JP2003169688A - HepNaDC GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sodium di- or tricarboxylic acid co-transporter gene (HepNaDC gene) expressed in a human hepatic cell carcinoma-derived cell HepG2, its expression vector, host cells holding the vector, an expressed product produced by the cells, an antibody against the expressed product and the utilization of the antibody as a medicine. <P>SOLUTION: Foe example, this DNA molecule containing a polynuleotide encoding a polypeptide consisting of an amino acid sequence expressed by a specific sequence, its expression vector, the host cells holding the vector, the expressed product produced by the cells, the antibody against the expressed product and the medicine consisting of the antibody as an active ingredient are provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel sodium-di- or tricarboxylic acid cotransporter gene (DNA molecule) which is specifically expressed in the liver and its use.

[0002]

The sodium-dicarboxylate cotransporter (cotransporter, NADC) is expressed on the cell membrane of renal proximal tubules. The NADC is sodium ion-dependent (2
Secondary active transport) is responsible for the reabsorption of dicarboxylic acids and tricarboxylic acids, which are intermediate products of the Krebs cycle including succinate and citrate, from the tubular membrane so as to be taken up into the cell and recycled. There is. It is known that sodium-dicarboxylate symportation by NADC is a potential formation process in which sodium ions bind to each divalent negative ion substrate.

On the other hand, urinary citrate is known to suppress calcium calcification, and if the urinary citrate concentration is lowered by the action of NADC, renal stones may be formed. . For example, hypocitrateuria has been reported to be associated with a tendency to form kidney stones.

The following reports have so far been made regarding the proteins constituting NADC and the genes encoding them.
That is, Pajor has reported the isolation of a cDNA encoding human SLC13A2 by screening a human kidney cDNA library with rabbit kidney NADC1 cDNA (Pajor, AM, Am. J. Physiol., 270. : F642-F64
8 (1996)). The sequence of the human cDNA predicts a protein of 592 amino acids encompassing at least 11 transmembrane regions and two potential N-glycosylation sites. The protein having this amino acid sequence is named NADC1. The deduced amino acid sequence of human NADC1 is rabbit NAD
It is 78% identical to that of C1 and 47% identical to that of the rat kidney sodium sulfate transporter NaSi1.
In addition, in the above report, in vitro transcription of human NADC1
It produces a protein of approximately 48 kD, which has been shown to increase in molecular weight to approximately 59 kD in the presence of pancreatic microsomes, leading to core glycosylation.

Regarding recombinant human NADC1 expressed in Xenopus egg cells, it has been confirmed that it has an action of transporting both succinate and citrate. This transport action has a low affinity for succinate, is sodium dependent, and is pH insensitive. It has also been reported that the transport function of citrate is stimulated by acidic pH. Northern blot analysis detects approximately 2.8 kb NADC1 in human kidney and small intestine, but not in liver.

Pajol is also a low-affinity NADC.
We report that a rabbit NADC1 resembling 1 was found on the apical membrane of the renal proximal tubule of rabbits and mapped the SLC13A2 gene to human chromosome 17 by somatic cell hybrid analysis.

[0007] Mann, et al. Have reported by FISH and radiation hybrid mapping to 17p11.1-q11.1 between markers D17S2123 and D17S841, SLC1.
Reported localization of 3A (Mann, SS, et al., Cytog
enet. Cell Genet. 84: 89-90 (1999)).

[Rogina, B., et al., Scienc
e, 290, 2137-2140 (2000) was used to study human and rat renal sodium-
Indy (I'm not d, which is 50% similar to the dicarboxylate cotransporter
We have reported the discovery of a gene called ead yet). It can extend the average adult lifespan by nearly two-fold without diminished fertility or decreased physical activity, due to a single allele mutation that inserts five independent P elements within a single gene. . This Indy is also most abundantly expressed at the major sites of intermediate metabolism in flies, namely the fat pad, midgut and enosite. The above report also shows that normalization of the Indy gene by deletion of the P element reverts lifespan to an average length.

Succinic acid, citric acid, α-ketoglutaric acid, etc., which NADC takes up into cells, are substrates of the citric acid cycle, among which succinic acid is a complex II in the electron transport system.
It is a substrate of succinate dehydrogenase that constitutes Citrate is not only an important substrate of the citric acid cycle in mitochondria, but also acetyl-C in the cytoplasm.
It has the effect of activating oA carboxylase and promoting fatty acid synthesis. Α-Ketoglutarate is also an important metabolite of carbohydrates, lipids and amino acids. The increase of these substrates is thought to increase the membrane potential of mitochondria and increase the amount of reactive oxygen species (ROS) generated (FEBS Let).
ters, 416, 15-18 (1997)).

It has long been suggested that oxidative stress is deeply involved in aging, and it has been demonstrated that the life span of nematodes is extended by the addition of a drug that eliminates ROS (Sci).
ence, 289, 1567-1569 (2000)). Recently, it has been reported that ROS is a fundamental cause of glucose toxicity causing complications of diabetes (Nature, 404, 787-790 (2000); PNA).
S, 97, 12222-12226 (2000)).

Therefore, it is considered in the art that an inhibitor of NADC can promote the extension of life cycle, and expectations for the possibility of such a drug are increasing.

[0012]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel gene (DNA molecule) related to the NADC gene and specifically expressed in the liver.

In particular, the present invention suppresses the activity of NADC and promotes the prolongation of the life cycle of a candidate compound (NAD).
(C inhibitor), a technique useful for screening candidate compounds exhibiting lipid metabolism-improving action, fatty liver-improving action, etc. and utilized in the technique, for example, recombinant cells expressing NADC gene, expression product of NADC gene It is intended to provide a specific antibody against (recombinant protein) and the like.

As a result of diligent research, the present inventors succeeded in isolating a novel DNA molecule having homology to NADC named HepNaDC, which is specifically expressed in liver. The present invention has been completed based on this success.

[0015]

The present invention provides the following (1)-(18).
The invention of the gist of is provided.

(1) A DNA molecule containing the following polynucleotide (a), (b) or (c): (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or those Complementary strand, (b) the polynucleotide of (a) or a polynucleotide having at least 95% homology to the complementary strand thereof, (c) in the amino acid sequence represented by SEQ ID NO: 1,
It consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and has a sodium-dicarboxylic acid cotransporter activity or a sodium-tricarboxylic acid cotransporter activity (hereinafter, these activities are simply referred to as `` sodium-dicarboxylic acid transtransport activity ''. Porter activity "
A polynucleotide encoding a polypeptide having

(2) The DNA molecule according to (1) above, which is a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or its complementary strand.

(3) A DNA molecule comprising the following polynucleotide (a) or (b): (a) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 2 or its complementary strand, (b) the above (a) (4) or a complementary strand thereof, which hybridizes under stringent conditions and encodes a polypeptide having sodium-dicarboxylic acid transporter activity.

(4) A polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 2 or its complementary strand (3)
DNA molecule according to.

(5) The DNA molecule according to any one of (1) to (4) above, which is a polynucleotide encoding a polypeptide having citrate cotransporter activity.

(6) A DNA molecule consisting of an amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted or added, and which encodes a polypeptide having a life cycle extending action. .

(7) A DNA molecule which is a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

(8) The DNA according to any one of (1) to (7) above
The expression product of the molecule.

(9) The DNA according to any one of (1) to (7) above
A recombinant expression vector having a molecule inserted therein.

(10) A host cell or transformant having the recombinant expression vector according to (9) above.

(11) An antibody capable of binding to the expression product of the DNA molecule according to (8) above or a partial fragment thereof.

(12) A medicament containing the antibody according to (11) above as an active ingredient.

(13) A polypeptide comprising the amino acid sequence of (a), (b) or (c) below: (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, (b) SEQ ID NO: A polynucleotide having at least 95% homology to the polypeptide consisting of the amino acid sequence represented by 1, (c) in the amino acid sequence represented by SEQ ID NO: 1, 1 or several amino acids are deleted, substituted or A polypeptide comprising an added amino acid sequence and having sodium-dicarboxylic acid cotransporter activity.

(14) The polypeptide according to (13) above, which is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

(15) The polypeptide according to (13) or (14) above, which is a polypeptide having citrate cotransporter activity.

(16) An antibody capable of binding to the polypeptide according to any one of (13) to (15) above or a partial fragment thereof.

(17) A medicament containing the antibody according to (16) above as an active ingredient.

(18) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids are deleted, substituted or added, and having a life cycle extending action.

The present invention also provides the inventions of the following (19)-(25).

(19) The DNA according to any one of (1) to (6) above
A method for screening a candidate compound that suppresses sodium-dicarboxylic acid cotransporter activity in an expression product of a molecule, comprising: a) under physiological conditions, the DNA molecule according to any one of (1) to (6) above. Of presenting the test substances to these expression products in an appropriate reaction medium / solution for a sufficient time for suppressing the sodium-dicarboxylic acid cotransporter activity of the expression products of ), B) comparing the sodium-dicarboxylic acid cotransporter activity in the presence of the test substance with that in the absence of the test substance, detecting the inhibition of the activity by the test substance and causing this inhibition A screening method comprising a step of identifying a candidate compound (detection step).

(20) The DNA according to any one of (1) to (6) above
A method for screening a candidate compound that suppresses intracellular uptake of dicarboxylic acid or tricarboxylic acid in an expression product of a molecule, comprising: a) sufficient to suppress intracellular uptake of dicarboxylic acid or tricarboxylic acid under physiological conditions. For a long period of time, a test substance is presented to the expression product of the DNA molecule according to any one of (1) to (6) above in a suitable reaction medium / solution (contact step), b) Comparing the intracellular uptake of the dicarboxylic acid or tricarboxylic acid in the presence of the test substance with that in the absence of the test substance, detecting the suppression of the uptake by the test substance, a candidate compound with this suppression A screening method comprising a step of identifying (detection step).

(21) The DNA according to any one of (1) to (6) above
A method for screening a candidate compound that suppresses the production of reactive oxygen species (ROS) in an expression product of a molecule, comprising: a) a sufficient time for suppressing the production of reactive oxygen species (ROS) under physiological conditions. During the step, the step of presenting the test substance to the expression product of the DNA molecule according to any one of (1) to (6) above in a suitable reaction medium / solution (contact step), b) test The amount of ROS produced in the presence of the substance was compared with that in the absence of the substance, and
A screening method comprising a step (detection step) of detecting a suppression of ROS production and identifying a candidate compound that induces the suppression of ROS production.

(22) A candidate compound having at least one action selected from the group consisting of lipid metabolism improving action, fatty liver improving action, antidiabetic action, antiobesity action and antiaging action is specified. ) -The screening method according to any one of (21).

(23) At least one action selected from the group consisting of lipid metabolism improving action, fatty liver improving action, antidiabetic action, antiobesity action and antiaging action selected by the screening method described in (22) above. A compound having:

(24) A detection probe or primer for detecting the DNA molecule according to (1) above.

(25) The polypeptide according to (13) above (Hep
NaDC) or the DNA molecule according to (1) (HepNaDC gene) is a screening method for identifying a candidate compound that stimulates or suppresses the function of the expression product, the following (a)-(d)
Any method selected from the group consisting of: (a) a method of measuring the binding between a candidate compound and the above-mentioned polypeptide or gene expression product by directly or indirectly binding a labeling substance to the candidate compound, (b ) A method of measuring the binding between a candidate compound and the polypeptide or gene expression product in the presence of a labeled competitor, (c) a signal generated by the candidate compound activating or suppressing the polypeptide or gene expression product. Is examined by a detection system suitable for cells or cell membranes carrying the polypeptide or gene expression product, and (d) a mixture of the candidate compound and a solution containing the polypeptide or gene expression product is prepared. Measuring the activity of the polypeptide or gene expression product in the mixture, and using the obtained measured value as a standard containing no candidate compound Methods for comparing the same activity in.

The polypeptide or gene product in the method described in (25) above may be in the form of cells or cell membranes carrying them. When a polypeptide is used, the measurement in each method may be measurement of mRNA encoding the polypeptide.

The abbreviations for amino acids, peptides, nucleic acid sequences, nucleic acids and the like in the present specification refer to the rules of IUPAC-IUB [IUPAC-IUB Communication on Biological Nomencla
ture, Eur. J. Biochem., 138: 9 (1984)], “Guidelines for Preparation of Descriptions Including Nucleotide Sequence or Amino Acid Sequence” (Edited by the Patent Office) and symbols conventionally used in the field. .

[0044]

BEST MODE FOR CARRYING OUT THE INVENTION As a specific example of the gene of the present invention, a P named "HepNaDC" will be shown in Examples described later.
Those deduced from the nucleic acid sequences of CR products can be mentioned. The nucleic acid sequence is as shown in SEQ ID NO: 3 (consisting of 3270 base length).

The gene is a human cDNA encoding a novel HepNaDC (also referred to as HepNaDC protein) consisting of the 568 amino acid sequence shown in SEQ ID NO: 1, and the entire length of the coding region is 1704 bases.

HepNaDC encoded by the HepNaDC gene of the present invention
The protein is based on the FASTA program (Person W. R., et al., Pro
c. Natl. Acad. Sci., USA., 85, 2444-2448 (1988)) GenBank / EMBL database search results, human
Homology is detected with NADC1, human NADC3, Drosophila Indy and Drosophila Indy2. Further, as a result of RT-PCR, the gene of the present invention was confirmed to be expressed specifically in the liver, although it was slightly expressed in the kidney and the pancreas.

The fact that the gene of the present invention is specifically expressed in the liver considers that the liver is a tissue in which gluconeogenesis occurs, and the Indy gene strongly affects the fat body corresponding to the liver of Drosophila. Considering the expression, a strong association between the gene of the present invention and the Indy gene is inferred. On the other hand, the gene of the present invention has been confirmed to have high specificity for citric acid as a substrate (strong citric acid cotransporter activity), and thus the human NADC1
It is speculated that, unlike human NADC3, it is strongly associated with fat metabolism and fatty liver related to fatty acid synthesis.

[0048] In the present specification, the term "gene" is intended to include not only double-stranded DNA but also single-stranded DNA consisting of a sense strand and an antisense strand constituting the double-stranded DNA, and its length is limited. Not a thing. Therefore, the gene of the present invention
Unless otherwise specified, (DNA molecule) includes double-stranded DNA containing human genomic DNA and single-stranded DNA containing cDNA (sense strand) and single-stranded DNA having a sequence complementary to the sense strand (antisense DNA). Chain) and fragments thereof.

In the present specification, the gene (DNA molecule) does not matter whether it is a functional region or not, and may include at least one of an expression suppressing region, a coding region, a leader sequence, an exon and an intron. . Polynucleotides include RNA and DNA. DNA molecules include cDNA, genomic DNA and synthetic DNA. Polypeptides having a specific amino acid sequence and polynucleotides encoding the same include fragments, homologues,
Derivatives and variants are included.

Polynucleotide variants include naturally occurring allelic variants; non-naturally occurring variants; deletions,
Substitutions, additions, insertions, and the like are included. However, these mutants should not substantially change the function of the polypeptide encoded by the polynucleotide before mutation.

Mutation of polypeptide (modification of amino acid sequence)
Does not have to occur in nature, for example, due to mutation or post-translational modification, and is artificially generated using a naturally-derived gene (for example, HepNaDC gene of the present invention). Good. Variants of the above-mentioned polypeptide include alleles, homologs, natural variants, etc., and at least 90%, preferably 95%, more preferably 98%, even more preferably 99% of the pre-mutation polypeptide Included are those that are homologous to peptides (polypeptides that encode the HepNaDC protein). Polypeptide homology was determined using known sequence analysis software, such as the FASTA program (Clustal, V., Methods Mol. Biol., 2
5, 307-318 (1994)), and also SWISSPLOTS
Can be analyzed with.

It is important that variants of the above-mentioned polypeptides also have structural features that are commonly conserved with the pre-mutation polypeptide. In particular, the biological activity of the expression product of the HepNaDC gene of the present invention, that is, the action of taking in and recycling dicarboxylic acid, tricarboxylic acid, etc. as a transporter active factor of the citric acid cycle into cells; To increase the production of ROS; to enhance the production of reactive oxygen species (ROS) affected by the increase of substrates such as succinic acid, citric acid and α-ketoglutarate; to promote fatty acid synthesis in the liver and to promote gluconeogenesis in the liver Must have one selected from.

Further, it is important that the mutation of the polynucleotide in the coding region is silent with respect to amino acid substitution (the amino acid residue encoded by the mutated nucleic acid sequence does not change) or is a conserved mutation. Amino acid residues that are conserved mutations are shown below.

Original amino acid residue Conserved mutant amino acid residue Ala Ser Arg Lys Asn Gln or His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn or Gln Ile Leu or Val Leu Ile or Val Lys Arg or Glu Met Leu or Ile Phe Met, Leu or Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp or Phe Val Ile or Leu One or more codons, which generally encode a Cys residue, affect the disulfide bond of a particular polypeptide.

Substitutions of amino acid residues which are generally considered to change the properties of proteins are as follows. a) Hydrophilic residues such as Ser or Thr hydrophobic residues such as Leu, Ile, Phe, Val or Ala, b) Substitution of Cys or Pro with other amino acid residues, c) Electrical Residues with positive side chains, eg Lys, Arg, His
Substitution of electronegative residues of, for example, Val or Asp, d) amino acid residues with very large side chains, for example Phe
Substitution of an amino acid residue having no side chain of, for example, Gly.

The gene of the present invention and its expression product provide useful information for elucidating and grasping the sodium-dicarboxylic acid cotransporter active factor, and for diagnosing, preventing and treating diseases in which the factor is involved. With this information,
Effective means for diagnosing and treating related diseases can be developed.

Further, the gene of the present invention exerts an action of interacting with the HepNaDC gene or an expression product thereof to exert the action of suppressing the biological activity possessed by these, and an action of suppressing the induction of the expression of the gene of the present invention. It can be suitably used in the development of drugs and the like. Gene of the present invention The gene of the present invention (DNA molecule) is specifically a DNA molecule of the nucleic acid sequence represented by SEQ ID NO: 2 which encodes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or the SEQ ID NO: DNA that is the complementary strand of the DNA of the nucleic acid sequence shown by 2
Shown as a molecule. Not particularly limited thereto, for example, D having a certain homology with a nucleic acid sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (for example, the nucleic acid sequence represented by SEQ ID NO: 2 or its complementary strand)
It may be an NA molecule. Further, it may be a DNA molecule encoding a polypeptide having an amino acid sequence having a certain modification in the amino acid sequence represented by SEQ ID NO: 1.

Examples of the DNA molecule having a certain homology include homologues of the HepNaDC gene of the present invention including a polynucleotide encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. . Examples of the "certain homology" in the homologue include at least 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more. The above "homologue" means the present invention.
Those with a certain homology to the HepNaDC gene, which are recognized as belonging to one gene family, namely, NADC (Na +, due to the common structural features and gene expression patterns, and similar biological activities. / dicarboxylate co
A series of related genes recognized as belonging to the transporter) family are included. This homologue naturally includes alleles (alleles) of the HepNaDC gene.

The polypeptide consisting of the amino acid sequence having the above-mentioned certain modifications has a deletion, substitution or substitution of at least 1, more specifically 1 or several amino acids in the amino acid sequence represented by SEQ ID NO: 1. It is a polypeptide having an added amino acid sequence (modified amino acid sequence).

Here, the degree of amino acid modification, that is, "deletion, substitution or addition" and their positions are such that the polypeptide of the modified amino acid sequence consists of the amino acid sequence represented by SEQ ID NO: 1. There is no particular limitation as long as it has the same effect as the (HepNaDC protein). The above-mentioned degree of modification is usually about 1 to several, which is preferable as it has the same function as the HepNaDC protein.

"Similar function" means a function as an NADC activator. This includes, for example, sodium-dicarboxylic acid cotransporter activating action, succinic acid, citric acid, α-ketoglutaric acid and other dicarboxylic acid and tricarboxylic acid intracellular uptake increasing action, reactive oxygen species (RO
S) production increasing action, hepatic fatty acid synthesis promoting action and hepatic gluconeogenesis promoting action are included. Below this
The function (action) as a NADC activator is defined as "NADC activity", "NA
Also referred to as "DC polypeptide activity" or "biological activity of NADC." The function (action) as the NADC activator is as described above.
Also included are the antigenic and immunogenic activities of the HepNaDC protein, particularly the antigenic and immunogenic activities of the polypeptide of SEQ ID NO: 1. The polypeptide of the present invention has only to have at least one of the above specific actions or activities.

Furthermore, the gene encoding a polypeptide having an amino acid sequence having a certain modification can detect the HepNaDC gene of the present invention encoding a polypeptide having an amino acid sequence before modification by its use. Good.

As described above, the gene of the present invention includes all modified genes having the above NADC activity, regardless of the cause and means of the modification or mutation.

As an artificial means for the above modification, for example, site-specific mutagenesis [Method
s in Enzymology, 154, 350, 367-382 (1987); ibid. 100,
468 (1983); Nucleic Acids Res., 12, 9441 (1984); Continuation Biochemistry Laboratory 1 "Gene Research Method II", edited by The Biochemical Society of Japan,
p105 (1986)] and other genetic engineering techniques, and the phosphate synthesis method such as the phosphate triester method and the phosphate amidite method [J.
Am. Chem. Soc., 89, 4801 (1967); ibid. 91, 3350 (196
9); Science, 150, 178 (1968); Tetrahedron Lett., 2
2, 1859 (1981); 24, 245 (1983)] and their combination methods.

More specifically, DNA can also be synthesized by a chemical synthesis method utilizing the phosphoramidite method or the triester method, or can be synthesized using a commercially available automatic oligonucleotide synthesizer or the like. You can also The double-stranded fragment is prepared by synthesizing a complementary strand and annealing the strand under appropriate conditions with the chemically synthesized single strand, or
Alternatively, it can be synthesized by adding the strand to a chemically synthesized single strand using a DNA polymerase together with an appropriate primer sequence.

Specific examples of the DNA molecule of the present invention include those having the nucleic acid sequence represented by SEQ ID NO: 2. This nucleic acid sequence (coding region) shows an example of one combination of codons representing each amino acid residue of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. The gene of the present invention has such a specific nucleic acid sequence
Not limited to a DNA molecule, it is also possible to combine any codon for each amino acid residue and have a selected nucleic acid sequence. Selection of codons can be performed according to a conventional method. The selection can be performed in consideration of the codon usage frequency of the host to be used [Ncleic Acids Res., 9, 43 (198
1)].

As described above, the DNA molecule of the present invention includes a polynucleotide (complementary strand) consisting of a nucleic acid sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 2 and a nucleic acid sequence having a certain homology with the complementary strand. A polynucleotide consisting of is also included. The polynucleotide having this homology has at least 90 nucleotides with the complementary strand of the nucleic acid sequence represented by SEQ ID NO: 2.
% Identity, more preferably at least 95% identity,
Most preferably it refers to polynucleotides that have 98% identity.

The homologous polynucleotide (complementary strand) can be isolated under stringent conditions, that is, at 50 ° C. in 0.2 × SSC containing 0.1% SDS or 60 ° C. in 1 × SSC containing 0.1% SDS.
Under the conditions of D consisting of the nucleic acid sequence shown in SEQ ID NO: 2
A polynucleotide (DNA molecule) that hybridizes with an NA molecule and encodes a polypeptide having NADC activity can be exemplified.

Further, the DNA molecule of the present invention also includes a polynucleotide (DNA molecule) encoding a polypeptide which hybridizes with the above-mentioned complementary strand under the above-mentioned stringent conditions and has NADC activity. Production of the Gene of the Present Invention The gene of the present invention can be easily produced and obtained by a general genetic engineering method based on the sequence information about the specific examples of the gene of the present invention disclosed in the present specification [Molecular Cloning 2d Ed, Cold Spring Harbor Lab.
Press (1989); Sequel Biochemistry Laboratory "Gene Research Methods I, I
I, III ”, edited by the Japanese Biochemical Society (1986), etc.].

Specifically, a cDNA library is prepared from a suitable source in which the DNA molecule of the present invention is expressed by a conventional method, and an appropriate probe or antibody specific to the gene of the present invention is used from the library. It can be produced by selecting a desired clone [Proc. Natl. Acad. Sci.,
USA., 78, 6613 (1981); Science, 222, 778 (1983)].

In the above, examples of the origin of cDNA include various cells and tissues expressing the gene of the present invention, cultured cells derived from these, and the like. Total RNA from these sources
Isolation of mRNA, isolation and purification of mRNA, acquisition of cDNA and cloning thereof, etc. can all be performed according to a conventional method. In addition, a cDNA library is commercially available, and in the production of the DNA molecule of the present invention, those commercially available cDN
A library, such as Clontech La
b. Inc.) and other commercially available cDNA libraries can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, and various ordinary methods can be used. As a specific method, for example, cD
CD corresponding to the protein produced by NA by immunological screening using a specific antibody of the protein
Examples thereof include a method of selecting an NA clone, a plaque hybridization method using a probe that selectively binds to a nucleic acid sequence of interest, a colony hybridization method, and the like, and combinations thereof.

As the probe used in the above method, DNA chemically synthesized based on the information on the nucleic acid sequence of the gene of the present invention can be generally used. In addition, the already obtained DNA molecule of the present invention and fragments thereof can also be advantageously used as the probe. Furthermore, the DNA of the present invention
The sense primer and antisense primer set based on the nucleic acid sequence information of the molecule can be used as a screening probe.

The nucleic acid sequence as the probe is a partial nucleic acid sequence corresponding to SEQ ID NO: 2 and has at least 15
Including those having 20 consecutive nucleic acid sequences, preferably 20 consecutive nucleic acid sequences, more preferably 30 consecutive nucleic acid sequences, most preferably 50 consecutive nucleic acid sequences. In addition, the above-mentioned positive clone itself for producing the gene of the present invention can also be used as this probe. The nucleic acid sequence used as a probe in the present invention is, for example, a partial sequence corresponding to SEQ ID NO: 2, and it is essential that the nucleic acid sequence is different from the nucleic acid sequences of other NADC family genes and Indy (s). It goes without saying that it is a requirement of.

For obtaining the gene of the present invention, PCR method [S
Cience, 230, 1350 (1985)] can be preferably used. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method [Rapid amplificat
ion of cDNA ends; Experimental Medicine, 12 (6), 35 (1994)], especially 5'-RACE method [MA Frohman, et al., Proc. Natl. Aca.
d. Sci., USA., 8, 8998 (1988)] is preferable.

The primers used in adopting such a PCR method can be appropriately set based on the sequence information of the gene of the present invention clarified by the present invention, and can be synthesized by a conventional method. Amplified DNA / R
The isolation and purification of the NA fragment can be carried out by a conventional method as described above. For example, gel electrophoresis can be used.

The gene of the present invention or various DNA fragments obtained according to the above method can be prepared by a conventional method such as the dideoxy method [Proc.
atl. Acad. Sci., USA., 74, 5463 (1977)], Maxam-Gilbert method (Methods in Enzymology, 65, 499 (19)
80)] and the like, and the nucleic acid sequence thereof can be determined. In addition, the nucleic acid sequence thereof can be easily determined using a commercially available sequencing kit or the like. Utilization of the Gene of the Present Invention (1) Utilizing the nucleic acid sequence of a part or all of the DNA molecule of the present invention, HepNaDC gene (DNA
The presence or absence of the expression of a molecule) can be specifically detected.

This detection can be carried out according to a conventional method. Specifically, for example, the detection is performed by RT-PCR [Reverse
transcribed-Polymerase chain reaction; ES Kawasa
ki, et al., Amplification of RNA. In PCR Protocol,
A Guide to methods and applications, Academic Pres
s, Inc., SanDiego, 21-27 (1991)], RNA amplification, Northern blotting analysis [Molecular Cloning, Cold
Spring Harbor Lab. (1989)], in situ RT-PCR (Nucl.
Acids Res., 21, 3159-3166 (1993)], a measurement method at the cell level using in situ hybridization, the NASBA method [Nucleic acid sequence-based amplifica
tion, Nature, 350, 91-92 (1991)] and the like. As a more suitable detection method,
The detection method by RT-PCR can be mentioned.

When the PCR method is used for the above detection, the primer used in the method is not particularly limited as long as it is unique to the DNA molecule capable of specifically amplifying only the DNA molecule of the present invention. The primer can be appropriately set based on the sequence information of the DNA molecule of the present invention. Usually, the primer has a nucleotide length of about 10-35, preferably about 15-30. The nucleic acid sequence is
It preferably corresponds to a partial sequence of the gene of the present invention.

The gene of the present invention also includes a DNA fragment used as a specific primer and / or a specific probe for detecting the HepNaDC gene. It is essential that this DNA fragment has a nucleic acid sequence different from the nucleic acid sequences of other NADC family genes and Indy (s), like the above-mentioned primer.

This DNA fragment can be defined as a DNA that hybridizes with the polynucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 2 under stringent conditions. Here, the stringent conditions may be ordinary conditions under which this kind of primer or probe is used. For example, including 0.1% SDS as described above
In 0.2 x SSC at 50 ° C or in 1 x SSC containing 0.1% SDS
The conditions of 60 ° C. can be mentioned. Use of the gene of the present invention (2) The gene of the present invention is an expression product (protein) of the gene of the present invention (DNA molecule) by a conventional genetic engineering technique utilizing this.
Or the protein containing this can be easily, in large quantities, stable,
It can be manufactured.

Accordingly, the present invention includes, for example, a protein (polypeptide) encoded by the gene of the present invention and a method for producing the protein, and a vector containing the gene of the present invention, a vector containing the gene of the present invention, for producing the protein. Also provided are transformed host cells and the like.

As a specific embodiment of the protein of the present invention, a HepNaDC polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 1 can be mentioned. The protein of the present invention includes
HepNaDC polypeptides are included as well as homologs thereof. The homologue is at least 95% of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
Polypeptides having homology of are included. Furthermore, the protein of the present invention comprises a modified amino acid sequence in which at least 1, usually 1 or several or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and NADC Polypeptides having activity are included. Here, NADC activity is a sodium-dicarboxylic acid cotransporter activator as a transporter activating action, succinic acid, citric acid, α-ketoglutaric acid and other dicarboxylic acid and tricarboxylic acid intracellular uptake increasing action, active oxygen At least one selected from the action of increasing the production of species (ROS), the action of promoting fatty acid synthesis in the liver, and the action of promoting gluconeogenesis in the liver can be exemplified. As a specific example of the above homolog, a gene product of a homolog of the HepNaDC gene (a HepNaDC equivalent gene including an allele) can be mentioned.

Further, a homologue of the protein of the present invention includes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (He
pNaDC protein), a mammalian protein having the same activity as
For example, humans, horses, sheep, cows, dogs, monkeys, cats,
Also included are proteins such as bears and rodents such as rats and rabbits. Genetically engineered production of the protein of the present invention The protein of the present invention is based on the sequence information of the HepNaDC gene provided by the present invention, and a conventional gene recombination technique (for example, Science, 224, 1431 (1984); Biochem. Biophys.
Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sc
i., USA., 80, 5990 (1983), etc.].

The protein is more particularly a recombinant DN capable of expressing a gene encoding the desired protein in a host cell.
A (expression vector) can be prepared, introduced into a host cell for transformation, the transformant is cultured, and then recovered from the resulting culture.

In the above, as the host cell, either a prokaryote or a eukaryote can be used. As prokaryotic hosts, commonly used hosts such as Escherichia coli and Bacillus subtilis can be widely used. Preferably E. coli,
In particular, those contained in the Escherichia coli K12 strain are used.

Eukaryotic host cells include cells such as vertebrates, yeasts. Examples of the former include COS cells that are monkey cells [Cell, 23: 175 (1981)], Chinese hamster ovary cells and dihydrofolate reductase-deficient strains [Proc. Natl. Acad. Sci., USA., 77:
4216 (1980)] and the like can be preferably used. As the latter, yeast cells of the genus Saccharomyces can be preferably used. Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a vector capable of replicating in the host cell is used, and a promoter and SD (Shine and Shine) are provided upstream of the gene so that the gene of the present invention can be expressed in this vector.・ Dalgarno) nucleic acid sequence, and also the initiation codon (eg A
An expression plasmid added with (TG) can be preferably used. As the above vector, a plasmid generally derived from Escherichia coli,
For example, pBR322, pBR325, pUC12, pUC13 and the like are often used, but not limited to these and various known vectors can be used. Examples of commercially available products of the above vector used in an expression system utilizing E. coli include pGEX-4T (Am
ersham Pharmacia Biotech), pMAL-C2, pMAl-P2 (New
England Biolabs), pET21, pET21 / lacq (Invitrogen
, PBAD / His (Invitrogen) and the like.

An expression vector in the case of using a vertebrate cell as a host is usually a promoter located upstream of the gene of the present invention to be expressed, an RNA splice site,
Examples thereof include those carrying a polyadenylation site and a transcription termination sequence, which may optionally have an origin of replication. Specific examples of the expression vector include:
For example, pSV2dhfr [Mo, which carries the SV40 early promoter,
l. Cell. Biol., 1: 854 (1981)] and the like.
In addition to the above, various known commercially available vectors can be used. Commercially available products of such a vector used in an expression system utilizing animal cells include, for example, pEGFP-N and pEGFP-C (C
lontrech), pIND (Invitrogen), pcDNA3.1 / His (Invi
vector for animal cells such as trogen), pFastBac HT (Gib
ciBRL), pAcGHLT (PharMingen), pAc5 / V5-His, pMT /
Examples include vectors for insect cells such as V5-His and pMT / Bip / V5-his (above Invitrogen).

Further, as a specific example of the expression vector when yeast cells are used as a host, for example, pAM82 [Proc. Natl.
Acad. Sci., USA., 80: 1 (1983)] and the like. Commercially available yeast cell expression vectors include, for example, pPICZ
(Invitrogen) and pPICZα (Invitrogen).

The promoter is not particularly limited, and when Escherichia bacterium is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. As a promoter when yeast is used as a host, for example, pH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be preferably used. Further, as a preferred promoter when using animal cells as a host, SV40-derived promoter,
Examples include retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters and SRα promoters.

As the expression vector of the gene of the present invention, a usual fusion protein expression vector can also be preferably used. Specific examples of the vector include pGE for expressing as a fusion protein with glutathione-S-transferase (GST).
Examples include X (Promega).

Examples of the nucleic acid sequence in which the coding sequence of the mature polypeptide assists the expression and secretion of the polypeptide from the host cell include a secretory sequence and a leader sequence. These sequences can also be used to produce the protein of the present invention. Further, in the production of the protein of the present invention, a marker sequence used for purification of the fused mature polypeptide against a bacterial host (hexahistidine tag, histidine tag),
In the case of mammalian cells, hemagglutinin (HA) tag and the like can also be used.

The method for introducing a desired recombinant DNA (expression vector) into a host cell and the method for transforming the same are not particularly limited, and various general methods can be adopted.

The transformant can be cultured according to a conventional method, and the desired protein of the present invention encoded by a gene designed as desired by the culture is expressed intracellularly, extracellularly or on the cell membrane of the transformant. It is produced (accumulated, secreted).

As the medium used for culturing, various kinds of medium commonly used depending on the host cell employed can be appropriately selected and used. Culturing can also be carried out under conditions suitable for growing host cells.

The recombinant protein of the present invention thus obtained is, if desired, subjected to various separation procedures utilizing its physical properties, chemical properties, etc. ["Biochemical Data Book II",
1175-1259, 1st edition, 1st print, June 23, 1980, Tokyo Kagaku Dojin; Biochemistry, 25 (25), 8274 (1986);
Eur. J. Biochem., 163, 313 (1987), etc.].

Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography ( Examples thereof include gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, various liquid chromatography such as high performance liquid chromatography (HPLC), dialysis method, and combinations thereof. As a particularly preferable method, affinity chromatography using a column to which an antibody specific to the protein of the present invention is bound can be exemplified.

In designing a desired gene encoding the protein of the present invention, the nucleic acid sequence of the HepNaDC gene shown in SEQ ID NO: 2 can be used as a suitable one.
In the gene, if desired, the codon indicating each amino acid residue can be appropriately selected and used.

When a partial amino acid residue or amino acid sequence of the amino acid sequence encoded by the HepNaDC gene is modified by substitution, deletion, addition, etc., various methods described above such as cytospecific mutagenesis are adopted. be able to. Production of Protein of the Present Invention by Chemical Synthesis Method The protein of the present invention can also be produced by a general chemical synthesis method according to the amino acid sequence shown in SEQ ID NO: 1. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

More specifically, the peptide synthesis method is as follows.
Based on the amino acid sequence information, so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance, and then each fragment is subjected to a coupling reaction. The synthesis of the protein of the present invention may be carried out by any of the methods including fragment condensation method.

The condensation method used for the peptide synthesis is also as follows:
Conventional methods can be followed, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive (additive: 1-
Hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornene-2,3-dicarboximide, etc.) method, Woodward method, etc.

The solvent which can be used in each of these methods can also be appropriately selected from the well-known general solvents used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof.

In the above peptide synthesis reaction, the carboxyl group in the amino acid or peptide not involved in the reaction is generally esterified to form a lower alkyl ester such as methyl ester, ethyl ester or tertiary butyl ester such as benzyl ester. , P-methoxybenzyl ester, p-nitrobenzyl ester and the like aralkyl ester and the like.

Further, an amino acid having a functional group in the side chain, for example, a hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc., but such protection is not always required. You don't have to. Further, for example, the guanidino group of the arginine residue is a suitable nitro group, tosyl group, p-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group, or the like. Can be protected by various protecting groups.

Deprotection reactions of the above protecting groups in the amino acids, peptides having the above protecting groups and the finally obtained protein of the present invention can also be carried out by a conventional method such as catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, It can be carried out according to a method using hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

The protein of the present invention thus obtained is appropriately purified according to various methods described above, for example, ion exchange resin, partition chromatography, gel chromatography, countercurrent partition method and other methods generally used in the field of peptide chemistry. It can be carried out. Antibody of the Present Invention The protein of the present invention can be suitably used as an immunogen for producing a specific antibody against the protein. By using this antigen, desired antiserum (polyclonal antibody) and monoclonal antibody can be obtained.

The method for producing the antibody itself is well understood by those skilled in the art. The production of the antibody of the present invention can also be carried out according to these conventional methods [see, for example, the Second Biochemistry Laboratory, "Immunobiochemistry Research Method", edited by the Japanese Biochemical Society (1986)].

The obtained antibody can be advantageously used, for example, for purification of HepNaDC protein, measurement or identification thereof by an immunological method, and the like.

As an example of the measurement or discrimination by the immunological method using the antibody of the present invention, there can be mentioned the antibody measurement method of wild-type HepNaDC and / or mutant HepNaDC described later. As described above, the antibody of the present invention is used in the pharmaceutical field,
It is useful as a drug for detecting and measuring HepNaDC and as a diagnostic drug for various diseases related to HepNaDC.

As will be described later, the antibody of the present invention is
It is also useful for screening candidate compounds that suppress (inhibit) activity. Compounds that suppress NADC activity NADC takes up into cells succinic acid, citric acid and α-
Ketoglutarate is a substrate of the citric acid cycle, succinate is a substrate of the succinate dehydrogenase of complex II in the electron transport chain, citric acid is an important substrate of the citric acid cycle in mitochondria, and Activates acetyl-CoA carboxylase in the cytoplasm,
It also has the action of promoting fatty acid synthesis, and α-ketoglutarate is utilized as an important metabolite in the metabolism of sugars, lipids and amino acids. The increase in these substrates is
It is thought to increase the mitochondrial membrane potential and increase the amount of reactive oxygen species (ROS) generated (FEBS Letters, 4
16, 15-18 (1997)).

On the other hand, it has long been suggested that oxidative stress is deeply involved in aging, and it has been demonstrated that the addition of a drug that eliminates ROS prolongs lifespan in nematodes.
(Science, 289, 1567-1569 (2000)).

Recently, it has also been reported that ROS is a fundamental cause of glucose toxicity causing complications of diabetes (Nature, 404, 787-790 (2000); PNAS, 97, 12222-1).
2226 (2000)).

From these facts, a compound which exerts an action of suppressing the NADC activity possessed by the expression product of the gene of the present invention or the polypeptide of the present invention and fragments thereof, or the compound which suppresses the expression of the gene of the present invention is It has a prolonging action, and by utilizing it, it is possible to prolong the biological life by removing active oxygen species, and based on this,
It can be expected to be used for pharmaceuticals such as antiaging agents, antidiabetic agents and antiobesity agents.

The expression product of the gene of the present invention has an action of promoting fatty acid synthesis in the liver, and has a high specificity for citric acid as a substrate. Metabolic disorders and fatty liver may occur. Accordingly, the expression product of the gene of the present invention or its activity, that is, the compound that exerts the action of suppressing the NADA activity and the compound that suppresses the expression of the gene of the present invention can be expected to have pharmaceutical uses as lipid metabolism disorder improving agents and fatty liver improving agents. .

[0116] Further, there is also a report that if the NADC activity is suppressed, a calorie intake suppressing effect is recognized, and there is also a report that the life span is extended as a result of the suppression of caloric intake. From these, it is considered that the above compounds are useful as agents for preventing or improving lipid metabolism disorders, fatty liver, diabetes, obesity and the like, and are also effective as pharmaceuticals for preventing aging.

Compounds having an action of suppressing NADC activity include low molecular weight compounds, high molecular weight compounds, variants of HepNaDC protein, antibodies against HepNaDC protein (polyclonal antibody and monoclonal antibody), antisense DNA molecules and the like. . Pharmaceutical composition The compound (including protein) used as an active ingredient in the pharmaceutical composition includes a pharmaceutically acceptable salt of the compound, for example, an alkali metal salt such as sodium, potassium, lithium, calcium, magnesium, barium or ammonium. ,
Alkaline earth metal salts, ammonium salts and the like are included. These salts can be prepared by methods well known in the art. Further, when the active ingredient compound has a functional group such as an amino group, the salts include acid addition salts obtained by reacting the active ingredient compound with a suitable organic acid or inorganic acid.
Representative acid addition salts include, for example, hydrochloride, hydrochlorate, hydrobromide, sulfate, sulfite, acetate, oxalate, valerate, oleate, laurate, borate,
Benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, ascorbic acid Examples thereof include salts, benzene sulfonates, napsilate and the like.

The pharmaceutical composition (pharmaceutical formulation) is, for example, the above-mentioned NADC.
A pharmaceutically effective amount of an active ingredient compound (active ingredient) such as a compound which exerts an activity suppressing effect is contained together with a suitable pharmaceutical carrier or diluent.

Here, the pharmaceutical carrier is a diluent such as a filler, a filler, a binder, a moisturizer, a disintegrant, a surface active agent, a lubricant or the like, which is usually used depending on the use form of the preparation. Examples include excipients. These are appropriately selected and used according to the dosage unit form of the obtained preparation.

When a protein is used as an active ingredient, the pharmaceutical preparation preferably contains various components used in ordinary protein preparations such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, and pH adjusters. It is prepared by appropriately using agents, surfactants and the like.

Examples of the stabilizer include human serum albumin, L-amino acid, saccharide, cellulose derivative and the like. These can be used alone or in combination with a surfactant and the like. In particular, this combination may be able to further improve the stability of the active ingredients.

L-amino acids include, for example, glycine, cystine, glutamic acid and the like.

Examples of sugars include monosaccharides such as glucose, mannose, galactose and fructose; mannitol,
Sugar alcohols such as inositol and xylitol; disaccharides such as sucrose, maltose and lactose; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate and hyaluronic acid, and derivatives thereof can be used.

The surfactant may be either an ionic surfactant or a nonionic surfactant, and examples thereof include polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type and sorbitan monoacyl ester type. , Fatty acid glycerides, etc. are included.

As the cellulose derivative, for example, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

The amount of the above saccharides added is about 1 μg of the active ingredient.
It is appropriate that the amount is about 0.0001 mg or more, preferably about 0.01-10 mg. The amount of the surfactant added is about 0.00001 mg or more per 1 μg of the active ingredient, preferably about 0.0001-
It is suitable to set it in the range of about 0.01 mg. The amount of human serum albumin added is appropriately about 0.0001 mg or more, preferably about 0.001-0.1 mg per 1 μg of the active ingredient. About 0.001-10mg of amino acid per 1μg of active ingredient
It is appropriate to set the degree. The amount of the cellulose derivative added is appropriately in the range of about 0.00001 mg or more, preferably about 0.001-0.1 mg per 1 μg of the active ingredient.

The amount of active ingredient contained in the pharmaceutical preparation is appropriately selected from a wide range. Usually, it is suitable to be selected from the range of about 0.00001-70% by weight, preferably about 0.0001-5% by weight.

Various additives such as buffers, isotonic agents, chelating agents and the like can be added to the pharmaceutical preparation. Here, as the buffer, boric acid, phosphoric acid, acetic acid,
Examples thereof include citric acid, ε-aminocaproic acid, glutamic acid, and / or their corresponding salts (for example, their alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and alkaline earth metal salts). Examples of the isotonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of chelating agents include sodium edetate and citric acid.

The pharmaceutical preparation of the present invention can be used not only as a solution preparation, but also after being lyophilized to a storable state, it is dissolved in a buffer solution containing water, a saline solution or the like before use. It is also possible to prepare and use it at an appropriate concentration.

[0130] As the dosage unit form of the pharmaceutical preparation, various forms can be selected according to the therapeutic purpose, and typical examples thereof include solids such as tablets, pills, powders, powders, granules and capsules. Dosage forms, solutions, suspensions, emulsions, syrups, elixirs and other liquid dosage forms are included, and depending on the route of administration, oral, parenteral, nasal, vaginal, suppository, tongue They are classified into laxatives, ointments, and the like, and can be compounded, molded, or prepared according to ordinary methods.

For example, in the case of molding in the form of tablets, as the above-mentioned pharmaceutical carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate and the like are shaped. Agent, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose,
Methyl cellulose, binders such as polyvinylpyrrolidone, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, low-substituted hydroxypropyl cellulose, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate,
Disintegrants such as calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, surfactants such as stearic acid monoglyceride, sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium base, Absorption enhancers such as sodium lauryl sulfate, moisturizers such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, purified talc, stearate, boric acid powder, and polyethylene glycol. You can use a drug such as a drug.

Further, the tablets may be tablets coated with a usual coating, if necessary, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, and double tablets or multilayer tablets. You can also

For shaping into a pill form, as a pharmaceutical carrier, for example, glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, disintegrating agents such as laminaran and agar can be used.

Capsules are usually prepared by mixing the active ingredient of the present invention with the various pharmaceutical carriers exemplified above and filling hard gelatin capsules, soft capsules and the like according to a conventional method.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs, etc. containing conventional inert diluents such as water, and further wetting agents, Auxiliary agents such as emulsions and suspensions can be included, and these are prepared according to a conventional method.

For preparing liquid dosage forms for parenteral administration, such as sterile aqueous or non-aqueous solutions, emulsions, suspensions, etc., diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxylated. Isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester and vegetable oil such as olive oil can be used, and injectable organic esters such as ethyl oleate can be added. Further, a usual solubilizing agent, buffering agent, wetting agent, emulsifying agent, suspending agent, preservative, dispersing agent and the like can be added to these.

Sterilization can be carried out, for example, by a filtering operation through a bacteria-retaining filter, blending of a sterilizing agent, irradiation treatment and heat treatment. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

In the case of molding in the form of suppositories or vaginal preparations, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic glycerides can be used as the drug carrier.

When the paste, cream, gel or other ointment is formed, as a diluent, for example, white petrolatum, paraffin, glycerin, cellulose derivative, propylene glycol, polyethylene glycol,
Silicone, bentonite and vegetable oils such as olive oil can be used.

The composition for intranasal or sublingual administration can be prepared by a conventional method using well-known standard excipients.

If necessary, the pharmaceutical composition of the present invention may contain a colorant, a preservative, a flavor, a flavoring agent, a sweetening agent, and other pharmaceuticals.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example tablets, pills,
Solutions, suspensions, emulsions, granules and capsules are administered orally, injections are administered alone or mixed intravenously with a normal replacement fluid such as glucose and amino acids, and further intramuscularly alone if necessary. Administered intradermally, subcutaneously or intraperitoneally,
Suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered orally, and ointments are administered transdermally topically.

The amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may depend on the desired therapeutic effect, administration method, treatment period, patient's age, sex and other conditions. It is appropriately selected from a wide range. Generally, the dose is usually about 0.
The dose may be about 01 μg-10 mg, preferably about 0.1 μg-1 mg, and the preparation can be administered once a day or in several divided doses. Antisense DNA molecule against the DNA molecule of the present invention.
, Showed homology in the amino acid sequence with human NADC, and was found to have liver-specific expression. This is
Antisense DNA molecule for HepNaDC gene
It suggests that it may interact with HepNaDC gene and influence not only the citric acid cycle but also carbohydrate, lipid and amino acid metabolism. Therefore, the antisense DNA molecule of the HepNaDC gene of the present invention itself or a vector expressing the antisense DNA molecule is used to suppress the function of the HepNaDC gene in the cell tissue of an organism or to the cells of the HepNaDC gene. Expression can be suppressed (gene therapy), thus improving lipid metabolism,
It can be expected to have an effect of improving fatty liver, an antidiabetic effect, an antiobesity effect, an antiaging effect, a calorie intake suppressing effect, etc., and also an effect of prolonging the life of an organism.

For example, an arbitrary gene expression vector including an antisense strand of all or part of the HepNaDC gene of the present invention is prepared, and the expression vector is introduced into cells or tissues of an organism to force the expression of the HepNaDC gene. If suppressed to, the action of NADC activator, that is, NADC activation action, for example, the action of increasing the intracellular concentration of dicarboxylic acid and tricarboxylic acid, the action of increasing the production of reactive oxygen species, the action of promoting lipid metabolism,
It is possible to suppress the action of promoting calorie intake.
Therefore, the antisense strand is considered to be effective as an active ingredient of a gene therapy composition or a gene therapy agent having an inhibitory action on these NADC activities.

The present invention provides a gene therapy vector containing an antisense strand of all or part of such a HepNaDC gene, and a gene therapy using a cell into which the antisense strand of the HepNaDC gene has been introduced as an active ingredient. A composition is provided.

More specifically, the present invention provides a gene therapy vector containing an antisense strand of the nucleic acid sequence represented by SEQ ID NO: 2 (all or part), and a cell into which the antisense strand is introduced by the vector, Also provided are gene therapy agents containing these gene therapy vectors and cells as active ingredients.

The present invention also provides a gene therapy vector containing an antisense strand of the nucleic acid sequence represented by SEQ ID NO: 2 (all or part) or a cell into which the antisense strand has been introduced as an active ingredient. The present invention provides a drug for suppressing the progression of disease in a diabetic patient or a drug for suppressing NADC activity or an antidiabetic drug.

The inhibitor or antidiabetic agent of the present invention is
For example, by administering to the hepatic or vascular tissue site of the diabetic patient to interact with cells expressing HepNaDC of the diabetic patient, based on the NADC activity inhibitory action in these tissues, etc., it is possible to exert an antidiabetic effect. Alternatively, the same antidiabetic effect can be exerted based on the suppression of HepNaDC gene expression level in these cells.

Further, the present invention provides a lipid metabolism improving agent, a fatty liver improving agent, etc., which contains the cells into which the antisense strand has been introduced as an active ingredient. In particular, citric acid has an action of activating acetyl-CoA carboxylase in the cytoplasm and promoting fatty acid synthesis, and HepNaDC has a cotransporter activity specific to the citric acid, and thus the NaDC activity of the present invention. Inhibitors can be expected to be effective in improving lipid metabolism disorders such as fatty liver and antilipidemia.

Furthermore, the present invention provides a life-extending drug such as an antiobesity drug, an antiaging drug, a caloric intake suppressor drug, etc., which contains the cell into which the antisense strand has been introduced as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition containing, as an active ingredient, a gene therapy vector containing the antisense strand, in particular, an effect of increasing NADC activity or intracellular uptake of dicarboxylic acid and tricarboxylic acid, Provided is a pharmaceutical composition for use in treatment for suppressing gene transcription based on the action of increasing active oxygen species or the action of inhibiting caloric intake promoting action. Gene Therapy Hereinafter, the gene therapeutic agent of the present invention will be described in detail. In carrying out the following gene therapy, conventional methods in chemistry, molecular biology, microbiology, recombinant DNA technology, genetics and immunology can be used. These methods are described in, for example, Maniatis, T., et al., Molecular cl
oning: A laboratory manual (Cold Spring Harbor Lab
oratory, Cold Spring Harbor, New York (1982)), Sambrook (J., et al., Molecular cloning:
A laboratory manual, 2nd Ed. (Cold Spring Harbor L
aboratory, Cold Spring Harbor, New York (1981)),
Ausbel (FM, et al., Current Protocols
in Molecular Biology, John Wiley and Sons, New Yor
k, New York, (1992)), Glover (D., DNAClon
ing, I and II (Oxford Press) (1985)), Anand (Anand,
Techniques for the Analysis of Complex Genomes, (Aca
demic Press (1992)), Guthrie, G., et al., G
uide to Yeast Genetics and Molecular Biology, (Aca
demic Press) (1991)), Fink (Fink, et al., Hum. Ge
ne Ther., 3, 11-19 (1992).

The gene therapy of the present invention is an antisense method for producing HepNaDC gene-expressing cells of the present invention, which produces an antagonistic sequence for mRNA in the cells, inhibits translation, and suppresses HepNaDC gene expression. It is based on the provision of a pharmaceutical composition comprising an oligonucleotide as an active ingredient. The pharmaceutical composition suppresses NADC activity or transcription activation activity, more specifically, increases intracellular concentration of dicarboxylic acid and tricarboxylic acid, promotes fatty acid synthesis in liver, promotes gluconeogenesis in liver, It is used for gene transcription inhibitory treatment based on inhibitory action such as action of increasing active oxygen species and action of promoting calorie intake.

Gene therapy for diabetic patients using antisense oligonucleotides includes, for example, retrovirus, adenovirus, adeno-associated virus vector (A
By incorporating an antisense oligonucleotide into a vector derived from AV, adeno-associated virus, etc., and infecting NADC activity-expressing cells with this to overexpress the antisense oligonucleotide, a desired antidiabetic effect can be obtained. Obtainable.

[0154] When the antisense oligonucleotide is introduced into cells having the HepNaDC gene of the present invention to suppress the expression of HepNaDC protein, the antisense oligonucleotide need not correspond to the full length of the corresponding HepNaDC gene. None, for example, as long as it retains substantially the same function as the function of suppressing the expression function of the HepNaDC gene, even in the above-mentioned modified form, it is also an oligonucleotide consisting of a partial sequence retaining a specific function. It may be.

Vectors for both recombination with antisense oligonucleotides and extrachromosomal maintenance include:
Any such known vector already known in the art can be used in the present invention. For example, there may be mentioned a viral vector or a plasmid vector containing a copy of the HepNaDC antisense oligonucleotide linked to an expression control element and capable of expressing the antisense oligonucleotide product in a target cell. As such a vector, the gene expression vector described above can also be used, but preferably,
For example, as the origin vector, the vectors (pWP-7A, pwP-19, pWU-1, pWP-8A, pWP-21 and / or the vectors disclosed in US Pat. No. 5,252,479 and PCT International Publication WO93 / 07282 are disclosed. pRSVL) or pRC / CMV (Invitrogen)
The vector prepared by using More preferably, various viral vectors described below can be used.

As the promoter used in the vector used in gene transfer therapy, those specific to the affected tissue to be treated for various diseases can be preferably used.

Specific examples thereof include, for liver, albumin, α-fetoprotein, α1-antitrypsin, transferrin, transstyrene and the like. For the colon, carboxylic acid anhydrase I, carcinoembrogen antigen and the like can be exemplified.
For the uterus and placenta, estrogen, aromatase cytochrome P450, cholesterol side chain cleavage P450,
17-alpha hydroxylase P450 etc. can be illustrated. Examples of the prostate include prostate antigen, gp91-fox gene, and prostate-specific kallikrein.
For breast, erb-B2, erb-B3, β-casein, β-lactoglobin, whey protein and the like can be exemplified. For the lung, the activator protein C uroglobulin and the like can be exemplified. For the skin, K-14-keratin, human keratin 1 or 6, leucrin and the like can be exemplified. For the brain,
Examples include glial fibrillary acidic protein, mature astrocyte-specific protein, myelin basic protein and tyrosine hydroxylase. Examples of the pancreas include villin, glucagon, islets of Langerhans amyloid polypeptide, and the like. Examples of thyroid gland include thyroglobulin and calcitonin. Examples of bone include α1 collagen, osteocalcin, and bone sialoglycoprotein. Examples of the kidney include renin, liver / bone / kidney alkaline phosphatase, erythropoietin and the like. For the pancreas, examples include amylase and PAP1.

In the production of a vector for introducing an antisense oligonucleotide, the introduced antisense oligonucleotide (all or part of the reverse complementary sequence corresponding to the HepNaDC gene sequence) is the nucleic acid sequence information of the HepNaDC gene of the present invention. Based on the above, it can be easily produced and obtained by a general genetic engineering method as described above.

Introduction of a vector for introducing an antisense oligonucleotide into a cell has already been carried out in the field of introducing DNA into a cell, such as electroporation, calcium phosphate coprecipitation method, virus transduction, and gene gun. It can be performed according to various known methods. The cells transformed with the antisense oligonucleotide of the HepNaDC gene can be used by themselves as a drug for suppressing NADC activity, or as a model system for therapeutic research.

In gene therapy, the vector for introducing an antisense oligonucleotide can be introduced into a target cell of a patient by locally or systemically injecting and administering it to a target tissue site of the patient. At this time, systemic administration enables the target vector to reach any cell capable of expressing HepNaDC mRNA. If the transduced gene is not permanently incorporated into the chromosome of each target cell, the administration can be repeated periodically to achieve the desired incorporation.

The gene therapy method of the present invention comprises an in vivo method of directly administering the above-mentioned material for introducing an antisense oligonucleotide (vector for introducing an antisense oligonucleotide) into the body, and Once the target cell is taken out, the gene is introduced outside the body, and then the cell is returned to the body ex vivo
Includes both laws. In the gene therapy method of the present invention, it is also possible to directly introduce the antisense oligonucleotide of the HepNaDC gene into cells and perform gene therapy using ribozyme, which is an active molecule that cleaves an RNA chain.

The target cell into which the antisense oligonucleotide is introduced can be appropriately selected depending on the target of gene therapy (treatment). As the target cells, for example, brain / nerve tissue, brain cells, cerebral nerve cells and the like are preferable. Without being limited to these, other tissues and cells in which expression of HepNaDC is recognized, for example, heart, placenta, lung, liver, pancreas, spleen, small intestine, cells of peripheral blood tissue, and nerve cells, lymphocytes, fibroblasts, Cells such as hepatocytes and hematopoietic stem cells can also be targeted cells.

The method of introducing an antisense oligonucleotide in gene therapy includes a viral introduction method and a non-viral introduction method.

As a viral introduction method, a method using a retrovirus vector as a vector can be mentioned in view of the fact that the antisense oligonucleotide of the HepNaDC gene is a foreign substance expressed in normal cells. Other viral vectors include, for example, adenovirus vector, HIV (human immunodeficiency viru
s) vector, adeno-associated virus vector (AAV, adeno-
associated virus), herpes virus vector, herpes simplex virus (HSV) vector and Epstein-
A bar virus (EBV, Epstein-Barr virus) vector or the like can also be used.

[0165] As a non-viral gene transfer method,
Calcium phosphate co-precipitation method: Membrane-fused liposomes in which DNA-encapsulated liposomes are fused with inactivated Sendai virus whose gene has been previously destroyed by ultraviolet light to create membrane-fused liposomes, which are directly fused with cell membranes to introduce DNA into cells. Method [Kato, K., et al., J. Biol. Chem., 266, 22071-2207
4 (1991)]; a method of coating plasmid DNA with gold and physically introducing the DNA into cells by high-voltage discharge [Yang, N.
S. et al., Proc.Natl.Acad.Sci., 87,9568-9572 (199
0)]; naked DNA method in which plasmid DNA is directly injected into an organ or tumor in vivo [Wolff, JA, et al. S
cience, 247, 1465-1467 (1990)]; Cationic to introduce a gene embedded in multilamellar positively charged liposomes into cells
Liposome method [Kunio Yagi, Ayumi of Medicine, Vol.175, No.
9,635-637 (1995)]; introducing a gene only into specific cells,
The ligand-DNA complex method [Frindei] in which a ligand that binds to a receptor expressed in a target cell is bound to DNA and administered in order to prevent it from entering other cells [Frindei
s, et al., Trends Biotechnol., 11,202 (1993); Miller,
et al., FASEB J., 9, 190 (1995)].

In the ligand-DNA complex method, for example, a method using asialoglycoprotein as a ligand and a target asialoglycoprotein receptor expressed by hepatocytes [Wu, et al., J. Biol. Chem., 266] is used. , 14338 (1991); Ferkol,
et al., FASEB J., 7, 1081-1091 (1993)] and the like.

In the gene therapy of the present invention, a method for introducing a desired gene into a target cell or target tissue is typically
Two methods are included.

In the first method, after collecting target cells from a patient to be treated, the cells are cultured in vitro with the addition of, for example, interleukin-2 (IL-2) or the like to prepare a retroviral vector. This is a method (ex vivo method) of retransplanting the cells obtained after introducing the target antisense oligonucleotide of the HepNaDC gene contained therein.

The second method is a direct gene transfer method (direct method) in which a desired antisense oligonucleotide is directly injected into a target site such as the liver, portal vein, or vascular tissue of a patient.

More specifically, the first method of gene therapy is carried out as follows, for example. That is, mononuclear cells collected from a patient are sorted from monocytes using a blood separator, and the sorted cells are cultured in an appropriate medium such as AIM-V medium in the presence of IL-2 for about 72 hours, A vector containing the antisense oligonucleotide to be introduced (HepNaDC antisense oligonucleotide) is added. Antisense
In order to improve the efficiency of introducing the oligonucleotide, centrifugation may be performed at 2500C for 1 hour at 32 ° C in the presence of protamine, and then the cells may be cultured at 37 ° C under 10% carbon dioxide gas for 24 hours. After repeating this operation several times, AI was further added in the presence of IL-2.
Incubate for 48 hours in MV medium, wash the cells with physiological saline, calculate the number of viable cells, measure the antisense oligonucleotide introduction efficiency by RT-PCR etc., and introduce the target antisense oligonucleotide. Check the effect.

[0171] In addition, after confirming safety by conducting bacterial / fungal culture in cultured cells, presence / absence of infection with mycoplasma, search for endotoxin, etc., and confirming safety, the expected effective dose of antisense oligonucleotide (Hep
The cultured cells introduced with NaDC antisense oligonucleotide) are returned to the patient by intravenous drip. Gene therapy is performed by repeating this method, for example, at intervals of several weeks to several months.

The dose of the viral vector is appropriately selected depending on the target cells to be introduced. Usually, the virus titer is, for example, 1 × 10 ³ cfu for 1 × 10 ⁸ target cells.
It is preferable to adopt a dose in the range of 1 × 10 ⁸ cfu.

As an alternative to the above-mentioned first method, a virus-producing cell containing a retroviral vector containing the target antisense oligonucleotide (HepNaDC antisense oligonucleotide) is co-cultured with, for example, a patient's cell. Alternatively, a method of introducing an antisense oligonucleotide (HepNaDC antisense oligonucleotide) into a target cell can be adopted.

In carrying out the second method (direct method) of gene therapy, the target antisense oligonucleotide (HepNaDC antisense oligonucleotide) is actually introduced by the gene transfer method by preliminary experiments in vitro. PCR of vector gene cDNA
It is desirable to confirm the desired therapeutic effect based on the introduction of the target antisense oligonucleotide (HepNaDC antisense oligonucleotide) by the method, in situ PCR method, or the like.

When a viral vector is used,
Safety by introducing antisense oligonucleotides during gene therapy by conducting a PCR search for proliferative retroviruses, measuring reverse transcriptase activity, or monitoring the membrane protein (env) gene by PCR Needless to say, it is important to confirm sex.

The gene therapy composition of the present invention can be prepared by a conventional method using various carriers exemplified in the above-mentioned pharmaceutical composition section, depending on the use form of the preparation.

The gene therapy composition (pharmaceutical preparation containing a vector for introducing an antisense oligonucleotide)
Is prepared in a form in which the vector is embedded in a liposome or in the form of cultured cells infected with a virus containing a retrovirus vector in which a desired antisense oligonucleotide is included, and is practically used.

These are phosphate buffered saline (pH 7.
4), it can be prepared in the form of being mixed in Ringer's solution, injectable solution for intracellular composition liquid, or in the form of being administered together with a substance such as protamine which enhances gene transfer efficiency.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like.

The amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may depend on the desired therapeutic effect, administration method, treatment period, patient's age, sex and other conditions. It is appropriately selected from a wide range.

Generally, the dose of the desired antisense oligonucleotide-containing retrovirus vector as a pharmaceutical preparation is about 1 × 10 ³ pfu to 1 × 10 ¹⁵ as a retrovirus titer per 1 kg of adult body weight per day. It should be about pfu. For cells into which the desired antisense oligonucleotide for introduction has been introduced, 1 × 10 ⁴ cells / body
To 1 × 10 ¹⁵ cells / body is suitable.

The preparation can be administered once a day, can be administered in several divided doses, or can be administered intermittently at intervals of 1 to several weeks. In addition, preferably, it can be co-administered with a substance such as protamine that enhances gene transfer efficiency or a preparation containing the substance. Detection of HepNaDC Gene The present invention is a method for detecting the presence of the HepNaDC gene, which comprises preparing a biological sample such as blood or serum, optionally extracting the nucleic acid, wherein the HepNaDC gene is present in these samples. Provided is a method for detecting HepNaDC gene, which is analyzed for the presence or absence.

In the detection method, first, a DNA fragment of HepNaDC gene is designed and prepared so as to be used for screening of HepNaDC gene and / or its amplification. The DNA
More specifically, the fragment has a property as a probe in, for example, plaque hybridization, colony hybridization, Southern blotting, Northern blotting and the like. It may also be used as a probe in order to obtain a DNA fragment of all or part of the HepNaDC gene amplified by polymerase chain reaction (PCR) in which a nucleic acid sequence is amplified by a polymerase. To create these DNA fragments, first of all, HepNa
Create a primer with the same sequence as the partial sequence of the DC gene.

Then, the presence of the HepNaDC gene in the sample is confirmed by reacting the sample with a biological sample (nucleic acid sample) using the primer as a screening probe. The nucleic acid sample may be prepared by various methods that facilitate detection of the target sequence, such as denaturation, restriction digestion, electrophoresis, dot blotting and the like.

As a screening method for confirming the presence of the target gene, the PCR method is particularly preferable from the viewpoint of sensitivity, but is not limited thereto, and various known methods using a HepNaDC gene fragment as a primer (for example, Sci
ence, 230, 1350-1354 (1985), for example, Yoshiyuki Sakaki, et al., Yodosha, experimental medicine, special edition, 8 (9) (1990); protein / nucleic acid / enzyme, special edition, Kyoritsu Publishing ( Co., Ltd., 35 (17) (19
The method described in 90)) can also be used.

The DNA fragment used as a primer is
It is chemically synthesized oligo DNA, and these oligo DNAs are
An automatic DNA synthesizer, such as a DNA synthesizer (Pharmacia LK
B Gene Assembler Plus: manufactured by Pharmacia) can be used. The length of the synthesized primer (sense primer or antisense primer) is
It is preferably in the range of about 10-30 nucleotides.
The probe used for the screening is usually a labeled probe, but it may be unlabeled. Also,
It is also possible to bind directly or indirectly to the labeled ligand. Suitable labeling methods, including methods for labeling probes and ligands, are known in the art. The labeling substance used also includes various substances that can be incorporated by known methods such as nick translation, random priming, and kinase treatment, for example, radiolabel, biotin, fluorescent group, chemiluminescent group, Any enzyme, antibody, etc. may be used.

Examples of the PCR method used for detection include the RT-PCR method. The method encompasses various variations used in the art.

According to the use of the HepNaDC gene of the present invention, Hep
The expression level of the NaDC gene and the polymorphism (polymorphism: individual difference) of the HepNaDC gene can be examined (gene diagnosis of HepNaDC). It becomes possible to select.

The detection of the HepNaDC gene in a sample according to the present invention is carried out by utilizing a reagent kit therefor.
It can be carried out easily. The present invention provides such a reagent kit, particularly a reagent kit for detecting the HepNaDC gene, which comprises the above-mentioned DNA fragment of the HepNaDC gene as an essential component.

The reagent kit comprises at least SEQ ID NO: 2
It is important to include, as an essential component, a DNA fragment that hybridizes with a part or all of the nucleic acid sequence shown in or the complementary nucleic acid sequence thereof. The kit can also include, as other components, for example, a labeling agent, a reagent essential for the PCR method (eg, Taq DNA polymerase, deoxynucleotide triphosphate, primer) and the like.

Examples of the labeling agent include radioisotopes, chemically modified substances such as fluorescent substances, and the like. Instead of including these labeling agents, a conjugate obtained by previously conjugating the DNA fragment itself with these labeling agents can be used as a component of the kit. Further, the reagent kit can also contain an appropriate reaction diluent, standard antibody, buffer solution, detergent, reaction stop solution, etc. for making the measurement simpler and more reliable.

The present invention also provides a diagnostic agent and a diagnostic kit used for detecting polymorphisms in the HepNaDC gene.

Further, by utilizing the detection method of the present invention, the HepNaDC sequence obtained from the test sample can be directly or indirectly determined, and from this sequence, a new HepNaDC gene highly homologous to the wild-type HepNaDC gene can be obtained. It is also possible to find genes related to. Therefore, according to the present invention, a method for determining the sequence of the HepNaDC gene in a test sample and screening a gene related to the human HepNaDC gene in the test sample can also be provided.

From the protein encoded by the human HepNaDC gene represented by SEQ ID NO: 1 or the amino acid sequence in which at least one amino acid has been deleted, substituted or added in SEQ ID NO: 1, for example, 1 or several or more. The antibody of the present invention obtained by using the polypeptide or a fragment thereof is also useful for measuring wild-type HepNaDC and / or mutant HepNaDC. Therefore, the present invention also provides an antibody measurement method for wild-type HepNaDC and / or mutant HepNaDC.

Wild-type HepNaDC and / or mutant HepNaDC
The measurement can be performed by sequence analysis of the HepNaDC gene according to the above-mentioned conventional technique, but is preferably performed according to the above-mentioned antibody measurement method using an antibody (polyclonal or monoclonal antibody). This antibody assay is HepN
Along with the detection of the presence or absence of aDC protein, changes in specific amino acids or amino acid sequences in HepNaDC protein can also be detected.

More specifically, the antibody measuring method of the present invention comprises
HepNaDC protein, which may be present in a solution containing a sample of biomaterial collected from human, such as blood or serum, is immunoreacted with the antibody of the present invention to cause precipitation, or on Western blot or immunoblot of polyacrylamide gel. The reaction is carried out by reacting the above HepNaDC protein with the antibody of the present invention.

The use of the antibodies of the invention also allows the detection of HepNaDC proteins which may be present in paraffin or in frozen tissue sections according to known immunohistochemical techniques.

Techniques for producing and purifying antibodies are well known in the art, and these techniques can be appropriately selected and used in the present invention.

Wild type HepNaDC or mutants thereof were examined.
More preferred embodiments related to the method of delivery include monoc
Using local and / or polyclonal antibodies
Enzyme-linked immunosorbent assay including sandwich method
Assay (ELISA), radioimmunoassay (RIA), immunoradiation
Assay methods (IRMA), immunoenzymatic methods (IEMA), etc. are included. Drug screening The present invention screens candidate compounds that suppress (inhibit) NADC activity.
Provide a way to lean.

The screening method is, for example, HepNaD.
C gene expression product or HepNaDC protein (hereinafter referred to as H
epNaDC protein) or an antibody against a fragment peptide thereof, labeled HepNaDC protein bound to the antibody by competitively reacting the labeled HepNaDC protein, a fragment peptide or a salt thereof with a candidate compound. It is carried out by measuring (quantifying) the ratio of proteins and the like.

Further, the screening method is the same as the above HepN.
A test solution containing aDC protein, a fragment peptide thereof or a salt thereof, the antibody insolubilized on a carrier, and a labeled antibody against HepNaDC protein are simultaneously or successively reacted with each other and then insolubilized. It can also be carried out by measuring the labeling activity on the carrier.

Further, the HepNaDC protein, its fragment peptide or a salt thereof is contacted with a substrate to measure the activity (NADC activity) of the HepNaDC protein, its fragment peptide or a salt thereof, and the obtained measurement value is used as a candidate compound. It is also possible to screen a compound that suppresses or inhibits NADC activity by comparing with a measurement value obtained by performing the same operation in the presence of a test liquid containing the same.

According to the invention, also for the development of more active or stable forms of derivatives of HepNaDC protein or for the development of agents which enhance or interfere with the function of HepNaDC protein eg in vivo. In addition, it is possible to produce biologically active proteins or structural analogs with which they interact, such as HepNaDC agonists, HepNaDC antagonists, HepNaDC inhibitors and the like.

The above structural analogs include, for example, three-dimensional structure of a complex of HepNaDC protein and other proteins, X-ray crystallography,
This can be confirmed by computer modeling or a combination of these methods. Information on the structure of the structural analog can also be obtained by modeling a protein based on the structure of a homologous protein.

The more active or stable forms of HepN described above
The method for obtaining the aDC protein derivative includes, for example, an alanine scan analysis method. The method is a method in which amino acid residues other than Ala constituting the target peptide are replaced with Ala and the effect on the activity of the peptide is measured.
By analyzing each amino acid residue of the peptide in this manner, a region important for the activity and stability of the peptide can be determined. By this method, more active or stable HepNaDC derivatives can be designed.

In this way, a drug having an action as an inhibitor, an agonist, an antagonist or the like having an improved NADC activity can be designed and developed.

Using any of the drug screening methods exemplified above, the desired HepNaDC protein and succinic acid,
By screening a candidate compound that inhibits the interaction with a substrate such as citric acid or α-ketoglutarate, a compound that suppresses the expression amount of reactive oxygen species (ROS), a compound that cancels the action of ROS, etc. can be obtained. it can. These are useful as antidiabetic agents, fatty liver improving agents, aging preventive agents, and the like.

According to the present invention, by creating a HepNaDC gene-containing knockout mouse (mutant mouse), which site of the HepNaDC gene sequence affects various NADC activities in vivo, namely, HepNaD
It is possible to confirm what functions the C gene product and the modified HepNaDC gene product have in vivo.

The method is a technique for intentionally modifying the genetic information of an organism by utilizing homologous recombination of genes. This method includes a method using mouse embryonic stem cells (ES cells) (Capeccchi, MR, Science, 244, 1288-12).
92 (1989)). Techniques for producing knockout mice are known in this field (edited by Tetsuo Noda, Experimental Medicine, special edition, 14
(20) (1996), Yodosha). In the present invention, by adapting the human wild-type HepNaDC gene and mutant HepNaDC gene of the present invention to this knockout mouse, a mutant mouse can be easily prepared, thus, an inhibitor of the improved HepNaDC activity or a stable HepNaDC activity, an agonist. It is possible to design and develop a drug having an action as an antagonist.

[0210]

INDUSTRIAL APPLICABILITY According to the present invention, a novel NADC gene that is specifically expressed in the liver is provided. By using the gene, a HepNaDC gene expression product or HepNaDC polypeptide can be produced, and an antibody using this as an antigen can be produced.

The present invention also provides a method for screening a candidate compound that suppresses the expression of a HepNaDC gene expression product or suppresses the biological activity of a HepNaDC polypeptide, and a compound obtained by the screening method. The compound is useful as an active ingredient of a pharmaceutical composition for anti-diabetes, anti-obesity, suppression of caloric intake, improvement of lipid metabolism due to suppression of fatty acid synthesis or suppression of fatty liver, and prevention or treatment of aging.

Further according to the present invention, there is provided a method for genetically engineering HepNaDC protein. The resulting protein is NADC
It is useful for studying activity and functions such as HepNaDC protein binding activity.

According to the use of the gene of the present invention, HepNaD
A C variant polypeptide can be produced, and the variant is useful as a drug having a life cycle prolonging action.

According to the present invention, a vector for gene transfer useful for gene therapy, which further contains an antisense oligonucleotide of the HepNaDC gene, a cell into which the antisense oligonucleotide has been introduced, and the vector or cell as an active ingredient. And a gene therapy method using the same.

According to the present invention, there is provided a method for screening an interaction using a HepNaDC protein or gene expression product and a substrate such as succinic acid, citric acid or α-ketoglutarate.

[0216]

EXAMPLES Examples will be given below to explain the present invention in more detail.

[0217]

Example 1 (1-1) Isolation of clones from human hepatocellular carcinoma-derived cells HepG2-cDNA library Human hepatocellular carcinoma-derived cells HepG2 were cultured, and isogens (Isogen: Nippon Gene Co., Ltd.) 2 mg of total RNA was isolated using and the oligotex-dT30 super (oligotex-dT30s
upper (Daiichiyaku Co., Ltd.) was used to purify poly (A) + RNA-dT30. Oligocapping method using 50 μg of poly (A) + RNA (Szuki Y, and Sugano S, et al., Gene, 200, 149-
156 (1997)) to create a full-length cDNA library.
As a vector, pME18-FL3 (Genebank accession
No. AB009864) was used.

[0218] From the cDNA library thus obtained, random sequence was performed using ABI3700 automatic sequencer (manufactured by Applied Biosystems),
A database was constructed based on the sequence data. (1-2) Selection of clones The clones Hep04917, Hep02334 sequenced from the database obtained in (1-1) above were cloned in silico (in si
ligation) to construct a new 3270 bp full-length cDNA sequence encoding 568 amino acids.

Hep04917 and Hep02334 are cDNs derived from HepG2 cells.
It was cloned from the A library. Since the protein encoded by the full-length cDNA has homology with sodium-dicarboxylic acid cotransporter (Na + / dicarboxylate cotransporter (NADC)), the present inventors have identified this gene as HepN.
We named it aDC.

BLAST program (Altschu, SF et al. Nuc
GenBank using leic Acid Res, 25, 3389-3402 (1997))
As a result of searching the database, clones predicted to be a family were obtained. (1-3) Nucleic acid sequence and deduced amino acid sequence of clones Regarding the clones selected in (1-2) above, FASTA program (Person, WR, et al., Proc. Natl. Acad. Sci., USA, 8
5, 2444-2448 (1988)) using GenBank / EMBL data
Comparison of the nucleic acid sequence in the base with the nucleotide data revealed that the gene has no homology with any other known nucleic acid sequence.

Thus, an open reading frame (ORF, consisting of 568 amino acids shown in SEQ ID NO: 1
A cDNA having a total length of about 3.3 kb containing the nucleic acid sequence of SEQ ID NO: 2 (shown as 1704 bp) was obtained. This gene was named HepNaDC gene. The full length nucleic acid sequence of this is SEQ ID NO: 3.
It consists of 3270 bases as shown in.

FIG. 1 shows an alignment comparison diagram of the amino acid sequences encoded by the HepNaDC gene of the present invention, two NADCs family genes (NADC1, NADC3) and the dIndy gene of Drosophila, respectively. In the figure, the top row (described as dIndyaa) shows the amino acid sequence encoded by the dIndy gene,
The second step (described as Hep HADCaa) shows the amino acid sequence encoded by the gene of the present invention, the third step (described as NADC1aa U26209)
Indicates the amino acid sequence encoded by NADC1, and the bottom row (NA
DC3aa XM_017841) indicates the amino acid sequence encoded by the NADC3 gene.

The amino acid similarity between the gene of the present invention and each of these genes was 54.4%, 47.5% and 3%, respectively.
It was 6.3%.

HepNaDC has an amino acid sequence 498- in the C-terminal region.
At 514th sodium sulfate entrained transport family
It had a motif (see SEQ ID NO: 4). (1-4) Analysis of the position and genomic structure of the HepNaDC of the present invention on the chromosome HepNaDC, as a result of the BLAST search, the genomic sequence of human chromosome 17 (GenBank No. AC004706), was divided into 12 fragments. It was found that they were almost aligned (> 99%) and aligned. As a result of comparing the sequences of both, it was found that the ORF of HepNaDC is composed of 12 exons (EXON) and has a genome structure as shown in FIG. In addition, in FIG. 2, I to XII represent the above 12 exons.

The presence of the following four polymorphisms was confirmed in this protein. 1. Mutation of 838C to T (R269W, substitution of Arg at 269th position with Trp) In NADC1 and NADC3, this position is Trp,
It is also considered that rp is mutated to Arg in HepNaDC of the present invention. 2. 1038T mutation to C (synonymous, no amino acid substitution) 3. 1125T mutation to C (synonymous, no amino acid substitution) 4. 1160G mutation to A (R376K, 376th Arg to Lys) Replacement).

[0226]

[Example 2] RT-PCR analysis In order to examine the expression profile of HepNaDC in tissues, RT-PCR analysis was performed using various human tissues.

For RT-PCR analysis, human Fastland cD
NA multi-panel I and II (manufactured by Clonetech) were used.

Using each of the following primers prepared using an automatic synthesizer (for HepNaDC, two kinds of primer sets (F1-R1 and F2-R2) were used), according to the manual attached to the multiple panel. RT-PCR was performed. The PCR reaction was performed at an annealing temperature of 60 ° C. for 35 cycles. Hep NaDC F1: SEQ ID NO: 5 Hep NaDC R1: SEQ ID NO: 6 (1515-1718, 204bp) Hep NaDC F2: SEQ ID NO: 7 Hep NaDC R2: SEQ ID NO: 8 (1008-1239, 232bp) NADC1 F1: Sequence NO: 9 NADC1 R1: SEQ ID NO: 10 (GenBank No. U26209, 129
7-1496, 200bp) NADC3 F1: SEQ ID NO: 11 NADC3 R1: SEQ ID NO: 12 (GenBank No. XM_017841,
1626-1825, 202bp) The results obtained are shown in FIG.

In FIG. 3, the top row shows He in each human tissue obtained by using the Hep NaDC F1 and R1 primer sets.
The expression pattern of p NADC is shown. The second row from the top is Hep N
The expression pattern of Hep NADC in each human tissue obtained using aDCF2 and R2 primer set is shown. The third row from the top shows a similar expression pattern of NADC1. The fourth row from the top shows a similar expression pattern of NADC3. The bottom row shows a similar expression pattern of G3PDH of interest.

The human tissues used were brain and heart.
t), Kidney (Kidney), Liver (Liver), Lung (Lung), Pancreas (Panc
reas), placenta (Placenta), skeletal muscle (S. muscle), colon (Col
on), ovary (Ovary), peripheral blood leukocytes (Peripheral blood leve)
ukocyte; PBL), prostate (Prostate), small intestine (Small int
estine), spleen (Spleen), testis (Testis), thymus (Thymus)
And mammary gland.

From FIG. 3, it is understood that HepNaDC-mRNA expression is almost liver-specific, although it is also observed in the kidney and pancreas in 17 human adult tissues, although it is slightly observed.

It is also clear that this tissue specificity of HepNaDC expression pattern is different from those expressed in NADC1 and NADC3 with sequence homology. That is, NADC1 is expressed in kidney, pancreas and mammary gland, and NAD1
C3 is expressed in kidney, liver, pancreas, placenta and mammary gland.

[0233]

Example 3 Construction of HepNaDC Gene Expression Vector The HepNaDC gene expression vector is an ORF of the HepNaDC gene.
Was constructed by incorporating into the EcoRI-NotI site of the mammalian cell expression vector pME18S a fragment containing the fragment (obtained by PCR using human normal liver cDNA purchased from Clontech) as a template.

[0234]

Example 4 Transient expression of HepNaDC gene product in COS1 cells COS1 cells were cultured in a 24-well dish, and 24 hours later,
Transfection was performed. 1.0 μg of the above plasmid
In contrast, Lipofectamine 2000 (manufactured by Lifetech Co., Ltd.)
2.5 μl and OPTI-MEM serum-free medium (Lifetech)
A transfection solution containing 100 μl was used. The operation was performed according to the protocol attached to the reagent.

Expression of the HepNaDC gene expression product was confirmed 48 hours after transfection by measurement of sodium-dicarboxylic acid transporter activity and Western blotting.

[0236]

Example 5 Confirmation of Expression of HepNaDC Gene Product by Western Blotting Confirmation of expression of HepNaDC-FLAG gene expression product by Western blotting was performed as follows. That is, the transfected COS1 cells were dissolved in SDS-sample buffer to prepare a sample. This sample is SDS-PA
After developing with GE, transfer to PDEF membrane, blocking for 2 hours, primary antibody treatment for 1 hour, secondary antibody treatment for 1 hour.
After that, color development processing was performed. A mouse anti-FLAG antibody (manufactured by Sigma) was used as the primary antibody. Anti-mouse alkaline phosphatase fusion antibody as secondary antibody
(Manufactured by Promega) was used. Western Blue for coloring
(Manufactured by Promega) was used.

[0237]

[Example 6] Establishment of CHO stable transformed cells that constitutively express HepNaDC gene 2.5 μl of Lipofectamine 2000 (manufactured by Lifetech) and 100 μl of OPTI-MEM serum-free medium (manufactured by Lifetech) The solution contained HepNa prepared in Example 3.
DC gene expression vector 0.8μg and neomycin resistance gene expression vector (pCDNA3.1) 0.1μg was added, HepNaDC gene and neomycin resistance gene in CHO cells cultured in a 24-well dish using the same solution as in Example 4 Was co-transfected. After 1 day, passivate the cells for 2
Geneticin was added on the day. The selective antibiotic resistant strain was cloned by the limiting dilution method, thus establishing a CHO cell line that constitutively expresses the HepNaDC gene expression product.

It was confirmed by Western blotting that this cell line constitutively expresses the HepNaDC gene expression product.

[0239]

[Example 7] Establishment of a constitutively expressing strain of HepNaDC gene expression product in Hela cells Vector pCDNA3.1 containing the HepNaDC gene expression vector and neomycin resistance gene prepared in Example 3 was used in Example 6
Was prepared in the same manner as above, and this was cotransfected into Hela cells under the same conditions as in Example 4. By performing drug selection in a medium containing 500 mg / ml neomycin, HepNaD
A cell line that constitutively expresses the C gene expression product was obtained.

It was confirmed by Western blotting that this cell line constitutively expresses the HepNaDC gene expression product.

[0241]

[Example 8] Measurement of sodium-dicarboxylic acid transporter activity Using COS1 cells transiently expressing the HepNaDC gene obtained in Example 4, the sodium-dicarboxylic acid transporter activity of the expression product was measured as follows. It was measured.

As the buffer solution for activity measurement, 140 mM NaCl (N
a buffer) or choline Cl (choline buffer); 2 mM KCl;
A buffer consisting of 1 mM CaCl ₂ ; 1 mM MgCl ₂ ; 10 mM HEPES-Tris (pH 7.4) was used.

HepNaDC-expressing COS1 cells cultured in a 24-well dish (culture plate manufactured by Packard) were washed twice with 1 ml of choline buffer, and then [ ¹⁴ C] succinic acid was added to the cells.
¹⁴ C) citric acid and ( ¹⁴ C) α-ketoglutaric acid either 0.1 mM Na buffer solution (indicated as Na + in Figure 4-6) or choline buffer solution (Figure 4-6, Na-and (Indicated) 250 μl was added. After standing for 15 minutes, the reaction solution was discarded, and 3 ml of choline buffer solution cooled with ice water was added. Furthermore, the culture dish was washed three times with cold choline buffer, and 0.51 ml of Microscinti 20 (manufactured by Packard) was added, and [ ¹⁴ C] succinic acid incorporated into cells, [ ¹⁴ C] citrate and [ ¹⁴ C] α-ketoglutarate top-counting any radioactivity of NT
It was measured by X (manufactured by Packard).

The results obtained (labeled “HepNaDC”) are shown in FIG.
([ ¹⁴ C] succinic acid uptake activity), FIG. 5 ([ ¹⁴ C] citric acid uptake activity) and FIG. 6 ([ ¹⁴ C] α-ketoglutarate uptake activity).

In each figure, HepNaDC prepared in Example 3 was used.
Instead of the gene expression vector, the same test results for COS1 cells obtained by transfection in the same manner as in Example 3 using the mammalian cell expression vector pME18S in which the HepNaDC gene was not introduced as a control vector, the control ( Displayed as "control").

[0246] From the results shown in Fig. 4-6, in COS1 cells in which the HepNaDC gene of the present invention was expressed, the intracellular uptake of dicarboxylic acid increased in a sodium-dependent manner. It is apparent that it has dicarboxylic acid transporter activity.

In particular, as shown in FIG. 4, the amount of succinic acid taken up by COS1 cells expressing the HepNaDC gene of the present invention was about 2.5 times that of the control. Also, as shown in FIG.
The amount of citric acid taken up by COS1 cells expressing the HepNaDC gene of the present invention is about 20 times that of the control. From this, it is clear that the HepNaDC of the present invention has high specificity for citric acid. The fact that intracellular uptake of dicarboxylic acid is sodium-dependent indicates that Na in each figure
It is clear from the comparison between the data at + and the data at Na-.

The transporter activity measurement system shown in this example can also be used as a screening system for candidate compounds that inhibit the uptake of dicarboxylic acid and tricarboxylic acid by HepNaDC by allowing the candidate compound to coexist in the reaction solution. it can.

[0249]

Example 9 Intracellular Uptake Assay of TCA Circuit Substrate in CHO Cells This assay uses the CHO cells established in Example 6,
The same procedure as in Example 8 was carried out as follows. That is, HepNaDC-expressing CHO cells cultured in a 96-well dish (LIA white plate manufactured by Greiner) were mixed with choline buffer 1
After washing twice with ml, the cells contained [ ¹⁴ C] citrate containing 0.1 mM of N
a buffer (labeled Na + in Figure 7) or choline buffer (Figure 7)
(Indicated as Na-) 50 μl was added. After standing for 60 minutes, the reaction solution was discarded, the culture dish was washed 4 times with ice-cold choline buffer, 0.1 ml of Microscint 20 (manufactured by Packard) was added, and incorporated into cells [ ¹⁴ C]. The radioactivity of citric acid was measured by Topcount NTX (made by Packard).

The results obtained (labeled “HepNaDC”) are shown in FIG.
Shown in. Incidentally, in each figure, instead of the HepNaDC gene expression vector prepared in Example 3, using the mammalian cell expression vector pME18S not introduced HepNaDC gene as a control vector, cotransfection in the same manner as in Example 6. The same test results for CHO cells obtained in this way are also shown as a control (denoted as "control").

The following is clear from FIG. That is, H
[ ¹⁴ C] citrate uptake activity of CHO cells constitutively expressing epNaDC is strong in the presence of Na, and disappears in the absence of Na. Therefore, citrate uptakes citrate in a Na-dependent manner.

[0252]

[Example 10] Measurement of ROS production-increasing action COS1 cells transfected with the HepNaDC gene expression vector described in Example 4 were added to 10 µl of the fluorescent probe CM-H ₂ DC.
Succinic acid is added together with FDA (manufactured by Molecular Probes Co., Ltd.), and the mixture is allowed to stand at 37 ° C. for 45 minutes, and then the fluorescence amount is measured with a fluorescence plate reader. The amount of ROS generated can be calculated using a standard curve created with H ₂ O ₂ .

The ROS measurement system described above can also be used as a screening system for candidate compounds that suppress the ROS production caused by the incorporation of dicarboxylic acids and tricarboxylic acids by HepNaDC by allowing a candidate compound to coexist in the reaction solution.

[0254]

[Sequence list]                                SEQUENCE LISTING <110> Otsuka Parmaceutical Co. Ltd. <120> HepNaDC gene <130> 43302JP <140> <141> <150> JP 2001-299433 <151> 2001-9-28 <160> 12 <170> PatentIn Ver. 2.1 <210> 1 <211> 568 <212> PRT <213> human hepatocellular carcinoma dirived cell HepG2 <400> 1 Met Ala Ser Ala Leu Ser Tyr Val Ser Lys Phe Lys Ser Phe Val Ile   1 5 10 15 Leu Phe Val Thr Pro Leu Leu Leu Leu Pro Leu Val Ile Leu Met Pro              20 25 30 Ala Lys Phe Val Arg Cys Ala Tyr Val Ile Ile Leu Met Ala Ile Tyr          35 40 45 Trp Cys Thr Glu Val Ile Pro Leu Ala Val Thr Ser Leu Met Pro Val      50 55 60 Leu Leu Phe Pro Leu Phe Gln Ile Leu Asp Ser Arg Gln Val Cys Val  65 70 75 80 Gln Tyr Met Lys Asp Thr Asn Met Leu Phe Leu Gly Gly Leu Ile Val                  85 90 95 Ala Val Ala Val Glu Arg Trp Asn Leu His Lys Arg Ile Ala Leu Arg             100 105 110 Thr Leu Leu Trp Val Gly Ala Lys Pro Ala Arg Leu Met Leu Gly Phe         115 120 125 Met Gly Val Thr Ala Leu Leu Ser Met Trp Ile Ser Asn Thr Ala Thr     130 135 140 Thr Ala Met Met Val Pro Ile Val Glu Ala Ile Leu Gln Gln Met Glu 145 150 155 160 Ala Thr Ser Ala Ala Thr Glu Ala Gly Leu Glu Leu Val Asp Lys Gly                 165 170 175 Lys Ala Lys Glu Leu Pro Gly Ser Gln Val Ile Phe Glu Gly Pro Thr             180 185 190 Leu Gly Gln Gln Glu Asp Gln Glu Arg Lys Arg Leu Cys Lys Ala Met         195 200 205 Thr Leu Cys Ile Cys Tyr Ala Ala Ser Ile Gly Gly Thr Ala Thr Leu     210 215 220 Thr Gly Thr Gly Pro Asn Val Val Leu Leu Gly Gln Met Asn Glu Leu 225 230 235 240 Phe Pro Asp Ser Lys Asp Leu Val Asn Phe Ala Ser Trp Phe Ala Phe                 245 250 255 Ala Phe Pro Asn Met Leu Val Met Leu Leu Phe Ala Arg Leu Trp Leu             260 265 270 Gln Phe Val Tyr Met Arg Phe Asn Phe Lys Lys Ser Trp Gly Cys Gly         275 280 285 Leu Glu Ser Lys Lys Asn Glu Lys Ala Ala Leu Lys Val Leu Gln Glu     290 295 300 Glu Tyr Arg Lys Leu Gly Pro Leu Ser Phe Ala Glu Ile Asn Val Leu 305 310 315 320 Ile Cys Phe Phe Leu Leu Val Ile Leu Trp Phe Ser Arg Asp Pro Gly                 325 330 335 Phe Met Pro Gly Trp Leu Thr Val Ala Trp Val Glu Gly Glu Thr Lys             340 345 350 Tyr Val Ser Asp Ala Thr Val Ala Ile Phe Val Ala Thr Leu Leu Phe         355 360 365 Ile Val Pro Ser Gln Lys Pro Arg Phe Asn Phe Arg Ser Gln Thr Glu     370 375 380 Glu Glu Arg Lys Thr Pro Phe Tyr Pro Pro Pro Leu Leu Asp Trp Lys 385 390 395 400 Val Thr Gln Glu Lys Val Pro Trp Gly Ile Val Leu Leu Leu Gly Gly                 405 410 415 Gly Phe Ala Leu Ala Lys Gly Ser Glu Ala Ser Gly Leu Ser Val Trp             420 425 430 Met Gly Lys Gln Met Glu Pro Leu His Ala Val Pro Pro Ala Ala Ile         435 440 445 Thr Leu Ile Leu Ser Leu Leu Val Ala Val Phe Thr Glu Cys Thr Ser     450 455 460 Asn Val Ala Thr Thr Thr Leu Phe Leu Pro Ile Phe Ala Ser Met Ser 465 470 475 480 Arg Ser Ile Gly Leu Asn Pro Leu Tyr Ile Met Leu Pro Cys Thr Leu                 485 490 495 Ser Ala Ser Phe Ala Phe Met Leu Pro Val Ala Thr Pro Pro Asn Ala             500 505 510 Ile Val Phe Thr Tyr Gly His Leu Lys Val Ala Asp Met Val Lys Thr         515 520 525 Gly Val Ile Met Asn Ile Ile Gly Val Phe Cys Val Phe Leu Ala Val     530 535 540 Asn Thr Trp Gly Arg Ala Ile Phe Asp Leu Asp His Phe Pro Asp Trp 545 550 555 560 Ala Asn Val Thr His Ile Glu Thr                 565 <210> 2 <211> 1704 <212> DNA <213> human hepatocellular carcinoma dirived cell HepG2 <400> 2 atggcctcgg cgctgagcta tgtctccaag ttcaagtcct tcgtgatctt gttcgtcacc 60 ccgctcctgc tgctgccact cgtcattctg atgcccgcca agtttgtcag gtgtgcctac 120 gtcatcatcc tcatggccat ttactggtgc acagaagtca tccctctggc tgtcacctct 180 ctcatgcctg tcttgctttt cccactcttc cagattctgg actccaggca ggtgtgtgtc 240 cagtacatga aggacaccaa catgctgttc ctgggcggcc tcatcgtggc cgtggctgtg 300 gagcgctgga acctgcacaa gaggatcgcc ctgcgcacgc tcctctgggt gggggccaag 360 cctgcacggc tgatgctggg cttcatgggc gtcacagccc tcctgtccat gtggatcagt 420 aacacggcaa ccacggccat gatggtgccc atcgtggagg ccatattgca gcagatggaa 480 gccacaagcg cagccaccga ggccggcctg gagctggtgg acaagggcaa ggccaaggag 540 ctgccaggga gtcaagtgat ttttgaaggc cccactctgg ggcagcagga agaccaagag 600 cggaagaggt tgtgtaaggc catgaccctg tgcatctgct acgcggccag catcgggggc 660 accgccaccc tgaccgggac gggacccaac gtggtgctcc tgggccagat gaacgagttg 720 tttcctgaca gcaaggacct cgtgaacttt gcttcctggt ttgcatttgc ctttcccaac 780 atgctggtga tgctgctgtt cgcccggctg tggctccagt ttgtttacat gagattcaat 840 tttaaaaagt cctggggctg cgggctagag agcaagaaaa acgagaaggc tgccctcaag 900 gtgctgcagg aggagtaccg gaagctgggg cccttgtcct tcgcggagat caacgtgctg 960 atctgcttct tcctgctggt catcctgtgg ttctcccgag accctggctt catgcccggc 1020 tggctgactg ttgcctgggt ggagggtgag acaaagtatg tctccgatgc cactgtggcc 1080 atctttgtgg ctaccctgct attcattgtg ccttcacaga agcccaggtt taacttccgc 1140 agccagactg aggaagaaag gaaaactcca ttttatcccc ctcccctgct ggattggaag 1200 gtaacccagg agaaagtgcc ctggggcatc gtgctgctac tagggggcgg atttgctctg 1260 gctaaaggat ccgaggcctc ggggctgtcc gtgtggatgg ggaagcagat ggagcccttg 1320 cacgcagtgc ccccggcagc catcaccttg atcttgtcct tgctcgttgc cgtgttcact 1380 gagtgcacaa gcaacgtggc caccaccacc ttgttcctgc ccatctttgc ctccatgtct 1440 cgctccatcg gcctcaatcc gctgtacatc atgctgccct gtaccctgag tgcctccttt 1500 gccttcatgt tgcctgtggc cacccctcca aatgccatcg tgttcaccta tgggcacctc 1560 aaggttgctg acatggtgaa aacaggagtc ataatgaaca taattggagt cttctgtgtg 1620 tttttggctg tcaacacctg gggacgggcc atatttgact tggatcattt ccctgactgg 1680 gctaatgtga cacatattga gact 1704 <210> 3 <211> 3270 <212> DNA <213> human hepatocellular carcinoma dirived cell HepG2 <220> <221> CDS <222> (34) .. (1740) <400> 3 actcgtctcg cccgccagtc tccctcccgc gcg atg gcc tcg gcg ctg agc tat 54                                      Met Ala Ser Ala Leu Ser Tyr                                        1 5 gtc tcc aag ttc aag tcc ttc gtg atc ttg ttc gtc acc ccg ctc ctg 102 Val Ser Lys Phe Lys Ser Phe Val Ile Leu Phe Val Thr Pro Leu Leu          10 15 20 ctg ctg cca ctc gtc att ctg atg ccc gcc aag ttt gtc agg tgt gcc 150 Leu Leu Pro Leu Val Ile Leu Met Pro Ala Lys Phe Val Arg Cys Ala      25 30 35 tac gtc atc atc ctc atg gcc att tac tgg tgc aca gaa gtc atc cct 198 Tyr Val Ile Ile Leu Met Ala Ile Tyr Trp Cys Thr Glu Val Ile Pro  40 45 50 55 ctg gct gtc acc tct ctc atg cct gtc ttg ctt ttc cca ctc ttc cag 246 Leu Ala Val Thr Ser Leu Met Pro Val Leu Leu Phe Pro Leu Phe Gln                  60 65 70 att ctg gac tcc agg cag gtg tgt gtc cag tac atg aag gac acc aac 294 Ile Leu Asp Ser Arg Gln Val Cys Val Gln Tyr Met Lys Asp Thr Asn              75 80 85 atg ctg ttc ctg ggc ggc ctc atc gtg gcc gtg gct gtg gag cgc tgg 342 Met Leu Phe Leu Gly Gly Leu Ile Val Ala Val Ala Val Glu Arg Trp          90 95 100 aac ctg cac aag agg atc gcc ctg cgc acg ctc ctc tgg gtg ggg gcc 390 Asn Leu His Lys Arg Ile Ala Leu Arg Thr Leu Leu Trp Val Gly Ala     105 110 115 aag cct gca cgg ctg atg ctg ggc ttc atg ggc gtc aca gcc ctc ctg 438 Lys Pro Ala Arg Leu Met Leu Gly Phe Met Gly Val Thr Ala Leu Leu 120 125 130 135 tcc atg tgg atc agt aac acg gca acc acg gcc atg atg gtg ccc atc 486 Ser Met Trp Ile Ser Asn Thr Ala Thr Thr Ala Met Met Val Pro Ile                 140 145 150 gtg gag gcc ata ttg cag cag atg gaa gcc aca agc gca gcc acc gag 534 Val Glu Ala Ile Leu Gln Gln Met Glu Ala Thr Ser Ala Ala Thr Glu             155 160 165 gcc ggc ctg gag ctg gtg gac aag ggc aag gcc aag gag ctg cca ggg 582 Ala Gly Leu Glu Leu Val Asp Lys Gly Lys Ala Lys Glu Leu Pro Gly         170 175 180 agt caa gtg att ttt gaa ggc ccc act ctg ggg cag cag gaa gac caa 630 Ser Gln Val Ile Phe Glu Gly Pro Thr Leu Gly Gln Gln Glu Asp Gln     185 190 195 gag cgg aag agg ttg tgt aag gcc atg acc ctg tgc atc tgc tac gcg 678 Glu Arg Lys Arg Leu Cys Lys Ala Met Thr Leu Cys Ile Cys Tyr Ala 200 205 210 215 gcc agc atc ggg ggc acc gcc acc ctg acc ggg acg gga ccc aac gtg 726 Ala Ser Ile Gly Gly Thr Ala Thr Leu Thr Gly Thr Gly Pro Asn Val                 220 225 230 gtg ctc ctg ggc cag atg aac gag ttg ttt cct gac agc aag gac ctc 774 Val Leu Leu Gly Gln Met Asn Glu Leu Phe Pro Asp Ser Lys Asp Leu             235 240 245 gtg aac ttt gct tcc tgg ttt gca ttt gcc ttt ccc aac atg ctg gtg 822 Val Asn Phe Ala Ser Trp Phe Ala Phe Ala Phe Pro Asn Met Leu Val         250 255 260 atg ctg ctg ttc gcc cgg ctg tgg ctc cag ttt gtt tac atg aga ttc 870 Met Leu Leu Phe Ala Arg Leu Trp Leu Gln Phe Val Tyr Met Arg Phe     265 270 275 aat ttt aaa aag tcc tgg ggc tgc ggg cta gag agc aag aaa aac gag 918 Asn Phe Lys Lys Ser Trp Gly Cys Gly Leu Glu Ser Lys Lys Asn Glu 280 285 290 295 aag gct gcc ctc aag gtg ctg cag gag gag tac cgg aag ctg ggg ccc 966 Lys Ala Ala Leu Lys Val Leu Gln Glu Glu Tyr Arg Lys Leu Gly Pro                 300 305 310 ttg tcc ttc gcg gag atc aac gtg ctg atc tgc ttc ttc ctg ctg gtc 1014 Leu Ser Phe Ala Glu Ile Asn Val Leu Ile Cys Phe Phe Leu Leu Val             315 320 325 atc ctg tgg ttc tcc cga gac cct ggc ttc atg ccc ggc tgg ctg act 1062 Ile Leu Trp Phe Ser Arg Asp Pro Gly Phe Met Pro Gly Trp Leu Thr         330 335 340 gtt gcc tgg gtg gag ggt gag aca aag tat gtc tcc gat gcc act gtg 1110 Val Ala Trp Val Glu Gly Glu Thr Lys Tyr Val Ser Asp Ala Thr Val     345 350 355 gcc atc ttt gtg gct acc ctg cta ttc att gtg cct tca cag aag ccc 1158 Ala Ile Phe Val Ala Thr Leu Leu Phe Ile Val Pro Ser Gln Lys Pro 360 365 370 375 agg ttt aac ttc cgc agc cag act gag gaa gaa agg aaa act cca ttt 1206 Arg Phe Asn Phe Arg Ser Gln Thr Glu Glu Glu Arg Lys Thr Pro Phe                 380 385 390 tat ccc cct ccc ctg ctg gat tgg aag gta acc cag gag aaa gtg ccc 1254 Tyr Pro Pro Pro Leu Leu Asp Trp Lys Val Thr Gln Glu Lys Val Pro             395 400 405 tgg ggc atc gtg ctg cta cta ggg ggc gga ttt gct ctg gct aaa gga 1302 Trp Gly Ile Val Leu Leu Leu Gly Gly Gly Phe Ala Leu Ala Lys Gly         410 415 420 tcc gag gcc tcg ggg ctg tcc gtg tgg atg ggg aag cag atg gag ccc 1350 Ser Glu Ala Ser Gly Leu Ser Val Trp Met Gly Lys Gln Met Glu Pro     425 430 435 ttg cac gca gtg ccc ccg gca gcc atc acc ttg atc ttg tcc ttg ctc 1398 Leu His Ala Val Pro Pro Ala Ala Ile Thr Leu Ile Leu Ser Leu Leu 440 445 450 455 gtt gcc gtg ttc act gag tgc aca agc aac gtg gcc acc acc acc ttg 1446 Val Ala Val Phe Thr Glu Cys Thr Ser Asn Val Ala Thr Thr Thr Leu                 460 465 470 ttc ctg ccc atc ttt gcc tcc atg tct cgc tcc atc ggc ctc aat ccg 1494 Phe Leu Pro Ile Phe Ala Ser Met Ser Arg Ser Ile Gly Leu Asn Pro             475 480 485 ctg tac atc atg ctg ccc tgt acc ctg agt gcc tcc ttt gcc ttc atg 1542 Leu Tyr Ile Met Leu Pro Cys Thr Leu Ser Ala Ser Phe Ala Phe Met         490 495 500 ttg cct gtg gcc acc cct cca aat gcc atc gtg ttc acc tat ggg cac 1590 Leu Pro Val Ala Thr Pro Pro Asn Ala Ile Val Phe Thr Tyr Gly His     505 510 515 ctc aag gtt gct gac atg gtg aaa aca gga gtc ata atg aac ata att 1638 Leu Lys Val Ala Asp Met Val Lys Thr Gly Val Ile Met Asn Ile Ile 520 525 530 535 gga gtc ttc tgt gtg ttt ttg gct gtc aac acc tgg gga cgg gcc ata 1686 Gly Val Phe Cys Val Phe Leu Ala Val Asn Thr Trp Gly Arg Ala Ile                 540 545 550 ttt gac ttg gat cat ttc cct gac tgg gct aat gtg aca cat att gag 1734 Phe Asp Leu Asp His Phe Pro Asp Trp Ala Asn Val Thr His Ile Glu             555 560 565 act tag gaagagccac aagaccacac acacagccct taccctcctc aggactaccg 1790 Thr aaccttctgg cacaccttgt acagagtttt ggggttcaca ccccaaaatg acccaacgat 1850 gtccacacac caccaaaacc cagccaatgg gccacctctt cctccaagcc cagatgcaga 1910 gatggacatg ggcagctgga gggtaggctc agaaatgaag ggaacccctc agtgggctgc 1970 tggacccatc tttcccaagc cttgccatta tctctgtgag ggaggccagg tagccgaggg 2030 atcaggatgc aggctgctgt acccgctctg cctcaagcat cccccacaca gggctctggt 2090 tttcactcgc ttcgtcctag atagtttaaa tgggaatcag atcccctggt tgagagctaa 2150 gacaaccacc taccagtgcc catgtccctt ccagctcacc ttgagcagcc tcagatcatc 2210 tctgtcactc tggaagggac accccagcca gggacggaat gcctggtctt gagcaacctc 2270 ccactgctgg agtgcgagtg ggaatcagag cctcctgaag cctctgggaa ctcctcctgt 2330 ggccaccacc aaaggatgag gaatctgagt tgccaacttc aggacgacac ctggcttgcc 2390 acccacagtg caccacaggc caacctacgc ccttcatcac ttggttctgt tttaatcgac 2450 tggccccctg tcccacctct ccagtgagcc tccttcaact ccttggtccc ctgttgtctg 2510 ggtcaacatt tgccgagacg ccttggctgg caccctctgg ggtccccctt ttctcccagg 2570 caggtcatct tttctgggag atgcttcccc tgccatcccc aaatagctag gatcacactc 2630 caagtatggg cagtgatggc gctctggggg ccacagtggg ctatctaggc cctccctcac 2690 ctgaggccca gagtggacac agctgttaat ttccactggc tatgccactt cagagtcttt 2750 catgccagcg tttgagctcc tctgggtaaa atcttccctt tgttgactgg ccttcacagc 2810 catggctggt gacaacagag gatcgttgag attgagcagc gcttggtgat ctctcagcaa 2870 acaacccctg cccgtgggcc aatctacttg aagttactcg gacaaagacc ccaaagtggg 2930 gcaacaactc cagagaggct gtgggaatct tcagaagccc ccctgtaaga gacagacatg 2990 agagacaagc atcttctttc ccccgcaagt ccattttatt tccttcttgt gctgctctgg 3050 aagagaggca gtagcaaaga gatgagctcc tggatggcat tttccagggc aggagaaagt 3110 atgagagcct caggaaaccc catcaaggac cgagtatgtg tctggttcct tgggtgggac 3170 gattcctgac cacactgtcc agctcttgct ctcattaaat gctctgtctc ccgcggaaaa 3230 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3270 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: motif sequence <400> 4 Ala Ser Phe Ala Phe Met Leu Pro Val Ala Thr Pro Pro Asn Ala Ile   1 5 10 15 Val <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence HepNaDC F1 <400> 5 taccctgagt gcctcctttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence HepNaDC R1 <400> 6 ttagcccagt cagggaaatg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence HepNaDC F2 <400> 7 gvtggtcatc ctgtggttct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence HepNaDC R2 <400> 8 ggttaccttc caatccagca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence NADC1 F1 <400> 9 ggaagacggt gaaccagaag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence NADC1 R1 <400> 10 gttgctagtg cactcggtca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence NADC3 F1 <400> 11 ctggacactt gctggtcaaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer sequence NADC3 R1 <400> 12 gagggtccga aatgtgtcat 20

[Brief description of drawings]

FIG. 1 is an alignment comparison diagram of amino acid sequences encoded by the HepNaDC gene of the present invention, two NADCs family genes (NADC1 and NADC3), and the dIndy gene of Drosophila, respectively.

FIG. 2 is a schematic diagram showing the genomic structure of the ORF of the HepNaDC gene of the present invention.

FIG. 3 is a drawing showing the expression pattern of HepNaDC in each tissue obtained by performing RT-PCR analysis according to Example 2 using various human tissues.

FIG. 4 is a graph showing the results of [ ¹⁴ C] succinic acid uptake activity in COS1 cells expressing HepNaDC according to Example 8.

FIG. 5 is a graph showing the results of [ ¹⁴ C] citrate uptake activity in HepNaDC-expressing COS1 cells according to Example 8.

FIG. 6 is a graph showing the results of determining [ ¹⁴ C] α-ketoglutarate uptake activity in HepNaDC-expressing COS1 cells according to Example 8.

FIG. 7 is a graph showing the results of [ ¹⁴ C] citrate uptake activity in HepNaDC-expressing CHO cells according to Example 9.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 A61K 31/7088 4H045 5/10 35/76 // A61K 31/7088 45/00 35/76 48/00 45/00 A61P 1/16 48/00 3/04 A61P 1/16 3/06 3/04 3/10 3/06 39/06 3/10 43/00 39/06 111 43/00 C12N 15 / 00 ZNAA 111 5/00 A (72) Inventor Sumio Sugano 4-8-13 Minamiogikubo, Suginami-ku, Tokyo (72) Inventor Yutaka Obuchi 7-2-2, Aoki, Otsu, Shiga Prefecture Yagi Heights 201 F term ( Reference) 4B024 AA01 AA11 BA45 BA61 CA01 CA04 CA06 CA11 DA02 DA03 DA20 EA04 FA10 GA11 GA13 HA17 4B065 AA90X AA93X AA93Y AC14 BA02 BA05 BA25 CA24 CA44 CA46 4C084 AA02 AA03 A53 A59 MA35 MA23 MA13 CA53 MA41 MA43 MA52 MA56 MA57 MA59 MA60 MA63 MA6 6 NA14 ZA702 ZA752 ZC332 ZC352 ZC412 ZC802 4C086 AA01 AA02 AA03 EA16 MA13 MA17 MA22 MA23 MA28 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA56 MA57 MA59 MA60 MA63 MA66 NA14 ZA70 ZA75 ZC33 ZC35 ZC41 ZC80 4C087 AA01 AA02 BC83 CA12 MA13 MA17 MA22 MA23 MA28 MA31 MA35 MA36 MA37 MA41 MA43 MA52 MA56 MA57 MA59 MA60 MA63 MA66 NA14 ZA70 ZA75 ZC33 ZC35 ZC41 ZC80 4H045 AA10 AA11 AA30 BA10 CA41 DA75 DA86 EA20 EA27 EA50 FA72 FA74 HA06

Claims (18)

[Claims] A DNA molecule comprising the following polynucleotide (a), (b) or (c): (a) A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or its complementary strand (b) At least 95% homology to the polynucleotide of (a) above or its complementary strand Polynucleotide (c) Amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having sodium-di- or -tricarboxylic acid cotransporter activity. Polynucleotide encoding peptide 2. The DNA molecule according to claim 1, which is a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a complementary strand thereof. A DNA molecule comprising the following polynucleotide (a) or (b): (a) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 2 or its complementary strand (b) hybridizing with the polynucleotide of (a) above or its complementary strand under stringent conditions, and sodium-di or -Polynucleotide encoding a polypeptide having tricarboxylic acid cotransporter activity 4. The DNA molecule according to claim 3, which is a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 2 or a complementary strand thereof. 5. The DNA molecule according to claim 1, which is a polynucleotide encoding a polypeptide having citrate cotransporter activity. 6. A DNA molecule encoding a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids are deleted, substituted or added, and having a life cycle extending action. 7. A DN, which is a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
A molecule. 8. An expression product of the DNA molecule according to any one of claims 1-7. 9. A recombinant expression vector into which the DNA molecule according to any one of claims 1 to 7 is inserted. 10. A host cell or a transformant having the recombinant expression vector according to claim 9. 11. An antibody capable of binding to the expression product of the DNA molecule according to claim 8 or a partial fragment thereof. 12. A medicine containing the antibody according to claim 11 as an active ingredient. 13. A polypeptide comprising the following amino acid sequence (a), (b) or (c). (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (b) a polynucleotide having at least 95% homology to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (c) the SEQ ID NO: : A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by 1, and having sodium-di- or -tricarboxylic acid cotransporter activity 14. The polypeptide according to claim 13, which is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. 15. The polypeptide according to claim 13 or 14, which is a polypeptide having citrate cotransporter activity. 16. An antibody having a binding property to the polypeptide according to any one of claims 13 to 15 or a partial fragment thereof. 17. A medicine containing the antibody according to claim 16 as an active ingredient. 18. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids are deleted, substituted or added, and having a life cycle extending action.