WO2000049149A1 - Novel tnf receptor-like proteins - Google Patents
Novel tnf receptor-like proteins Download PDFInfo
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- WO2000049149A1 WO2000049149A1 PCT/JP2000/000940 JP0000940W WO0049149A1 WO 2000049149 A1 WO2000049149 A1 WO 2000049149A1 JP 0000940 W JP0000940 W JP 0000940W WO 0049149 A1 WO0049149 A1 WO 0049149A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
Definitions
- the present invention relates to a novel receptor protein, an MA encoding the protein, a vector containing the MA, a host cell carrying the vector, an antibody against the protein, a method for screening a compound using the protein, It relates to a compound that can be isolated by a screening method. North
- Cytokines and their receptors that act in the brain are important targets for the development of new brain-related drugs.
- cytokines that act on the brain include nerve growth factor? (P 01138), brain-derived neurotrophic factor (P23560), neurotrophin-3 (P207 83), neurotrophin-4 (P34130), and neurotrophin-6 (P34132).
- Nutritional factors such as neurotrophin-6 ⁇ (P34133) and neurotrophin-6r (P34134) and hormone-like cytokines such as leptin (P41160) have been reported.
- An object of the present invention is to provide a novel TNF receptor-like protein and its gene. Another object of the present invention is to provide a vector into which the gene has been inserted, a host cell having the vector, and an antibody that binds to the protein. Sa Furthermore, an object of the present invention is to provide a method of screening for a compound that binds to the protein, such as a ligand, using the protein.
- the present invention relates to novel receptor proteins and their genes and their uses, more specifically,
- a method for producing the protein according to (5) comprising culturing the host cell according to (4), and collecting a protein expressed from the host cell or a culture supernatant thereof.
- nucleotide having a length of at least 15 bases which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, 8, or 10 or a complementary strand thereof,
- ligand refers to a protein that activates its function by binding to the protein of the present invention expressed on a cell membrane.
- agonist refers to a protein that can cause a phenomenon similar to the binding of the protein of the present invention to a ligand (activation of the protein of the present invention).
- ligand activation of the protein of the present invention.
- molecules that can bind refers to molecules that can bind.
- angigonist refers to a molecule that specifically binds to the protein of the present invention to suppress its function.
- the present invention provides a novel membrane secreted protein.
- the nucleotide sequence of the mouse-derived cDNA (named mouse Troy) isolated by the present inventors is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the mouse Troy cDNA is shown in SEQ ID NO: 2.
- the nucleotide sequence of human cDNA (named human Troy) corresponding to mouse Troy cDNA is shown in SEQ ID NO: 10, and the amino acid sequence of the protein encoded by human Troy cDNA is shown in SEQ ID NO: 11.
- These proteins are compatible with known proteins belonging to the TNF receptor superfamily, have a signal sequence at the N-terminus, and have a transmembrane region in the middle that is expected to be a highly hydrophobic amino acid region.
- these proteins are novel proteins belonging to the TNF receptor superfamily (Beulter.B, and Huffel.C.V (1994) SCIENCE 264, 667-668 Unraveling function in the TNF ligand and receptor knew s). It is believed that there is.
- mouse dTroy a cDNA clone encoding a protein lacking most of the intracellular domain of the mouse Troy protein (designated mouse dTroy).
- the nucleotide sequence of mouse dTroy cDNA is shown in SEQ ID NO: 8
- the amino acid sequence of the protein encoded by mouse dTroy cDNA is shown in SEQ ID NO: 9. Since the protein lacks most of the intracellular domain and does not show NF-KB activation ability, it is expressed on the cell membrane and binds to the ligand to convert the complete ligand to Troy. It is considered that binding is inhibited, and thereby signal transmission is inhibited.
- TNFR members form homotrimers and transmit signals, but may inhibit signal transmission by forming heterotrimers containing dTroy.
- proteins having such functions for example, TRAIL-R3 (TRAIL decoy-type receptor) and DcR3 (FasL decoy-type receptor) are known.
- the invention also includes proteins that are functionally equivalent to the mouse and human Troy proteins.
- “functionally equivalent” means that the target protein is extracellular.
- the function as such a receptor protein includes, for example, the ability to induce NF-kB activity.
- the ability to induce NF-kB activity can be detected by the method described in Example 6.
- the invention also includes proteins functionally equivalent to the mouse dTroy protein.
- “functionally equivalent” means that the target protein functions as a decoy receptor protein.
- the function as a decoy-type receptor protein includes, for example, a function of inhibiting the ability of a full-type receptor protein (for example, Troy protein) to induce NF-kB activity.
- proteins having one or more mutated amino acids in the amino acid sequence of the mouse and human Troy protein or Pomatus dTroy protein, and functionally equivalent to these proteins are also present in the present invention. Included in the proteins of the invention.
- the number of amino acids to be mutated in such a mutant is generally considered to be within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, and further preferably within 3 amino acids.
- the amino acid residue to be mutated is mutated to another amino acid in which the properties of the amino acid side chain are conserved.
- the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl-containing side chains (S, ⁇ , ⁇ ), sulfur atom-containing side Amino acids (D, N, E, Q) with carboxylic acids and amide-containing side chains, amino acids with side chains containing bases (R, K, ⁇ ), aromatic-containing Amino acids having a chain ( ⁇ , F, Y, W) can be mentioned (all brackets indicate one letter of amino acids).
- Examples of the protein to which one or more amino acid residues are added include a fusion protein containing Troy protein or dTroy protein of the present invention.
- the fusion protein is a fusion of these proteins with other peptides or proteins, and is included in the present invention.
- the method for producing a fusion protein is as follows: a DNA encoding the protein of the present invention and another peptide or a DNA encoding the protein are ligated so that their frames coincide with each other, and this is introduced into an expression vector; And a known method can be used.
- Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.
- Hybridization to isolate DNA encoding a functionally equivalent protein can be performed, for example, under conditions where the stringency is 10% formamide, 5xSSPE, lx Denhardt's solution, and lx salmon sperm MA. . More preferred conditions (more stringent conditions) are 25% formamide, 5xSSPE, lx Denhardt's solution, and lx salmon sperm DNA, and more preferred conditions (more stringent conditions) are 50% The conditions are formamide, 5xSSPE, lx Denhardt's solution, and lx salmon sperm DNA.
- the DNA encoded by the DNA isolated by the hybridization technique or the gene amplification technique usually has a high homology in amino acid sequence with the protein of the present invention described in SEQ ID NO: 2, 9, or 11.
- High homology usually means a homology of 70% or more, preferably 85% or more, more preferably 95% or more.
- the literature Wang, WJ an d Lipman, DJ Proc. Natl. Acad. Sci. USA (1983) 80, 726-730).
- the protein of the present invention can be prepared as a recombinant protein or as a natural protein by methods known to those skilled in the art.
- a recombinant protein for example, a portion other than the region required for membrane binding of the protein of the present invention is extracellularly secreted as a soluble protein.
- the culture supernatant of the cells is collected and concentrated, and then subjected to chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which the antibody against the protein of the present invention is immobilized on a column. Purification can be carried out by combining them or by combining a plurality of these columns.
- the protein of the present invention is expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion protein with a glutathione S-transferase protein or as a recombinant protein to which a plurality of histidines are added.
- the recombinant protein is purified using a glutathione column or a nickel column. Thereafter, if necessary, a region other than the target protein in the fusion protein can be prepared by a method of cleaving with thrombin or Factor-Xa and removing it.
- the protein is a natural protein, it is purified by, for example, allowing an extract of cells expressing the protein of the present invention to act on an affinity column to which the antibody of the present invention described later is bound. Can be isolated.
- the present invention also includes partial peptides of the protein of the present invention.
- the partial peptide of the present invention has an amino acid sequence of at least 8 amino acids or more, preferably 15 amino acids or more, more preferably 50 amino acids or more (for example, 100 amino acids or more).
- the partial peptide is used, for example, for preparing an antibody against the protein of the present invention, screening a compound such as a ligand that binds to the protein of the present invention, and preparing a competitive inhibitor of the protein of the present invention. obtain.
- the partial peptide of the present invention includes, for example, the protein of the present invention in which the signal peptide is removed from the protein and the protein of the present invention.
- soluble proteins from which the region required for membrane binding has been removed and proteins in the form of corn by removing the intracellular region.
- a partial peptide (which functions as a competitive inhibitor of the receptor of the present invention) that has the ability to bind to a ligand but does not have the ability to transmit signals into cells is included.
- the partial peptide 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.
- the present invention also provides a DNA encoding the protein of the present invention.
- the DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, whether it is cDNA synthesized from mRNA, genomic DNA, or chemical synthesis! It does not matter whether it is NA or not. Also, as long as it can encode the protein of the present invention, MA having an arbitrary base sequence based on the degeneracy of the genetic code is included.
- the DNA of the present invention can be prepared by a method known to those skilled in the art.
- a cDNA library is prepared from cells expressing the protein of the present invention, and a part of the DNA sequence of the present invention (for example, the DNA sequence of SEQ ID NO: 1, 8, or 10) is probed. And by performing hybridization.
- RNA is prepared from cells expressing the protein of the present invention, and oligo DNA is synthesized based on the sequence of the DNA of the present invention (for example, the DNA sequence described in SEQ ID NO: 1, 8, or 10).
- it can also be prepared by performing a PCR reaction using this as a primer and amplifying a cDNA encoding the protein of the present invention.
- the DNA of the present invention can be used to produce the protein of the present invention as a recombinant protein. If the DNA encoding the protein of the present invention is defective, application to antisense function inhibition, gene therapy for replacement with a normal gene, and the like can be considered.
- the present invention also provides a vector into which the DNA of the present invention has been inserted.
- the vector of the present invention is not particularly limited as long as it can express the DNA of the present invention in a host cell.
- the vector is For example, JM109, DH5, and HB10U XLlBlue
- JM109, DH5, and HB10U XLlBlue have an “ori” to be amplified in Escherichia coli for large-scale amplification and preparation in Escherichia coli
- a selection gene for transformed Escherichia coli for example, any drug (A drug resistance gene that can be identified by ampicillin, tetracycline, kanamycin, chloramphenicol) is not particularly limited.
- M13 system vector For example, M13 system vector, pUC system vector, pBR322, pBluescript, pCR-Script, and the like.
- pGEM-T For the purpose of cDNA subcloning and excision, pGEM-T, pDIRECT, pT7, etc. may also be used in addition to the above vectors.
- an expression vector is particularly useful.
- an expression vector for example, when the purpose is expression in E. coli, in addition to having the above characteristics such that the vector is amplified in E. coli, when the host is E. coli such as JM109, DH5, HB10K XLlBlue, etc.
- a promoter eg, lac, T7, etc.
- a vector examples include pGEX, pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 MA polymerase) in addition to the above vectors.
- the promoters required for expression in cells (SV40, MMLV-LT1, EF1, CMV It is essential to have a promoter, etc., and it is more preferable to have a gene for selecting transformation into cells (for example, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)).
- Vectors having such characteristics include, for example, bandits, pDR2, pBK-RSV, pBK-CMV ⁇ pOPRSV, and pOP13.
- a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell eg, , PCHOI, etc.
- amplifying with methotrexate (MTX) MTX
- COS cells having the SV40 T antigen on the chromosome are used to transform the cells with the replication mechanism of SV40 (such as pcD ").
- the DNA of the present invention is incorporated into an appropriate vector, and the retroviral method, the ribosome method, the cationic liposome method, the adenovirus method, and the like are used to express the DNA in vivo.
- the vector to be used is not particularly limited, and examples include pAdexlcw and pZIPneo.
- General genetic manipulation such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.6, 5.63).
- the present invention also provides a host cell into which the vector of the present invention has been introduced.
- the host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used. Examples of Escherichia coli include JM109, DH5 and HB101, and examples of animal cells include CH0 cells, COS cells, 3T3 cells, and HeLa cells. In animal cells, when large-scale expression is intended, CH0 cells are particularly preferred.
- the introduction of the vector into a host cell can be carried out by, for example, a calcium phosphate method, a DEAE dextran method, electo-portation, lipofection, and the like.
- the present invention also provides an antibody that binds to the protein of the present invention.
- the form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody as well as a polyclonal antibody. Also included are antisera obtained by immunizing egrets and the like with the protein of the present invention, all classes of polyclonal and monoclonal antibodies, and human antibodies and humanized antibodies obtained by genetic recombination.
- the antibody of the present invention can be prepared by the following method.
- a polyclonal antibody for example, a small animal such as a heron is immunized with the protein of the present invention to obtain a serum, which is then subjected to an affinity column in which the protein of the present invention is coupled. Of immunoglobulin G or M from this fraction was applied to a protein A or protein G column. It can be prepared by further purification.
- a monoclonal antibody a small animal such as a mouse is immunized with the protein of the present invention, the spleen is excised from the mouse, and the spleen is crushed into cells, and mouse myeloma cells and reagents such as polyethylene glycol are used.
- a clone that produces an antibody against the protein of the present invention is selected from the resulting fused cells (hybridomas).
- the obtained hybridoma was transplanted into a mouse intraperitoneal cavity, ascites was recovered from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purifying the protein of the present invention using a coupled affinity column or the like.
- the antibody of the present invention is used for purification and detection of the protein of the present invention, and is also a candidate for an agonist and an agonist of the protein of the present invention. It is also conceivable to apply the antibody of the present invention to antibody therapy for neurological diseases. When used for antibody therapy, human or humanized antibodies are preferred to reduce immunogenicity.
- a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing the antibody-producing cell with myeloma cells is used.
- a human antibody against the protein see International Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735 and W096-34096).
- the antibody of the present invention may be an antibody fragment or modified antibody thereof as long as it binds to the protein of the present invention.
- an antibody fragment Fab, F (ab r ) 2, Fv or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker
- the antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector.
- Expression in host cells eg, Co, M. S.
- modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can be used.
- PEG polyethylene glycol
- the “antibody” of the present invention 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.
- the present invention also provides a nucleotide that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 8, or 10, or a complementary strand thereof, and has a length of at least 15 bases.
- a nucleotide is preferably a nucleotide that specifically hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 8, or 10, or a complementary strand thereof.
- the term "specifically hybridize” as used herein refers to a state in which hybridization with DNA encoding another protein is carried out under ordinary hybridization conditions, preferably under stringent hybridization conditions. It means that no hybridization occurs significantly.
- nucleotides include probes and primers capable of specifically hybridizing with the DNA encoding the protein of the present invention or its complementary strand, nucleotides or nucleotide derivatives (for example, antisense oligonucleotides and ribozymes). .
- the present invention also provides a method for screening for a compound that binds to the protein of the present invention, using the protein of the present invention.
- the protein of the present invention used for screening may be any of a recombinant type, a natural type and a partial peptide.
- purified proteins and their partial peptides Or a form expressed on the cell surface or a form as a membrane fraction.
- This screening method comprises: (a) a step of bringing a test sample into contact with the protein of the present invention or a partial peptide thereof; (b) a step of selecting a compound having an activity of binding to the protein of the present invention or a partial peptide thereof; including.
- the test sample used in this screening method is not particularly limited, and examples include a cell extract, a cell culture supernatant, a protein, a peptide, and a synthetic low-molecular compound.
- the protein of the present invention that makes a test sample come into contact with the test sample can be, for example, a purified protein, a form expressed on a cell membrane, or a cell membrane fraction.
- a method for isolating a ligand to the protein using the protein of the present invention many methods known to those skilled in the art can be used.
- a cDNA library using a phage vector eg, gtll, ZAP, etc.
- the expressed protein is fixed, the protein of the present invention is purified as a fusion protein with a biotin label or a GST protein, and the purified protein is reacted with the above-mentioned filter, and a plaque expressing the protein to be bound is obtained.
- a cDNA library is prepared that can be expressed in the form of a cell, and is introduced into the yeast cells described above.
- the cDNA derived from the library is isolated from the positive clones detected and introduced into E. coli for expression.
- “Two hybrid system” (“ MATCHMARKER Two-Hybrid Systemj, "Ma thigh alian MATC HMAKER Two-Hybrid Assay Kitj,” MATC thigh KER One-Hybrid Systemj (all manufactured by clontech), "HybriZAP Two-Hybrid Vector Systemj (stratagene), literature”
- screening for a ligand that specifically binds to the protein of the present invention comprises screening the extracellular domain of the protein of the present invention and the intracellular domain containing the transmembrane domain of a receptor protein such as a hemopoietin receptor having a known signal transduction ability. And expressed on the cell surface of an appropriate cell line, preferably a cell line that can survive and grow only in the presence of an appropriate growth factor (growth factor-dependent cell line). Thereafter, the cell line can be cultured by adding a material expected to contain various growth factors, cytodynamic factors, hematopoietic factors, and the like.
- This method utilizes the fact that the above-mentioned growth factor-dependent cell line can survive and proliferate only when the test material contains a ligand that specifically binds to the extracellular domain of the protein of the present invention.
- Known hemopoietin receptors include, for example, thrombopoietin receptor, erythropoietin receptor, G-CSF receptor, gpl30, and the like.Partners of the chimeric receptor used in the screening system of the present invention include those known The receptor is not limited to the hemopoietin receptor, and any receptor having a structure necessary for signal transduction activity in the cytoplasmic domain may be used.
- the 0X40 receptor belonging to the same TNF receptor superfamily as the protein of the present invention can be selected as a partner of the chimeric receptor (Mallet, S. et al., EMB 0 J., 19, 1063). -1068 (1990)). Since the 0X40 receptor can transmit a positive signal into cells, screening for ligands using proliferative response etc. as an index (Baum, PR et al., EMBO J. 5 13, 3992-4001 (1994)). Proliferation factors As resident cell lines, for example, IL3-dependent cell lines such as BaF3 and FDC-P1 can be used.
- the protein of the present invention is expressed in a cell that does not express the ligand, and then an expressed cDNA library constructed from cells that are expected to express the ligand is expressed in the cell.
- “Direct expression cloning method” in which the culture supernatant obtained by introduction into cells is added, and ligands are searched based on the functional or morphological changes of the cells (Yokota T, Otsuka T, Mosmann II, Banchereau J, DeFrance T, Blanchard D, De Vries JE, Lee F, and Arai K.
- the culture supernatant of cells expected to express the ligand of the present invention is placed on an affinity column on which the protein of the present invention is immobilized, and the protein that specifically binds to the column is purified.
- the DNA encoding the ligand can be obtained by analyzing the amino acid sequence of the obtained protein (ligand), synthesizing oligo DNA based on the amino acid sequence, and screening a cDNA library using the DNA as a probe. It is possible.
- an antibody-like chimeric protein can be used as an agonist.
- Methods for isolating an agonist and an engonist against the protein using the protein of the present invention include, for example, a compound, a natural product bank, or a random phage in the immobilized protein of the present invention.
- Screening for binding molecules using peptide display libraries and high-throughput screening methods using combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mu lcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (UNITE D STATES) Jul 26 1996, 273 p458-64, Verdine GL., The Nature (ENGLAND) Nov 7 1996, 384 pi 13, Hogan JC Jr., Di rected combinatorial chemistry.Nature (ENGLAND) Nov 7 1996, 384 pl 7-9) are known to those skilled in the art.
- the present invention also provides a method for screening for a compound that regulates the activity of the protein of the present invention.
- This screening method comprises the steps of: (a) covering cells containing a vector that expresses a DNA encoding the protein of the present invention and a vector that expresses a repo allele in response to binding of NF-KB; Contacting a test sample, (b) detecting the repo overnight activity in the cells, and (c) comparing the repo activity to the test sample without contacting the cells. Decrease or increase the activity overnight Selecting a compound.
- pCOSl or pME18 can be used as a vector for expressing DNA encoding the protein of the present month.
- a vector that expresses a reporter gene in response to activation of NF-kB for example, kB-luc described in Examples can be used.
- kB-luc expresses a reporter gene according to the following principle. That is, when a signal is transmitted to the protein of the present invention, kinases downstream thereof are activated via TRAF (TNFR associated factor). This kinase activates the IkB kinase complex and phosphorylates the inhibitory protein IkB bound to NF-kB. Then, IkB is degraded, and NF-kB is released and translocated into the nucleus. When this NF-kB binds to the binding site on kB-luc, the repo overnight gene is expressed accordingly.
- TRAF TNFR associated factor
- the HEK293T cell line is preferable.
- Introduction of a vector into a cell can be performed by a method known to those skilled in the art.
- a test sample is brought into contact with cells into which a vector has been introduced, and the repo overnight activity is detected.
- the test sample is not particularly limited, and includes, for example, a cell extract, a cell culture supernatant, a protein, a peptide, and a synthetic low-molecular compound. It is also conceivable to use a compound isolated by screening for a compound that binds to the protein of the present invention.
- the test sample used inhibits the activity of the protein of the present invention if the repo overnight activity is lower than when the test sample is not brought into contact with the cells.
- the reporter activity is increased, it is determined that the test sample used induces or increases the activity of the protein of the present invention.
- the ligand, agonist or aminogonist isolated by the screening of the present invention can be applied to the treatment of a disease associated with the protein of the present invention (for example, a nerve-related disease).
- Compound isolated by the screening method of the present invention When used as a drug, it can be formulated into a drug by a known pharmaceutical manufacturing method. For example, it is administered to a patient together with a pharmacologically acceptable carrier or vehicle (eg, saline, vegetable oils, suspensions, surfactants, stabilizers, etc.). Administration will be transdermal, intranasal, transbronchial, intramuscular, intravenous, or oral, depending on the nature of the compound. The dose varies depending on the patient's age, body weight, symptoms, administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
- a pharmacologically acceptable carrier or vehicle eg, saline, vegetable oils, suspensions, surfactants, stabilizers, etc.
- Administration will be transdermal
- the dosage of the compound having the binding activity to the protein of the present invention may vary depending on the condition. It is believed to be 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
- the single dose varies depending on the subject of administration, target organ, symptoms, and administration method.
- parenteral injections usually for adults (with a body weight of 60 kg)
- the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.
- FIG. 1 shows the results of analyzing the ability of mouse Troy and dTroy to induce NF-kB activity.
- Example 1 Mouse brain cDNA library by signal sequence trap (SST) method Bally Screening
- BstXI adapter-1 Invitrogen was added, and a cDNA of 400 bp or more was fractionated using a SizeSep 400 Spun Column (Pharmacia). Separately, the mixture was mixed with BstXI (Takara Shuzo) and pMXGM (-) v-mpl M2 (see Japanese Patent Application No.
- Plasmids extracted from the recombinant Escherichia coli were purified using a JETstar column (GEN0M ED). Acad. Sci. USA, vol. 90: 8392-8396, 1993) was used to transfect a plasmid cDNA library using LipofectMINE (LIFE TECHNOLOGIES).
- B0SC23 is seeded on a 6 cm dish (Corning) with Dulbecco's Modified Eagel Medium (DM EM, LIFE TECHNOLOGIES) containing 10% fetal calf serum (FCS, JRH BIOSCIENCES), and after 16 hours, washed with DMEM and washed first.
- DM EM Dulbecco's Modified Eagel Medium
- FCS fetal calf serum
- JRH BIOSCIENCES fetal calf serum
- IL-3 Mouse interleukin-3
- 10 g / ml hexadimethrine bromide were added to the culture supernatant containing the recombinant virus, and Ba / F3 cells were suspended and infected with the mixture. Twenty-four hours after the infection, the cells were washed three times with a phosphate buffer, and cultured with RPMI1640 containing 10% FCS.
- a chromosomal DNA was extracted from a clone grown in the absence of IL-3, and a primer (5, -gggggtggaccatcctcta-3 '/ SEQ ID NO: 3, which was set so as to sandwich the cDNA insertion site, And 5, -cgcgcagctgtaaacggtag-3, / SEQ ID NO: 4), and a cDNA fragment was recovered by PCR.
- PCR 500 ng chromosomal DNA, 500 pM each primer, TaKaRa LA Taq (Takara Shuzo) 2.5 units, 2.5 mM MgCl 2 , 0.3 mM dNTPs and enzyme attached buffer went.
- a mouse brain cDNA library was synthesized using oligo dT primers, and the above cDNA fragment was screened as a probe.
- 0.1 ⁇ g of the DNA fragment previously obtained by the SST method was converted into type III and its internal primer (50 pmoles) (55S; 5, one caaggtcctacctctacaca—3 // Rooster column numbers: 6, and 511A; 5′-aaggtt caccttgctggtac-3, / SEQ ID NO: 7), 2.5 mM MgCl 2 , 0.2 mM dATP, dGTP, dCTP ⁇ 0.
- a cDNA fragment obtained using a mouse Multiple Tissue Northern (MTN) Blot was hybridized to a probe, A strong signal was observed in the brain, a weak signal was observed in the heart 'lung' and liver, and an extremely weak signal was also observed in the skeletal muscle and kidney 'testes', suggesting that the receptor was expressed specifically in the brain.
- MTN Multiple Tissue Northern
- Example 4 A clone containing almost no intracellular domain “Isolation of mouse dTroyj” A skin cDNA library of a mouse embryo on day 17 was constructed using a random hexamer as a primer, and the extracellular domain of mouse cDNA was constructed. Using the portion as a probe, clones were isolated by the RecA method, and as a result, a clone dTroy containing almost no intracellular domain was isolated, and the nucleotide sequence of the dTroy cDNA was represented by SEQ ID NO: 8, and the cDNA was coded. The amino acid sequence of the protein is shown in SEQ ID NO: 9.
- a human gliosarcoma cell line (gliosarcoma) GI-1 expression of a human homologous molecule of the mouse Troy gene was observed in a Northern plot. Therefore, a cDNA library derived from the human glial sarcoma cell line GI-1 was constructed using random hexamers as primers, and the RecA method was performed using the extracellular domain portion of mouse cDNA as a probe. As a result, a human clone corresponding to the mouse Troy gene was isolated.
- the nucleotide sequence of human Troy cDNA is shown in SEQ ID NO: 10
- the sequence of the protein encoded by the cDNA is shown in FIG.
- the amino acid sequence is shown in SEQ ID NO: 11.
- TNFR-associated factor 2
- TNFR-associated factor 2
- FuGene6 FuGene6
- the repo overnight plasmid kB-luc contains five NF-kB binding sites upstream of the basic HTLV-1 promoter dN55 (cancer, 79, 800-804 (1988)) and a luciferase gene downstream. Having. After 24 hours, the cells were washed with PBS, suspended in 250 mM Tris buffer (PH7.8), and frozen and thawed three times to break the cells. Using 0-nitrophenyl -.- D-galactobyranoside as a substrate using the centrifuged supernatant as a sample, the galactosidase activity was measured as described in the literature (Molecular Cloning).
- luciferase activity was measured using Pitka Gene (manufactured by Toyo Ink). As a result, when mouse Troy was expressed compared to Mock, luciferase activity was induced 3.5 times or more, suggesting that NF-kB was activated by the receptor. On the other hand, expression of the short molecule dTroy hardly induced luciferase activity (Fig. 1). Industrial applicability
- TNF receptor-like protein and its gene have been provided. These proteins and genes can be used as useful tools for purifying and cloning new factors involved in brain function and skin development, and for screening drug candidate compounds.
Abstract
Description
Claims
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AU25742/00A AU2574200A (en) | 1999-02-19 | 2000-02-18 | Novel tnf receptor-like proteins |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001038526A1 (en) * | 1999-11-23 | 2001-05-31 | Glaxo Group Limited | Human tnf receptor |
JP2006230356A (en) * | 2005-02-28 | 2006-09-07 | Chugai Pharmaceut Co Ltd | Transgenic non-human animal inhibiting troy signal |
US7223852B2 (en) | 1997-09-12 | 2007-05-29 | Biogen Idec Ma Inc. | Nucleic acids encoding TRAIN-R: a cysteine rich member of the TNF-receptor family |
US7846438B2 (en) | 2004-08-03 | 2010-12-07 | Biogen Idec Ma Inc. | Methods of promoting neurite outgrowth with soluble TAJ polypeptides |
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2000
- 2000-02-18 AU AU25742/00A patent/AU2574200A/en not_active Abandoned
- 2000-02-18 WO PCT/JP2000/000940 patent/WO2000049149A1/en active Application Filing
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7223852B2 (en) | 1997-09-12 | 2007-05-29 | Biogen Idec Ma Inc. | Nucleic acids encoding TRAIN-R: a cysteine rich member of the TNF-receptor family |
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US7838639B2 (en) | 1997-09-12 | 2010-11-23 | Biogen Idec Ma Inc. | Antibodies to train R: a cysteine-rich member of the TNF-receptor family, and methods of treating tumors expressing said receptor |
WO2001038526A1 (en) * | 1999-11-23 | 2001-05-31 | Glaxo Group Limited | Human tnf receptor |
US7846438B2 (en) | 2004-08-03 | 2010-12-07 | Biogen Idec Ma Inc. | Methods of promoting neurite outgrowth with soluble TAJ polypeptides |
JP2006230356A (en) * | 2005-02-28 | 2006-09-07 | Chugai Pharmaceut Co Ltd | Transgenic non-human animal inhibiting troy signal |
Also Published As
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WO2000049149A9 (en) | 2001-03-01 |
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