Probe set, recombinant cell, medicine screening system and method for high-throughput screening of TFEB nuclear transfer regulation and control medicine

Abstract

The invention discloses a probe set. The probe set comprises a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a nuclear localization sequence and a first protein molecule, and the nuclear localization sequence is connected with the first protein molecule; the second fusion protein comprises a TFEB protein and a second protein molecule, the TFEB protein is connected with the second protein molecule, the second protein molecule is used for being combined with the first protein molecule, and a detectable signal is generated after the first protein molecule and the second protein molecule are combined. The probe set can rapidly, accurately and sensitively detect the TFEB nuclear transfer condition, can be used for high-throughput screening of TFEB regulation and control drugs, and reduces the drug screening cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug screening, and particularly relates to a probe set for high-throughput screening of drugs for regulating nuclear translocation of TFEB, a recombinant cell, a drug screening system and a method. BACKGROUND

[0002] Transcription factor EB (TFEB) is a member of the basic helix-loop-helix leucine zipper transcription factor MiT / TFE family and is a key regulator in the autophagy / lysosome-to-nucleus signaling pathway, playing a very important role in lysosome biogenesis and material metabolism. Studies have shown that intervention in autophagy and lysosome function by regulating TFEB can remove damaged organelles and abnormally accumulated disease-related proteins in cells, thereby relieving diseases related to autophagy and lysosome disorders, such as lysosomal storage diseases, neurodegenerative diseases, cancer and metabolic disorders. Therefore, TFEB has become an important target for drug development.

[0003] Under normal conditions, TFEB is mainly expressed in the cytoplasm, but when the cell encounters a stimulus, such as drug induction, starvation or lysosome dysfunction, it will cause TFEB to transfer from the cytoplasm into the nucleus (referred to as nuclear translocation) and bind to target genes to promote the transcription of target genes. Therefore, the nuclear translocation of TFEB can be used as a marker for the activation of TFEB.

[0004] Therefore, there is an urgent need to develop a method for high-throughput screening of drugs for regulating nuclear translocation of TFEB. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a probe set for high-throughput screening of drugs for regulating nuclear translocation of TFEB. The use of the probe set allows high-throughput screening of drugs for regulating nuclear translocation of TFEB, which is easy to detect, has a short detection period, and the detection results are intuitive and accurate.

[0006] The present application is based on the following findings of the inventors:

[0007] Currently, the detection of nuclear translocation of TFEB is usually carried out by using a fluorescence microscope to perform microscopic imaging of cells expressing TFEB with a fluorescent label (such as TFEB-GFP fusion protein) to detect the nuclear translocation of TFEB. The main defects are:

[0008] 1) The results obtained using a fluorescence microscope need to be analyzed cell by cell, which is suitable for small-scale experimental verification but not suitable for high-throughput drug screening;

[0009] 2) In the unactivated state, TFEB is mainly expressed in the cytoplasm, and after activation, TFEB is expressed in both the cytoplasm and the nucleus. The inconsistent criteria for distinguishing whether to enter the nucleus lead to inaccurate analysis results.

[0010] 3) Detecting the nuclear transfer of TFEB requires the use of a high-power fluorescence microscope, which has high requirements for instruments and cannot be popularized.

[0011] Therefore, the method of using a fusion fluorescent protein TFEB to screen drugs for nuclear transfer of TFEB has the disadvantages of inaccurate analysis results, high requirements for instruments, and inability to perform high-throughput screening.

[0012] Therefore, in one aspect of the present application, a probe set is provided. According to embodiments of the present application, the probe set comprises: a first fusion protein and a second fusion protein; wherein the first fusion protein comprises a nuclear localization sequence and a first protein molecule, the nuclear localization sequence being connected to the first protein molecule; the second fusion protein comprises a TFEB protein and a second protein molecule, the TFEB protein being connected to the second protein molecule, the second protein molecule being used to bind to the first protein molecule, and the first protein molecule and the second protein molecule having a detectable signal after binding. The probe set of the present application can quickly, accurately and sensitively detect the nuclear transfer of TFEB, and can be used for high-throughput screening of drugs for regulating TFEB, thereby reducing the cost of drug screening.

[0013] In another aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the above-mentioned probe set. The nucleic acid molecule according to embodiments of the present application can encode the above-mentioned probe set.

[0014] In another aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the above-mentioned nucleic acid molecule. The expression vector according to embodiments of the present application can express the above-mentioned probe set.

[0015] In another aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell carries the above-mentioned nucleic acid molecule or the above-mentioned expression vector; or the recombinant cell expresses the above-mentioned probe set. The use of the above-mentioned recombinant cell can perform high-throughput screening of drugs for regulating TFEB, thereby reducing the cost of drug screening.

[0016] In another aspect of the present application, a drug screening system is provided, the drug being used for regulating TFEB, and the system comprising the above-mentioned recombinant cell. The use of the above-mentioned drug screening system can perform high-throughput screening of drugs for regulating TFEB, thereby reducing the cost of drug screening.

[0017] In yet another aspect of the present application, the present application provides a method for screening a drug for modulating TFEB, comprising: screening a drug by using the recombinant cell as described above; and determining whether the drug is a target drug based on the detectable signal in the recombinant cell. The method can be used for high-throughput screening of drugs for modulating TFEB, thereby reducing the cost of drug screening.

[0018] In yet another aspect of the present application, the present application provides a use of the probe set as described above, the nucleic acid molecule as described above, the expression vector as described above, or the recombinant cell as described above in the preparation of a high-throughput screening drug for modulating TFEB.

[0019] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0020] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0021] Figure 1 A schematic diagram of the TFEB nuclear drug screening probe of the present application;

[0022] Figure 2 Fluorescence detection results in HeLa cells expressing TFEB-GFP 11 and / or GFP 1-10 -NLS of Example 1 of the present application;

[0023] Figure 3 Fluorescence detection results and fluorescence intensity in HeLa cells co-expressing TFEB-GFP 11 and GFP 1-10 -NLS under starvation or Rapamycin treatment of Example 1 of the present application;

[0024] Figure 4 Fluorescence detection results in HeLa cells co-expressing GFP 1-10 -NLS and TFEB-mCherry-GFP 11 of Example 2 of the present application;

[0025] Figure 5 Fluorescence detection results and fluorescence intensity in HeLa cells co-expressing TFEB-mCherry-GFP 11 and GFP 1-10 -NLS under starvation or Rapamycin treatment of Example 2 of the present application;

[0026] Figure 6The results of fluorescence detection in HeLa cells expressing EGFP-NLS, EGFP-2xNLS or EGFP-3xNLS respectively in Example 3 of the present application;

[0027] Figure 7 The results of fluorescence detection in HeLa cells co-expressing TFEB-mCherry-GFP 11 and GFP 1-10 -3xNLS under the condition of starvation or Rapamycin treatment in Example 4 of the present application and their fluorescence intensity;

[0028] Figure 8 The fluorescence intensity in HeLa cells co-expressing TFEB-mCherry-GFP 11 and GFP 1-10 -3xNLS under the condition of Rapamycin treatment in Example 5 of the present application detected by microplate reader;

[0029] Figure 9 The results of fluorescence detection in HeLa cells co-expressing one of TFEB-linker-GFP 11 , TFEB-3xlinker-3xGFP 11 and TFEB-5xlinker-5xGFP 11 with GFP 1-10 -NLS in Example 6 of the present application and their fluorescence intensity. DETAILED DESCRIPTION

[0030] The examples of the present application are described in detail below. The examples described below are exemplary only and are set forth to explain the present application and should not be used to limit the present application.

[0031] It should be noted that the terms "first", "second" are used only for the purpose of description and should not be understood as indicating or implying relative importance or implying that the indicated technical features are limited to one or more. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0032] The present application is described in detail

[0033] Definitions and general terms

[0034] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless explicitly described otherwise, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art in the field to which this application belongs. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids (i.e., amino acids in the left-handed L-configuration).

[0035] In the present document, the terms "comprising" or "including" or "having" are open-ended, that is, the listed steps or options need not be present for the claimed application to be useful.

[0036] In the present document, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0037] In the present document, the term "expression vector" generally refers to a nucleic acid molecule capable of replicating itself in a suitable host into which it is inserted, which transfers the inserted nucleic acid molecule into and / or between cells or hosts. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, the vector can produce a desired expression product by culturing a suitable cell or host containing the vector.

[0038] In the present document, the term "cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, which has a unique trait with stable heredity. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The term "transformed" or "transfected" used herein means introducing a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0039] The present application proposes a probe set, recombinant cell, screening system and method for high-throughput screening of drugs regulating nuclear translocation of TFEB, which will be described in detail below.

[0040] probe group

[0041] In one aspect of this application, a probe set is proposed. According to an embodiment of this application, the probe set includes: a first fusion protein and a second fusion protein; wherein the first fusion protein includes a nuclear localization sequence and a first protein molecule, the nuclear localization sequence being linked to the first protein molecule; the second fusion protein includes a TFEB protein and a second protein molecule, the TFEB protein being linked to the second protein molecule, the second protein molecule being used to bind to the first protein molecule, and the binding of the first protein molecule and the second protein molecule having a detectable signal.

[0042] The first fusion protein in the probe set described in this application contains a nuclear localization sequence and can be expressed in the cell nucleus, while the second fusion protein is initially expressed in the cytoplasm. When cells encounter stimulation (e.g., drug induction, starvation, or lysosomal dysfunction), because the second fusion protein contains TFEB protein, it can enter the cell nucleus from the cytoplasm, causing the first protein molecule of the first fusion protein and the second protein molecule of the second fusion protein to bind and generate a detectable signal. Furthermore, the presence or absence of a detectable signal can determine whether the second fusion protein has entered the cell nucleus, or the strength of the detectable signal can determine the degree of entry into the cell nucleus, especially enabling high-throughput detection. Therefore, the probe set of this application can rapidly, accurately, and sensitively detect the nuclear translocation of TFEB, and can be used for high-throughput screening of drugs that regulate TFEB, reducing the cost of drug screening.

[0043] In this document, the term "probe" refers to a detection molecule capable of detecting a target protein, including tracking and observing the presence or absence of the target protein, or its location, dynamics, and interactions within the cell. The probe set in this application is used to observe the nuclear translocation of the target protein (i.e., TFEB protein), specifically, the detection of a detectable signal after the binding of a second protein molecule to a first protein molecule, used to determine the entry of the target protein (i.e., TFEB protein) from the cytoplasm into the nucleus.

[0044] In this paper, the term "detectable signal" refers to a physical or chemical indicator that can be used to monitor the nuclear entry status of the TFEB protein, including but not limited to fluorescence signals, luminescence signals, etc. For example, a detectable signal is a fluorescence signal.

[0045] According to an embodiment of this application, the C-terminus of the first protein molecule is connected to the N-terminus of the nuclear localization sequence, or the C-terminus of the nuclear localization sequence is connected to the N-terminus of the first protein molecule.

[0046] In this document, the term "Nuclear Localization Sequence (NLS)" refers to a specific type of amino acid sequence present in proteins and responsible for guiding the protein through the nuclear pore complex from the cytoplasm into the nucleus. In this application, the specific type of NLS is not limited, as long as it can guide the first fusion protein into the nucleus, it is within the scope of protection of this application. In one example of the invention, NLSs typically contain 4 to 8 amino acid residues and are rich in positively charged amino acids, such as lysine (Lys) and arginine (Arg), for example, PKKKRKV (SEQ ID NO:1).

[0047] In one optional embodiment of this application, the C-terminus of the first protein molecule is connected to the N-terminus of the nuclear localization sequence.

[0048] According to embodiments of this application, the nuclear localization sequence has an amino acid sequence as shown in any one of SEQ ID NO:1 to 15.

[0049]

[0050]

[0051] According to embodiments of this application, the C-terminus of the TFEB protein is connected to the N-terminus of the second protein molecule, or the C-terminus of the second protein molecule is connected to the N-terminus of the TFEB protein.

[0052] In one optional embodiment of this application, the C-terminus of the TFEB protein is connected to the N-terminus of the second protein molecule.

[0053] According to embodiments of this application, the TFEB protein has an amino acid sequence as shown in SEQ ID NO:16.

[0054] MTASSGWEPAPAATMASRIGLRMQLMREQAQQEEQRERMQQQAVMHYMQQQQQQQQQLGGPPTPAINTPVHFQSPPPVPGEVLKVQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHIS PAQGSPKPPPAASPGVRAGHVLSSSAGNSAPNSPMAMLHIGSNPERELDDVIDNIMRLDDVLGYINPEMQMPNTLPLSSSHLNVYSSDPQVTASLVGVTSSSSCPADLTQKRELTDAESRALAKE RQKKDNNHNLIERRRRFNINDRIKELGMLIPKANDLDVRWNKGTILKASVDYIRRMQKDLQKSRELENHSRRLEMTNKQLWLRIQELEMQARVHGLPTTSPSGMNMAELAQQVVKQELPSEEGP GEALMLGAEVPDPEPLPALPPQAPLPLPTQPPSPFHHLDFSHSLSFGGREDEGPPGYPEPLAPGHGSPFPSLSKKDLDLMLLDDSLPLASDPLLSTMSPEASKASSRRSSFSMEEGDVL(SEQ ID NO:16).

[0055] According to embodiments of this application, the first protein molecule and the second protein molecule are fluorescent bimolecular complementary proteins.

[0056] In this document, the term "fluorescent bicomponent protein" refers to a protein fragment containing two different proteins that are not fluorescent when used alone, but can form a complete fluorescent protein when combined. In this application, as long as the first and second protein molecules are not fluorescent when used alone, but can form a complete fluorescent protein when combined, the specific type is not limited and is within the scope of protection of this application.

[0057] According to embodiments of this application, the first protein molecule and the second protein molecule are selected from any one of the following groups:

[0058] 1)GFP 1-10 EBFP2 1-10 Cerulean 1-10 One of them, and GFP 11 ,

[0059] 3)mNG2 1-10 mNG3K 1-10 mNG3A 1-10One of them, and mNG2 11 ,

[0060] 4) mRuby4 1-10 sfCherry2 1-10 One of them, and sfCherry2 11 ,

[0061] 5) GAF protein and PAS protein.

[0062] According to embodiments of this application, the GFP 1-10 It has the amino acid sequence shown in SEQ ID NO:17.

[0063] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTR AVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK(SEQ IDNO:17).

[0064] According to an embodiment of this application, the EBFP2 1-10 It has the amino acid sequence shown in SEQ ID NO:18.

[0065] MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGKYKTR AVVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIKVNFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQTVLSKDPNEK(SEQID NO:18).

[0066] According to embodiments of this application, the Cerulean 1-10 It has the amino acid sequence shown in SEQ ID NO:19.

[0067] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTR AVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNANSDNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK(SEQ IDNO:19).

[0068] According to embodiments of this application, the GFP 11 It has the amino acid sequence shown in SEQ ID NO:20.

[0069] RDHMVLHEYVNAAGIT (SEQ ID NO:20).

[0070] According to an embodiment of this application, the mNG2 1-10 It has the amino acid sequence shown in SEQ ID NO:21.

[0071] MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVMGTGFPADGPVMTNTLTAADWCMSKKTYPNDKTIISTFKWSYTTVNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSM(SEQ IDNO:21).

[0072] According to an embodiment of this application, the mNG3K 1-10 It has the amino acid sequence shown in SEQ ID NO:22.

[0073] MVSKGEEDNMASLPATHELHIFGSINDVDFDMVGQGTGNPNEGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVIGTGFPADGPVMTNTLTAADWCMSKMTYPNDKTIISTFKWSYITVNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSM(SEQ IDNO:22).

[0074] According to an embodiment of this application, the mNG3A 1-10 It has the amino acid sequence shown in SEQ ID NO:23.

[0075] MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVMGTGFPADGPVMTNTLTAADLCVSKMTYPNDKTIISTFKWSYTTVNGKRYRSTARTTYTFAKPMAAKYLKNQPMYVLRKTELKHSM(SEQ IDNO:23).

[0076] According to an embodiment of this application, the mNG2 11 It has the amino acid sequence shown in SEQ ID NO:24.

[0077] TELNFKEWQKAFTDMM (SEQ ID NO:24).

[0078] According to an embodiment of this application, the mRuby4 1-10 It has the amino acid sequence shown in SEQ ID NO:43.

[0079] MVSKGEELIKENMRTKVVMEGSVNGHHFKCIGEGEGRPYEGVQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGVIT VTQDTCLEDGELVYNVKVSGVNFPSNGPVMQKKTKGWEPNTEMMYPVDGGLRGYTDIALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDN(SEQ IDNO:43).

[0080] According to an embodiment of this application, the sfCherry2 1-10 It has the amino acid sequence shown in SEQ ID NO:44.

[0081] KLATMEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFEDGG VVTVTQDSSLQDGQFIYKVKLLGINFPSDGPVMQKKTMGWEASTERMYPEDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDITSHNED(SEQ IDNO:44).

[0082] According to an embodiment of this application, the sfCherry2 11 It has the amino acid sequence shown in SEQ ID NO:45.

[0083] YTIVEQYERAEARHST (SEQ ID NO: 45).

[0084] According to embodiments of this application, the PAS protein has an amino acid sequence as shown in SEQ ID NO:46.

[0085] MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGA LIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELE PP (SEQ ID NO: 46).

[0086] According to embodiments of this application, the GAF protein has an amino acid sequence as shown in SEQ ID NO:47.

[0087] PPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGYDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPVPVTPYLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACELVAQVLARQIGVMEE(SEQ ID NO:47).

[0088] According to embodiments of this application, the first protein molecule and the second protein molecule are selected from any one of the following groups:

[0089] First protein molecule Second protein molecule 1 GFP 1-10 ]] GFP 11 ]] 2 EBFP2 1-10 ]]> GFP 11 ]]> 3 Cerulean 1-10 ]] GFP 11 ]] 4 mNG2 1-10 ]] mNG2 11 ]] 5 mNG3K 1-10 ]]> mNG2 11 ]]> 6 mNG3A 1-10 ]]> mNG2 11 ]]> 7 mRuby4 1-10 ]]> sfCherry2 11 ]] 8 sfCherry2 1-10 ]]> sfCherry2 11 ]] 9 GAF protein PAS protein .

[0090] In one exemplary embodiment of the present invention, the first protein molecule is selected from GFP. 1-10 The second protein molecule is selected from GFP. 11 .like Figure 1 As shown, the first fusion protein in the probe group of this application contains a nuclear localization sequence, and this first fusion protein can be expressed in the cell nucleus, while the second fusion protein is initially expressed in the cytoplasm. When the cell encounters stimulation (e.g., drug induction, starvation, or lysosomal dysfunction), because the second fusion protein contains TFEB protein, it can enter the cell nucleus from the cytoplasm, causing the GFP of the first fusion protein to be expressed. 1-10 GFP of the second fusion protein 11 The fusion protein binds and generates a detectable signal. Furthermore, the presence or absence of a detectable signal can determine whether the second fusion protein has entered the cell nucleus, or the strength of the detectable signal can determine the degree to which the second fusion protein has entered the cell nucleus, especially enabling high-throughput detection.

[0091] According to an embodiment of this application, the first fusion protein contains one or more nuclear localization sequences.

[0092] According to embodiments of this application, the first fusion protein contains 1, 2, 3, 4, or 5 nuclear localization sequences.

[0093] In an optional embodiment of the present invention, the first fusion protein contains multiple nuclear localization sequences.

[0094] In one optional embodiment of the present invention, the first fusion protein contains two nuclear localization sequences. In another optional embodiment of the present invention, the first fusion protein contains three nuclear localization sequences.

[0095] According to an embodiment of this application, the first fusion protein further includes a first linker peptide, wherein the C-terminus of the first protein molecule is linked to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is linked to the N-terminus of a nuclear localization sequence.

[0096] In an optional embodiment of the present invention, when multiple nuclear localization sequences are provided, the first fusion protein, from N-terminus to C-terminus, consists of a first protein molecule, a first linker peptide, and multiple nuclear localization sequences. In an optional embodiment of the present invention, when two nuclear localization sequences are provided, the first fusion protein, from N-terminus to C-terminus, consists of a first protein molecule, a first linker peptide, a nuclear localization sequence, and a nuclear localization sequence. In an optional embodiment of the present invention, when three nuclear localization sequences are provided, the first fusion protein, from N-terminus to C-terminus, consists of a first protein molecule, a first linker peptide, a nuclear localization sequence, a nuclear localization sequence, and a nuclear localization sequence.

[0097] According to an embodiment of this application, the first linker peptide is selected from (GGGGS)n, where n is any integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0098] According to an embodiment of this application, the first linker peptide is selected from GGGGS (SEQ ID NO: 48).

[0099] According to an embodiment of this application, the second fusion protein contains one or more of the following components:

[0100] According to embodiments of this application, the second fusion protein contains 1, 2, 3, 4, or 5 molecules.

[0101] In an optional embodiment of the present invention, the second fusion protein comprises a plurality of second protein molecules.

[0102] According to an embodiment of this application, the second fusion protein further includes one or more second linker peptides, wherein the C-terminus of the TFEB protein is linked to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is linked to the N-terminus of the second protein molecule.

[0103] According to an embodiment of this application, the second linker peptide is selected from flexible linkers peptides, and the number of amino acids in the second linker peptide is 5 to 20, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any two of these values ​​as a range between endpoint values.

[0104] According to an embodiment of this application, the second linker peptide is selected from (GGGGS)n, where n is any integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0105] According to an embodiment of this application, the second linker peptide is selected from GGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0106] According to embodiments of this application, the second linker peptide is set to 1, 2, 3, 4, or 5.

[0107] According to an embodiment of this application, the number of the second protein molecule and the second linker peptide are the same, and the C-terminus of each second linker peptide is linked to the N-terminus of the second protein molecule.

[0108] According to an embodiment of this application, the second fusion protein further includes a fluorescent protein. The C-terminus of the TFEB protein is linked to the N-terminus of the fluorescent protein, and the C-terminus of the fluorescent protein is linked to the N-terminus of the second protein molecule. The fluorescence color of the fluorescent protein is different from the fluorescence color after the first and second protein molecules bind. Therefore, the first fluorescence intensity of the fluorescent protein can be used as the total TFEB expression, and the change in second fluorescence intensity after the first and second protein molecules bind following TFEB nuclear translocation can be used as the nuclear translocation status of TFEB. After drug-induced TFEB nuclear translocation, the ratio of the change in second fluorescence intensity to the first fluorescence intensity can be calculated to more accurately reflect the nuclear translocation status of TFEB.

[0109] According to an embodiment of this application, the probe set includes:

[0110] The first fusion protein as shown in any one of SEQ ID NO:26, 41 and 42, and the second fusion protein as shown in any one of SEQ ID NO:30, 34, 38 to 40.

[0111] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTL VNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGSGGGGSPKKKRKVPKKKRKV(SEQ ID NO: 41);

[0112] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRI ELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGSGGGGSPKKKRKVPKKKRKVPKKKRKV(SEQ ID NO: 42).

[0113] According to an embodiment of this application, the probe set includes:

[0114] The first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:30; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:34; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:38; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:39; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:40; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:30; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:34; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:38; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:30. The second fusion protein shown in NO:39; and / or the first fusion protein shown in SEQ ID NO:41 and the second fusion protein shown in SEQ ID NO:40; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:30; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:34; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:38; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:39; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:40.

[0115] Nucleic acid molecules, expression vectors, recombinant cells

[0116] In another aspect, this application provides a nucleic acid molecule. According to an embodiment of this application, the nucleic acid molecule encodes the aforementioned probe set. The nucleic acid molecule according to an embodiment of this application can encode the aforementioned probe set.

[0117] According to embodiments of this application, the nucleic acid molecule includes DNA.

[0118] It should be noted that, for the nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0119] In another aspect, this application proposes an expression vector. According to an embodiment of this application, the expression vector carries the aforementioned nucleic acid molecule. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the nucleic acid molecule and the control elements only need to be operably linked.

[0120] In this article, "operable ligation" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0121] According to embodiments of this application, the expression vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vectors used in this application are not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used. After the expression vector according to some specific embodiments of this application is introduced into suitable cells, the expression of the aforementioned probe set can be effectively achieved under the mediation of a regulatory system, thereby obtaining recombinant cells containing the probe set.

[0122] According to embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0123] According to an embodiment of this application, the expression vector is a plasmid expression vector.

[0124] In another aspect, this application proposes a recombinant cell. According to embodiments of this application, the recombinant cell carries the aforementioned nucleic acid molecule or expression vector; or the recombinant cell expresses the aforementioned probe set. Thus, recombinant cells containing the aforementioned probe set can be obtained, i.e., the first fusion protein in the recombinant cell contains a nuclear localization sequence, and the first fusion protein can be expressed in the nucleus of the recombinant cell, while the second fusion protein is initially expressed in the cytoplasm of the recombinant cell. As previously known, using this recombinant cell allows for high-throughput screening of drugs regulating TFEB, reducing the cost of drug screening.

[0125] Furthermore, the TFEB-regulating drug to be screened (hereinafter referred to as the screening drug) is co-cultured with recombinant cells. Since the second fusion protein contains TFEB protein, in regulating the stimulatory effect of the TFEB drug, the second fusion protein can enter the nucleus from the cytoplasm, causing the first protein molecule of the first fusion protein and the second protein molecule of the second fusion protein to bind and generate a detectable signal. The nuclear translocation of TFEB can be determined through the detectable signal in the recombinant cells, thereby enabling high-throughput screening of TFEB-regulating drugs.

[0126] According to an embodiment of this application, the recombinant cells are obtained by introducing the above-described expression vector into host cells.

[0127] In one optional embodiment of this application, the host cell is a eukaryotic cell, and the specific type is not limited, as long as it enables the nuclear transfer abbreviation TFEB. For example, the host cell is a mammalian eukaryotic cell.

[0128] Drug screening system

[0129] In another aspect of this application, a drug screening system is proposed, wherein the drug is used to regulate TFEB, and the system includes the aforementioned recombinant cells. As previously known, when the TFEB-regulating drug to be screened (hereinafter referred to as the screening drug) is co-cultured with recombinant cells, since the second fusion protein contains TFEB protein, in regulating the stimulatory effect of the TFEB drug, the second fusion protein can enter the cell nucleus from the cytoplasm, causing the first protein molecule of the first fusion protein and the second protein molecule of the second fusion protein to bind, generating a detectable signal. Therefore, using a system containing the aforementioned recombinant cells allows for high-throughput screening of TFEB-regulating drugs, reducing the cost of drug screening.

[0130] method

[0131] In another aspect of this application, a method for screening drugs used to regulate TFEB is proposed. The method includes: screening the drug using the aforementioned recombinant cells; and determining whether the drug is a target drug based on detectable signals in the recombinant cells. As previously known, co-culturing the TFEB-regulating drug (hereinafter referred to as the drug to be screened) with recombinant cells allows the second fusion protein, containing TFEB protein, to enter the nucleus from the cytoplasm, binding the first protein molecule of the first fusion protein to the second protein molecule of the second fusion protein, thus generating a detectable signal. Therefore, the method of this application utilizes recombinant cells to perform high-throughput screening of TFEB-regulating drugs, reducing the cost of drug screening.

[0132] According to some optional embodiments of this application, the target drug is a drug that promotes TFEB nuclear transfer or a drug that inhibits TFEB nuclear transfer.

[0133] According to some optional embodiments of this application, the drug is determined to be a target drug based on the changes in detectable signals before and after the recombinant cells come into contact with the drug.

[0134] According to some optional embodiments of this application, the increase in the detectable signal of the recombinant cells after contact with the drug, compared to the detectable signal before contact with the drug, is an indication that the drug is a drug that promotes TFEB nuclear transfer.

[0135] According to some optional embodiments of this application, the decrease in the detectable signal of the recombinant cells after contact with the drug, compared to the detectable signal before contact with the drug, is an indication that the drug is a drug that inhibits TFEB nuclear transfer.

[0136] In this document, unless otherwise specified, the screening of target drugs can be determined based on increased or decreased content. Exemplarily, it can be determined by comparing increased or decreased content with a predetermined threshold, which can be determined using conventional methods in the art through a large number of samples; no specific method is limited, and all are within the scope of protection of this application.

[0137] use

[0138] In another aspect of this application, the application proposes the use of the above-described probe set, the above-described nucleic acid molecule, the above-described expression vector, or the above-described recombinant cell in the preparation of a high-throughput screening drug, said drug being used to regulate TFEB.

[0139] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0140] Example 1:

[0141] In this embodiment, the nucleic acid molecule encoding the fusion protein was constructed into the pcDNA3.1(+) vector, and the pcDNA3.1(+) vector carrying the nucleic acid molecule encoding the fusion protein was introduced into HeLa or HEK293T cells. The constructed fusion protein was then expressed in HeLa or HEK293T cells. The specific method is as follows:

[0142] GFP was synthesized using gene synthesis.1-10 The cDNA sequence of linker-NLS (nucleotide sequence as shown in SEQ ID NO:25, amino acid sequence as shown in SEQ ID NO:26) was constructed and inserted into the HindIII and EcoRI restriction sites of the pcDNA3.1(+) vector to obtain the plasmid pcDNA3.1(+)-GFP. 1-10 -NLS.

[0143] (SEQ ID NO:25);

[0144] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEG DTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGSGGGGSPKKKRKV(SEQ ID NO:26).

[0145] Linker-GFP was synthesized using gene synthesis. 11 The cDNA sequence was obtained and inserted into the EcoRI and XhoI restriction sites of the pcDNA3.1(+) vector to obtain the plasmid pcDNA3.1(+)-GFP. 11 The cDNA fragment of TFEB (the TFEB gene was derived from a commercially available plasmid, the amino acid sequence of which is shown in SEQ ID NO:16) was amplified by PCR using the following primers (nucleotide sequences shown in SEQ ID NO:27 and 28).

[0146] TFEB-For:cttggtaccgagctcggatccgccaccatgacagcaagctcaggc(BamHI)(SEQ IDNO:27);

[0147] TFEB-Rev: gctgcctccgccaccgaattccagcacatcgccctcctccatgctg(EcoRI) (SEQ ID NO: 28).

[0148] BamHI and EcoRI were used to target pcDNA3.1(+)-GFP. 11 Enzyme digestion was performed, and the amplified TFEB cDNA fragment was fused into the digested pcDNA3.1(+)-GFP using homologous recombination. 11 From the plasmid, we obtained plasmid pcDNA3.1(+)-TFEB-linker-GFP. 11 (The nucleotide sequence is shown in SEQ ID NO:29, and the amino acid sequence is shown in SEQ ID NO:30).

[0149]

[0150] MTASSGWEPAPAATMASRIGLRMQLMREQAQQEEQRERMQQQAVMHYMQQQQQQQQQLGGPPTPAINTPVHFQSPPPVPGEVLKVQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHISPAQGSP KPPPAASPGVRAGHVLSSSAGNSAPNSPMAMLHIGSNPERELDDVIDNIMRLDDVLGYINPEMQMPNTLPLSSSHLNVYSSDPQVTASLVGVTSSCPADLTQKRELTDAESRALAKERQKKDNHNLIE RRRRFNINDRIKELGMLIPKANDLDVRWNKGTILKASVDYIRRMQKDLQKSRELENHSRRLEMTNKQLWLRIQELEMQARVHGLPTTSPSGMNMAELAQQVVKQELPSEEGPGEALMLGAEVPDPEPLP ALPPQAPLPLPTQPPSPFHHLDFSHSLSFGGREDEGPPGYPEPLAPGHGSPFPSLSKKDLDLMLLDSLLPLASDPLLSTMSPEASKASSRRSSFSMEEGDVLEFGGGGSRDHMVLHEYVNAAGIT(SEQ ID NO:30).

[0151] Plasmid pcDNA3.1(+)-GFP 1-10 -NLS and / or plasmid pcDNA3.1(+)-TFEB-GFP 11 The cells were transfected separately or co-transfected into HeLa cells or HEK293T cells. Histone H2A was used to label the cell nuclei. Then, starvation or rapamycin was used to induce autophagy to promote the nuclear translocation of TFEB. Fluorescence microscopy was used to detect the fluorescence in HeLa cells and HEK293T cells, and the effectiveness of the probe (i.e., co-expression of GFP1-10-NLS and TFEB-NLS) in reflecting the nuclear translocation of TFEB was observed. This example demonstrates the results of transfection into HeLa cells. The results are as follows: Figure 2 and Figure 3 As shown. Figure 2The results showed that there was no obvious fluorescence in the cells when GFP1-10-NLS and TFEB-NLS were expressed alone. When GFP1-10-NLS and TFEB-NLS were co-expressed, there was obvious green fluorescence in the cells, and the expression was mainly in the nucleus. The fluorescence expressed in the nucleus had good co-localization with histone H2A. Figure 3 The results showed that the fluorescence intensity of the TFEB probe increased significantly under starvation or rapamycin treatment, indicating that the probe could sensitively translate the nuclear translocation of TFEB under induced stimulation.

[0152] Unless otherwise specified, the control group in this embodiment or other embodiments is a control group that has not been induced with HeLa cells or HEK293T cells.

[0153] Example 2:

[0154] The cDNA fragment of TFEB-mCherry (the TFEB-mCherry gene is derived from a commercially available plasmid) was amplified using PCR with the following primers (nucleotide sequences are shown in SEQ ID NO: 31 and 32).

[0155] TFEB-For: 5-cttggtaccgagctcggatccgccaccatgacagcaagctcaggc-3 (BamHI) (SEQ ID NO: 31);

[0156] TFEB-mCherry-Rev: 5-gctgcctccgccaccgaattccttgtacagctcgtccatgccgcc-3 (EcoRI) (SEQ ID NO: 32).

[0157] BamHI and EcoRI were used to target pcDNA3.1(+)-GFP. 11 Enzyme digestion was performed, and the amplified TFEB-mCherry cDNA fragment was fused into the digested pcDNA3.1(+)-GFP using homologous recombination. 11 From the plasmid, we obtained plasmid pcDNA3.1(+)-TFEB-mCherry-GFP. 11 Among them, TFEB-mCherry-GFP 11 The nucleotide sequence is shown in SEQ ID NO:33, and the amino acid sequence is shown in SEQ ID NO:34.

[0158]

[0159] (SEQ ID NO:34).

[0160] Plasmid pcDNA3.1(+)-GFP 1-10 -NLS (construction method see Example 1) and plasmid pcDNA3.1(+)-TFEB-mCherry-GFP 11 Co-transfected into HeLa cells, autophagy was induced using either starvation or rapamycin to promote the nuclear translocation of TFEB, and fluorescence was detected using fluorescence microscopy. The results are as follows:Figure 4 and Figure 5 As shown.

[0161] Figure 4 As can be seen, co-expression of GFP 1-10 -NLS and TFEB-mCherry-GFP 11 Red and green fluorescence have good co-localization. The fluorescence intensity of mCherry can be used as the total expression of TFEB, and the change in fluorescence intensity after TFEB nuclear translocation can be used as the nuclear entry of TFEB. The ratio of the change in fluorescence intensity after drug-induced TFEB nuclear translocation to the intensity of mCherry can more accurately reflect the nuclear entry of TFEB.

[0162] Figure 5 The study used starvation or rapamycin to induce autophagy, promoting the nuclear translocation of TFEB, and observed whether the probe could effectively reflect the nuclear translocation of TFEB. Statistical results showed that under starvation or rapamycin treatment, the fluorescence intensity of the TFEB probe significantly increased, indicating that the probe could sensitively translate the nuclear translocation of TFEB under induced stimulation. Example 3:

[0163] In Example 2, it was unexpectedly discovered that GFP 1-10 -NLS is expressed in greater quantities in the cytoplasm, which helps reduce GFP. 1-10 -NLS expression levels in the cytoplasm were assessed using different numbers of nuclear localization signals for GFP in this embodiment. 1-10 For nuclear localization, EGFP is used instead of GFP in this embodiment for ease of detection. 1-10 Conduct the experiment.

[0164] In this embodiment, the nucleic acid molecule encoding the fusion protein was constructed into the pcDNA3.1(+) vector, resulting in pcDNA3.1(+)-EGFP-NLS, pcDNA3.1(+)-EGFP-2×NLS, and pcDNA3.1(+)-EGFP-3×NLS, respectively. These plasmids were then transfected into HeLa cells to express the fusion protein. Specific experimental methods are described in Example 1. The amino acid sequence of EGFP-NLS is shown in SEQ ID NO:35, the amino acid sequence of EGFP-2×NLS is shown in SEQ ID NO:36, and the amino acid sequence of EGFP-3×NLS is shown in SEQ ID NO:37.

[0165] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGGGGSPKKKRKV(SEQ ID NO:35);

[0166] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGGGGSPKKKRKVPKKKRKV(SEQ ID NO:36);

[0167] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGGGGSPKKKRKVPKKKRKVPKKKRKV(SEQ ID NO:37).

[0168] The nucleus was labeled with histone H2A, and fluorescence was detected using a fluorescence microscope. The results are as Figure 6 shown.

[0169] Figure 6The results showed that as the number of nuclear localization sequences increased, the expression of EGFP in the cytoplasm decreased, while the expression of EGFP in the nucleus increased. When the number of nuclear localization sequences increased to three, EGFP was completely localized in the nucleus and had very good co-localization with histone H2A.

[0170] Example 4:

[0171] By comparison Figure 5 and Figure 3 Unexpectedly, TFEB-mCherry fused with GFP was discovered. 11 The intensity should increase more significantly after starvation or drug induction, indicating greater sensitivity for TFEB nuclear entry detection. This may be because TFEB and GFP... 11 The intermediate interval is longer, therefore this embodiment explores TFEB and GFP. 11 The impact of linker lengths on detection sensitivity.

[0172] In this embodiment, the nucleic acid molecule encoding the fusion protein is constructed into the pcDNA3.1(+) vector, resulting in pcDNA3.1(+)-TFEB-3×linker-GFP. 11 The constructed pcDNA3.1(+)-TFEB-3×linker-GFP 11 With pcDNA3.1(+)-GFP 1-10 -NLS (construction method see Example 1) was transfected into HeLa cells and HEK293T cells, enabling the fusion protein TFEB-3×linker-GFP to be expressed. 11 and GFP 1-10 -NLS was expressed in HeLa cells and HEK293T cells; specific experimental methods are described in Example 1. Among them, TFEB-3×linker-GFP... 11 The amino acid sequence is shown in SEQ ID NO:38.

[0173] (SEQ ID NO:38).

[0174] Then, in HEK293T and HeLa cells, autophagy was induced by starvation or rapamycin to promote the nuclear translocation of TFEB. Fluorescence microscopy was used to detect the fluorescence in HEK293T and HeLa cells, observing the probe (TFEB-3×linker-GFP). 11 and GFP 1-10 Whether -NLS can effectively reflect the nuclear translocation of TFEB, see the results. Figure 7 .

[0175] The results show that, with Figure 3 In comparison, under conditions of starvation or rapamycin treatment, Figure 7 The fluorescence intensity was significantly increased, indicating that the probe could sensitively translate the nuclear translocation of TFEB under induced stimulation. Furthermore, the optimized probe was mainly expressed in the cell nucleus, and no fluorescence was observed in the cytoplasm.

[0176] Example 5:

[0177] Currently, the traditional TFEB nuclear entry detection probes in this field involve fusing TFEB with fluorescent proteins (such as EGFP and mCherry) and expressing them into cells. After drug induction, the increase in nuclear fluorescence intensity and the decrease in cytoplasmic fluorescence are observed under a laser confocal microscope. The obtained data needs to be manually processed one by one, which is generally suitable for laboratory validation work but not for large-scale drug screening.

[0178] Fluorescent microplate readers can rapidly read fluorescence intensity values ​​in 96-384 well plates, with short processing time and no need for secondary data processing, making them a popular choice for drug screening. Based on this, this embodiment uses TFEB-3×linker-GFP co-expressed in Example 4. 11 and GFP 1-10 -NLS used rapamycin to induce autophagy in HeLa and HEK293T cells to promote the nuclear translocation of TFEB, and the changes in fluorescence intensity of the TFEB nuclear translocation probe under rapamycin treatment were detected by microplate reader. The results are as follows: Figure 8 As shown.

[0179] Figure 8 The results showed that the results of the ELISA reader and Figure 3 , Figure 5 , Figure 7 The results were consistent, indicating that the probe constructed in this invention is suitable for high-throughput screening using an ELISA reader. Therefore, it can be further stated that the probe of this application can be used to detect TFEB nuclear translocation, and can be used for high-throughput screening of TFEB nuclear translocation drugs, with short processing time and low screening cost.

[0180] Example 6:

[0181] To further optimize the fluorescence intensity changes of the TFEB probe in untreated cells, this embodiment fuses 3×GFP after the TFEB gene. 11 (TFEB-3×linker-3×GFP 11 ) or 5×GFP 11 (TFEB-5×linker-5×GFP 11 ), and with TFEB-linker-GFP 11 Compare them.

[0182] In this embodiment, the nucleic acid molecule encoding the fusion protein is constructed into the pcDNA3.1(+) vector to obtain pcDNA3.1(+)-TFEB-linker-GFP. 11 , pcDNA3.1(+)-TFEB-3×linker-3×GFP 11 , pcDNA3.1(+)-TFEB-5×linker-5×GFP11 The constructed pcDNA3.1(+)-TFEB-linker-GFP 11 , pcDNA3.1(+)-TFEB-3×linker-3×GFP 11 , pcDNA3.1(+)-TFEB-5×linker-5×GFP 11 With pcDNA3.1(+)-GFP respectively 1-10 -NLS (construction method see Example 1) was transfected into HeLa cells to induce the fusion protein TFEB-linker-GFP. 11 (TFEB-3×linker-3×GFP 11 or TFEB-5×linker-5×GFP 11 ) and GFP 1-10 -NLS is expressed in HeLa cells; specific experimental methods are described in Example 1. Among them, TFEB-3×linker-3×GFP... 11 The amino acid sequence is shown in SEQ ID NO:39, TFEB-5×linker-5×GFP. 11 The amino acid sequence is shown in SEQ ID NO:40.

[0183] MTASSGWEPAPAATMASRIGLRMQLMREQAQQEEQRERMQQQAVMHYMQQQQQQQQQQLGGPPTPAINTPVHFQSPPPVPGEVLKVQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHISPAQGSPKPPPAASPGVRAGHVLSSSAGNSAPNSPMAMLHIGSNPERELDDVIDNIMRLDDVLGYINPEMQMPNTLPLSSSHLNVYSSDPQVTASLVGVTSSSCPADLTQKRELTDAESRALAKERQKKDNHNLIERRRRFNINDRIKELGMLIPKANDLDVRWNKGTILKASVDYIRRMQKDLQKSRELENHSRRLEMTNKQLWLRIQELEMQARVHGLPTTSPSGMNMAELAQQVVKQELPSEEGPGEALMLGAEVPDPEPLPALPPQAPLPLPTQPPSPFHHLDFSHSLSFGGREDEGPPGYPEPLAPGHGSPFPSLSKKDLDLMLLDDSLLPLASDPLLSTMSPEASKASSRRSSFSMEEGDVLEFMGGGGSGGGGSGGGGSRDHMVLHEYVNAAGITGGGGSGGGGSRDHMVLHEYVNAAGITGGGGSGGGGSRDHMVLHEYVNAAGIT(SEQ ID NO:39);

[0184] (SEQ ID NO:40).

[0185] Fluorescence in uninduced HeLa cells was detected using fluorescence microscopy. The results are shown in [link to results]. Figure 9 The results showed that, due to the low level of autophagy in the cells, some TFEB was present in the nucleus under uninduced conditions, and nuclear fluorescence was observed. Because the basal fluorescence of the probe was weak in the early stages, the fluorescence intensity after drug induction was not very high either. However, with the increase of GFP... 11 With increasing quantity, the fluorescence intensity of the TFEB probe gradually increases under uninduced conditions. Therefore, increasing the baseline fluorescence value allows for a more direct observation of the fluorescence change. This result further illustrates the effectiveness of using multiple GFP probes. 11 It is more conducive to the screening of TFEB cores.

[0186] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0187] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A probe array, comprising: First fusion protein and second fusion protein; The first fusion protein includes a nuclear localization sequence and a first protein molecule, wherein the nuclear localization sequence is linked to the first protein molecule; The second fusion protein includes a TFEB protein and a second protein molecule, wherein the TFEB protein is linked to the second protein molecule, the second protein molecule is used to bind to the first protein molecule, and the binding of the first protein molecule and the second protein molecule produces a detectable signal.

2. The probe assembly according to claim 1, characterized in that, The C-terminus of the nuclear localization sequence is linked to the N-terminus of the first protein molecule; And / or, the C-terminus of the TFEB protein is connected to the N-terminus of the second protein molecule, or the C-terminus of the second protein molecule is connected to the N-terminus of the TFEB protein; And / or, the first protein molecule and the second protein molecule are fluorescent bimolecular complementary proteins; And / or, the nuclear localization sequence has an amino acid sequence as shown in any one of SEQ ID NO:1 to 15; And / or, the TFEB protein has an amino acid sequence as shown in SEQ ID NO:

16.

3. The probe assembly according to claim 2, characterized in that, The first protein molecule and the second protein molecule are selected from any one of the following groups: 1)GFP 1-10 EBFP2 1-10 Cerulean 1-10 One of them, and GFP 11 , 2)mNG2 1-10 mNG3K 1-10 mNG3A 1-10 One of them, and mNG2 11 , 3) mRuby4 1-10 sfCherry2 1-10 One of them, and sfCherry2 11 , 4) GAF protein and PAS protein; And / or, the GFP 1-10 It has the amino acid sequence shown in SEQ ID NO:17; And / or, the EBFP2 1-10 It has the amino acid sequence shown in SEQ ID NO:18; And / or, the Cerulean 1-10 It has the amino acid sequence shown in SEQ ID NO:19; And / or, the GFP 11 It has the amino acid sequence shown in SEQ ID NO:20; And / or, the mNG2 1-10 It has the amino acid sequence shown in SEQ ID NO:21; And / or, the mNG3K 1-10 It has the amino acid sequence shown in SEQ ID NO:22; And / or, the mNG3A 1-10 It has the amino acid sequence shown in SEQ ID NO:23; And / or, the mNG2 11 It has the amino acid sequence shown in SEQ ID NO:24; And / or, the mRuby4 1-10 It has the amino acid sequence shown in SEQ ID NO:43; And / or, the sfCherry2 1-10 It has the amino acid sequence shown in SEQ ID NO:44; And / or, the sfCherry2 11 It has the amino acid sequence shown in SEQ ID NO:45; And / or, the PAS protein has the amino acid sequence shown in SEQ ID NO:46; And / or, the GAF protein has an amino acid sequence as shown in SEQ ID NO:

47.

4. The probe assembly according to claim 3, characterized in that, The first protein molecule and the second protein molecule are selected from any one of the following groups:

5. The probe assembly according to any one of claims 1 to 4, characterized in that, In the first fusion protein, one or more nuclear localization sequences are provided, preferably 1, 2, 3, 4 or 5; And / or, the first fusion protein further includes a first linker peptide, wherein the C-terminus of the first protein molecule is linked to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is linked to the N-terminus of the nuclear localization sequence. Optionally, the first linker peptide is selected from (GGGGS)n, where n is any integer between 1 and 10; Optionally, the first linker peptide is selected from GGGGS; And / or, in the second fusion protein, one or more second protein molecules are provided, preferably 1, 2, 3, 4 or 5; And / or, the second fusion protein further includes one or more second linker peptides, wherein the C-terminus of the TFEB protein is linked to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is linked to the N-terminus of the second protein molecule; Optionally, the second linker peptide is selected from (GGGGS)n, where n is any integer between 1 and 10; Optionally, the second linker peptide is selected from (GGGGS)2; Optionally, the second linker peptide is set to 1, 2, 3, 4 or 5; Optionally, the number of the second protein molecule and the second linker peptide are the same, and the C-terminus of each second linker peptide is linked to the N-terminus of the second protein molecule; Optionally, the second fusion protein further includes a fluorescent protein, wherein the C-terminus of the TFEB protein is linked to the N-terminus of the fluorescent protein, the C-terminus of the fluorescent protein is linked to the N-terminus of the second protein molecule, and the fluorescence color of the fluorescent protein is different from the fluorescence color of the first protein molecule and the second protein molecule after binding.

6. The probe assembly according to any one of claims 1 to 4, characterized in that, The probe set includes a first fusion protein as shown in any one of SEQ ID NO:26, 41 and 42, and a second fusion protein as shown in any one of SEQ ID NO:30, 34, 38 to 40; Optionally, the probe set includes: The first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:30; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:34; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:38; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:39; and / or the first fusion protein as shown in SEQ ID NO:26 and the second fusion protein as shown in SEQ ID NO:40; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:30; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:34; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:38; and / or the first fusion protein as shown in SEQ ID NO:41 and the second fusion protein as shown in SEQ ID NO:

30. The second fusion protein shown in NO:39; and / or the first fusion protein shown in SEQ ID NO:41 and the second fusion protein shown in SEQ ID NO:40; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:30; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:34; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:38; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:39; and / or the first fusion protein shown in SEQ ID NO:42 and the second fusion protein shown in SEQ ID NO:

40.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the probe set according to any one of claims 1 to 6.

8. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule according to claim 7; Optionally, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage, preferably a plasmid expression vector.

9. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of claim 7 or the expression vector of claim 8; or The recombinant cells express the probe set according to any one of claims 1 to 6; Optionally, the recombinant cells are obtained by introducing the expression vector of claim 8 into a host cell.

10. A drug screening system, wherein the drug is used to regulate TFEB, characterized in that, The system includes the recombinant cells of claim 9.

11. A method for screening drugs, said drugs being used to regulate TFEB, characterized in that, The method includes: The recombinant cells described in claim 9 are used to screen for drugs; Based on the detectable signals in the recombinant cells, it is determined whether the drug is the target drug.

12. Use of the probe set according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, or the recombinant cell according to claim 9 in the preparation of a high-throughput screening drug, wherein the drug is used to regulate TFEB.

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