC1 fluorescent probe, characterized in that The structure of the probe includes a sensing domain p70S6K1 protein or a fragment thereof and two fluorescent modules at both ends of the sensing domain.

2. The mTORC1 fluorescent probe according to claim 1, characterized in that The amino acid sequence of the sensing domain is shown in SEQ ID NO. 3 or SEQ ID NO.

16.

3. The mTORC1 fluorescent probe according to claim 1 or 2, characterized in that The nucleotide sequence encoding the probe sensing domain is shown in SEQ ID NO. 4 or SEQ ID NO.

17.

4. The mTORC1 fluorescent probe according to any one of claims 1 to 3, characterized in that The probe further comprises TOS Motif, and the sequence of the TOS Motif is shown in SEQ ID NO.19, and preferably the TOS Motif is located at the N-terminus.

5. A method for preparing an mTORC1 fluorescent probe, characterized in that: The mTORC1 downstream substrate p70S6K1 protein or its fragment is used as the sensing domain, and its two ends are respectively connected to the amino terminal part and the carboxyl terminal part of the fluorescent protein to prepare the mTORC1 fluorescent probe; Preferably, the mTORC1 fluorescent probe is further connected to a TOS Motif, and more preferably, the TOS Motif is located at the N-terminus, and the sequence of the TOS Motif is shown in SEQ ID NO.

19.

6. The method according to claim 5, characterized in that: The arginine (R) at position 367 of the amino acid sequence of the p70S6K1 protein is replaced by proline (P); Preferably, the amino acid sequence of the p70S6K1 protein is as shown in SEQ ID NO. 3; preferably, the fragment of the p70S6K1 protein is a Linker-Hydrophobic Motif, and its amino acid sequence is as shown in SEQ ID NO.

16.

7. The method according to claim 5 or 6, characterized in that: The fluorescent protein is yellow fluorescent protein EYFP.

8. The method according to any one of claims 5 to 7, characterized in that: The sequence of the p70S6K1 protein is the sequence of S6K1 without the nuclear localization sequence, namely, amino acids 25-525.

9. Use of the mTORC1 fluorescent probe defined in any one of claims 1 to 4 in detecting mTORC1 signals, characterized in that: The mTORC1 fluorescent probe is used to detect the activity of mTORC1 in living cells, preferably to detect changes in the activity state of mTORC1.

10. A fluorescence imaging detection method capable of characterizing the activity state of mTORC1 in living cells, characterized in that: The following steps are involved: (1) transfecting the mTORC1 fluorescent probe defined in any one of claims 1 to 4 into mammalian cells; (2) performing imaging analysis using a fluorescence microscope to detect changes in the fluorescence intensity of the mTORC1 fluorescent probe, wherein the single-photon excitation wavelength used in the imaging analysis is 488 nm; Preferably, the steps include: (1) transfecting the mTORC1 fluorescent probe defined in any one of claims 1 to 4 into mammalian cells; (2) Stimulate mammalian cells to increase or decrease intracellular mTORC1 activity; (3) Performing imaging analysis using a fluorescence microscope to detect changes in the fluorescence intensity of the mTORC1 fluorescent probe before and after the stimulation, wherein the single-photon excitation wavelength used in the imaging analysis is 488 nm.

11. Use of an mTORC1 fluorescent probe in preparing a kit for disease detection or diagnosis, characterized in that: The kit comprises the mTORC1 fluorescent probe defined in any one of claims 1-4.

12. The use according to claim 11, characterized in that: The diseases are diseases affected by the expression of mTORC1, including cancer, neurodegenerative diseases, and metabolic diseases.

Images