JP2003334088A - HUMAN-DERIVED NEW Klotho-LIKE PROTEIN AND ITS GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a human-derived new Klotho-like protein and its gene. <P>SOLUTION: A gene cDNA encoding a human-derived new Klotho-like protein is prepared by VTS (virtual transcribed sequence) approach. The invention provides the human-derived new Klotho-like protein, a Klotho-like gene encoding the protein, a recombinant vector, a transformant containing the recombinant vector and a method for producing the Klotho-like protein based on the gene cDNA. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel human-derived Kl.
Otho-like senescence control protein (also called Klotho-like protein), Klotho-like senescence control gene (also called Klotho-like gene) encoding the protein, recombinant vector containing the gene, transformation containing the recombinant vector The present invention relates to a body, a method for producing the Klotho-like protein, and the like.

[0002]

2. Description of the Related Art The aging phenomenon means deterioration of an individual in the functional and appearance changes of the individual that progress with aging, and it is known that the frequency of occurrence of various lifestyle-related diseases increases with aging. ing. Therefore, it is expected that a drug capable of controlling aging in some way will be a therapeutic or preventive drug for lifestyle-related diseases, and a protective or preventive drug against functional / appearance deterioration. So far, few drugs have been scientifically proven to have an aging control effect. Genetic level research on individual aging has just started worldwide, and there is almost no molecular genetic information on individual aging. However, several genetic premature aging diseases are known, such as Turner syndrome, Werner syndrome, Hutchinson-Gilford syndrome (Progeria).
In particular, the causative gene has been identified for Werner syndrome [Science 272: 258 (1996)]. From the above, the existence of genes involved in aging is a definite fact. If aging is controlled by a gene, it can be regulated by controlling the activity of that gene, and can be used for the treatment or prevention of various diseases that are closely related to aging. it is conceivable that.

In 1997, Nabeshima et al. Discovered a mouse having a mutant phenotype that resembles various human aging symptoms.
The causative gene Klotho has been identified [Kuro-o M. et al .:
Nature 390: 45-51 (1997)]. Mice with dysfunction of this gene exhibit marked and various premature aging symptoms such as shortened lifespan, calcification of various organs, arteriosclerosis, and atrophy of reproductive organs. Using this mouse Klotho gene, human Klotho
The gene has also been cloned and its structure has been elucidated. In addition, analysis of human Klotho cDNA has revealed that two types of mRNA are transcribed and two types of protein are translated from the human Klotho gene due to the difference in splicing. Of these two types of Klotho proteins, one protein is a type 1 membrane protein having a structure having an N-terminal signal sequence region, an extracellular domain region and a C-terminal transmembrane domain region, and the extracellular domain is a bacterial domain. Alternatively, it is composed of two domains (KL1, KL2) homologous to plant β-glucosidase, while the other protein is a secretory protein having an N-terminal signal sequence region and a KL1 domain region. It was revealed [Mat
sumura H. et al .: Bichem. Biophys. Res. Commun. 24
2: 626 (1998)]. Furthermore, a search of mouse and human gene databases found a hit sequence similar to the Klotho gene, and using it as a clue, a gene encoding a β-Klotho protein having a high degree of homology with the Klotho protein was found [Ito S. et al: Mechanisms of De
velopment 98: 115 (2000)].

[0004] As in the case of the mouse β-Klotho protein, unknown Klotho-like proteins other than the known Klotho protein also exist in the human body, and they are combined with various diseases that develop with aging. It is thought that they are involved in this. Therefore, it is important to clarify the whole repertoire of Klotho proteins existing in the human body from the viewpoint of prevention and treatment of the above diseases.

[0005]

The present invention encodes a novel Klotho-like protein of human origin and the protein.
Klotho-like gene, recombinant vector containing the gene,
It is an object to provide a transformant containing the recombinant vector, a method for producing the Klotho-like protein, and the like.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that VTS (virtual tr
We succeeded in obtaining a gene cDNA encoding a novel human-derived Klotho-like protein by the anscribed sequence) approach, and completed the present invention. That is, the present invention is the following (1) to (15).

(1) An aging control protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. (2) A peptide or a salt thereof, which comprises a partial amino acid sequence of the aging control protein of (1) above. (3) A polynucleotide comprising a nucleotide sequence encoding the aging control protein or the partial peptide thereof according to (1) above. (4) The polynucleotide according to (3) above, wherein the nucleotide sequence encoding the aging control protein according to (1) above is the nucleotide sequence represented by SEQ ID NO: 1. (5) The polynucleotide of (3) or (4) above, wherein the polynucleotide is DNA or RNA.

(6) A recombinant vector containing the polynucleotide according to any one of (3) to (5) above. (7) A transformant transformed with the recombinant vector of (6) above. (8) The transformant of (7) above is cultured in a medium, and the protein or salt thereof of (1) above, or the peptide of (2) or salt thereof is collected from the resulting culture. A method for producing the protein according to (1) or a salt thereof or the peptide according to (2) or a salt thereof. (9) The protein of the above (1) or a salt thereof, or the above (2)
An antibody against the peptide or a salt thereof. (10) The protein of the above (1) or a salt thereof or the above (2)
A drug for treating premature aging or a drug for suppressing aging, which comprises the peptide or salt thereof as an active ingredient.

(11) An agent for improving or preventing a pathological condition due to deterioration of vascular endothelial function, which comprises the protein of (1) or a salt thereof or the peptide of (2) or a salt thereof as an active ingredient. (12) The agent for improving or preventing a pathological condition due to deterioration of vascular endothelial function according to (11), wherein the pathological condition due to deterioration of vascular endothelial function is hypertension or arteriosclerosis. (13) A gene therapy vector for treating premature aging, which comprises the polynucleotide according to any one of (3) to (5) above. (14) A gene therapy vector for improving or preventing a pathological condition due to deterioration of vascular endothelial function, which comprises the polynucleotide according to any one of (3) to (5) above. (15) The gene therapy vector for improving or preventing the pathological condition due to the deterioration of vascular endothelial function according to the above (14), wherein the pathological condition due to the deterioration of vascular endothelial function is hypertension or arteriosclerosis. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The novel Klotho-like protein of the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. The present protein can be obtained as described below and has various industrial applicability described below.

1. Cloning of the gene encoding the Klotho-like protein of the present invention The gene encoding the Klotho-like protein of the present invention is VT
It can be cloned by the S (virtual transcribed sequence) approach. Here, the "VTS approach" is a VTS that has not been confirmed as an EST by predicting and listing gene coding regions from a human genome sequence data by a gene prediction program.
Was detected by RT-PCR from various RNA sources, the presence or absence of splicing was confirmed by determining the nucleotide sequence of the detected fragments, and the evaluation as a real gene gave a new expression. A method of identifying a gene. Hereinafter, the method for cloning the gene encoding the Klotho-like protein of the present invention will be specifically described with reference to FIG.

First, a gene coding region such as GENESCAN is used to predict gene coding regions on a human genome sequence, and those are listed to construct a VTS database. Note that the VTS database is already commercially available and can be used. Next, with respect to the virtual transcription sequence (VTS) group, VTS whose expression has not been confirmed as EST in the database and which is considered to encode a Klotho-like protein is searched. Here, an amino acid sequence existing in a known human or mouse Klotho protein can be used as a query sequence (query sequence) for the search.

Based on the exons and introns predicted on the genome sequence of the obtained VTS, a primer sequence is designed and synthesized on the exons so as to sandwich one or more introns as shown in FIG. 1 (a). Here, the design of the primer can be performed using commercially available primer design software such as OLIGO (National Biosciences). Perform RT-PCR with various RNA sources using the set primers. Here, examples of the source of RNA include human-derived hepatocytes, mammary gland cells, and the like. The cells can also be prepared from humans who have aborted or aborted spontaneously. RNA can be prepared by a method usually used in the art.

A DNA fragment obtained by RT-PCR [see FIG.
(c): Demonstration exon sequence]. This confirms that the DNA fragment is the same as the spliced product predicted from the genomic sequence [(b) in FIG. 1: predicted exon sequence]. Then, using the primers designed based on the confirmed sequence, 5'-RACE and 3'-RACE
Is executed [(d) in FIG. 1]. This makes the Klo of the present invention
cDNA encoding the full-length amino acid sequence of tho-like protein
The base sequence of
(e)].

In the present invention, a novel Klotho-like protein cloned from human and a gene cDNA encoding the protein are exemplified as follows. That is, SEQ ID NO: 2 is the amino acid sequence of a human-derived Klotho-like protein, and SEQ ID NO: 1 is the base sequence of a gene cDNA encoding the protein. Once the nucleotide sequence of the gene cDNA of the present invention has been determined, the polynucleotide of the present invention can be obtained thereafter by chemical synthesis or by PCR. For example, the Klotho of the present invention
The DNA (SEQ ID NO: 1) encoding the full-length amino acid sequence (SEQ ID NO: 2) of the protein-like protein is 5'-atgaagccaggctgtgcggcagga using human hepatocyte or mammary gland cell-derived cDNA as a template.
t-3 '(SEQ ID NO: 3) and 5'-ttagctaacaactctcttgcctttc
It can be easily prepared by PCR by using a primer having a base sequence of -3 '(SEQ ID NO: 4). However, in the present invention, the primer is not limited to these.

2. Klotho-like protein of the present invention and partial peptides thereof The Klotho-like protein of the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. Here, the "protein containing substantially the same amino acid sequence" means about 50% or more, preferably about 70% or more, more preferably about 80% or more, and further preferably, the amino acid sequence represented by SEQ ID NO: 2. About 90
%, Most preferably about 95% or more homology, and refers to a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 2. The “substantially the same activity” means that the activity is the same as the physiological activity originally possessed by the Klotho protein, such as the activity of controlling aging.

A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is:
1 or 2 or more (preferably about 1 to 30, more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 2
1 or 2 or more (preferably 1 to 30 and more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 2 in which an amino acid sequence of about 1, preferably 5 to 10 amino acids is deleted. About 1 piece, more preferably 1 to 5 pieces)
Amino acid sequence having the amino acid of SEQ ID NO: 2 and SEQ ID NO: 2
An amino acid in which 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10 and further preferably 1 to 5) amino acids in the amino acid sequence represented by are substituted with other amino acids. Examples include proteins that include sequences. The amino acid deletion, addition and substitution can be performed by modifying the gene encoding the Klotho-like protein by a method known in the art. For example, substitution of a specific amino acid residue can be performed by using a commercially available kit [for example, Mutan ^™ -G (TAKARA), Mutan ^™ -K (TA
KARA Co., Ltd.) and the like, and the base substitution can be achieved by a known method such as the Gupped duplex method or the Kunkel method or a method similar thereto.

The C-terminal of the Klotho-like protein of the present invention is usually a carboxyl group (-COOH), and the carboxyl group is an amide (-CONH ₂ ) or ester (-COO).
R) and the like may be chemically modified. Here, R in the ester is a C _1-6 alkyl group (eg, methyl,
Ethyl, n-propyl, isopropyl, n-butyl), C _3-
8 cycloalkyl group (eg, cyclopentyl, cyclohexyl), C _6-12 aryl group (eg, phenyl, α)
-Naphthyl), phenyl-C _1-2 alkyl group (eg, benzyl, phenethyl), α-naphthyl-C _1-2 alkyl group (eg, α-naphthylmethyl) and the like. In addition, pivaloyloxymethyl ester, which is widely used as an oral ester, can be used. Of the present invention
When the Klotho-like protein has a carboxyl group in the polypeptide chain in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. Examples of the ester in this case include the above-mentioned respective esters. Similarly, the invention
The N-terminus of Klotho-like protein is usually an amino group (-NH ₂ )
However, the amino group may be chemically modified with a C _1-6 acyl group such as a formyl group and an acetyl group. In addition, the glutamyl group produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, and the substituents on the side chains of amino acids in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group). , Guanidino groups, etc.) are chemically modified with an appropriate functional group (eg, formyl group, acetyl, etc.) and sugar chains are also included in the protein of the present invention.

A peptide containing a partial amino acid sequence in any of the above Klotho-like proteins (also referred to as a partial peptide) is also included in the scope of the present invention. That is, the partial peptide of the present invention may be any one as long as it includes a partial amino acid sequence of the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. Among the partial peptides of the present invention, the portion exposed outside the cell membrane when the Klotho-like protein is naturally present is particularly useful in controlling the aging phenomenon. Examples of such partial peptides include a membrane protein analysis program for the amino acid sequence represented by SEQ ID NO: 2 (for example, TM
In the analysis by the HMM program etc.), a portion predicted to be an extracellular region can be mentioned. The number of amino acids constituting the partial peptide of the present invention is at least 10 or more, preferably 30 or more, more preferably 80 or more. Usually, the C-terminal of the partial peptide of the present invention has a carboxyl group (-COO
H), but N-terminal is an amino group (-NH _2), these are as in the case of the Klotho-like proteins, may be chemically modified.

The Klotho-like protein of the present invention or its partial peptide can be provided in the form of a salt, preferably a physiologically acceptable acid addition salt, if necessary. Such salts include salts of inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, Examples thereof include salts of citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

[0021] The Klotho-like protein of the present invention or a salt thereof can be obtained by extraction / separation from cultured cells or tissues of humans or mammals expressing the Klotho-like protein of the present invention.
Alternatively, it can also be produced by culturing a transformant containing a DNA encoding the Klotho-like protein of the present invention as described below. When produced from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction with acid or the like, the resulting extract is subjected to hydrophobic chromatography, reverse phase chromatography,
It can be isolated and purified by combining various chromatographys such as ion exchange chromatography.

The partial peptide of the present invention or a salt thereof can be produced by a known peptide synthesis method or by cleaving the Klotho-like protein with an appropriate peptidase (eg, trypsin, chymotrypsin, arginyl endopeptidase). it can. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the Klotho-like protein of the present invention with the residual portion and removing the protecting group when the product has a protecting group. . After the synthesis reaction, the partial peptide of the present invention can be isolated and purified by combining conventional purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, and recrystallization. Obtained by the above method
When the Klotho-like protein or its partial peptide is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted into a free form by a known method. You can

3. Polynucleotide encoding the Klotho-like protein of the present invention or its partial peptide The polynucleotide encoding the Klotho-like protein of the present invention or its partial peptide contains a base sequence encoding the Klotho-like protein of 2 or its partial peptide. Any material may be used. Examples of the polynucleotide include DNA and RNA (for example, mRNA) encoding the Klotho-like protein of the present invention, and may be double-stranded or single-stranded. When it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). Using a polynucleotide encoding the Klotho-like protein of the present invention, for example, by the method described in Experimental Medicine special number "New PCR and its application" 15 (7), 1997 or a method similar thereto, the Klotho-like protein of the present invention mRNA can be quantified. The DNA encoding the Klotho-like protein of the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the cell of 1 above, or prepared from the cDNA library derived from the cell or tissue of 1 above, or synthetic DNA. But it's okay. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the total RNA or mRNA fraction prepared from the cell or tissue described in 1 above, the RT-PCR method (Reverse Transcrib
It can also be amplified by the ptasePolymerase Chain Reaction. Specifically, examples of the polynucleotide encoding the Klotho-like protein of the present invention include a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 as described in 1 above. Any polynucleotide that hybridizes under stringent conditions and that encodes a Klotho-like protein having substantially the same activity as the Klotho-like protein of the present invention (for example, senescence control activity) can be used. Good.

As the DNA hybridizable with the polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, the DNA represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more. , More preferably about 90% or more, and most preferably about 95% or more homology-containing DNA sequences.
Here, the "stringent conditions" refer to conditions where the sodium concentration is about 20-40 mM, preferably about 20-25 mM, and the temperature is about 50-70 ° C, preferably about 60-65 ° C.

4. Preparation of Recombinant Vector and Transformant (1) Preparation of Recombinant Vector The recombinant vector of the present invention is a suitable vector of the present invention.
It can be obtained by ligating a polynucleotide (eg, DNA) encoding a Klotho-like protein or a partial peptide thereof. The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. As the plasmid DNA, a plasmid derived from Escherichia coli (for example, pBR322, pBR325, pUC118, pUC119, pUC18,
pUC19, pCBD-C, etc.), a plasmid derived from Bacillus subtilis (for example, pU
B110, pTP5, pC194, etc.), a yeast-derived plasmid (for example,
YEp13, YEp24, YCp50, YIp30 and the like), and the phage DNA includes λ phage and the like. further,
Animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus and togavirus can also be used.

The ligation of the polynucleotide of the present invention to a vector is carried out by directly or directly digesting the DNA encoding the Klotho-like protein cloned in 1 above, or by digesting it with a restriction enzyme, or adding a linker to the vector DN.
It can be carried out by inserting into the restriction enzyme site of A or the multiple cloning site. The DNA to be linked is
It has ATG as a translation initiation codon on the 5'end side, and also has 3 '
The terminal side may have TAA, TGA or TAG as a translation stop codon. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter. The DNA to be ligated needs to be incorporated into a vector so that the Klotho-like protein of the present invention encoded in the DNA can be expressed in a host cell. Therefore, in addition to the Klotho-like protein coding sequence, the recombinant vector of the present invention includes a promoter, a selection marker, a terminator, an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (S
D sequence) etc. can be linked.

Here, the promoter used in the present invention
Is suitable for the host used for gene expression.
The promoter is not particularly limited as long as it is a promoter. For example, an animal
When using cells as hosts, SRα promoter, CM
V promoter, SV40 promoter, LTR promoter,
Examples include HSV-TK promoter. The host is E. coli
The trp promoter, lac promoter, recA
Promoter, λP _LPromoter, lpp promoter, etc.
However, when the host is Bacillus subtilis, the SPO1 promoter,
The host is yeast such as SPO2 promoter and penP promoter.
, PHO5 promoter, PGK promoter
, GAP promoter, ADH promoter, etc.
It If the host is an insect cell, polyhedrin promoter
, P10 promoter and the like are preferable. In addition, select
Ampicillin resistance gene, neomycin
Examples include syn resistance gene and dihydrofolate reductase gene.
To be

(2) Preparation of Transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the Klotho-like gene of the present invention can be expressed. . Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia col
i ) such as Escherichia, Bacillus subtilis ( Bacil
bacterium belonging to the genus Bacillus such as lus subtilis, the genus Pseudomonas such as Pseudomonas putida , the bacterium belonging to the genus Rhizobium meliloti such as Rhizobium meliloti , and Saccharomyces cerevisiae ( Sa)
Ccharomyces cerevisiae ), yeast such as Schizosaccharomyces pombe , monkey cell C
Examples include OS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, animal cells such as human GH3 and human FL cells, and insect cells such as Sf9 and Sf21.

As a method for introducing the recombinant vector into Escherichia coli, a method using calcium ion [Cohen, SN et
al .: Proc. Natl. Acad. Sci., USA, 69: 2110 (197
2)], electroporation method and the like. As a method for introducing a recombinant vector into yeast, an electroporation method [Becker, DM et al .: Methods. Enzymo
l., 194: 182 (1990)], spheroplast method [Hinnen, A.
et al .: Proc. Natl. Acad. Sci., USA, 75: 1929 (19
78)], lithium acetate method [Itoh, H .: J. Bacteriol., 153:
163 (1983)] and the like. Examples of the method for introducing the recombinant vector into animal cells include the electroporation method, calcium phosphate method, lipofection method and the like. As a method of introducing the recombinant vector into insect cells,
For example, the calcium phosphate method, lipofection method, electroporation method and the like can be mentioned.

5. Production of Klotho-Like Protein of the Present Invention The Klotho-like protein of the present invention can be produced by culturing the transformant obtained in the above 4 and collecting from the culture. The "culture" means any of the culture supernatant, the cultured cells or cultured cells, or the disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism, and efficiently cultivates the transformant. Either a natural medium or a synthetic medium may be used as long as it can be performed. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As a nitrogen source, ammonia,
In addition to ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As inorganic salts, potassium dibasic phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate,
Sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,
Calcium carbonate or the like is used.

The culture is carried out under conditions suitable for the host cell.
For example, as a medium for culturing E. coli, LB medium,
M9 medium and the like are preferable. If desired, a drug such as isopropyl-1-thio-β-D-galactoside, 3β-indolylacrylic acid can be added in order to work the promoter efficiently. For E. coli, the culture is usually about 15-43
It may be carried out at ℃ for about 3 to 24 hours, and if necessary, aeration and stirring may be added. If the host is Bacillus subtilis, the culture is usually about
It is carried out at 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. Examples of the medium for culturing yeast include SD medium and YPD medium. The pH of the medium is about 5
It is preferable to adjust to 8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and agitation are added if necessary. When culturing a transformant whose host is an insect cell or an insect, examples of the medium include Grace's insect medium containing bovine serum. The pH of the medium is preferably adjusted to about 6.2-6.4. Culturing is usually carried out at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, about 5
MEM medium containing ~ 20% fetal bovine serum, DMEM medium, RPMI 16
40 medium or the like is used. The pH is preferably about 6-8. Culturing is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added if necessary. As described above, the Klotho-like protein of the present invention can be produced on the cell membrane or the like of the transformant. Klotho of the present invention from the above culture
The protein-like protein can be separated and purified by the following method, for example.

When the Klotho-like protein of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, and the cells or cells are suspended in an appropriate buffer solution, followed by ultrasonication, lysozyme and After destroying the cells or cells by freezing and thawing (if necessary, treating with a surfactant), centrifuge or filtration to Klotho
A method for obtaining a crude extract of such protein is appropriately used. When the Klotho-like protein is secreted into the culture medium, after the completion of the culture, the bacterial cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The Klotho-like protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation / purification methods. As the known separation and purification methods thereof, a method utilizing solubility such as salting out or solvent precipitation method, a dialysis method, an ultrafiltration method, a gel filtration method, and mainly a difference in molecular weight such as SDS-PAGE are used. Method, method utilizing difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric point A method utilizing a difference in isoelectric point such as an electrophoresis method is used.

6. Antisense Polynucleotide of the Present Invention The antisense polynucleotide capable of inhibiting the replication or expression of the gene encoding the Klotho-like protein of the present invention is used as the nucleotide sequence information of the DNA encoding the Klotho-like protein cloned in the above 1. It can be designed and synthesized based on the above. Specifically, the antisense polynucleotide of the present invention includes SEQ ID NO: 2
Which is complementary to a base sequence encoding a Klotho-like protein or a partial peptide of the protein containing the same or substantially the same amino acid sequence as that represented by, or hybridizes under stringent conditions. And a polynucleotide capable of Such antisense polynucleotides are Kl of the invention.
It is a polynucleotide having a nucleotide sequence complementary to at least a part of the mRNA of the otho-like gene, and can hybridize to the mRNA to regulate or control the expression of the Klotho-like gene. The antisense polynucleotide is useful for treating or diagnosing a disease associated with the Klotho-like gene of the present invention, in addition to regulating or controlling the expression of the Klotho-like protein of the present invention.

The antisense polynucleotide is 2-
Deoxy-D-ribose-containing polydeoxyribonucleotides, D-ribose-containing polyribonucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other with non-nucleotide backbones Polymers (eg commercially available peptide nucleic acids and synthetic sequence-specific nucleic acid polymers)
Or other polymers containing special bonds (however,
The polymer contains nucleotides having a configuration that allows pairing of bases and attachment of bases such as found in DNA or RNA). They are double-stranded DNA,
Single-stranded DNA, double-stranded RNA, single-stranded RNA, and may be a hybrid of DNA and RNA, further unmodified polynucleotide (or unmodified oligonucleotide), further with known modifications added, For example, one having a label known in the art, one having a cap, one having a methylation, one having one or more natural nucleotides substituted with an analogue, one having an intramolecular nucleotide modification, for example, a non-nucleotide Those having a charged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), a charged bond or a sulfur-containing bond (eg,
Those having side chains such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides, etc.) Having, having an intercurrent compound (for example, acridine, psoralen, etc.), containing a chelate compound (for example, metal, radioactive metal, boron, oxidizing metal, etc.), alkylating agent Or a compound having a modified bond (eg, α-anomeric nucleic acid, etc.).

The antisense polynucleotide of the present invention is
It may be provided in a special form such as liposome or microsphere, or may be provided in a form in which a specific compound or a modifying group is attached. Specifically, as the substance to be bound to the antisense polynucleotide, when the antisense polynucleotide is DNA, a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate skeleton of Crude aqueous substances such as phospholipids and cholesterol that enhance the interaction between nucleoti and cell membrane and increase the uptake of antisense polynucleotide into cells, polyethylene glycol, tetraethylene glycol, etc. that prevent the degradation of antisense polynucleotide by nuclease. But not limited thereto.

The activity of the antisense polynucleotide of the present invention to inhibit the expression of Klotho-like gene is determined by the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the Klotho-like protein of the present invention. It can be examined using the translation system in vivo or in vitro.

7. Antibody against Klotho-Like Protein of the Present Invention The antibody of the present invention comprises Klotho containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2.
Like protein or a salt thereof, or a peptide containing a partial amino acid sequence of the Klotho-like protein or a salt thereof. The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the Klotho-like protein of the present invention or a partial peptide thereof. By using the antibody of the present invention, the Klotho of the present invention in a sample of blood, a part of an organ, cells, etc.
Such proteins can be detected and quantified. ELISA using microtiter plate is the preferred method
Method, fluorescent antibody method, Western blotting method, etc., and can be used for immunohistological staining using a pathological tissue section. Therefore, the present antibody is useful for diagnosing premature aging and various diseases related to aging associated with a decrease in the expression of the Klotho-like protein of the present invention. The antibody of the present invention is the Klot of the present invention.
By using ho-like protein or its partial peptide as an antigen, it can be produced according to a method for producing an antibody or antiserum known in the art.

(1) Preparation of Monoclonal Antibody (i) Preparation of Monoclonal Antibody-Producing Cell The Klotho-like protein or its partial peptide of the present invention is itself or a carrier at a site capable of producing an antibody upon administration to a mammal. , With a diluent.
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually once every 2 to 6 weeks for a total of 2 to 10
It is performed about once. Examples of mammals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, an individual with an antibody titer is selected from mice and the final immunization is performed.
By collecting spleen or lymph node after -5 days and fusing the antibody-producing cells contained therein with myeloma cells,
A monoclonal antibody-producing hybridoma can be prepared.

The antibody titer in the antiserum can be measured, for example, by reacting the labeled Klotho-like protein or a partial peptide thereof described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. Can be done. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
Preferably PEG is used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0 and the like, and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is 1: 1 to 20 :.
1 or less, PEG (preferably PEG1000 to PEG6000)
Is added at a concentration of about 10 to 80%, and cell fusion can be efficiently performed by incubating at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes.

Various methods can be used for screening a hybridoma producing a monoclonal antibody. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then, A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A , Hybridoma culture supernatant is added to the solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, and Klotho-like protein or its partial peptide labeled with radioactive substance or enzyme is added to detect the monoclonal antibody bound to the solid phase. There are ways to do it. The monoclonal antibody can be selected according to a method known per se or a method analogous thereto,
It can be performed in a medium for animal cells or the like supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example,
RPMI containing 1-20%, preferably 10-20% fetal calf serum
1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.), serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) and the like can be used. Cultivation temperature is usually 20 ° C to 40 ° C, preferably about 37 ° C
Is. Cultivation time is usually 5 days to 3 weeks, preferably 1
Weeks to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(Ii) Purification of Monoclonal Antibody The isolation and purification of the monoclonal antibody can be carried out in the same manner as the isolation and purification of ordinary polyclonal antibodies (eg, salting out method, alcohol precipitation method, isoelectric precipitation method, etc.). Electrophoresis method, adsorption / desorption method with ion exchanger, ultracentrifugation method, gel filtration method, antigen-binding solid phase or an active adsorbent such as protein A or protein G is used to collect only the antibody and dissociate the bond to obtain the antibody. Specific purification method).

(2) Preparation of Polyclonal Antibody The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, by forming a complex between the antigen and the carrier protein,
It can be produced by immunizing a mammal in the same manner as the above-mentioned method for producing a monoclonal antibody, collecting an antibody-containing substance against the Klotho-like protein of the present invention or a partial peptide thereof from the immunized animal, and separating and purifying the antibody. . Regarding a complex of an antigen and a carrier protein used for immunizing a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that if the antibody can be efficiently against the hapten immunized by crosslinking with the carrier, What kind of thing may be crosslinked at any ratio, but for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. in a weight ratio of about 0.1 to 20, preferably about 0.1 to 20, About 1
A method of coupling at a ratio of ~ 5 is used.

Various coupling agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group and dithiopyridyl group, etc. are used. The condensation product is a carrier itself or a carrier at a site capable of producing an antibody, for mammals,
Administered with diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from blood, ascites, etc., preferably blood of a mammal immunized by the above method. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

8. Application of the present invention to medical treatment and others The aging can be suppressed by administering the full-length or partial fragment of the Klotho-like protein of the present invention to a living body. Therefore, it is useful as a therapeutic or preventive drug for various diseases that are closely related to the progress of aging, such as arteriosclerosis, hypertension, and osteoporosis. It is also useful for the effect of prolonging the life due to the suppression of aging.

The Klotho-like gene (aging suppressing gene) of the present invention can also be incorporated into a viral vector such as retrovirus or adenovirus or other vector and used for treatment by a gene therapy method. Furthermore, by using the Klotho-like gene or oligonucleotide of the present invention, by the Northern hybridization method or the PCR method, the expression level of the Klotho-like gene is measured, and aging is diagnosed, and the Klotho-like gene or its partial oligonucleotide is aged. It can also be used for suppression. Further, due to the congenital deletion of the Klotho-like gene of the present invention, an individual in which aging is promoted and various diseases are more likely to occur is
A Klotho-like gene or a partial oligonucleotide thereof may be used for detection by Southern blot hybridization or PCR, and genetic diagnosis may be performed based on the detected nucleic acid sequence information of the individual. Furthermore, the Klotho-like gene or its partial oligonucleotide is extremely useful as a reagent for gene research.

Furthermore, the Klotho-like gene of the present invention or livestock such as cows, sheep, goats, pigs, horses and chickens can be used.
Administration of a Klotho-like protein or the Kl of the invention
It is also a preferred embodiment of the present invention to prolong the life of livestock and suppress aging by expressing the otho-like gene in an individual using a vector such as a virus suitable for gene therapy. As a result, breeding in good condition for a long time,
It is possible to collect milk, eggs, and offspring over a long period of time. Alternatively, by introducing the Klotho-like gene of the present invention into a fertilized egg, by inserting and expressing the Klotho-like gene in the chromosome of all cells in the body, by producing a so-called transgenic animal,
Similar effects can be expected, and it is considered highly useful in the field of livestock breeding in terms of breeding. In addition, using a vector containing the Klotho-like gene of the present invention, a known homologous recombination method for the senescence suppressor gene on the chromosome in embryonic stem cells (ES cells) [C
ell 51 (3): 503 (1987), etc.] can be used to prepare a mutant clone inactivated or replaced with an arbitrary sequence.

Using the thus-produced embryonic stem cell clone, a chimeric individual consisting of the embryonic stem cell clone and normal cells is prepared by a method such as a chimera method of injecting a fertilized egg of an animal into a blastocyst or an assembled chimera method. can do. By crossing this chimeric individual with a normal individual, an individual having an arbitrary mutation in the cell senescence suppressor gene of the whole body can be obtained, and further, by the crossing of the individual, both homologous chromosomes have a mutation, a homologous individual. Can be obtained.
In this way, it is possible to introduce a mutation into any position of the Klotho-like gene of the present invention in an individual animal. For example,
The activity of the product can be changed by introducing mutations such as base substitutions, deletions and insertions into the translation region of the Klotho-like gene of the present invention. It is also possible to modify the degree of expression, timing, tissue specificity, etc. by introducing similar mutations into the expression control region.

Further, by combining with the Cre-loxP system, it is possible to more positively control the expression time, the expression site, the expression amount and the like. As such an example, a promoter expressed in a specific region of the brain is used to delete a target gene only in that region [Cell 87 (7): 1317 (199
6)] or an adenovirus expressing Cre, in which the target gene was deleted in an organ-specific manner at the target time [Scien
ce 278: 5335 (1997)] is known. Therefore, according to the present invention
Expression of the Klotho-like gene can be controlled in this manner at any time and tissue. Alternatively, it is possible to prepare an animal individual having any insertion, deletion, or substitution in its translation region or expression control region. Such an animal can induce symptoms of aging and various diseases associated with aging at any time, at any degree, or at any site. Therefore, it becomes an extremely useful animal model in the treatment and prevention of aging and various diseases related thereto. In particular, it is very useful as a model for evaluation of therapeutic agents, preventive agents and the like.

The Klotho-like protein of the present invention is useful as a reagent for searching for and determining a ligand that specifically binds to the protein. In addition, using the Klotho-like protein of the present invention and the identified ligand, the Klho of the present invention
It can be used to search for and identify compounds that inhibit the specific binding of otho-like proteins and ligands. Moreover,
By using the Klotho-like protein of the present invention and its ligand, it is possible to search and identify a molecule induced as a result of binding of the Klotho-like protein of the present invention to the ligand. The ligand of the Klotho-like protein of the present invention, a compound that inhibits the specific binding between the Klotho-like protein of the present invention of the present invention and its ligand, and the molecule derived as a result of the binding of the Klotho-like protein of the present invention to the ligand, Klotho of the present invention
It is considered to substitute or assist the aging inhibitory function of such proteins, and agents containing these molecules are useful as therapeutic agents for premature aging, aging inhibitors and the like. Furthermore, a compound that increases the expression of the gene encoding the Klotho-like protein of the present invention of the present invention (expression-enhancing compound), which is useful as a therapeutic agent for premature aging and an aging inhibitor, is similar to the Klotho-like protein of the present invention. It is useful for treating / preventing aging and suppressing aging.

[0050]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. [Example 1] Cloning of Klotho-like gene of the present invention First, a gene prediction program GENE SCAN was used to broadly list gene candidates from known human genome sequences, that is, virtual transcription sequences (VTS), and a VTS database was constructed. . Next, among each VTS in the VTS database, a VTS whose nucleotide sequence was not registered in the EST database, that is, the expression in human cells was not confirmed, was searched. As a result, we found multiple VTSs that differ in sequence from any of the known ESTs. Then, according to the usual method,
It was investigated whether or not each VTS found above was actually expressed in cells. As a result, it was confirmed that some of the VTSs were actually expressed in human cells. Regarding the VTS whose expression was confirmed, homology analysis with a known gene registered in the gene database was performed, and it was found that one of them has similarity to the mouse β-Klotho gene, We tried full-length cDNA cloning.

Since it seems that the VTS sequence already covers the entire length, the following primers were set outside the open reading frame. g6vts68545-1f: 5'-atcctcagtctcccagttcaa-3 '(SEQ ID NO: 5) g6vts68545-1r: 5'-ggaactgggatgaatttttaa-3' (SEQ ID NO: 6) PCR is performed using the RNA fractions of liver and mammary gland as templates. It was As a result, an amplified fragment of the expected size was obtained. A band of the expected size was excised and cloned into pGEM-T Easy Vector. Then
The nucleotide sequence was determined for colonies subjected to PCR to give a band of an appropriate size. As a result, this gene has 10
It was found to encode a protein consisting of 44 amino acids. The determined nucleotide sequence is shown in SEQ ID NO: 1. This gene was named Klotho-like gene.

[Example 2] Computer analysis of Klotho-like gene and protein of the present invention Klotho-like gene product, using all 1044 amino acids as a query
A BLAST search was performed on the nr database. As a result, the Klotho-like protein of the present invention is known to be a known human Klotho.
The amino acid identity with the protein (SEQ ID NO: 7) was 43% (FIG. 2 and FIG. 3 show pairwise alignment between the Klotho-like protein of the present invention and the known human Klotho protein ClustalW). On the other hand, the Klotho-like protein of the present invention is a mouse β-Klotho protein (SEQ ID NO: 8).
And the degree of amino acid identity was 79% (Figs. 4 and 5 show pairwise alignments of the Klotho-like protein of the present invention and the known mouse β-Klotho protein ClustalW).

Next, motif analysis of this sequence was performed. The analysis result is shown in FIG. The amino acid numbers are shown below. The 1st to 52nd amino acids are the signal peptide region [SignalP [CB
S (Center for Biological Sequence Analysis)] and Sig
cleave [EMBOSS (The EuropeanMolecular Biology Open S
of the Attware Suite)], amino acids 77 to 512 and amino acids 517 to 971 are glycosyl hydrolase family 1 motif regions (analyzed by Pfam), and amino acids 996 to 1018 are transmembrane helix regions. [Analyzed by TMHMM (CBS)], the 241, 416, 691 and 887 amino acids correspond to four E (Glu) responsible for the active center of β-glucosidase, among them, the 241 amino acid The 691st amino acid is similar to mouse β-Klotho protein, human Klotho protein and mouse Klotho protein, and N (Asn), A (A
each was replaced by la). All of these motifs are common to all four Klotho-related proteins. From the above, the Klotho-like protein of the present invention is mouse β-Klotho
It is considered a human homologue of the protein.

Example 3 Confirmation of Expression Site of Klotho-Like Gene of the Present Invention Using the primers of Example 1, the expression site was confirmed by RT-PCR. The results are shown in Fig. 7. As is clear from FIG. 7, high expression was observed in the liver and mammary gland.

[0055]

INDUSTRIAL APPLICABILITY According to the present invention, a novel human Klotho-like aging regulatory protein and Klot encoding the protein
ho-like senescence control gene, recombinant vector containing the gene, transformant containing the recombinant vector, and the Klotho
A method for producing such a protein is provided. INDUSTRIAL APPLICABILITY The present invention is useful for the development of preventive and / or therapeutic agents for aging-related diseases.

[0056]

[Sequence list]                                SEQUENCE LISTING        <110> PharmaDesign, Inc. <120> A Novel Klotho like Protein and Gene thereof. <130> PDP-0019 <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 3135 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (3135) <400> 1 atg aag cca ggc tgt gcg gca gga tct cca ggg aat gaa tgg att ttc 48 Met Lys Pro Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe   1 5 10 15 ttc agc act gat gaa ata acc aca cgc tat agg aat aca atg tcc aac 96 Phe Ser Thr Asp Glu Ile Thr Thr Arg Tyr Arg Asn Thr Met Ser Asn              20 25 30 ggg gga ttg caa aga tct gtc atc ctg tca gca ctt att ctg cta cga 144 Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile Leu Leu Arg          35 40 45 gct gtt act gga ttc tct gga gat gga aga gct ata tgg tct aaa aat 192 Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp Ser Lys Asn      50 55 60 cct aat ttt act ccg gta aat gaa agt cag ctg ttt ctc tat gac act 240 Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu Tyr Asp Thr  65 70 75 80 ttc cct aaa aac ttt ttc tgg ggt att ggg act gga gca ttg caa gtg 288 Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thr Gly Ala Leu Gln Val                  85 90 95 gaa ggg agt tgg aag aag gat gga aaa gga cct tct ata tgg gat cat 336 Glu Gly Ser Trp Lys Lys Asp Gly Lys Gly Pro Ser Ile Trp Asp His             100 105 110 ttc atc cac aca cac ctt aaa aat gtc agc agc acg aat ggt tcc agt 384 Phe Ile His Thr His Leu Lys Asn Val Ser Ser Thr Asn Gly Ser Ser         115 120 125 gac agt tat att ttt ctg gaa aaa gac tta tca gcc ctg gat ttt ata 432 Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Ser Ala Leu Asp Phe Ile     130 135 140 gga gtt tct ttt tat caa ttt tca att tcc tgg cca agg ctt ttc ccc 480 Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro 145 150 155 160 gat gga ata gta aca gtt gcc aac gca aaa ggt ctg cag tac tac agt 528 Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln Tyr Tyr Ser                 165 170 175 act ctt ctg gac gct cta gtg ctt aga aac att gaa cct ata gtt act 576 Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr             180 185 190 tta tac cac tgg gat ttg cct ttg gca cta caa gaa aaa tat ggg ggg 624 Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys Tyr Gly Gly         195 200 205 tgg aaa aat gat acc ata ata gat atc ttc aat gac tat gcc aca tac 672 Trp Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn Asp Tyr Ala Thr Tyr     210 215 220 tgt ttc cag atg ttt ggg gac cgt gtc aaa tat tgg att aca att cac 720 Cys Phe Gln Met Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His 225 230 235 240 aac cca tat cta gtg gct tgg cat ggg tat ggg aca ggt atg cat gcc 768 Asn Pro Tyr Leu Val Ala Trp His Gly Tyr Gly Thr Gly Met His Ala                 245 250 255 cct gga gag aag gga aat tta gca gct gtc tac act gtg gga cac aac 816 Pro Gly Glu Lys Gly Asn Leu Ala Ala Val Tyr Thr Val Gly His Asn             260 265 270 ttg atc aag gct cac tcg aaa gtt tgg cat aac tac aac aca cat ttc 864 Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asn Thr His Phe         275 280 285 cgc cca cat cag aag ggt tgg tta tcg atc acg ttg gga tct cat tgg 912 Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp     290 295 300 atc gag cca aac cgg tcg gaa aac acg atg gat ata ttc aaa tgt caa 960 Ile Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe Lys Cys Gln 305 310 315 320 caa tcc atg gtt tct gtg ctt gga tgg ttt gcc aac cct atc cat ggg 1008 Gln Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly                 325 330 335 gat ggc gac tat cca gag ggg atg aga aag aag ttg ttc tcc gtt cta 1056 Asp Gly Asp Tyr Pro Glu Gly Met Arg Lys Lys Leu Phe Ser Val Leu             340 345 350 ccc att ttc tct gaa gca gag aag cat gag atg aga ggc aca gct gat 1104 Pro Ile Phe Ser Glu Ala Glu Lys His Glu Met Arg Gly Thr Ala Asp         355 360 365 ttc ttt gcc ttt tct ttt gga ccc aac aac ttc aag ccc cta aac acc 1152 Phe Phe Ala Phe Ser Phe Gly Pro Asn Asn Phe Lys Pro Leu Asn Thr     370 375 380 atg gct aaa atg gga caa aat gtt tca ctt aat tta aga gaa gcg ctg 1200 Met Ala Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Glu Ala Leu 385 390 395 400 aac tgg att aaa ctg gaa tac aac aac cct cga atc ttg att gct gag 1248 Asn Trp Ile Lys Leu Glu Tyr Asn Asn Pro Arg Ile Leu Ile Ala Glu                 405 410 415 aat ggc tgg ttc aca gac agt cgt gtg aaa aca gaa gac acc acg gcc 1296 Asn Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp Thr Thr Ala             420 425 430 atc tac atg atg aag aat ttc ctc agc cag gtg ctt caa gca ata agg 1344 Ile Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln Ala Ile Arg         435 440 445 tta gat gaa ata cga gtg ttt ggt tat act gcc tgg tct ctc ctg gat 1392 Leu Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Ser Leu Leu Asp     450 455 460 ggc ttt gaa tgg cag gat gct tac acc atc cgc cga gga tta ttt tat 1440 Gly Phe Glu Trp Gln Asp Ala Tyr Thr Ile Arg Arg Gly Leu Phe Tyr 465 470 475 480 gtg gat ttt aac agt aaa cag aaa gag cgg aaa cct aag tct tca gca 1488 Val Asp Phe Asn Ser Lys Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala                 485 490 495 cac tac tac aaa cag atc ata cga gaa aat ggt ttt tct tta aaa gag 1536 His Tyr Tyr Lys Gln Ile Ile Arg Glu Asn Gly Phe Ser Leu Lys Glu             500 505 510 tcc acg cca gat gtg cag ggc cag ttt ccc tgt gac ttc tcc tgg ggt 1584 Ser Thr Pro Asp Val Gln Gly Gln Phe Pro Cys Asp Phe Ser Trp Gly         515 520 525 gtc act gaa tct gtt ctt aag ccc gag tct gtg gct tcg tcc cca cag 1632 Val Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser Ser Pro Gln     530 535 540 ttc agc gat cct cat ctg tac gtg tgg aac gcc act ggc aac aga ctg 1680 Phe Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly Asn Arg Leu 545 550 555 560 ttg cac cga gtg gaa ggg gtg agg ctg aaa aca cga ccc gct caa tgc 1728 Leu His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ala Gln Cys                 565 570 575 aca gat ttt gta aac atc aaa aaa caa ctt gag atg ttg gca aga atg 1776 Thr Asp Phe Val Asn Ile Lys Lys Gln Leu Glu Met Leu Ala Arg Met             580 585 590 aaa gtc acc cac tac cgg ttt gct ctg gat tgg gcc tcg gtc ctt ccc 1824 Lys Val Thr His Tyr Arg Phe Ala Leu Asp Trp Ala Ser Val Leu Pro         595 600 605 act ggc aac ctg tcc gcg gtg aac cga cag gcc ctg agg tac tac agg 1872 Thr Gly Asn Leu Ser Ala Val Asn Arg Gln Ala Leu Arg Tyr Tyr Arg     610 615 620 tgc gtg gtc agt gag ggg ctg aag ctt ggc atc tcc gcg atg gtc acc 1920 Cys Val Val Ser Glu Gly Leu Lys Leu Gly Ile Ser Ala Met Val Thr 625 630 635 640 ctg tat tat ccg acc cac gcc cac cta ggc ctc ccc gag cct ctg ttg 1968 Leu Tyr Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu Pro Leu Leu                 645 650 655 cat gcc gac ggg tgg ctg aac cca tcg acg gcc gag gcc ttc cag gcc 2016 His Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala Phe Gln Ala             660 665 670 tac gct ggg ctg tgc ttc cag gag ctg ggg gac ctg gtg aag ctc tgg 2064 Tyr Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val Lys Leu Trp         675 680 685 atc acc atc aac gag cct aac cgg cta agt gac atc tac aac cgc tct 2112 Ile Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr Asn Arg Ser     690 695 700 ggc aac gac acc tac ggg gcg gcg cac aac ctg ctg gtg gcc cac gcc 2160 Gly Asn Asp Thr Tyr Gly Ala Ala His Asn Leu Leu Val Ala His Ala 705 710 715 720 ctg gcc tgg cgc ctc tac gac cgg cag ttc agg ccc tca cag cgc ggg 2208 Leu Ala Trp Arg Leu Tyr Asp Arg Gln Phe Arg Pro Ser Gln Arg Gly                 725 730 735 gcc gtg tcg ctg tcg ctg cac gcg gac tgg gcg gaa ccc gcc aac ccc 2256 Ala Val Ser Leu Ser Leu His Ala Asp Trp Ala Glu Pro Ala Asn Pro             740 745 750 tat gct gac tcg cac tgg agg gcg gcc gag cgc ttc ctg cag ttc gag 2304 Tyr Ala Asp Ser His Trp Arg Ala Ala Glu Arg Phe Leu Gln Phe Glu         755 760 765 atc gcc tgg ttc gcc gag ccg ctc ttc aag acc ggg gac tac ccc gcg 2352 Ile Ala Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ala     770 775 780 gcc atg agg gaa tac att gcc tcc aag cac cga cgg ggg ctt tcc agc 2400 Ala Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly Leu Ser Ser 785 790 795 800 tcg gcc ctg ccg cgc ctc acc gag gcc gaa agg agg ctg ctc aag ggc 2448 Ser Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu Leu Lys Gly                 805 810 815 acg gtc gac ttc tgc gcg ctc aac cac ttc acc act agg ttc gtg atg 2496 Thr Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr Arg Phe Val Met             820 825 830 cac gag cag ctg gcc ggc agc cgc tac gac tcg gac agg gac atc cag 2544 His Glu Gln Leu Ala Gly Ser Arg Tyr Asp Ser Asp Arg Asp Ile Gln         835 840 845 ttt ctg cag gac atc acc cgc ctg agc tcc ccc acg cgc ctg gct gtg 2592 Phe Leu Gln Asp Ile Thr Arg Leu Ser Ser Pro Thr Arg Leu Ala Val     850 855 860 att ccc tgg ggg gtg cgc aag ctg ctg cgg tgg gtc cgg agg aac tac 2640 Ile Pro Trp Gly Val Arg Lys Leu Leu Arg Trp Val Arg Arg Asn Tyr 865 870 875 880 ggc gac atg gac att tac atc acc gcc agt ggc atc gac gac cag gct 2688 Gly Asp Met Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp Asp Gln Ala                 885 890 895 ctg gag gat gac cgg ctc cgg aag tac tac cta ggg aag tac ctt cag 2736 Leu Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys Tyr Leu Gln             900 905 910 gag gtg ctg aaa gca tac ctg att gat aaa gtc aga atc aaa ggc tat 2784 Glu Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile Lys Gly Tyr         915 920 925 tat gca ttc aaa ctg gct gaa gag aaa tct aaa ccc aga ttt gga ttc 2832 Tyr Ala Phe Lys Leu Ala Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe     930 935 940 ttc aca tct gat ttt aaa gct aaa tcc tca ata caa ttt tac aac aaa 2880 Phe Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln Phe Tyr Asn Lys 945 950 955 960 gtg atc agc agc agg ggc ttc cct ttt gag aac agt agt tct aga tgc 2928 Val Ile Ser Ser Arg Gly Phe Pro Phe Glu Asn Ser Ser Ser Arg Cys                 965 970 975 agt cag acc caa gaa aat aca gag tgc act gtc tgc tta ttc ctt gtg 2976 Ser Gln Thr Gln Glu Asn Thr Glu Cys Thr Val Cys Leu Phe Leu Val             980 985 990 cag aag aaa cca ctg ata ttc ctg ggt tgt tgc ttc ttc tcc acc ctg 3024 Gln Lys Lys Pro Leu Ile Phe Leu Gly Cys Cys Phe Phe Ser Thr Leu         995 1000 1005 gtt cta ctc tta tca att gcc att ttt caa agg cag aag aga aga aag 3072 Val Leu Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys Arg Arg Lys    1010 1015 1020 ttt tgg aaa gca aaa aac tta caa cac ata cca tta aag aaa ggc aag 3120 Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys Lys Gly Lys 1025 1030 1035 1040 aga gtt gtt agc taa 3135 Arg Val Val Ser                1045    <210> 2 <211> 1044 <212> PRT <213> Homo sapiens <400> 2 Met Lys Pro Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe   1 5 10 15 Phe Ser Thr Asp Glu Ile Thr Thr Arg Tyr Arg Asn Thr Met Ser Asn              20 25 30 Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile Leu Leu Arg          35 40 45 Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp Ser Lys Asn      50 55 60 Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu Tyr Asp Thr  65 70 75 80 Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thr Gly Ala Leu Gln Val                  85 90 95 Glu Gly Ser Trp Lys Lys Asp Gly Lys Gly Pro Ser Ile Trp Asp His             100 105 110 Phe Ile His Thr His Leu Lys Asn Val Ser Ser Thr Asn Gly Ser Ser         115 120 125 Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Ser Ala Leu Asp Phe Ile     130 135 140 Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro 145 150 155 160 Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln Tyr Tyr Ser                 165 170 175 Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr             180 185 190 Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys Tyr Gly Gly         195 200 205 Trp Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn Asp Tyr Ala Thr Tyr     210 215 220 Cys Phe Gln Met Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His 225 230 235 240 Asn Pro Tyr Leu Val Ala Trp His Gly Tyr Gly Thr Gly Met His Ala                 245 250 255 Pro Gly Glu Lys Gly Asn Leu Ala Ala Val Tyr Thr Val Gly His Asn             260 265 270 Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asn Thr His Phe         275 280 285 Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp     290 295 300 Ile Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe Lys Cys Gln 305 310 315 320 Gln Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly                 325 330 335 Asp Gly Asp Tyr Pro Glu Gly Met Arg Lys Lys Leu Phe Ser Val Leu             340 345 350 Pro Ile Phe Ser Glu Ala Glu Lys His Glu Met Arg Gly Thr Ala Asp         355 360 365 Phe Phe Ala Phe Ser Phe Gly Pro Asn Asn Phe Lys Pro Leu Asn Thr     370 375 380 Met Ala Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Glu Ala Leu 385 390 395 400 Asn Trp Ile Lys Leu Glu Tyr Asn Asn Pro Arg Ile Leu Ile Ala Glu                 405 410 415 Asn Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp Thr Thr Ala             420 425 430 Ile Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln Ala Ile Arg         435 440 445 Leu Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Ser Leu Leu Asp     450 455 460 Gly Phe Glu Trp Gln Asp Ala Tyr Thr Ile Arg Arg Gly Leu Phe Tyr 465 470 475 480 Val Asp Phe Asn Ser Lys Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala                 485 490 495 His Tyr Tyr Lys Gln Ile Ile Arg Glu Asn Gly Phe Ser Leu Lys Glu             500 505 510 Ser Thr Pro Asp Val Gln Gly Gln Phe Pro Cys Asp Phe Ser Trp Gly         515 520 525 Val Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser Ser Pro Gln     530 535 540 Phe Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly Asn Arg Leu 545 550 555 560 Leu His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ala Gln Cys                 565 570 575 Thr Asp Phe Val Asn Ile Lys Lys Gln Leu Glu Met Leu Ala Arg Met             580 585 590 Lys Val Thr His Tyr Arg Phe Ala Leu Asp Trp Ala Ser Val Leu Pro         595 600 605 Thr Gly Asn Leu Ser Ala Val Asn Arg Gln Ala Leu Arg Tyr Tyr Arg     610 615 620 Cys Val Val Ser Glu Gly Leu Lys Leu Gly Ile Ser Ala Met Val Thr 625 630 635 640 Leu Tyr Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu Pro Leu Leu                 645 650 655 His Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala Phe Gln Ala             660 665 670 Tyr Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val Lys Leu Trp         675 680 685 Ile Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr Asn Arg Ser     690 695 700 Gly Asn Asp Thr Tyr Gly Ala Ala His Asn Leu Leu Val Ala His Ala 705 710 715 720 Leu Ala Trp Arg Leu Tyr Asp Arg Gln Phe Arg Pro Ser Gln Arg Gly                 725 730 735 Ala Val Ser Leu Ser Leu His Ala Asp Trp Ala Glu Pro Ala Asn Pro             740 745 750 Tyr Ala Asp Ser His Trp Arg Ala Ala Glu Arg Phe Leu Gln Phe Glu         755 760 765 Ile Ala Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ala     770 775 780 Ala Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly Leu Ser Ser 785 790 795 800 Ser Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu Leu Lys Gly                 805 810 815 Thr Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr Arg Phe Val Met             820 825 830 His Glu Gln Leu Ala Gly Ser Arg Tyr Asp Ser Asp Arg Asp Ile Gln         835 840 845 Phe Leu Gln Asp Ile Thr Arg Leu Ser Ser Pro Thr Arg Leu Ala Val     850 855 860 Ile Pro Trp Gly Val Arg Lys Leu Leu Arg Trp Val Arg Arg Asn Tyr 865 870 875 880 Gly Asp Met Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp Asp Gln Ala                 885 890 895 Leu Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys Tyr Leu Gln             900 905 910 Glu Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile Lys Gly Tyr         915 920 925 Tyr Ala Phe Lys Leu Ala Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe     930 935 940 Phe Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln Phe Tyr Asn Lys 945 950 955 960 Val Ile Ser Ser Arg Gly Phe Pro Phe Glu Asn Ser Ser Ser Arg Cys                 965 970 975 Ser Gln Thr Gln Glu Asn Thr Glu Cys Thr Val Cys Leu Phe Leu Val             980 985 990 Gln Lys Lys Pro Leu Ile Phe Leu Gly Cys Cys Phe Phe Ser Thr Leu         995 1000 1005 Val Leu Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys Arg Arg Lys    1010 1015 1020 Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys Lys Gly Lys 1025 1030 1035 1040 Arg Val Val Ser    <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 3 atgaagccag gctgtgcggc aggat 25    <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 4 ttagctaaca actctcttgc ctttc 25    <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 5 atcctcagtc tcccagttca a 21    <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 6 ggaactggga tgaattttta a 21    <210> 7 <211> 1012 <212> PRT <213> Homo sapiens <400> 7 Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro Pro Ser   1 5 10 15 Leu Ser Leu Leu Leu Val Leu Leu Gly Leu Gly Gly Arg Arg Leu Arg              20 25 30 Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Phe Ser Arg Pro          35 40 45 Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly Thr Phe Pro Asp Gly      50 55 60 Phe Leu Trp Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu Gly Gly Trp  65 70 75 80 Gln Gln His Gly Lys Gly Ala Ser Ile Trp Asp Thr Phe Thr His His                  85 90 95 Pro Leu Ala Pro Pro Gly Asp Ser Arg Asn Ala Ser Leu Pro Leu Gly             100 105 110 Ala Pro Ser Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser Asp Ser         115 120 125 Tyr Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg Glu Leu Gly Val     130 135 140 Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro Asn Gly 145 150 155 160 Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg Arg Leu                 165 170 175 Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Pro Val Val Thr Leu Tyr             180 185 190 His Trp Asp Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly Gly Trp Ala         195 200 205 Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu Cys Phe     210 215 220 Arg His Phe Gly Gly Gln Val Lys Tyr Trp Ile Thr Ile Asp Asn Pro 225 230 235 240 Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala Pro Gly                 245 250 255 Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val Ala His Asn Leu Leu             260 265 270 Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe Arg Pro         275 280 285 Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His Trp Ile Asn     290 295 300 Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu Cys Gln Lys Ser Leu 305 310 315 320 Asp Phe Val Leu Gly Trp Phe Ala Lys Pro Val Phe Ile Asp Gly Asp                 325 330 335 Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro Asp Phe             340 345 350 Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp Phe Phe Ala         355 360 365 Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp Pro His Met     370 375 380 Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu Ser Trp 385 390 395 400 Ile Asp Leu Glu Phe Asn His Pro Gln Ile Phe Ile Val Glu Asn Gly                 405 410 415 Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr Met Tyr             420 425 430 Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys Leu Asp         435 440 445 Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp Gly Phe     450 455 460 Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr Val Asp 465 470 475 480 Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser Ser Ala Leu Phe                 485 490 495 Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe Pro Pro Leu Pro Glu Asn             500 505 510 Gln Pro Leu Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly Val Val         515 520 525 Asp Asn Tyr Ile Gln Val Asp Thr Thruu Ser Gln Phe Thr Asp Leu     530 535 540 Asn Val Tyr Leu Trp Asp Val His His Ser Lys Arg Leu Ile Lys Val 545 550 555 560 Asp Gly Val Val Thr Lys Lys Arg Lys Ser Tyr Cys Val Asp Phe Ala                 565 570 575 Ala Ile Gln Pro Gln Ile Ala Leu Leu Gln Glu Met His Val Thr His             580 585 590 Phe Arg Phe Ser Leu Asp Trp Ala Leu Ile Leu Pro Leu Gly Asn Gln         595 600 605 Ser Gln Val Asn His Thr Ile Leu Gln Tyr Tyr Arg Cys Met Ala Ser     610 615 620 Glu Leu Val Arg Val Asn Ile Thr Pro Val Val Ala Leu Trp Gln Pro 625 630 635 640 Met Ala Pro Asn Gln Gly Leu Pro Arg Leu Leu Ala Arg Gln Gly Ala                 645 650 655 Trp Glu Asn Pro Tyr Thr Ala Leu Ala Phe Ala Glu Tyr Ala Arg Leu             660 665 670 Cys Phe Gln Glu Leu Gly His His Val Lys Leu Trp Ile Thr Met Asn         675 680 685 Glu Pro Tyr Thr Arg Asn Met Thr Tyr Ser Ala Gly His Asn Leu Leu     690 695 700 Lys Ala His Ala Leu Ala Trp His Val Tyr Asn Glu Lys Phe Arg His 705 710 715 720 Ala Gln Asn Gly Lys Ile Ser Ile Ala Leu Gln Ala Asp Trp Ile Glu                 725 730 735 Pro Ala Cys Pro Phe Ser Gln Lys Asp Lys Glu Val Ala Glu Arg Val             740 745 750 Leu Glu Phe Asp Ile Gly Trp Leu Ala Glu Pro Ile Phe Gly Ser Gly         755 760 765 Asp Tyr Pro Trp Val Met Arg Asp Trp Leu Asn Gln Arg Asn Asn Phe     770 775 780 Leu Leu Pro Tyr Phe Thr Glu Asp Glu Lys Lys Leu Ile Gln Gly Thr 785 790 795 800 Phe Asp Phe Leu Ala Leu Ser His Tyr Thr Thr Ile Leu Val Asp Ser                 805 810 815 Glu Lys Glu Asp Pro Ile Lys Tyr Asn Asp Tyr Leu Glu Val Gln Glu             820 825 830 Met Thr Asp Ile Thr Trp Leu Asn Ser Pro Ser Gln Val Ala Val Val         835 840 845 Pro Trp Gly Leu Arg Lys Val Leu Asn Trp Leu Lys Phe Lys Tyr Gly     850 855 860 Asp Leu Pro Met Tyr Ile Ile Ser Asn Gly Ile Asp Asp Gly Leu His 865 870 875 880 Ala Glu Asp Asp Gln Leu Arg Val Tyr Tyr Met Gln Asn Tyr Ile Asn                 885 890 895 Glu Ala Leu Lys Ala His Ile Leu Asp Gly Ile Asn Leu Cys Gly Tyr             900 905 910 Phe Ala Tyr Ser Phe Asn Asp Arg Thr Ala Pro Arg Phe Gly Leu Tyr         915 920 925 Arg Tyr Ala Ala Asp Gln Phe Glu Pro Lys Ala Ser Met Lys His Tyr     930 935 940 Arg Lys Ile Ile Asp Ser Asn Gly Phe Pro Gly Pro Glu Thr Leu Glu 945 950 955 960 Arg Phe Cys Pro Glu Glu Phe Thr Val Cys Thr Glu Cys Ser Phe Phe                 965 970 975 His Thr Arg Lys Ser Leu Leu Ala Phe Ile Ala Phe Leu Phe Phe Ala             980 985 990 Ser Ile Ile Ser Leu Ser Leu Ile Phe Tyr Tyr Ser Lys Lys Gly Arg         995 1000 1005 Arg Ser Tyr Lys    1010    <210> 8 <211> 1043 <212> PRT <213> Mus musculus <400> 8 Met Lys Thr Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe   1 5 10 15 Phe Ser Ser Asp Glu Arg Asn Thr Arg Ser Arg Lys Thr Met Ser Asn              20 25 30 Arg Ala Leu Gln Arg Ser Ala Val Leu Ser Ala Phe Val Leu Leu Arg          35 40 45 Ala Val Thr Gly Phe Ser Gly Asp Gly Lys Ala Ile Trp Asp Lys Lys      50 55 60 Gln Tyr Val Ser Pro Val Asn Pro Ser Gln Leu Phe Leu Tyr Asp Thr  65 70 75 80 Phe Pro Lys Asn Phe Ser Trp Gly Val Gly Thr Gly Ala Phe Gln Val                  85 90 95 Glu Gly Ser Trp Lys Thr Asp Gly Arg Gly Pro Ser Ile Trp Asp Arg             100 105 110 Tyr Val Tyr Ser His Leu Arg Gly Val Asn Gly Thr Asp Arg Ser Thr         115 120 125 Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Leu Ala Leu Asp Phe Leu     130 135 140 Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro 145 150 155 160 Asn Gly Thr Val Ala Ala Val Asn Ala Gln Gly Leu Arg Tyr Tyr Arg                 165 170 175 Ala Leu Leu Asp Ser Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr             180 185 190 Leu Tyr His Trp Asp Leu Pro Leu Thr Leu Gln Glu Glu Tyr Gly Gly         195 200 205 Trp Lys Asn Ala Thr Met Ile Asp Leu Phe Asn Asp Tyr Ala Thr Tyr     210 215 220 Cys Phe Gln Thr Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His 225 230 235 240 Asn Pro Tyr Leu Val Ala Trp His Gly Phe Gly Thr Gly Met His Ala                 245 250 255 Pro Gly Glu Lys Gly Asn Leu Thr Ala Val Tyr Thr Val Gly His Asn             260 265 270 Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asp Lys Asn Phe         275 280 285 Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp     290 295 300 Ile Glu Pro Asn Arg Thr Asp Asn Met Glu Asp Val Ile Asn Cys Gln 305 310 315 320 His Ser Met Ser Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly                 325 330 335 Asp Gly Asp Tyr Pro Glu Phe Met Lys Thr Gly Ala Met Ile Pro Glu             340 345 350 Phe Ser Glu Ala Glu Lys Glu Glu Val Arg Gly Thr Ala Asp Phe Phe         355 360 365 Ala Phe Ser Phe Gly Pro Asn Asn Phe Arg Pro Ser Asn Thr Val Val     370 375 380 Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Gln Val Leu Asn Trp 385 390 395 400 Ile Lys Leu Glu Tyr Asp Asp Pro Gln Ile Leu Ile Ser Glu Asn Gly                 405 410 415 Trp Phe Thr Asp Ser Tyr Ile Lys Thr Glu Asp Thr Thr Ala Ile Tyr             420 425 430 Met Met Lys Asn Phe Leu Asn Gln Val Leu Gln Ala Ile Lys Phe Asp         435 440 445 Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Thr Leu Leu Asp Gly Phe     450 455 460 Glu Trp Gln Asp Ala Tyr Thr Thr Arg Arg Gly Leu Phe Tyr Val Asp 465 470 475 480 Phe Asn Ser Glu Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala His Tyr                 485 490 495 Tyr Lys Gln Ile Ile Gln Asp Asn Gly Phe Pro Leu Lys Glu Ser Thr             500 505 510 Pro Asp Met Lys Gly Arg Phe Pro Cys Asp Phe Ser Trp Gly Val Thr         515 520 525 Glu Ser Val Leu Lys Pro Glu Phe Thr Val Ser Ser Pro Gln Phe Thr     530 535 540 Asp Pro His Leu Tyr Val Trp Asn Val Thr Gly Asn Arg Leu Leu Tyr 545 550 555 560 Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ser Gln Cys Thr Asp                 565 570 575 Tyr Val Ser Ile Lys Lys Arg Val Glu Met Leu Ala Lys Met Lys Val             580 585 590 Thr His Tyr Gln Phe Ala Leu Asp Trp Thr Ser Ile Leu Pro Thr Gly         595 600 605 Asn Leu Ser Lys Val Asn Arg Gln Val Leu Arg Tyr Tyr Arg Cys Val     610 615 620 Val Ser Glu Gly Leu Lys Leu Gly Val Phe Pro Met Val Thr Leu Tyr 625 630 635 640 His Pro Thr His Ser His Leu Gly Leu Pro Leu Pro Leu Leu Ser Ser                 645 650 655 Gly Gly Trp Leu Asn Met Asn Thr Ala Lys Ala Phe Gln Asp Tyr Ala             660 665 670 Glu Leu Cys Phe Arg Glu Leu Gly Asp Leu Val Lys Leu Trp Ile Thr         675 680 685 Ile Asn Glu Pro Asn Arg Leu Ser Asp Met Tyr Asn Arg Thr Ser Asn     690 695 700 Asp Thr Tyr Arg Ala Ala His Asn Leu Met Ile Ala His Ala Gln Val 705 710 715 720 Trp His Leu Tyr Asp Arg Gln Tyr Arg Pro Val Gln His Gly Ala Val                 725 730 735 Ser Leu Ser Leu His Cys Asp Trp Ala Glu Pro Ala Asn Pro Phe Val             740 745 750 Asp Ser His Trp Lys Ala Ala Glu Arg Phe Leu Gln Phe Glu Ile Ala         755 760 765 Trp Phe Ala Asp Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ser Val Met     770 775 780 Lys Glu Tyr Ile Ala Ser Lys Asn Gln Arg Gly Leu Ser Ser Ser Val 785 790 795 800 Leu Pro Arg Phe Thr Ala Lys Glu Ser Arg Leu Val Lys Gly Thr Val                 805 810 815 Asp Phe Tyr Ala Leu Asn His Phe Thr Thr Arg Phe Val Ile His Lys             820 825 830 Gln Leu Asn Thr Asn Arg Ser Val Ala Asp Arg Asp Val Gln Phe Leu         835 840 845 Gln Asp Ile Thr Arg Leu Ser Ser Pro Ser Arg Leu Ala Val Thr Pro     850 855 860 Trp Gly Val Arg Lys Leu Leu Ala Trp Ile Arg Arg Asn Tyr Arg Asp 865 870 875 880 Arg Asp Ile Tyr Ile Thr Ala Asn Gly Ile Asp Asp Leu Ala Leu Glu                 885 890 895 Asp Asp Gln Ile Arg Lys Tyr Tyr Leu Glu Lys Tyr Val Gln Glu Ala             900 905 910 Leu Lys Ala Tyr Leu Ile Asp Lys Val Lys Ile Lys Gly Tyr Tyr Ala         915 920 925 Phe Lys Leu Thr Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe Phe Thr     930 935 940 Ser Asp Phe Arg Ala Lys Ser Ser Val Gln Phe Tyr Ser Lys Leu Ile 945 950 955 960 Ser Ser Ser Gly Leu Pro Ala Glu Asn Arg Ser Pro Ala Cys Gly Gln                 965 970 975 Pro Ala Glu Asp Thr Asp Cys Thr Ile Cys Ser Phe Leu Val Glu Lys             980 985 990 Lys Pro Leu Ile Phe Phe Gly Cys Cys Phe Ile Ser Thr Leu Ala Val         995 1000 1005 Leu Leu Ser Ile Thr Val Phe His His Gln Lys Arg Arg Lys Phe Gln    1010 1015 1020 Lys Ala Arg Asn Leu Gln Asn Ile Pro Leu Lys Lys Gly His Ser Arg 1025 1030 1035 1040 Val Phe Ser

[0057]

[Sequence list free text]

SEQ ID NO: 3 synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of gene cloning by the VTS approach.

FIG. 2 is a diagram showing an alignment between a human Klotho protein and a Klotho-like protein of the present invention.

FIG. 3 is a diagram showing an alignment between a human Klotho protein and a Klotho-like protein of the present invention.

FIG. 4 is a diagram showing an alignment of mouse βKlotho protein and Klotho-like protein of the present invention.

FIG. 5 is a diagram showing an alignment between mouse βKlotho protein and Klotho-like protein of the present invention.

FIG. 6 is a diagram showing a motif comparison of four Klotho-related proteins.

FIG. 7 is a photograph showing the result of amplifying the Klotho-like gene of the present invention by RT-PCR using RNA derived from each organ as a template.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/12 C07K 14/47 43/00 107 16/18 C07K 14/47 C12N 1/15 16/18 1 / 19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12N 15/00 ZNAA 5/10 5/00 A C12P 21/02 A61K 37/02 F term (reference) 4B024 AA01 CA04 CA11 GA16 HA01 HA17 4B064 AG01 CA19 CC24 DA01 4B065 AB01 BA02 CA24 CA44 4C084 AA02 AA07 AA13 BA01 BA08 BA22 CA18 CA53 DC50 NA14 ZA36 ZA42 ZA45 ZB22 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA75 EA23

Claims (15)

[Claims] 1. An aging control protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. 2. A peptide comprising the partial amino acid sequence of the aging control protein according to claim 1 or a salt thereof. 3. A polynucleotide comprising a nucleotide sequence encoding the aging control protein or the partial peptide thereof according to claim 1. 4. The polynucleotide according to claim 3, wherein the nucleotide sequence encoding the aging control protein according to claim 1 is the nucleotide sequence represented by SEQ ID NO: 1. 5. The polynucleotide according to claim 3 or 4, wherein the polynucleotide is DNA or RNA. 6. A recombinant vector containing the polynucleotide according to any one of claims 3 to 5. 7. A transformant transformed with the recombinant vector according to claim 6. 8. The transformant according to claim 7 is cultured in a medium, and the protein or salt thereof according to claim 1 or the peptide or salt thereof according to claim 2 is collected from the resulting culture. The method for producing the protein or salt thereof according to claim 1 or the peptide or salt thereof according to claim 2. 9. An antibody against the protein according to claim 1 or a salt thereof or the peptide according to claim 2 or a salt thereof. 10. A therapeutic agent for premature aging or an antiaging agent, which comprises the protein or salt thereof according to claim 1 or the peptide or salt thereof according to claim 2 as an active ingredient. 11. An agent for improving or preventing a pathological condition caused by deterioration of vascular endothelial function, which comprises the protein according to claim 1 or a salt thereof or the peptide according to claim 2 or a salt thereof as an active ingredient. 12. The agent for improving or preventing a pathological condition due to deterioration of vascular endothelial function according to claim 11, wherein the pathological condition due to deterioration of vascular endothelial function is hypertension or arteriosclerosis. 13. A gene therapy vector for treating premature aging, which comprises the polynucleotide according to any one of claims 3 to 5. 14. A gene therapy vector for improving or preventing a pathological condition due to vascular endothelial function deterioration, which comprises the polynucleotide according to any one of claims 3 to 5. 15. The gene therapy vector for ameliorating or preventing a disease state caused by vascular endothelial function deterioration according to claim 14, wherein the disease state caused by vascular endothelial function deterioration is hypertension or arteriosclerosis.