WO2002101034A1 - Leptin-induced gene - Google Patents
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- WO2002101034A1 WO2002101034A1 PCT/JP2002/002766 JP0202766W WO02101034A1 WO 2002101034 A1 WO2002101034 A1 WO 2002101034A1 JP 0202766 W JP0202766 W JP 0202766W WO 02101034 A1 WO02101034 A1 WO 02101034A1
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Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a gene induced by Leptin. Background art
- Leptin is a cytokine isolated by positional cloning from ob / ob mice, which are obesity disease model mice.
- Ob / ob mice do not produce wild-type lebutin due to mutations in the lebutin gene, and exhibit phenotypes such as overeating, obesity, and type I diabetes.
- Administration of leptin to these mice ameliorates these symptoms, indicating that leptin acts in regulating the energy balance in vivo.
- Leptin is produced by mature adipocytes. Its blood volume decreases during fasting (hunger) and conversely increases upon eating. Like other cytokines, lebutin transmits its signal into cells via a unique receptor. Leptin receptors have been shown to be present in various organs in the body. Among them, the part that seems to be directly involved in appetite control and energy balance control is the hypothalamus in the brain.
- lebutin rapidly activates a transcription factor called Stat3 in the hypothalamus.
- the administration of lebutin to ob / ob mice requires several weeks of treatment to improve the pathological symptoms.
- changes in the expression levels of various neuropeptides at the transcription level are observed by administration of lebutin. Specifically, a few days after the administration of lebutin, pro-opiomelanocortin (P0MC) increases, In addition, a decrease in neuropeptide Y (NPY) and agouti-related peptide (AGRP) is observed.
- P0MC pro-opiomelanocortin
- NPY neuropeptide Y
- An object of the present invention is to provide a novel leptin-responsive gene whose expression level changes in response to leptin administration, a protein encoded by this gene, and methods for producing and using them.
- the present inventors used ob / ob mice, which are obese because the signal pathway on the receptor side is normal but the synthesis of the ligand, levulin, cannot be performed normally. Since ob / ob mice respond to leptin administered from the outside, it is thought that the difference in gene expression caused by leptin administration can be increased.
- PCR-select subtrac from Clontech was used. A subtracted cDNA was prepared using the tion kit. Furthermore, to screen for those with truly differential expression, the insert of the subtracted cDNA was amplified by PCR, and this was arranged on a nylon membrane.
- a digoxigenin (DIG) -labeled DNA probe was used to detect rare cMA.
- DIG digoxigenin
- the present invention relates to the following polynucleotides, proteins encoded by the polynucleotides, and screening methods using them.
- amino acids in the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 have an amino acid sequence in which substitution, deletion, insertion, and / or addition has been performed;
- [3] A vector into which the polynucleotide of [1] has been inserted.
- [5] a method for producing the protein according to [2], comprising the step of culturing the transformed cell according to [4] and collecting a protein or peptide expressed from the transformed cell or a culture supernatant thereof; .
- a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a continuous nucleotide sequence of at least 15 nucleotides complementary to a complementary strand thereof.
- [8] A method for evaluating the activity of regulating the expression of the polynucleotide according to [1], comprising the following steps.
- a pharmaceutical composition comprising at least one component selected from the group consisting of the polynucleotide according to [1], the protein according to [2], and the vector according to [3].
- a method for measuring the protein according to [2] and / or a partial peptide thereof in a biological sample comprising the following steps:
- kits for measuring the protein according to [2] and / or a partial peptide thereof, comprising the antibody according to [6].
- a method for measuring the polynucleotide according to [1] in a biological sample comprising the following steps.
- the present invention provides a novel leptin-inducible gene.
- the nucleotide sequence of the leptin-inducible gene of the present invention is shown in SEQ ID NO: 1 (mouse) and SEQ ID NO: 3 (human), and the amino acid sequence of the protein encoded by the leptin-inducible gene of the present invention is represented by SEQ ID NO: 2 ( Mouse) and SEQ ID NO: 4 (human).
- ESTs AK002535, RIKEN full length cDNA project, cDNA from mouse kidney
- amino acid sequence predicted from its ORF were known. However, it was unknown whether it actually existed in living organisms as a gene product.
- the base sequence As a result of morphological search, only human homolog (GenBank Acc. No. Z84479) is found as a homologous gene. Moreover, the nucleotide sequence showing homology is an antisense sequence, and it is not possible to derive a correct nucleotide sequence and amino acid sequence. Furthermore, no known motif or the like could be confirmed in the amino acid sequence encoded by the lebutin-inducible gene of the present invention. Therefore, induction of these genes by lebutin is a novel finding provided by the present invention.
- the expression of the gene of the present invention is induced in response to lebutin, it is useful as an indicator of levulin responsiveness. That is, it can be used as an index for screening for a drug that alters leptin-dependent glycogen storage in hepatic hepatocytes that express the leptin receptor.
- immunochemical staining analysis confirmed that the expression of this gene was found in intertesticular cells (Lomme cells), and the administration of leptin inhibited the production of testosterone.
- leptin deficiency was responsible for obesity.
- obesity due to levulin deficiency is very rare, and blood levels of levutin increase with the degree of obesity in many obese patients. This fact indicates that obesity cannot be ameliorated by simply administering lebutin to humans with obesity.
- Such a state can be referred to as a state in which the signal of lebutin cannot be transmitted correctly, that is, a state of leptin resistance.
- the decrease in responsiveness to lebutin, rather than the administration of lebutin It is necessary to release the resistance.
- a therapeutic strategy for obesity can be selected based on the expression level of the gene of the present invention. According to the present invention, there is provided a method for diagnosing lebutin responsiveness, comprising the following steps.
- a polynucleotide comprising the coding region of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
- iii one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 having an amino acid sequence with substitution, deletion, insertion, and / or addition;
- the expression level of each polynucleotide can be evaluated by measuring any one of the expression intensity of the polynucleotide and the expression level of the protein encoded by each polynucleotide.
- the expression intensity of a polynucleotide having a given base sequence can be measured by using hybridization or PCR.
- the expression level of a protein having a given amino acid sequence can be measured by using an immunoassay or the like.
- the measured value of the expression level indicates the subject's responsiveness to lebutin. For example, when the measured value is low, it is determined that the responsiveness to lebutin is low.
- the leptin responsiveness is low. Can be considered low.
- the diagnostic method of the present invention more appropriate results can be expected by considering the leptin concentration in blood.
- the diagnostic method of the present invention is useful for evaluating levulin responsiveness in a subject receiving lebutin.
- a combination of elements necessary for carrying out the diagnostic method of the present invention can constitute a lebutin-responsive diagnostic kit.
- a kit for measuring the polynucleotide or protein of the present invention which will be described later, is useful as a diagnostic kit for responsiveness to lebutin.
- the gene of the present invention responds to leptin stimulation in a short time in the signal transduction pathway by leptin. It is known that the transcription of various neuropeptide genes such as P0MC, NPY, or AGRP is affected by administration of lebutin. However, changes in the transcription levels of these neuropeptides appear only a few days after leptin administration. On the other hand, the gene of the present invention responds rapidly to lebutin administration. That is, it can be considered that the gene of the present invention functions upstream of the above-described neurobeptide in the signal transduction pathway of lebutin. Therefore, elucidation of the relationship between this gene and these neuropeptides will lead to a clear picture of the lebutin signaling pathway.
- the gene of the present invention is a gene that responds promptly to lebutin, the relationship between the present gene and leptin resistance can be examined using the movement of the gene as an index. Furthermore, if there is a difference in the expression level of this gene according to the degree of obesity, it can be used as an indicator of obesity.
- the gene of the present invention can be used for antiobesity drug screening. That is, since the expression of the gene of the present invention increases in response to lebutin, a compound that increases the expression of the present gene in the presence of lebutin is used. Is expected to release lebutin resistance and suppress obesity. Since lebutin resistance is considered to be one of the important causes of obesity in humans, the gene of the present invention is an important target in developing therapeutics for obesity and elucidating the molecular biology of obesity.
- the present invention also relates to a substantially pure protein encoded by the polynucleotide according to any one of the above (a) to (e).
- a substantially pure protein means that the protein is substantially free of a large molecule derived from an organism in addition to the protein.
- a substantially pure protein in the present invention refers to a protein having a purity of at least 70% or more, usually 80%, preferably 90%, more preferably 95% or more, for example, 99%. To tell. Techniques for measuring protein purity are known. As a method for knowing the purity of the protein, for example, polyacrylamide gel electrophoresis, HPLC, or the like can be shown.
- the protein of the present invention can be prepared as a recombinant protein or as a natural protein.
- the recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention has been inserted into an appropriate host cell and purifying the protein expressed in the transformant, as described below. It is.
- a natural protein can be prepared, for example, using an affinity column to which an antibody against the protein of the present invention described later is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) ) Publish. John Wiley & Sons Section 16.1-16.19).
- the antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody.
- the protein of the present invention can be prepared by such methods.
- the present invention relates to the mouse or human-derived protein identified in this example.
- Functionally equivalent proteins are included.
- Such proteins include, for example, SEQ ID NO:
- variants, homologs, variants, etc. of the protein consisting of the amino acid sequence of 2 (mouse) or SEQ ID NO: 4 (human).
- “functionally equivalent” means that the target protein is induced to be expressed in vivo by administration of lebutin. C Such a function can be inferred by the disappearance or decrease of the expression.
- proteins functionally equivalent to the protein identified in this example for example, by introducing a mutation into an amino acid sequence in the protein (for example, by site-directed mutagenesis (Current Protocols). (1987) Publish. John Wiley & Sons Section 8.1-8.5)).
- such proteins may be produced by amino acid mutations in nature.
- one or more amino acids may be substituted or deleted in the amino acid sequence (SEQ ID NO: 2 or SEQ ID NO: 4) as long as it has a function equivalent to the protein identified in this example.
- the insertion and / or insertion include different proteins due to addition and the like.
- the number and location of amino acid mutations in proteins are not limited as long as their functions are maintained.
- the number of mutations is typically within 30 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids).
- the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution, from the viewpoint of maintaining the function of the protein. Substitution of an amino acid constituting a protein with an amino acid having similar properties is called conservative substitution. For example, Ala s Val, Leu, l ie ⁇ Pro, Met, Phe, Trp are all classified into non-polar amino acid, it is thought to have similar properties to each other.
- examples of non-charged ones include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin.
- acidic amino acids include Asp and Glu
- basic amino acids include Lys, Arg, and His.
- Proteins functionally equivalent to the proteins identified in this example are well known to those of skill in the art. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) or gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.
- polynucleotide encoding the protein identified in this example SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof as a probe, or specific to the polynucleotide.
- a polynucleotide that hybridizes with the polynucleotide can be isolated. Further, based on the isolated polynucleotide, a protein encoded by the polynucleotide can be prepared.
- the present invention includes proteins encoded by polynucleotides that hybridize with polynucleotides encoding these proteins, as long as they have a function equivalent to the proteins identified in the present examples.
- Organisms for isolating functionally equivalent proteins include, but are not limited to, for example, vertebrates such as rats, magpies, bushes, and magpies.
- the stringent conditions for hybridization to isolate a polynucleotide encoding a functionally equivalent protein are usually ⁇ lxSS 0.13 ⁇ 4SDS, 37 ° C ''. Is about 0.5xSSC, 0.1% SDS, 42 ° C, and the more severe condition is about 0.1xSSC, 0.1% SDS, 65 ° C. Isolation of a polynucleotide having high homology to the probe sequence can be expected.
- the combination of the SSC, SDS, and temperature conditions described above is merely an example, and those skilled in the art will recognize the above and other factors that determine the stringency of the hybridization (eg, probe concentration, probe length, The same stringency as described above can be realized by appropriately combining the hybridization reaction time and the like.
- the isolated protein usually has higher homology in its amino acid sequence as compared to the protein of the present invention described in SEQ ID NO: 2 or SEQ ID NO: 4.
- High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, more preferably 90% or more (eg, 95% or more).
- Amino acid sequence homology can be determined by homology search using BLASTX.
- the present invention also provides a partial peptide of the protein of the present invention.
- the partial peptide of the protein of the present invention can be used, for example, for preparing an antibody that binds to the protein of the present invention.
- an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and specifically found in the amino acid sequence is useful as an immunogen for obtaining an antibody that binds to the protein of the present invention. It is.
- An amino acid sequence that is specifically found refers to a contiguous amino acid sequence that is different from the amino acid sequences that constitute other proteins.
- the other protein refers to a protein having an amino acid sequence different from the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4.
- the partial peptide of the present invention has a continuous amino acid sequence of at least 7 amino acids, preferably at least 9 amino acids, more preferably at least 12 amino acids, and more preferably at least 15 amino acids.
- the partial peptide of the present invention can be produced, for example, 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 polynucleotide encoding the protein of the present invention.
- the polynucleotide of the present invention is not particularly limited in its form as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, MA and the like in addition to cDNA.
- a polynucleotide having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included as long as it can encode the protein of the present invention.
- the polynucleotide encoding the protein of the present invention can be obtained by the hybridization method using the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof as a probe. It can be isolated by a conventional method such as a gene amplification method (PCR) using primers designed based on the information of these nucleotide sequences.
- PCR gene amplification method
- the polynucleotide of the present invention includes an isolated polynucleotide.
- the polynucleotide isolated in the present study refers to a polynucleotide structurally different from a naturally occurring polynucleotide or a fragment thereof.
- Specific examples of the isolated polynucleotide include the following polynucleotides.
- Isolated DNA including cDNA, genomic fragment, restriction enzyme fragment, or PCR product
- the present invention also provides a vector into which a polynucleotide encoding the protein of the present invention has been inserted.
- the vector of the present invention is not particularly limited as long as it stably retains the inserted polynucleotide.
- Escherichia coli is used as a host
- a pBluescript vector manufactured by Stratagene
- an expression vector is particularly useful. Examples of expression vectors include PBEST vector (Promega) for expression in vitro, pET vector (Invitrogen) for expression in E. coli, and PME18S for expression in cultured cells.
- -FL3 vector (GenBank Accession No. AB009864) and PME18S vector (Mol Cell Biol. 8: 466_472 (1988)) can be suitably used for expression in living organisms. Insertion of a polynucleotide encoding the protein of the present invention into a vector can be carried out by a conventional method, for example, a ligase reaction using a restriction enzyme site (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish) John Wiley & Sons. Section 11.4_11. 11).
- the present invention also provides a transformant carrying a polynucleotide encoding the protein of the present invention or a vector into which the polynucleotide has been inserted.
- the host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention. Production systems for protein production include in Wiro and i / j production systems. Examples of the in F / iro production system include a production system using eukaryotic cells and a production system using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts.
- the host cells of the present invention also include cells used for analyzing the function of a lebutin-induced protein and screening for a function inhibitor or a function promoter using the lebutin-induced protein.
- Vector introduction into host cells can be performed, for example, by calcium phosphate precipitation, electroless breeding (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.Section 9.1-9.9), It can be performed by a method such as the ribofect-min method (GIBC0-BRL) or the microinjection method.
- Preparation of a lebutin-derived protein from a transformant can be carried out by a protein separation / purification method known to those skilled in the art.
- the present invention also provides a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a polynucleotide comprising a continuous nucleotide sequence of at least 15 bases complementary to a complementary strand thereof.
- the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T, G: C base pairs with respect to the other strand.
- complementary is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably Should have at least 95% homology on the base sequence.
- the algorithm for determining homology may use the algorithm described in this specification.
- Such a polynucleotide detects DNA encoding the protein of the present invention, It can be used as a probe for isolation and as a primer for amplifying the polynucleotide of the present invention.
- a primer When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp.
- a polynucleotide having at least a part or the entire sequence of the polynucleotide of the present invention and having a chain length of at least 15 bp is used.
- the 3'-side region needs to be complementary, but a restriction enzyme-recognition sequence or evening fragment can be added to the 5'-side.
- the polynucleotide of the present invention includes an antisense for suppressing the expression of the lebutin-inducing protein of the present invention.
- the antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect.
- Such an antisense can be obtained, for example, by the phosphorothione method (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. 1 (1988)).
- the polynucleotide of the present invention can be applied to, for example, gene therapy.
- a disease targeted for gene therapy using the polynucleotide of the present invention for example, obesity and diabetes are suitable. That is, when lebutin resistance has been formed due to mutation or abnormal expression of the gene of the present invention, there is a possibility that lebutin resistance can be released by expressing the gene of the present invention in vivo.
- a non-viral vector such as a retroviral vector, an adenovirus vector, an adeno-associated virus vector, a non-viral vector such as a liposome, etc. It may be administered to patients by the or in'O method.
- the antisense of the polynucleotide of the present invention is useful for constructing a model of lebutin resistance.
- the dominant negative of the polynucleotide of the present invention When the polynucleotide of the present invention causes obesity due to a severe mutation, suppression of the expression of the polynucleotide of the present invention can be used as a leptin resistance model.
- 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 polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. In addition, antibodies of all classes are included.
- the antibodies of the present invention also include special antibodies such as humanized antibodies.
- the antibody of the present invention can be obtained by a known method.
- a polyclonal antibody it can be obtained by immunizing a rabbit with an immunogen according to a conventional method (Current protocols in Molecular Biology edit.
- a mouse in the case of a monoclonal antibody, a mouse can be immunized with an immunogen according to a conventional method and obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells (Current protocols in Molecular Biology edit). . Ausubel et al. (1987) Publ ish.
- an oligopeptide comprising an amino acid sequence selected from the amino acid sequence of the present invention, and a recombinant protein obtained by expressing and purifying the gene of the present invention in Escherichia coli or the like can be used.
- an amino acid sequence conserved between SEQ ID NO: 2 and SEQ ID NO: 4 is used as an amino acid sequence constituting an oligopeptide, an antibody that can recognize the protein of the present invention across species can be obtained. .
- an oligopeptide comprising an amino acid sequence different between the two sequences is used as an immunogen, a species-specific antibody can be obtained.
- Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of these proteins, in addition to the purification of the protein of the present invention.
- proteins are extracted from tissues, blood, cells, or the like, and the proteins of the present invention are detected by methods such as Western blotting, immunoprecipitation, and ELISA.
- the presence or absence of abnormal expression or structure can be examined and diagnosed.
- Antibodies that bind to the protein of the present invention may be used for the purpose of treating diseases related to the protein of the present invention.
- a human antibody or a humanized antibody is preferred because of its low immunogenicity.
- Human antibodies are obtained by replacing the immune system with that of a human mouse (for example, "" Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146-156 ”).
- a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).
- the present invention also provides a method for evaluating the activity of regulating the expression of the polynucleotide of the present invention.
- the method includes the following steps (a) and (b).
- the method for evaluating the activity of regulating the expression of the polynucleotide of the present invention is useful for a method of screening for a compound having this activity. That is, the present invention relates to a method for screening a compound having an activity of regulating the expression of the polynucleotide of the present invention, comprising the following steps.
- a cell expressing the gene of the present invention used in the screening method of the present invention for example, a cell line derived from kidney tubule or liver hepatocytes can be used.
- a specific method of screening in the presence of the test compound Lebutin is brought into contact with the cells. After culturing for a certain period of time, the change in the expression level of the gene of the present invention is measured and compared with the expression level in a control cultured by contact with leptin in the absence of the test compound.
- the expression level of a gene can be measured using the amount of the gene or protein as an expression product as an index.
- the gene can be quantified by a known method such as quantitative PCR.
- the amount of protein is measured by a technique such as ELISA.
- test sample used for screening Any compound can be used as the test sample used for screening.
- Specific examples include cell extracts, expression products of gene libraries, synthetic low-molecular compounds, synthetic peptides, and natural compounds.
- Compounds isolated by this screening are candidates for compounds that regulate responsiveness to lebutin.
- it is a candidate for a compound that inhibits the interaction between the protein of the present invention and a molecule that interacts with the protein of the present invention in vivo.
- the gene of the present invention its protein, a compound that regulates the expression of the gene, or a compound that regulates the activity of the protein
- the drug itself can be used as a drug
- it may be used in the form of a formulation by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like.
- Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection.
- the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
- the polynucleotide may be incorporated into a vector for gene therapy and administered to a patient.
- the dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but those skilled in the art will be able to select as appropriate.
- the expression level of the gene can be determined by measuring the transcription product mMA, the translation product protein, and fragments thereof.
- the present invention Provides a method for measuring the following polynucleotide, or a method for measuring a protein and / or a partial peptide thereof, and a kit for the method.
- the polynucleotide of the present invention can be measured by hybridization with a probe having a complementary sequence to the base sequence shown in SEQ ID NO: 1.
- the expression level of the gene of the present invention can also be measured by a PCR method using an oligonucleotide consisting of at least 15 bases complementary to the base sequence shown in SEQ ID NO: 1 and its complementary strand as a primer. .
- the RT-PCR method using mRNA as type II is also known.
- the protein that is a translation product of the gene of the present invention can be measured based on the immunoassay principle using an antibody that recognizes the protein of the present invention.
- the method for obtaining an antibody that recognizes the protein of the present invention is as described above.
- kits comprising a primer for performing RT-PCR using mRNA as a type III, reverse transcriptase, DNA polymerase, and a buffer suitable for the reaction.
- a kit comprising a labeled antibody recognizing the protein of the present invention, a reagent for measuring the label, and a buffer suitable for an immune reaction can also be constituted.
- the present invention also provides a non-human vertebrate engineered to alter the expression of the protein of the present invention.
- alteration of expression includes enhancement and attenuation of expression.
- Modification of protein expression includes modification of both transcription and translation steps.
- Such non-human vertebrates include animals that have been engineered to stop or reduce expression of the endogenous protein of the invention (knockout animals) and those that express the exogenous protein of the invention. Includes animals (transgenic animals) into which the coding gene has been introduced. Such knockout and transgenic non-human vertebrates are described in the literature "Neuroscience 'Lab Manual.
- protein translation can also be modified by deleting a part of the exon or replacing it with a stop codon by introducing a point mutation into the translation region.
- the expression of the gene of the present invention can be controlled by expressing antisense RNA or ribozyme. Introduction of these mutations can be performed by a known method.
- Such non-human vertebrates are useful for studying transcriptional functions, elucidating the mechanisms of transcription-related diseases, and developing disease model animals used for drug screening and the like.
- the gene of the present invention is considered to be a gene constituting the upstream of the signal transduction pathway by lebutin. Therefore, animals and cells in which the expression of the gene of the present invention has been suppressed are useful for screening for a drug that releases lebutin resistance. Among them, embryonic stem cells (embryonic stem eel 1; ES cells) lacking the expression of the gene of the present invention are useful, for example, for producing knockout animals in which the expression of the gene is suppressed.
- embryonic stem cells embryonic stem eel 1; ES cells
- a known method can be applied to suppress the expression of the gene of the present invention in a non-human animal.
- exons that constitute the protein coding region particularly the exons that constitute the protein coding region in the nucleotide sequence shown in SEQ ID NO: 1 are mapped on the nucleotide sequence of the genome, and a mutation is introduced into at least a part of the nucleotide sequence to obtain a protein.
- a stop codon is generated in the exon, for example, by substitution, deletion, addition, or insertion of a part of the exon base sequence.
- Homologous recombination techniques are used to introduce mutations in the genome sequence.
- a knockout mouse can be obtained by using the thus constructed ES cells in which the expression of the gene of the present invention has been deleted.
- the ES cells are first injected into the blastocyst stage of the mouse and transplanted into the uterus of the foster parent.
- the egg injected with the ES cells produces a chimeric mouse (Faunda-1), which consists of the original fertilized egg and cells derived from the ES cells. If the genome that has been knocked out can be confirmed in the offspring produced by crossing Huawunda with wild mice, the offspring is a knockout animal that has the knocked-out gene heterozygously. By crossing heterozygous offspring further, a knockout mouse (homo) can be obtained.
- the present inventors have shown that the expression of the gene of the present invention in mice can be almost completely suppressed by deleting the region including the initiation codon in the protein coding region of the nucleotide sequence shown in SEQ ID NO: 1. confirmed. Therefore, as a non-human animal in which the expression of the gene of the present invention is suppressed, a knockout animal having a mutation in the initiation codon of the gene of the present invention is preferable. Alternatively, as a cell in which the expression of the gene of the present invention is suppressed, a cell having a mutation in the initiation codon of the gene of the present invention is preferable. Mutations in the initiation codon can be caused by base substitutions, deletions, additions, or insertions.
- FIG. 1 is a photograph showing the results of Northern blot analysis of induction of the gene of the present invention in the hypothalamus of ob / ob mice by administration of lebutin.
- A9 is the gene of the present invention
- BRI is a control.
- the lane marked with levbutin (-) shows the results when PBS was administered, and the (+) shows the results when mouse lebutin was administered.
- FIG. 2 is a photograph showing the results of Northern blot analysis of the tissue distribution of the gene of the present invention. Each lane corresponds to the tissue shown in the figure.
- FIG. 3 is a photograph showing the result of Western blot analysis of the protein of the present invention. Each lane corresponds to the following proteins or cell extracts.
- Lane 1 purified protein-GST fusion protein of the present invention (immunogen) 5 ng
- Lane 2 purified protein-GST fusion protein of the present invention (immunogen) 15 ng
- Lane 3 10 zg mouse kidney whole cell extract
- Lane 5 C0S7 cells overexpressing the gene of the present invention (approximately ag of whole cell extract)
- FIG. 4 shows the results of comparing the amino acid sequences of the protein of the present invention between mouse and human.
- the mouse amino acid sequence is shown above and the human amino acid sequence is shown below.
- FIG. 5 is a photograph showing the results of immunohistochemical analysis of human liver tissue. A (top) is 1 5
- B (bottom) is 300 times.
- FIG. 6 is a photograph showing the results of immunohistochemical analysis of human kidney cortical tissue. A (top) is
- B (bottom) is 300 times.
- FIG. 7 is a photograph ( ⁇ 300) showing the results of immunohistochemical analysis of human adrenocortical tissue.
- FIG. 8 is a photograph ( ⁇ 300) showing the results of immunohistochemical analysis of human testis tissue.
- FIG. 9 is a photograph ( ⁇ 300) showing the results of immunohistochemical analysis of human gastric epithelial tissue.
- FIG. 10 is a set of photographs and photographs showing the steps of constructing ES cells in mice that suppress the expression of the levulin-inducible gene of the present invention. Genomic DNA with the best targeting vector
- Ob / ob mice have normal signaling pathways on the receptor side, but exhibit obesity due to inability to synthesize ligand leptin normally.
- leptin-induced genes can be found. It was thought that such a condition could increase the difference in gene expression caused by the presence or absence of lebutin administration.
- the subtraction method was used to obtain genes with different expression levels. The specific operation is as follows.
- mice were purchased from the Jackson Laboratory, USA. 1 g of mouse lebutin (R & D) or PBS was administered by intravenous injection, and 1 hour later, the hypothalamus was collected, and total RNA was extracted by the guanidine monothiocyanate method. Then, NA was purified using an Oligo d T cell lulose column from Roche. The PCR subtraction method was performed using this mMA.
- cDNA was synthesized from 2 ⁇ g of each mRNA using AMV reverse transcriptase.
- the cDNA after administration of lebutin was used as a tester, and the cDNA after administration of PBS was used as a driver.
- the cDNA was digested with Rsal and fragmented. The Rsal fragment has blunt ends.
- first hybridization an excess amount of driver cDNA was added to each tester, heat denaturation was performed at 98 ° C for 1.5 minutes, and then incubation was performed at 68 ° C for 8 hours.
- second hybridization samples obtained from the first hybridization were mixed without denaturation, and a denatured cDNA driver was added thereto, followed by incubation at 68 ° C. After the reaction, the hybridization solution was diluted with a buffer for dilution, and incubated at 75 ° C. for 5 minutes to extend a part of the single-stranded adapter into a double-stranded DNA, which was used for PCR type II.
- PCR was performed using PCR primer 1, which is a primer corresponding to the adapter, to selectively amplify only cDNAs having different adapters at both ends. At this time, since no adapter is bound to the end of the driver cDNA, the tester cDNA hybridized with the driver cDNA is not amplified.
- Nested PCR primers 1 and 2R which are primers inside the adapter primers 1 and 2R, to obtain a product with further increased selectivity.
- This product was cloned into pT-Adv (Clontech) vector, Transform using BRL's E1 ctroMaxDHlOB cell and extract the subtraction library. Created.
- a cDNA probe incorporating DIG-dUTP was prepared by a reverse transcriptase reaction.
- the preparation was performed according to Roche's 'The DIG system user's guide for filter hybridization PP20-21, Preparing cDNA with digoxigenin-ll-dUTP'.
- the product was dissolved in a standard hybridization buffer (5x SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 1% blocking reagent ⁇ Roche 3 ⁇ 4 Blocking Reagen t ⁇ ) at a concentration of 51 O ng / ml, and hybridized. Used for one shot. From the subtraction library obtained using the mRNA after PBS-administration as a driver, 384-well, 3 colonies (total of 1152 clones) were selected arbitrarily.
- the cDNA inserted into the pT-Adv vector was amplified by PCR from these E. coli stocks and spotted as duplicates on Amersham-Pharmacia's positively charged Nylon membrane (subtraction cDNA array). Four sets of these arrays were prepared, and each was hybridized with a DIG-labeled cDNA probe after administration of PBS- and lebutin at 68 ° C for 1 ⁇ . Thereafter, the plate was washed twice with a 2x SSC 0.1% SDS solution at room temperature for 5 minutes, and twice with an O.lx SSC, 0.1% SDS solution at 68 ° C for 20 minutes.
- cRNA was prepared using the inserts of the clones having a difference in the expression level on the array as a probe, and a Northern plot was performed to examine whether or not the expression level actually differs.
- a positive clone having a difference in the expression level was selected as a mouse leptin-inducible gene, and the nucleotide sequence of the insert was determined. Since this subtraction library had been cut with Rsal, full-length cDNA could not be obtained with this library. As a result of Northern blot analysis, the expression of this gene was found to be high in kidney and liver, so a cDNA library was prepared using mRNA from mouse kidney.
- a cDNA library was prepared using the Superscript PI asniid System of BRL (now In Vitrogen), and the cDNA was inserted into the pSPORTl vector. The library was screened using the previously identified gene fragment as a probe to obtain the full length.
- the nucleotide sequence of the mouse lebutin-inducible gene is described in SEQ ID NO: 1, and the amino acid sequence predicted from the open reading frame is described in SEQ ID NO: 2.
- Example 2 Gene induction in the hypothalamus of ob / ob mice with lebutin 1 g of mouse leptin (+) or PBS (-) per 1 g of body weight was administered to ob / ob mice via vein, and 1 hour later, thalamus The lower part was excised to extract Total MA and mMA. 0.4 ⁇ g of the extracted mRNA was separated on an agarose gel and transferred to a positively charged Nylon membrane of Amersham Pharmacia Co., Ltd.
- the DIG-labeled cRNA prepared from the full-length cDNA of the gene of the present invention was hybridized as a probe, and washed under stringent conditions (0.1 ⁇ SSC, 0.1% SDS, 68 ° C.). Thereafter, signals were detected using an alkaline phosphatase-labeled anti-DIG antibody and CDP-Star. The chemiluminescence signal was quantified using an ATTO CCD camera.
- Hybridization was performed using DIG-labeled cRNA of the gene of the present invention as a probe. Washing was carried out under very harsh conditions as in Example 2, and signal detection was carried out in the same manner. The results are shown in FIG.
- the size of the transcript is estimated to be 800-90 Obp. This size matches the length (827b) of the nucleotide sequence shown in SEQ ID NO: 1. Among the organs, expression was highest in the kidney, followed by strong expression in the liver. It was confirmed that the gene of the present invention was also expressed in wild-type mice.
- the specificity of the anti-A9 antibody and the presence of the protein encoded by the gene of the present invention were confirmed by Western blotting.
- the following proteins or cell extracts were used as antigens.
- the cell extract of C0S7 overexpressing the gene of the present invention was prepared as follows. That is, the gene of the present invention was introduced into pM18S having an SRa promoter, and transfected into C0S7 cells by the ribonucleoside method. On the third day after gene transfer, the cells were lysed with ⁇ buffer to obtain a cell extract.
- Kidney cortex (Figure 6) Gastric epithelium ( Figure 9)
- Adrenal cortex ( Figure 7)
- the results of the analysis are shown in Figs.
- the anti-A9 antibody was shown to recognize a protein encoded by the human levulin-inducible gene.
- the following functions were presumed for the lebutin-inducible gene in humans.
- a strong signal was observed in the liver hepatocyte (Fig. 5). It was considered that the protein of the present invention may be involved in lebutin-dependent glycogen storage.
- the localization of the protein of the present invention was also found in the globular layer of the adrenal cortex (FIG. 7).
- the globular layer of the adrenal cortex is a tissue involved in steroid hormone production, suggesting a relationship between lebutin and the steroid hormone-producing system.
- the localization of the protein of the present invention in intertesticular cells (Lomme cells) (FIG. 8) was considered to be a result supporting the inhibitory effect of the administration of lebutin on the male hormone production system.
- signals in gastric epithelial cells ( Figure 9) suggested that leptin controls digestive function.
- the localization of the protein of the present invention in cells was examined using an antibody and a confocal microscope. After immobilizing the cells in which the gene of the present invention was strongly expressed in Hela cells, using the same anti-A9 antibody as in Example 4 and a fluorescently labeled secondary antibody, The intracellular localization was analyzed. In the same preparation, the localization of calnexin, a marker protein of the endoplasmic reticulum, was compared. As a result, it was confirmed that the protein of the present invention was localized in the same portion as calnexin. From these results, it was clarified that the protein of the present invention was abundantly present in the endoplasmic reticulum.
- the gene of the present invention has no known motifs and has no homology to any known proteins. Therefore, the role of the gene-deficient mouse (knockout mouse) of the present invention in clarifying its physiological function is significant. If a knockout mouse can be obtained, the physiological function of the gene of the present invention can be clarified by observing a change in the phenotype in the mouse.
- genomic DNA of this gene was obtained in order to create knockout mice.
- a genomic library derived from Stratagene 129SvJ was screened using a probe designed based on the nucleotide sequence of the gene of the present invention.
- the probe used was a 29 mer DIG-labeled oligonucleotide. The base sequence of the probe is shown below.
- Probe sequence 5, -GGCTCTTTAGAGCAGCAGGACACCTGCTC-3 '(SEQ ID NO: 12)
- a positive clone was subcloned into the Notl site of the pKS (+) vector (Stratagene) and the 6-base recognition restriction enzyme was used to clone the gene of the present invention.
- Genomic mapping was performed. The mapping results are shown as “genomic DNA” in FIG.
- the Spel and Asp718 sequences were used for the 5 'arm of the evening targeting vector.
- the Notl-Scal site was used as the 3 'arm.
- the tk-Neo gene ("neogene" in Fig.
- tk-Neo gene was designed to replace the exon containing ATG (from Asp718 to Notl site). Evening target Fig. 10 shows the relationship between the structure of the vector and the genomic DNA.
- the evening-targeting vector was introduced into ES cells by electro-boration.
- One hundred and ten G418 resistant clones were selected, and Southern blotting was performed using the sequence (3 'side) outside the vector arm used as a probe. If the tk-Neo gene is correctly integrated on the genomic DNA of the gene of the present invention, the genomic DNA loses the BamHI site containing the start codon ATG. As a result, the pattern of restriction enzyme fragments by BamHI changes. In other words, as shown in Fig. 10, if two bands of 4.2 kbp and 8 kbp were observed when the 3 'probe was used, recombination was correctly performed by the targeting vector in the evening. It turns out that it is an ES cell.
- the gene of the present invention is useful as an indicator of a lebutin response.
- the expression of the gene of the present invention is rapidly induced by administration of lebutin. Therefore, by using the gene of the present invention as an index, it is possible to evaluate the degree of lebutin response of a cell or a living body.
- the gene of the present invention is useful as a diagnostic index for lebutin resistance. That is, by measuring the expression level of the gene of the present invention and comparing it with a normal level, the degree of lebutin resistance can be known. Since the expression level of the gene of the present invention increases in a short period of time in response to lebutin, it is desirable as an indicator of lebutin responsiveness.
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Abstract
A novel leptin-induced gene which is induced by administering leptin. Since this gene is located immediately downstream of leptin signal, leptin-resistance can be evaluated by monitoring this gene. Also, a drug capable of ameliorating leptin-resistance can be screened by using this gene as an indication.
Description
明細書 レブチン誘導遺伝子 Description Lebutin-inducible gene
技術分野 Technical field
本発明は、 レブチン（Leptin)によって誘導される遺伝子に関する。 背景技術 The present invention relates to a gene induced by Leptin. Background art
レプチンは肥満疾患モデルマウスである ob/ob マウスから positional cloni ngによって単離されたサイ トカインである。 ob/obマウスはレブチン遺伝子の 変異によって野生型のレブチンが産生されず、 過食、 肥満、 I I型糖尿病などの 表現型を呈する。 このマウスにレブチンを投与するとこれらの症状は改善される, このことから、 レプチンが生体内でエネルギーバランスの調節に働いていること が明らかである。 Leptin is a cytokine isolated by positional cloning from ob / ob mice, which are obesity disease model mice. Ob / ob mice do not produce wild-type lebutin due to mutations in the lebutin gene, and exhibit phenotypes such as overeating, obesity, and type I diabetes. Administration of leptin to these mice ameliorates these symptoms, indicating that leptin acts in regulating the energy balance in vivo.
レブチンは成熟脂肪細胞より産生される。 その血中量は絶食 （空腹） において 減少し、 反対に摂食により増大する。 他のサイ トカインと同様に、 レブチンはそ のシグナルを固有の受容体を介して細胞内へと伝達している。 レブチン受容体は 生体内のさまざまな臓器に存在することが明らかにされている。 その中で、 特に 食欲制御、 エネルギーバランスの調節に直接関わっていると考えられる部位は、 脳内視床下部である。 Leptin is produced by mature adipocytes. Its blood volume decreases during fasting (hunger) and conversely increases upon eating. Like other cytokines, lebutin transmits its signal into cells via a unique receptor. Leptin receptors have been shown to be present in various organs in the body. Among them, the part that seems to be directly involved in appetite control and energy balance control is the hypothalamus in the brain.
現在までにレブチン投与により、 Stat3と呼ばれる転写因子が視床下部におい て急速に活性化されるのが確認されている。 しかし現在のところ、 この活性化の 生理的意義は定かではない。 ob/obマウスにレブチンを投与して先の病的症状を 改善させるのには、 数週間に渡る投与が必要である。 さらにレブチン投与により 転写レベルで各種ニューロぺプチドの発現レベルの変化が観察される。 具体的に は、 レブチンの投与後数日してから、 pro- opiomelanocortin (P0MC)の上昇、
並びに neuropeptide Y (NPY)や agouti- related peptide (AGRP )の減少が観察 される。 このことから、 視床下部においてはレブチン投与により何らかの遺伝子 が活性化もしくは不活性化されて P0MC、 NPY, AGRPなどの転写調節、 ひいては 肥満、 I I型糖尿病などの解消へと導くカスケードが想定される。 そこで、 レブ チン投与後に活性化または不活性化される遺伝子を同定できれば P0MC、 NPY, AG RPなどへの転写のカスケードの解明につながる。 更に、 このカスケードを構成 する因子は、 肥満や I I型糖尿病などの新たな創薬の夕ーゲッ卜となることが考 えられる。 発明の開示 To date, it has been confirmed that the administration of lebutin rapidly activates a transcription factor called Stat3 in the hypothalamus. However, at present, the physiological significance of this activation is uncertain. The administration of lebutin to ob / ob mice requires several weeks of treatment to improve the pathological symptoms. In addition, changes in the expression levels of various neuropeptides at the transcription level are observed by administration of lebutin. Specifically, a few days after the administration of lebutin, pro-opiomelanocortin (P0MC) increases, In addition, a decrease in neuropeptide Y (NPY) and agouti-related peptide (AGRP) is observed. This suggests that in the hypothalamus, some genes are activated or inactivated by the administration of lebutin, leading to a cascade leading to the regulation of transcription of P0MC, NPY, AGRP, etc., and eventually obesity, type II diabetes, etc. Therefore, identification of genes that are activated or inactivated after administration of levulin will lead to elucidation of the cascade of transcription to P0MC, NPY, AGRP, and the like. Furthermore, the factors that make up this cascade are considered to be a target for new drug discovery such as obesity and type II diabetes. Disclosure of the invention
本発明の課題は、 レブチン投与に応答して発現レベルが変化する新規なレプチ ン応答性遺伝子、 この遺伝子によってコードされる蛋白質、 並びにそれらの製造 方法および用途を提供することにある。 An object of the present invention is to provide a novel leptin-responsive gene whose expression level changes in response to leptin administration, a protein encoded by this gene, and methods for producing and using them.
マウスなどの実験動物を用いてレブチンによって早期に誘導される遺伝子を取 得する試みは、 米国 Mil lenium pharmacuetical社で行われたが成功に到らな かった。 この原因として彼等は、 視床下部に存在する neuronが全てレブチン受 容体を有するものでなく、 レブチンに反応するものがあってもノイズに埋もれて しまうことを指摘している（White, D. W. Zhou, J. Strieker- Krongrad, A. Ge, P. Morgenstern, J. P. Dembski, M. Tartagl ia, L. A. 2000. Identification of leptin - induced transcripts in the mouse hypothalamus. Diabetes 49， pp 1443-1450)。 Attempts to obtain genes induced early by lebutin in laboratory animals such as mice have been unsuccessful at Millenium pharmacuetical, USA. As the cause, they point out that not all neurons present in the hypothalamus have lebutin receptors, and that some that respond to lebutin are buried in noise (White, DW Zhou, J. Strieker-Krongrad, A. Ge, P. Morgenstern, JP Dembski, M. Tartaglia, LA 2000. Identification of leptin-induced transcripts in the mouse hypothalamus. Diabetes 49, pp 1443-1450).
これに対して本発明者らは、 受容体側のシグナル経路は正常であるが、 リガン ドであるレブチンの合成が正常にできないため肥満になっている ob/obマウス を実験に用いた。 ob/obマウスは外部から投与したレブチンに反応することから、 レプチン投与の有無によってもたらされる遺伝子発現の差異を大きくできると考 えた。 また差異発現遺伝子を取得するため、 Clontech社の PCR-select subtrac
tion kitを使用して、 subtracted cDNAを作成した。 さらに真に発現差異のあ るものをスクリーニングするため、 subtracted cDNAの insertを PCRによって 増幅してこれをナイロン膜上に並べ、 希少 cMAを検出できるように、 ジゴキシ ゲニン（DIG)標識した DNAプローブを作成することによってレブチン誘導遺伝子 をクローン化することに成功した。 すなわち本発明は、 以下のポリヌクレオチド、 このポリヌクレオチドによってコ一ドされるタンパク質、 並びにそれらを用いた スクリーニング方法に関する。 On the other hand, the present inventors used ob / ob mice, which are obese because the signal pathway on the receptor side is normal but the synthesis of the ligand, levulin, cannot be performed normally. Since ob / ob mice respond to leptin administered from the outside, it is thought that the difference in gene expression caused by leptin administration can be increased. To obtain differentially expressed genes, PCR-select subtrac from Clontech was used. A subtracted cDNA was prepared using the tion kit. Furthermore, to screen for those with truly differential expression, the insert of the subtracted cDNA was amplified by PCR, and this was arranged on a nylon membrane. A digoxigenin (DIG) -labeled DNA probe was used to detect rare cMA. By constructing the gene, we succeeded in cloning the lebutin-inducible gene. That is, the present invention relates to the following polynucleotides, proteins encoded by the polynucleotides, and screening methods using them.
〔 1〕 下記 （a ) から （e ) のいずれかに記載のポリヌクレオチド。 [1] The polynucleotide according to any one of the following (a) to (e):
( a ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列からなる蛋白 質をコードするポリヌクレオチド。 (a) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
( b ) 配列番号： 1または配列番号： 3に記載の塩基配列のコード領域を含 むポリヌクレオチド。 (b) a polynucleotide comprising the coding region of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
( c ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列において 1若 しくは複数のアミノ酸が置換、 欠失、 挿入、 および/または付加したァミノ 酸配列を有し、 配列番号： 2または配列番号： 4に記載のアミノ酸配列から なる蛋白質と機能的に同等な蛋白質をコードするポリヌクレオチド。 (c) one or more amino acids in the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 have an amino acid sequence in which substitution, deletion, insertion, and / or addition has been performed; A polynucleotide encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4.
( d ) 配列番号： 1または配列番号： 3に記載の塩基配列からなるポリヌク レオチドとストリンジヱン卜な条件下でハイブリダィズし、 配列番号： 2ま たは配列番号： 4に記載のアミノ酸配列からなる蛋白質と機能的に同等な蛋 白質をコードするポリヌクレオチド。 (d) a protein which hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 and which comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A polynucleotide encoding a protein functionally equivalent to the above.
( e ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列からなる蛋白 質に特異的に見出される部分アミノ酸配列を含む蛋白質をコードするポリヌ クレオチド。 (e) A polynucleotide encoding a protein comprising a partial amino acid sequence specifically found in a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
〔2〕 〔1〕 に記載のポリヌクレオチドによりコードされる蛋白質。 [2] A protein encoded by the polynucleotide of [1].
〔3〕 〔1〕 に記載のポリヌクレオチドが挿入されたベクター。 [3] A vector into which the polynucleotide of [1] has been inserted.
〔4〕 〔1〕 に記載のポリヌクレオチドまたは 〔3〕 に記載のベクタ一を保持す
る形質転換細胞。 (4) the polynucleotide of (1) or the vector of (3) Transformed cells.
〔5〕 〔4〕 に記載の形質転換細胞を培養し、 該形質転換細胞またはその培養上 清から発現させた蛋白質またはペプチドを回収する工程を含む、 〔2〕 に記 載の蛋白質の製造方法。 [5] a method for producing the protein according to [2], comprising the step of culturing the transformed cell according to [4] and collecting a protein or peptide expressed from the transformed cell or a culture supernatant thereof; .
〔6〕 〔2〕 に記載の蛋白質に結合する抗体。 [6] an antibody that binds to the protein of [2];
〔7〕 配列番号： 1または配列番号： 3に記載の塩基配列からなるポリヌクレオ チドまたはその相補鎖に相補的な少なくとも 15塩基の連続した塩基配列を 含むポリヌクレオチド。 [7] A polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a continuous nucleotide sequence of at least 15 nucleotides complementary to a complementary strand thereof.
〔8〕 次の工程を含む、 〔1〕 に記載のポリヌクレオチドの発現を調節する活性 の評価方法。 [8] A method for evaluating the activity of regulating the expression of the polynucleotide according to [1], comprising the following steps.
( a ) 被験化合物の存在下で 〔 1〕 に記載のポリヌクレオチドを発現する細 胞にレブチンを接触させる工程、 および (a) contacting lebutin with a cell expressing the polynucleotide of (1) in the presence of a test compound, and
( b ) 前記ポリヌクレオチドの発現レベルを測定する工程、 (b) measuring the expression level of the polynucleotide,
〔9〕 次の工程を含む、 〔 1〕 に記載のポリヌクレオチドの発現を調節する活性 を有する化合物のスクリーニング方法。 [9] A method for screening a compound having an activity of regulating the expression of the polynucleotide according to [1], comprising the following steps.
( a ) 〔8〕 に記載の方法によって、 被験化合物の 〔1〕 に記載のポリヌク レオチドの発現を調節する活性を評価する工程、 および (a) evaluating the activity of the test compound to regulate the expression of the polynucleotide according to (1), by the method according to (8), and
( b ) 被験化合物非存在下でレプチンを接触させた対照と比較して前記ポリ ヌクレオチドの発現レベルを変化させる化合物を選択する工程 (b) selecting a compound that changes the expression level of the polynucleotide as compared to a control that has been contacted with leptin in the absence of the test compound
〔 1 0〕 〔1〕 に記載のポリヌクレオチド、 〔2〕 に記載の蛋白質、 および 〔3〕 に記載のベクタ一からなる群から選択された少なくとも 1つの成分を 含有する医薬組成物。 [10] A pharmaceutical composition comprising at least one component selected from the group consisting of the polynucleotide according to [1], the protein according to [2], and the vector according to [3].
〔 1 1〕 次の工程を含む、 生体試料中の 〔2〕 に記載の蛋白質、 および/または その部分べプチドの測定方法。 [11] A method for measuring the protein according to [2] and / or a partial peptide thereof in a biological sample, comprising the following steps:
( 1 )生体試料を 〔6〕 に記載の抗体と接触させる工程、 および (1) contacting the biological sample with the antibody according to (6), and
(2 ) 〔2〕 に記載の蛋白質、 および/またはその部分ペプチドに結合する、
〔6〕 に記載の抗体を検出する工程 (2) binds to the protein according to (2), and / or a partial peptide thereof, Step of detecting the antibody according to [6].
〔1 2〕 〔6〕 に記載の抗体を含む、 〔2〕 に記載の蛋白質、 および/またはそ の部分べプチドの測定用キット。 [12] A kit for measuring the protein according to [2] and / or a partial peptide thereof, comprising the antibody according to [6].
〔1 3〕 次の工程を含む、 生体試料中の 〔 1〕 に記載のポリヌクレオチドの測定 方法。 [13] A method for measuring the polynucleotide according to [1] in a biological sample, comprising the following steps.
( 1 )生体試料を 〔7〕 に記載のポリヌクレオチドと接触させる工程、 および (1) contacting the biological sample with the polynucleotide according to (7), and
(2 )生体試料中に含まれる、 〔7〕 に記載のポリヌクレオチドとハイブリダ ィズするポリヌクレオチドを検出する工程 (2) a step of detecting a polynucleotide that is contained in the biological sample and hybridizes with the polynucleotide of (7).
〔 1 4〕 〔7〕 に記載のポリヌクレオチドを含む、 〔 1〕 に記載のポリヌクレオ チドの測定用キツト。 [14] A kit for measuring the polynucleotide according to [1], comprising the polynucleotide according to [7].
〔1 5〕 〔2〕 に記載の蛋白質の発現が改変されるように操作された非ヒト脊椎 動物。 [15] A non-human vertebrate animal which has been engineered to alter the expression of the protein of [2].
〔 1 6〕 ノックアウト動物またはトランスジエニック動物である、 〔 1 5〕 に記 載の非ヒト脊椎動物。 [16] The non-human vertebrate according to [15], which is a knockout animal or a transgenic animal.
〔 1 7〕 配列番号： 2に記載のアミノ酸配列からなるタンパク質の発現が抑制さ れているマウス胚性幹細胞。 [17] a mouse embryonic stem cell in which expression of a protein consisting of the amino acid sequence of SEQ ID NO: 2 is suppressed.
〔1 8〕 配列番号： 1に記載された塩基配列を構成することができるェキソンに おいて、 少なくとも開始コドンに変異を有する 〔 1 7〕 に記載のマウス胚性 幹細胞。 [18] The mouse embryonic stem cell according to [17], wherein the exon capable of constituting the base sequence described in SEQ ID NO: 1 has a mutation in at least an initiation codon.
本発明は、 新規なレブチン誘導遺伝子を提供する。 本発明のレブチン誘導遺伝 子の塩基配列配列番号： 1 (マウス） 、 および配列番号： 3 (ヒト） に、 そして 本発明のレプチン誘導遺伝子によってコードされる蛋白質のァミノ酸配列を配列 番号： 2 (マウス） 、 および配列番号： 4 (ヒト） に示す。 本発明の遺伝子の E STs (AK002535, Riken ful l length cDNA project, cDNA from mouse kidney)、 ならびにその ORFより予想されるアミノ酸配列は知られていた。 しかし、 遺伝 子産物として実際に生体中に存在するか否かは不明であった。 また塩基配列のホ
モロジ一検索の結果、 ホモロジ一のある遺伝子としてはヒトのォ一ソログ（GenB ank Acc . No. Z84479 )が見出されるのみである。 しかもホモロジ一を示した塩基 配列は、 アンチセンス配列となっており、 正しい塩基配列とアミノ酸配列を導き 出すことはできない。 更に、 本発明のレブチン誘導遺伝子によってコードされる アミノ酸配列には、 既知のモチーフなどは確認できなかった。 したがって、 これ らの遺伝子がレブチンによって誘導されることは、 本発明によってもたらされた 新規な知見である。 The present invention provides a novel leptin-inducible gene. The nucleotide sequence of the leptin-inducible gene of the present invention is shown in SEQ ID NO: 1 (mouse) and SEQ ID NO: 3 (human), and the amino acid sequence of the protein encoded by the leptin-inducible gene of the present invention is represented by SEQ ID NO: 2 ( Mouse) and SEQ ID NO: 4 (human). ESTs (AK002535, RIKEN full length cDNA project, cDNA from mouse kidney) of the gene of the present invention, and the amino acid sequence predicted from its ORF were known. However, it was unknown whether it actually existed in living organisms as a gene product. In addition, the base sequence As a result of morphological search, only human homolog (GenBank Acc. No. Z84479) is found as a homologous gene. Moreover, the nucleotide sequence showing homology is an antisense sequence, and it is not possible to derive a correct nucleotide sequence and amino acid sequence. Furthermore, no known motif or the like could be confirmed in the amino acid sequence encoded by the lebutin-inducible gene of the present invention. Therefore, induction of these genes by lebutin is a novel finding provided by the present invention.
加えて本遺伝子産物が実際に生体内に存在することをノーザンブロッ ト、 in s/ii;ハイブリダィゼーシヨン、 ウエスタンプロット、 そして免疫化学染色によ つて証明した。 ノーザンプロッ トおよび免疫化学染色解析の結果、 本発明の遺伝 子は脳にも発現がみられるが、 腎、 肝臓に強い発現が見られた。 In addition, the existence of this gene product in vivo was demonstrated by Northern blot, ins / ii; hybridization, Western plot, and immunochemical staining. As a result of Northern blot analysis and immunochemical staining analysis, the gene of the present invention was also expressed in the brain, but was strongly expressed in the kidney and liver.
本発明の遺伝子は、 レブチンに応答して発現が誘導されることから、 レブチン 応答性の指標として有用である。 すなわちレブチン受容体を発現する肝臓 hepat ocytesにおけるレプチン依存的なグリコーゲンの貯蔵を変化させる薬剤のスク リーニングのための指標として使用できる。 あるいは、 免疫化学染色解析より睾 丸の間細胞 (Leidig cell )にこの遺伝子の発現が確認されたこと、 そしてレプチ ンの投与によりテストステロンの産生が阻害されることなどから、 これらの男性 ホルモン産生経路を調節する化合物のスクリーニングの指標としても使用可能で あ o Since the expression of the gene of the present invention is induced in response to lebutin, it is useful as an indicator of levulin responsiveness. That is, it can be used as an index for screening for a drug that alters leptin-dependent glycogen storage in hepatic hepatocytes that express the leptin receptor. Alternatively, immunochemical staining analysis confirmed that the expression of this gene was found in intertesticular cells (Leidig cells), and the administration of leptin inhibited the production of testosterone. Can also be used as an index for screening compounds that regulate
ob/obマウスでは、 レプチンの欠損が肥満の原因となっていた。 一方ヒトにお いては、 レブチンの欠損によって肥満になっている場合は非常に稀で、 多くの肥 満患者では血中のレブチン濃度が肥満の程度とともに上昇している。 この事実は、 肥満を有するヒ卜に単にレブチンを投与するだけでは、 肥満を改善できないこと を示している。 このような状態は、 レブチンのシグナルが正しく伝達できない状 態、 すなわちレブチン抵抗性の状態と言うことができる。 ヒトの肥満においては、 レブチンの投与ではなく、 レブチンに対する反応性の低下、 すなわちレブチン抵
抗性を解除する必要がある。 このように、 本発明の遺伝子の発現レベルに基づい て、 肥満の治療方針を選択することができる。 本発明に基づいて、 次の工程を含 む、 レブチン応答性を診断する方法が提供される。 In ob / ob mice, leptin deficiency was responsible for obesity. In humans, on the other hand, obesity due to levulin deficiency is very rare, and blood levels of levutin increase with the degree of obesity in many obese patients. This fact indicates that obesity cannot be ameliorated by simply administering lebutin to humans with obesity. Such a state can be referred to as a state in which the signal of lebutin cannot be transmitted correctly, that is, a state of leptin resistance. In human obesity, the decrease in responsiveness to lebutin, rather than the administration of lebutin, It is necessary to release the resistance. Thus, a therapeutic strategy for obesity can be selected based on the expression level of the gene of the present invention. According to the present invention, there is provided a method for diagnosing lebutin responsiveness, comprising the following steps.
( a ) 被検者から採取された生体試料における以下の指標 i )〜iv)のいずれ かに記載のポリヌクレオチドの発現レベルを測定する工程、 および i )配列番号： 2または配列番号： 4に記載のアミノ酸配列からなる蛋白質 をコードするポリヌクレオチド、 (a) a step of measuring the expression level of the polynucleotide according to any one of the following indices i) to iv) in a biological sample collected from a subject; and i) the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. A polynucleotide encoding a protein consisting of the amino acid sequence described above;
ii )配列番号： 1または配列番号： 3に記載の塩基配列のコード領域を含む ポリヌクレオチド、 ii) a polynucleotide comprising the coding region of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
i i i )配列番号： 2または配列番号： 4に記載のアミノ酸配列において 1若 しくは複数のアミノ酸が置換、 欠失、 挿入、 および/または付加したァミノ 酸配列を有し、 配列番号： 2または配列番号： 4に記載のアミノ酸配列から なる蛋白質と機能的に同等な蛋白質をコードするポリヌクレオチド、 および iv) 配列番号： 1または配列番号： 3に記載の塩基配列からなるポリヌク レオチドとストリンジェン '卜な条件下でハイブリダィズし、 配列番号： 2ま たは配列番号： 4に記載のアミノ酸配列からなる蛋白質と機能的に同等な蛋 白質をコードするポリヌクレオチド iii) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 having an amino acid sequence with substitution, deletion, insertion, and / or addition; A polynucleotide encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4; and iv) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a stringent polynucleotide. Under the following conditions, and which encodes a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
( b ) ( a ) の測定値をレブチン応答性と関連付ける工程 (b) associating the measured value of (a) with lebutin responsiveness
上記診断方法において、 各ポリヌクレオチドの発現レベルとは、 ポリヌクレオ チドの発現強度、 および各ポリヌクレオチドによってコードされる蛋白質の発現 レベルのいずれかを測定することによって評価することができる。 与えられた塩 基配列を有するポリヌクレオチドの発現強度は、 ハイブリダィゼ一シヨンや P C Rを利用して測定することができる。 また、 与えられたアミノ酸配列を有する蛋 白質の発現レベルは、 ィムノアツセィなどによつて測定することができる。 本発明において前記発現レベルの測定値は、 被検者のレブチン応答性を示して いる。 たとえば測定値が低い場合には、 レブチン応答性が低いと判断される。 特
に、 血中のレプチン濃度に異常が無い場合、 あるいは +分量のレブチンの投与を 受けているのにもかかわらず、 前記測定値が健常者に比較して低い場合には、 レ プチン応答性が低いと見なすことができる。 本発明の診断方法は、 血中のレプチ ン濃度を考慮することによって、 より適切な結果が期待できる。 あるいは本発明 の診断方法は、 レブチンの投与を受けている被検者における、 レブチン応答性の 評価に有用である。 In the above diagnostic method, the expression level of each polynucleotide can be evaluated by measuring any one of the expression intensity of the polynucleotide and the expression level of the protein encoded by each polynucleotide. The expression intensity of a polynucleotide having a given base sequence can be measured by using hybridization or PCR. In addition, the expression level of a protein having a given amino acid sequence can be measured by using an immunoassay or the like. In the present invention, the measured value of the expression level indicates the subject's responsiveness to lebutin. For example, when the measured value is low, it is determined that the responsiveness to lebutin is low. Special In addition, if there is no abnormality in the leptin concentration in the blood, or if the above measurement value is lower than that of a healthy subject despite receiving a plus amount of leptin, the leptin responsiveness is low. Can be considered low. In the diagnostic method of the present invention, more appropriate results can be expected by considering the leptin concentration in blood. Alternatively, the diagnostic method of the present invention is useful for evaluating levulin responsiveness in a subject receiving lebutin.
本発明の診断方法を実施するために必要な要素を組み合せて、 レブチン応答性 の診断用キッ トを構成することができる。 たとえば後に述べるような、 本発明の ポリヌクレオチドや蛋白質を測定するためのキッ卜は、 レブチン応答性の診断用 キッ 卜として有用である。 A combination of elements necessary for carrying out the diagnostic method of the present invention can constitute a lebutin-responsive diagnostic kit. For example, a kit for measuring the polynucleotide or protein of the present invention, which will be described later, is useful as a diagnostic kit for responsiveness to lebutin.
本発明の遺伝子は、 レブチンによるシグナルの伝達経路の中で、 レプチンの刺 激に対して短時間のうちに応答する。 P0MC、 NPY、 あるいは AGRPなどの各種の ニューロペプチド遺伝子の転写が、 レブチンの投与によって影響を受けることは 公知である。 しかし、 これらのニューロペプチドの転写レベルに変化が現れるの は、 レブチン投与後数日してからである。 他方、 本発明の遺伝子は、 レブチン投 与に速やかに応答する。 つまり、 本発明の遺伝子は、 レブチンのシグナル伝達経 路において上記ニューロべプチドより上流において機能しているものと考えるこ とができる。 したがって、 本遺伝子とこれらのニューロペプチドとの関連性の解 明は、 レブチンのシグナル伝達経路の全体像を明らかにすることにつながる。 ま た本発明の遺伝子は、 レブチンによって速やかに応答する遺伝子であることから、 本遺伝子の動きを指標として、 本遺伝子とレプチン抵抗性との関連を調べること ができる。 更に、 肥満の程度に対応して本遺伝子の発現レベルに差異があれば、 肥満の指標として用いることもできる。 The gene of the present invention responds to leptin stimulation in a short time in the signal transduction pathway by leptin. It is known that the transcription of various neuropeptide genes such as P0MC, NPY, or AGRP is affected by administration of lebutin. However, changes in the transcription levels of these neuropeptides appear only a few days after leptin administration. On the other hand, the gene of the present invention responds rapidly to lebutin administration. That is, it can be considered that the gene of the present invention functions upstream of the above-described neurobeptide in the signal transduction pathway of lebutin. Therefore, elucidation of the relationship between this gene and these neuropeptides will lead to a clear picture of the lebutin signaling pathway. Further, since the gene of the present invention is a gene that responds promptly to lebutin, the relationship between the present gene and leptin resistance can be examined using the movement of the gene as an index. Furthermore, if there is a difference in the expression level of this gene according to the degree of obesity, it can be used as an indicator of obesity.
加えて、 これらの事実により、 本発明の遺伝子は、 抗肥満薬のスクリーニング に使用することができる。 すなわち、 本発明の遺伝子がレブチンに応答して発現 が上昇することから、 レブチンの存在下で本遺伝子の発現を増加させる化合物に
は、 レブチン抵抗性を解除し、 肥満を抑制する作用が期待できる。 レブチン抵抗 性は、 ヒトにおける肥満の重要な原因の一つと考えられることから、 本発明の遺 伝子は、 肥満の治療薬開発や肥満の分子生物学的解明において、 重要な標的とな る。 In addition, due to these facts, the gene of the present invention can be used for antiobesity drug screening. That is, since the expression of the gene of the present invention increases in response to lebutin, a compound that increases the expression of the present gene in the presence of lebutin is used. Is expected to release lebutin resistance and suppress obesity. Since lebutin resistance is considered to be one of the important causes of obesity in humans, the gene of the present invention is an important target in developing therapeutics for obesity and elucidating the molecular biology of obesity.
また本発明は、 前記 （a ) ― ( e ) のいずれかに記載されたポリヌクレオチド によってコードされる実質的に純粋な蛋白質に関する。 本発明において、 実質的 に純粋な蛋白質とは、 当該蛋白質の他に生物に由来する大型分子を実質的に含ま ない状態にあることを言う。 本発明における実質的に純粋な蛋白質は、 少なくと も 7 0 %以上、 通常 8 0 %、 好ましくは 9 0 %、 より好ましくは 9 5 %以上、 た とえば 9 9 %の純度を有する蛋白質を言う。 蛋白質の純度を測定する手法は公知 である。 蛋白質の純度を知るための手法として、 たとえば、 ポリアクリルアミ ド ゲル電気泳動や HPLC等を示すことができる。 The present invention also relates to a substantially pure protein encoded by the polynucleotide according to any one of the above (a) to (e). In the present invention, a substantially pure protein means that the protein is substantially free of a large molecule derived from an organism in addition to the protein. A substantially pure protein in the present invention refers to a protein having a purity of at least 70% or more, usually 80%, preferably 90%, more preferably 95% or more, for example, 99%. To tell. Techniques for measuring protein purity are known. As a method for knowing the purity of the protein, for example, polyacrylamide gel electrophoresis, HPLC, or the like can be shown.
本発明の蛋白質は、 組み換え蛋白質として、 また天然の蛋白質として調製する ことが可能である。 組み換え蛋白質は、 例えば、 後述するように本発明の蛋白質 をコードする DNAを挿入したベクタ一を適当な宿主細胞に導入し、 形質転換体 内で発現した蛋白質を精製することにより調製することが可能である。 一方、 天 然の蛋白質は、 例えば、 後述する本発明の蛋白質に対する抗体を結合したァフィ 二ティ一カラムを利用して調製することができる（Current Protocols in olec ular Biology edit. Ausubel et al . ( 1987) Publ ish. John Wi ley & Sons Sec tion 16. 1-16. 19 )。 ァフィ二ティ一精製に用いる抗体は、 ポリクローナル抗体 であってもモノクローナル抗体であってもよい。 また、 インビトロ卜ランスレー シヨン (例えば、 「0n the fidelity of mRNA translation in the nuclease - 1 reated rabbit reticulocyte lysate system. Dasso,M. C. , Jackson, R. J. ( 198 9) Nucleic Acids Res. 17: 3129-3144」 参照） などにより本発明の蛋白質を調 製することも可能である。 The protein of the present invention can be prepared as a recombinant protein or as a natural protein. The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention has been inserted into an appropriate host cell and purifying the protein expressed in the transformant, as described below. It is. On the other hand, a natural protein can be prepared, for example, using an affinity column to which an antibody against the protein of the present invention described later is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) ) Publish. John Wiley & Sons Section 16.1-16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. Also, in vitro translation (for example, see “0n the fidelity of mRNA translation in the nuclease-1 reated rabbit reticulocyte lysate system. Dasso, MC, Jackson, RJ (198 9) Nucleic Acids Res. 17: 3129-3144”) For example, the protein of the present invention can be prepared by such methods.
本発明には、 本実施例において同定されたマウス、 またはヒト由来の蛋白質と
機能的に同等な蛋白質が含まれる。 このような蛋白質には、 例えば、 配列番号：The present invention relates to the mouse or human-derived protein identified in this example. Functionally equivalent proteins are included. Such proteins include, for example, SEQ ID NO:
2 (マウス） または配列番号： 4 (ヒト） に記載のアミノ酸配列からなる蛋白質 の変異体、 ホモログ、 バリアントなどが含まれる。 ここで 「機能的に同等」 とは、 対象となる蛋白質がレブチンの投与によって生体内で発現誘導されることを言う c このような機能は、 その発現の消失または低下により推測することができる。 これら本実施例において同定された蛋白質と機能的に同等な蛋白質は、 当業者 であれば、 例えば、 蛋白質中のアミノ酸配列に変異を導入する方法 （例えば、 部 位特異的変異誘発法（Current Protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publ ish. John Wi ley & Sons Section 8. 1-8.5 )) を利用して 調製することができる。 また、 このような蛋白質は、 自然界におけるアミノ酸の 変異により生じることもある。 本発明には、 このように本実施例において同定さ れた蛋白質と同等の機能を有する限り、 そのアミノ酸配列 （配列番号： 2または 配列番号： 4 ) において 1もしくは複数のアミノ酸が置換、 欠失、 挿入および /もしぐは付加などにより異なる蛋白質が含まれる。 Includes variants, homologs, variants, etc. of the protein consisting of the amino acid sequence of 2 (mouse) or SEQ ID NO: 4 (human). Here, “functionally equivalent” means that the target protein is induced to be expressed in vivo by administration of lebutin. C Such a function can be inferred by the disappearance or decrease of the expression. Those skilled in the art can use these proteins functionally equivalent to the protein identified in this example, for example, by introducing a mutation into an amino acid sequence in the protein (for example, by site-directed mutagenesis (Current Protocols). (1987) Publish. John Wiley & Sons Section 8.1-8.5)). In addition, such proteins may be produced by amino acid mutations in nature. In the present invention, one or more amino acids may be substituted or deleted in the amino acid sequence (SEQ ID NO: 2 or SEQ ID NO: 4) as long as it has a function equivalent to the protein identified in this example. The insertion and / or insertion include different proteins due to addition and the like.
蛋白質におけるアミノ酸の変異数や変異部位は、 その機能が保持される限り制 限はない。 変異数は、 典型的には、 30アミノ酸以内であり、 好ましくは 10アミ ノ酸以内であり、 さらに好ましくは 5アミノ酸以内 （例えば、 3アミノ酸以内） である。 置換されるアミノ酸は、 蛋白質の機能の保持の観点から、 置換前のアミ ノ酸と似た性質を有するアミノ酸であることが好ましい。 蛋白質を構成するアミ ノ酸を似た性質のアミノ酸に置換することは、 保存的置換（conservative subst itution)と呼ばれている。 例えば、 Alas Val、 Leu、 l ieヽ Pro、 Met, Phe、 Trp は、 共に非極性アミノ酸に分類されるため、 互いに似た性質を有すると考えられ る。 また、 非荷電性としては、 Gly、 Ser、 Thr、 Cys、 Tyr、 Asn、 Ginが挙げられ る。 また、 酸性アミノ酸としては、 Aspおよび Gluが、 塩基性アミノ酸としては、 Lys、 Arg、 Hisが挙げられる。 The number and location of amino acid mutations in proteins are not limited as long as their functions are maintained. The number of mutations is typically within 30 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids). The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution, from the viewpoint of maintaining the function of the protein. Substitution of an amino acid constituting a protein with an amino acid having similar properties is called conservative substitution. For example, Ala s Val, Leu, l ieヽPro, Met, Phe, Trp are all classified into non-polar amino acid, it is thought to have similar properties to each other. In addition, examples of non-charged ones include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin. In addition, acidic amino acids include Asp and Glu, and basic amino acids include Lys, Arg, and His.
本実施例において同定された蛋白質と機能的に同等な蛋白質は、 当業者に周知
のノヽイブリダィゼ一シヨン技術 (Current Protocols in Molecular Biology edi t. Ausubel et al . ( 1987) Publish. John Wi ley & Sons Section 6.3-6.4)、 あるいは 遺伝子増幅技術 (PCR) (Current protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publ ish. John Wi ley & Sons Section 6. 1-6.Proteins functionally equivalent to the proteins identified in this example are well known to those of skill in the art. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) or gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.
4) を利用して単離することも可能である。 即ち、 当業者であれば、 本実施例に おいて同定された蛋白質をコードするポリヌクレオチド （配列番号： 1、 または 配列番号： 3 ) またはその一部をプローブとして、 あるいは該ポリヌクレオチド と特異的にハイプリダイズするオリゴヌクレオチドをプライマーとして、 該ポリ ヌクレオチドとハイブリダイズするポリヌクレオチドを単離することができる。 さらに単離したポリヌクレオチドを基に、 該ポリヌクレオチドによりコードされ る蛋白質を調製することができる。 本発明には、 本実施例において同定された蛋 白質と同等の機能を有する限り、 これら蛋白質をコードするポリヌクレオチドと ハイブリダィズするポリヌクレオチドによりコードされる蛋白質が含まれる。 機 能的に同等な蛋白質を単離するための生物としては、 例えば、 ラット、 ゥサギ、 ブ夕、 ゥシ等の脊椎動物が挙げられるが、 これらに制限されない。 It is also possible to isolate using 4). That is, those skilled in the art can use the polynucleotide encoding the protein identified in this example (SEQ ID NO: 1 or SEQ ID NO: 3) or a part thereof as a probe, or specific to the polynucleotide. Using an oligonucleotide that hybridizes with the polynucleotide as a primer, a polynucleotide that hybridizes with the polynucleotide can be isolated. Further, based on the isolated polynucleotide, a protein encoded by the polynucleotide can be prepared. The present invention includes proteins encoded by polynucleotides that hybridize with polynucleotides encoding these proteins, as long as they have a function equivalent to the proteins identified in the present examples. Organisms for isolating functionally equivalent proteins include, but are not limited to, for example, vertebrates such as rats, magpies, bushes, and magpies.
機能的に同等な蛋白質をコードするポリヌクレオチドを単離するためのハイブ リダィゼ一シヨンのストリンジェン卜な条件は、 通常 「lxSS 0. 1¾ SDS、 3 7°C」 程度であり、 より厳しい条件としては 「0.5xSSC、 0.1% SDS、 42°C」 程度 であり、 さらに厳しい条件としては 「0. 1xSSC、 0. 1% SDS、 65°C」 程度であり、 ハイプリダイゼーシヨンの条件が厳しくなるほどプローブ配列と高い相同性を有 するポリヌクレオチドの単離を期待しうる。 但し、 上記 SSC、 SDSおよび温度の 条件の組み合わせは例示であり、 当業者であれば、 ハイブリダィゼ一シヨンのス トリンジヱンシ一を決定する上記若しくは他の要素 （例えば、 プローブ濃度、 プ ローブの長さ、 ハイブリダィゼ一シヨン反応時間など） を適宜組み合わせること により、 上記と同様のストリンジエンシーを実現することが可能である。 The stringent conditions for hybridization to isolate a polynucleotide encoding a functionally equivalent protein are usually `` lxSS 0.1¾SDS, 37 ° C ''. Is about 0.5xSSC, 0.1% SDS, 42 ° C, and the more severe condition is about 0.1xSSC, 0.1% SDS, 65 ° C. Isolation of a polynucleotide having high homology to the probe sequence can be expected. However, the combination of the SSC, SDS, and temperature conditions described above is merely an example, and those skilled in the art will recognize the above and other factors that determine the stringency of the hybridization (eg, probe concentration, probe length, The same stringency as described above can be realized by appropriately combining the hybridization reaction time and the like.
このようなハイブリダイゼーション技術あるいは遺伝子増幅技術を利用して単
離される蛋白質は、 配列番号： 2または配列番号： 4に記載の本発明の蛋白質と 比較して、 通常、 そのアミノ酸配列において高い相同性を有する。 高い相同性と は、 少なくとも 50%以上、 さらに好ましくは 70%以上、 さらに好ましくは 90% 以上 （例えば、 95 %以上） の配列の同一性を指す。 アミノ酸配列の相同性は、 B LASTXによる相同性検索により決定することができる。 Simply using such a hybridization technique or gene amplification technique, The isolated protein usually has higher homology in its amino acid sequence as compared to the protein of the present invention described in SEQ ID NO: 2 or SEQ ID NO: 4. High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, more preferably 90% or more (eg, 95% or more). Amino acid sequence homology can be determined by homology search using BLASTX.
本発明は、 また、 本発明の蛋白質の部分ペプチドを提供する。 本発明の蛋白質 の部分ペプチドは、 例えば、 本発明の蛋白質に結合する抗体の調製に利用するこ とができる。 特に、 配列番号： 2または配列番号： 4に記載のアミノ酸配列から 選択され、 該アミノ酸配列に特異的に見出されるアミノ酸配列は、 本発明の蛋白 質に結合する抗体を得るための免疫原として有用である。 特異的に見出されるァ ミノ酸配列とは、 他の蛋白質を構成するァミノ酸配列とは異なる連続したァミノ 酸配列を言う。 なお他の蛋白質とは、 配列番号： 2または配列番号： 4に記載の アミノ酸配列とは異なるアミノ酸配列からなる蛋白質を言う。 したがって、 配列 番号： 2および配列番号： 4に記載のアミノ酸配列の間で共有されたアミノ酸配 列は、 本発明の部分ペプチドに含まれる。 本発明の部分ペプチドは、 少なくとも 7アミノ酸、 好ましくは 9アミノ酸以上、 より好ましくは 12アミノ酸以上、 よ り好ましくは 15アミノ酸以上の連続したアミノ酸配列からなる。 本発明の部分 ペプチドは、 例えば、 遺伝子工学的手法、 公知のペプチド合成法、 あるいは本発 明の蛋白質を適当なぺプチダ一ゼで切断することによって製造することができる。 また本発明は、 本発明の蛋白質をコードするポリヌクレオチドを提供する。 本 発明のポリヌクレオチドとしては、 本発明の蛋白質をコードしうるものであれば、 その形態に特に制限はなく、 cDNAの他、 ゲノム DNA、 化学合成 DNA、 MAなども 含まれる。 また、 本発明の蛋白質をコードしうる限り、 遺伝暗号の縮重に基づく 任意の塩基配列を有するポリヌクレオチドが含まれる。 本発明の蛋白質をコ一ド するポリヌクレオチドは、 上記のように、 配列番号： 1または配列番号： 3に記 載の塩基配列もしくはその一部をプローブとしたハイブリダイゼーシヨン法やこ
れら塩基配列の情報に基づき設計したプライマーを用いた遺伝子増幅法 （PCR) 等の常法により単離することが可能である。 The present invention also provides a partial peptide of the protein of the present invention. The partial peptide of the protein of the present invention can be used, for example, for preparing an antibody that binds to the protein of the present invention. In particular, an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and specifically found in the amino acid sequence is useful as an immunogen for obtaining an antibody that binds to the protein of the present invention. It is. An amino acid sequence that is specifically found refers to a contiguous amino acid sequence that is different from the amino acid sequences that constitute other proteins. The other protein refers to a protein having an amino acid sequence different from the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4. Therefore, the amino acid sequence shared between the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4 is included in the partial peptide of the present invention. The partial peptide of the present invention has a continuous amino acid sequence of at least 7 amino acids, preferably at least 9 amino acids, more preferably at least 12 amino acids, and more preferably at least 15 amino acids. The partial peptide of the present invention can be produced, for example, 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 polynucleotide encoding the protein of the present invention. The polynucleotide of the present invention is not particularly limited in its form as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, MA and the like in addition to cDNA. In addition, a polynucleotide having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included as long as it can encode the protein of the present invention. As described above, the polynucleotide encoding the protein of the present invention can be obtained by the hybridization method using the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof as a probe. It can be isolated by a conventional method such as a gene amplification method (PCR) using primers designed based on the information of these nucleotide sequences.
本発明のポリヌクレオチドは、 単離されたポリヌクレオチドを含む。 本究明に おいて単離されたポリヌクレオチドとは、 天然に存在するポリヌクレオチドやそ の断片とは構造的に異なるポリヌクレオチドを言う。 単離されたポリヌクレオチ ドとして、 具体的には、 たとえば次のようなポリヌクレオチドを示すことができ る。 The polynucleotide of the present invention includes an isolated polynucleotide. The polynucleotide isolated in the present study refers to a polynucleotide structurally different from a naturally occurring polynucleotide or a fragment thereof. Specific examples of the isolated polynucleotide include the following polynucleotides.
ベクタ一やゲノムに人為的に組み込まれた状態にある D N A DNA that is artificially integrated into the vector or genome
分離された D N A ( c D N A、 ゲノム断片、 制限酵素断片、 あるいは P C R の合成産物等を含む） Isolated DNA (including cDNA, genomic fragment, restriction enzyme fragment, or PCR product)
本発明の蛋白質に他の蛋白質を融合させた融合蛋白質、 あるいはタグを付加 した蛋白質などをコードする D N A DNA encoding a fusion protein obtained by fusing another protein to the protein of the present invention, or a tag-added protein, etc.
本発明は、 また、 本発明の蛋白質をコ一ドするポリヌクレオチドが揷入された ベクターを提供する。 本発明のベクターとしては、 挿入したポリヌクレオチドを 安定に保持するものであれば特に制限されず、 例えば宿主に大腸菌を用いるので あれば、 クローニング用べクタ一としては pBluescriptベクタ一（Stratagene社 製）などが好ましい。 本発明の蛋白質を生産する目的においてベクターを用いる 場合には、 特に発現ベクターが有用である。 発現べクタ一としては、 例えば、 試 験管内発現であれば PBESTベクタ一 （プロメガ社製） を、 大腸菌における発現 であれば pETベクタ一 （Invitrogen社製） を、 培養細胞における発現であれば PME18S- FL3ベクター (GenBank Accession No. AB009864) を、 生物個体におけ る発現であれば PME18Sベクター (Mol Cell Biol . 8 :466_472( 1988)) を、 好適 に用いることができる。 本発明の蛋白質をコードするポリヌクレオチドのべクタ —への挿入は常法、 例えば、 制限酵素サイ トを用いたリガ一ゼ反応 （Current p rotocols in Molecular Biology edit. Ausubel et al . ( 1987) Publish. Joh n Wi ley & Sons. Section 11.4_11. 11) により行うことができる。
本発明は、 また、 本発明の蛋白質をコードするポリヌクレオチドまたは該ポリ ヌクレオチドが挿入されたベクターを保持する形質転換体を提供する。 本発明の ベクターが導入される宿主細胞としては特に制限はなく、 目的に応じて種々の宿 主細胞が用いられる。 宿主細胞は、 例えば、 本発明のタンパク質の製造のために 使用することができる。 タンパク質製造のための産生系は、 in Wiroおよび i/j の産生系がある。 in F/iroの産生系としては、 真核細胞を使用する産生 系や原核細胞を使用する産生系が挙げられる。 真核細胞を使用する場合、 例えば、 動物細胞、 植物細胞、 真菌細胞を宿主に用いることができる。 The present invention also provides a vector into which a polynucleotide encoding the protein of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it stably retains the inserted polynucleotide. For example, if Escherichia coli is used as a host, a pBluescript vector (manufactured by Stratagene) may be used as a cloning vector. Are preferred. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. Examples of expression vectors include PBEST vector (Promega) for expression in vitro, pET vector (Invitrogen) for expression in E. coli, and PME18S for expression in cultured cells. -FL3 vector (GenBank Accession No. AB009864) and PME18S vector (Mol Cell Biol. 8: 466_472 (1988)) can be suitably used for expression in living organisms. Insertion of a polynucleotide encoding the protein of the present invention into a vector can be carried out by a conventional method, for example, a ligase reaction using a restriction enzyme site (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish) John Wiley & Sons. Section 11.4_11. 11). The present invention also provides a transformant carrying a polynucleotide encoding the protein of the present invention or a vector into which the polynucleotide has been inserted. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention. Production systems for protein production include in Wiro and i / j production systems. Examples of the in F / iro production system include a production system using eukaryotic cells and a production system using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts.
また、 本発明の宿主細胞には、 レブチン誘導蛋白質の機能解析やレブチン誘導 蛋白質を利用したその機能阻害剤や機能促進剤のスクリーニングのために用いる 細胞も含まれる。 宿主細胞へのベクター導入は、 例えば、 リン酸カルシウム沈殿 法、 電気ノ レス穿孑し法 (Current protocols in Molecular Biology edit. Ausu bel et al . ( 1987) Publish. John Wiley & Sons. Section 9.1-9.9) 、 リボフ ェク夕ミン法 （GIBC0-BRL社製） 、 マイクロインジェクション法などの方法で行 うことが可能である。 形質転換体からのレブチン誘導蛋白質の調製は、 当業者に 公知の蛋白質の分離 ·精製法を利用して行なうことができる。 The host cells of the present invention also include cells used for analyzing the function of a lebutin-induced protein and screening for a function inhibitor or a function promoter using the lebutin-induced protein. Vector introduction into host cells can be performed, for example, by calcium phosphate precipitation, electroless breeding (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.Section 9.1-9.9), It can be performed by a method such as the ribofect-min method (GIBC0-BRL) or the microinjection method. Preparation of a lebutin-derived protein from a transformant can be carried out by a protein separation / purification method known to those skilled in the art.
本発明はまた、 配列番号： 1または配列番号： 3に記載の塩基配列からなるポ リヌクレオチドまたはその相補鎖に相補的な、 少なくとも 15塩基の連続した塩 基配列を含むポリヌクレオチドを提供する。 ここで 「相補鎖」 とは、 A: T、 G:C の塩基対からなる 2本鎖 DNAの一方の鎖に対する他方の鎖を指す。 また、 「相 補的」 とは、 少なくとも 15個の連続したヌクレオチド領域で完全に相補配列で ある場合に限られず、 少なくとも 70%、 好ましくは少なくとも 80%、 より好まし くは 90%、 さらに好ましくは 95% 以上の塩基配列上の相同性を有すればよい。 相同性を決定するためのアルゴリズムは本明細書に記載したものを使用すればよ い。 The present invention also provides a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a polynucleotide comprising a continuous nucleotide sequence of at least 15 bases complementary to a complementary strand thereof. Here, the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T, G: C base pairs with respect to the other strand. The term "complementary" is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably Should have at least 95% homology on the base sequence. The algorithm for determining homology may use the algorithm described in this specification.
このようなポリヌクレオチドは、 本発明の蛋白質をコードする DNAを検出、
単離するためのプローブとして、 また、 本発明のポリヌクレオチドを増幅するた めのプライマーとして利用することが可能である。 プライマ一として用いる場合 には、 通常、 15bp-100bp、 好ましくは 15bp- 35bpの鎖長を有する。 また、 プロ —ブとして用いる場合には、 本発明のポリヌクレオチドの少なくとも一部若しく は全部の配列を有し、 少なくとも 15bpの鎖長のポリヌクレオチドが用いられる。 プライマーとして用いる場合、 3'側の領域は相補的である必要があるが、 5'側 には制限酵素認識配列や夕グなどを付加することができる。 Such a polynucleotide detects DNA encoding the protein of the present invention, It can be used as a probe for isolation and as a primer for amplifying the polynucleotide of the present invention. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a polynucleotide having at least a part or the entire sequence of the polynucleotide of the present invention and having a chain length of at least 15 bp is used. When used as a primer, the 3'-side region needs to be complementary, but a restriction enzyme-recognition sequence or evening fragment can be added to the 5'-side.
また、 本発明のポリヌクレオチドには、 本発明のレブチン誘導蛋白質の発現を 抑制するためのアンチセンスが含まれる。 アンチセンスは、 アンチセンス効果を 引き起こすために、 少なくとも 15bp以上、 好ましくは 100bp、 さらに好ましく は 500bp以上の鎖長を有し、 好ましくは 2000bp以内の鎖長を有する。 このよう なアンチセンスは、 例えば、 配列番号： 1または配列番号： 3に記載の塩基配列 情報を基にホスホロチォネ一卜法 (Stein, 1988 Physicochemical properties o f phosphorothioate ol igodeoxynucleotides. Nucleic Acids Res 16, 3209-2 1 ( 1988) ) などにより調製することができる。 Further, the polynucleotide of the present invention includes an antisense for suppressing the expression of the lebutin-inducing protein of the present invention. The antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect. Such an antisense can be obtained, for example, by the phosphorothione method (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. 1 (1988)).
本発明のポリヌクレオチドには、 例えば、 遺伝子治療への応用が考えられる。 本発明のポリヌクレオチドを利用した遺伝子治療の標的となる疾患としては、 例 えば、 肥満や糖尿病が好適である。 すなわち、 本発明の遺伝子の変異や発現異常 によって、 レブチン抵抗性が形成されているときには、 本発明の遺伝子を生体内 で発現させることによって、 レブチン抵抗性を解除できる可能性がある。 これら 分子を遺伝子治療に用いる場合には、 例えば、 レトロウイルスベクタ一、 アデノ ウィルスベクタ一、 アデノ随伴ウィルスベクタ一などのウィルスベクタ一ゃリポ ゾームなどの非ウィルスベクターなどを利用して、 ex vivo法や in ' O法など により患者へ投与すればよい。 The polynucleotide of the present invention can be applied to, for example, gene therapy. As a disease targeted for gene therapy using the polynucleotide of the present invention, for example, obesity and diabetes are suitable. That is, when lebutin resistance has been formed due to mutation or abnormal expression of the gene of the present invention, there is a possibility that lebutin resistance can be released by expressing the gene of the present invention in vivo. When these molecules are used for gene therapy, for example, a non-viral vector such as a retroviral vector, an adenovirus vector, an adeno-associated virus vector, a non-viral vector such as a liposome, etc. It may be administered to patients by the or in'O method.
また、 本発明のポリヌクレオチドのアンチセンスは、 レブチン抵抗性のモデル の構築に有用である。 例えば、 本発明のポリヌクレオチドのドミナントネガティ
ブな変異により、 本発明のポリヌクレオチドが肥満をもたらす場合には、 本発明 のポリヌクレオチドの発現の抑制によって、 レプチン抵抗性モデルとすることが できる。 Further, the antisense of the polynucleotide of the present invention is useful for constructing a model of lebutin resistance. For example, the dominant negative of the polynucleotide of the present invention When the polynucleotide of the present invention causes obesity due to a severe mutation, suppression of the expression of the polynucleotide of the present invention can be used as a leptin resistance model.
本発明は、 また、 本発明の蛋白質に結合する抗体を提供する。 本発明の抗体の 形態には特に制限はなく、 ポリクローナル抗体やモノクローナル抗体または抗原 結合性を有するそれらの一部も含まれる。 また、 全てのクラスの抗体が含まれる さらに、 本発明の抗体には、 ヒ卜化抗体などの特殊抗体も含まれる。 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 polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. In addition, antibodies of all classes are included. The antibodies of the present invention also include special antibodies such as humanized antibodies.
本発明の抗体は、 公知の方法によって得ることができる。 たとえばポリクロー ナル抗体の場合には、 常法に従い免疫原を家兎に免疫することにより得ることが 可能である (Current protocols in Molecular Biology edit. Ausubel et al . The antibody of the present invention can be obtained by a known method. For example, in the case of a polyclonal antibody, it can be obtained by immunizing a rabbit with an immunogen according to a conventional method (Current protocols in Molecular Biology edit.
( 1987) Publ ish. John Wiley & Sons. Section 11.12— U . 13) 。 一方、 モノク ローナル抗体の場合には、 常法に従い免疫原でマウスを免疫し、 脾臓細胞と骨髄 腫細胞を細胞融合させたハイプリ ドーマ細胞の中から得ることができる （Curre nt protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publ ish.(1987) Publish. John Wiley & Sons. Section 11.12—U. 13). On the other hand, in the case of a monoclonal antibody, a mouse can be immunized with an immunogen according to a conventional method and obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells (Current protocols in Molecular Biology edit). . Ausubel et al. (1987) Publ ish.
John Wiley & Sons. Section 11.4—11.11) 。 John Wiley & Sons. Section 11.4—11.11).
免疫原には、 本発明のアミノ酸配列から選択されたアミノ酸配列からなるオリ ゴぺプチドゃ、 本発明の遺伝子を大腸菌などで発現し精製した組換え蛋白質を用 いることができる。 オリゴペプチドを構成するアミノ酸配列として、 配列番号： 2と配列番号： 4の間で保存されたアミノ酸配列を用いれば、 種を越えて本発明 の蛋白質を認識することができる抗体を得ることができる。 また、 両配列の間で 相違するァミノ酸配列からなるオリゴぺプチドを免疫原とすれば、 種特異的な抗 体とすることができる。 As the immunogen, an oligopeptide comprising an amino acid sequence selected from the amino acid sequence of the present invention, and a recombinant protein obtained by expressing and purifying the gene of the present invention in Escherichia coli or the like can be used. When an amino acid sequence conserved between SEQ ID NO: 2 and SEQ ID NO: 4 is used as an amino acid sequence constituting an oligopeptide, an antibody that can recognize the protein of the present invention across species can be obtained. . In addition, if an oligopeptide comprising an amino acid sequence different between the two sequences is used as an immunogen, a species-specific antibody can be obtained.
本発明の蛋白質に結合する抗体は、 本発明の蛋白質の精製に加え、 例えば、 こ れら蛋白質の発現異常や構造異常の検査 ·診断に利用することも考えられる。 具 体的には、 例えば組織、 血液、 または細胞などから蛋白質を抽出し、 ウエスタン ブロッテイング、 免疫沈降、 ELISA等の方法による本発明の蛋白質の検出を通し
て、 発現や構造の異常の有無を検査，診断することができる。 Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of these proteins, in addition to the purification of the protein of the present invention. Specifically, for example, proteins are extracted from tissues, blood, cells, or the like, and the proteins of the present invention are detected by methods such as Western blotting, immunoprecipitation, and ELISA. Thus, the presence or absence of abnormal expression or structure can be examined and diagnosed.
本発明の蛋白質に結合する抗体は、 本発明の蛋白質に関連した疾患の治療など の目的に利用することも考えられる。 抗体を患者の治療目的で用いる場合には、 ヒト抗体またはヒト化抗体が免疫原性の少ない点で好ましい。 ヒト抗体は、 免疫 系をヒ卜のものと入れ換えたマウス （例えば、 ""Functional transplant of me gabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, M.J. et al . ( 1997) Nat. Genet. 15 : 146- 156」 参照） に免疫 することにより調製することができる。 また、 ヒト化抗体は、 モノクローナル抗 体の超可変領域を用いた遺伝子組み換えによって調製することができる（Method s in Enzymology 203, 99-121( 1991 ) )。 Antibodies that bind to the protein of the present invention may be used for the purpose of treating diseases related to the protein of the present invention. When an antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies are obtained by replacing the immune system with that of a human mouse (for example, "" Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146-156 ”). In addition, a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).
また本発明は、 本発明のポリヌクレオチドの発現を調節する活性を評価する 方法を提供する。 この方法は、 次の工程 （a ) および （b ) を含む。 The present invention also provides a method for evaluating the activity of regulating the expression of the polynucleotide of the present invention. The method includes the following steps (a) and (b).
( a ) 被験化合物の存在下で本発明ののポリヌクレオチドを発現する細胞に レブチンを接触させる工程、 および (a) contacting cells expressing the polynucleotide of the present invention with lebutin in the presence of the test compound, and
( b ) 前記ポリヌクレオチドの発現レベルを測定する工程、 (b) measuring the expression level of the polynucleotide,
本発明のポリヌクレオチドの発現を調節する活性を評価する方法は、 この活性 を有する化合物のスクリーニング方法に有用である。 すなわち本発明は、 次の工 程を含む、 本発明のポリヌクレオチドの発現を調節する活性を有する化合物のス クリーニング方法に関する。 The method for evaluating the activity of regulating the expression of the polynucleotide of the present invention is useful for a method of screening for a compound having this activity. That is, the present invention relates to a method for screening a compound having an activity of regulating the expression of the polynucleotide of the present invention, comprising the following steps.
( a ) 前記評価方法によって、 被験化合物の本発明のポリヌクレオチドの発 現を調節する活性を評価する工程、 および (a) evaluating the activity of the test compound to regulate the expression of the polynucleotide of the present invention by the evaluation method; and
( b ) 被験化合物非存在下でレプチンを接触させた対照と比較して前記ポリ ヌクレオチドの発現レベルを変化させる化合物を選択する工程 (b) selecting a compound that changes the expression level of the polynucleotide as compared to a control that has been contacted with leptin in the absence of the test compound
本発明のスクリーニング方法に用いる、 本発明の遺伝子を発現する細胞には、 たとえば腎臓の尿細管もしくは肝臓の hepatocytes由来の細胞株などを用いる ことができる。 スクリーニングの具体的な方法としては、 被験化合物の存在下で
レブチンと前記細胞とを接触させる。 一定時間の培養の後に、 本発明の遺伝子の 発現レベルの変化を測定し、 被験化合物非存在下でレプチンと接触させて培養し た対照における発現レベルと比較する。 As a cell expressing the gene of the present invention used in the screening method of the present invention, for example, a cell line derived from kidney tubule or liver hepatocytes can be used. As a specific method of screening, in the presence of the test compound Lebutin is brought into contact with the cells. After culturing for a certain period of time, the change in the expression level of the gene of the present invention is measured and compared with the expression level in a control cultured by contact with leptin in the absence of the test compound.
遺伝子の発現レベルは、 遺伝子や発現産物である蛋白質の量を指標として測定 することができる。 遺伝子は定量的 PCR等の公知の手法により定量することが できる。 また蛋白質の量は、 ELISAなどの手法によって測定される。 The expression level of a gene can be measured using the amount of the gene or protein as an expression product as an index. The gene can be quantified by a known method such as quantitative PCR. The amount of protein is measured by a technique such as ELISA.
スクリーニングに用いる被験試料には、 あらゆる化合物を用いることができる 具体的には、 例えば、 細胞抽出液、 遺伝子ライブラリーの発現産物、 合成低分子 化合物、 合成ペプチド、 天然化合物などが挙げられる。 Any compound can be used as the test sample used for screening. Specific examples include cell extracts, expression products of gene libraries, synthetic low-molecular compounds, synthetic peptides, and natural compounds.
このスクリーニングにより単離される化合物は、 レブチンに対する応答性を調 節する化合物の候補となる。 また、 生体内において、 本発明の蛋白質とこれと相 互作用する分子との該相互作用を阻害する化合物の候補となる。 Compounds isolated by this screening are candidates for compounds that regulate responsiveness to lebutin. In addition, it is a candidate for a compound that inhibits the interaction between the protein of the present invention and a molecule that interacts with the protein of the present invention in vivo.
本発明の遺伝子、 その蛋白質、 該遺伝子の発現を制御する化合物、 あるいは該 蛋白質の活性を制御する化合物を医薬品として用いる場合には、 それ自体を医薬 品として用いることも可能であるが、 公知の製剤学的方法により製剤化して用い ることも可能である。 例えば、 薬理学上許容される担体もしくは媒体、 具体的に は、 滅菌水や生理食塩水、 植物油、 乳化剤、 懸濁剤などと適宜組み合わせて製剤 化して用いることが考えられる。 患者への投与は、 例えば、 動脈内注射、 静脈内 注射、 皮下注射など当業者に公知の方法により行いうる。 投与量は、 患者の体重 や年齢、 投与方法などにより変動するが、 当業者であれば適当な投与量を適宜選 択することが可能である。 また、 ポリヌクレオチドを治療薬として使用する場合 には、 該ポリヌクレオチドを遺伝子治療用べクタ一に組込み、 患者に投与するこ とも考えられる。 投与量、 投与方法は、 患者の体重や年齢、 症状などにより変動 するが、 当業者であれば適宜選択することが可能であろう。 When the gene of the present invention, its protein, a compound that regulates the expression of the gene, or a compound that regulates the activity of the protein is used as a drug, the drug itself can be used as a drug, It is also possible to formulate and use it by a pharmaceutical method. For example, it may be used in the form of a formulation by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. When a polynucleotide is used as a therapeutic agent, the polynucleotide may be incorporated into a vector for gene therapy and administered to a patient. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but those skilled in the art will be able to select as appropriate.
遺伝子の発現レベルは、 転写生成物である mMAや、 翻訳産物である蛋白質、 ならびにそれらの断片を測定することにより明らかにすることができる。 本発明
は、 以下のポリヌクレオチドの測定方法、 あるいは蛋白質および/またはその部 分ペプチドの測定方法と、 該測定方法のためのキットを提供する。 The expression level of the gene can be determined by measuring the transcription product mMA, the translation product protein, and fragments thereof. The present invention Provides a method for measuring the following polynucleotide, or a method for measuring a protein and / or a partial peptide thereof, and a kit for the method.
まず本発明のポリヌクレオチドは、 配列番号： 1に示す塩基配列の相補配列を 有するプローブとのハイプリダイゼーシヨンにより測定することができる。 ある いは、 配列番号： 1に示す塩基配列およびその相補鎖に相補的な少なくとも 1 5 塩基からなるオリゴヌクレオチドをプライマ一とする PCR法によって、 本発明 の遺伝子の発現レベルを測定することもできる。 mRNAを鎵型とする RT- PCR法も 公知である。 First, the polynucleotide of the present invention can be measured by hybridization with a probe having a complementary sequence to the base sequence shown in SEQ ID NO: 1. Alternatively, the expression level of the gene of the present invention can also be measured by a PCR method using an oligonucleotide consisting of at least 15 bases complementary to the base sequence shown in SEQ ID NO: 1 and its complementary strand as a primer. . The RT-PCR method using mRNA as type II is also known.
また本発明の遺伝子の翻訳生成物である蛋白質は、 本発明の蛋白質を認識する 抗体を使って、 ィムノアツセィの原理に基づいて測定することができる。 本発明 の蛋白質を認識する抗体を取得する方法は既に述べたとおりである。 In addition, the protein that is a translation product of the gene of the present invention can be measured based on the immunoassay principle using an antibody that recognizes the protein of the present invention. The method for obtaining an antibody that recognizes the protein of the present invention is as described above.
上記測定方法に必要な成分を、 予め組み合わせてキットとすることができる。 たとえば、 mRNAを錡型として RT-PCRを行うためのプライマ一、.逆転写酵素、 DN Aポリメラーゼ、 および反応に好適な緩衝液からなるキットを構成することがで きる。 また、 本発明の蛋白質を認識する標識抗体、 標識を測定するための試薬、 そして免疫反応に好適な緩衝液からなるキットを構成することもできる。 Components necessary for the above-mentioned measurement method can be combined in advance to form a kit. For example, it is possible to construct a kit comprising a primer for performing RT-PCR using mRNA as a type III, reverse transcriptase, DNA polymerase, and a buffer suitable for the reaction. Further, a kit comprising a labeled antibody recognizing the protein of the present invention, a reagent for measuring the label, and a buffer suitable for an immune reaction can also be constituted.
また、 本発明は、 本発明の蛋白質の発現が改変されるように操作された非ヒト 脊椎動物を提供する。 ここで 「発現の改変」 には、 発現の増強および減弱が含ま れる。 また、 「蛋白質の発現の改変」 は、 転写と翻訳のいずれのステップの改変 も含まれる。 このような非ヒト脊椎動物には、 内因性の本発明の蛋白質の発現を 停止または減少させるように操作された動物 （ノックアウト動物） および外来性 の本発明の蛋白質を発現するように該蛋白質をコ一ドする遺伝子が導入された動 物 （トランスジエニック動物） が含まれる。 このようなノックアウトおよびトラ ンスジエニック非ヒト脊椎動物は、 文献 「ニューロサイエンス 'ラボマニュアル The present invention also provides a non-human vertebrate engineered to alter the expression of the protein of the present invention. Here, “alteration of expression” includes enhancement and attenuation of expression. “Modification of protein expression” includes modification of both transcription and translation steps. Such non-human vertebrates include animals that have been engineered to stop or reduce expression of the endogenous protein of the invention (knockout animals) and those that express the exogenous protein of the invention. Includes animals (transgenic animals) into which the coding gene has been introduced. Such knockout and transgenic non-human vertebrates are described in the literature "Neuroscience 'Lab Manual.
3、 神経生物学のための胚と個体の遺伝子操作法 （編集 ·近藤寿人、 シュプリン ガー 'フエアラーク東京株式会社） 」 に従って作製することができる。
例えば、 本発明の蛋白質をコードする DNAが染色体に組込まれたトランスジ エニック動物を作製することにより、 これらの蛋白質の発現を上昇させたり、 発 現パターンや分布の改変を行うことができる。 また、 これらの内因性遺伝子の発 現制御領域に変異を導入したり、 他の発現制御領域を付加または置換することな どにより、 本来の遺伝子の発現レベルと比較して人工的に転写レベルを上昇、 下 降、 または発現パターンや分布の改変を行うことができる。 一方、 ェキソンの一 部を欠損させたり、 翻訳領域への点突然変異の導入により終止コドンへ置換する ことにより、 タンパク質への翻訳を修飾することもできる。 また、 アンチセンス RNAやリボザィムを発現させることで、 本発明の遺伝子の発現を制御することも 可能である。 これらの変異の導入は、 公知の方法により行うことができる。 3. Genetic manipulation of embryos and individuals for neurobiology (edited by Toshihito Kondo, Springer 'Fairlark Tokyo Co., Ltd.). For example, by preparing a transgenic animal in which a DNA encoding the protein of the present invention has been integrated into a chromosome, it is possible to increase the expression of these proteins or to modify the expression pattern or distribution. In addition, by introducing mutations into the expression control regions of these endogenous genes or adding or replacing other expression control regions, the transcription level is artificially increased as compared to the expression level of the original gene. Up, down, or alter expression patterns and distributions. On the other hand, protein translation can also be modified by deleting a part of the exon or replacing it with a stop codon by introducing a point mutation into the translation region. The expression of the gene of the present invention can be controlled by expressing antisense RNA or ribozyme. Introduction of these mutations can be performed by a known method.
このような非ヒト脊椎動物は、 転写機能の研究、 転写に関連する疾患のメカ二 ズムの解明、 医薬品のスクリーニング等に用いる疾患モデル動物の開発に有用で ある。 Such non-human vertebrates are useful for studying transcriptional functions, elucidating the mechanisms of transcription-related diseases, and developing disease model animals used for drug screening and the like.
前に述べたように、 本発明の遺伝子は、 レブチンによるシグナル伝達経路の上 流を構成する遺伝子と考えられる。 したがって、 本発明の遺伝子の発現を抑制し た動物や細胞は、 レブチン抵抗性を解除する薬剤のスクリ一ニングに有用である。 中でも、 本発明の遺伝子の発現を欠失させた胚性幹細胞 （embryonic stem eel 1 ; ES細胞） は、 たとえば当該遺伝子の発現を抑制したノックアウト動物の作成 に有用である。 As described above, the gene of the present invention is considered to be a gene constituting the upstream of the signal transduction pathway by lebutin. Therefore, animals and cells in which the expression of the gene of the present invention has been suppressed are useful for screening for a drug that releases lebutin resistance. Among them, embryonic stem cells (embryonic stem eel 1; ES cells) lacking the expression of the gene of the present invention are useful, for example, for producing knockout animals in which the expression of the gene is suppressed.
非ヒト動物における、 本発明の遺伝子の発現抑制には、 公知の方法を応用する ことができる。 たとえばマウスであれば、 配列番号： 1に示した塩基配列中、 と くに蛋白質コード領域を構成するェキソンをゲノムの塩基配列上にマツビングし、 その塩基配列の少なくとも一部に変異を導入して蛋白質への翻訳を正常に行えな いようにする。 具体的には、 たとえばェキソンの一部の塩基配列の置換、 欠失、 付加、 あるいは挿入などにより、 ェキソンの途中にストップコドンを生じるよう にする。 その結果、 変異を導入された細胞においては、 本発明の遺伝子の正常な
発現が行えなくなる。 ゲノムの塩基配列に変異をもたらすには、 相同組み換えの 技術が用いられる。 A known method can be applied to suppress the expression of the gene of the present invention in a non-human animal. For example, in the case of a mouse, exons that constitute the protein coding region, particularly the exons that constitute the protein coding region in the nucleotide sequence shown in SEQ ID NO: 1, are mapped on the nucleotide sequence of the genome, and a mutation is introduced into at least a part of the nucleotide sequence to obtain a protein. To be unable to translate correctly. Specifically, a stop codon is generated in the exon, for example, by substitution, deletion, addition, or insertion of a part of the exon base sequence. As a result, in the cells into which the mutation was introduced, the normal Expression cannot be performed. Homologous recombination techniques are used to introduce mutations in the genome sequence.
こうして構築された、 本究明の遺伝子の発現を欠失させた ES細胞によって、 ノックアウトマウスを得ることができる。 ノックアウトマウスを得るには、 まず この ES細胞をマウスの胚盤胞期の卵子に注入し、 仮親の子宮に移植する。 ES細 胞を注入した卵子からは、 本来の受精卵と ES細胞由来の細胞からなるキメラマ ウス （フアウンダ一） が生まれる。 フアウンダ一と野生マウスの交配によって生 まれてくる子孫にノックアウトされたゲノムが確認できれば、 その子孫は、 ノッ クァゥ卜された遺伝子をへテロに持つノックァゥト動物である。 ヘテロの子孫を 更に交配することによって、 ノヅクアウトマウス （ホモ） を得ることができる。 本発明者らは、 配列番号： 1に示す塩基配列の蛋白質コード領域中、 特に開始 コドンを含む領域を欠失させることにより、 マウスにおける本発明の遺伝子の発 現を、 ほぼ完全に抑制できることを確認した。 したがって、 本発明の遺伝子の発 現を抑制した非ヒト動物として、 本発明の遺伝子における開始コドンに変異を有 するノックアウト動物は好ましい。 あるいは本発明の遺伝子の発現を抑制した細 胞として、 本発明の遺伝子における開始コドンに変異を有する細胞は好ましい。 開始コドンの変異は、 塩基の置換、 欠失、 付加、 あるいは揷入によってもたらさ れる。 A knockout mouse can be obtained by using the thus constructed ES cells in which the expression of the gene of the present invention has been deleted. To obtain a knockout mouse, the ES cells are first injected into the blastocyst stage of the mouse and transplanted into the uterus of the foster parent. The egg injected with the ES cells produces a chimeric mouse (Faunda-1), which consists of the original fertilized egg and cells derived from the ES cells. If the genome that has been knocked out can be confirmed in the offspring produced by crossing Huawunda with wild mice, the offspring is a knockout animal that has the knocked-out gene heterozygously. By crossing heterozygous offspring further, a knockout mouse (homo) can be obtained. The present inventors have shown that the expression of the gene of the present invention in mice can be almost completely suppressed by deleting the region including the initiation codon in the protein coding region of the nucleotide sequence shown in SEQ ID NO: 1. confirmed. Therefore, as a non-human animal in which the expression of the gene of the present invention is suppressed, a knockout animal having a mutation in the initiation codon of the gene of the present invention is preferable. Alternatively, as a cell in which the expression of the gene of the present invention is suppressed, a cell having a mutation in the initiation codon of the gene of the present invention is preferable. Mutations in the initiation codon can be caused by base substitutions, deletions, additions, or insertions.
なお本明細書において引用された全ての先行技術文献は、 参照として本明細書 に組み入れられる。 図面の簡単な説明 All prior art documents cited in the present specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 レブチン投与による、 ob/obマウス視床下部における本発明の遺伝子 の誘導をノーザンブロッ卜によって調べた結果を示す写真。 A 9が本発明の遺伝 子、 B R Iが対照である。 図中、 レブチン （―） と示したレーンが P B Sを投与 した場合、 （+ ) がマウスレブチンを投与した場合の結果である。
図 2は、 本発明の遺伝子の組織分布をノーザンブロッ卜によって調べた結果を 示す写真。 各レーンは図中に示した組織に対応する。 FIG. 1 is a photograph showing the results of Northern blot analysis of induction of the gene of the present invention in the hypothalamus of ob / ob mice by administration of lebutin. A9 is the gene of the present invention, and BRI is a control. In the figure, the lane marked with levbutin (-) shows the results when PBS was administered, and the (+) shows the results when mouse lebutin was administered. FIG. 2 is a photograph showing the results of Northern blot analysis of the tissue distribution of the gene of the present invention. Each lane corresponds to the tissue shown in the figure.
図 3は、 本発明の蛋白質のウエスタンプロット解析の結果を示す写真。 各レ一 ンは、 以下の蛋白質、 または細胞抽出液に対応する。 FIG. 3 is a photograph showing the result of Western blot analysis of the protein of the present invention. Each lane corresponds to the following proteins or cell extracts.
レーン 1 ：精製本発明の蛋白質- GST融合タンパク質 （免疫原） 5 ng Lane 1: purified protein-GST fusion protein of the present invention (immunogen) 5 ng
レーン 2 ：精製本発明の蛋白質- GST融合タンパク質 （免疫原） 15 ng Lane 2: purified protein-GST fusion protein of the present invention (immunogen) 15 ng
レーン 3 ： 10 zgのマウス腎臓の全細胞抽出液 Lane 3: 10 zg mouse kidney whole cell extract
レーン 4 ： 30 zgのマウス腎臓の全細胞抽出液 Lane 4: 30 zg mouse kidney whole cell extract
レーン 5 ：本発明の遺伝子を過剰発現させた C0S7細胞 （全細胞抽出液約 a g) Lane 5: C0S7 cells overexpressing the gene of the present invention (approximately ag of whole cell extract)
レーン 6 ： 30//gのマウス腎臓の全細胞抽出液 （レーン 4とは別のマウス） 図 4は、 マウスとヒトにおける本発明の蛋白質のアミノ酸配列を比較した結果 を示す図。 マウスのアミノ酸配列を上に、 ヒトのアミノ酸配列を下に示した。 図 5は、 ヒト肝臓組織の免疫組織化学的解析結果を示す写真。 A (上） が 1 5Lane 6: 30 // g mouse kidney whole cell extract (mice different from lane 4) FIG. 4 shows the results of comparing the amino acid sequences of the protein of the present invention between mouse and human. The mouse amino acid sequence is shown above and the human amino acid sequence is shown below. FIG. 5 is a photograph showing the results of immunohistochemical analysis of human liver tissue. A (top) is 1 5
0倍、 B (下） が 3 0 0倍。 0 times, B (bottom) is 300 times.
図 6は、 ヒト腎臓皮質組織の免疫組織化学的解析結果を示す写真。 A (上） が FIG. 6 is a photograph showing the results of immunohistochemical analysis of human kidney cortical tissue. A (top) is
1 5 0倍、 B (下） が 3 0 0倍。 150 times, B (bottom) is 300 times.
図 7は、 ヒト副腎皮質組織の免疫組織化学的解析結果を示す写真 （3 0 0倍） 。 図 8は、 ヒト睾丸組織の免疫組織化学的解析結果を示す写真 （3 0 0倍） 。 図 9は、 ヒト胃上皮組織の免疫組織化学的解析結果を示す写真 （3 0 0倍） 。 図 1 0は、 マウスにおける本発明のレブチン誘導遺伝子の発現を抑制した ES 細胞の構築工程を示す図と写真。 ゲノミック DNAに夕ーゲティングベクタ一で FIG. 7 is a photograph (× 300) showing the results of immunohistochemical analysis of human adrenocortical tissue. FIG. 8 is a photograph (× 300) showing the results of immunohistochemical analysis of human testis tissue. FIG. 9 is a photograph (× 300) showing the results of immunohistochemical analysis of human gastric epithelial tissue. FIG. 10 is a set of photographs and photographs showing the steps of constructing ES cells in mice that suppress the expression of the levulin-inducible gene of the present invention. Genomic DNA with the best targeting vector
「ネオマイシン耐性遺伝子（neogene )」 を導入することにより、 レブチン誘導遺 伝子の開始コドンを含む BamHIサイ トを失う （上） 。 その結果、 サザンブロッ 卜で 3，probeによる 8 kbのバンドが現れる （下） 。
発明を実施するための最良の形態 By introducing the neomycin resistance gene (neogene), the BamHI site containing the initiation codon of the lebutin-inducible gene is lost (top). As a result, an 8 kb band due to 3, probe appears in the Southern blot (bottom). BEST MODE FOR CARRYING OUT THE INVENTION
以下、 実施例により本発明をさらに詳細に説明するが、 本発明はこれら実施例 に制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[実施例 1 ] マウスのレプチン誘導遺伝子の単離 [Example 1] Isolation of mouse leptin-inducible gene
ob/obマウスにおけるレブチン投与の有無によって発現レベルが変動する遺伝 子を、 マウスレブチン誘導遺伝子として単離した。 ob/obマウスは、 受容体側の シグナル経路は正常であるが、 リガンドであるレプチン合成が正常にできないた め肥満を呈する。 このマウスにレブチンを投与すれば、 レプチンによって誘導さ れる遺伝子を見出すことができる。 このような条件とすることにより、 レブチン 投与の有無によってもたらされる遺伝子発現の差異を大きくできると考えた。 発 現レベルの異なる遺伝子の取得には、 サブトラクシヨン法を利用した。 具体的な 操作は以下のとおりである。 A gene whose expression level fluctuated depending on the presence or absence of lebutin administration in ob / ob mice was isolated as a mouse lebutin-inducible gene. Ob / ob mice have normal signaling pathways on the receptor side, but exhibit obesity due to inability to synthesize ligand leptin normally. When leptin is administered to these mice, leptin-induced genes can be found. It was thought that such a condition could increase the difference in gene expression caused by the presence or absence of lebutin administration. The subtraction method was used to obtain genes with different expression levels. The specific operation is as follows.
まず ob/obマウスにレプヂンを投与した。 ob/obマウスは米国ジャクソン研究 所より購入した。 体重 l gにっき 1 gのマウスレブチン（R & D 社） 、 もしく は PBSを静脈注射によって投与し、 1時間後に視床下部を採取し、 Total RNA をグァニジン一チオシァネート法によって抽出した。 その後 Roche社の ol igo d T cel lulose column を用いて NAを精製した。 この mMAを用いて PCRサブト ラクシヨン法を実施した。 First, leppan was administered to ob / ob mice. Ob / ob mice were purchased from the Jackson Laboratory, USA. 1 g of mouse lebutin (R & D) or PBS was administered by intravenous injection, and 1 hour later, the hypothalamus was collected, and total RNA was extracted by the guanidine monothiocyanate method. Then, NA was purified using an Oligo d T cell lulose column from Roche. The PCR subtraction method was performed using this mMA.
PCRサブトラクシヨン法には、 市販のキット 「PCR-Select™ cDNA Subtractio n kitj (CLONTECH製）を用いた。 A commercially available kit “PCR-Select ™ cDNA Subtraction kit” (manufactured by CLONTECH) was used for the PCR subtraction method.
まず各 mRNA 2〃gから AMV逆転写酵素を用いて cDNAを合成した。 レブチン投 与後の cDNAをテスター、 PBS投与後の cDNAをドライバ一とした。 cDNAは Rsal で消化し断片化した。 Rsalによる断片は、 平滑末端を有する。 First, cDNA was synthesized from 2 µg of each mRNA using AMV reverse transcriptase. The cDNA after administration of lebutin was used as a tester, and the cDNA after administration of PBS was used as a driver. The cDNA was digested with Rsal and fragmented. The Rsal fragment has blunt ends.
次に、 テスター cDNAを 2群に分け、 それぞれに異なる cDNAアダプタ一 1、 2R を結合した。 各アダプターの塩基配列は次のとおりである。 なおドライバ一 cDN Aにはアダプタ一を結合しない。
アダプター 1 Next, the tester cDNAs were divided into two groups, and different cDNA adapters-11 and 2R were connected to each. The nucleotide sequence of each adapter is as follows. Note that an adapter is not connected to the driver / cDN A. Adapter 1
5， CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT 3，（配列番号： 5 ) 3， GGCCCGTCCA 5，（配列番号： 6 ) 5, CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT 3, (SEQ ID NO: 5) 3, GGCCCGTCCA 5, (SEQ ID NO: 6)
アダプター 2R Adapter 2R
5' CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT 3， （配列番号： 7 ) 5 'CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT3, (SEQ ID NO: 7)
3' GCCGGCTCCA 5' (配列番号： 8 ) 3 'GCCGGCTCCA 5' (SEQ ID NO: 8)
次に、 1stハイブリダイゼーシヨンと 2ndハイブリダイゼーシヨンの 2回のハ イブリダィゼ一シヨンを行った。 1stハイブリダィゼ一シヨンでは、 各テスター に過剰量ドライバ一 cDNAを加え、 98°C1.5分間の熱変性を行った後、 68°Cで 8 時間インキュベーションした。 2ndハイブリダィゼ一シヨンでは、 1stハイプリ ダイゼーシヨンから得られたサンプルを変性せずに混合し、 さらに変性 cDNA ド ライバーを加え、 68°Cでー晚インキュベーションした。 反応後のハイプリグイ ゼ一シヨン溶液を希釈用バッファ一で希釈し、 75°Cで 5分インキュベートして 1本鎖のアダプタ一部を 2本鎖に伸長し、 PCRの鍊型に用いた。 アダプターに対 応するプライマ一である PCRプライマ一 1を用いた PCRを行い、 両端に異なる アダプタ一を持った cDNAのみを選択的に増幅した。 このとき、 ドライバー cDNA の末端にはアダプターが結合していないので、 ドライバ一 cDNAとハイブリダィ ズしたテス夕一 cDNAは増幅されない。 Next, two hybridizations, a first hybridization and a second hybridization, were performed. In the first hybridization, an excess amount of driver cDNA was added to each tester, heat denaturation was performed at 98 ° C for 1.5 minutes, and then incubation was performed at 68 ° C for 8 hours. In the second hybridization, samples obtained from the first hybridization were mixed without denaturation, and a denatured cDNA driver was added thereto, followed by incubation at 68 ° C. After the reaction, the hybridization solution was diluted with a buffer for dilution, and incubated at 75 ° C. for 5 minutes to extend a part of the single-stranded adapter into a double-stranded DNA, which was used for PCR type II. PCR was performed using PCR primer 1, which is a primer corresponding to the adapter, to selectively amplify only cDNAs having different adapters at both ends. At this time, since no adapter is bound to the end of the driver cDNA, the tester cDNA hybridized with the driver cDNA is not amplified.
PCRプライマー 1 5， CTAATACGACTCACTATAGGGC 3' (配列番号： 9 ) PCR primer 15, CTAATACGACTCACTATAGGGC 3 '(SEQ ID NO: 9)
これの一部を銃型とし、 アダプタープライマー 1、 2 Rの内側のプライマーで ある Nested PCRプライマ一 1と 2 Rを用いて PCRを行い、 さらに選択性を増し た生成物を得た。 A part of this was made into a gun form, and PCR was performed using Nested PCR primers 1 and 2R, which are primers inside the adapter primers 1 and 2R, to obtain a product with further increased selectivity.
Nested PCRプライマ一 1 55 TCGAGCGGCCGCCCGGGCAGGT 3，（配列番号 : 1 0 ) Nested PCRプライマ一 2R 5' AGGGCGTGGTGCGGAGGGCGGT 3，（配列番号： 1 1 ) この生成物を pT-Adv (Clontech社）ベクターにクローニングし、 BRL社の E1 ctroMaxDHlOBセルを用いて形質転換を行い、 サブトラクシヨンライブラリーを
作成した。 Nested PCR Primer 1 5 5 TCGAGCGGCCGCCCGGGCAGGT 3, (SEQ ID NO: 10) Nested PCR Primer 2R 5 ′ AGGGCGTGGTGCGGAGGGCGGT 3, (SEQ ID NO: 11) This product was cloned into pT-Adv (Clontech) vector, Transform using BRL's E1 ctroMaxDHlOB cell and extract the subtraction library. Created.
ライブラリーの各クローン cDNAのィンサ一トを PCi こよって増幅し、 ナイ口 ンメンプレン上にスポットしてアレイを作製した。 次に、 レブチン投与および P BS投与マウス cDNAから調製したジゴキゲニン標識プローブを用いて、 ハイプリ ダイズを行った。 検出には Roche社の抗ジゴキゲニン抗体、 CDP- starを用いた。 ハイブリダイゼーションの結果、 発現量に差が見られたクローンをスクリーニン グした。 An insert of each cDNA clone in the library was amplified by PCi and spotted on a nylon membrane to produce an array. Next, hybridization was performed using a digokigenin-labeled probe prepared from mouse cDNAs administered with lebutin and PBS. Roche anti-digokigenin antibody, CDP-star, was used for detection. As a result of hybridization, clones showing a difference in the expression level were screened.
サブトラクシヨンライブラリ一のスクリ一二ング Screening of Subtraction Library
レブチン、 もしくは PBS投与後の視床下部より調整した各 mRNA 1 gを使用 して逆転写酵素反応により DIG-dUTPを取り込んだ cDNAプローブを作成した。 作成は Roche社 'The DIG system user' s guide for filter hybridization PP20-21 , Preparing cDNA with digoxigenin-ll-dUTP' .に従った。 生成物を 5 一 1 O ng/mlの濃度で standard hybridization buffer (5x SSC, 0. 1 % N-laur oylsarcosine, 0.02 % SDS, 1% blocking reagent{Roche ¾ Blocking Reagen t} )に溶解し、 ハイブリダィゼ一シヨンに用いた。 PBS-投与後の mRNAをドライ バーとして得られたサブトラクシヨンライブラリ一より 384 wel l， 3枚分 （計 1 1 5 2クローン） のコロニーを任意に選択した。 Using 1 g of each mRNA prepared from the hypothalamus after administration of lebutin or PBS, a cDNA probe incorporating DIG-dUTP was prepared by a reverse transcriptase reaction. The preparation was performed according to Roche's 'The DIG system user's guide for filter hybridization PP20-21, Preparing cDNA with digoxigenin-ll-dUTP'. The product was dissolved in a standard hybridization buffer (5x SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 1% blocking reagent {Roche ¾ Blocking Reagen t}) at a concentration of 51 O ng / ml, and hybridized. Used for one shot. From the subtraction library obtained using the mRNA after PBS-administration as a driver, 384-well, 3 colonies (total of 1152 clones) were selected arbitrarily.
これらの大腸菌のストツクより pT- Advベクタ一に挿入されている cDNAを PCR 法によって増幅し、 Amersham - Pharmacia社の positively charged Nylon膜上 に duplicateでスポッ トした （サブトラクシヨン cDNAアレイ） 。 これらのァレ ィを 4組作成し、 それぞれ PBS -， レブチン投与後の DIG標識 cDNAプローブで 6 8 °C 1晚ハイブリダィズした。 その後、 2x SSC 0.1 % SDS 溶液を用いて室温 で五分ずつ二回洗浄し、 6 8 °Cの O. lx SSC、 0. 1 % SDS溶液で 2 0分ずつ二回 洗浄した。 その後アレイ上で発現量に差の見られたクローンのィンサートをプロ ーブとして cRNAを作成し、 ノーザンプロットを行い、 実際に発現量に差異があ るか否かを検討した。
次に発現量に差異があるポジティブクローンをマウスのレプチン誘導遺伝子と して選択し、 そのインサートの塩基配列を決定した。 本サブトラクシヨンライブ ラリーは Rsalによる切断を経ているためこのライブラリーでは全長 cDNAが得 られなかった。 ノーザンプロット解析により本遺伝子は腎臓、 肝臓にその発現が 多く見られたことからマウス腎臓由来の mRNAを使用して cDNAライブラリ一を 作成した。 cDNAライブラリーは BRL社 （現 In Vitrogen社） の Superscript PI asniid Systemを使用して作成し、 pSPORTlベクタ一に cDNAを挿入した。 先に同 定された遺伝子断片をプローブとしてライブラリーをスクリーニングして全長を 取得した。 マウスレブチン誘導遺伝子の塩基配列を配列番号： 1に、 また、 ォー プンリーディングフレームから予測されるアミノ酸配列を配列番号： 2に記載す る。 The cDNA inserted into the pT-Adv vector was amplified by PCR from these E. coli stocks and spotted as duplicates on Amersham-Pharmacia's positively charged Nylon membrane (subtraction cDNA array). Four sets of these arrays were prepared, and each was hybridized with a DIG-labeled cDNA probe after administration of PBS- and lebutin at 68 ° C for 1 晚. Thereafter, the plate was washed twice with a 2x SSC 0.1% SDS solution at room temperature for 5 minutes, and twice with an O.lx SSC, 0.1% SDS solution at 68 ° C for 20 minutes. After that, cRNA was prepared using the inserts of the clones having a difference in the expression level on the array as a probe, and a Northern plot was performed to examine whether or not the expression level actually differs. Next, a positive clone having a difference in the expression level was selected as a mouse leptin-inducible gene, and the nucleotide sequence of the insert was determined. Since this subtraction library had been cut with Rsal, full-length cDNA could not be obtained with this library. As a result of Northern blot analysis, the expression of this gene was found to be high in kidney and liver, so a cDNA library was prepared using mRNA from mouse kidney. A cDNA library was prepared using the Superscript PI asniid System of BRL (now In Vitrogen), and the cDNA was inserted into the pSPORTl vector. The library was screened using the previously identified gene fragment as a probe to obtain the full length. The nucleotide sequence of the mouse lebutin-inducible gene is described in SEQ ID NO: 1, and the amino acid sequence predicted from the open reading frame is described in SEQ ID NO: 2.
[実施例 2 ] レブチンによる ob/obマウス視床下部における遺伝子誘導 ob/obマウスに体重 1 g当たり 1 gのマウスレプチン （+ ) 、 または PBS ( - ) を静脈経由で投与し、 1時間後に視床下部を摘出して Total MAおよび mMAを抽出した。 抽出した mRNA0.4〃gをァガロースゲル上で分離し、 Amersha m_Pharmacia社の positively charged Nylon 膜—ヒにキヤビラリ一卜ランスファ 一した。 本発明の遺伝子の全長 cDNAより調製した D I G標識 cRNAをプローブと してハイブリダィズさせ、 ストリンジェン卜な条件下（0. 1 X SSC, 0. 1 % SDS, 68°C )で洗浄した。 その後アルカリフォスファタ一ゼ標識抗 DIG抗体、 および CD P- Starを用いてシグナルを検出した。 化学発光シグナルは ATT0社の CCDカメラ で定量した。 [Example 2] Gene induction in the hypothalamus of ob / ob mice with lebutin 1 g of mouse leptin (+) or PBS (-) per 1 g of body weight was administered to ob / ob mice via vein, and 1 hour later, thalamus The lower part was excised to extract Total MA and mMA. 0.4 μg of the extracted mRNA was separated on an agarose gel and transferred to a positively charged Nylon membrane of Amersham Pharmacia Co., Ltd. The DIG-labeled cRNA prepared from the full-length cDNA of the gene of the present invention was hybridized as a probe, and washed under stringent conditions (0.1 × SSC, 0.1% SDS, 68 ° C.). Thereafter, signals were detected using an alkaline phosphatase-labeled anti-DIG antibody and CDP-Star. The chemiluminescence signal was quantified using an ATTO CCD camera.
その結果レプチン投与によりこの転写産物量は約 2倍になることが明らかにな つた。 さらにネガチイブコントロールとして同量の mRNAがのせられたメンブレ ンを別のプローブ （BRI遺伝子， AF272044) でプロ一ビングを行った。 この場合 にはレブチン、 PBS投与による遺伝子の mRNAレベルの差異は観察されなかった。
以上の結果を図 1に示した。 この実験により、 本発明の遺伝子 (A9 )が、 ob/obマ ウスの視床下部において、 レブチンの投与 1時間後に顕著に発現が増強すること が裏付けられた。 As a result, it was clarified that the amount of this transcript was approximately doubled by leptin administration. In addition, a membrane on which the same amount of mRNA was applied as a negative control was probed with another probe (BRI gene, AF272044). In this case, no difference in the mRNA level of the gene due to the administration of lebutin and PBS was observed. The above results are shown in FIG. This experiment confirmed that the expression of the gene of the present invention (A9) was significantly enhanced in the hypothalamus of ob / ob mice 1 hour after administration of lebutin.
[実施例 3 ] マウスレプチン誘導遺伝子の組織特異的発現 [Example 3] Tissue-specific expression of mouse leptin-induced gene
マウスレプチン誘導遺伝子の組織特異的発現をノーザンプロッ トにより解析し た。 Tissue-specific expression of the mouse leptin-inducible gene was analyzed by Northern blot.
野生型マウス各組織より Total RNAをグァニジン一チオシァネート法によつ て抽出した。 この Total MAを各 8〃gずつ使用し、 該遺伝子の DIG標識 c A をプローブとしてノーザンブロッ卜を行った。 Total RNAはァガロースゲルによ つて分離した後 Amersham-Pharmacia社の positively charged Nylon 莫上にキ ャピラリートランスファ一によってプロッティングした。 Total RNA was extracted from each tissue of the wild-type mouse by the guanidine-thiocyanate method. Using this total MA in an amount of 8 μg each, Northern blotting was performed using DIG-labeled cA of the gene as a probe. Total RNA was separated by agarose gel and then plotted on a positively charged Nylon from Amersham-Pharmacia by capillary transfer.
本発明の遺伝子の DIG標識 cRNAをプローブとしてハイプリダイズさせた。 洗 浄は実施例 2の場合と同様に非常にきつい条件で行い、 シグナル検出も同様の方 法で行った。 結果を図 2に示した。 転写産物の大きさは 8 0 0 - 9 0 O bpと推 定される。 このサイズは、 配列番号： 1に示した塩基配列の長さ（827b)と一致 する。 各臓器中で腎臓での発現が最も高く、 次いで肝臓でも強い発現が見られた。 本発明の遺伝子が野生型マウスにおいても発現していることが確認できた。 Hybridization was performed using DIG-labeled cRNA of the gene of the present invention as a probe. Washing was carried out under very harsh conditions as in Example 2, and signal detection was carried out in the same manner. The results are shown in FIG. The size of the transcript is estimated to be 800-90 Obp. This size matches the length (827b) of the nucleotide sequence shown in SEQ ID NO: 1. Among the organs, expression was highest in the kidney, followed by strong expression in the liver. It was confirmed that the gene of the present invention was also expressed in wild-type mice.
[実施例 4 ] マウスレプチン誘導遺伝子によってコードされる蛋白質を認識 する抗体 [Example 4] Antibody recognizing protein encoded by mouse leptin-inducible gene
配列番号： 2のアミノ酸配列からなる蛋白質を認識する抗体の調製を試みた。 該遺伝子の 0RFから予測される全長部分を PCR反応によって増幅し、 Amersham- Pharmacia社の pGEX4T- 1の EcoRI-XhoIに組み込んだ。 このベクターを大腸菌に 形質転換し、 IPTGで誘導したところ約 50 kDaのタンパク質 （GSTタンパク質と の融合産物） の産生増幅が観察された。 組み換えタンパク質は大腸菌を超音波に
よって破壊し、 Glutathione Sepharose 4Bを用いて精製した。 このタンパク質 に対するポリクローナル抗体を作成するため精製組み換えタンパク質 2 mgをゥ サギに 3回に分けて免疫し、 抗血清を得た。 抗血清を更に Protein Aカラムで 精製して、 抗 A9抗体として用いた。 An attempt was made to prepare an antibody that recognizes a protein consisting of the amino acid sequence of SEQ ID NO: 2. The full-length portion predicted from 0RF of the gene was amplified by PCR, and incorporated into EcoRI-XhoI of pGEX4T-1 from Amersham-Pharmacia. Escherichia coli was transformed with this vector and induced with IPTG. As a result, production and amplification of a protein of about 50 kDa (fusion product with GST protein) was observed. Recombinant protein transforms E. coli into ultrasound Therefore, it was disrupted and purified using Glutathione Sepharose 4B. In order to prepare a polyclonal antibody against this protein, 2 mg of the purified recombinant protein was immunized in three times against a egret to obtain an antiserum. The antiserum was further purified on a Protein A column and used as an anti-A9 antibody.
抗 A9抗体の特異性と、 本発明の遺伝子によってコ一ドされる蛋白質の存在を ウエスタンプロット法によって確認した。 抗原としては、 次の蛋白質、 あるいは 細胞抽出液を用いた。 The specificity of the anti-A9 antibody and the presence of the protein encoded by the gene of the present invention were confirmed by Western blotting. The following proteins or cell extracts were used as antigens.
• GSTと本発明の蛋白質の融合蛋白質 • Fusion protein of GST and the protein of the present invention
•マウス腎臓の全細胞抽出液 • Mouse kidney whole cell extract
•本発明の遺伝子を過剰発現させた C0S7の細胞抽出液 • Cell extract of C0S7 overexpressing the gene of the present invention
本発明の遺伝子を過剰発現させた C0S7の細胞抽出液は、 次のようにして調製 した。 すなわち、 本発明の遺伝子を SR aのプロモーターを持つ pM18Sに揷入し、 C0S7細胞にリボフヱク夕ミン法でトランスフヱクトした。 遺伝子導入後 3日目 に細胞を ΪΠΡΑバッファーで溶かして細胞抽出液とした。 The cell extract of C0S7 overexpressing the gene of the present invention was prepared as follows. That is, the gene of the present invention was introduced into pM18S having an SRa promoter, and transfected into C0S7 cells by the ribonucleoside method. On the third day after gene transfer, the cells were lysed with ΪΠΡΑ buffer to obtain a cell extract.
これらの蛋白質、 あるいは細胞抽出液を、 SDSサンプルバッファ一に溶解し、 熱処理後、 SDSポリアクリルアミ ド電気泳動によって分離した。 分離した蛋白質 をブロッテイングしたメンブランに、 抗 Α9抗体を反応させた。 抗 Α9抗体は TBS Τ-5 % non-fat mi lkで 1 / 1 0 0 0に薄めて使用し、 HRP (ホースラデシュパー ォキシダーゼ）標識二次抗体 （抗ゥサギ IgG 1 / 3 0 0 0 ) でインキュベート後、 NEB社 Renaissance試薬で検出した。 結果を図 3に示した。 These proteins or cell extracts were dissolved in SDS sample buffer, heat-treated, and separated by SDS polyacrylamide electrophoresis. An anti-antibody 9 was reacted with the membrane on which the separated proteins were blotted. Anti-Α9 antibody was diluted to 1/1000 with TBS Τ-5% non-fat milk and used with HRP (horseradish peroxidase) -labeled secondary antibody (anti-Egret IgG 1/300). After incubation, detection was performed using Renaissance reagent from NEB. The results are shown in FIG.
その結果、 抗 A9抗体は、 融合タンパク質 （約 5 0 kDa) への反応性は確認さ れたが、 GSTに対する反応性は検出できなかった。 また本発明の遺伝子を過剰発 現させた C0S7細胞の細胞抽出液 （レーン 5 ) では、 配列番号： 2のアミノ酸配 列からなる蛋白質に相当する約 2 3 kDaのバンドが確認された。 以上の結果から、 抗 A9抗体は、 本発明の蛋白質に対し、 高い特異性を有することが確認された。 更に、 マウス腎臓の全細胞抽出液 （レーン 3— 4、 および 6 ) において、 レ一
ン 5と同じ約 2 3 kDaのバンドが確認された。 このことは、 マウスの腎臓におい て、 配列番号： 2のアミノ酸配列からなる蛋白質が存在していることを示してい る。 As a result, the reactivity of the anti-A9 antibody to the fusion protein (about 50 kDa) was confirmed, but the reactivity to GST could not be detected. In the cell extract of C0S7 cells overexpressing the gene of the present invention (lane 5), a band of about 23 kDa corresponding to the protein consisting of the amino acid sequence of SEQ ID NO: 2 was confirmed. From the above results, it was confirmed that the anti-A9 antibody had high specificity for the protein of the present invention. In addition, in the mouse kidney whole cell extract (lanes 3-4 and 6), A band of about 23 kDa, which is the same as that of step 5, was confirmed. This indicates that a protein consisting of the amino acid sequence of SEQ ID NO: 2 is present in the mouse kidney.
[実施例 5 ] ヒトのレプチン誘導遺伝子 [Example 5] Human leptin-inducible gene
実施例 1において決定したマウスに由来するレブチン誘導遺伝子の塩基配列 (配列番号： 1 ) を queryとして、 GenBankのデ一夕をホモロジ一サーチした。 その結果、 あるヒト ESTの塩基配列（GenBank Acc . No. Z84479 )が見出された。 Z 84479は、 ヒト 1 6番染色体 16pl3. 3にマッピングされた ESTである。 公知の情 報によれば、 Z84479にいくつかの repeat regionが見出されているが、 0RFは 明らかでない。 両者の塩基配列を更に詳細に比較解析したところ、 Z84479の塩 基配列と、 配列番号： 1の塩基配列は、 逆向き相補配列となっていた。 そこで、 Z84479の塩基配列に基づいて逆向き相補配列を導き、 更に 0RFを解析した。 Using the base sequence (SEQ ID NO: 1) of the mouse-derived lebutin-inducible gene determined in Example 1 as a query, a homology search was performed on GenBank data. As a result, a nucleotide sequence of a certain human EST (GenBank Acc. No. Z84479) was found. Z84479 is an EST mapped to human chromosome 16 16pl3.3. According to known information, several repeat regions are found in Z84479, but 0RF is not clear. When the base sequences of both were compared in more detail, the base sequence of Z84479 and the base sequence of SEQ ID NO: 1 were inverted complementary sequences. Therefore, an inverted complementary sequence was derived based on the base sequence of Z84479, and 0RF was further analyzed.
最終的に、 Z84479の逆向き相補配列 （配列番号： 3 ) によって、 配列番号： 4に示すアミノ酸配列がコードされていることが明らかになった。 そして配列番 号： 4のアミノ酸配列は、 配列番号： 2のアミノ酸配列 （マウス） と高い相同性 を示した （図 4 ) 。 これらの結果に基づいて、 配列番号： 3に示す塩基配列から なる遺伝子を、 レブチン誘導遺伝子のヒトにおけるォーソログであると判断した。 念のため、 Z84479に基づくプローブで antisense cRNAのプローブを作成しノ ーザンブロット解析を行ったところ全くシグナルが検出できなかった。 Z84479 はアンチセンス配列となっているため、 mRNAの検出ができなかったためと考え られた。 Finally, it was revealed that the reverse complementary sequence of Z84479 (SEQ ID NO: 3) encodes the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 showed high homology with the amino acid sequence of SEQ ID NO: 2 (mouse) (FIG. 4). Based on these results, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 3 was determined to be the ortholog of the lebutin-induced gene in humans. As a precaution, an antisense cRNA probe was prepared using a probe based on Z84479, and Northern blot analysis was performed. No signal was detected. It was considered that mRNA could not be detected because Z84479 had an antisense sequence.
[実施例 6 ] 抗 A 9抗体による免疫組織化学的な解析 [Example 6] Immunohistochemical analysis using anti-A9 antibody
抗 A 9抗体を用いて、 様々な組織を免疫組織化学的に解析した。 配列番号： 2 (マウス） のアミノ酸配列と、 配列番号： 4 (ヒトのアミノ酸配列） は、 図 4に
示すように高いホモロジ一を有する。 したがって、 マウスの蛋白質を免疫原とし て得た抗 A 9抗体は、 ヒ卜の蛋白質を認識する可能性が高い。 Various tissues were analyzed immunohistochemically using anti-A9 antibody. The amino acid sequence of SEQ ID NO: 2 (mouse) and SEQ ID NO: 4 (human amino acid sequence) are shown in FIG. It has a high homology as shown. Therefore, an anti-A9 antibody obtained using mouse protein as an immunogen is highly likely to recognize human protein.
実施例 4と同じ抗 A 9抗体、 および二次抗体を用いて、 免疫組織化学的解析を 行った。 解析に用いたヒト組織は次のとおりである。 これらの組織の切片を常法 にしたがってスライ ドガラスに固定し解析に用いた。 Using the same anti-A9 antibody and secondary antibody as in Example 4, immunohistochemical analysis was performed. The human tissues used for the analysis are as follows. Sections of these tissues were fixed on slide glass according to a conventional method and used for analysis.
肝臓 （図 5 ) 睾丸 （図 8 ) Liver (Figure 5) Testicle (Figure 8)
腎臓皮質 （図 6 ) 胃上皮 （図 9 ) Kidney cortex (Figure 6) Gastric epithelium (Figure 9)
副腎皮質 （図 7 ) Adrenal cortex (Figure 7)
解析の結果を図 5—図 9に示す。 抗 A 9抗体は、 ヒトのレブチン誘導遺伝子に よってコ一ドされるタンパク質を認識することが示された。 またヒト組織におけ る局在から、 ヒトにおけるレブチン誘導遺伝子には、 以下のような機能が推測さ れた。 The results of the analysis are shown in Figs. The anti-A9 antibody was shown to recognize a protein encoded by the human levulin-inducible gene. In addition, from the localization in human tissues, the following functions were presumed for the lebutin-inducible gene in humans.
まず肝臓のへパトサイ 卜に、 強いシグナルが見られた （図 5 ) 。 レブチン依存 的なグリコーゲンの貯蔵に本発明の蛋白質が関与する可能性が考えられた。 また 腎臓皮質においては尿細管への局在が見られた （図 6 ) 。 副腎皮質の球状層にも 本発明の蛋白質の局在が見られた （図 7 ) 。 副腎皮質の球状層はステロイ ドホル モンの産生に関与する組織であることから、 レブチンとステロイ ドホルモン産生 系との関連が示唆された。 また睾丸の間細胞 (Leidig cell )における本発明の蛋 白質の局在 （図 8 ) は、 レブチン投与による男性ホルモン産生系の阻害作用を裏 付ける結果と思われた。 更に、 胃上皮細胞におけるシグナル （図 9 ) は、 レプチ ンによる消化機能の制御を示唆していた。 First, a strong signal was observed in the liver hepatocyte (Fig. 5). It was considered that the protein of the present invention may be involved in lebutin-dependent glycogen storage. In the renal cortex, localization to tubules was observed (Fig. 6). The localization of the protein of the present invention was also found in the globular layer of the adrenal cortex (FIG. 7). The globular layer of the adrenal cortex is a tissue involved in steroid hormone production, suggesting a relationship between lebutin and the steroid hormone-producing system. In addition, the localization of the protein of the present invention in intertesticular cells (Leidig cells) (FIG. 8) was considered to be a result supporting the inhibitory effect of the administration of lebutin on the male hormone production system. Furthermore, signals in gastric epithelial cells (Figure 9) suggested that leptin controls digestive function.
[実施例 7 ] 本発明の蛋白質の細胞内局在 [Example 7] Subcellular localization of the protein of the present invention
本発明の蛋白質の細胞内における局在について、 抗体を使用しコンフォーカル 顕微鏡を用いて検討した。 Hela細胞に本発明の遺伝子を強発現させた細胞を固 定化後、 実施例 4と同じ抗 A 9抗体、 および蛍光標識された二次抗体を用いて、
細胞内局在を解析した。 また同様に調製した標本について、 小胞体のマーカー夕 ンパク質である calnexinの局在と比較した。 その結果、 本発明の蛋白質は caln exinと同じ部分に局在していることが確認できた。 この結果から、 本発明の蛋 白質は、 細胞質の小胞体に多く存在することが明らかになつた。 The localization of the protein of the present invention in cells was examined using an antibody and a confocal microscope. After immobilizing the cells in which the gene of the present invention was strongly expressed in Hela cells, using the same anti-A9 antibody as in Example 4 and a fluorescently labeled secondary antibody, The intracellular localization was analyzed. In the same preparation, the localization of calnexin, a marker protein of the endoplasmic reticulum, was compared. As a result, it was confirmed that the protein of the present invention was localized in the same portion as calnexin. From these results, it was clarified that the protein of the present invention was abundantly present in the endoplasmic reticulum.
[実施例 8 ] 本発明の蛋白質の発現を抑制した E S細胞 [Example 8] ES cells in which expression of the protein of the present invention was suppressed
. 先に述べたように本発明の遺伝子には既知のモチーフが存在せず、 いかなる既 知のタンパク質とも相同性を有しない。 したがって、 その生理機能を明らかにす る上で、 本発明の遺伝子の欠損マウス （ノックアウトマウス） が果たす役割は大 きい。 ノックアウトマウスを得ることができれば、 このマウスにおける表現形の 変化を観察することによって、 本発明の遺伝子の生理機能を明らかにすることが できる。 As mentioned above, the gene of the present invention has no known motifs and has no homology to any known proteins. Therefore, the role of the gene-deficient mouse (knockout mouse) of the present invention in clarifying its physiological function is significant. If a knockout mouse can be obtained, the physiological function of the gene of the present invention can be clarified by observing a change in the phenotype in the mouse.
ノックァゥトマウスを作成するため、 まず本遺伝子のゲノミック DNAを取得 した。 Stratagene社の 129SvJ由来のゲノミックライブラリーを、 本発明の遺伝 子の塩基配列に基づいてデザインしたプローブを用いてスクリーニングした。 プ ローブには 29 merの DIG標識オリゴヌクレオチドを用いた。 プローブの塩基配 列を以下に示す。 First, genomic DNA of this gene was obtained in order to create knockout mice. A genomic library derived from Stratagene 129SvJ was screened using a probe designed based on the nucleotide sequence of the gene of the present invention. The probe used was a 29 mer DIG-labeled oligonucleotide. The base sequence of the probe is shown below.
プローブ配列： 5，- GGCTCTTTAGAGCAGCAGGACACCTGCTC- 3' (配列番号 : 1 2 ) ポジティブクローンを pKS( + ) ベクター（Stratagene社）の Notl siteにサブ クローン化し 6塩基認識制限酵素を使用して本発明の遺伝子のゲノミックマツピ ングを行った。 マッピングの結果は図 1 0の 「ゲノミック DNA」 として示したと おりである。 次にマッピングの結果に基づいて夕一ゲティングベクタ一を構築し た。 夕ーゲティングベクタ一の 5'アームには Spel と Asp718の配列を用いた。 また 3 'アームとしては Notl-Scalの部位を使用した。 tk- Neo遺伝子 （図 1 0の 「neogene」 ) をこれらの配列で挟み込み、 tk-Neo遺伝子が ATGを含むェキソン (Asp718から Notl部位まで） と入れ替わるように設計した。 夕ーゲティングべ
クタ一の構造とゲノミック DNAとの関係を、 図 1 0に示した。 Probe sequence: 5, -GGCTCTTTAGAGCAGCAGGACACCTGCTC-3 '(SEQ ID NO: 12) A positive clone was subcloned into the Notl site of the pKS (+) vector (Stratagene) and the 6-base recognition restriction enzyme was used to clone the gene of the present invention. Genomic mapping was performed. The mapping results are shown as “genomic DNA” in FIG. Next, based on the mapping results, we constructed a targeting vector. The Spel and Asp718 sequences were used for the 5 'arm of the evening targeting vector. The Notl-Scal site was used as the 3 'arm. The tk-Neo gene ("neogene" in Fig. 10) was flanked by these sequences, and the tk-Neo gene was designed to replace the exon containing ATG (from Asp718 to Notl site). Evening target Fig. 10 shows the relationship between the structure of the vector and the genomic DNA.
夕ーゲティングベクターは、 エレク卜ロボレ一シヨンによって ES細胞に導入 した。 G418耐性クローンを 1 0 0 0ケ選択し、 用いたベクターアームの外側の 配列 （3'側 ) をプローブとしサザンプロヅトを行った。 tk- Neo遺伝子が本発明 の遺伝子のゲノム DNA上に正確に組み込まれれば、 ゲノミック DNAは、 開始コ ドン ATGを含む BamHIサイ トを失う。 その結果、 BamHIによる制限酵素断片のパ 夕一ンは変化する。 つまり、 図 1 0に示されるように、 3'側のプローブを使用 したとき 4.2 kbpと 8 kbpの二本のバンドが観察されれば、 夕一ゲティングべ クタ一によつて正しく組み換えの起った ES細胞であることがわかる。 1 0 0 0 ケの G418耐性クローンより 4ケの組み換えが起った ES細胞を取得することが できた。 またこれらのクローンは tk-Neo遺伝子をプロ一ブとしたサザンプロッ 卜で 8 kbpのバンドを与えることを確認している。 産業上の利用の可能†生 The evening-targeting vector was introduced into ES cells by electro-boration. One hundred and ten G418 resistant clones were selected, and Southern blotting was performed using the sequence (3 'side) outside the vector arm used as a probe. If the tk-Neo gene is correctly integrated on the genomic DNA of the gene of the present invention, the genomic DNA loses the BamHI site containing the start codon ATG. As a result, the pattern of restriction enzyme fragments by BamHI changes. In other words, as shown in Fig. 10, if two bands of 4.2 kbp and 8 kbp were observed when the 3 'probe was used, recombination was correctly performed by the targeting vector in the evening. It turns out that it is an ES cell. From the 1000 G418-resistant clones, four recombinant ES cells could be obtained. In addition, it has been confirmed that these clones give an 8 kbp band in a Southern plot using the tk-Neo gene as a probe. Industrial use
本発明の遺伝子は、 レブチン応答の指標として有用である。 本発明の遺伝子は、 レブチンの投与によって、 速やかにその発現が誘導される。 したがって、 本発明 の遺伝子を指標とすることにより、 細胞や、 生体のレブチン応答の程度を評価す ることができる。 既に述べたように、 ヒトにおいてはレブチンの発現レベルでは なく、 レブチンに対する応答性のレベルが、 肥満と密接に関連していることが推 測されている。 したがって、 レブチンに対する応答性の指標となる遺伝子は重要 である。 The gene of the present invention is useful as an indicator of a lebutin response. The expression of the gene of the present invention is rapidly induced by administration of lebutin. Therefore, by using the gene of the present invention as an index, it is possible to evaluate the degree of lebutin response of a cell or a living body. As already mentioned, it has been estimated that the level of responsiveness to lebutin, but not the level of expression of lebutin in humans, is closely linked to obesity. Therefore, genes that are indicators of responsiveness to lebutin are important.
また本発明の遺伝子は、 レブチン抵抗性の診断指標として有用である。 すなわ ち、 本発明の遺伝子の発現レベルを測定し正常なレベルと比較することにより、 レブチン抵抗性の程度を知ることができる。 本発明の遺伝子は、 レブチンに応答 して短期間にその発現レベルが上昇するので、 レブチン応答性の指標として望ま しい。
Further, the gene of the present invention is useful as a diagnostic index for lebutin resistance. That is, by measuring the expression level of the gene of the present invention and comparing it with a normal level, the degree of lebutin resistance can be known. Since the expression level of the gene of the present invention increases in a short period of time in response to lebutin, it is desirable as an indicator of lebutin responsiveness.
Claims
請求の範囲 下記 （a ) から （e ) のいずれかに記載のポリヌクレオチド。 Claims The polynucleotide according to any one of (a) to (e) below.
( a ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列からなる蛋白 質をコードするポリヌクレオチド。 (a) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
( b ) 配列番号： 1または配列番号： 3に記載の塩基配列のコード領域を含 むポリヌクレオチド。 (b) a polynucleotide comprising the coding region of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
( c ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列において 1若 しくは複数のアミノ酸が置換、 欠失、 挿入、 および/または付加したァミノ 酸配列を有し、 配列番号： 2または配列番号： 4に記載のアミノ酸配列から なる蛋白質と機能的に同等な蛋白質をコードするポリヌクレオチド。 (c) one or more amino acids in the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 have an amino acid sequence in which substitution, deletion, insertion, and / or addition has been performed; A polynucleotide encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4.
( d ) 配列番号： 1または配列番号： 3に記載の塩基配列からなるポリヌク レオチドとストリンジェントな条件下でハイブリダイズし、 配列番号： 2ま たは配列番号： 4に記載のアミノ酸配列からなる蛋白質と機能的に同等な蛋 白質をコードするポリヌクレオチド。 (d) hybridizes with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A polynucleotide that encodes a protein functionally equivalent to a protein.
( e ) 配列番号： 2または配列番号： 4に記載のアミノ酸配列からなる蛋白 質に特異的に見出される部分アミノ酸配列を含む蛋白質をコードするポリヌ クレオチド。 (e) A polynucleotide encoding a protein comprising a partial amino acid sequence specifically found in a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
請求項 1に記載のポリヌクレオチドによりコードされる蛋白質。 A protein encoded by the polynucleotide according to claim 1.
請求項 1に記載のポリヌクレオチドが揷入されたベクター。 A vector into which the polynucleotide according to claim 1 has been inserted.
請求項 1に記載のポリヌクレオチドまたは請求項 3に記載のベクターを保 持する形質転換細胞。 A transformed cell carrying the polynucleotide according to claim 1 or the vector according to claim 3.
請求項 4に記載の形質転換細胞を培養し、 該形質転換細胞またはその培養 上清から発現させた蛋白質またはペプチドを回収する工程を含む、 請求項 2 に記載の蛋白質の製造方法。 The method for producing a protein according to claim 2, comprising a step of culturing the transformed cell according to claim 4, and recovering the expressed protein or peptide from the transformed cell or a culture supernatant thereof.
請求項 2に記載の蛋白質に結合する抗体。
配列番号： 1または配列番号： 3に記載の塩基配列からなるポリヌクレオ チドまたはその相補鎖に相補的な少なくとも 15塩基の連続した塩基配列を 含むポリヌクレオチド。 An antibody that binds to the protein according to claim 2. A polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a continuous nucleotide sequence of at least 15 nucleotides complementary to a complementary strand thereof.
次の工程を含む、 請求項 1に記載のポリヌクレオチドの発現を調節する活 性の評価方法。 The method for evaluating the activity of regulating the expression of the polynucleotide according to claim 1, comprising the following steps.
( a ) 被験化合物の存在下で請求項 1に記載のポリヌクレオチドを発現する 細胞にレブチンを接触させる工程、 および (a) contacting a cell expressing the polynucleotide of claim 1 with lebutin in the presence of a test compound, and
( b ) 前記ポリヌクレオチドの発現レベルを測定する工程、 (b) measuring the expression level of the polynucleotide,
次の工程を含む、 請求項 1に記載のポリヌクレオチドの発現を調節する活 性を有する化合物のスクリーニング方法。 A method for screening a compound having an activity of regulating the expression of the polynucleotide according to claim 1, comprising the following steps.
( a ) 請求項 8に記載の方法によって、 被験化合物の請求項 1に記載のポリ ヌクレオチドの発現を調節する活性を評価する工程、 および (a) evaluating the activity of the test compound to regulate the expression of the polynucleotide according to claim 1, by the method according to claim 8, and
( b ) 被験化合物非存在下でレプチンを接触させた対照と比較して前記ポリ ヌクレオチドの発現レベルを変化させる化合物を選択する工程 (b) selecting a compound that changes the expression level of the polynucleotide as compared to a control that has been contacted with leptin in the absence of the test compound
. 請求項 1に記載のポリヌクレオチド、 請求項 2に記載の蛋白質、 および請 求項 3に記載のベクタ一からなる群から選択された少なくとも 1つの成分を 含有する医薬組成物。A pharmaceutical composition comprising the polynucleotide according to claim 1, the protein according to claim 2, and at least one component selected from the group consisting of the vector according to claim 3.
. 次の工程を含む、 生体試料中の請求項 2に記載の蛋 (3質、 および/または その部分べプチドの測定方法。 A method for measuring the protein (3) and / or its partial peptide according to claim 2 in a biological sample, comprising the following steps:
( 1 )生体試料を請求項 6に記載の抗体と接触させる工程、 および (1) contacting a biological sample with the antibody according to claim 6, and
(2)請求項 2に記載の蛋白質、 および/またはその部分べプチドに結合する、 請求項 6に記載の抗体を検出する工程 (2) a step of detecting the antibody according to claim 6, which binds to the protein according to claim 2 and / or a partial peptide thereof;
. 請求項 6に記載の抗体を含む、 請求項 2に記載の蛋白質、 および/または その部分べプチドの測定用キッ ト。 A kit for measuring the protein according to claim 2 and / or a partial peptide thereof, comprising the antibody according to claim 6.
. 次の工程を含む、 生体試料中の請求項 1に記載のポリヌクレオチドの測定 方法。
(1)生体試料を請求項 7に記載のポリヌクレオチドと接触させる工程、 およ び The method for measuring the polynucleotide according to claim 1 in a biological sample, comprising the following steps. (1) contacting a biological sample with the polynucleotide according to claim 7, and
(2)生体試料中に含まれる、 請求項 7に記載のポリヌクレオチドとハイプリ ダイズするポリヌクレオチドを検出する工程 (2) a step of detecting a polynucleotide that hybridizes with the polynucleotide according to claim 7, which is contained in a biological sample.
14. 請求項 7に記載のポリヌクレオチドを含む、 請求項 1に記載のポリヌクレ ォチドの測定用キット。 14. The kit for measuring a polynucleotide according to claim 1, comprising the polynucleotide according to claim 7.
15. 請求項 2に記載の蛋白質の発現が改変されるように操作された非ヒト脊椎 動物。 15. A non-human vertebrate that has been engineered to alter the expression of the protein of claim 2.
16. ノックアウト動物またはトランスジエニック動物である、 請求項 15に記 載の非ヒト脊椎動物。 16. The non-human vertebrate of claim 15, which is a knockout animal or a transgenic animal.
17. 配列番号： 2に記載のアミノ酸配列からなるタンパク質の発現が抑制され ているマウス胚性幹細胞。 17. A mouse embryonic stem cell in which expression of a protein consisting of the amino acid sequence of SEQ ID NO: 2 is suppressed.
18. 配列番号： 1に記載された塩基配列を構成することができるェキソンにお いて、 少なくとも開始コドンに変異を有する請求項 17に記載のマウス胚性 幹細胞。
18. The mouse embryonic stem cell according to claim 17, which has a mutation in at least an initiation codon in an exon capable of constituting the base sequence described in SEQ ID NO: 1.
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| WO2000007014A2 (en) * | 1998-07-28 | 2000-02-10 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Leptin-mediated gene-induction |
| WO2000015790A2 (en) * | 1998-09-10 | 2000-03-23 | Millennium Pharmaceuticals, Inc. | Leptin induced genes |
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| WO2000007014A2 (en) * | 1998-07-28 | 2000-02-10 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Leptin-mediated gene-induction |
| WO2000015790A2 (en) * | 1998-09-10 | 2000-03-23 | Millennium Pharmaceuticals, Inc. | Leptin induced genes |
Non-Patent Citations (2)
| Title |
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| KAWAI J. ET AL.: "Functional annotation of a full-length mouse cDNA collection", NATURE, vol. 409, 8 February 2001 (2001-02-08), pages 685 - 690, XP001009930 * |
| Y. MORIKAWA ET AL.: "Isolation of novel genes induced leptin in the brain of ob/ob mice", SOCIETY FOR NEUROSCIENCE, vol. 25, no. 1-2, 1999, pages 412, XP002951577 * |
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