WO2000012712A1 - Novel nerve differentiation factor - Google Patents

Abstract

A novel gene Tbr-2 encoding 688 amino acids having a DNA-binding region called T-box which has been found out by searching a gene showing high expression in fetal mouse brain by the subtraction method in order to obtain a novel nerve differentiation factor relating to the mouse brain nerve construction; and a novel human gene Tbr-2 corresponding thereto.

Description

 Description New differentiation factor Technical field

 The present invention relates to a novel neuronal differentiation factor protein Tbr-2, a gene encoding the same, a method for producing them, and use thereof. Background art

 The formation of the brain, composed of a wide variety of cells, is controlled by various genes that are expressed in a region-specific manner during the early stages of its development. However, the developmental processes of morphologically complex mammalian cranial nerves are still largely unknown.

 Clochy of the Brachyury (T) gene (Herrmann, BG et al., 1990, Nature. 343, 617-622) as a causative gene for a mouse mutant with abnormal chordal differentiation and tail formation. During this period, the search for factors involved in morphogenesis was actively conducted. As a result, Tbx3 (Bamshad, M. et al., 1995, Hum. Mol. Genet. 4, 1973-1977), which is the causative gene of ulnar-mammary syndrome, and Holt-Ollam syndrome Tbx5 (Basson, CT et al., 1994, Nat. Genet. 15, 30-35, Li, QY et al., 1997, Nat. Genet. 15, 21-29), which is the gene responsible for (Holt-Oram syndrome) A series of genes, called T-box gene families, have been identified.

The T-box gene family has been identified from various species, is expressed during fetal development, and is known to be an important gene in the formation process of individuals (Bollag, RJ et al., 1994, Nat. Genet. 7, 383-389, Chapman, DL et al., 1996, Dev Dyn. 206, 379-390 Chapman, DL et al., 1996, Dev. Biol. 180, 534-542, Gibson-Brown, JJ et al., 1996, ech. Dev. 56, 93-101). As a structural feature, the T-box gene It has been shown that the genes that make up the family are very conserved, from nematodes to mammals, a DNA-binding domain called T-box (Kispert,, and Herrmann, BG, 1993). , EMBO. J. 12, 3211-3220). Furthermore, it is considered that the T-box gene family constitutes several sub-families based on the difference in amino acid sequences in T-box (Papaioannou, VE, 1997, Trends. Genet. 13, 212-). 213).

 Brachyury, Tbxl to 6, and Tbr-1 are currently known as T-box gene families cloned in mice.Functional analysis of morphogenesis during development has been performed, but brain morphogenesis has been performed. The only gene reported to be involved in Tbr-1 is currently Tbr-1.

 Therefore, elucidating the existence of a gene having a new T-box, finding genes involved in brain morphogenesis, and elucidating the effects of these neuronal differentiation factors would be a new possibility for diseases caused by cranial nerves. It has the potential to provide an effective treatment. Disclosure of the invention

 The present invention provides a novel neuronal differentiation factor Tbr-2, and a DNA encoding the same. The present invention also provides a vector into which the DNA has been inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. The present invention further provides a method for screening for a compound that binds to the protein.

In order to obtain novel genes related to the construction of mouse cranial nerves, we searched for genes that are highly expressed in the brain of fetal mice by the subtraction method. As a result, a novel gene encoding 688 amino acids including a DNA binding region called T-boX was isolated. This clone was highly homologous to Tbr-1 in mice and Eomesodermin in Drosophila, and was expected to be a novel molecule constituting the T-box gene family, and was named Tbr-2. Furthermore, the inventors succeeded in isolating a human gene having high homology to this mouse Tbr-2. Comparison of the expression distribution of Tbr-1 and Tbr-2 in the brain from embryonic days 12.5 to 14.5 by whole mount in situ hybridization (ISH) showed that Tbr-1 was expressed mainly in the telencephalon. In contrast, expression of Tbr-2 was observed in the midbrain and rhombic brain. Furthermore, specific expression was also observed in the limb buds formed from around embryonic day 9.5. The expression site of Tbr-2 in the brain at 14.5 days of fetal embryo was identified by I SH in brain slices. As a result, expression in specific neurons was observed. Therefore, it was suggested that proteins encoded by these genes are particularly involved in neuronal differentiation.

 As a result of comparing the expression patterns of Tbr-2, Tbr-1 and Brachyury genes in the early mouse development, the expression of Tbr-1 was observed around embryonic day 8.5 and increased gradually thereafter. Tbr-2 showed a bilayer expression pattern. The first expression observed in the early stage of Tbr-2 development is earlier than the expression of the Brachyury gene, which is a very important gene for mesoderm formation. It is suggested that this molecule is deeply involved in the early formation of the germ layer.

 In addition, conA stimulation, which is known to activate T lymphocytes, increased the expression of Tbr-2 in the thymus. Therefore, it was suggested that Tbr-2 has some function in T lymphocyte activation or in activated T lymphocytes. The present invention relates to a novel neuronal differentiation factor Tbr-2 and its gene, and their use.

 (1) a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence modified by deletion, addition, and / or substitution of another amino acid in one or more amino acids in the amino acid sequence in the protein; A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1,

(2) a protein consisting of the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence modified by deletion, addition, and / or substitution of another amino acid in one or more amino acids in the amino acid sequence in the protein; No, SEQ ID NO: A protein functionally equivalent to the protein consisting of the amino acid sequence according to 3,

 (3) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 2; a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1;

 (4) a protein encoded by a DNA that hybridizes to MA consisting of the nucleotide sequence of SEQ ID NO: 4; a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 3;

 (5) a fusion protein comprising the protein according to any of (1) to (4) above and another peptide or polypeptide;

 (6) a DNA encoding the protein according to any one of (1) to (5),

(7) A vector into which the DNA according to (6) is inserted,

 (8) a transformant capable of expressing the DNA of (6) above,

 (9) The method for producing a protein according to any one of (1) to (5), comprising a step of culturing the transformant according to (8).

 (10) A method for screening a compound that binds to a protein according to any one of (1) to (5) above,

 (a) contacting a test sample with the protein according to any of (1) to (5) above;

 (b) selecting a compound having an activity of binding to the protein according to any one of the above (1) to (5);

 (11) an antibody that specifically binds to the protein according to any one of (1) to (5);

(12) Contacting the antibody according to (11) with a sample expected to contain the protein according to any of (1) to (5), and immunizing the antibody with the protein. A method for detecting or measuring the protein, comprising detecting or measuring the formation of a complex; (13) a DNA which specifically hybridizes with a DNA consisting of the nucleotide sequence of any of SEQ ID NO: 2 or SEQ ID NO: 4, and has a chain length of at least 15 bases,

(14) A partial peptide of a protein comprising the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 3.

 In the present invention, the “peptide” refers to a compound in which amino acids are bonded to each other by peptide bonds. Therefore, a so-called polypeptide II protein having a long peptide chain is also included in the “peptide” of the present invention.

 The present invention relates to a novel nerve differentiation factor protein. The amino acid sequence of a mouse protein named “T br -2” contained in the protein of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of a human protein is shown in SEQ ID NO: 3, and the cDNA encoding the protein is The base sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 4.

 Tbr-2, which was found by the present inventors, is very highly expressed in the embryonic brain, and its expression gradually decreases as it matures, and it is hardly expressed in the completed adult brain (Example 4). ). The brain around the 14th day of fetal life is the time when the mobile neurons proliferate actively in the ventricles (Ventikiura zone; VZ) and are sent out to the mantle zone. The expression region of Tbr-2 was mainly distributed from the midbrain to the rhombic region. In the midbrain, expression was strong from the gray matter where many neurons gathered to the reticulum, and particularly strong expression was observed in various developing cerebral nuclei existing from the midbrain to the diencephalon. This expression disappeared in the brain at 18.5 days of embryonic period (Example 5). At this time, the cranial nerve group has already moved to a specific position and has undergone terminal differentiation, and is now in the phase of cell elongation that forms synaptic connections. It strongly suggests that this is a gene that is expressed only.

In Example 4, expression of Tbr-2 was slightly observed in the completed adult brain by RT-PCIU, but the expression site was in the hippocampus and the olfactory bulb. These two organs are adult mammals, and the only organs that continue to supply new neurons This fact also strongly indicates that Tbr-2 is a very important molecule for the differentiation of two eurones. The function of Tbr-2 may be independent of temporal factors, whether it is fetal neuron or adult neuron.

 Tbr-1 (Bulfone, et al., 1995, Jul. Neuron. 15, 63-78) has been expressed from E10, but its expression site is near the E10 mantle zone in the telencephalon. MZ), which is restricted to the cortical layer where cells that have stopped growing are gathered. Thereafter, from E16.5 to E18.5, a gradient expression was observed in the telencephalic cortex. During this period, cells migrate from the ventricular zone (VZ) to the cortical layer while maturing, and the formation of a six-layered brain cortex is occurring. Therefore, it is expected that T br-1 is a molecule involved in the formation of nerve fiber connections from the thalamus to the cerebral cortex.

 Based on the above facts, expression is not observed in many neural stem cells that are proliferative in both Tbr-1 and Tbr-2, and post-mitotic (non-dividing) neurons that stop proliferation and differentiate during or after differentiation Expression is lost in the formation of neural networks, meaning that both genes have a common property, that is, they are involved only when neurons differentiate and are unnecessary for subsequent maturation. Was found to have The major difference is that Tbr-1 is expressed in neurons that go to the cortex of the telencephalon, whereas Tbr-2 is expressed in neurons that go from the midbrain to the rhombic brain. This suggests that the expression of both genes is involved in the region specificity of neurons.

 Therefore, Tbr-2 is thought to act as a molecule that promotes regeneration after neurodegeneration, and uses Tbr-2 itself and genes located upstream and downstream thereof to treat various neurological diseases, for example, It is expected to be applied to the treatment of senile dementia, the promotion of regeneration after nerve damage, and the treatment of various neurodegenerative diseases.

The Tbr-2 protein of the present invention can be produced as a natural protein or as a recombinant protein using gene recombination technology. The natural protein of the present invention The protein can be prepared by extracting a protein from a tissue or a cell (for example, fetal brain) in which the protein is considered to exist, and performing affinity chromatography using the antibody of the present invention described later. In order to produce a recombinant protein, a DNA encoding the protein of the present invention is incorporated into an expression vector so that it can be expressed under the control of an expression control region, for example, an enhancer or a promoter. . Next, host cells are transformed with this expression vector to express the protein. Specifically, the following may be performed. When using mammalian cells, construct a useful promoter / enhancer commonly used, a DNA encoding the protein of the present invention, a DNA having a polyA signal operably linked to its 3 ′ downstream, or a vector containing the same. I do. For example, the promoter / enhancer can be a human cytomegalovirus i promoter / early promoter / enhancer.

 Other promoters / enhancers that can be used for protein expression include retroviruses, poliovirus, adenovirus, simian virus 40 (SV40), and other virus promoters / enhancers. A promoter / enhancer derived from a mammalian cell of HEFla may be used 1.

 For example, the method of Mulligan et al. (Nature (1979) 277, 108) when using the SV40 Promoter / Enhansa, and the method of Mizushima et al. (Using the HEF1 Hypro Promoter / Enhansa) ( It can be easily carried out according to Nucleic Acids Res. (1990) 18, 5322).

When Escherichia coli is used, useful promoters commonly used, signal sequences for polypeptide secretion, and genes to be expressed can be functionally linked and expressed. For example, promoters include lacZ promoter overnight and araB promoter overnight. When using the lacZ promoter overnight, the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB pro When using Mo'ichi, the method of Better et al. (Science (1988) 240, 1041-1043) may be used.

 As a signal sequence for protein secretion, the pelB signal sequence (Lei, SP. Et al J. Bacterid. (1987) 169, 4379) may be used when produced by the periplasm of Escherichia coli.

 As the origin of replication, those derived from SV40, poliovirus, adenovirus, パ paspiravirus (BPV) and the like can be used. In addition, the expression vector should be selected for the purpose of gene copy number amplification in the host cell system, such as the aminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyltrans. It can contain the ferragase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, and the like.

 The expression vector for producing the protein of the present invention may be any expression vector that is suitably used in the present invention. Examples of the expression vector of the present invention include expression vectors derived from mammals (eg, pEF and pCDM8), expression vectors derived from insect cells (eg, pBacPAK8), and expression vectors derived from plants (eg, pMH1, pH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIpneo), yeast-derived expression vectors-(eg, pNVll, SP-Q01), Bacillus subtilis And expression vectors derived from Escherichia coli (eg, pQE, pGEAPP, pGEMEAPP, pMALp2), etc., but are not limited thereto. The vectors of the present invention include not only the proteins of the present invention produced in vivo and in vitro, but also vectors used for gene therapy of mammals, for example, humans.

 Known methods for introducing the expression vector of the present invention into a host constructed as described above include, for example, the calcium phosphate method (Virology (1973) 52, 456-467) and the electroporation method (EMB0). J. (1982) 1, 841-845).

In the present invention, any production system can be used for protein production. Wear. Production systems for protein production include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

 When eukaryotic cells are used, there are production systems using animal cells, plant cells, and fungal cells. Animal cells include mammalian cells, such as CH0 (J. Exp. Med. (1995) 108, 945), COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, such as African Megafrog oocytes (Valle, et al., Nature (1981) 291, 358-340), or insect cells such as sf9, sf21, and Tn5. CΗ0 cells include DHfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CH0 Kl (Proc. Natl. Acad. Sci.) USA (1968) 60, 1275) can be suitably used.

 Known plant cells are cells derived from Nicotiana tabacum, which may be cultured in virulent. Fungal cells include yeast, such as the genus Sacc haromyces, such as Saccharomyces cerevis iae, and filamentous fungi, such as the genus Aspergilus, such as Aspergilu lus niger. It has been known.

 When prokaryotic cells are used, there are production systems using bacterial cells. As bacterial cells, E. coli and Bacillus subtilis are known.

 The protein is obtained by transforming these cells with the target DNA and culturing the transformed cells in vitro. Culture is performed according to a known method. C For example, DMEM, MEM, RPI 1640, IMDM can be used as a culture solution. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during the culturing is preferably about 6 to 8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.

On the other hand, examples of in vivo production systems include production systems using animals and production systems using plants. The DNA of interest is introduced into these animals or plants, Produce and recover proteins in the body of the plant. The “host” in the present invention includes these animals and plants.

 When using animals, there are production systems that use mammals and bizotes. As mammals, goats, bushes, sheep, mice, and mice can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used. For example, a DNA of interest is inserted into a gene encoding a protein that is specifically produced in milk, such as goat's casein, to prepare a fusion gene. A DNA fragment containing the fusion gene into which the DNA is inserted is injected into a goat embryo, and the embryo is introduced into a female goat. A protein is obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a descendant thereof. Hormones may be used in transgenic goats as appropriate to increase the amount of milk containing proteins produced by transgenic goats (Ebert, KM et al., Bio / Technology (1994) 12 , 699-702). In addition, silkworms can be used as insects, for example. When a silkworm is used, the silkworm is infected with a baculovirus into which a target DNA has been inserted, and a desired protein is obtained from the body fluid of the chicken (Susumu, M. et al., Nature (1985) 315, 592-594. ).

 Further, when using plants, for example, tobacco can be used. When tobacco is used, the desired MA is inserted into a plant expression vector, for example, pMON530, and the vector is introduced into a bacterium such as AgrobacteriuiD tumefaciens. This bacterium is infected to tobacco, for example, Nicotiana tabacum, to obtain the desired polypeptide from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J.I. thigh unol. (1994) 24 , 131-138).

The present invention also encompasses a protein functionally equivalent to the above Tbr-2 protein. Here, "functionally equivalent" means having the same neuronal differentiation factor biological activity as the Tbr-2 protein. The biological activity of a neural differentiation factor is, for example, It is an activity that controls the differentiation of vesicles.

 As a method for obtaining such a protein, a method of introducing a mutation into the amino acid sequence of the protein has been used. In order to obtain such a protein, the desired mutation can be introduced, for example, by site-directed mutagenesis using synthetic oligonucleotide primers (Kramer,. And Fritz, HJ Methods in Enzymol. (1987) 154, 350-367), or a site-directed mutagenesis system by PCR (GIBC0-BRL) can be used. According to these methods, one or more amino acids are deleted, added and / or added to a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3 so as not to affect its biological activity. A protein functionally equivalent to the protein of the present invention, modified by substitution with another amino acid, can be obtained.

 Specific examples of the protein functionally equivalent to the protein of the present invention include, in addition to the protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, SEQ ID NO: 1 or SEQ ID NO: 3 1 or 2 or more, preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 1 or 2 or more, preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids added to the amino acid sequence shown; amino acid shown in SEQ ID NO: 1 or SEQ ID NO: 3 One or more amino acids in the sequence, preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids are substituted with other amino acids.

It is already known that a protein having an amino acid sequence modified by deleting, adding, and / or substituting one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Aca d. Sci. USA (1982) 79, 6409-6413).

 For example, a fusion protein is one example of a protein in which one or more amino acid residues are added to the protein of the present invention. The fusion protein is a fusion of the protein of the present invention and another peptide, and is included in the present invention. A method for preparing a fusion protein is as follows: DNA encoding the protein of the present invention and MA encoding another peptide are ligated in such a way that their frames match each other, introduced into an expression vector, and expressed in a host. It is only necessary to use a method known to those skilled in the art. Other peptides to be fused with the protein of the present invention are not particularly limited.

 For example, peptides include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 x His consisting of 6 His (histidine) residues, 10 x His, influenza agglutinin (HA) , Human C-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, hy-tubul in fragment, B-tag, Known peptides such as fragments of Protein C are used. Proteins include, for example, GST (glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, galactosidase, MBP (maltosis binding protein) and the like. Can be A commercially available DNA encoding these can be used.

 The protein functionally equivalent to the Tbr-2 protein of the present invention also includes a DNA encoding the Tbr-2 protein (a DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4) and a stringent protein. Includes proteins that are encoded by DNA that hybridizes under simple conditions and that have a biological activity equivalent to that of the Tbr-2 protein.

Also included are proteins having the biological activity of the Tbr-2 protein and having homology with the protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. A protein having homology is defined as at least 70, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% or more, the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Has amino acid sequence homology Protein. To determine the homology of proteins, the algorithm described in Wilbur, WJ and Lipman, DJ Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be used.

 The protein of the present invention differs in the amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains depending on the cell or host producing the protein or the purification method. However, as long as the obtained protein is functionally equivalent to the Tbr-2 protein of the present invention, it is included in the present invention.

For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein. When expressed in a eukaryotic cell, for example, a mammalian cell, the N-terminal signal sequence is removed. _C The protein of the present invention also includes such a protein.

The present invention also includes partial peptides of the Tbr-2 protein. Such a peptide can be used, for example, as an immunogen for obtaining an antibody that binds to the Tbr-2 protein. In this case, the peptide has a chain length of at least 12 amino acids or more, and preferably has a chain length of 20 amino acids or more. Further, the partial peptide of the present invention includes a partial peptide consisting of an active center of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 _; Examples of the active center include the amino acid at position 457 from amino acid 278 in SEQ ID NO: 1, or the amino acid at position 455 from amino acid 276 in SEQ ID NO: 3. The partial peptide of the protein of the present invention may include all or a part of the amino acid sequence of the active center. Furthermore, the active center may not be contained depending on the purpose, and such a partial peptide is also included in the present invention.

The partial peptide of the protein of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. As the peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. The protein of the present invention or a partial peptide thereof expressed in a host using a genetic engineering technique can be isolated from inside and outside a cell and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be carried out using the separation and purification methods used in ordinary protein purification, and is not limited in any way. For example, chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization The proteins can be separated and purified by appropriately selecting and combining them.

 Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies or Protein Purification and Characterization: A Laboratory Course Manu al. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HP LC and FPLC. The present invention also includes highly purified proteins using these purification methods.

 The protein can be arbitrarily modified or partially removed by reacting the protein with a suitable protein modifying enzyme before or after purification. As the protein modifying enzyme, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase, protein kinase, and glucosidase are used.

 The present invention also relates to a DNA encoding the protein of the present invention. The dienomic DNA or cDNA encoding the protein of the present invention can be obtained by screening a human cDNA library using the probe described herein.

Using the obtained cDNA or cDNA fragment as a probe and screening the cDNA library, obtain genes from different cells, tissues, organs or species 0 1

Fifteen

be able to. A cDNA library is described, for example, in Sambrook, J. et al., Molecular.

It may be prepared by the method described in Cloning, Cold Spring Harbor Laboratory Press (1989), or a commercially available DNA library may be used.

 Further, by determining the nucleotide sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. Further, by screening the dienomic DNA library using the obtained cDNA as a probe, dienomic DNA can be isolated.

 Specifically, the following may be performed. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. The isolation of mMA is performed by a known method, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-529 9) ヽ AGPC method (Chomczynski, P. and Sacchi, N. , Anal. Biochem. (1987) 162,

156-159) Prepare total RNA using the method described above, and purify mRNA from total A using mRNA Purification Kit (Pharmacia). Also, QuickPrep mRNA Purification Kit

(Pharmacia) to directly prepare mRNA.

 CDNA is synthesized from the obtained fflMA using reverse transcriptase. cDNA synthesis can also be carried out using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Shi-Dai-gaku Kogyo) or the like. In addition, using the probe described in the present specification, a 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and a polymerase chain reaction (PCR) using a 5, -RACE method (Frohman, A. et al., Pro c. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al.,

According to Nucleic Acids Res. (1989) 17, 2919-2932), cDNA can be synthesized and amplified.

A target DNA fragment is prepared from the obtained PCR product and ligated to a vector DNA. Furthermore, a recombinant vector is prepared from this, introduced into E. coli, etc., and a colony is selected to prepare a desired recombinant vector. Confirm the base sequence of the target DNA by a known method, for example, the dideoxynucleotide chain method. do it.

 In addition, the DNA of the present invention can be designed to have a higher expression efficiency in consideration of the codon usage of the host used for expression (Grantham, R. et al, Nuclear Acids Research (1981)). ) 9, r43-r74). Further, the DNA of the present invention can be modified by a commercially available kit or a known method. Examples include digestion with a restriction enzyme, insertion of a synthetic oligonucleotide or an appropriate DNA fragment, addition of a linker, insertion of a start codon (ATG) and / or a stop codon (ATT, TGA or TAG), and the like.

 Specifically, the DNA of the present invention is a DNA consisting of the base A at position 444 to the base C at position 2507 in the base sequence of SEQ ID NO: 2 and the base sequence A at position 2058 from base A at position 1 Includes DNA consisting of base C.

 The DNA of the present invention is a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of either SEQ ID NO: 2 or SEQ ID NO: 4, and the DNA of the Tbr-2 protein of the present invention. Includes DNA encoding proteins having biological activity. Stringent conditions can be appropriately selected by those skilled in the art, and include, for example, low stringent conditions. The conditions of low stringency (for example, 42 ° C., 2 × SSC, 0.1% SDS, and the like, preferably 50 ° C., 2 × SSC, 0.1% SDS. Further more preferably High stringent conditions include, for example, 65 ° C, 2 × SSC, and 0.1% SDS Under these conditions, the higher the temperature, the higher the temperature. DNA with homology can be obtained.

The hybridizing DNA is preferably a naturally occurring DNA, for example, cDNA or chromosomal DNA. As shown in the Examples, it was found that the mRNA that hybridizes with the cDNA encoding the protein of the present invention, mMA, was specifically distributed in fetal brain and adult brain. In particular, it was found to be distributed from the midbrain to the rhombic brain of the fetus. Therefore, the naturally occurring DNA described above is, for example, It may be a cDNA or a dienomic DNA derived from a tissue or a nerve cell in which mA that hybridizes with the cDNA encoding the light protein is detected. Moreover, DNA encoding the蛋white matter of the present invention, other cDNA Ya Jienomikku MA, DNA for a protein and the protein co Solo de also be _£ the present invention a synthetic MA is to be used for screening Useful. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound that binds to the protein, and a compound having an activity of binding to the protein of the present invention is selected. It is used in the method for screening for a compound that binds to the protein of the present invention. As a method for selecting a compound having an activity of binding to the protein of the present invention, many methods usually used by those skilled in the art can be used.

 The protein of the present invention used in these screening methods may be any of a recombinant type, a natural type and a partial peptide. Further, the compound having an activity of binding to the protein of the present invention may be a protein having a binding activity, or may be a chemically synthesized compound having a binding activity.

The method of screening for a protein that binds to the protein of the present invention can be performed, for example, using the West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90). CMA was isolated from cells, tissues, and organs that are expected to express a protein that binds to the protein of the present invention, and introduced into a phage vector, for example, Agtll, ZAPII, etc., to produce a cDNA library. This was expressed on a plate from which the medium was drawn, and the protein expressed at the filter was fixed, and the protein of the present invention purified and labeled and reacted with the filter at the filter, and the protein of the present invention was reacted with the protein of the present invention. Plaques expressing the bound protein may be detected by labeling. As a method for labeling the protein of the present invention, a method utilizing the binding property of biotin and avidin, and an antibody which specifically binds to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention are used. Method, a method using a radioisotope, a method using fluorescence, and the like. One specific example of the screening system provided in the present invention uses a two-hybrid system using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292). Can be done.

 An expression vector containing DNA encoding a fusion protein of the protein of the present invention and one of the transcription regulators comprising a heterodimer and a transcription regulator comprising a heterodimer and a cDNA desired as a test sample. An expression vector containing DNA obtained by ligating DNA coding for one of the subunits is introduced into cells and expressed, and the protein of the present invention binds to the protein whose cDNA is bound to the protein of the present invention. When a heterodimer is formed, a two-hybrid, lid system can be used that expresses a repo overnight gene constructed in advance in a cell. When a protein that binds to the protein of the present invention is present, the protein can be selected by detecting and / or measuring the expression level of the repo overnight gene.

 Specifically, the following may be performed. That is, the DNA encoding the protein of the present invention and the gene encoding the DNA binding domain of LexA are ligated in frame so as to prepare an expression vector. Next, an expression vector is prepared by ligating the desired cDNA to a gene encoding a GAL4 transcription activation domain. Cells transformed with the HIS3 gene, whose transcription is regulated by the promoter containing the LexA-binding motif, are transformed using the two-hybrid system expression plasmid described above, and then transformed on a histidine-free synthetic medium. As a result, cell growth is observed only when protein interaction is observed. Thus, the increase in the expression level of the reporter gene can be examined depending on the degree of growth of the transformants.

 As the repo gene, luciferase gene, ADE2 gene, LacZ gene, CDC25H gene and the like can be used in addition to the HI S3 gene.

The hybrid system may be constructed by a commonly used method, or a commercially available kit may be used. Commercially available two-hybrid system kits include: For example, ATCH ARKER Two-Hybrid System, Mammalian MATCHMARKER Two-Hybrid Assay Kit (both from CLONTECH), HybriZAP Two-Hybrid Vector System (from Stratagene), CytoTrap two-hybrid system (from Stratagene) ₀ The screening method provided in the present invention can be performed using affinity chromatography. That is, the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied here. Test samples in this case include cell culture supernatants, cell extracts, cell lysates, and the like. After applying the test sample, the column is washed to obtain a protein bound to the protein of the present invention. Test samples used in the screening method of the present invention include, for example, peptides, purified or purified proteins, non-peptide compounds, synthetic compounds, fermentation products of microorganisms, marine organism extracts, plant extracts, and cells. Extracts and animal tissue extracts.

 The compound isolated by this screening method is a candidate for a drug for promoting or inhibiting the activity of the protein of the present invention. Compounds obtained by using the screening method of the present invention, in which a part of the structure of the compound having the activity of binding to the protein of the present invention is converted by addition, deletion and / or substitution, are also compounds of the present invention. -Included in compounds obtained by using the method.

 Compounds obtained using the screening method of the present invention can be used in humans, mammals, for example, mice, rats, guinea pigs, rabbits, chicks, cats, dogs, sheep, bush, sea lions, monkeys, baboons, and chimpanzees. When used as a medicament, it can be carried out according to commonly used means.

For example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as required, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids Can be used parenterally in the form of injections. For example, a physiologically recognized simple substance of a compound having a binding activity with the protein of the present invention, It can be prepared by mixing together flavoring agents, excipients, vehicles, preservatives, stabilizers and binders in unit dosage forms generally required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

 Excipients that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used. When the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid simple substance such as oil and fat. Sterile compositions for injection can be formulated according to the normal formulation practice of dissolving or suspending the active substances in vehicles, such as distilled water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing pudose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. For example, alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), and HC0-50 may be used in combination.

Oily liquids include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. It may also be combined with a buffer, such as a phosphate buffer, a sodium acetate buffer, a soothing agent, such as benzalkonium chloride, pro-hydrochloride, or a stabilizer, such as benzyl alcohol, phenol, or an antioxidant. Good. The prepared injection solution is usually filled into an appropriate ampoule. The dose of the compound having the binding activity to the protein of the present invention varies depending on the symptom. However, in the case of oral administration, generally, in an adult (with a body weight of 60 kg), the dose is 1 day. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

 For parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, and administration method. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

 Further, since the protein of the present invention has a DNA-binding domain called a T-box, it can be used for screening of DNA binding to the domain. The method for screening a compound that binds to the protein of the present invention includes a method for screening DNA that binds to the protein of the present invention. The screening of the DNA binding to the protein of the present invention is performed, for example, in accordance with the principle of “Genomic binding-site cloning method” described in the literature (Experimental Medicine Separate Volume, Bio Manual Series II, Transcription Factor Research Method, p209-214 Yodosha). It can be carried out. It can also be isolated by the method described in the literature (J. F. Morris et al., Molecular and Cellular Biology, 1994, 14, pl786-1795). DNA that can be isolated by these methods is a candidate for a transcription control region of a gene whose expression is controlled by the protein of the present invention and a gene under the control. The regulatory region and the gene under its control are expected to be used for controlling neuronal differentiation, and for treating and diagnosing diseases associated with neuronal differentiation.

The present invention also relates to an antibody that binds to the protein of the present invention. Such an antibody can be used for detection or purification of the protein of the present invention. The antibody can be obtained as a monoclonal antibody or a polyclonal antibody using known means. An antibody that specifically binds to the protein of the present invention can be obtained by immunizing the protein using the protein as a sensitizing antigen in accordance with a usual immunization method and obtaining the obtained immune cells by a conventional cell fusion method. Fusing with parental cells for normal screening It can be prepared by screening antibody-producing cells.

 Specifically, a monoclonal antibody or a polyclonal antibody that specifically binds to the protein of the present invention may be prepared as follows.

 For example, the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to an animal species from which the protein is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human. preferable. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed in the present specification. In the present invention, proteins used as sensitizing antigens include all proteins described in the present specification. Can be used. Further, a partial peptide of a protein can also be used. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” refers to an antibody that specifically reacts with the full length or fragment of a protein.

 After the gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system and the host cell described in this specification is transformed, the desired protein can be transferred inside or outside the host cell or from the host. Alternatively, a fragment thereof may be obtained by a known method, and this protein may be used as a sensitizing antigen. Alternatively, a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen.

 The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.ゥ Egrets and primates are used.

As rodent animals, for example, mice, rats, hams, and the like are used.動物 As an heronoid animal, for example, a heron is used. For example, monkeys are used as primates. As the monkeys, monkeys of the lower nose (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used. Immunization of an animal with the sensitizing antigen can be performed according to a known method. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an ordinary adjuvant, such as Freund's complete adjuvant, if desired. After emulsification, it is preferably administered to mammals, and thereafter, an appropriate amount of sensitizing antigen mixed with Freund's incomplete adjuvant is administered several times every 4 to 21 days. In addition, a suitable carrier can be used during immunization of the sensitizing antigen. Immunization is performed in this manner, and an increase in the level of a desired antibody in the serum is confirmed by a conventional method.

 Here, in order to obtain a polyclonal antibody against the protein of the present invention, after confirming that the level of the desired antibody in the serum has increased, the blood of a mammal sensitized with the antigen is taken out. The serum is separated from the blood by a known method. Serum containing a polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.

 In order to obtain a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of a mammal sensitized with the above antigen, the immune cells are removed from the mammal and subjected to cell fusion. Good. At this time, spleen cells are particularly preferable as the immune cells used for cell fusion. The other parent cell to be fused with the immune cell is preferably a mammalian myeloma cell.

 The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be performed according to it.

The hybridoma obtained by cell fusion is selected by culturing in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT culture solution requires a sufficient time for the cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks. Continue for a while. Next, a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.

 In addition, in addition to obtaining the above-mentioned hybridoma by immunizing animals other than humans with the antigen, human lymphocytes, for example, human lymphocytes infected with EB virus, are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. Alternatively, the sensitized lymphocytes can be fused with a human-derived myeloma cell having permanent division ability, for example, U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity (see No. 63-17688).

 Furthermore, a transgenic animal having a human antibody gene liver bird was immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, which were then fused with myeoma cells. Human antibodies to the protein may be obtained using a dormer (WO 92-03918, WO 93-2227, WO 94-02602, WO 94-25585, WO 96-33735). And International Publication No. 96-34096).

In addition to producing antibodies with High Priestess dormer, antibody immune cell such as sensitized lymphocytes that produce an oncogene (oncogene) may be used cells immortalized by _c monoclonal antibodies obtained in the Hare this J Can also be obtained as a recombinant antibody produced using a genetic recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MCMILLAN PUBL ISHERS LTD, 1990 reference). Recombinant antibodies are produced by cloning DNA encoding the same from immune cells such as hybridomas or sensitized lymphocytes that produce the antibody, incorporating the DNA into an appropriate vector, and introducing it into the host. The present invention includes this recombinant antibody.

The antibody of the present invention may be an antibody fragment or an antibody modification thereof as long as it binds to the protein of the present invention. For example, antibody fragments include Fab, F (ab ') 2, Fv or single chain Fv (scFv) (Huston, J. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, the antibody is treated with an enzyme, for example, papain or pepsin, to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. (For example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horitz, AH, Methods Enzymol. (1989) 178, 476-496) Pluckthun, A. and Skerra, Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (198 6) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

 Antibodies bound to various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies. The “antibody” referred to in the claims of the present application also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

 In addition, the antibody of the present invention can be prepared by using a chimeric antibody composed of a variable region derived from a non-human antibody and a constant region derived from a human antibody or a CDR derived from a non-human antibody (complementarity determining region) and a human antibody. Can be obtained as a humanized antibody consisting of the FR (framework region) and constant region.

 The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins, and is not limited at all. The concentration of the antibody obtained as described above can be measured by absorbance measurement, enzyme-linked immunosorbent assay (ELISA), or the like.

As a method for measuring the antigen-binding activity of the antibody of the present invention, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody method can be used. For example, when ELISA is used, the protein of the present invention is added to a plate on which the antibody of the present invention has been immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. Add the following antibody to recognize the antibody labeled with an enzyme, for example, alfa phosphatase, and incubate and wash the plate.Add an enzyme substrate such as p-nitrophenyl phosphate and measure the absorbance for antigen binding. Activity can be assessed. As the protein, a protein fragment, for example, a C-terminal fragment or an N-terminal fragment thereof may be used. For the activity evaluation of the antibody of the present invention, BIAcore (Pharmacia) can be used.

 By using these techniques, the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention contained in the sample, and an immune complex of the antibody and the protein is detected or measured. Or the method of detecting or measuring the protein of the present invention comprising:

 Since the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins.

 In addition, the present invention includes a DNA having a chain length of at least 15 bases, which specifically hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a DNA complementary to the DNA. I do. That is, a probe, a nucleotide or a nucleotide derivative capable of selectively hybridizing with a DNA encoding the protein of the present invention or a DNA complementary to the DNA, for example, an antisense oligonucleotide, a ribozyme and the like are included.

The present invention includes, for example, an antisense oligonucleotide which hybridizes at any position in the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The antisense oligonucleotide is preferably an antisense oligonucleotide corresponding to at least 15 or more consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. More preferably, the antisequence wherein at least 15 or more consecutive nucleotides include a translation initiation codon. Oligonucleotide.

 As the antisense oligonucleotide, derivatives and modifications thereof can be used. Examples of such a modified product include a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified product or a phosphoroamidate modified product.

 As used herein, the term “antisense oligonucleotide” includes not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary, but also those in which DNA or mRNA and an oligo nucleotide are represented by SEQ ID NO: One or more nucleotide mismatches may be present as long as they can selectively and selectively hybridize to the base sequence shown in 1.

 Selectively and stably hybridizing means that under normal hybridization conditions, preferably under stringent hybridization conditions, cross-hybridization with DNA encoding other proteins is significant. Does not occur in the future. Such DNAs include at least 15 contiguous nucleotide sequence regions having at least 70, preferably at least 80%, more preferably 90%, and even more preferably 95% or more nucleotide sequence homology. Is shown. Note that the algorithm described in this specification may be used as an algorithm for determining homology. Such a DNA is useful as a probe for detecting or isolating a DNA encoding the protein of the present invention, or as a primer for amplification, as described in Examples below.

 The antisense oligonucleotide derivative of the present invention acts on a cell producing the protein of the present invention to bind to DNA or mMA encoding the protein, thereby inhibiting its transcription or translation or inhibiting the degradation of mRNA. By promoting or suppressing the expression of the protein of the present invention, it has the effect of suppressing the action of the protein of the present invention.

The antisense oligonucleotide derivatives of the present invention are inactive against them An external preparation such as a coating agent or a poultice can be prepared by mixing with an appropriate base. If necessary, excipients, isotonic agents, solubilizing agents, stabilizing agents, preservatives, soothing agents, etc. may be added to tablets, splinters, granules, capsules, ribosome capsules, It can be a lyophilized agent such as a propellant, a liquid, a nasal drop and the like. These can be prepared according to a conventional method.

 The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so that it can reach the affected area as a result of intravenous administration or the like. Furthermore, an antisense-encapsulated material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.

 The dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.

 The antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention, and is therefore useful in suppressing the biological activity of the protein of the present invention. Expression inhibition containing the antisense oligonucleotide of the present invention The agent is useful in that it can suppress the biological activity of the white matter of the present invention. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows a comparison of the mouse and human Tbr-2 coding region cDNA sequences with the predicted amino acid sequence (1-232 amino acids of mouse Tbr-2 and 1-230 amino acids of human Tbr-2). It is a schematic diagram. Matching bases are shaded. Gaps are represented by hyphens (-). Amino acids are represented by one letter code. Amino acids that differ between mouse and human are underlined and italicized.

Figure 2 shows the cDNA sequence of the mouse and human Tbr-2 coding region and the predicted amino acid sequence (amino acids 233-472 of mouse Tbr-2 and 231-470 amino acids of human Tbr-2). 3 is a schematic diagram showing a comparison of Matching bases are shaded. Gaps are represented by hyphens (-). Amino acids are represented by one letter code. Amino acids that differ between mouse and human are shown in underlined italics.

 Figure 3 compares the cDNA sequences of the mouse and human Tbr-2 coding regions with the predicted amino acid sequence (amino acids 473-688 of mouse Tbr-2 and 471-680 amino acids of human Tbr-2). FIG. Matching bases are shaded. Gaps are represented by hyphens (-). Amino acids are represented by one letter code. Amino acids that differ between mouse and human are shown in underlined italics.

 Fig. 4 shows homologs of human Tbr-2 (hTbr2), mouse Tbr-2 (mTbr2), Eomes ヽ human Tbr-1 (hTbrl) and mouse Tbr-1 (mTbrl) belonging to the T-box gene family. It is a schematic diagram which shows a gee comparison. Amino acids are represented by one letter code. Matching amino acids are shaded. Gaps are represented by hyphens (-). The T-box area is represented by an asterisk (*).

 Figure 5 shows the evolutionary phylogenetic tree of the mouse T-box gene family.

 FIG. 6 is a diagram when the expression of Tbr-2 in each of the fetal brain and adult tissues was analyzed by RT-PCR.

 FIG. 7 is a diagram when changes in the expression level of Tbr2 in E14.5, E18.5, P2.0, P14.0, and adult brain were examined by Northern blot analysis. The upper row shows the Northern blot signal, and the lower row shows the RNA staining image.

 Figure 8 shows that the fetuses at each stage (E9.5: A, B, E10 ^ iC, D, £ 11.5: £) show Tbr-1 (A, C, E) and Tbr-2 (B, D, FIG. 2 is a diagram showing the expression of F) analyzed by whole mount in situ hybridization.

 Figure 9 shows that the whole brain at £ 14.5, 8, (:, 0) is the same for the hindlimb (E, F) as Tbr-1 (A, C, E) and Tbr-2 ( FIG. 9 shows the results of analyzing the expression of (B, D, F).

Figure 10 shows the coronal section (a, b, It is a figure which shows the result of having hybridized c, d, e, f) and the sagittal section (Sagital section) (g, h, I, j, k, 1) with a Tbr-2 antisense probe. a and g are the olfactory bulb, b and h are from the striatum of the telencephalon to the hippocampus, c is the periphery of the hippocampus, i is the antiflexion of the suprathalamus, d, e, f and j, k, l In the area from the midbrain to the pons, many cell populations (neural nuclei) are stained. In the figure, `` AQ (Aqueduct of sylvius) '' is the middle cerebral aqueduct, `` HA '' (Habenular recess of third ventricle) is the inter-rein legway, `` ISS '' (Intermediate subpallial sulcus) is the inside of the mantle groove. "1 ventricle)" is the lateral ventricle, "ph (Posterior horn)" is the dorsal horn, "ΠΙν (Τ hird ventricle)" is the third ventricle, and "IVv (Fourth ventricle)" is the fourth ventricle.

 Fig. 11 shows Tbr in the olfactory bulb (a, b, c) and hippocampus (d, e, f) in E18.5 (a, d), P4.0 (b, e) adult (c, f). FIG. 3 is a diagram when the expression of -2 was analyzed by in situ hybridization.

 FIG. 12 shows the results of expression analysis of the Tbr-2 gene in adult mouse brain. Embryonic 18.5 days (a, d), 4 days after birth (b, e), and Tbr-2 in the olfactory bulb (a, b, c) and hippocampus (d, e, f) of adult mice (c, f) Expression was analyzed by the in situ hybridization method. The open triangles and arrowheads indicate the gonulla single cell layer and the neutral cell layer of the olfactory bulb, respectively. Tbr-2 is expressed in the olfactory bulb and hippocampus in the adult mouse brain.

 FIG. 13 shows a comparison of the expression patterns of Tbr-2, Tbr-1, and Brachyury genes in early mouse development.

 FIG. 14 shows the results of RT-PCIU detecting an increase in Tbr-2 expression in the thymus due to conA stimulation. BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. [Example 1] Cloning of mouse Tbr-2 cDNA

Embryonic 13. Each than 5 days mice telencephalon telencephalon and adult mice were purified poly A RNA, PCR- Select cDNA Subtraction Kit and _c obtained cDNA fragment obtained by subtracting using (CLONTECH Corp.) pGEM- T vector (Promega Subcloning was performed, and cDNA highly expressed in embryonic day 13.5 brain was selected by dot plotting and sequenced. The obtained clone # 61 contained a 531 bp gene fragment (Tbr-2) containing a DNA-binding region called T-box. mouse 17-day Embryo tr-stretch plus c brittle (CLON TECH) was obtained by plaque hybridization. The resulting mouse Tbr-2 cDNA was 2883 bp and encoded 688 amino acids.

 [Example 2] Cloning of human Tbr-2 cDNA

First, a primer h61-up (SEQ ID NO: 5 / 5'-gagccctcaaagacccaga-3 ', 1243-1261) was synthesized based on a human EST sequence very similar to the mouse Tbr-2 cDNA sequence. Using h6 up as the upstream primer and primer SIS '(SEQ ID NO: 6/5, -ctagggacttgtgtaaaaagc-3') containing the termination codon of mouse Tbr-2 as the downstream primer, the human fetal brain cDNA was The DNA was transformed into type III and subjected to PCR (94 ° C for 30 seconds, 62 ° C for 90 seconds, 35 cycles) to obtain an approximately 800 bp cDNA fragment of human Tbr-2 on the third side. Based on this sequence, primer -h61-4 (SEQ ID NO: 7 / 5'-aatatcggtgttttggtagg-3 ', 1301-1320) was synthesized. Next, Primer 61-5, (SEQ ID NO: 8 / 5'-atgcagttgggagagcag-3 ') containing the initiation codon of mouse Tbr-2 was synthesized with the upstream primer based on the sequence of mouse Tbr-2. Primer 61-N (SEQ ID NO: 9 / 5'-catggagccgtaggggta-3 ', 554-571) に し て Set the downstream primer to human fetal brain cDNA and set it to PCR in the presence of 5% DMS0 (94 ° C 30 sec, 60 ° C for 30 sec, 72 ° C for 30 sec, 30 cycles) to obtain an approximately 570 bp cDNA fragment of human Tbr-2 on the 5, side. Based on this sequence, a primer h61-3 (SEQ ID NO: 10 / 5'-gtcctcaggggtcggagc-3 ', 449-466) was synthesized. Finally, use h61-3 for the upstream primer and h6 to 4 for the downstream primer, and perform PCR (94 ° C 3 0 sec, 58 ° C 30 sec, 72 ° C 30 sec, 30 cycles) to obtain a fragment of about 750 bp. These were all connected to determine the sequence of the coding region of human Tbr-2 cDNA.

 [Example 3] Comparison of amino acid sequences of human and mouse Tbr-2

 The obtained mouse and human-derived Tbr-2 were compared (FIGS. 1 to 3). Human Tbr-2 cDNA encoded 686 amino acids. Both genes had a homology of more than 90% at the amino acid level as a whole. The amino acid sequence encoding the T-box region (mouse; 278-457 amino acids, human; 276-455 amino acids) in the center was 100-matched. On the other hand, in the N-terminal region, 10 amino acids were found in mouse Tbr-2 (amino acids 28-37) and 8 amino acids were found in human Tbr-2 (amino acids 123-130).

 As a result of comparing the amino acid sequences of Tbr-2 and other proteins (Fig. 4), one of the T-box gene families, Xenopus Eomesodermin (Eomes) (Ryan, K. et al. 1996 Cell 87 879-1000) and mouse and human Tbr-1 genes (Buifone A. et al., 1995, Jul. Neuron. 15, 63-78). Comparing each region, the T-box region had 92.7% homology with Eomes and 83.8% homology with mouse Tbr-1. At the C-terminal side (amino acids 458-688) downstream of that, 55.6 14.6¾-matched, respectively. On the other hand, in the upstream N-terminal side (amino acids 1-277), Eomes hardly matched with 11,4% Tbr-1. However, other T-box gene families (i.e., Brae hyury (Herrmann, BG et al., 1990, Nature, 343, 617-622)) and Tbxl-6 (Bol lag, RJ et al., 1994, Nat. Genet. , 7, 383- 389)) has little homology except for the T-box, and its homology is not as high as that of Eomes and Tbr-1.Tbr-2 is one of the T-box gene families. However, it was inferred that the gene belongs to the same subfamily as Eomes and Tbr-1 (Fig. 5).

Recently, a cDNA fragment encoding the T-box region of Tbr-2 has been partially cloned from a mouse and registered (Accession No. AF013281). Since this region is highly homologous to Eomes, it has been named 'MEomes' (Wattler S. et al., 1998, Genomics, 15, 2 ot4-3 o o3 /), but the homology of the N-terminal region of both proteins is high. Was low. [Example 4] Tbr-2 gene tissue expression analysis

 Tbr-2 expression was examined by RT-PCR in fetal, adult and adult tissues (Figure 6). As a result, the expression of Tbr-2 was overwhelmingly high in the fetal brain and only slightly detectable in the adult brain. No expression was observed in adult tissues (spinal cord, kidney, heart, liver).

 To further examine brain expression from fetal to adult, Northern blots were performed on the brains of mice at each age. As a result, the expression of Tbr-2 was very high during fetal life and decreased sharply in the postnatal brain (Fig. 7). This suggests that Tbr-2, like Tbr-1, is a gene that is very involved in fetal brain architecture.

 [Example 5] Tissue expression analysis of Tbr-2 gene by whole mount in situ hybridization

 Both genes are highly expressed in the embryonic brain and are similar in structure. Whole mount in situ hybridization is used to determine where in the developing brain the two genes, Tbr-1 and Tbr-2, are expressed. The comparison was made by Whole mount in situ hybridization (ISH).

Tbr-1 is a cDNA encoding 164 amino acids from the initiation codon, and Tbr-2 is a cDNA encoding 184 amino acids from the initiation codon. Alternatively, antisense digigogenin (DIG) -labeled RNA was synthesized. For whole mount in situ hybridization, this RNA was used as a probe, and for in situ hybridization for sections, this A was hydrolyzed and used as a 150 base fragment. . For whole-mount in situ hybridization, the embryos at each stage were left as they were, or the extracted brains were fixed with 4% paraformaldehyde / PBT, and hybridization was performed using the above-mentioned probe according to a standard method. In situ hybridization was performed on 4% paraformal brains from fetal brains directly or from perfused fixed mice. The cells were fixed with dehyde and hybridized with the above probe according to a standard method (Sumiharu Noji, "Immunostaining, in situ hybridization", Yodosha). Then, after reacting with anti-DIG antibody labeled with alkaline phosphatase, a substrate was added and the signal was detected.

 Embryos at 9.5 days, 10.5 days and 11.5 days were stained with antisense RNA probes of ¾1-1 and Tbr-2. Both Tbr-1 and Tbr-2 were already expressed in the fetal brain region on embryonic day 9.5 (Fig. 8). Interestingly, Tbr-2 expression was also observed in the limb buds (limbbud) formed from embryonic day 9.5 (B in FIG. 8). This expression moved to the tip along with the formation of the limb (limb), and its expression was localized at the fingertip in the limb (limb) at 14.5 days of embryogenesis when the finger was formed (E and F in Fig. 9). ).

 To examine the distribution in the brain, embryos at 13.5 and 14.5 days of embryonic age were removed and stained with Tbr-1 and Tbr-2 antisense RNA proteins. As a result, expression of Tbr-1 was observed in the telencephalon (A, C in Fig. 9), while expression of Tbr-2 was observed in the midbrain and rhomboid to medulla (B, D in Fig. 9). ). In the olfactory bulb, expression of both genes was observed (C and D in Fig. 9).

 Next, in order to examine in detail which region of the developing brain expresses Tbr-2, ISH was performed on a section of the brain from day 14.

In the olfactory bulb, there were sparse cells with positive expression. The olfactory bulb at this stage is in the process of formation, and a clear stratified structure cannot be confirmed. However, at this stage, it is not the neuroepitherium that has many undifferentiated cells, but the future mitral cell layer (mitral cell layer). ) And Tbr2 expression was observed at the site forming the glomerular cell 1 ater (a, g in Fig. 10). In the forebrain region, expression was observed in the amygdala from the striatum (cpu; caudateputamen) (Fig. 10, b, h). _{0 The} hippocampus was not yet formed, but was slightly expressed in the hippocampus primordium ( C, h in Fig. 10). Some expression was also observed in the retina nucleus, which starts the anti-bending bundle, which is a nerve bundle that crosses the thalamus (Fig. 10-1). In the midbrain region, central gray matter (central gray), expression was observed in the mesencephalic reticulum, and this expression continued to the caudal rhomboencephalic region (from the pons (hindbrain) to the medulla oblongata; the cerebellum had not yet been formed). Expression was also observed in multiple developing nuclei (the oculomotor nucleus, the red nucleus, the trigeminal nucleus, the vestibular nucleus, the facial nucleus, the hypoglossal nucleus) distributed from the midbrain to the pons (Fig. 1 0 d, e, f,, k, l) o

 Sections of E18.5, P4 and adult mouse brains were also prepared to determine the localization of Tbr-2. In the brain after E18.5, no specific expression in various cerebral nuclei observed in the brain at E14.5 was observed. On the other hand, high expression was observed in the hippocampus, which was rarely seen in E14.5. At E 18.5, expression was observed over a wide area of the hippocampus, but the expression range narrowed as the brain matured, and its expression was more restricted in the adult brain (Fig. 11). d, e, f). On the other hand, even in the olfactory bulb where the expression site did not stick out in E14.5, on E18.5 days, the expression cells were restricted to the glomerular layer and the mitrasolole cell layer (mitral cell layer). Was. This expression, like that in the hippocampus, narrowed with maturation of the brain and was barely localized in a very small area in adults (a, b, c in Fig. 11).

 [Example 6] Expression analysis of Tbr-2 gene in adult mouse brain by in situ hybridization

Mouse Tbr-2 cDNA (encoding amino acid at position 184) was subcloned into pBluescript II (SK-). This was transcribed in vitro into a type II to synthesize antisense and sense RNA labeled with digoxigenin (DIG). The RM of this was treated with alkaline hydrolysis buffer one _{(40mM NaHC0 3, 60mM Na 2} C0 3, pH 10.2), it was used as probes. The brain was removed from a mouse at 18.5 days of age, 4 days after birth, and 10 weeks old, and a 10- / m section was prepared. These were air-dried, fixed with 4% paraformaldehyde for 15 minutes, and hybridized overnight with l〃g / ml DIG-labeled RNA probe. Then, treat it with RnaseA, wash it several times, and Signal was detected using IG antibody.

 As a result, Tbr-2 was expressed only in the olfactory bulb and hippocampus in adult mice (Fig. 12). These two tissues are the only sites in the adult mouse brain where the euron supplied from the ependymal / subependymal zone is differentiating. This expression pattern suggests that Tbr-2 is a molecule involved in neuronal differentiation.

 [Example 7] Comparison of expression patterns of Tbr-2, Tbr-1, and Brachyury genes in early mouse development.

 Total RNA was prepared from embryos (WE) from embryos 6.5 to 11.5 days and fetal brain (BR) from embryos 13.5 and 14.5 days. After synthesizing cDNAs from l〃g of total RNA, they were transformed into type III using primers for the Tbr-2, Tbr-1, Brachyury, and G3PDH genes (control), 30, 30, 25, and 22, respectively. A cycle of PCR was performed.

 As a result, expression of Tbr-1 was observed around embryonic day 8.5, and gradually increased thereafter. On the other hand, Tbr-2 showed a bilayer expression pattern. The first peak of expression was observed around embryonic day 6.5, and once disappeared on embryonic day 9.5, expression was observed again after embryonic day 10.5 (Fig. 13). The first expression observed early in development is earlier than the expression of the Brachyury gene, which is a very important gene for mesoderm formation, indicating that Tbr-2 is deeply involved in the early formation of mesoderm in addition to the differentiation of cranial nerves. It is expected to be a related molecule.

 [Example 8] Increase of Tbr-2 expression in thymus by conA stimulation

 The spleen and thymus were surgically removed from adult mice. Cells from these tissues were disintegrated and seeded in petri dishes. After stimulating the cells with 2 ug / m 1 conA, the culture was continued, and the cells were collected after 1 day, 2 days, and 3 days. Total RNA was extracted from these and RT-PCR was performed with primers specific for Tbr-2 and G3PDH in the same manner as described above.

As a result, even when the spleen was stimulated with conA, the expression level of Tbr-2 did not change, but in the thymus, the expression level of Tbr-2 was increased by stimulation with conA. conA is T lymphocyte It is known to activate spheres, but this result suggests that Tbr-2 activates T lymphocytes or has some function in activated T lymphocytes. Industrial applicability

According to the present invention, a Tbr-2 protein and its gene, a method for producing them, and uses thereof are provided. The protein and gene of the present invention are thought to be involved in the differentiation of neurons, and are used for the treatment of various neurological diseases, for example, the treatment of senile dementia, the promotion of regeneration after nerve injury, and the treatment of various neurodegenerative diseases. Application is expected.

Claims

The scope of the claims

1. From a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence in the protein obtained by modifying one or more amino acids in the amino acid sequence by deletion, addition and / or substitution with another amino acid A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1.

 A protein comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence modified by deletion, addition, and / or substitution of another amino acid in the amino acid sequence of the protein, A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 3

 3. A protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 2, which is functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1.

 4. A protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 4, which is functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 3.

 5. A fusion protein comprising the protein according to any one of claims 1 to 4 and another peptide.

 6. A DNA encoding the protein according to any one of claims 1 to 5.

 7. A vector into which the DNA according to claim 6 has been inserted.

 8. A transformant that retains the DNA of claim 6 so that it can be expressed.

 9. The method for producing a protein according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 8.

 10. A method for screening a compound that binds to the protein according to any one of claims 1 to 5, wherein

(a) contacting a test sample with the protein according to any one of claims 1 to 5, (b) selecting a compound having an activity of binding to the protein according to any one of claims 1 to 5.

 11. An antibody that specifically binds to the protein according to any one of claims 1 to 5.

 12. Contacting the antibody according to claim 11 with a sample expected to contain the protein according to any one of claims 1 to 5, to form an immune complex between the antibody and the protein. Or a method for detecting or measuring said protein.

13. A DNA that specifically hybridizes with a DNA consisting of the nucleotide sequence of either SEQ ID NO: 2 or SEQ ID NO: 4, and has a chain length of at least 15 bases.

14. A partial peptide of a protein comprising the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 3.