WO2001038529A1 - Novel polypeptide, novel dna, novel anitbody and novel transgenic animal - Google Patents

Abstract

A polypeptide having an activity of inhibiting animal aging; a DNA encoding this polypeptide; a method of preventing diseases associating aging and a method of treating these diseases by using the above-described polypeptide or the DNA encoding the polypeptide; a drug having an interaction with the polypeptide; a method of searching for the above-described drug by using the above-described polypeptide or DNA; and a non-human animal with the deletion or partial modification of the above-described DNA which is essentially required as a model animal in evaluating the above-described drug.

Description

 Detail

 New polypeptides, new DNAs, new antibodies and new genetically modified animals

 The present invention relates to a novel polypeptide having an activity of suppressing aging of an animal, a DNA encoding the polypeptide, a recombinant DNA containing the DNA, a transformant having the recombinant DNA, A method for producing a peptide, an antibody that recognizes the polypeptide, a ligand for the polypeptide, a compound that inhibits specific binding between the polypeptide and the ligand, aging that encodes the polypeptide The present invention relates to a compound that increases the expression of a suppressor gene, a method for searching for the compound, and a method for producing and using an animal in which the aging suppressor gene is deficient or partially modified. Background art

 The aging phenomenon refers to the deterioration of an individual in the functional and appearance changes of the individual, which usually progresses with aging, and it is known that the frequency of various adult diseases increases with aging. Therefore, drugs that can control aging in some way are expected to be therapeutic and prophylactic drugs for adult diseases, and protective and preventive drugs against functional and cosmetic deterioration.

 At the genetic level, research on aging of individuals has only just begun in the world, and there has been no molecular genetic information on aging of individuals. However, several types of hereditary premature aging are known, including Werner syndrome, Hutchinson's Guilford syndrome (Progeria), Down syndrome, Evening syndrome, Louis Barr syndrome, Rossmond-Thomson syndrome, etc. (Edited by Daisaburo Fujimoto: Mechanism and control of aging, IPC 1993).

For Werner syndrome, the causative gene has been identified! : Science, 272, 258 (1996)]. The gene responsible for Werner's syndrome is thought to be helicase, Various mutations have been observed in the genes of patients with Werner syndrome. In such patients, it is considered that normal helicase protein is not produced due to the gene mutation and the function is not expressed, thereby causing early aging symptoms.

 From the above, it was suggested that there is a gene involved in aging, and that mutation of this gene promotes aging. It is thought that multiple genes are involved in aging, since the causative genes are also present in other genetic premature aging.

 Based on this idea, the present inventors conducted a search for genes involved in aging and succeeded in cloning the klotho gene encoding a polypeptide having aging inhibitory activity (Nature, 390, 45 (1997), W098 / 29544]. Mice with reduced expression due to mutations in the klotho gene show various premature aging symptoms as shown in the following observation examples.

 [Observation example 1] External observation: Aged mice (8 to 9 weeks old) are about 4 cm long and considerably smaller than wild-type mice of the same age (about 6 cm). The ratio of the head to the length of the torso is large. In aged mice (8-week-old, male), the body is dwarf, but no abnormality is found in the hair, nails, etc. of the limbs. Behavior is also normal.

 Regarding lifespan, 1) Growth stops around 3 weeks after birth, and activity gradually decreases.

 2) Reduction of life expectancy: The average life expectancy is 8.0 ± 0.9 weeks (n = 13) for females and 9.3 ± 0.9 weeks (n = 13) for females. It is characterized by premature aging, with death occurring.

Below, 5 weeks each of homo females and females around 8 weeks of age, and 4 female females and 4 females each of their siblings' wild type mice were dissected and visually observed, followed by standard methods. (Keiichi Watanabe, Kazuho Nakane, Enzyme-Linked Immunosorbent Assay, Interdisciplinary Project, 3rd revised edition, 1992) Formalin fixation, paraffin embedding, sectioning, HE staining, and pathological observation (Hereinafter, observation was performed by the same method up to observation example 12).

 [Observation Example 2] Pathological observation of the pituitary gland: decrease in eosinophilic cells was observed. The eosinophilic cells of the pituitary gland are cells that produce growth hormone (GH) or prolactin (PRL), but their numbers are decreasing. In the elderly

Reduced GH secretion is known.

 [Observation example 3] Pathological observation of gonads: The gonads were found to be significantly atrophied and infertile. In os, the testes are macroscopically markedly atrophied. The diameter of the seminiferous tubules is atrophy to about 1/3 of that of the wild type, sperm maturation has progressed only to pachytene spermatocytes, and no sperm is observed. In females, the ovaries' uterus is also significantly macroscopically atrophied. Oocyte maturation has progressed only to the primary follicle, and no secondary follicle or luteal body is observed.

 [Observation example 4] Pathological observation of the submandibular gland: Normal osseous cells have a large amount of eosinophilic granules in the linear part, but in homozygous cells, the cells in the linear part are low in length, Almost no.

 [Observation example 5] Pathological observation of kidney: Calcification is observed in small artery wall, tubular epithelium, Bowman's capsule wall, etc. Bowman's capsule wall is squamous epithelium.

[Observation example 6] Pathological observation of vascular system: First, atherosclerosis was remarkable in homozygous individuals. The diameter of the thoracic aorta was irregular, severe media calcification was present, and intimal thickening was also observed. Atherosclerosis mainly due to medial calcification and intimal hyperplasia was observed in small blood vessels in medium vessels and parenchymal organs such as renal arteries. In particular, calcification of the vascular media in the renal parenchyma was remarkable, but glomeruli and tubules were almost normal. Kossa staining confirmed that the deposits were indeed ^{Ca2 +} . Aortic media calcification is a common finding in the elderly.

 [Observation example 7] Pathological observation of lung tissue: The destruction of the alveolar structure accompanying the destruction of the alveolar wall was remarkable. Calcification is observed on the alveolar wall.

[Observation example 8] Soft tissue · Cartilage observation: lamina propria of the tracheal mucosa, lamina propria of the stomach Calcification was observed in the plate and alveolar epithelium. Calcification was observed in fundic gland cells, submucosal connective tissue, and muscle layer. Calcification of the gastric mucosa is similar to that observed in long-term rats and mice. In some individuals, mitral annulus calcification of the heart was observed. Mitral annulus calcification is a characteristic finding in the elderly. Calcification was also observed in articular cartilage, tracheal cartilage, and costal cartilage. Calcification of these hyaline cartilages is a common finding in the elderly.

 [Observation example 9] Bone histopathology and X-ray observation: Increased bone density at the metaphysis at the distal end of the femur and thickening of the epiphyseal cartilage are observed. Articular cartilage has incorrect calcification of chondrocytes. X-ray findings of the femur and tibia show that the X-ray permeability is increased, indicating a decrease in bone mineral density. When bone mineral density is measured, the diaphyseal part, distal end of tibia and proximal end of femur are reduced to about half that of the wild type. Histologically, cortical bone is significantly reduced. However, in the metaphysis at the proximal end of the tibia and the distal end of the femur, the primary cancellous bone is exceptionally increased, and the bone mineral density increases only in this area.

 [Observation example 10] Pathological observation of the cerebellum: Decrease in the number of Purkinje cells in the cerebellar nerve cell layer, isolated necrosis, and swelling of axons were observed. In the cerebellum, it is known that Purken cells decrease with aging, and neurites swell accordingly.

 [Observation Example 1 1] Pathological observation of liver: Glycogen granules that stain eosinophilic are hardly observed. The cytoplasm of hepatocytes is slightly smaller than normal.

 [Observation example 1 2] Pathological observation of the thymus: Some individuals had markedly atrophy of the thymus and could not be visually confirmed.

By expressing the klotho gene in the mouse showing the above-mentioned premature aging-like symptoms, the premature aging symptom was suppressed, and thus it was revealed that the gene is a suppressor gene for the premature aging symptom in the premature aging mouse. . In addition, when the adenovirus expressing the klotho gene was inoculated into mice exhibiting the premature aging symptoms, weight gain and survival benefit were clearly observed, and the premature aging symptoms were resolved (Nature, In addition, a human-derived anti-aging gene highly homologous to the klotho gene has also been cloned [Nature, 390, 45 (1997), WO 98/29544].

 From the above, the existence and usefulness of polypeptides, which have an aging inhibitory effect, were clarified.However, as described above, since there are multiple genetic premature aging diseases, there are also multiple causal genes, and aging Symptoms may be related to multiple genes.

 Therefore, it is sufficiently possible that there are other genes related to aging other than the klotho gene. However, other than the klotho gene and the above-mentioned human-derived klotho homolog gene, an aging inhibitory gene has not yet been known. Disclosure of the invention

 If premature aging symptoms occur due to the loss of the function of a gene involved in aging, treatment for complementing the gene function, that is, administering a protein product encoded by the gene or expressing the gene therapeutically. Method is useful. Also, drugs that interact with the gene product or drugs that can regulate the expression of the gene are extremely useful. Furthermore, if aging can be controlled in aging other than genetic premature aging, it can be used for the treatment and prevention of various adult diseases closely related to aging.

 At the same time, the evaluation system for such drugs in animals is an indispensable factor in drug development, and model animals, particularly animals in which the gene has been deleted or partially modified, are very useful for such drug development. .

The present invention relates to a method for treating the above-mentioned premature senescence, a drug that regulates the expression of a gene involved in aging and a method for searching for the same, a drug and a method effective for the treatment, prevention, and diagnosis of adult diseases associated with aging, and the drug. It is an object of the present invention to provide an aging model animal necessary for the development of an animal. The present inventors have conducted intensive studies to solve the above-mentioned problems, and are of human origin. The present inventors have obtained DNA encoding a polypeptide having a high homology to the klotho gene homolog and having an activity of suppressing aging different from that of the klotho gene, thereby completing the present invention.

 That is, the present invention relates to the following (1) to (40).

 (1) A polypeptide having an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 1, 2, and 3 and having an activity of suppressing aging.

 (2) having an amino acid sequence in which one or more amino acids have been deleted, replaced or added in the amino acid sequence of a polypeptide having an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, and A polypeptide that has inhibitory activity.

 (3) DNA encoding the polypeptide according to (1) or (2).

 (4) A DNA represented by the nucleotide sequence of any one of SEQ ID NOs: 4 to 6.

(5) a DNA having a base sequence in which one or more bases are deleted, substituted or added in the DNA selected from the DNA described in (4), and which encodes a polypeptide having an activity of suppressing aging. DNA.

 (6) a DNA that hybridizes with a DNA selected from the DNAs of any one of (3) to (5) under stringent conditions and encodes a polypeptide having an activity of suppressing aging DNA.

 (7) A recombinant DNA obtained by incorporating a DNA selected from the DNA according to any one of (3) to (6) into a vector.

 (8) An antibody that recognizes the polypeptide according to (1) or (2).

 (9) An agent for treating premature aging or adult disease, comprising the polypeptide according to (1) or (2).

 (10) An aging inhibitor comprising the polypeptide according to (1) or (2).

(11) An immunological quantification method for the polypeptide according to (1) or (2), using the antibody according to (8). (12) An aging diagnostic method using the quantitative method according to (11).

 (13) A gene therapy vector for treating premature aging or adult disease, comprising the DNA according to any one of (3) to (6).

 (14) A gene therapy vector for suppressing aging, comprising the DNA according to any one of (3) to (6).

 (15) A transformant having the recombinant DNA of (7).

 (16) A transformant having the recombinant DNA described in (7) is cultured in a culture medium, and a polypeptide encoded by the recombinant DNA is produced and accumulated in the culture medium. The method for producing a polypeptide according to (1) or (2), wherein the polypeptide is collected from a liquid.

 (17) The polypeptide of (1) or (2) is brought into contact with a test sample, and a ligand for the polypeptide of (1) or (2) is selected from the test sample, A method for searching for a ligand for the polypeptide according to (1) or (2).

 (18) A ligand obtained by the method according to (17).

 (19) [a] When the polypeptide according to (1) or (2) is brought into contact with the ligand according to (18), and [b] When the polypeptide according to (1) or (2) is brought into contact with the polypeptide according to (1) or (2). Inhibiting the specific binding of the polypeptide according to (1) or (2) with the ligand according to (18), which is compared with the case where the ligand and test compound described above are brought into contact with each other. To search for a compound.

 (20) A compound obtained by the method of (19) or a pharmacologically acceptable salt thereof.

(21) Contacting cells expressing the polypeptide of (1) or (2) with a test sample to enhance the expression of the gene encoding the polypeptide of (1) or (2) from the test sample Selecting a compound, wherein the compound that enhances the expression of a gene encoding the polypeptide according to (1) or (2) is Search method.

 (22) The search method according to (21), wherein the enhanced expression is detected using the quantification method according to (11).

 (23) The search method according to (21), wherein the enhanced expression is detected by measuring the amount of mRNA encoding the polypeptide according to (1) or (2).

 (24) A compound or a pharmacologically acceptable salt thereof obtained by the search method according to any one of (21), (22) and (23).

 (25) A transformant transformed with a plasmid containing DNA linked to a repo overnight gene downstream of a region controlling transcription of the gene encoding the polypeptide according to (1) or (2). And a test sample, and selecting a compound that enhances the expression of the gene encoding the polypeptide described in (1) or (2) from the test sample, wherein (1) or (2) A method for searching for a compound that enhances the expression of a gene encoding the described polypeptide.

 (26) The (25) wherein the transcription controlling region according to (25) is a DNA having a nucleotide sequence represented by nucleotide numbers 1-2177 in the nucleotide sequence shown in SEQ ID NO: 7. Search method.

 (27) A compound obtained by the search method according to (25) or (26) or a pharmacologically acceptable salt thereof.

 (28) A therapeutic agent for premature aging or adult disease, comprising the compound according to any one of (20), (24) and (27) or a pharmacologically acceptable salt thereof.

(29) An aging inhibitor comprising the compound according to any one of (20), (24) and (27) or a pharmacologically acceptable salt thereof.

 (30) A method for improving livestock using a DNA containing the DNA according to any one of (3) to (6).

(31) The DNA according to any one of (3) to (6) may be overexpressed. (30) The method for improving livestock according to (30).

 (32) Expression of a polypeptide having an activity of suppressing aging by deleting or substituting all or a part of the gene containing the DNA according to any one of (3) to (6). Genetically deleted or replaced non-human animals with altered amounts, timing or tissue specificity.

 (33) The gene-deleted or substituted non-human animal according to (32), wherein the change in the expression level is a change in which the expression is reduced or not expressed.

 (34) The animal according to (32) or (33), wherein the non-human animal is a non-human mammal.

 (35) The animal according to (33), wherein the non-human mammal is a mouse.

(36) The animal according to any one of (32) to (35), or an organ, tissue or cell of the animal is brought into contact with a test compound, and a therapeutic agent for premature aging or an adult disease is selected from the test compound. A method for searching for a therapeutic agent for premature aging or adult disease characterized by the following.

 (37) The method according to any one of (32) to (35), wherein the test compound is brought into contact with the animal, or an organ, tissue or cell of the animal, and an aging inhibitor is selected from the test compound. To search for antiaging drugs.

 (38) A compound obtained by the method according to (36) or (37) or a pharmacologically acceptable salt thereof.

 (39) A therapeutic agent for premature aging or adult disease, comprising the compound according to (38) or a pharmacologically acceptable salt thereof.

(40) An aging inhibitor comprising the compound according to (39) or a pharmacologically acceptable salt thereof. The DNA of the present invention encodes a polypeptide having an activity of suppressing aging DNA, for example,

 (1) a DNA encoding a polypeptide having an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 1, 2, and 3,

 (2) the amino acid sequence of a polypeptide having an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 1, 2, and 3 has an amino acid sequence in which one or more amino acids have been deleted, substituted or added, DNA encoding a polypeptide having an activity of suppressing aging and

 (3) a DNA represented by the nucleotide sequence of SEQ ID NO: 4, 5, or 6,

 (4) In the nucleotide sequence of DNA represented by the nucleotide sequence of SEQ ID NO: 4, 5 or 6, one or more bases have a deleted, substituted or added base sequence and suppresses aging DNA encoding an active polypeptide,

 (5) a DNA encoding a polypeptide that hybridizes with a DNA selected from any one of (1) to (4) under stringent conditions and has an activity of suppressing aging. it can.

In the above description, the term "DNA encoding a polypeptide having an activity of inhibiting aging and hybridizing under stringent conditions" refers to a DNA encoding a polypeptide having an activity of inhibiting aging or a chromosomal gene fragment thereof. As a lobe, DNA obtained by using the colony 'hybridization method, plaque' hybridization method or Southern blot 'hybridization method, etc., specifically, from the colony or plaque After performing hybridization at 42 to 68 ° C in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA was immobilized, 0.1 to 2 times concentration of SSC solution ( The composition of a 1x concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate). It is possible to increase the DNA that can be identified by Kiyoshi. Hybridization is performed by Molecular Cloning, A laboratory manual, Second Edition. (1989) (Hereinafter abbreviated as Molecular 'Cloning 2nd edition), Current Protocols in Molecular Biology, John and Wily & Sons (1987-1997)

• Protocols 'in' abbreviated as 'molecule' biology), MA cloning

1: Can be performed according to the method described in Core Techniques, APractical Approach, Second Edition, Oxford University (1995). Specifically, DNA that can be hybridized is calculated using BLAST [J. Mol. Biol., 215, 403 (1990), FASTA Qiethod in Enzymology, 183, 63 (1990)]. A DNA having at least 60% or more homology with the nucleotide sequence of a DNA encoding a polypeptide having an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, and preferably at least 80% DNA having homology of more than 95%, more preferably DNA having homology of 95% or more.

 Examples of the polypeptide of the present invention include polypeptides encoded by the above-mentioned DNAs. Specifically, polypeptides having an amino acid sequence that is hesitant to the amino acid sequences described in SEQ ID NOs: 1, 2, and 3 Amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of a peptide or a polypeptide having an amino acid sequence selected from the amino acid sequences described in SEQ ID NOs: 1, 2 and 3. And peptides having the activity of suppressing aging.

 In the above description, deletion, substitution or addition of one or more amino acids refers to deletion, substitution or addition of a sufficient number of amino acids that can be deleted, substituted or added by a well-known method such as site-directed mutagenesis. Means, for example, an amino acid sequence in which any number of 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 5 amino acids have been deleted, substituted or added. .

A peptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of a polypeptide, and which has an activity of suppressing aging. Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Proc. Natl. Acad. Sci., USA, 81,5662 (1984), Science , 224, 1431 (1984), WO 85/00817, Nature, 316, 601 (1985), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Current Protocols in 'Molecular. It can be prepared using a mutant DNA obtained by deleting, substituting or adding a base contained in the DNA encoding the polypeptide according to the method described in Biology and the like.

 Recombinant DNA containing DNA encoding the aging inhibitory polypeptide of the present invention can be obtained by ligating a DNA selected from the DNA of the present invention to a vector according to the method described in Molecular Cloning, Second Edition.

 The method for producing the polypeptide of the present invention is a method for producing a cell transformed by using the recombinant DNA prepared by the method described above according to the method described in Molecular Cloning, 2nd edition. Examples of the method include culturing in a suitable culture solution, accumulating the polypeptide of SEQ ID NO: 1, 2 or 3 in the culture solution, and collecting the polypeptide from the culture solution. Further, to collect the polypeptide from the culture solution, after solubilizing the polypeptide, ion exchange, gel filtration, hydrophobic mouth chromatography, etc., or a combination of these methods can be used. Separation and purification can be mentioned.

 Examples of the antibody of the present invention include an antibody that recognizes the polypeptide of the present invention.

 As the vector DNA for gene therapy that ligates the DNA of the present invention, a DNA selected from the DNA of the present invention, such as a retrovirus vector, an adenovirus vector, or a derivative of the virus vector, is introduced into animal cells. And vectors that can be expressed.

The ligand of the present invention means a substance that specifically binds to the polypeptide of the present invention, and any substance that specifically binds to the polypeptide of the present invention. It can be used as the ligand of the present invention.

 Examples of the compound selected from the polypeptides of the present invention that enhances the expression of a polypeptide include a substance that specifically increases the transcription amount of DNA encoding the polypeptide.

 The genetically modified non-human animal of the present invention may be any animal other than a human, but is preferably a mammal, for example, a horse, a bush, a sheep, a dog, a cat, a hampus, a guinea pig. , Mice, rats and the like are used. In particular, rodents are preferred as model animals because they have a relatively short life cycle and are easy to breed. Rats and mice are particularly preferred. The genetically modified non-human animal produced using the animal is a polynucleotide encoding the polypeptide of the present invention having the effect of suppressing aging using the DNA of the present invention and the chromosome gene containing the DNA. For alleles in which all or part of the gene containing DNA to be deleted is deleted, an embryonic stem cell clone having the replaced gene is created, and a chimeric individual is created by crossing the embryonic stem cell clone with normal cells by the chimera method. Thereafter, the chimeric individual and the normal individual are multiplied to obtain an individual having a mutation in the DNA of all cells, and then the homologous chromosome is mutated in both homologous chromosomes by crossing the individuals. Can be created by the method. Examples of the chromosome DNA containing DNA of the present invention include a fragment of the chromosomal gene shown in FIG. 1 containing DNA represented by the nucleotide sequence of SEQ ID NO: 7. An individual thus prepared, which has a gene having a mutation in the gene encoding the polypeptide having the activity of suppressing aging, can express the polypeptide having the activity of inhibiting aging according to the present invention. It is useful as a wound and an aging model animal.

 Examples of the method of searching for a therapeutic agent for early senescence, a therapeutic agent for adult disease, and an aging inhibitor using the genetically modified non-human animal of the present invention include a method using the above-mentioned aging model animal.

Specifically, the animals are treated with the test compound and compared to untreated control animals. Thus, the effect of the compound can be obtained by observing changes in organs, tissues, cells, etc. of the animal or changes in the pathological conditions of premature aging, adult diseases or aging symptoms. Examples of the method of treating the animal with the test compound include a method of orally administering the animal, or a method of administering the compound by intravenous injection, and the like.However, the method is not particularly limited to these methods. Can be selected as appropriate.

 1) Cloning of DNA encoding a polypeptide having aging-inhibiting activity

 (1) Cloning of a DNA fragment encoding a polypeptide that has aging-inhibiting activity

A DNA fragment encoding a polypeptide having an activity of suppressing aging can be obtained by PCR using a primer prepared based on nucleotide sequence information such as the klotho gene described in W098 / 29544 (PCR Protocol, Humana Press ( 1993)] can be obtained by the following method. Examples of the primer include primers prepared based on the nucleotide sequence of the human aging-derived aging inhibitory gene encoded in pRYHH02 described in W098 / 29544, for example, as shown in SEQ ID NO: 9 and SEQ ID NO: 10. And a set of primers having a modified base sequence. Using this set of primers, PCR is performed using chromosomal DNA as type III to amplify gene fragments. Examples of the chromosomal DNA include animal-derived chromosomal DNA, such as mouse-derived chromosomal DNA. The conditions for the PCR reaction are not particularly limited, as long as the specific amplification fragment is obtained by the PCR method using the designed primers. For example, using DNA consisting of the nucleotide sequences of SEQ ID NOS: 9 and 10 at 97 ° C for 10 minutes, at 94 ° C for 1 minute, at 65 ° C (0.5 ° C every cycle) 20 minutes at 72 ° C for 1 minute), 20 cycles of (1 minute at 94 ° C, 1 minute at 65 ° C, 1 minute at 72 ° C) and 5 cycles at 72 ° C. Min, then hold at 4 ° C. it can. It is preferable that the PCR conditions are appropriately changed according to the properties of the Taq polymerase used in the PCR. The nucleotide sequence of the DNA fragment amplified by the PCR method can be used, for example, by the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] For example, it is determined using 373 A DNA Sequencer (Applied Biosystems). As the nucleotide sequence of DNA encoding a part of the polypeptide having the activity of suppressing aging determined by the above method, for example, the nucleotide sequence of SEQ ID NO: 8 can be mentioned. Also, by comparing the nucleotide sequence of the chromosome gene fragment with the nucleotide sequence of the human aging-derived aging inhibitory gene encoded in pRYHH02 described in W098 / 29544, the chromosomal gene fragment It can be confirmed that the DNA contains. (2) Cloning of a DNA fragment encoding a polypeptide having activity to suppress senescence from a chromosomal gene cosmid library

 Using the DNA encoding a part of the polypeptide having the activity of suppressing senescence obtained in (1) above, a longer chromosomal gene fragment containing the DNA fragment can be obtained, for example, from a chromosomal gene cosmid library (1). ) Can be obtained by selecting a cosmid clone containing the DNA fragment obtained in step (1). Examples of the selection method include a colony hybridization method using the chromosomal DNA partial fragment obtained in (1) as a probe, a plaque * hybridization method (described in Molecular Cloning, Second Edition), or (1) And a method using PCR in which a synthetic DNA having a sequence selected from the nucleotide sequence of the partial chromosomal DNA fragment obtained in step 1 is used as a primer.

 A specific method using PCR includes the following method.

A primer having a sequence selected from the base sequence of the chromosomal DNA fragment of SEQ ID NO: 8 obtained in (1) is synthesized. Examples of the primer include a set of primers having the nucleotide sequences of SEQ ID NOS: 11 and 12. Can be. A chromosomal gene cosmid library is screened by PCR using the primer set. The chromosome gene cosmid library can be prepared by the method described in Molecular Cloning Second Edition, but a commercially available chromosome gene cosmid library (for example, manufactured by KURAB0) can also be used. A specific method for selecting a clone containing the gene from the obtained chromosomal DNA cosmid library by PCR is performed by selecting a cosmid clone giving an amplified fragment by PCR using the primer. The conditions of the PCR reaction are not particularly limited as long as specific DNA amplification can be amplified by the PCR reaction using the primer.

Next, a restriction map was prepared for the chromosome DNA contained in the clone that gave the amplified fragment by the PCR reaction, and the Southern hybridization method using the chromosome partial fragment obtained in (1) as a probe was performed. By combining these, the position of the partial chromosomal DNA fragment cloned in (1), that is, the restriction enzyme fragment containing the partial chromosomal fragment (exon region) can be identified. In the case of mice, a restriction map of mouse chromosome DNA as shown in FIG. 1 can be prepared according to a conventional method. Next, the DNA fragment obtained by digesting the mouse chromosomal DNA with an appropriate restriction enzyme was subjected to Southern analysis using a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 8 as a probe, and the DNA fragment of SEQ ID NO: 8 on the mouse chromosome DNA was analyzed. By determining the location of the DNA, the exon site can be determined (see Fig. 1). The partial base sequence of the chromosomal DNA fragment containing the exogene region is determined by the conventional method described in (1), and the homology is compared with that of the human senescence-derived aging inhibitory gene, thereby selecting from the cosmid library. It is confirmed that the mouse chromosomal DNA fragment contained in the obtained cosmid clone is a fragment containing a mouse aging suppressor gene. An example of the partial base sequence is the base sequence shown in SEQ ID NO: 7. Using the same method for other animals, a chromosomal DNA fragment containing DNA encoding a part of a polypeptide having an activity of suppressing aging can be obtained. Escherichia coli EMRYHH02-S10 harboring the plasmid pEMRYHH02-S10 into which the chromosomal DNA fragment was inserted was designated as FERM BP-6935 on January 10, 2001 It has been deposited with 1-3-3 Tsukuba East Higashi, Ibaraki, Japan (zip code 305-8566).

 (3) Cloning of cDNA encoding a polypeptide having activity to suppress aging

 As a method for cloning cDNA encoding a polypeptide having the activity of suppressing aging, a part of the peptide having the activity of suppressing aging, which is identified by the method described in (2) above, may be used. A method for screening a cDNA library by Southern hybridization or plaque hybridization using a DNA having a sequence selected from the base sequence of the exon part on the chromosome DNA as a probe, There is a method in which a primer is designed based on the partial sequence, and screening is performed from the cDNA library by PCR.

As a method using PCR, for example, a primer set is created based on the nucleotide sequence of the exon portion of the aging inhibitory gene contained in DNA having the nucleotide sequence of SEQ ID NO: 7, and a cDNA library is screened by PCR. There are ways to do it. Braima can be any sequence and combination as long as it can specifically amplify the sequence in the exon of the aging inhibitory gene, and preferably DNAs described in SEQ ID NO: 13 and SEQ ID NO: 14. The cDNA library is not limited as long as an amplified fragment specifically appears in the primer set, that is, a tissue that expresses the aging suppression gene. In the case of mice, those produced using 19-day-old embryos can be mentioned. The cDNA library can be prepared by the method described in Molecular'Cloning 2nd edition using the embryo, but a commercially available product may be purchased. From commercially available cDNA libraries As a specific preferred example of the PCR method of PCR described above, in the case of mice, a method using a 19-day-old embryo library that is subdivided into microtiter well plates can be used (RAP ID- SCREEN ™ cDNA LIBRARY PANELS (OriGene Technologies)].

 PCR was performed on the sample in the well of the evening plate using a set of primers having the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14, and the library corresponding to the well in which the amplified fragment was recognized was obtained. The subpool is further screened by PCR. The PCR conditions are not particularly limited as long as they give specific amplified fragments to the primers used.Preferably, the primer set described in SEQ ID NO: 13 and SEQ ID NO: 14 is used at 95 ° C. The reaction can be carried out for 9 minutes at 95 ° C for 30 seconds, at 65 ° C for 30 seconds at 72 ° C for 30 seconds, and 33 times at 72 ° C for 5 minutes. Next, a positive gel, that is, a colony of Escherichia coli containing the cDNA in the gel to which the amplified fragment was given was formed on an agar medium, and a primer set having the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14 was prepared. A colony in which an amplified fragment is observed is selected by the colony PCR method used, the plasmid contained therein is recovered, and the gene sequence of the cDNA portion contained in the plasmid is determined by the usual method described in (1).

 Examples of the senescence-suppressing gene sequence thus determined include, for example, DNA having the sequence shown in SEQ ID NO: 4, 5, or 6. Escherichia coli EMRYHH02—F1 containing the plasmid pEMRYHH02—F1 having the DNA of SEQ ID NO: 4 contains the plasmid pEMRYHH02—T1 having the DNA of SEQ ID NO: 6 as FERM BP-6934. E. coli EMRYHH02-T1 is FERM BP-6936 as of FERM BP-6936, respectively, dated January 10, 2001, Research Institute of Biotechnology, Industrial Science and Technology, 1-3-1 Tsukuba East, Ibaraki, Japan (postal service) Deposit No. 305-8566).

Examples of the polypeptide encoded by the aging inhibitory gene include: Examples thereof include polypeptides having the amino acid sequence shown in 1, 2, or 3.

 2) Production of anti-aging polypeptide

 In order to express the aging repressor gene obtained as described above in a host cell and produce the polypeptide of the present invention, molecular cloning second edition, Current 'Protocols in Molecular Biology. Etc. can be used.

 That is, a recombinant DNA having the DNA of the present invention inserted downstream of a promoter of an appropriate expression vector is constructed, and the vector is introduced into a host cell adapted to the vector to express the polypeptide of the present invention. The transformant can be produced by culturing the transformant in a culture solution and accumulating the polypeptide of the present invention in the culture solution.

 Any host cell that can express the gene of interest can be used. For example, prokaryotes belonging to the genera Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., Kluybium Myces, Saccharomyces, Schizosaccharomyces, Trichosporone Yeasts, animal cell hosts, etc. belonging to the genus Genus, Pseudomonas genus and the like.

 As the expression vector, those which are capable of autonomous replication in the above-mentioned host cell or capable of being integrated into a chromosome, and which contain a promoter at a position where the aging inhibitory gene can be transcribed are used.

 When a prokaryote such as a bacterium is used as a host cell, the senescence inhibitor gene expression vector can replicate autonomously in the procaryote, and at the same time, the promoter, ribosome binding sequence, senescence inhibitor gene, transcription termination sequence, More preferably, it is composed of A gene that controls the promoter may be included.

Examples of expression vectors include, for example, p BT rp 2, p BT acl, p BT ac 2 (all manufactured by Boehringer Mannheim), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP IO (JP-A-58-110600), pKYP 200 CAgric. Biol. Chem., 48, 669 (1984)], pLSA1 Agric. Biol. Chem., 53, 277 (1989)), pGEL1

Natl. Acad. Sci., USA, 82, 4306 (1985)), pBluescript (STRATAGENE), pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 ( FERM BP-400), pGKA2 (FE RM B-6798), pTe rm2 (JP-A-3-22979, US 4686

191, US4939094, US5160735), pKK233-2 (Pharmacia), pGEX (Pharmacia), pET system (Novagen), Supex pUB110, pTP5, pC194, and the like.

Any promoter can be used as long as it can be expressed in a host cell such as Escherichia coli. For example, ^ promoter (P ^ £), promoter (Plac), P _L promoter evening one, P _R promoter evening one, Pletl promo Isseki one, promoter one derived from the P _SE promoter evening of the first class, E. coli or phage, etc. Evening, SP01 Promo Overnight, SPO2 promoter, penP Promo Overnight etc. can be mentioned. Also, a promoter (P ^ x2) in which two PtrDs are connected in series, and a promoter designed and modified artificially, such as a tac promoter, can be used.

 The ribosome binding sequence is not particularly limited as long as it can be expressed in a host cell such as Escherichia coli. An appropriate distance between the Shine-Dalgarno sequence and the initiation codon (eg, 6 to 18) It is preferable to use a plasmid adjusted to (base).

 Although a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, it is preferable to arrange the transcription termination sequence immediately below a structural gene.

Host cells include the genera Escherichia, Serratia, Corynebacterium, Microorganisms belonging to the genus Brevibacterium, Pseudomonas, Bacillus, etc., for example, Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli , Escherichia coli HB10 Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Bacillus subtil is. Bacillus amyloliquefacines. Brevibacterium anmoniagenes Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Brevibacterium f lavum ATCC14067, Brevibacterium lactofermentum ATCC 13869 Corynebacterium glutamicum ATCC13032, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354 and the like.

 As a method for introducing the recombinant vector, any method for introducing DNA into the above host cells can be used. For example, a method using calcium ions CProc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP-A-63-248394), Gene, 17, 107 (1982), and the method described in Molecular & General Genetics, 168, 111 (1979).

 When a yeast strain is used as a host cell, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

 Any promoter can be used as long as it can be expressed in a yeast strain. For example, PH05 promoter overnight, PGK promoter, GAP promoter, ADH promoter overnight, gal 1 promoter, gal 10 promoter, heat shock protein promoter overnight, MF 1 promoter, CUP 1 promoter, etc. You can give a one-day promotion.

As host cells, Saccharomyces cerevisae (Saccharomyces cerevisae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kruybe Mouth Mrs 'Lactis (Kluyveromyces lactis), Trichosporon' Pullrance (Trichosporon pul lulans, Schwanniomyces alluvius) and the like.

 As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, the elect opening method (Methods.EnzymoL, 194, 182 (1990)), the spheroplast method (Proc. Natl. Acad. Sei. USA, 84, 1929 (1978)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 ( 1978).

 When animal cells are used as host cells, expression vectors include, for example, pcDNAI ヽ pcDM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Application Laid-Open No. 3-22979, Cytotechnology, 3, 133 (1990)]. , PAS3-3 (Japanese Unexamined Patent Publication No. 2-227075), pCDM8 CNature, 329, 840 (1987)], pcDNA I / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen) pAGE 103 [J Biochem., 101, 1307 (1987)], pAGE 210 and the like.

 Any promoter can be used as long as it can be expressed in animal cells. For example, the promoter of the IE (immediate early) gene of cytomegalovirus (human CMV) and the early promoter of SV40 can be used. One or metallothionein promoter, a retrovirus promoter, a meta-thickinin promoter, a heat shock promoter, an SR promoter, and the like. Alternatively, the enhancer of the IE gene of human CMV may be used together with the promoter.

 Examples of the host cell include Namalba cell, HBT 5637 (Japanese Patent Laid-Open No. 63-299), COS 1 cell, COS 7 cell, CHO cell and the like.

As a method for introducing the recombinant vector into animal cells, any method capable of introducing DNA into animal cells can be used. For example, elect mouth poration Method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 227075/1990), Lipofexion method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)], Virology, 52, 456. (1973). Obtaining and culturing the transformant is described in Japanese Patent Application Laid-Open No. H2-227075 or

It can be carried out according to the method described in 257891.

 When insect cells are used as hosts, for example, Baculovirus Expression-Vectors, A Laboratory Manual, current 'protocols in' molec The polypeptide of the present invention can be expressed by the method described in Yura's Biology, Bio / Technology, 6, 47 (1988) and the like.

 That is, the recombinant gene transfer vector and the baculovirus are co-introduced into insect cells to obtain recombinant viruses in the culture supernatant of insect cells. The peptide can be expressed. The gene transfer vector used in the method includes, for example, pVLl

392, pVL1393, pBlueBacIII (both manufactured by Invitrogen) and the like.

 As the baculovirus, for example, autographa californica nuclear polyhedrosis virus, which is a virus infecting night moth insects, can be used.

Insect cells include Spodoptera frugiperda ovarian cells, Sf9 and Sf21 [baculovirus 'Expression Vectors', 'Laboratory One', Manual, Double H'Eich 'Freeman' and 'Campaine' (WH Freeman) and Company), New York (New York), (1992)], and Trichoplusiail ovary cells, High 5 (manufactured by Invitrogen), and the like. The above-described recombinant gene transfer vector into insect cells for preparing a recombinant virus. As a method of co-introducing Yuichi and the above baculovirus, for example, calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), ribofusion method [Proc. Natl. Acad. Sci. USA, 8, 7413 (1987)] and the like can be used. I can give it.

As the expression method of gene, in addition to direct expression, in accordance with the method or the like that are described in Molecular 'black-learning Second Edition, secretory production, fusion protein expression and the like can fi ¹ Ukoto.

 When expressed by yeast, animal cells or insect cells, a sugar or a sugar chain-added polypeptide can be obtained.

 Culturing a transformant having the recombinant DNA into which the DNA of the present invention has been incorporated in a medium, producing and accumulating the polypeptide of the present invention in the culture, and collecting the polypeptide from the culture; Thus, the polypeptide can be produced. When the transformant for producing a polypeptide is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, a medium for culturing these organisms may be a carbon source, a nitrogen source, an inorganic Either a natural medium or a synthetic medium may be used as long as the medium contains salts and the like and can efficiently culture the transformant.

 Any carbon source can be used as long as each microorganism can assimilate it. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol and propanol are used.

 Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., various ammonium and ammonium salts of organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof are used.

Inorganic salts include potassium (II) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, and sulfuric acid. Copper acid, calcium carbonate and the like are used.

 The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 to 96 hours. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.

 If necessary, an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.

 When culturing a microorganism transformed in an expression vector using an inducible promoter as a promoter, an Indian user may be added to the medium as necessary. For example, when culturing microorganisms transformed with an expression vector using the i ^ promoter, use isopropyl-1 /?-D-thiogalactovyranoside (IPTG) or the like with the ^ promoter. When culturing a microorganism transformed with a vector, indoleacrylic acid (IAA) or the like may be added to the medium. As a medium for culturing a transformant obtained using animal cells as host cells, commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eag 1 e MEM medium [Science, 122, 501 (1952)], DMEM medium Virology, 8, 396 (1959)], 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)) A medium or the like to which fetal serum or the like is added is used.

Cultivation is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and the presence of 5% CO ₂ .

 If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

As a medium for culturing a transformant obtained by using insect cells as host cells, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCe ll 400, ExCe ll 405 (both JRH Biosciences), Grace's Insect Medium Nature, 195, 788 (1962)] and the like.

 The pH is 6-7, the culture temperature is 25-30 ° C, and the culture time is usually 1-5 days.

 If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.

 In order to isolate and purify the polypeptide of the present invention from the culture of the transformant for producing the polypeptide of the present invention, a usual enzyme isolation and purification method may be used.

 For example, when the polypeptide of the present invention is expressed in a lysed state in a cell, after completion of the culture, the cell is recovered by centrifugation, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a mantongaulin. Crush cells with a homogenizer, dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, getylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using resin such as DIAION HPA-75 (Mitsubishi Kasei), resin such as S-Sepharose FF (Pharmacia) Chromatography using resins such as cation-exchange chromatography, butyl-sepharose, and phenylsepharose, gel filtration using molecular sieve, affinity chromatography, and chromatography. A purified sample can be obtained by using one or a combination of electrophoresis methods such as the one-casing method and isoelectric focusing.

When the polypeptide is expressed by forming an insoluble substance in the cells, the cells are similarly recovered, crushed, and the polypeptide is purified by a conventional method from the precipitate fraction obtained by centrifugation. After recovering the peptide, the insoluble form of the polypeptide is solubilized with a polypeptide denaturing agent. The lysate is converted to a solution containing no polypeptide denaturing agent or a solution in which the concentration of the polypeptide denaturing agent is so low that the polypeptide is not denatured. After dilution or dialysis to form the polypeptide into a normal three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above.

 When the polypeptide of the present invention or a derivative such as a modified sugar thereof is secreted extracellularly, the derivative such as the polypeptide or a sugar chain adduct thereof can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. Can be obtained.

 Further, the polypeptide expressed by the above method can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). . Kuwawa Trading (AdvancedchemTech), Perkin-Elmer Japan (Perkin-Elmer), Pharmacia Biotech (Pharmacia Biotech), Aroka (Protein Technology Instrument), Kurabo (Synthecell-Vega) ), Nippon Perceptive 'Limited (manufactured by PerSeptive), Shimadzu Corporation, and other peptide synthesizing machines.

3) Production of antibodies that recognize aging-inhibiting polypeptides

 (1) Preparation of polyclonal antibody

 ゥ Purified full-length or partial fragments of the aging-suppressing polypeptide of the present invention (antigen) obtained in 2) above, which were obtained in 50 to 1 week, in a heron, a goat or a 30 to 20-week-old rat, mouse or hamster. About 100 mg / animal, subcutaneously, intravenously or intraperitoneally of the animal together with an appropriate adjuvant (for example, Complete Freund's Adjuvant or aluminum hydroxide gel and pertussis vaccine) Administer.

The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus on days 3 to 7 after each administration, and the reaction of the serum with the antigen used for immunization is determined by enzyme immunoassay [enzyme immunoassay (ELISA): published by The Medical Shoin 1976 , Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory (1988) ] And confirm.

 A serum was obtained from a heron, a goat, a mouse, a rat or a hamster whose serum showed a sufficient antibody titer against the antigen used for immunization, and a salting out method using 40-50% saturated ammonium sulfate was obtained from the serum. Purified antibody is obtained by a conventional method such as chromatography using a polyacrylic acid precipitation method, DEAE-Sepharose column, Protein A-column or gel filtration column.

 (2) Preparation of monoclonal antibody

 (a) Preparation of antibody-producing cells

 Rats whose serum shows a sufficient antibody titer against the aging inhibitory fragment polypeptide used for immunization are used as a source of antibody-producing cells.

 Three to seven days after the final administration of the antigenic substance to the rat showing the antibody titer, the spleen is removed.

 The spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.

 The spleen cells in the resulting precipitate fraction are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, and then washed three times with MEM medium. Used as antibody-producing cells.

 (b) Preparation of myeloma cells

As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3_U1) [Curr. Topics Microbiol. Immunol., 81, 1 (1978), Europ. J Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) CNature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 ( 1979)], P3-X63-Ag8 (X63) (Nature, 256, 495 (1975)), etc. These cell lines can be used in an 8-azaguanine medium [glutamine (1.5 mmol / l L), 2-mercaptoethanol (5xlO- ⁵ mol / L), Jiwen evening puromycin (10〃G / ml) and a medium supplemented with fetal calf serum (FCS) (manufactured by CSL, 10%) (hereinafter referred to as normal medium), followed by a medium supplemented with 8-azaguanine (15 µg / ml)] However, 3 to 4 days before cell fusion, the cells are cultured in a normal medium, and 2 × 10 ⁷ or more cells are used for fusion.

 (c) Preparation of hybrid dorma

 The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were combined with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of salt, distilled) Wash well with 1 liter of water, pH 7.2), mix the cells so that the number of antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuge at 1,200 rpm for 5 minutes, Discard the Qing.

Thoroughly loosened The resulting precipitated fraction of cell groups, the said group of cells, with stirring, at 37, 10 ⁸ antibody-producing cells per polyethylene glyco one Le - 1000 (PE G- 1000) 2g , MEM 2 m 1 Then add 0.2 to 1 ml of a solution obtained by mixing 0.7 ml of dimethyl sulfoxide (DMSO), and then add 1 to 2 ml of MEM medium several times every 1 to 2 minutes.

 After addition, add MEM medium to adjust the total volume to 50 ml.

 After centrifuging the preparation at 900 rpm for 5 minutes, discard the supernatant.

After the precipitated fraction of the cells obtained was gently loosened, intake included by a measuring pipette, hypoxanthine (10_ ⁴ mol / L) to gently HAT medium [normal medium in blowing, thymidine (1. 5 x 10- ⁵ mol / L) and aminopterin (4x 10- ⁷ mol / L) was added medium] is suspended in 100 ml.

The suspension is dispensed at 100 1 / well into a 96-well culture plate, and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator.

After culturing, a part of the culture supernatant is taken to specifically bind to the senescence-inhibiting partial fragment polypeptide by the enzyme immunoassay described in Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter H (1988). Select Hypri-Doma to react I do.

 The following method can be given as a specific example of the enzyme immunoassay.

 At the time of immunization, the senescence-inhibiting partial fragment polypeptide used as the antigen is coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below is reacted as the first antibody. An anti-rat immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like is reacted as an antibody, and then a reaction is performed according to the labeling substance.Anti-aging that specifically reacts with the anti-aging polypeptide is performed Select as hybridoma producing an inhibitory polypeptide monoclonal antibody.

 Cloning was repeated twice by limiting dilution method using the hybridoma.

[The first time, use HT medium (medium in which aminopterin is removed from HAT medium), the second time, use a normal medium.] The one with a stable and strong antibody titer is the anti-aging inhibitory polypeptide antibody Select as a producing hybridoma strain.

 (d) Preparation of monoclonal antibody

To 8-10 week old mice or nude mice that had been treated with pristane [0.5 ml of 2,6,10,14-tetramethylpenedecane (Pristane) administered intraperitoneally and bred for 2 weeks], (c Intraperitoneally inject 5 to 20 × 10 ⁶ cells / animal-producing anti-aging polypeptide monoclonal antibody-producing hybridoma cells obtained in). Hypridoma develops ascites cancer in 10 to 21 days.

 Ascites is collected from the mouse with ascites cancer, and the solid content is removed by centrifugation at 3,000 rpm for 5 minutes.

The obtained supernatant was subjected to a salting-out method using 40-50% saturated ammonium sulfate, a capryprilic acid precipitation method (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, (1988)), DE AE-Sepharose column, protein A —Collect IgG or IgM fractions using a column or column chromatography using Cellulofine GSL2000 (manufactured by Seikagaku Corporation) and use it as a purified monoclonal antibody. The antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein content is calculated by the Lowry method or from the absorbance at 280 nm.

 4) Immune cell staining using monoclonal antibodies

 When performing immune cell staining using adherent cells, it is preferable to use cells that have been detached from the culture flask by performing the following treatment in advance.

 That is, the cultured adherent cells were washed with PBS buffer, and 3 ml of PBS buffer containing 0.05% trypsin and 0.02% EDTA (ethylenediamine tetraacetic acid) was added. After removing the excess solution, 37 ° C The cells are detached from the flask by incubating for 5 minutes (hereinafter, this operation is referred to as trypsin-EDTA treatment). Cultured cells can be used as they are for suspension cells.

Cells immune cell staining buffer to perform immunocytochemistry were suspended in such (1% BSA, 0. 02% EDTA, 0. PBS containing 0.05% sodium azide), 1~20 X 10 ⁵ cells each round Dispense into a 96-well plate.

 The culture supernatant of the anti-aging inhibitory polypeptide monoclonal antibody-producing hybridoma obtained in (c), the purified monoclonal antibody obtained in (d), or the monoclonal antibody obtained by the method (enzyme antibody method: Dilute the antibody labeled with biotin as described in (Interdisciplinary Planning, 1985) with an immunocell staining buffer or an immunocell staining buffer containing 10% animal serum to a concentration of 0.1 to 50 μg / ml. Dispense the solution so that the volume becomes 20-500 1 / well, and leave under ice-cooling for 30 minutes.

In the above, when the culture supernatant of the anti-aging inhibitory polypeptide monoclonal antibody-producing hybridoma obtained in (c) or the purified monoclonal antibody obtained in (d) is used, the plate for immunocell staining is used. After adding a buffer and washing the cells, add 0.1-50 μl of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with a fluorescent dye such as FITC or phycoerythrin. Dispense 50-500〃1 / well of an immunocell staining buffer containing a concentration of about g / ml, and leave it on ice for 30 minutes in the dark.

 When the monoclonal antibody labeled with biotin is used, 50 to 501 / well of streptavidin is dispensed into the above-mentioned plate, and the mixture is allowed to stand under ice-cooling for 30 minutes under ice-cooling.

 In both cases, after standing, add a buffer solution for immunocytochemistry to the plate, wash the cells well, and analyze with a cell saw.

 5) Immunoprecipitation of aging inhibitory polypeptide

 CH0 cells or bizoa cells expressing the aging inhibitory polypeptide of the present invention are cultured in a culture vessel such as a dish.

 Add PBS to the culture vessel and wash the cells.

 Solubilize the cell membrane in an ice-cooled buffer containing 1% Triton X100, 20 mmol / LT ris-HC1, and 150 mmol / L NaC1 (hereinafter referred to as buffer 1) in the culture vessel. Add 100-500〃1 of the buffer solution, leave on ice for 30 minutes, and gently shake at 5 minute intervals to solubilize.

 The treated solution is collected in a 1.5 ml centrifuge tube, and centrifuged at 14,000 rpm for 30 minutes.

 After adding 10 to 501 of protein G-sepharose or protein A-sepharose equilibrated with the buffer solution used above to the obtained supernatant and shaking at 4 ° C for 1 hour or more, Centrifuge at 5,000 rpm for 2 minutes and collect the supernatant.

The culture supernatant of the anti-aging inhibitory polypeptide monoclonal antibody-producing hybridoma obtained in (c) or the purified monoclonal antibody obtained in (d) is added to the supernatant at 0.01 to 50 μg / ml. And shake at 4 ° C for at least 1 hour. Add 10 to 50〃1 of Protein G-Sepharose or Protein A-Sepharose to the shaker, shake at 4 ° C for 1 hour or more, Centrifuge for minutes.

 To the obtained precipitate fraction, 2001 of a buffer solution for solubilizing the cell membrane is added to suspend the precipitate. Repeat the same procedure at least three times to wash the precipitate fraction. To the precipitate is added a sample buffer for SDS-polyacrylamide gel electrophoresis, and the mixture is heated using a heat block, followed by SDS-PAGE.

 After completion of SDS-PAGE, the polypeptide in the obtained gel is transferred to a PVDF membrane or the like, and the anti-aging polypeptide is subjected to Western blotting or the like using the anti-aging suppressing partial fragment polypeptide polyclonal antibody of the present invention. Detect the peptide.

 6) Search and identification of ligands that specifically bind to the aging inhibitory polypeptide of the present invention

 A ligand can be searched for and identified by bringing the test sample into contact with the aging inhibitory polypeptide of the present invention and selecting a substance that specifically binds to the polypeptide of the present invention from the test sample.

 Test samples include, for example, urine, body fluids, and tissue extracts from mammals (eg, mice, rats, guinea pigs, Hamus, hummus, higgs, horses, dogs, dogs, cats, monkeys, humans, etc.) Products, cell culture supernatants, cell extracts and the like can be used.

 For example, urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, etc. are appropriately diluted, concentrated, fractionated, etc., and brought into contact with the aging inhibitory polypeptide of the present invention, and the cell stimulating activity, etc. Further fractionation into the indicator can ultimately isolate a single ligand.

Specifically, a test sample is brought into contact with both a cell that does not originally express the senescence inhibitor gene and a cell that has the senescence inhibitor gene introduced and expressed in the cell, for example, intracellular calcium, cAMP Changes in intracellular signaling molecules such as cGMP, phosphorylation of intracellular proteins, changes in expression of early transcription factor genes, changes in cell membrane potential, changes in intracellular pH, release of extracellular signaling molecules Various cell stimulating activities, such as changes in cell morphology, were measured in both cells, and the differences between the two cells were compared and analyzed in detail. By doing so, ligands can be searched and identified.

 Synthetic compounds, naturally occurring or artificially synthesized proteins, carbohydrates, lipids and their modified products and derivatives are labeled with a radioisotope or the like, and the expression of the labeled compound of the aging inhibitory polypeptide of the present invention is performed. By measuring the amount of binding to the cell, the cell membrane fraction, or the aging-inhibiting polypeptide of the present invention solidified on a microplate or the like, it is determined whether or not the ligand is the ligand of the present invention. Identification is possible.

 In addition, a known method in which the aging-inhibiting polypeptide of the present invention or a partially modified or partial peptide thereof is covalently bound to a BIAcore (Pharmacia Biotech) sensor chip and contacted with a test sample [Nature, 368, 558 ( 1994)] can search for and identify ligands.

 Further, using the labeled aging inhibitory gene polypeptide of the present invention or the unlabeled polypeptide and a labeled antibody against the polypeptide, a ligand specifically associated with the polypeptide is searched for and identified by the following method. It is possible. That is, after appropriate fractionation of urine, body fluid, tissue extract, cell culture supernatant, cell extract, etc., electrophoresis such as polyacrylamide electrophoresis, agarose electrophoresis, two-dimensional electrophoresis, etc. Fractionation by techniques such as column chromatography such as HPLC, thin-layer chromatography, etc., and individuals expressing the present aging inhibitory gene and those not expressing the aging inhibitory gene, individuals, or expressing the present aging inhibitory gene. By comparing and analyzing the differences between live cells and non-expressing cells in detail, band-spots and beaks that are observed (or disappear) specifically correlated with the expression of the senescence-suppressing gene are identified. Identify.

The bands, spots, and peaks were transferred from a gel or a thin plate to a support such as a nitrocellulose membrane, nylon membrane, or PVDF membrane using a blotting technique, and then labeled with the labeled anti-aging gene. Using the peptide or the unlabeled polypeptide and a labeled antibody against the polypeptide, A band or spot that specifically binds can be searched for, and a ligand can be extracted from the band or spot and identified.

 7) Search for and identify compounds that inhibit the specific binding between the aging inhibitory polypeptide of the present invention and the ligand of the present invention

 By comparing the case of contacting the anti-aging polypeptide of the present invention with the ligand of the present invention and the case of contacting the present anti-aging polypeptide, the ligand of the present invention and the test compound, Compounds that inhibit the specific binding between the aging inhibitory polypeptide and the ligand (eg, proteins, peptides, carbohydrates, lipids, non-peptidic compounds, synthetic compounds, fermentation products, biological components, etc.) can be screened. Compounds obtained by the screening include a compound having an activity of inhibiting the specific binding of a ligand to an aging-inhibiting polypeptide, an activity of inhibiting the activity of the aging-inhibiting polypeptide (an evening gonist), and a specific binding agent. Compounds that inhibit but have an activity similar to or replace the ligand (agonist) are included.

 As test samples, in addition to synthetic compounds, naturally occurring or artificially synthesized proteins, carbohydrates, lipids and their modified products and derivatives, for example, mammals (eg, mouse, rat, guinea pig, Urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, and other fermented products, plants and other hamsters, bushes, sheep, wedges, horses, horses, dogs, cats, monkeys, humans, etc.) It can provide biological extracts.

 Examples of the method for screening the compound include the following methods.

A test sample is brought into contact with a ligand of the present invention alone or a test sample with a cell into which the present senescence suppressor gene has been introduced or expressed or a cell originally expressing the present senescence suppressor gene. Changes in the concentration of intracellular signaling molecules such as cGMP, phosphorylation of intracellular proteins, and changes in expression of early transcription factor genes Various cell stimulating activities, such as activation, changes in cell membrane potential, changes in intracellular pH, release of extracellular signaling molecules, changes in cell morphology, etc. By comparing the above, there can be mentioned a method for searching for and identifying a compound that inhibits the specific binding between the aging inhibitory gene polypeptide of the present invention and the ligand of the present invention.

 In addition, the ligand of the present invention is labeled with a radioisotope or the like, and the labeled aging of the present invention in which the labeled ligand is immobilized on the senescence-inhibiting polypeptide-expressing cells of the present invention, the cell membrane fraction, or a micro-type plate or the like. The amount of binding to the inhibitory polypeptide was measured using the label, and the aging inhibitory gene polypeptide of the present invention was compared with the ligand by comparing the ligand alone and the ligand in contact with the test sample. Compounds that inhibit the binding of can be searched for and identified.

8) Search and identification of compounds induced as a result of binding of the aging inhibitory polypeptide of the present invention to the ligand of the present invention

 The culture supernatant, cells, and cytoplasmic fractions of the cells expressing the polypeptide of the present invention and the cells not expressing the polypeptide are respectively contacted with and without the ligand of the present invention. Fractions, cell membrane fractions, etc., are subjected to polyacrylamide electrophoresis, agarose electrophoresis, two-dimensional electrophoresis, etc., column chromatography such as HPLC, thin-layer chromatography, etc., and each component is fractionated. Then, by comparing each component, a band, spot, peak, etc., which is specific when the ligand is brought into contact and which specifically appears or disappears in correlation with the expression of the polypeptide, is identified. The target molecule can be obtained from the spot, the peak, and the peak.

In addition, in the individual expressing the polypeptide of the present invention, it is considered that the specific binding between the polypeptide and the ligand is constantly occurring, and the individual not expressing the polypeptide (specific examples It is considered that this interaction does not occur in the gene-disrupted mouse described in this patent specification). Thus normal mice and premature aging as described in this patent After appropriate fractionation of various mouse organs, tissues, body fluids, blood, urine, etc., electrophoresis such as polyacrylamide electrophoresis, agarose electrophoresis, two-dimensional electrophoresis, and columns such as HPLC Fractionation by techniques such as chromatography and thin-layer chromatography and comparison are performed to search for bands, spots, peaks, etc., which exhibit different behaviors between the two, and molecules are recovered from the bands, spots, peaks, etc. The target compound can be identified from the band, spot, or peak. 9) Search and identification of compounds that enhance the expression of the aging inhibitory polypeptide of the present invention (hereinafter abbreviated as expression enhancing compounds)

 (1) Search and identification using an antibody that recognizes the aging inhibitory polypeptide of the present invention After contacting a cell expressing the aging inhibitory polypeptide of the present invention with a test sample, the aging inhibitory polypeptide of the present invention By quantifying the polypeptide using an antibody recognizing the expression, an expression-enhancing compound present in the cell or in the cell culture supernatant can be searched for and identified.

 Cells expressing the aging inhibitory polypeptide of the present invention include, for example, cells derived from mouse gland, fetal liver, and mammary gland. For example, as for fetal liver, those prepared using a known method (primary culture hepatocyte experiment method, Gakkai Shuppan Senyuichi, 1987) can be used.

 As test samples, in addition to synthetic compounds, naturally occurring or artificially synthesized proteins, peptides, non-peptidic compounds, carbohydrates, lipids, and modified and derivative forms thereof, for example, mammals ( For example, urine, body fluid, tissue extract, cell culture supernatant, cell extract, and fermentation of mice, rats, guinea pigs, hamsters, bush, hibiscus, hidge, horses, horses, dogs, cats, monkeys, humans, etc.) Examples include, but are not limited to, products, extracts of plants and other organisms.

The cells expressing the aging inhibitory polypeptide of the present invention are suspended, for example, in a medium capable of growing the cells, a test sample is added to the medium, and the cells are brought into contact with each other. The anti-aging polypeptide content of the polyclonal Quantification according to the method described in 4) above, using a monoclonal antibody or a monoclonal antibody. An expression-enhancing compound can be identified by searching for a test sample capable of increasing the aging-inhibiting polypeptide content as compared with a system without the test sample.

 (2) Search and Identification Using a Quantitative System for Transcripts of the Senescence Inhibitor Gene of the Present Invention After Contacting Cells Expressing the Antioxidant Polypeptide of the Present Invention with a Test Sample, Transcription of the Senescence Repressor Gene of the Present Invention By quantifying the product, an expression enhancing compound can be searched and identified.

 As the cells expressing the aging inhibitory polypeptide of the present invention and the test sample, those described in the above (1) can be used.

 The cells expressing the aging inhibitory polypeptide of the present invention are suspended, for example, in a medium capable of growing the cells, a test sample is added to the medium, and the cells are brought into contact with each other. The amount of the transcript of the aging repressor gene can be quantified by the usual Northern blot blot hybridization method, RNA dot blot blot hybridization method, RT-PCR method and the like.

 Examples of the probe that can be used for the hybridization method and the like and the primer that can be used for the RT-PCR method and the like include the senescence-suppressing gene fragment of the present invention, and specifically, SEQ ID NO: 4, 5 or A DNA fragment having a sequence selected from the DNA sequences described in 6 can be suitably used.

 An expression-enhancing compound can be identified by searching for a test sample capable of increasing the transcript content of the aging-suppressing gene of the present invention, as compared with a system in which the test sample is not added.

 (3) Search and identification using repo overnight gene

A transformant transformed with a plasmid containing DNA linked to a repo allele downstream of a region that regulates transcription of a gene encoding the aging inhibitory polypeptide of the present invention (hereinafter, abbreviated as a transcription control region). After contacting the test sample with By quantifying the expression level of the polypeptide encoded by the overnight gene, an expression enhancing compound can be searched and identified.

 Examples of the transcription control region include the region of base numbers 1-2177 described in SEQ ID NO: 7. In addition, a fragment obtained by cutting this region using an appropriate restriction enzyme into an appropriate length can be used as a transcription control region.

 As the repo overnight gene, any gene can be used as long as the translation product of the gene is stable in cells and the amount of the translation product can be easily quantified. For example, chloramphene Genes encoding nicole acetyltransferase (CAT),? -Galactosidase (? -Ga1), luciferase (1 uc), green fluorescein protein (GFP), etc. .

 The test sample described above (1) can be used.

 A reporter gene is ligated to the downstream of the transcription control region by a conventional method, and a host cell is transformed by a conventional method using the prepared plasmid.

 The transformant is suspended, for example, in a medium in which the cells can grow, a test sample is added to the medium, and the cells are contacted. Then, the amount of the transcript of the reporter gene expressed in the cells is measured. It is detected and quantified by a method suitable for the transcript.

As a detection and quantification method, in the case of CAT, for example, the method described in Molecular Cloning, Second Edition, Chapter 16, page 60, is used. — In the case of ga1, for example, Molecular 'cloning, Second Edition In the case of luc, for example, the method described in Experimental Medicine Separate Volume Bio Manual Series 4, Gene Transfer and Expression 'Analysis, 81 (1994) Natl. Acad. Sci., USA, 94, 4653 (1997). An expression-enhancing compound can be identified by searching for a test sample capable of increasing the transcript content of the aging-suppressing gene of the present invention, as compared with a system to which no test sample is added. 10) Anti-aging polypeptide, DNA encoding the polypeptide, antibody recognizing the polypeptide, ligand of the present invention and use of expression enhancing compound

 (1) By using the anti-aging inhibitory polypeptide antibody of the present invention, it is possible to detect and quantify the aging inhibiting polypeptide in a sample of blood, a part of an organ, a cell or the like.

 Specific preferred methods include the ELISA method using a micro-tie plate, a fluorescent antibody method, and a Western blot method, and the method can also be used for immunohistological staining using a pathological tissue section. Therefore, the antibody is useful for diagnosing the possibility of the occurrence of premature aging and various adult diseases accompanying the decrease in the expression of the aging inhibitory polypeptide. Similarly, it is also useful as a research reagent in research targeting the peptide. (2) Aging can be suppressed by administering a full-length or partial fragment of the aging-inhibiting polypeptide of the present invention to a living body. Therefore, it is useful as a therapeutic or prophylactic for various adult diseases closely related to the progress of aging, such as arteriosclerosis, hypertension, and osteoporosis. It is also useful for prolonging the life based on the suppression of aging.

(3) The aging inhibitory gene of the present invention can be incorporated into viral vectors such as retroviruses and adenoviruses and other vectors and used for therapy by a gene therapy method.

 (4) Using the aging inhibitory gene of the present invention, the expression level of the gene is measured by the Northern hybridization method or the PCR method to diagnose aging and adult diseases, and aging the aging inhibiting gene. It can be used for suppression and adult disease onset. In addition, aging is promoted due to the congenital deficiency of the aging suppressor gene, and individuals that are susceptible to adult disease are subjected to Southern blot hybridization using the aging suppressor gene, PCR method or SSCP method. Detected, and genetic diagnosis can be performed based on the detected nucleic acid sequence information of the individual. Further, the aging suppression gene is extremely useful as a reagent for gene research.

(5) In addition, the target livestock animals provided by the present invention, for example, Aging inhibitory genes derived from germ, goat, bush, horse, chicken, etc. are administered to the corresponding animals, or the genes are expressed in individuals using a vector such as a virus suitable for gene therapy. By doing so, it is possible to further extend the lifespan of the target livestock and suppress senescence by a cloning method by nuclear transfer using a nucleus of a cell that strongly expresses the gene. This allows for long-term good breeding and long-term collection of milk, eggs and offspring. Alternatively, the same effect can be expected by introducing the gene into a fertilized egg and inserting the gene into the chromosomes of all cells in the body and expressing it, so-called transgenic animals. However, it is considered to be highly useful in terms of breeding.

 (6) The anti-aging polypeptide of the present invention is useful as a reagent for searching for and determining a ligand that specifically binds to the polypeptide.

 (7) The aging inhibitory polypeptide of the present invention and the ligand of the present invention can be used for searching and identifying a compound that inhibits specific binding between the polypeptide and the ligand.

 (8) By using the aging inhibitory polypeptide of the present invention and the ligand of the present invention, it is possible to search and identify a molecule induced as a result of binding of the polypeptide to the ligand.

 (9) The ligand of the present invention, a compound that inhibits the specific binding between the aging inhibitory polypeptide of the present invention and the ligand of the present invention, and a molecule derived as a result of binding of the polypeptide of the present invention to the ligand include: It is thought to replace or assist the aging inhibitory function of the aging inhibitory gene polypeptide of the present invention, and drugs containing these molecules are useful as therapeutic drugs for early aging, adult diseases, and aging inhibitors.

(10) A compound that increases the expression of an aging inhibitory gene that encodes the aging inhibitory polypeptide of the present invention, which is useful as a drug for the treatment of premature aging, an adult disease, or an aging inhibitor (expression enhancement) The compound is useful for inhibiting aging, treating and preventing adult diseases, like the aging-inhibiting polypeptide. 1 1) Method for producing and using genetically modified non-human animals in which the aging suppressor gene has been deleted or replaced

 (1) All or part of the aging inhibitory gene is deleted or replaced, and a chromosomal aging inhibitory gene is known in embryonic stem cells using a vector containing an inactive or substituted aging inhibitory gene. Mutant clones that have been inactivated or replaced with an arbitrary sequence can be prepared by homologous recombination techniques (for example, Nature, 326, 295 (1987), Cell, 51, 503 (1987), etc.) [Nature, 350, 243 (1991)].

 Specifically, the chromosomal gene fragment shown in FIG. 1 or SEQ ID NO: 7 can be used. Evening targeting vectors can be constructed as follows, but are not limited thereto.

 The fragment of Fig. 1 (short arm: about 2.3 kb), PGKneobpA cassette [PGK probe-neomycin resistance gene-gamma growth hormone gene neomycin resistance gene expression unit consisting of polyadenylation signal sequence, Cell, 64 , 693 (1991)], the I- ^ I fragment of Figure 1 (lomg arm: about 4.3 kb), MC1 / DT-A cassette [MCI promoter and diphtheria toxin A chain expression unit consisting of diphtheria toxin A chain, Analytical Biochemistry, 214, 77 (1993)] as shown in FIG. 1 as a Targeting vector to construct pBluescriptlSK (-). The vector is cut into a linear form by cutting it with 1 ^ 1, etc., and then introduced into an ES cell line such as TT2 cells (Analytical Biochemistry, 214, 70 (1993)). A clone in which exon 1 of the aging repressor gene has been destroyed by homologous recombination is selected by the dilution method.

Using the embryonic stem cell clone thus prepared, a chimeric individual consisting of an embryonic stem cell clone and normal cells is prepared by a method such as injection chimera method or assembly chimera method of injecting an animal fertilized egg into a blastocyst (blastcyst). can do. By crossing this chimeric individual with a normal individual, mutations are made An individual having the homologous chromosome can be obtained by crossing the individuals.

 In this way, it is possible to introduce a mutation into any position of the aging suppressor gene in an animal individual. For example, the activity of the product can be changed by introducing a mutation such as a base substitution, deletion, or insertion into the translation region of the aging suppressor gene. Also, by introducing a similar mutation into the expression control region, the degree, timing, tissue specificity, etc. of the expression can be altered. In addition, it is also possible to more positively control the expression timing, expression site, expression amount, etc., in combination with the Cre-loxP system (; J. Clin. Invest., 98, 600 (1996)). Examples include the use of promoters expressed in specific regions of the brain to delete target genes only in those regions [Cell, 87, 1317 (1996)] and adenoviruses expressing Cre An example is known in which the target gene is specifically deleted at the target time using the method [Science, 278, 5335 (1997)]. It is possible to create an individual animal whose expression can be controlled at the time or in the tissue, or that has an arbitrary insertion, deletion, or substitution in its translation region or expression control region. At any degree or at any site, with aging and with aging It is capable of inducing the symptoms of various diseases such as cheating adult diseases, etc. Therefore, it is an extremely useful animal model in the treatment and prevention of various diseases such as aging and adult diseases. It is also very useful as a model for evaluating functional foods, health foods, etc.

FIG. 1 is a diagram showing a restriction enzyme map of a chromosome gene clone of an aging suppressor gene and a structure of a targeting vector for creating the present aging suppressor gene deletion and substitution by homologous recombination. In the figure, bp is base pairs (ヽ base pairs) ヽ kb is kilobase pairs (kilobase pairs), PGKneobpA is PGK pro overnight-neomycin resistance gene- ゥ growth hormone Neomycin resistance gene expression unit MC1 / DT-A consisting of a polyadenylation signal sequence: Represents a diphtheria toxin A chain expression unit consisting of MCI promoter overnight and diphtheria toxin A chain. BEST MODE FOR CARRYING OUT THE INVENTION

The examples are shown below. In the examples, unless otherwise specified, the genetic engineering method was performed by the method described in the well-known molecular cloning second edition. Example 1 Cloning of the chromosome gene of the aging suppression gene mouse counterpart (Step 1) Cloning of the chromosome fragment of the mouse aging suppression gene Mouse chromosome DNA 10 Ong, sense primer 1 O ^ having the nucleotide sequence shown in SEQ ID NO: 9 molZL 1/1, antisense primer having the base sequence shown in SEQ ID NO: 10 1 mol / L 1〃1, 10X PCR buffer (use the one attached to the enzyme) 4〃1 , 2.5 mmol / L dNTP 3.21, mixed with water to bring the total volume to 40〃1, and then mixed with Taq DNA polymerase (Takara Shuzo, 5 units / ί1) 0.5 zl did. 20 cycles of 97 ° C for 10 minutes, [94 ° C for 1 minute, 65 ° C for 1 minute at 0.5 ° C per cycle, 72 ° C for 1 minute], ( PCR was performed under the following conditions: 20 cycles of 94 ° C for 1 minute, 65 ° C for 1 minute, 72 ° C for 1 minute), 72 ° C for 5 minutes, and 4 ° C hold. As a result of analysis by agarose gel electrophoresis, a fragment of about 0.3 kb was observed. The amplified fragment was purified from the agarose gel, and placed in a pT7Blue T-vector (Novagen) using the DNA ligation kit ver. 2 (Takara Shuzo) (Made by the company). The method followed the instructions in the kit instructions. Using this solution, Escherichia coli XL1-Blue strain was transformed to obtain a transformant having ampicillin resistance. Plasmid was extracted from the transformant using a plasmid extractor P1-100 (manufactured by Kurabo Industries), and the nucleotide sequence was determined using ABI Prism BigDyeTerminator Cycle Sequencing Ready Reaction Kit (manufactured by Applied Biosystem). One 377 CPerkin Elmer SEQ ID NO: showing homology to the human aging suppressor gene

A clone having the nucleotide sequence described in 8 was found.

 (Step 2) Cloning of mouse aging inhibitory gene chromosomal gene SEQ ID NO: 11 as a primer that specifically and completely matches the sequence of the clone isolated in Step 1

(The base sequence of SEQ ID NO: 8, base numbers 31 to 58) and SEQ ID NO: 12 (the complementary sequence of base numbers 198 to 219 in the base sequence of SEQ ID NO: 8) were synthesized. Using a set of primers having the nucleotide sequences of SEQ ID NOS: 11 and 12, a chromosomal gene BAC library of the 129Svj mouse-derived ES cell line was searched, and a specific band was amplified by this set of primers. That is, a BAC clone containing the mouse aging suppression gene, clone 19348, was isolated. clone 19348 had an imported fragment of about 14 Okb.

The obtained clones were analyzed by various restriction enzyme digestions and by Southern blot using the clone fragments obtained in step 1 as probes. As a probe for Southern blot analysis, a probe obtained by labeling the clone fragment obtained in step (1) with digoxigenin (DIG) using a DIG DNA labeling kit (Boehringer Mannheim) was used. The labeling method followed the instructions attached to the kit. The BAC clone was cleaved with various restriction enzymes, fractionated by agarose electrophoresis, and then subjected to nylon membrane fining-HybondN + (manufactured by Amersham) (this was set in the mouth. The protocol of Boehringer Mannheim) was used. The DNA was denatured, immobilized, hybridized with the probe, and washed with a filter in accordance with the DIG Luminescent Ditection Kit for Nucleic Acids.The hybridizing solution was 5XSSC; 1% Blocking solution [DIG Luminescent Ditection] Kit for Nucleic Acids (manufactured by Berlinka Mannodim Co.)], 0.1% Sacrcosyl, 0.02% SDS, and overnight at 68. Washing was carried out using 0 Anti-DIG antibody after 2 x 10 min in 233〇 with 1% 303 at room temperature and 0.1 x 2x in SSC with 0.1% SDS for 15 min at 68 ° C DIG detection kit using It was confirmed that a region hybridizing with the probe was present on a fragment of about 1 O kb when digested with Boehringer Mannheim).

 Then, clone 19348 was digested with ^ I, and the resulting fragment of about 1 O kb was cloned into the ^ I site of pBluescript SK (-). The fragment was digested with various restriction enzymes to prepare a restriction map shown in FIG. By subcloning fragments generated by various restriction enzyme digestions, and simultaneously, a deletion series was prepared by sequential digestion using Exo Mung Bean Deletion kit (Stratagene) according to the instruction manual attached to the kit. The nucleotide sequence was determined by the method described in Step 1. As a result, the nucleotide sequence could be determined within a range of about 4 kb from the left end of the restriction enzyme map in FIG. 1, and a region showing high homology with human RYHH02 cDNA, that is, a region considered to be exon 1 was identified. The position shown in Figure 1 could be identified. The determined nucleotide sequence is shown in SEQ ID NO: 7. Therefore, it was confirmed that the exon 1 of the mouse aging suppressor gene was contained on the approximately 10 kb fragment.

Example 2 Cloning of mouse aging suppressor gene cDNA

 For screening of mouse aging suppressor gene cDNA, Swiss Webster mouse 19-day-old embryo cDNA library RAP ID-SCREEN ™ cDNA LIBRARY PANELS (OriGene

Technologies) (SEQ ID NO: 13 (in the base sequence described in SEQ ID NO: 7, base numbers 2389 to 2411)) and SEQ ID NO: 14 (in the base sequence described in SEQ ID NO: 7, base number 29) (Complementary strand of 51 to 295) The PCR was carried out using a set of primers as described. All screening conditions and the like were in accordance with the manual of OriGene Technologies. That is, using the plasmid solution of each plate on the plate, 1 X PCR buffer, 0.2 band ols L each dNTPs, and 1 lmol of the primer having the base sequence shown in SEQ ID NO: 13 in 2 reaction systems / L, 1 mol / L of a primer having the nucleotide sequence of SEQ ID NO: 14, 1.25 U Amli TaqR Go Id DNA Polymerase (Perkin Elmer), 95. 3 cycles of the reaction for 9 minutes at C, 30 seconds at 95 ° C, 30 seconds at 65 ° C, and 30 seconds at 72 ° C, 5 minutes at 72 ° C The PCR reaction was performed under the temperature conditions between. As a result of analysis by agarose gel electrophoresis, a specific amplification band was detected in the reaction solution derived from the six wells.A 96-well subplate corresponding to the six wells was purchased, and a PCR reaction was performed in the same manner. Finally, a positive reaction was found in 2 ml. Escherichia coli glycerol per 1-well stock corresponding to the 2-well was diluted 1000-fold with LB medium, spread on an agar medium, and colonies appeared.

 Appearing colonies were randomly searched by colony PCR, and plasmids were extracted from the obtained positive colonies. Then, the entire nucleotide sequence of the cDNA portion contained in the plasmid was determined. The obtained nucleotide sequences are shown in SEQ ID NOs: 4, 5, and 6, and the amino acid sequences encoded by the nucleotide sequences are shown in SEQ ID NOs: 1, 2, and 3, respectively.

Example 3 Tissue expression distribution of aging suppressor gene

 The human aging-derived aging inhibitory polypeptide encoded by pRYHH02 described in W098 / 29544 shows about 45% homology in amino acid sequence with the polypeptide encoded by the klotho gene, and thus belongs to the klotho family. It is an aging inhibitory polypeptide.

 The anti-aging polypeptide encoded by the DNA of the present invention is a mouse counterpart of an anti-aging polypeptide derived from human lentin, which is about 44% of the polypeptide encoded by the klotho gene and derived from human lentin. Since it has 76% homology with the aging inhibitory polypeptide, the aging inhibitory polypeptide of the present invention is also a polypeptide belonging to klotho family and is considered to have a similar function to klotho. In predicting the function of the aging inhibitory polypeptide of the present invention, it is an effective means to compare the tissue expression distribution of the polypeptide encoded by the klotho gene and the human aging-derived aging inhibitory polypeptide. it is conceivable that.

Based on the base sequence of the human anti-aging gene encoded by pRYHH02 described in W098 / 29544, a DNA consisting of the base sequence of SEQ ID NO: 15 and the base sequence of SEQ ID NO: 16 A DNA consisting of the described base sequence was synthesized.

 On the other hand, single-stranded cDNA was synthesized from mRNA (Clontech) derived from various human organs using a kit (SUPERSCRIPTTM Preamplification System; BRL). Single-stranded cDNA was synthesized from l〃g of mRNA, diluted 240-fold with water, and used as PCR type I. An oligo (dT) primer was used as a primer. mRNA includes adrenal gland, brain, caudate nucleus, hippocampus, substantia nigra, thalamus, kidney, england, pituitary gland, small intestine, bone marrow, amygdala, cerebellum, corpus callosum, fetal brain, fetal kidney, fetal liver, fetal Lung, heart, liver, lung, lymph node, mammary gland, placenta, prostate, salivary gland, skeletal muscle, spinal cord, spleen, girdle, testis, thymus, thyroid, trachea, uterus .

 RT-PCR reaction was performed for single-stranded cDNA (0.2 ng / 〃l) 5〃1, 10 x Ex-Taq buffer 2〃1, 2.5 mmol / L dNTPs 1.6〃1, 20 mol / L SEQ ID NO: 1

Primer having the base sequence described in 5, primer 1 having the base sequence described in SEQ ID NO: 16 of 1〃1, 20〃mo 1 / L, DMSO l

, Ex-Taq (Takara Shuzo Co., Ltd.) 0.5 1〃1, H ₂ 0 8. consisting 3〃1 solution 9 8 ° C for 5 minutes, 3 0 seconds (94 ° C, 1 in 6 5 ° C The reaction was carried out for 30 cycles at 72 ° C for 10 minutes at 72 ° C for 10 minutes. The reaction products were analyzed by agarose gel electrophoresis, and the expression intensity in each tissue was analyzed based on the intensity of the resulting band.

 As a result, strong expression was observed in the tissues of the kidney, fetal liver, and mammary gland, and weak expression was observed in the caudate nucleus, bone marrow, liver, and testis, but expression was confirmed in other tissues within the range examined. Did not. Therefore, the expression of the human senile gland-derived senescence inhibitory gene is tissue-specific, and it is clear that the expression distribution is distinctly different from that of the klotho gene, which is strongly expressed in the kidney. It is apparently different.

Therefore, the aging inhibitory polypeptide of the present invention, which has higher homology to the human kidney-derived aging inhibitory polypeptide, also has an effect different from that of klotho in its role. It is thought that. Industrial applicability

 The present invention relates to a novel polypeptide that suppresses animal aging, DNA encoding the polypeptide, a chromosomal DNA fragment containing the DNA, an antibody recognizing the polypeptide, a ligand for the polypeptide of the present invention, A compound that inhibits specific binding between the polypeptide of the present invention and a ligand; a compound that increases the expression of an aging inhibitory gene encoding the aging-inhibiting polypeptide of the present invention; a method of searching for the compound and the aging Provided are methods for producing and using animals in which a suppressor gene has been deleted or partially modified.

"Sequence List Free Text"

SEQ ID NO: 9 Sequence description: Synthetic DNA

SEQ ID NO: 10—Description of sequence: Synthetic DNA

SEQ ID NO: 1 1—Sequence description: synthetic DNA

SEQ ID NO: 12—Description of sequence: Synthetic DNA

SEQ ID NO: 13—Description of sequence: Synthetic DNA

SEQ ID NO: 14—Sequence description: synthetic DNA

SEQ ID NO: 15—Sequence description: synthetic DNA

SEQ ID NO: 16—Sequence description: synthetic DNA

Claims

The scope of the claims

 1. A polypeptide having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1, 2, and 3 and having an activity of suppressing aging.

 2. A polypeptide having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1, 2 and 3 has an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and suppresses aging. A polypeptide having the activity of:

 3. A DNA encoding the polypeptide of claim 1 or 2.

 4. DNA represented by the nucleotide sequence of any one of SEQ ID NOs: 4 to 6.

5. A DNA encoding a polypeptide which is a DNA having a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence of the DNA according to claim 4, and which has an activity of suppressing aging. .

 6. A DNA that hybridizes with a DNA selected from the DNA according to any one of claims 3 to 5 under a stringent condition and encodes a polypeptide having an activity of suppressing aging.

 7. A recombinant DNA obtained by incorporating a DNA selected from the DNA according to any one of claims 3 to 6 into a vector.

 8. An antibody that recognizes the polypeptide of claim 1 or 2.

 9. A therapeutic agent for premature aging or adult disease, comprising the polypeptide of claim 1 or 2.

 10. An aging inhibitor comprising the polypeptide according to claim 1 or 2.

 11. An immunological method for quantifying the polypeptide according to claim 1 or 2, wherein the antibody according to claim 8 is used.

 12. An aging diagnostic method using the quantitative method according to claim 11.

13. A gene therapy vector for treating premature aging or adult disease, comprising the DNA according to any one of claims 3 to 6.

14. A gene therapy vector for suppressing aging, comprising the DNA of any one of claims 3 to 6.

 15. A transformant having the recombinant DNA of claim 7.

 16. culturing a transformant having the recombinant DNA according to claim 7 in a culture medium, producing and accumulating a polypeptide encoded by the recombinant DNA in the culture medium; 3. The method for producing a polypeptide according to claim 1, wherein the polypeptide is collected.

 17. The polypeptide according to claim 1 or 2, wherein the polypeptide according to claim 1 or 2 is brought into contact with a test sample, and a ligand for the polypeptide according to claim 1 or 2 is selected from the test sample. A method for searching for a ligand for a polypeptide.

18. A ligand obtained by the method according to claim 17.

 19. (a) When the polypeptide according to claim 1 or 2 is brought into contact with the ligand according to claim 18; and (b) When the polypeptide according to claim 1 or 2 is brought into contact with the ligand according to claim 18 and the test. A method for searching for a compound that inhibits specific binding between the polypeptide according to claim 1 or 2 and the ligand according to claim 18, wherein the comparison is made with the case where the compound is brought into contact with the compound. .

 20. A compound obtained by the method according to claim 19 or a pharmacologically acceptable salt thereof.

 21. Contacting cells expressing the polypeptide of claim 1 or 2 with a test sample and selecting a compound that enhances the expression of the gene encoding the polypeptide of claim 1 or 2 from the test sample. A method for searching for a compound that enhances the expression of a gene encoding the polypeptide according to claim 1 or 2, which is characterized by:

22. The search method according to claim 21, wherein the enhanced expression is detected using the quantitative method according to claim 11.

23. The search according to claim 21, wherein the enhanced expression is detected by measuring the amount of mRNA encoding the polypeptide according to claim 1 or 2. Law.

 24. A compound or a pharmacologically acceptable salt thereof obtained by the search method according to any one of claims 21, 22, and 23. .

 25. A transformant transformed with a plasmid containing a DNA linked to a reporter gene downstream of a region controlling transcription of the gene encoding the polypeptide according to claim 1 or 2. 3. The polypeptide according to claim 1 or 2, wherein the polypeptide is brought into contact with a test sample, and a compound that enhances expression of the gene encoding the polypeptide according to claim 1 or 2 is selected from the test sample. A method for searching for a compound that enhances the expression of a gene encoding

 26. The region that controls transcription according to claim 25 is a DNA having a base sequence represented by base numbers 1-217 in the base sequence set forth in SEQ ID NO: 7. The search method according to item 25.

 27. A compound obtained by the search method according to claim 25 or 26 or a pharmacologically acceptable salt thereof.

 28. A therapeutic agent for premature aging or adult disease, comprising the compound according to any one of claims 20, 24 and 27 or a pharmaceutically acceptable salt thereof.

 29. An anti-aging agent comprising the compound according to any one of claims 20, 24 and 27 or a pharmacologically acceptable salt thereof.

 30. A method for improving livestock using a DNA comprising the DNA according to any one of claims 3 to 6.

 31. The method for improving livestock according to claim 30, wherein the DNA according to any one of claims 3 to 6 is overexpressed.

32. The amount and timing of expression of a polypeptide having an activity to suppress aging by deleting or substituting all or part of the gene containing the DNA according to any one of claims 3 to 6 Or a gene-deleted or substituted non-human animal with altered tissue specificity.

33. The non-human animal animal of claim 32, wherein the change in the amount of expression is a change in which expression is reduced or not expressed.

 34. The animal according to claim 32 or 33, wherein the non-human animal is a non-human mammal.

 35. The animal according to claim 34, wherein the non-human mammal is a mouse.

36. Contacting the test compound with the animal according to any one of claims 32 to 35, or an organ, tissue or cell of the animal, and selecting a therapeutic agent for premature aging or adult disease from the test compound A method for searching for a drug for treating early aging or adult diseases characterized by the following.

 37. Contacting the animal according to any one of claims 32 to 35, or an organ, tissue or cell of the animal with a test compound, and selecting an aging inhibitor from the test sample. To search for antiaging drugs.

 38. A compound obtained by the method according to claim 36 or 37 or a pharmacologically acceptable salt thereof.

 39. A therapeutic agent for premature aging or adult disease, comprising the compound according to claim 38 or a pharmacologically acceptable salt thereof.

 40. An aging inhibitor comprising the compound according to claim 38 or a pharmacologically acceptable salt thereof.