Trimerization regulatory element, method for constructing same, and use thereof

Abstract

Disclosed are a trimerization regulatory element, a method for constructing same, and use thereof. In addition to a protein A and a protein B that are obtained by splitting the hepatitis C virus protease NS3a, the trimerization regulatory element comprises a protein C. The trimerization of the trimerization regulatory element can be mediated by means of a targeting drug specific to the protease NS3a (grazoprevir / danoprevir). The trimerization regulatory element provided by the present invention has an excellent small-molecule-mediated trimerization effect, and a drug approved by the National Medical Products Administration is used. The protein used does not exist in the human body; therefore, the protein can result in better biological orthogonality. Moreover, the protein can be used as a clinically effective drug regulatory switch, effectively avoiding problems such as serious cytotoxicity and off-target effects.

Description

A trimerization regulatory element, a method for constructing the same and applications thereof

[0001] This application claims priority to Chinese patent application 202410786398X with a filing date of 2024 / 6 / 18. This application incorporates the entire text of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the field of protein technology, in particular to a trimerization regulatory element, a method for constructing the same and applications thereof. BACKGROUND

[0003] Synthetic biology is a frontier research field that aims to develop complex, programmable controls to regulate cell behavior by using engineering concepts such as computer-aided design, modularization, abstraction, and feedback control, combined with specific biological rules, to establish new biological systems [1] For example, small molecule compounds mediated trimerization biological elements obtained through rational design can help further explore the physiological processes in cells and can serve as powerful tools for studying dynamic physiological processes in cells. Synthetic biology not only provides scientists in the field of life medicine research with diverse tools for cell biology research, but has also been applied to new clinical treatment models.

[0004] Techniques for transiently regulating protein co-localization play a very important role in studying and programming dynamic physiological processes in cells. Co-localization regulation of proteins using small molecules is a common method. Existing small molecule compounds mediated trimerization regulatory elements (a system based on FKBP-FRB-Rapamycin split design [2] ) have a sequence of binding of small molecules and different components, and are not completely independent trimerization systems. Moreover, the small molecules used usually interfere with endogenous signaling pathways in the human body, causing cytotoxicity, thus having certain limitations in subsequent clinical use [3] Designing based on target points of drugs approved by the National Medical Products Administration has better biological orthogonality, which can effectively avoid serious cytotoxicity and off-target problems, and has a broader application range and prospect.

[0005] REFERENCES

[0006] [1] Kitada T, DiAndreth B, Teague B, Weiss R. Science. 2018 Feb 9;359(6376).

[0007] [2]Wu HD, Kikuchi M, Dagliyan O, Aragaki AK, Nakamura H, Dokholyan NV, Umehara T, Inoue T. Nat Methods. 2020 Sep; 17(9): 928-936.

[0008] [3]Bayle JH, Grimley JS, Stankunas K, Gestwicki JE, Wandless TJ, Crabtree GR. Chem Biol. 2006 Jan; 13(1): 99-107. SUMMARY

[0009] To solve the technical problems of lack of using FDA-approved drugs as a trimerization system for regulating small molecules and easy to cause cytotoxicity or off-target in application in the prior art, the application provides a trimerization regulatory element, a method for constructing the same, and application thereof. The application is designed by rational disassembly of the serine protease NS3a of hepatitis C virus, and two protein components obtained after disassembly, and a Binder protein based on specific targeted drugs designed by computer-aided de novo design to form a trimerization element, and the trimerization is mediated by the specific targeted drug (grazoprevir / danoprevir) of the NS3a protease. The trimerization regulatory element provided by the application has excellent small molecule drug-mediated trimerization effect, and uses FDA-approved drugs, and the proteins used do not exist in the human body, which can bring better biological orthogonality, and can be used as an effective drug regulatory switch in clinic, which can effectively avoid serious cytotoxicity and off-target problems.

[0010] The application solves the above technical problems through the following technical solutions.

[0011] The application provides a trimerization regulatory element in the first aspect, wherein in addition to comprising protein A and protein B obtained by disassembly of the hepatitis C virus protease NS3a, the trimerization regulatory element further comprises protein C.

[0012] In the present application, the trimerization regulatory element is composed of three protein fragments. Two of the protein fragments, protein A and protein B, are also called dimerization regulatory elements, which are split from the hepatitis C virus protease. The third protein fragment, protein C, is also called binder protein, which includes GNCR1 or DNCR2. Small molecule drugs mediate the trimerization or dimerization of the trimerization regulatory element or the dimerization regulatory element through non-covalent binding. The protein C is a Binder protein designed from scratch based on specific targeting drugs through computer assistance, including Grazoprevir / NS3a complex reader 1 (GNCR1) or Danoprevir / NS3a complex reader 2 (DNCR2). After the dimerization regulatory element or the trimerization regulatory element is combined with the transcription regulatory element, it also has the function of activating downstream genes and regulating transcription.

[0013] In some embodiments of the present application, the protein A includes an amino acid sequence as shown in SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15, and / or the protein B includes an amino acid sequence as shown in SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16; the protein C is a drug / NS3a complex binding protein, for example, GNCR1 or DNCR2; the GNCR1 includes an amino acid sequence as shown in SEQ ID NO: 27, and the DNCR2 includes an amino acid sequence as shown in SEQ ID NO: 26.

[0014] In some embodiments of the present application, the protein A and the protein B are selected from any one of the following groups:

[0015] (1) the protein A includes an amino acid sequence as shown in SEQ ID NO: 11, and the protein B includes an amino acid sequence as shown in SEQ ID NO: 12;

[0016] (2) the protein A includes an amino acid sequence as shown in SEQ ID NO: 13, and the protein B includes an amino acid sequence as shown in SEQ ID NO: 14;

[0017] (3) the protein A includes an amino acid sequence as shown in SEQ ID NO: 15, and the protein B includes an amino acid sequence as shown in SEQ ID NO: 16.

[0018] In some specific embodiments of the present application, the trimerization regulatory element is selected from any one of the following groups:

[0019] (1) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 11, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 27;

[0020] (2) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 13, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 14, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 27;

[0021] (3) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 15, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 16, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 27;

[0022] (4) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 11, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 26;

[0023] (5) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 13, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 14, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 26;

[0024] (6) the protein A comprises an amino acid sequence as set forth in SEQ ID NO: 15, the protein B comprises an amino acid sequence as set forth in SEQ ID NO: 16, and the protein C comprises an amino acid sequence as set forth in SEQ ID NO: 26.

[0025] The second aspect of the present application provides a dimerization regulation system, which comprises the protein A and the protein B as defined in the trimerization regulation element of the first aspect, and further comprises a small molecule drug, i.e. Grazoprevir. The CAS number of Grazoprevir is 1350514-68-9.

[0026] In some embodiments of the present application, the dimerization of the protein A and the protein B is mediated by non-covalent binding of the Grazoprevir.

[0027] The elements in the dimerization regulation system of the present application can exist in the following states:

[0028] (1) the Grazoprevir, the protein A and the protein B are in an isolated state;

[0029] (2) the protein A is combined with the protein B through non-covalent bond, and the protein A and the protein B are in a dimerization state.

[0030] In some embodiments of the present application, the protein A and the protein B are selected from any one of the following groups:

[0031] (1) the protein A comprises an amino acid sequence as shown in SEQ ID NO: 11, and the protein B comprises an amino acid sequence as shown in SEQ ID NO: 12; or, (2) the protein A comprises an amino acid sequence as shown in SEQ ID NO: 13, and the protein B comprises an amino acid sequence as shown in SEQ ID NO: 14; or, (3) the protein A comprises an amino acid sequence as shown in SEQ ID NO: 15, and the protein B comprises an amino acid sequence as shown in SEQ ID NO: 16.

[0032] The third aspect of the present application provides a trimerization regulation system, which comprises the trimerization regulation element according to the first aspect.

[0033] In some embodiments of the present application, the trimerization regulation system further comprises a small molecule drug, for example, Grazoprevir (Grazo) or Danoprevir (Dano).

[0034] The CAS number of Grazoprevir is 1350514-68-9, and the CAS number of Danoprevir is 850876-88-9.

[0035] In some preferred embodiments of the present application, the small molecule drug mediates the trimerization of the trimerization regulation element according to the first aspect through non-covalent bond.

[0036] The fourth aspect of the present application provides an isolated nucleic acid, characterized in that the nucleic acid comprises a nucleotide sequence encoding the trimerization regulation element according to the first aspect.

[0037] The fifth aspect of the present application provides a recombinant expression vector, which comprises the nucleic acid according to the fourth aspect.

[0038] In some embodiments of the present application, the backbone plasmid of the recombinant expression vector is pcDNA3.1(+), pcDNA5 or pGEX-6P-1.

[0039] In some embodiments of the present application, the recombinant expression vector further comprises a tetR gene, a transcriptional activator, a self-cleavage peptide and / or an IRES element; the transcriptional activator is for example VP64, p65 and / or RtA; the self-cleavage peptide is for example P2A; and the IRES element is for example IRES2.

[0040] In some preferred embodiments of the present application, the sequence of the protein A, the protein B and the protein C are separated by the IRES element and / or the P2A on the recombinant expression vector; and / or, the protein A, the protein B and the protein C are connected with the tetR gene, VP64 and "p65 or RtA", respectively.

[0041] In some more preferred embodiments of the present application, the tetR gene is located at the 5' end of the nucleotide sequence encoding the protein A, the VP64 is located at the 3' end of the nucleotide sequence encoding the protein B, and the p65 or RtA is located at the 3' end of the nucleotide sequence encoding the protein C.

[0042] In some specific embodiments of the present application, the recombinant expression vector comprises, from 5' end to 3' end, the protein B, the VP64, the IRES element, the protein C, the p65, the P2A, the tetR and the protein A; or, the protein B, the VP64, the IRES element, the protein C, the RtA, the P2A, the tetR and the protein A. The Tet-off system used in the present application can also be other Tet-off systems commonly used in the art.

[0043] The sixth aspect of the present application provides an isolated cell, wherein the cell comprises the nucleic acid according to the fourth aspect or the recombinant expression vector according to the fifth aspect.

[0044] In some embodiments of the present application, the cell is a mammalian cell, for example, a HeLa-CCL2 or HEK-293T cell.

[0045] The seventh aspect of the present application provides a kit, wherein the kit comprises one or more of the trimerization regulatory element according to the first aspect, the dimerization regulatory system according to the second aspect, the trimerization regulatory system according to the third aspect, the nucleic acid according to the fourth aspect, the recombinant expression vector according to the fifth aspect, and the cell according to the sixth aspect.

[0046] The eighth aspect of the present application provides a method for preparing a trimerization regulatory element, wherein the method comprises the steps of culturing the cell according to the sixth aspect and obtaining the trimerization regulatory element.

[0047] The ninth aspect of the present application provides a method for constructing a dimerization regulatory system, wherein the method comprises the following steps:

[0048] (1) splitting the hepatitis C virus protease into protein A and protein B;

[0049] (2) adding a small molecule drug, wherein the small molecule drug mediates the dimerization of the protein A and the protein B through non-covalent binding, i.e., forming the dimerization regulatory system;

[0050] The protein A and the protein B are as defined in the trimerization regulatory element of the first aspect, and the small molecule drug is grazoprevir.

[0051] The tenth aspect of the present application provides a method for constructing a trimerization regulatory system, the method comprising the following steps:

[0052] (1) splitting the hepatitis C virus protease into protein A and protein B, and adding protein C;

[0053] (2) adding a small molecule drug, which mediates the trimerization of protein A, protein B and protein C through non-covalent binding, i.e. forming the trimerization regulatory system;

[0054] The protein A, the protein B and the protein C are as defined in the trimerization regulatory element of the first aspect, and the small molecule drug is as defined in the trimerization regulatory system of the third aspect.

[0055] The eleventh aspect of the present application provides a method for regulating the expression of a target gene by using the trimerization regulatory element of the first aspect, the dimerization regulatory system of the second aspect, the trimerization regulatory system of the third aspect, the nucleic acid of the fourth aspect, the recombinant expression vector of the fifth aspect, the cell of the sixth aspect or the kit of the seventh aspect to regulate the expression of a target gene.

[0056] In some embodiments of the present application, the method is a method for non-disease treatment purposes.

[0057] The twelfth aspect of the present application provides the use of the trimerization regulatory element of the first aspect, the dimerization regulatory system of the second aspect, the trimerization regulatory system of the third aspect, the nucleic acid of the fourth aspect, the recombinant expression vector of the fifth aspect, the cell of the sixth aspect or the kit of the seventh aspect in the preparation of a reagent or a drug for regulating cell signal transduction, gene editing, protein co-localization or cell therapy.

[0058] In some embodiments of the present application, the cell therapy is CAR-T cell therapy.

[0059] The thirteenth aspect of the present application provides the use of the trimerization regulatory element of the first aspect, the dimerization regulatory system of the second aspect, the trimerization regulatory system of the third aspect, the nucleic acid of the fourth aspect, the recombinant expression vector of the fifth aspect, the cell of the sixth aspect or the kit of the seventh aspect in regulating cell signal transduction, gene editing, protein co-localization or cell therapy.

[0060] In some embodiments of the present application, the cell therapy is CAR-T cell therapy.

[0061] The fourteenth aspect of the present application provides the trimerization regulatory element of the first aspect, the dimerization regulatory system of the second aspect, the trimerization regulatory system of the third aspect, the nucleic acid of the fourth aspect, the recombinant expression vector of the fifth aspect, the cell of the sixth aspect, or the kit of the seventh aspect for regulating cell signaling, gene editing, protein co-localization, or cell therapy.

[0062] In some embodiments of the present application, the cell therapy is CAR-T cell therapy.

[0063] In some embodiments of the present application, the regulation of cell signaling is the regulation of the expression of SEAP reporter gene. The method for regulating the expression of the gene is achieved by the trimerization of protein A, protein B, and protein C. For example, when the trimerization of protein A, protein B, and protein C does not occur, the gene is in a silent state, and when the trimerization occurs, the expression of the SEAP reporter gene is triggered.

[0064] In some embodiments of the present application, the regulation of cell signaling is the regulation of the activation and deactivation of Ras / MAPK signal.

[0065] The trimerization regulatory element, the trimerization regulatory system, the dimerization regulatory system, the nucleic acid, the recombinant expression vector, the cell, or the kit of the present application can be used for various biological and pre-transformation application researches, including the regulation of cell signaling pathway, epigenetic engineering, and cell therapy based on drug delivery, etc.

[0066] In the present application, "A element", "A protein" or "protein A", "B element", "B protein" or "protein B" respectively refer to the rational split design of the serine protease NS3a of hepatitis C virus, and the two protein components NS3a-A and NS3a-B obtained after splitting. The splitting site and specific sequence information are shown in Table 1. In the present application, "protein C", "C protein" or "element C" is a Binder protein designed from scratch with computer assistance, including GNCR1 and DNCR2, which can trimerize with protein A and protein B described in the present application under the mediation of small molecule drugs to form a complex. In some specific embodiments of the present application, the sequences of GNCR1 and DNCR2 are shown in SEQ ID NO: 27 and SEQ ID NO: 26, respectively.

[0067] The trimerization described in the present application refers to chemically induced trimerization (CIT), which is a strategy for regulating protein function and structural changes using exogenous ligands. Split protein fragments obtained after splitting the protein can be used for protein structural reorganization with the Binder protein, and the exogenous ligand acts as a "bridge" between the three fragments. Specifically, the present application is to mediate the close proximity of three proteins to form a complex through non-covalent binding of small molecule compounds.

[0068] The dimerization described in the present application refers to chemically induced dimerization (CID), which is a strategy for regulating protein function and structural changes using exogenous ligands. Split protein fragments obtained after splitting the protein can be used for protein structural reorganization, and the exogenous ligand acts as a "bridge" between the two fragments. The ligand contains a considerable free energy when it is tightly bound to the binding pocket of the protein, which can promote the dimerization of the two protein fragments. Specifically, the present application is to induce the close proximity of two proteins to form a complex through non-covalent binding between small molecule compounds and each protein. Specifically, the present application is to induce the close proximity of two proteins to form a complex through non-covalent binding between small molecule compounds and each protein.

[0069] On the basis of common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. each preferred example of the present application.

[0070] The reagents and raw materials used in the present application are commercially available.

[0071] The positive progress effect of the present application is that the trimerization regulatory element provided by the present application has excellent small molecule-mediated trimerization effect. The use of drugs approved by the State Drug Administration can bring better biological orthogonality, effectively avoid serious cytotoxicity and off-target problems in application, and can be widely used in biological and pre-transformation application research, and can be widely used in regulating cell signal transduction, gene editing, protein co-localization or cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0072] FIG. 1A-FIG. 1C are schematic diagrams of small molecule drug-mediated trimerization biological elements. FIG. 1A: Schematic diagram of small molecule drug-mediated trimerization biological elements; FIG. 1B: Molecular structure of the Hepatitis C Virus Serine Protease NS3a targeted drug Danoprevir, and its mediated structure of the corresponding Binder protein DNCR2 forming a complex with NS3a (PDB ID: 6N4N), which can be split into two parts A / B by site; FIG. 1C: Molecular structure of the Hepatitis C Virus Serine Protease NS3a targeted drug Grazoprevir, and its mediated structure of the corresponding Binder protein GNCR1 forming a complex with NS3a (predicted using Alphafold2), which can be split into two parts A / B by site.

[0073] FIG. 2A-FIG. 2C are basic information of each amino acid site in the NS3a protein sequence. FIG. 2A: Solvent accessible area (SAA, Solvent Accessible Area) of each site is considered, FIG. 2B: Conservation of each site is considered, and FIG. 2C: Split energy of each site is considered. The candidate sites are marked in black.

[0074] FIG. 3A-FIG. 3C are schematic diagrams of secreted alkaline phosphatase and preliminary screening expression results of 6#, 7#, 8# split sites in the SEAP system. FIG. 3A: Schematic diagram of secreted alkaline phosphatase, which can induce the trimerization of split proteins NS3a-A / B and Binder proteins by adding NS3a specific targeted drugs, thereby activating the transcription and secretory expression of SEAP gene. FIG. 3B: Preliminary screening from 12 candidate sites using Danoprevir, including the results of adding or not adding Binder protein DNCR2; FIG. 3C: Preliminary screening from 12 candidate sites using Grazoprevir, including the results of adding or not adding Binder protein GNCR1.

[0075] FIG. 4A and FIG. 4B are further expression verification results and action conditions in the SEAP system. FIG. 4A: Schematic diagram of a single plasmid for overexpression of trimerization elements by introducing IRES / P2A elements (from 5' end to 3' end); FIG. 4B: Further expression verification results and action conditions of 6#, 7#, 8# split sites in the SEAP system, PC is a NS3a full-length protein fused with tetR and VP64 at N and C termini, respectively.

[0076] FIGS. 5A-5D are VPR system splitting effect verification by Danoprevir-mediated 6# / 7# / 8# split site trimerization system. FIG. 5A is a schematic diagram of the secreted alkaline phosphatase system with the introduction of p65 / RtA transcription factor, which is fused to the C-terminus of Binder protein DNCR2; FIG. 5B is the result of the interaction of p65 and RtA with protein A and protein B obtained from 6# and 7# split sites, respectively; FIG. 5C is a schematic diagram of a plasmid further containing p65 on a single plasmid with IRES / P2A elements (from 5' end to 3' end); and FIG. 5D is the result of the SEAP system with the overexpression of p65 at 6#, 7# or 8# sites by a single plasmid containing IRES / P2A elements.

[0077] FIGS. 6 and 7A-7F are the effect of the trimerization system verified by fluorescence co-localization (taking the 6# split site as an example). FIG. 6 is a schematic diagram of fluorescence co-localization experiment, different fluorescence proteins are fused on A / B / C three biological elements to observe the co-localization; FIGS. 7A-7F are the fluorescence intensity changes of BFP, eGFP and mCherry three fluorescence in the cell membrane and cytoplasm of living cells at different time points after Danoprevir treatment under confocal microscope, FIGS. 7A and 7D are the fluorescence intensity changes at 0 min, FIGS. 7B and 7E are the fluorescence intensity changes at 10 min, and FIGS. 7C and 7F are the fluorescence intensity changes at 20 min; in FIGS. 7A-7C, the upper left small graph is a Merge graph formed by merging the upper right, lower left and lower right three small graphs, the upper right small graph shows the mcherry fluorescence intensity, the lower left small graph shows the eGFP fluorescence intensity, and the lower right shows the BFP fluorescence intensity. DETAILED DESCRIPTION

[0078] The present application is further illustrated by the following examples without limiting the present application to the described examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0079] Example 1 Selection of Split Sites

[0080] Based on the information of protein sequence conservation degree, protein structure solvent accessible area and protein splitting energy, a total of 12 candidate sites (FIGS. 2A-2C) were selected, and the hepatitis C virus protease NS3a (amino acid sequence as shown in SEQ ID NO: 25) was truncated into A / B two parts from the candidate sites for subsequent verification (FIG. 1A).

[0081] The detailed information of A / B protein sequences is shown in Table 1:

[0082] Table 1 Detailed information of A / B protein sequences

[0083] Construction of secreted alkaline phosphatase (SEAP) reporter system

[0084] The present application screens and preliminarily verifies the candidate sites by a secreted alkaline phosphatase (SEAP) reporter gene system (Figure 3A). SEAP is a mutant of human placental alkaline phosphatase, which can be secreted by the expression cells to the outside of the cells. The cell culture supernatant can be detected at any time point without destroying the cells. Finally, the expression and effect of the element are characterized by the strength of the enzyme activity. The SEAP reporter gene can be activated to express transcription under the joint action of tetR element (DNA recognition domain) and VP64 (transcription activation element) and p65 (transcription activation element). The SEAP protein after expression can be secreted to the outside of the cells to react with the exogenous substrate, and further quantitatively detected by an enzyme label instrument.

[0085] The application constructs plasmid A expressing N-terminal fusion tetR A protein, plasmid C expressing C protein (plasmid sequence as shown in SEQ ID NO: 30 or 31) and plasmid B expressing C-terminal fusion VP64 B protein based on the SEAP reporter gene for preliminary verification (principle schematic diagram as shown in FIG. 3A, results as shown in FIG. 3B and FIG. 3C), and plasmid A / B / C all use pcDNA3.1(+) as an expression vector. In the application, the C protein is a Binder protein designed from scratch based on specific targeting drugs by computer assistance, including Grazoprevir / NS3a complex binding protein 1 (Grazoprevir / NS3a complex reader 1, GNCR1) or Danoprevir / NS3a complex binding protein 2 (Danoprevir / NS3a complex reader 2, DNCR2). The SEAP gene is expressed by the plasmid pMF111 (pMF111 can refer to the literature Xue, S., Yin, J., Shao, J., Yu, Y., Yang, L., & Wang, Y., et al. (2017). A synthetic-biology-inspired therapeutic strategy for targeting and treating hepatogenous diabetes. Molecular Therapy, 25(2); the specific nucleotide sequence is shown in SEQ ID NO: 29). In the plasmid construction of further verification test, pcDNA3.1(+) (purchased from Addgene, catalog number #138209) is used as an expression vector, the C-terminal fusion VP64 B protein and the C protein are separated by an IRES element, and the C protein and the N-terminal fusion tetR A protein are separated by a P2A sequence, so that all elements are expressed on the same plasmid (FIG. 4A). Further, the SEAP reporter system of p65 is introduced, and the plasmid is constructed to fuse p65 at the C-terminal of the C protein based on the plasmid containing the IRES element and the P2A sequence (FIG. 5C).

[0086] The constructed plasmid is transfected into mammalian cells HEK-293T by transient transfection. In a 24-well plate, 1×10 5Each well was inoculated with cells at a certain concentration, and after 16-20 hours of incubation at 37°C, 300 μg of each of the four plasmids A / B / C / pMF111 was mixed with 1.5 μL of Lipo3000, and incubated at room temperature for 10-15 min before being added dropwise to the well. After transfection for 2-4 hours, the medium was replaced, and small molecule drugs Danoprevir (Danoprevir, Dano) or Grazoprevir (Grazoprevir, Grazo) were added to a final concentration of 10 μM for Danoprevir and 5 μM for Grazoprevir. After 48 hours of incubation at 37°C, the supernatant was collected for SEAP activity detection. The molecular structure of Danoprevir and its mediation of the formation of a complex between the corresponding Binder protein DNCR2 and NS3a are shown in Figure 1B, and the molecular structure of Grazoprevir and its mediation of the formation of a complex between the corresponding Binder protein GNCR1 and NS3a are shown in Figure 1C.

[0087] Example 3 Secreted alkaline phosphatase activity detection

[0088] After 48 hours of cell culture, the collected supernatant samples were placed in a 65°C water bath for 30 minutes, and then phosphatase activity was determined using the SEAP Reporter Gene Assay kit (Luminescence) (abcam, ab133077). This reaction can also use p-nitrophenyl phosphate (PNPP) (Macklin, N816055) as an exogenous substrate for phosphatase, and 60 μL of 20 mM PNPP substrate solution was added to 40 μL of sample. After the reaction, the absorbance value was measured at a wavelength of 405 nm. Enzyme activity detection used a Tecan Spark microplate multifunctional analyzer to measure the absorbance value of the reaction product for 10 minutes, and the strength of phosphatase activity was calculated by the rate of change in absorbance value. The strength of the SEAP enzyme activity represents the affinity of the trimerization biological element composed of different split sites of the hepatitis C virus serine protease NS3a protein.

[0089] Example 4 Verification of small molecule drug-mediated trimerization system

[0090] The application designs 12 candidate split sites in total, and the preliminary screening is carried out by using the hepatitis C virus serine protease targeted drug Danoprevir (TargetMol, T6025) and Grazoprevir (TargetMol, T4547). The results are shown in FIG. 3B and FIG. 3C. The 6th, 7th and 8th split sites have stronger signals, but Grazoprevir can induce the dimerization of the NS3a-A / B two-part element without the participation of the C protein, while Danoprevir cannot induce the dimerization of the NS3a-A / B two-part element. Therefore, the trimerization system mediated by Grazoprevir is different from the trimerization system mediated by Danoprevir, and the elements in the trimerization system mediated by Danoprevir are more independent.

[0091] Subsequently, the Danoprevir-induced trimerization of the A protein and the B protein obtained from the 6th, 7th or 8th split site and the DNCR2 three-part element is verified by introducing an IRES / P2A element into a single plasmid (the schematic diagram of plasmid construction is shown in FIG. 4A, and the results are shown in FIG. 4B) and a SEAP system fused with p65 (the schematic diagram of the action of the secreted alkaline phosphatase system of the p65 / RtA transcription factor is shown in FIG. 5A, and the verification results are shown in FIG. 5B and FIG. 5D).

[0092] As shown in FIG. 4B, when the 6th, 7th and 8th split sites are further verified in the SEAP system by using Danoprevir, the three split sites all have strong signals, indicating that Danoprevir can produce an ideal induction of co-localization effect.

[0093] Plasmid A expressing N-terminal fusion tetR A protein, plasmid C expressing C-terminal fusion p65 or RtA transcription factor C protein (DNCR2) and plasmid B expressing C-terminal fusion VP64 B protein were constructed respectively, co-transfected with pMF111, and Danoprevir was added after 2h of transfection for culture. The supernatant was taken for secreted alkaline phosphatase activity detection (in the method described in Examples 2 and 3). As shown in Figure 5B, the signal of the fusion p65 element in the trimerization system obtained by 6# and 7# split sites was significantly improved compared with the original design (plasmid expressing C protein without fusion p65 or RtA), further verifying the trimerization process involving the C protein element; at the same time, the signal of the sample group with fusion p65 element was higher than that with fusion RtA, indicating that the Danoprevir-mediated trimerization system can be used for screening of transcription element action and expanding its application scenarios. Plasmids containing IRES element and P2A sequence (as shown in Figure 4A) and further containing a single plasmid with p65 (as shown in Figure 5C) were constructed. The single plasmid with p65 was constructed by fusing p65 at the C-terminal of C protein based on the plasmid containing IRES element and P2A sequence. The above two plasmids were co-transfected with pMF111 respectively, and Danoprevir was added after 2h of transfection for culture. The supernatant was taken for secreted alkaline phosphatase activity detection (in the method described in Examples 2 and 3), and the results are shown in Figure 5D. The 6#, 7# and 8# split sites with fusion p65 element all showed trimerization effect.

[0094] Example 5 Confocal fluorescence co-localization verification

[0095] The present application verifies the trimerization of biological elements at 6#, 7# and 8# sites by observing the co-localization of fusion fluorescent proteins under confocal microscope. The experimental schematic diagram is shown in Figure 6.

[0096] The present application uses pCDNA5 as an expression vector to construct N-terminal fusion eGFP A protein, C-terminal fusion mCherry B protein (with CAAX membrane localization sequence) and C-terminal fusion TagBFP C protein, and uses IRES and P2A sequence to separate each component during protein expression.

[0097] The constructed plasmid was transferred into mammalian cell HeLa-CCL2 by transient transfection. In a 35mm confocal glass dish, 3.5x10 5Concentration of inoculated cells, 37℃ incubated cells for 24 hours, 1 μg plasmid mixed with 1.5 μL Lipo3000 by Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, incubated at room temperature for 10-15 min, then added to the dish, 3-4 hours after transfection, the liquid was changed. 37℃ incubated for 24 hours, using Nikon CSU-W1 Sora2Camera, observing the fluorescence protein co-localization in the living cells under Danoprevir treatment.

[0098] As shown in FIGS. 7A-7F, the fluorescence intensity on the same cell membrane was counted at 0 min (FIGS. 7A and 7D), 10 min (FIGS. 7B and 7E), and 20 min (FIGS. 7C and 7F), respectively. The eGFP and BFP originally located in the cytoplasm were co-localized to the cell membrane with the fused B / C protein at 10 min, resulting in a change in fluorescence intensity, and the fluorescence intensity change was more significant and stable at 20 min. It can be seen that the small molecule drug can mediate trimerization within 10 min.

[0099] Other main sequences involved in the present application:

[0100] DNCR2: (SEQ ID NO: 26)

[0101] GNCR1: (SEQ ID NO: 27)

[0102] IRES2 element: (SEQ ID NO: 28)

[0103] Although the specific embodiments of the present application are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present application. Therefore, the protection scope of the present application is defined by the appended claims.

Claims

1. A trimerization control element, characterized in that, The trimerization regulatory element includes protein A and protein B, which are obtained by the separation of hepatitis C virus protease NS3a, and also includes protein C; Wherein, protein A includes an amino acid sequence as shown in SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, and / or protein B includes an amino acid sequence as shown in SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16; The protein C is a drug / NS3a complex binding protein, such as GNCR1 or DNCR2; GNCR1 includes the amino acid sequence shown in SEQ ID NO:27, and DNCR2 includes the amino acid sequence shown in SEQ ID NO:

26.

2. The trimerization control element as described in claim 1, characterized in that, Protein A and protein B are selected from any one of the following groups: (1) Protein A includes the amino acid sequence shown in SEQ ID NO:11, and protein B includes the amino acid sequence shown in SEQ ID NO:12; (2) Protein A includes the amino acid sequence shown in SEQ ID NO:13, and protein B includes the amino acid sequence shown in SEQ ID NO:14; (3) Protein A includes the amino acid sequence shown in SEQ ID NO:15, and protein B includes the amino acid sequence shown in SEQ ID NO:

16.

3. A dimerization control system, characterized in that, The dimerization regulation system includes, in addition to the protein A and protein B as defined in the trimerization regulation element as described in claim 1, granivevir; Preferably, the grazovir mediates the dimerization of protein A and protein B via non-covalent binding; and / or, protein A and protein B are selected from any one of the following groups: (1) Protein A comprises the amino acid sequence shown in SEQ ID NO:11, and protein B comprises the amino acid sequence shown in SEQ ID NO:12; or, (2) Protein A comprises the amino acid sequence shown in SEQ ID NO:13, and protein B comprises the amino acid sequence shown in SEQ ID NO:14; or, (3) Protein A includes the amino acid sequence shown in SEQ ID NO:15, and protein B includes the amino acid sequence shown in SEQ ID NO:

16.

4. A trimerization control system, characterized in that, The trimerization control system includes the trimerization control element as described in claim 1 or 2; Preferably, the trimerization regulation system further includes a small molecule drug, such as grazoprevir or danoprevir; More preferably, the small molecule drug mediates the trimerization of the trimerization regulating element as described in claim 1 or 2 via non-covalent binding.

5. An isolated nucleic acid, characterized in that, The nucleic acid includes a nucleotide sequence encoding a trimerization regulatory element as described in claim 1 or 2.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 5; Preferably, the backbone plasmid of the recombinant expression vector is pcDNA3.1(+), pcDNA5, or pGEX-6P-1.

7. The recombinant expression vector as described in claim 6, characterized in that, The recombinant expression vector also includes the tetR gene, a transcription activator, a self-cleaving peptide, and / or an IRES element; the transcription activator is, for example, VP64, p65, and / or RtA; the self-cleaving peptide is, for example, P2A; and the IRES element is, for example, IRES2. Preferably, the sequences of protein A, protein B, and protein C are separated on the recombinant expression vector by the IRES element and / or the P2A, respectively; and / or, protein A, protein B, and protein C are linked to the tetR gene, VP64, and "p65 or RtA", respectively. More preferably, the tetR gene is located at the 5' end of the nucleotide sequence encoding protein A, VP64 is located at the 3' end of the nucleotide sequence encoding protein B, and p65 or RtA is located at the 3' end of the nucleotide sequence encoding protein C.

8. An isolated cell, characterized in that, The cells comprise the nucleic acid as described in claim 5, or the recombinant expression vector as described in claim 6 or 7; Preferably, the cells are mammalian cells, such as HeLa-CCL2 or HEK-293T cells.

9. A reagent kit, characterized in that, The kit comprises one or more of the following: the trimerization regulatory element as described in claim 1 or 2; the dimerization regulatory system as described in claim 3; the trimerization regulatory system as described in claim 4; the nucleic acid as described in claim 5; the recombinant expression vector as described in claim 6 or 7; and the cell as described in claim 8.

10. A method for preparing trimerization control elements, characterized in that, The method includes the step of culturing the cells as described in claim 8 to obtain trimerization regulatory elements.

11. A method for constructing a dimerization control system, characterized in that, The method includes the following steps: (1) The hepatitis C virus protease was separated into protein A and protein B; (2) Add a small molecule drug, wherein the small molecule drug mediates the dimerization of protein A and protein B through non-covalent binding, thereby forming the dimerization regulatory system; Protein A and protein B are defined in the dimerization regulation system as described in claim 3, and the small molecule drug is grazoprevir.

12. A method for constructing a trimerization control system, characterized in that, The method includes the following steps: (1) The hepatitis C virus protease was separated into protein A and protein B, and protein C was added; (2) Add a small molecule drug, wherein the small molecule drug mediates the trimerization of protein A, protein B and protein C through non-covalent binding, thereby forming the trimerization regulatory system; Protein A, protein B, and protein C are defined as trimerization regulatory elements as described in claim 1 or 2, and the small molecule drug is, for example, grazoprevir or danoprevir.

13. A method for regulating the expression of a target gene, characterized in that, The method regulates the expression of the target gene by using the trimerization regulatory element as described in claim 1 or 2, the dimerization regulatory system as described in claim 3, the trimerization regulatory system as described in claim 4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6 or 7, the cell as described in claim 8, or the kit as described in claim 9. Preferably, the method is a method for non-disease treatment purposes.

14. The use of the trimerization regulatory element as described in claim 1 or 2, the dimerization regulatory system as described in claim 3, the trimerization regulatory system as described in claim 4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6 or 7, the cell as described in claim 8, or the kit as described in claim 9 in the preparation of reagents or drugs for regulating cell signal transduction, gene editing, protein colocalization, or cell therapy; Preferably, the cell therapy is CAR-T cell therapy.

15. The use of the trimerization regulatory element as described in claim 1 or 2, the dimerization regulatory system as described in claim 3, the trimerization regulatory system as described in claim 4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6 or 7, the cell as described in claim 8, or the kit as described in claim 9 in regulating cell signal transduction, gene editing, protein colocalization, or cell therapy; Preferably, the cell therapy is CAR-T cell therapy.

16. The trimerization regulatory element as described in claim 1 or 2, the dimerization regulatory system as described in claim 3, the trimerization regulatory system as described in claim 4, the nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6 or 7, the cell as described in claim 8, or the kit as described in claim 9, for regulating cell signal transduction, gene editing, protein colocalization, or cell therapy; Preferably, the cell therapy is CAR-T cell therapy.

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