TW200307040A - Split-hybrid system - Google Patents

Abstract

A split-hybrid system that contains a nucleic acid including a promoter, a transcriptional regulatory sequence operably linked to the promoter and having a protein-binding domain, and a reporter gene operably linked to the promoter; a first recombinant polypeptide including a DNA-binding domain that binds to the protein- binding domain and a bait polypeptide; and a second recombinant polypeptide including a transcriptional repression domain and a prey polypeptide that interacts with the bait polypeptide. Interaction between the bait and prey polypeptides represses the expression of the reporter gene, and disruption of the interaction between the bait and prey polypeptides activates the expression of the reporter gene.

Description

200307040 (1) Description of the invention: [Technical field to which the invention belongs ^ 1 The present invention relates to a plug-in interference protein interaction & synthesizing system, which can be used to identify dry compounds. [Prior technology] Interactions between proteins-loops, these interactions are important in the reaction of each cell of Hachijo "slightly & externally (such as the interaction between ligands and receptors, cells Intercellular adhesion, antigen recognition, eyebrow allowance, 1 π 丄 recognition, and virus-to-host identification) and intracellular (such as the formation of protein complexes involved in the transmission of your field green mouth from η / 5, transcription , Translocation and DNA, s ^ ^ ^ ^ ^ ^ U complement and replication) will occur. The disease development process usually involves abnormal ^ ^,, protein-protein interactions. The parent-protein interaction between these proteins becomes It has a latent stomach as a target for treating diseases. [Summary of the Invention] A system comprising (1) a promoter linked to a regulatory sequence, and a nucleic acid that is a kind of split-hybrid system of the present invention. 'It includes: a promoter, a reporter gene linked to and acting on a segment of a protein containing a protein junction region; and (2) the first segment of a recombinant polypeptide chain' 丼 contains a protein DNA binding region 'combined with a "bait polipeptide"; (3) a second recombinant polypeptide chain' which contains a transcriptional repression region and a "prey" polypeptide bond (a Prey polypeptide), the "prey" polypeptide chain can be combined with the "bait" polypeptide bond. There is a 200307040 parent interaction between the "feed" polypeptide bond and the "wax" polypeptide chain. Inhibiting the performance of the reporter gene, when the interaction between the "bait" polypeptide chain and the "deprivation 1" is disturbed, the cousin of the reporter gene will be activated. The month or day will increase. D month, otherwise "interference" in the present invention An example of the term is #, Γ Γ '' s interaction field, bait "polypeptide chain and" prey "peptide chain interspersed with 5 A between" at private bait "polypeptide chain and" prey "polypeptide A strand "inhibits or increases its effect. The promoter day can be sloppy" refers to a nucleic acid sequence capable of initiating transcription. Or) (eg: thymidine kinase [TK] promoter and Roche but (eg, cytomegalovirus [CMV] promoter-A tumor virus [RSV] promoter).

Jinked turned. The regulatory sequence is "linked to the promoter and its role (" ㈣ ,,,,, t〇) '"refers to a nucleic acid sequence that can regulate (eg, the activity of the addon." Protein-binding domain ) b 4b 疋 Cai said a nucleic acid sequence that can bind to proteins, such as: Gal4 binding site. "Reporter gene" refers to a gene that can easily determine the extent of its expression, such as luciferase (Luc) gene, secretion Type alkaline phosphatase hydrolase (SEAP) gene, or a fluorescent protein (such as EGFP or RED). A reporter gene "linked to and interacts with the promoter" refers to the transcription of this gene The action is controlled by this promoter. "DNA binding domain" refers to an amino acid sequence that can bind DNA, such as the n-terminal region of the Gal4 protein (1-147 amino acids, abbreviated as Gal4N) 200307040 "transcriptional repressive region" amino acid sequence, for example: resting of the adenoid receptor > refers to a section capable of inhibiting the Max-binding protein (Mad) (Silencing mediato and thyroid hormone) receptor [SMRT]). A transcript-active, A-acid and thyroid retinoic acid "bait" polypeptide chain "prey" polypeptide chain is-the polypeptide chain that will interact with each other 'For example: A acid nuclear protein Receptor (retin〇id [RAR]) and its second interaction region (interacting domain π [IDII]); β-type cancerous growth factor type 1 receptor (transf〇rming

Growth facto (β type! receptor [TRI]) and ρκ5 06 binding protein (FKBP12), and β-catenin and τ cell factor (TCF). The interaction between the "bait" polypeptide chain and the "prey" polypeptide chain can be inhibited by a compound (eg, nucleic acid, polypeptide chain, small peptides, ligands, antibodies, small molecules, esters, sugars; or from animals Natural extracts from plants, fungi, microorganisms). This compound can achieve the purpose of inhibition whether it is combined with the "bait" polypeptide chain or the "prey" polypeptide chain.

Another aspect of the present invention is a cell (eg, mammalian cell), which comprises (1) a first nucleic acid, which includes a promoter, a segment connected to the promoter and a protein-binding region interacting with the promoter. Transcriptional regulatory sequences and a reporter gene linked to and acting on the promoter; (2) a second nucleic acid that encodes a first recombinant polypeptide chain, the first recombinant polypeptide chain contains a The DNA binding region combined with the protein binding region and a "bait" polypeptide chain; (3) — the third nucleic acid, which can encode to generate a second recombinant polypeptide chain, the second recombinant polypeptide chain 7 200307040

, Contains a transcriptional repression region and a "wolf" polypeptide chain that can be combined with the "poly" polypeptide chain. If there is an interaction between the polypeptide peptide and the iron iron polypeptide bond, the reporter gene table will be inhibited. See, the interaction between the "bait" polypeptide chain and the "prey" polypeptide chain will interfere with the reporter gene. Performance. "The present invention also relates to a method that can be used to identify compounds that interfere with the interaction between the" bait "polypeptide chain and the" prey "polypeptide chain. This method includes providing a dividing hybrid system or cell as described above, and contacting the system or cell with a compound. If the reported gene performs more in the presence of the compound than in the absence of the compound, it means that the compound inhibits the interaction between the "bait" polypeptide chain and the "prey" polypeptide chain. The present invention provides a system and a method for identifying compounds that can inhibit the interaction between a "_" polypeptide chain and a "prey" polypeptide chain, and also a high throughput screening. The compounds identified by this method can be used to treat diseases involving abnormal interactions between proteins. For example, in a gene therapy, a patient in need of treatment may be given an effective amount of a nucleic acid that encodes a protein that produces a therapeutic effect.

One or more embodiments of the present invention will be described in detail as follows. Other advantages, features, and motivations of the present invention will also become apparent from the detailed description and the scope of patent applications. [Embodiment] The present invention provides a method for identifying a chemical compound that can inhibit the interaction between a "bait" polypeptide chain and a "prey" polypeptide chain by using a split hybrid system. The system includes three elements: a segment The nuclear motif is related to Qiqin ^ It consists of a promoter linked to its function and contains a gene of the transcriptional regulation library protein junction region; one of its functions is a reporter \ recombinant polypeptide chain which contains one The peptide junction of the cytoplasmic junction is the same as the aforementioned protein ~ paragraph weight% capacity bait "polypeptide chain; a peptide chain, which contains a transcriptional suppression" bait "multi-team 13 system area and a section of the reconcilable The "spray ..." bond that combines the "recruitment" and "recruitment." Polypeptide shows that the interaction between peptide bonds will inhibit the interference of the reporter gene, ... the country chain and the "prey" polypeptide chain Interactions were expressed by stem-element metagenic genes. The following will describe in detail each system in the system and the method of the present invention: Μ 41 · ju A DNA binding region spot, "A library peptide" Any polypeptide chain that can bind to a specific DNA sequence can be used as a DNA operator The DNA binding region can be bound from the naturally occurring dnA ^ J y | y, orally, for example, a DNA binding protein of a prokaryotic or eukaryotic organism. In addition, the DNA is Γ-, 、 3 The region may also be a polypeptide chain obtained from a protein that has been artificially engineered to interact with a specific DNA sequence. Examples of DNA binding regions of natural eukaryotic DNA-binding proteins include the P53 protein, τ

Un protein, Fos protein, GCN4 protein and Gal4 protein; DNA. . Regions can also be produced from viral proteins, such as the papillomavirus E2 protein; "DNA binding regions can be obtained from prokaryotes, for example: LexA inhibitor protein or DetR inhibitor protein of the large intestine f_; or DNA binding region It is obtained from phages, such as: λ_ cardiomyocytes. Typical true 200307040

The DNA-binding region of nuclear organisms also includes the oNA-binding sites of P22 Arc repressor, MetJ, CENP-B, Rapl, Xyis / Ada / AraC, Bir5, and DtxR. The DNA-binding protein may also be a non-naturally occurring DNA-binding region produced by recombinant technology, a method for generating a completely new and selectively binding DNA-binding protein. This method is known in the art. See U.S. Patent No. 5,198,346.

The basic requirements of this recombinant protein include its ability to specifically bind to a specific nucleic acid sequence (ie, a DNA binding site) located upstream of the appropriate reporter gene, which itself should not or hardly alter the translation activity of the reporter gene; meanwhile, The 1 "polypeptide chain will not interfere with the ability of this DNA binding region to bind to the binding site on DNA.

This DNA-binding region may comprise an oligomeric configuration (oiiginerization motifs), and some translational regulators are known to polymerize dimers. Dimer polymerization can promote translational regulators to cooperate to bind to their homologous DNA binding sites. For example, when a DNA binding region of a LexA protein is used, it can include a DNA binding region of the LexA protein in the form of a dimer polymerization. This dimerized form can accelerate the formation of effective LexA protein dimers and optimize their binding to DNA (see Golemis and Brent (1992) Mol. Cell Biol. 12: 3006). Other representative oligomeric configurations include the tetrameric region of the p53 protein and the tetrameric region of the BCR-ABL protein. A "bait" polypeptide chain can be any protein of interest, including known, unknown, or suspected proteins of diagnostic, therapeutic, or pharmacological importance. For example, the protein we ’re interested in might be a pregnant 10 200307040

Proteins suspected of inhibiting or activating intracellular effects (eg, receptor message transmission, cell apoptosis, cell proliferation, cell differentiation, import or export of toxicants and nutrients). Examples of "feed:" polypeptide chains include oncogenic proteins such as myc protein, Ras protein, src protein, and Fos protein; tumor suppressor proteins such as p53 protein, p21 protein, p16 protein, Rb protein, and continuous activation and deletion Carbonated Rb proteins (see Knudsen et al. (1 999) Oncogene 18: 5239-45); proteins involved in regulating cell cycle, such as kinases (for example: TRI) and phosphohydrolase; or proteins involved in information transmission, For example: IDII protein, β-catenin, Zap-70 protein or SAM-68 protein. All or a fragment of the protein of interest can be used as a "bait" polypeptide chain. When the protein of interest is too large, such as a molecular weight of more than 20,000 channels, it is convenient to use fragments of the polypeptide chain. An unstructured polypeptide chain linker can exist between the DNA binding region and the "bait" polypeptide chain. The linker can accelerate the interaction between the DNA binding region and the corresponding binding site on the DNA and the "bait" polypeptide chain and the "prey" Interactions between polypeptide chains.

Recombinant polypeptide chains can be produced from a vector containing a nucleic acid sequence encoding a DNA-binding region and a "trans" polypeptide chain. Suitable expression vectors for the production of proteins in mammals are well known techniques, such as the vector p S G4 2 4 (see Sadowski and Ptashne (1989) Nucleic Acid Research, 17: 7539) and the vector pM (source: ciontech , Palo Alto, CA). The expression vector may include one or more regulatory sequences (for example, enhancers), which are linked to and function with a promoter that can direct the expression of the recombinant polypeptide chain; the expression vector also includes a selectable marker gene. 11 200307040 Due to the performance, you can use the young girl, the couple, and 1 、 ^ to pick and select the cells that contain the marker gene and those that do not contain the marker gene. Desert media are known as well-known technologies, such as neomycin, zeocin and blasticidin. This vector can escape & in cells The existence of DNA forms outside the chromosome can also be embedded in the chromosome.

Distinguish the interaction between the "skin bond" and the "prey" polypeptide chain: the interaction between the two egg mouths connects the DNA binding region fused to the "_" polypeptide chain and the translation inhibition region fused to the "poly" polypeptide chain to generate a A protein complex capable of directly inhibiting reporter gene expression. As described above, the recombinant polypeptide chain can be produced from a vector that contains a nucleic acid sequence that can produce a transition inhibitory region and a "prey" polypeptide chain. Carrier

It can include nuclear ligament sequences (such as sequences obtained from the Gal4 and MATα2 genes), and the nuclear localization sequence can achieve the maximum efficiency when the polypeptide chain reaches the reporter fragment located in the nucleus. ’If the reported gene is expressed, it means that there are more" bait "polypeptide chains and r prey. 12 200307040 Peptide chains will interact with each other. Any suitable reporter gene can be used in this cleavage hybrid system. Representative examples include chloramphenicol acetyl transferase C CAT. See Alton and Vapnek (1 979) Nature 282: 864-869 ); And other enzyme detection systems, such as β-galactosidase (β-galactosidase), firefly luciferase (see de Wet et al. (1 987) Mol. Cell. Biol · 7: 725- 737)), bacterial luciferase (see Engebrecht and Silverman (1 984) PNAS 1:41 54-41 58; Baldwin et al. (1 984) Biochemistry 23: 3 663-3 667), algal fluorescence Proteins (phycobiliproteins, especially phycoerythrin), alkaline phosphatase (see Toh et al. (1 989) Eur. J Biochem. 1 82:23 1-238; Hall et al. (1 983) J. Mol. Appl. Gen. 2: 101), secreted alkaline phosphatase (SEAP), see Cullen and Malim (1 992) Methods in Enzymol. 216: 362-3 68) or glorious protein (Eg · GFP). Other suitable reporter genes include genes encoding cell surface antigens (eg, CD 16) and drug resistance genes (eg, hypoxanthine guanopurine phosphorosylnucleoside converting enzyme [HPRT]). The amount of translation of a reporter gene can be measured by methods well known in the art. For example, the performance of specific information RNA can be detected by the northern blot method, and specific protein products can be identified using antibodies. The protein encoded by the reporter gene can also be detected by analyzing the intrinsic activity associated with this protein. For example, 'the reporter gene can encode a gene product, sex, trigger-can be detected mainly by glory, color or cold light. :; Enzyme, 13 200307040 It is better to provide two or more reporter gene fragments, both of which are regulated by the interaction between the "bait" polypeptide chain and the "prey" polypeptide chain, for example: GFP and BFP genes . Simultaneous expression of each individual reporter gene also provides a tool that can be used to distinguish whether the "bait" polypeptide chain and the "prey" polypeptide chain actually interact, for example, to identify mutations or other false positive events. Reported gene activation.

The reporter gene can be carried by a suitable vector that can remain in the form of a free genome in a cell (e.g., a mammalian cell) or remain embedded in the chromosome of the cell. Host Cell This split hybrid system can be used in vivo and in vitro. Any artificially cultured cells (such as primary cells, secondary cells, or immortalized mammalian cells) can be applied to the split hybrid system used in vivo. Typical mammalian cells are mice, hamsters, rats, rabbits , Dog, cattle, and primate cells, including humans, they can be of a variety of different tissue types, including giant cells, endothelial cells, liver cells, kidney cells, and other cell types. Φ "Primary cells" are cells isolated from mammals (for example, from tissue sources), which are the first cultured cells before being transferred to the subculture; "secondary cells" refer to All subsequent cultured cells; examples of mammalian primary and secondary cells that can be transfected include fibroblasts, keratinocytes, epithelial cells (eg, breast epithelial cells and intestinal epithelial cells), endothelial cells, glial cells, neurons 14 200307040 Cells, blood-derived components (such as lymphocytes and bone marrow cells), muscle cells, and precursors of these somatic cell types. "Immortalized cells" are cell lines that can extend life indefinitely. Examples of immortalized human cell lines that can be used in this mammalian hybrid system include, but are not limited to, the following: CV-1 cells, HT 1 080 cells (ATCC CCL 121), cervical cancer cells (HeLa cells) and their derivatives (ATCC CCL 2, 2.1 and 2.2), MCF-7 breast cancer

Cells (ATCC BTH 22), K-562 leukemia cells (ATCC CCL 243), KB tumor cells (ATCC CCL 17), 2780AD ovarian cancer cells (see Van der Blick et al. (1988) Cancer Res 48: 5927-5932) , Raji cells (ATCC CCL 86), Jurkat cells (ATCC TIB 152), Namalwa cells (ATCC CRL 1432), HL-60 cells (ATCC CCL 240), Daudi cells (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cells (ATCC CRL 1 593),

Bowes melanoma cells (ATCC CRL 9607), WI-38VA13

Line 2R4 cells (ATCC CLL 75.1) and MOLT-4 cells (ATCC CRL 1 5 82), and hybrid fusion tumor cells made of human cells and cells of other species may also be used. Secondary human fibroblast cell lines such as WI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171) can also be used. The above-mentioned plastids can be transfected into cells using procedures well known in the technical field. Examples of mammalian cell transfection methods include co-precipitation method of calcium phosphate and calcium vaporization, DEAE-glucosan-mediated transfection method, microlipid Infection, gene gun delivery and electroporation. Appropriate methods for in vitro transfection of cells can be obtained from the Molecular Reproduction-Laboratory Handbook Second Edition by Sambrook et al. 15 200307040 (Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Press, Cold Spring Harbor, New York) or other Laboratory operating manual found. Screening test This split-hybrid system can be used to identify compounds that interfere with protein-protein interactions. The compounds identified by this method can be used to treat diseases involving abnormal interactions between proteins.

The candidate compounds of the present invention can be obtained from any of a number of research methods of combinatorial library well-known in the art. These molecular databases include: nucleic acid molecular database, peptide molecular database, peptoid Molecular database (the molecules in this molecular database have the function of peptides, but have a new, non-peptide backbone that can resist the degradation of enzymes), spatially addressable parallel solid or liquid molecular databases (spatially addressable parallel solid phase or solution phase libraries), synthetic molecular databases obtained from deconvolution or affinity chromatography, and "one-bead one-compound" molecular databases. See Zuckermann et al.

(1994) J. Med. Chem. 37: 2678-85 and Lam (1997) Anticancer Drug Des. 12: 145 o Methods for synthesizing molecular libraries can be found in many examples in this technical field ', for example: De Witt et al. (1 993) PNAS USA 90: 6909; Erb et al (1994) PNAS USA 91: 1 1422; Zuckermann et al. (1994) J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; 16 200307040

Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061 and Gallop et al. (1994) J. Med. Chem. 37: 1 233.

Molecules of the molecular database can be in solution (see Houghten (1992) Biotechniques 13: 412-421), or on glass beads (see Lam (1991) Nature 354: 82-84), on wafers (see Fodor (1 993 ) Nature 3 64: 555-556), bacterial (see Ladner, US Patent No. 5,223,409), spores (see Ladner, supra), plastids (see Cull etal · (1992) PN AS USA 89: 1865- 1869) or on mycoplasma (see Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (1990) PNASUSA 87: 6378-6382; FeliciC 1991) J Mol. Biol. 222: 301-3 1 0 and Ladner, supra).

In order to identify compounds that can interfere with protein-protein interactions, the above-mentioned split-hybrid systems will be in contact with candidate compounds, and the reported gene expression will be evaluated relative to the degree of expression in the absence of this compound. If the degree of expression of the reporter gene is greater when there is a candidate compound than when there is no candidate compound, this candidate compound is considered a potential drug and can be used to treat diseases involving abnormal interactions between proteins. The four tests can be performed in vivo or in vitro. There are significant advantages to using cells for testing. For example, the starting material (ie, the cell) will replicate on its own, the biological situation is closer to the normal physiological environment, and it can provide insight into the biological availability (ie, whether the compound can enter the cell to act on the intracellular target). And toxicity (ie, whether the compound threatens cell growth). The following examples are only for explanation and illustration, and are not limited to the details of the 17 200307040 certificate. It is not intended to ^ anyone familiar with the technology and does not need to go deeper and more delicate work, ^ can use this document The narrative uses the invention in its entirety, and all publications listed here are incorporated in their entirety by reference.

Gal4N-SMRT > η λ Han1 Gal4N-Mad and Gal4N-SHP (short heterodimeric pairs), were used: mesh, 1 + surgery / bei test they are for 4 copies of Gal4 protein 、、、 口 口 位 t TK started? , The ability to inhibit its gene expression. The results show that

Gal4N-SMRT and Gal4N-Mad can inhibit the activity of the promoter, while

Gal4N-SHP cannot effectively inhibit the activity of this promoter. These results suggest that SMRT and Mad can be used to establish a split heterozygous system. The two fusion proteins of the cleavage heterozygous strand M of Λ 醆, VP16-RAr and Gal4N-IDII, are expressed together in cells containing the Gal-TK-Luc reporter gene fragment. The results showed that the interaction between RAR and IDII caused the reporter gene activity to be induced about 120 times. Conversely, in the presence of imM A acid, the interaction between RAR and IDII was hindered, resulting in the reporter gene activity being induced to decrease to only 7 These results suggest that the interaction between RAR and IDII is quite strong and will be disturbed by the added A acid. Next example: Two fusion proteins, NLS (nuclear localization sequence) -ΗA (hemagglutinin) -Mad-RAR and Gal4N-IDII, are expressed together in cells containing the Gal-TK-Luc reporter gene fragment. The interaction between RAR and IDII 18 200307040 caused the reporter gene activity to be inhibited approximately 7.5 times, and by adding A acid (ImM), this inhibition was completely reversed. A acid only specifically inhibits protein-protein interactions, and A acid itself or other control groups transfected together (ie Gal4N-Mad, Gal4N-IDII, and NLS.HA_Mad-RAR) will not change Gal-TK-Luc reports Gene activity. Use of gMV and RSV promoters Other promoters such as the CMV promoter and the RSV promoter were tested 4 to see if the daggers had two basic activities and could allow a wider range to measure the inhibitory effect. Five copies of the Gal4 protein binding site are placed at the 5 ’end of the CMV or RSV promoter, which is linked next to the luc reporter gene

(Gal-CMV-Luc and Gal-RSV-Luc). As a result, the basic activity of the CMV and RSV promoters is much higher than that of the TK promoter.

Gal4N-SMRT and Gal4N-Mad have similar inhibitory effects on Gal-TK-Luc. Compared with the inhibition of Gal-CMV-Luc, Gal4N-SMRT has very strong inhibition (about 30 times) of Gal-RSV-Lixc; on the other hand, Gal4N-Mad inhibits these two promoters Neither is as effective as the τκ promoter. Appropriate findings indicate that Gal-RSV-Luc plus SMRT inhibitory areas are a good combination for establishing a split-hybrid system. Next example: Two fusion proteins, SMRT-RAR and Gal4N-IDII, are expressed together in cells containing the Gal-RSV-Luc reporter gene fragment. It was found that the interaction between Gal4N-IDII and SMRT-RAR had an 8.5-fold inhibition effect. This inhibition effect was reversed after the addition of A acid treatment. Another pair of interacting proteins, TRI and FKBP12, were tested for use in the 19 200307040 split heterozygous system. It is known that the interaction between these two proteins will be inhibited by the small molecule FK506, the intracellular region of TRI is fused with the dNA junction region of Gal4 (Gal4_TRl), and FKBP12 is connected to the inhibition region of SMRT (SMRT-FKBP12), together with Gal4-RSV- Luc dimers were transfected into CVe 1 cells together. The results show that the interaction of Gal4-TRI and SMRT-FKBP12 results in approximately doubled inhibition of the reporter gene activity. When treated with FK506, the protein interaction was blocked, leading to the activation of promoter activity corresponding to the dose of FK506. The reporter genes SEAP and RED1 SEAP have been successfully used in high-throughput screening. A split-hybrid system was used to speed up high-throughput screening and construct a Gai-RSV-SEAP-containing plastid, Gal4N-SMRT and Gal-RSV-SEAP—transfected into CV-1 cells. The results showed that Gal4N-SMRT caused I # -fold inhibition of the reporter gene activity. In addition, the interaction between β-catenin and TCF had the same degree of inhibition of SEAp reporter gene activity as Luc reporter gene activity.

Gal4N-SMRT can effectively inhibit the performance of RED1, but Gal4N or the reporter gene alone still has a fluorescent signal, and the red glory generated from redi can be detected in the presence of A acid, RAR and IDII or FK506, TRI and FKBP12 . & Other embodiments The features disclosed in the description of the present invention may be combined in any combination. Each feature disclosed in the description of the present invention may be replaced by other features having the same, equal, or similar properties. Therefore, unless there are other expressions, each feature disclosed here is only a group of features with equal or similar properties. Single example. From the above description, those skilled in the art can easily understand the most important and fundamental characteristics of the present invention. Without departing from the spirit and scope of the present invention, different changes and modifications can be made to the present invention to adapt it to different Use and environment, therefore, other embodiments also fall within the scope of the scope of the accompanying patent application.

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Claims (1)

200307040 Scope of patent application: 1. A split hybrid system comprising: a nucleic acid, including a promoter, a transcriptional regulatory sequence connected to the promoter and containing a protein junction region, and connected to the promoter And a reporter gene that acts on it; the first recombinant polypeptide chain, which contains a DNA binding region that can be combined with the protein junction region, and a "bait" polypeptide chain; The second segment of the recombinant polypeptide chain contains a transcriptional repression region and a "prey" polypeptide chain that can be combined with the "I ear" polypeptide chain; there is an interaction between the "bait" polypeptide chain and the "prey" polypeptide chain The effect will inhibit the performance of the reporter gene, and the interaction between the "bait" polypeptide chain and the "prey" polypeptide chain will be disturbed to activate the performance of the reporter gene. 2. A cleavage hybrid as described in item 1 of the scope of patent application System, wherein the promoter is a thymine kinase (TK) promoter, a cytomegalovirus (CMV) promoter, or a Roche sarcoma virus (RSV) promoter. 3. A split-hybrid system according to item 1 of the scope of the patent application, wherein the protein-binding region is a Gal4 protein binding site and the DNA-binding region thereof is a Gal4N protein. 4. A split-hybrid system as described in item 3 of the patent application, wherein the promoter is a thymine kinase promoter, a cytomegalovirus promoter 22 200307040 promoter, or a Roche sarcoma virus promoter, and its translational suppression region is Max Binding protein (Mad) or A acid and the resting medium (SMRT) region of the thyroid hormone receptor. 5. A split hybrid system as described in item 1 of the scope of the patent application, wherein the "bait" polypeptide chain and the "prey" polypeptide chain can be a compound that binds to the "bait" polypeptide chain or "prey" polypeptide chain, Prevent them from interacting with each other. 6. A cell, comprising: a first nucleic acid comprising a promoter, a transcriptional regulatory sequence connected to the promoter and interacting with it comprising a protein junction region, and a reporter gene connected to the promoter and interacting with the promoter; The second nucleic acid, which can encode the first recombinant polypeptide chain, contains (1) a DNA binding region that can be combined with the protein junction region, and (2) a "bait" polypeptide chain; The third nucleic acid, which can encode a second recombinant polypeptide chain, includes (1) a transcriptional repression region, and (2) a "prey" polypeptide chain that can be combined with a "bait" polypeptide chain; wherein The interaction between the "bait" polypeptide chain and the "prey" polypeptide chain will inhibit the performance of the reporter gene, and the interaction between the "bait" polypeptide chain and the "prey" polypeptide chain will interfere with the performance of the reporter gene. . 23 200307040 7. The cell according to item 6 of the scope of the patent application, wherein the promoter is a thymic thymus kinase (TK) promoter, a cytomegalovirus (CMV) promoter, or a Roche sarcoma virus (RSV) promoter . 8. A cell according to item 6 of the scope of the patent application, wherein the protein-binding region is a Gal4 protein binding site and the DNA-binding region thereof is a Gal4N protein. 9. A cell as described in item 8 of the scope of the patent application, wherein the promoter 0 is a thymine kinase promoter, a cytomegalovirus promoter, or a Roche sarcoma virus promoter, and its translation inhibition region is a Max binding protein (Mad ) Or A acid and thyroid hormone receptor stationary medium (SMRT) zone 0 10 · A cell described in item 6 of the scope of the patent application, in which the "bait" polypeptide chain and the "prey" polypeptide chain are Compounds bound by "bait" polypeptide chains or "prey" polypeptide chains to prevent their interaction with each other. Φ Π. A cell according to item 6 of the patent application, wherein the cell is a mammalian cell. 12. A cell according to item 11 in the scope of application for a patent, wherein the promoter is a thymus jet kinase promoter, a cytomegalovirus promoter, and a Roche 24 200307040 gamma tumor virus promoter, and its translation inhibition region is Max binding Protein (Mad) or A-acid and thyroid hormone receptor stationary medium (smrT) 〇13. A cell as described in the scope of patent application 帛 11, wherein the protein junction region is a GaM protein binding site, and its DNA binding region For Gal4N protein. 14. A cell according to item 13 of the declared patent, wherein the promoter φ promoter is a thymus-mouth biting kinase promoter, a cytomegalovirus promoter, or a Roche sarcoma virus promoter, and its translation inhibition region is a Max binding protein ( Centrifuge) or octanoic acid and thyroid hormone receptor stationary medium (31 ^^) 15. A cell as described in item U of the patent application, wherein the "_" polypeptide chain and the "prey" polypeptide chain are A compound that can bind to a "bait" polypeptide chain or a "prison" polypeptide chain to prevent their interaction with each other. Pain 16. A method for recognizing a compound that inhibits parental interaction between a "bait" polypeptide chain and a "prey" polypeptide chain, which comprises providing a cleavage hybrid system as described in item 1 of the scope of the patent application, and The system is contacted with a compound, and if the expression level of the reported gene is more when there is a compound than when there is no compound, it means that the compound will interfere with the interaction between the "chain 25" and "prey" polypeptide chains. 17. A method for identifying a compound capable of inhibiting the interaction between a "bait" polypeptide chain and a "prey" polypeptide chain, comprising providing a cell as described in item 6 of the patent application scope, and contacting the cell with the compound Among them, if the degree of expression of the reported gene is greater when there is a compound than when there is no compound, it means that the compound will interfere with the interaction between the "_" polypeptide chain and the "prey" polypeptide chain. 18. A method as described in item 17 of the scope of patent application, wherein the cell is a mammalian cell. 26 200307040 (1) Designated representative map: (1) The designated representative map in this case is: Figure 2. (2) Brief description of the component representative symbols of this representative map: No designated representative map 捌 If there is a chemical formula in this case, please disclose the chemical formula that can best show the characteristics of the invention: No representative chemical formula 4