JP2000236885A - New protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein comprising a protein having a specific amino acid sequence, having Na+-HCO3- cotransporter activity and useful for developing a prophylactic and therapeutic drug, a diagnostic drug, or the like, for diseases such as myocardial infarction, unstable angina, hypertension, cerebral infarction, cerebral hemorrhage. SOLUTION: This protein has an amino acid sequence, which is same or practically same as the amino acid sequence represented by the formula, and Na+-HCO3- cotransporter activity. The protein is useful for developing a prophylactic drug and a therapeutic drug, a diagnostic drug, or the like, for diseases such as myocardial infarction, unstable angina, cardiac dysrhythmia, cardiac failure, hypertension, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, or the like, by screening a compound promoting or inhibiting the Na+-HCO3- cotransporter activity. The protein is obtained by screening a plasmid cDNA library originated from a human heart using a synthetic oligonucleotide comprising a partial sequence as a probe, incorporating the obtained gene into a vector, transducing the vector into a host cell such as CHO-K1 cell and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a protein (preferably Na ⁺ -HCO) having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
A protein having ₃ ^- cotransporter activity: hereinafter sometimes abbreviated as the protein of the present invention) or a salt thereof, a DNA encoding the same, and the like.

[0002]

2. Description of the Related Art Organisms maintain and regulate the ionic environment in cells so that cell functions such as proliferation, differentiation, contraction, and secretion can be maximized as necessary. In order to adjust the ionic environment to optimal conditions for survival, the cell plasma membrane contains Na ⁺ , C
_{^{a 2+, H +, HCO 3}} - transporter proteins that transport ions, such as there are a plurality. Na ⁺ belonging to such transporters
-HCO ₃ ^- cotransporter is driven by the concentration gradient of Na ⁺ inside and outside the cell membrane, and takes in one Na ⁺ into the cell together with one or more HCO ₃ ^- ions [translated by Keiko Nakamura et al., Molecular Biology of Cell Gaku 3rd edition education company]. Since the structure of proteins such as enzymes that function in cells is greatly affected by pH, the optimal pH for protein to function is determined. Therefore, maintenance and regulation of intracellular pH is extremely important for cells to maintain homeostasis of cell functions. Na ⁺ -HCO ₃ ^- cotransporter is present in the cell membrane, Na ⁺ -HCO ₃ ^- cotransporter HCO taken into the cell by ₃ ^- since neutralizes H ⁺ in the cytosol, Na
^{Like the +} -H ⁺ exchanger, it plays an important role in regulating intracellular pH [translated by Keiko Nakamura et al., Molecular Biology of Cells, 3rd Edition Educational Company]. Furthermore, Na ⁺ -HCO ₃ ^- cotransporter is HCO ₃ from there to the proximal convoluted tubules of the kidney, glomerular filtrate with such Na ⁺ -H ⁺ exchanger ^- working reabsorption [The Journal
of Biological Chemistry, 272, 19111 (1997)]. Na ⁺ −
HCO ₃ ^- cotransporter is during normal play an important role in the homeostasis of cells, at the time of ischemia is related to the occurrence of cytotoxicity. Ischemia causes intracellular acidosis,
Na ⁺ -HCO ₃ ^- cotransporters and Na ⁺ -H ⁺ exchangers function to regulate H lowering. As a result, intracellular Na ⁺ concentration increases, N
Ca2 ⁺ overload into cells occurs via the a ⁺ -Ca2 ⁺ exchanger. It is thought that this overloaded Ca ²⁺ is one of the factors that cause cell damage as a result [Basic Research in Cardiology, 91, 191 (1996)].
. The kidney-derived Na ⁺ -HCO ₃ ^- cotransporter gene was used for salamanders (amphibian) [Nature, 387, 409 (1997)], rats [American Journal of Physiology, 274, F425 (19)
98)], human [The Journal of Biological Chemistry, 2
72, 19111, (1997)]. However, the Na ⁺ -HCO ₃ ^- cotransporter gene that is mainly expressed in organs other than the kidney such as the heart and brain has not been known so far.

[0003]

SUMMARY OF THE INVENTION The present invention relates to SEQ ID NO:
A protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by
⁺ -HCO ₃ ^- proteins with cotransporter activity), its partial peptide or a salt thereof, wherein SEQ ID NO: protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by (preferably, Na ⁺ -HCO ₃ ^- cotransporter activity proteins with) or DNA encoding the partial peptide, a recombinant vector containing said DNA, transformants carrying the recombinant vector, the SEQ ID NO: represented by 1 For producing a protein (preferably a protein having Na ⁺ -HCO ₃ ^- cotransporter activity) or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 proteins having the same or substantially the same amino acid sequence and sequence (preferably, Na ⁺ -HCO ₃ ^- protein with co-transporter activity ), Antibody against a partial peptide thereof or a salt thereof, the SEQ ID NO: protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by (preferably, Na ⁺ -HCO ₃ ^- cotransporter activity Na ⁺ -HCO ₃ proteins) having ^- cotransporter activity change to method of screening a compound or its salt comprising a compound or a salt thereof and the compound or its salt obtained using the screening method medicament The purpose is to provide.

[0004]

Means for Solving the Problems The present inventors have made intensive studies and found that they are specifically expressed in human-derived hearts.
Na ⁺ -HCO ₃ ^- cDNA encoding cotransporter protein isolate, after analyzing total base sequence was successfully expressed in a cell. The present inventors have further studied based on these findings, and as a result, have completed the present invention. That is, the present invention relates to (1) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof;
The protein or salt thereof according to the above (1), which has Na ⁺ -HCO ₃ ^- cotransporter activity; (3) the partial peptide of the protein according to the above (1), its amide or its ester or its salt; (1) a DNA containing a DNA having a nucleotide sequence encoding the protein of (1), (5) a DNA of (4) having a nucleotide sequence represented by SEQ ID NO: 2, (6) a DNA of (5), A recombinant vector containing DNA, (7) a transformant transformed with the recombinant vector according to (6),
(8) Production of the protein or the salt thereof according to the above (1), wherein the transformant according to the above (7) is cultured, the protein according to the above (1) is produced, accumulated, and collected. (9) an antibody against the protein or salt thereof according to (1) or the partial peptide or amide or ester or salt thereof according to (3); (10) the protein or salt thereof according to (1) or The partial peptide according to (3) or an amide or ester thereof or a salt thereof, wherein the protein or a salt thereof according to the above (1) or the partial peptide or an amide or an ester thereof according to the above (3) is used. Na of the salt
⁺ -HCO ₃ ^- co test compound to transporter activity to promote or inhibit (11) above (1) protein or a salt thereof or the above described (3) a partial peptide, or its amide or ester, according Or promoting the Na ⁺ -HCO ₃ ^- cotransporter activity of the protein according to the above (1) or a salt thereof, or the partial peptide according to the above (3) or an amide or an ester thereof or a salt thereof comprising the salt thereof; A protein or a salt thereof according to the above (1), which can be obtained by using the screening kit for a compound to be inhibited or a salt thereof, (12) a screening method according to the above (10) or a screening kit according to the above (11). The partial peptide according to claim 3, or an amide or an ester thereof. Or its salt Na ⁺ -H
CO ₃ ^- cotransporter active compound or its salt that promotes or inhibits, can be obtained using (13) (10) the screening method or the (11) the screening kit according to, wherein said (1), wherein Or a partial peptide according to the above (3), an amide or an ester thereof, or a Na ^{+ of the} salt thereof.
-HCO ₃ ^- co transporter activity promoting or inhibiting compound or pharmaceutical agent comprising a salt thereof, (14) myocardial infarction and dysfunction accompanying therewith, unstable angina, restenosis after PTCA, arrhythmia, heart failure A medicament according to the above (13), which is a preventive / therapeutic agent for hypertension and associated tissue disorders, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and associated brain disorders;
(15) Myocardial infarction and its associated dysfunction, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and its associated tissue disorders, characterized by containing the antibody of (9) above, A diagnostic agent for infarction, cerebral hemorrhage, subarachnoid hemorrhage and associated brain damage, and (16) the above (1), which comprises using the antibody of the above (9).
A protein or a salt thereof, or a partial peptide or an amide or an ester or a salt thereof according to the above (3).

More specifically, (17) the protein
1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 9, more preferably several (1 or 2) in the amino acid sequence represented by SEQ ID NO: 1.
Amino acid sequence), the amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably about 1 to 30, more preferably 1 to 1)
About 0, more preferably several (1 or 2) amino acids added, 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1 More preferably, about 1 to 10,
More preferably several (1 or 2)) amino acids is a protein containing the amino acid sequence, or amino acid sequence comprising a combination thereof are substituted with other amino acids above (1), wherein the Na ⁺ -HCO ₃ ^- A cotransporter protein or a salt thereof, (18) a case where the test compound is brought into contact with the protein or the salt thereof described in the above (1) or the partial peptide or the amide or the ester or the salt thereof described in the above (3). A screening method according to the above (10), characterized in that a comparison is made with a case where the test compound is not brought into contact with a cell containing the protein according to the above (1); and performing a comparison of Na ⁺ -HCO ₃ ^- a method of screening a compound or its salt that alters the cotransporter activity, (20 By culturing the transformant according to (7) above, a comparison was made between the case where the test compound was brought into contact with the protein according to (1), which was expressed on the cell membrane of the transformant and the case where the test compound was not contacted. (1) the protein according Na ⁺ -HCO _3, wherein ^- cotransporter activity change to method of screening a compound or its salt obtained in (21) above (18) to (20) above, wherein the screening method is, the (1) Na ⁺ -HCO ₃ protein described ^- can be obtained by co-transporter activity is changed to a compound or a salt thereof, and (22) above (18) to (20) above, wherein the screening method, Myocardial infarction and dysfunction associated therewith, unstable angina, restenosis after PTCA, comprising a compound or a salt thereof that alters the function of the protein according to the above (1); Seimyaku, heart failure, high blood pressure and it involves tissue injury, cerebral infarction, cerebral hemorrhage, prevention of such subarachnoid hemorrhage and cerebral disorder accompanying therewith,
Provide therapeutic agents and the like.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (FIGS. 1 to 3). The protein of the present invention includes, for example, humans and warm-blooded animals (eg, guinea pigs, rats, mice, rabbits,
Pigs, sheep, cows, monkeys, etc.) Fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovium Cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), or any tissue in which these cells are present , For example, the brain, each part of the brain (eg,
Olfactory bulb, abdominal nucleus, basal cerebral sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, nigra , Spinal cord, pituitary, stomach,
Pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, It may be a protein derived from the prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle and the like (particularly, brain and various parts of the brain), or may be a synthetic protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 90% or more, preferably about 95% or more, and more preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 1. About 98% or more,
Most preferably, an amino acid sequence having about 99% or more homology can be mentioned. Examples of the protein of the present invention having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 is preferred. "Substantially the same" activity of a protein with, for example, co-transporter activity (Na ⁺ -HCO ₃ ^- such cotransporter activity), such as physiological properties, means substantially the same thing. Amino acid substitutions, deletions, additions or insertions often do not significantly alter the physiological or chemical properties of the polypeptide,
In such a case, the substituted, deleted, added or inserted protein will be substantially identical to the protein without such substituted, deleted, added or inserted. Substantially identical substitutions of amino acids in the amino acid sequence may be selected, for example, from other amino acids of the class to which the amino acid belongs. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine and the like. Glycine, serine, and polar (neutral) amino acids
Threonine, cysteine, tyrosine, asparagine, glutamine and the like. Examples of (basic) amino acids having a positive charge include arginine, lysine, histidine and the like. As negatively charged (acidic) amino acids,
Aspartic acid, glutamic acid and the like. The term “substantially the same” means that their activities are the same in nature (physiologically or pharmacologically).
Therefore, although their activities are preferably equivalent,
Quantitative factors such as the degree of these activities and the molecular weight of the protein may differ. The co-transporter activity can be measured according to a method known per se, for example, according to a screening method described later.

The protein of the present invention includes an amino acid sequence in which one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 have been deleted,
An amino acid sequence in which one or more amino acids are added to the amino acid sequence represented by 1, an amino acid sequence in which one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted with another amino acid, or A protein containing an amino acid sequence obtained by combining them is also used. Further, in the protein of the present invention, the amino group of the N-terminal methionine residue may be a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl). ), G formed by cutting the N-end side in vivo
ln is pyroglutamine-oxidized, and a substituent on the side chain of an amino acid in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) has an appropriate protecting group (for example, formyl group). Groups, C _1-6 acyl groups such as C _2-6 alkanoyl groups such as acetyl, etc., and complex proteins such as so-called glycoproteins to which sugar chains are bound. The partial peptide of the protein of the present invention (hereinafter sometimes abbreviated as a partial peptide) may be any peptide as long as it is a partial peptide of the protein of the present invention described above. Among them, a part exposed outside the cell membrane is used. In particular,
As a partial peptide of the protein having the amino acid sequence represented by SEQ ID NO: 1, in the hydrophobic plot analysis shown in FIG. 4, the extracellular region (hydrophilic (Hydrophili)
c) a peptide containing a moiety that was analyzed to be a site). Further, a peptide partially containing a hydrophobic (hydrophobic) site can also be used. A peptide containing individual domains separately may be used, but a peptide containing a plurality of domains simultaneously may be used. Further, the partial peptide of the present invention has one or more amino acids in the above amino acid sequence deleted, or one or more amino acids added to the amino acid sequence, or One or more amino acids may be replaced with another amino acid. In the protein or partial peptide of the present invention, the C-terminus usually has a carboxyl group (-CO
OH) or carboxylate (—COO ⁻ ), but the C-terminal may be amide (—CONH ₂ ) or ester (—COOR) as in the protein of the present invention described above. As R of the ester, for example, methyl,
C _1-6 alkyl groups such as ethyl, n-propyl, isopropyl or n-butyl, C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, phenyl, α-
C _6-12 aryl groups such as naphthyl, phenyl-C _1-2 alkyl such as benzyl, phenethyl, benzhydryl,
Or α-naphthyl-C such as α-naphthylmethyl
_In addition to C _7-14 aralkyl groups such as _1-2 alkyl and the like, pivaloyloxymethyl groups widely used as oral esters and the like can be mentioned. Furthermore, the partial peptide of the present invention includes
Also included are those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide. Since the partial peptide of the present invention can be used as an antigen for producing an antibody, it is not always necessary to have Na ⁺ -HCO ₃ ^- cotransporter activity.

The salt of the protein of the present invention or its partial peptide is preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). The protein of the present invention or a salt thereof, a partial peptide of the protein of the present invention, an amide thereof, an ester thereof, or a salt thereof may be produced from the human or mammalian cells or tissues described above by a purification method known per se,
It can also be produced by culturing a transformant containing a DNA encoding the protein of the present invention described below. Further, it can also be produced by the protein synthesis method described below or according to it. When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, perform extraction with an acid or the like,
The extract can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography. The partial peptide of the protein of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is,
The target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Examples of the known condensation method and elimination of the protecting group include the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis, Hirokawa Shoten The partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained as a salt, a known method or analogous method It can be converted into a free form or another salt by a method.

The DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention. Genomic DNA, genomic DNA library,
Any of the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from the cells and tissues described above,
Reverse Transcriptase Polymerase Chain React directly using prepared total RNA or mRNA fraction
It can also be amplified by ion (hereinafter abbreviated as RT-PCR method). Specifically, the DNA encoding the protein of the present invention includes, for example, a DNA containing the base sequence represented by SEQ ID NO: 2, or a DNA containing SEQ ID NO: 2
Has a hybridizing nucleotide sequence in the base sequence under highly stringent conditions represented, protein activity substantially equivalent to the present invention (eg, Na ⁺ -HCO ₃ ^- cotransporter activity, etc.) with a Any DNA may be used as long as it encodes a protein. Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 include, for example, about 90% or more, preferably about 95% or more, and more preferably 98% with the nucleotide sequence represented by SEQ ID NO: 2.
As described above, DNA containing a nucleotide sequence having a homology of 99% or more is most preferably used. Hybridization can be performed by a method known per se or a method analogous thereto, for example, molecular cloning (Molecular cloning).
Cloning) 2nd (J. Sambrook et al., Cold Spring Harb
or Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions. High stringency conditions include, for example, a sodium concentration of about 19-40.
mM, preferably about 19-20 mM, and a temperature of about 50-
The condition is 70 ° C, preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA having the base sequence represented by SEQ ID NO: 2 or the like is used.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the mRNA fraction prepared from the cells and tissues described above,
Transcriptase Polymerase Chain Reaction (hereinafter, RT
-PCR method). Specifically, D encoding the partial peptide of the present invention
As the NA, for example, a DNA having a partial base sequence of a DNA having the base sequence represented by SEQ ID NO: 2 or a base sequence hybridizing under high stringent conditions to the base sequence represented by SEQ ID NO: 2 Has substantially the same activity as the protein of the present invention (eg, Na
⁺ -HCO ₃ ^- such as a DNA having a partial nucleotide sequence of the DNA encoding a protein having a co-transporter activity, etc.) is used. Examples of the DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 2 include, for example, about 90% or more, preferably about 95% with the base sequence represented by SEQ ID NO: 2.
Or more, more preferably 98% or more, most preferably 99% or more.
For example, DNA containing a base sequence having a homology of at least% is used. The same hybridization method and high stringency conditions as described above are used. Means for cloning a DNA that completely encodes the protein of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as the protein of the present invention) include:
(1) Amplification by PCR using a synthetic DNA primer having a partial base sequence of the protein of the present invention, or (2) coding of a DNA incorporated into an appropriate vector into a part or the whole region of the protein of the present invention. Selection by hybridization with a labeled DNA fragment or a synthesized DNA. Hybridization methods include, for example, molecular cloning (Molecular Cloning) 2nd
(J. Sambrook et al., Cold SpringHarbor Lab. Pres
s, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
Conversion (deletion / addition / substitution) of a DNA base sequence can be performed by a known kit, for example, Mutan ^™ -G (Takara Shuzo Co., Ltd.), Mutan
^{Using TM-} K (Takara Shuzo Co., Ltd.) or the like, it can be carried out according to a method known per se, such as the gapped duplex method or the Kunkel method, or a method analogous thereto. The cloned DNA encoding the protein of the present invention can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA
Has ATG at the 5 'end as a translation initiation codon and TA at the 3' end as a translation stop codon.
It may have A, TGA or TAG. These translation initiation codons and translation stop codons are appropriately synthesized DNA
It can also be added using an adapter. The expression vector for the protein of the present invention is, for example, (a) cutting out a target DNA fragment from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC12) is used.
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as phage λ, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as hosts, the SRα promoter, SV40 promoter, L
TR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like can be mentioned. Among these, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter,
When the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter, etc., and when the host is a yeast, a PHO5 promoter, a PGK promoter, a GA
P promoters and ADH promoters are preferred.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to the above, the expression vector may optionally include an enhancer, a splicing signal, a polyA addition signal, a selection marker,
Those containing SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) or the like can be used. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r),
Neomycin resistance gene (hereinafter, sometimes abbreviated as Neo, G418 resistance) and the like. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, and the like. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used. A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
Escherichia coli K12 DH
1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160
(1968)], JM103 [Nucleic Acids
Research, (Nucleic Acids Research), 9, 309
(1981)], JA221 [Journal of Molecular Biolog
y)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus bacteria include, for example, Bacillus
Bacillus subtilis MI114 [Gene,
24, 255 (1983)], 207-21 [Journal of Biochemis
try), 95, 87 (1984)].
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used. As insect cells, for example, the virus is AcNPV.
In the case of larvae of the night roth moth (Spodoptera fru
giperda cell (Sf cell), MG1 cells derived from the midgut of Trichoplusia ni, High Five derived from eggs of Trichoplusia ni
^TM cells, cells derived from Mamestra brassicae or Estigmen
Cells derived from a acrea are used. Virus is Bm
In the case of NPV, a silkworm-derived cell line (Bombyx mori N; B
mN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL 1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 21).
3-217, (1977)). As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells),
Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, HEK293 cells,
C127 cells, BALB3T3 cells, Sp-2 cells and the like are used. Among these, CHO cells, CHO
(Dhfr ⁻ ) cells, HEK293 cells and the like are preferred.

To transform Escherichia, for example, Procagings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69 , 21
10 (1972) and Gene, 17, 107 (1)
982). For transformation of Bacillus sp., For example, Molecular and General Genetics (Mole
cular & General Genetics), 168, 111 (19
79) and the like.
To transform yeast, see, for example, Methods in Enzymology, 194.
Vol. 182-187 (1991).
Proc. Natl. Acad. Sc of the National Academy of Sciences of the USA
USA), Vol. 75, 1929 (1978). Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). Examples of a method for introducing an expression vector into animal cells include a calcium phosphate method [Graham, FL and van der Eb, AJ Virology 52, 456-467 (1973)], an electroporation method [Nu
emann, E. et al. Embo Journal (EMBO J.) 1,
841-845 (1982)]. In this way,
A transformant transformed with an expression vector containing a DNA encoding the protein of the present invention can be obtained. In addition, as a method for stably expressing the protein of the present invention using animal cells, there is a method of selecting, by clonal selection, cells in which the expression vector introduced into the animal cells is integrated into the chromosome. Specifically, a transformant is selected using the above-mentioned selection marker as an index. Furthermore, a stable animal cell line having a high expression ability of the protein of the present invention can be obtained by repeatedly performing clonal selection on the animal cells obtained using the selection marker. When the dhfr gene was used as a selection marker, the MTX concentration was gradually increased, and the cells were cultured. By selecting resistant strains, together with the dhfr gene, the DNA encoding the protein of the present invention was amplified in cells. Furthermore, an animal cell line with high expression can be obtained. The above-mentioned transformant is cultured under conditions in which a DNA encoding the protein of the present invention can be expressed, and the protein of the present invention or a salt thereof can be produced by producing and accumulating the protein of the present invention. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation. Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. Medium p
H is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is yeast, for example, Burkholder's minimum medium [Bostian, KL
Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 450
5 (1980)] and an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings of the.
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), 81, 5330 (1984)].
Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added. When culturing a transformant whose host is an insect cell or an insect, the medium used is Grac.
e's Insect Medium (Grace, TCC, Nature (Natur
e), 195, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. Medium pH
Is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and / or agitation are added as necessary. When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 12
2, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI
1640 medium [Journal of the American
Medical Association (The Journal of the A
American Medical Association) 199, 519 (19
67)], 199 medium [Proceeding of the Society for the Biological Medicine]
cine), 73, 1 (1950)]. p
H is preferably about 6-8. Culture is usually about 30 ° C
Perform at １５40 ° C. for about 15-72 hours, adding aeration and agitation as needed. As described above, the protein of the present invention can be expressed in a transformant. To separate and purify the protein of the present invention from the culture, for example,
It can be performed by the following method.

When the protein of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and then subjected to ultrasonication, lysozyme and / or lysozyme. After the cells or cells are destroyed by freeze-thawing or the like, a method of obtaining a crude extract of the protein of the present invention by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . The protein of the present invention contained in the extract thus obtained can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing. When the thus obtained protein of the present invention is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when the protein of the present invention is obtained in a salt, It can be converted to a free form or another salt by a method analogous thereto.
The protein of the present invention produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The thus produced protein of the present invention or a salt thereof can be measured by an enzyme immunoassay or the like using a specific antibody. An antibody against the protein of the present invention or a salt thereof, a partial peptide of the protein of the present invention, an amide thereof, an ester thereof, or a salt thereof (hereinafter, sometimes referred to as the protein of the present invention) is an antibody capable of recognizing the protein of the present invention. If so, any of a polyclonal antibody and a monoclonal antibody may be used. An antibody against the protein of the present invention can be produced by using the protein of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a mammal at a site capable of producing an antibody by administration to itself or together with a carrier and a diluent. . Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats.
Mice and rats are preferably used. When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the contained antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum is measured, for example, by reacting the labeled protein of the present invention described below with the antiserum,
It can be carried out by measuring the activity of the labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 2
56, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, A
Examples include myeloma cells of warm-blooded animals such as Pa, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably,
PEG1000-PEG6000) is added at a concentration of about 10-80%, and about 20-40 ° C., preferably about 30 ° C.
By incubating at -37 ° C for about 1-10 minutes, cell fusion can be carried out efficiently. Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the protein antigen of the present invention is directly or adsorbed together with a carrier, and then added. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A A method of adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein of the present invention labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase. Is raised. The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto.
(Thymidine) in a medium for animal cells or the like. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, 1-10%
Medium containing fetal calf serum (Wako Pure Chemical Industries, Ltd.)
Alternatively, a serum-free medium for hybridoma culture (SFM-10
1, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of monoclonal antibodies can be carried out in the same manner as in the separation and purification of normal polyclonal antibodies. , Electrophoresis, adsorption / desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or an active adsorbent such as protein A or protein G to collect only antibodies and bind Specific Purification Method for Obtaining Antibody by Dissociation].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (the protein antigen of the present invention) itself or a complex thereof with a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting the antibody-containing substance and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. Any one of them may be cross-linked at any ratio, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. in a weight ratio of about 0.1 to 1 hapten.
A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody. A DNA encoding the protein of the present invention or a salt thereof, a partial peptide of the protein of the present invention, an amide thereof, an ester thereof, or a salt thereof (hereinafter, sometimes referred to as the DNA of the present invention) may be obtained by obtaining antibodies and antisera, Construction of expression system of protein of the present invention, Na ⁺ using the expression system
-HCO ₃ ^- cotransporter screening activity measuring construct and drug candidate compounds systems, Na ⁺ -HCO ₃ ^- implementation of drug design based on the three-dimensional structure of the co-transporter, probes or PCR primers in gene diagnosis It can be used as a reagent for preparation, preparation of a transgenic animal, or a drug such as a gene prophylactic / therapeutic agent. In particular, Na ⁺ -HCO ₃ expression system using the protein of the present invention ^- by using a co-transporter activity assay system, human or mammal to a specific Na ⁺ -HCO ₃ ^- changing the cotransporter activity Compounds can be screened, and the compounds can be used as agents for preventing or treating various diseases.

The use of the protein of the present invention, the DNA of the present invention, the cells expressing the protein of the present invention, and the antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below. Will be described. (1) Screening method for compound that alters the activity of the protein of the present invention Using the protein of the present invention or constructing an expression system for the protein of the present invention, and using the expression system, Na ⁺ -HCO ₃ ^- cotransportation by using the body activity measurement system, Na ⁺ -HCO ₃ proteins of the present invention ^- compounds that alter the cotransporter activity (e.g., peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, etc.) or a The salt can be efficiently screened. Such compounds, of the protein of the present invention Na ⁺ -HCO ₃ ^- compound enhances cotransporter activity, or the protein of the present invention Na ⁺
-HCO ₃ ^-, and the like compounds which inhibit cotransporter activity. That is, the present invention provides a function of the protein of the present invention (specifically, Na ⁺ -HCO ₃ ^- providing a method of screening a compound or its salt that alters the cotransporter activity, etc.).
More specifically, the present invention provides a screening method comprising comparing a case where a protein of the present invention is brought into contact with a test compound and a case where a test compound is not brought into contact with the test compound. The function of the protein of the present invention, which is compared with the case where the test compound is brought into contact with the case where the test compound is not brought into contact (specifically, Na ⁺ -HC
O ₃ ^- test protein co-transporter activity) alter test compound to, and the present invention in which a transformant containing the DNA of the present invention expressed on the cell membrane of the transformant by culturing The function of the protein of the present invention, which is characterized by comparing the case where the compound is brought into contact with the case and the case where the compound is not brought into contact (specifically, Na ⁺
-HCO ₃ ^- provides a method of screening a compound or its salt that alters the cotransporter activity, etc.). Before the proteins of the present invention is obtained, for example, Na ⁺ -HCO ₃ ^- when screening compounds that alter cotransporter activity, Na ⁺ -HCO ₃ such first rat ^- cells comprising co-transporter protein, tissue obtaining a candidate compound with (primary screening), then the candidate compound is actually a human Na ⁺ -HCO ₃ ^-
A test (secondary screening) to confirm whether or not the compound alters the activity of the co-transporter was required. Cells, tissues other for transporter proteins also coexist be used as it is, Na ⁺ -HCO ₃ aims ^- it is difficult to actually screen the cotransporter activity altering compound of proteins. However, for example, by using cells expressing the human-derived protein of the present invention, the need for primary screening is eliminated, and Na ⁺ -HCO
Compounds that alter ₃ ^- cotransporter activity can be screened efficiently. The specific description of the screening method of the present invention is as follows. First, the protein of the present invention used in the screening method of the present invention may be any protein as long as it contains the above-described protein of the present invention. Are preferred. However, since it is extremely difficult to obtain human-derived organs in particular, cells expressing human-derived proteins are suitable for screening. The above-mentioned method is used to construct a cell in which the protein of the present invention has been expressed, but it is preferable to carry out the expression of the DNA of the present invention in mammalian cells or insect cells. A complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto.
For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding the protein of the present invention into host animal cells and express them efficiently, the D
Nuclear polyhedrosis virus (N) belonging to the baculovirus using the NA fragment as an insect host
It is preferable to incorporate the promoter into the downstream of the polyhedrin promoter of PV), the SV40-derived promoter, the retrovirus promoter, the metallothionein promoter, the human heat shock promoter, the cytomegalovirus promoter, and the SRα promoter. Cells used for expression may be any cells as long as expression of the protein of the present invention can be confirmed, but preferably,
Na ⁺ -HCO ₃ ^- low cotransporter activity cells are used. The quantity and quality of the expressed Na ⁺ -HCO ₃ ^- cotransporter can be examined by a method known per se. For example, a method for measuring intracellular pH (Journal of Biological Chemistry)
(The Journal of Biological Chemistry), Vol. 272,
19111-19114 (1997)), a method of measuring intracellular sodium ion concentration using a sodium ion indicator (Investigative Ophthalmology a
nd Visual Science), Vol. 33, pp. 3068-3079 (1992
Year)), a method for measuring the activity of incorporation of labeled sodium into cells (The Journal of Clinical Inves
tigation), Vol. 79, pp. 1276-1280 (1987)), a method for measuring membrane potential (Nature, Vol. 387, 409-41).
3 (1997)). More specifically, first, cells containing the protein of the present invention are cultured in a multiwell plate or the like. After replacing with a fresh medium or an appropriate buffer that is not toxic to cells, the cells are loaded with an intracellular pH indicator such as BCECF. Thereafter, the buffer is exchanged for a buffer containing ammonium chloride, and after a certain period of incubation, the buffer is exchanged for an appropriate buffer not containing ammonium chloride. Thereafter, screening can be performed by adding a test compound and the like, incubating for a certain period of time, adding a buffer containing sodium ions and bicarbonate ions, and measuring the subsequent increase in intracellular pH. Test compounds include, for example, peptides,
Proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like are used, and these compounds may be novel compounds or known compounds. Is also good.

Compounds that inhibit the activity of the protein of the present invention include, for example, ischemic diseases (eg, myocardial infarction and associated dysfunction, unstable angina), restenosis after PTCA,
It is useful as a safe and low-toxic drug for arrhythmia, heart failure, hypertension and associated tissue damage, ischemic brain disease (eg, cerebral infarction, cerebral hemorrhage, brain damage associated with subarachnoid hemorrhage, etc.), and the like. On the other hand, the compound that promotes the activity of the protein of the present invention is useful as a safe and low-toxic drug against metabolic acidosis associated with, for example, kidney disease. When a compound or a salt thereof obtained by using the screening method of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. The preparations thus obtained are safe and low toxic,
For example, it can be administered to humans and mammals (for example, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.For example, when an inhibitor of the protein of the present invention is orally administered for the purpose of treating myocardial infarction, it is generally used in adults. (As 60 kg) at about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg of the inhibitor per day.
mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound may vary depending on the administration subject, target disease, and the like. When administered to an adult (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the inhibitor per day is intravenously injected. It is convenient to administer. In the case of other animals, the dose can be administered in terms of 60 kg.

(2) Quantification of the protein of the present invention Since the antibody of the present invention can specifically recognize the protein of the present invention, quantification of the protein of the present invention in a test solution, particularly sandwich immunoassay It can be used for quantification by That is, the present invention, for example,
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. And (ii) reacting the test solution with the antibody of the present invention and the labeled antibody of the present invention insolubilized on a carrier simultaneously or continuously. Thereafter, the present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier. In the above (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. The protein of the present invention can be measured using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used. The measurement method using an antibody against the protein of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (eg, the amount of the protein of the present invention) in a test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used,
In terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [
¹⁴ C]. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing proteins or enzymes may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the protein of the present invention. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably For example, an antibody that recognizes an N-terminal other than the C-terminal is used. Measurement system other than the sandwich method of the monoclonal antibody of the present invention, for example, a competition method,
It can be used for immunometric methods or nephelometry. In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.

In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a certain amount of a labeled antibody, and then the solid phase and the liquid phase are separated. The antigen therein is reacted with an excessive amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In the nephelometry, the amount of insoluble precipitate generated as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used. In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, etc.
"Radio Immunoassay" edited by Hiroshi (Kodansha, published in 1974), "Improvement Radioimmunoassay" edited by Hiroshi Irie (published in 1974), Eiji Ishikawa et al. "Enzyme immunoassay" (Medical Publishing, published in 1978) ), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Medical Publishing, published in 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Medical Publishing, published in 1987) ), "Methods in ENZYMOLOGY" Vol. 70 (Immunoc
chemical Techniques (Part A)), ibid.Vol. 73 (Immunoc
chemical Techniques (Part B)), ibid.Vol. 74 (Immunoch
emical Techniques (Part C)), ibid.Vol. 84 (Immunoch
emical Techniques (Part D: Selected Immunoassays)),
Ibid. Vol. 92 (Immunochemical Techniques (Part E: Mon
oclonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: HybridomaTechnology and Monoclonal Antibodies))
(These are published by Academic Press Co., Ltd.). As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. further,
By quantifying the protein of the present invention in vivo using the antibody of the present invention, when overexpression of the protein of the present invention can be detected, myocardial infarction and associated dysfunction, unstable angina, PTCA It is possible to diagnose later restenosis, arrhythmia, heart failure, high blood pressure and associated tissue damage, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and associated brain damage or a high likelihood of suffering in the future. That is, the antibodies of the present invention are useful as diagnostic agents for the above diseases.

(3) Neutralizing antibody The antibody of the present invention binds to the extracellular region of the protein of the present invention, and functions as a protein of the present invention (eg, Na ⁺ -HCO
₃ ^- neutralizing antibodies capable of suppressing cotransporter activity), ischemic disease
(E.g., myocardial infarction and associated dysfunction, unstable angina, etc.), restenosis after PTCA, arrhythmia, heart failure, hypertension and associated tissue damage, ischemic brain disease (e.g., cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage) Treatment, such as accompanying brain damage)
It can be used as a prophylactic agent. The antibody of the present invention
It can be administered to humans or warm-blooded animals as it is, or formulated with physiologically acceptable carriers such as adjuvants for promoting uptake.

(4) Antisense DNA An antisense DNA that can complementarily bind to the DNA of the present invention and suppress the expression of the DNA can inhibit the function of the protein or DNA of the present invention in vivo. Ischemic disease (e.g., myocardial infarction and associated dysfunction, unstable angina, etc.), restenosis after PTCA, arrhythmia, heart failure, high blood pressure and associated tissue damage, ischemic brain disease (e.g., cerebral infarction) Cerebral hemorrhage, brain damage associated with subarachnoid hemorrhage, etc.). The above-mentioned antisense DNA is treated with
When used as a prophylactic agent, the antisense DN of the present invention
A can be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, and then administered to humans or warm-blooded animals according to conventional means. The antisense DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. Antisense DNA having a base sequence substantially complementary to the DNA of the present invention includes those having a base sequence substantially complementary to the DNA of the present invention and having an action capable of suppressing the expression of the DNA. Any antisense DNA may be used. The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 80% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 90%
%, More preferably about 95% or more, still more preferably about 98% or more, most preferably about 99% or more. In particular, the D of the present invention
Of the entire nucleotide sequence of the complementary strand of NA, about 80% or more, preferably about 80% or more of the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention or its partial peptide (eg, the nucleotide sequence near the start codon). Antisense DNA having a homology of about 90% or more, more preferably about 95% or more, still more preferably about 98% or more, and most preferably about 99% or more is suitable. These antisense D
NA can be produced using a known DNA synthesizer or the like.

(5) D encoding the protein of the present invention
Preparation of Non-Human Animal Having NA The transgenic non-human animal expressing the protein of the present invention can be prepared using the DNA of the present invention. Non-human animals include mammals (eg, rats,
Mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) (hereinafter abbreviated as animals).
In particular, mice, rats, and the like are suitable. The DN of the present invention
In transferring A to a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, when transferring the DNA of the present invention derived from a rat, a gene construct linked to downstream of various promoters that are highly homologous to the animal and that can express the DNA of the present invention in animal cells,
For example, a DNA-transferred animal that highly produces the protein of the present invention can be produced by microinjection into a fertilized rat egg. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or metallothionein may be used, but a gene promoter specifically expressed in the heart is preferably used. Transfer of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the protein of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the protein of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the gene have the protein of the invention in all of their germinal and somatic cells. The DN of the present invention
After confirming that the A-transferred animal stably retains the gene by mating, the animal can be bred and reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, a homozygous animal having the transgene on both homologous chromosomes is obtained,
By breeding the male and female animals, it is possible to breed the cells so that all offspring have the DNA. The animal to which the DNA of the present invention has been transferred has high expression of the protein of the present invention, and thus is useful as an animal for screening a drug acting on the protein of the present invention.

The DNA transgenic animal of the present invention can also be used as a cell source for tissue culture. For example, DNA or RNA in the tissue of the DNA-transferred rat of the present invention
The protein of the present invention can be analyzed by directly analyzing, or by analyzing the tissue in which the protein of the present invention expressed by the gene is present. Cells of a tissue having the protein of the invention can be cultured by standard tissue culture techniques and used to study the function of cells from commonly difficult tissues such as, for example, from brain or peripheral tissues. . In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. In addition, if there is a high expression cell line, the protein of the present invention can be isolated and purified therefrom. In the present specification and drawings, when bases, amino acids, and the like are represented by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in this field, examples of which are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: thyronine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group

Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy -4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the protein of the present invention. [SEQ ID NO: 2] This shows the base sequence of DNA encoding the protein of the present invention having the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 3] This shows the base sequence of a primer used for cloning DNA encoding the protein of the present invention derived from human heart. [SEQ ID NO: 4] This shows the base sequence of a primer used for cloning DNA encoding the protein of the present invention derived from human heart. [SEQ ID NO: 5] This shows the nucleotide sequence of a primer used for cloning DNA encoding the human heart-derived protein of the present invention. [SEQ ID NO: 6] This shows the base sequence of a primer used for cloning DNA encoding the human heart-derived protein of the present invention. [SEQ ID NO: 7] This shows the base sequence of the human heart-derived protein DNA fragment of the present invention used in Northern blotting in Example 2. [SEQ ID NO: 8] This shows the base sequence of the human retina-derived Na ⁺ -HCO ₃ ^- cotransporter DNA fragment used for Northern blotting in Example 2.

The transformant Escherichia coli JM109 / pMSRαN obtained in Example 1 described below is used.
BC26 neo has been deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) on November 24, 1998 under the deposit number FERM BP-6585 on November 5, 1998. ) At deposit number I
Deposited as FO 16215.

[0032]

The present invention will be described in more detail with reference to the following Examples, which do not limit the scope of the present invention. In addition, the genetic manipulation method using Escherichia coli followed the method described in Molecular cloning.

Example 1 Cloning of cDNA Encoding a Protein Derived from Human Heart and Determination of Nucleotide Sequence The cloning of the first half of the cDNA encoding a protein derived from human heart was performed using a Genetrapper positive selection system (manufactured by Life Technology). It was performed using. A plasmid cDNA library derived from a human heart (manufactured by Life Technology Co., Ltd.) was digested with GeneII and ExoIII to prepare a single-stranded cDNA library. On the other hand, a synthetic oligonucleotide (SEQ ID NO: 3) was used as a probe for c.
Used for screening DNA libraries. The probe was labeled by biotinylating the 3 'end with TdT, biotin-14-dCTP. After treating the single-stranded cDNA library at 95 ° C. for 1 minute, the mixture was rapidly cooled on an ice table, and a biotinylated probe was added, followed by hybridization at 37 ° C. for 1 hour and room temperature. After hybridization, add magnetic beads and stir at room temperature every 3 minutes for 3 minutes.
Left for 0 minutes. After that, put it in the magnet rack and
Let stand for minutes. The supernatant was discarded, and the magnetic beads were washed three times with a wash buffer. Then, the mixture was left in a magnet rack, the supernatant was discarded, an elution buffer was added, and the mixture was left at room temperature for 5 minutes. Put it in a magnet rack 5
After standing for minutes, the DNA solution of the supernatant was recovered. A synthetic oligonucleotide (SEQ ID NO: 3) was added as a primer to the obtained DNA solution and left at 95 ° C. for 1 minute. After adding the repair enzyme, the mixture was allowed to stand at 70 ° C for 15 minutes, and the double-stranded DNA was subjected to electroporation (manufactured by Bio-Rad).
It was introduced into E. coli DH10B strain. Using the obtained transformant, screening was performed by colony PCR using two oligonucleotides (SEQ ID NO: 3 and SEQ ID NO: 4) as primers. A colony in which an amplified fragment of 0.24 Kbp was formed by PCR was selected as a positive clone. After culturing the selected Escherichia coli, the plasmid was extracted, and the nucleotide sequence of cDNA was determined using a 377 DNA sequencer (manufactured by PerkinElmer). Cloning of the second half of cDNA encoding a protein derived from human heart was performed by PCR using a plasmid cDNA library derived from human heart (manufactured by Life Technology) as a template. PCR was performed using synthetic nucleotides (SEQ ID NO: 5, SEQ ID NO: 6) as primers. The PCR reaction was performed by the Hot Start method using Ampliwax PCR Gem 100 (Takara Shuzo). As the lower layer mixture, 2 μl of 10X LA taq Buffer (Takara Shuzo), 2.5 mM dNT
4 μl of the P solution, 1 μl of each 10 μM primer solution, and 12 μl of sterile distilled water were mixed. As the upper layer mixture, 0.7 μl of human brain heart-derived cDNA library solution, 10X LA taq B
uffer (Takara Shuzo) 3μl, 2.5mM dNTP solution 4μl, 20mM
Magnesium chloride 5μl, LA taq DNA polymerase (
0.5 μl of Takara Shuzo) and 16.8 μl of sterilized distilled water were mixed.
One Ampliwax PCR Gem 100 was added to the prepared lower layer mixture, treated at 94 ° C. for 1 minute and 4 ° C. for 5 minutes, and the upper layer mixture was added to prepare a PCR reaction solution. The reaction conditions were as follows: one cycle of 94 ° C. for 30 seconds was performed, and then a cycle of 94 ° C. for 30 seconds and 68 ° C. for 4 minutes was repeated 45 times. When the reaction solution was electrophoresed on a 1% agarose gel, a DNA fragment (about 0.6 kb) thought to encode the 3 ′ region was specifically amplified. The DNA fragment was recovered from an agarose gel according to a conventional method, and DNA ligation was performed with pT7Blue (promega).
It was subcloned with kit (Takara Shuzo). Plasmid containing the cDNA encoding the first half was transformed with the restriction enzyme HindI.
After digestion with II (Takara Shuzo) and EcoRI (Takara Shuzo) and electrophoresis on a 1% agarose gel, a 0.6 kb DNA fragment was recovered. In addition, the plasmid having the cDNA encoding the latter half was digested with restriction enzymes SalI (Takara Shuzo) and EcoRI (Takara Shuzo), and electrophoresed on a 1% agarose gel.
The DNA fragment was recovered. Plasmid pBluescript II SK +
Was digested with SalI (Takara Shuzo) and HindIII (Takara Shuzo) to obtain a 3.0 kb DNA fragment. The above 3.0 kb DNA fragment, a 2.6 kb DNA fragment encoding the first half of the previously prepared human heart-derived protein, a 0.6 kb DNA encoding the second half
The fragments were mixed, ligated with a DNA Ligation kit (Takara Shuzo), and transformed into competent cells of Escherichia coli JM109 (Takara Shuzo) to prepare a plasmid pNBC2b. cDNA
Was determined using a 377 DNA sequencer (manufactured by PerkinElmer). The obtained cDNA is SEQ ID NO: 2
And the base sequence represented by This cDNA fragment encoded a novel protein consisting of 971 amino acids represented by SEQ ID NO: 1.

Example 2 Confirmation of Expression Specificity in Each Tissue (Northern Hybridization) The specificity analysis of a gene expression tissue by Northern hybridization was performed using a membrane filter of a human multiple tissue northern blot (Clontech). Was. Use this membrane filter in prehybridization buffer (50% deionized formamide, 5X SSPE, 2X Denhardt's solution, 2% SDS, 1%
Prehybridization was performed at 50 ° C. for 3 hours in 00 μg / ml heat-denatured salmon sperm DNA). On the other hand, a DNA fragment specific to the gene encoding the protein of the present invention represented by SEQ ID NO: 7 was used as a probe with [α- ³² P] dCTP manufactured by Amersham) and Bca BEST labeling kit (manufactured by Takara Shuzo). Labeled. Hybridization was performed at 64 ° C. for 1 hour in an express hybridization buffer containing a labeled probe. The filter is 2X
Wash twice in SSC, 0.05% SDS solution at room temperature, and further add 0.
The plate was washed twice at 50 ° C. in a 1 × SSC, 0.1% SDS solution. BAS20
A band hybridized with the probe was detected by taking an autoradiogram of the filter using 00 (manufactured by Fuji Film Co., Ltd.). As a result, specific bands were detected in heart and skeletal muscle. [FIG. 5-A] On the other hand, a DNA fragment represented by SEQ ID NO: 8 was used as a probe specific to hNBC2, which is a protein derived from human retina, and hybridization was performed under the same conditions as described above. . As a result, hNBC2 is found in pancreas, kidney, skeletal muscle, lung,
Bands were detected in the placenta and heart. [Fig. 5-B]

Example 3 Construction of Expression Vector for cDNA Encoding a Protein Derived from Human Heart The SRα promoter derived from pTB1411 described in JP-A-5-076385 was digested with BglII (Takara Shuzo) and blunted. The DNA was ligated to a pCI vector (promega) digested with EcoRI (Takara Shuzo) using a DNA Ligation kit (Takara Shuzo) to prepare pCI-SRα. Next, this pCI-SRα was
After digestion with Takara Shuzo, the ends were blunt-ended by treatment with T4 DNA polymerase (Takara Shuzo). On the other hand, pGFP-C1
After digesting (Toyobo) with Bsu36I (Daiichi Pure Chemicals), T4
The ends were blunted by treatment with DNA polymerase (Takara Shuzo), the both were ligated with a DNA Ligation kit (Takara Shuzo), and the plasmid pMSRαneo was obtained by transforming E. coli JM109 competent cells (Takara Shuzo). Plasmid pM
SRαneo was digested with NotI (Takara Shuzo) and SalI (Takara Shuzo) to obtain a 5.4 kb DNA fragment. Plasmid pNBC2b was digested with NotI (Takara Shuzo), SalI (Takara Shuzo) and XmnI (New England Biolab) to obtain a 3.2 kb DNA fragment.
The previously prepared 5.4 kb DNA fragment and 3.2 kb DNA fragment were mixed, ligated with a DNA Ligation kit (Takara Shuzo), and
The plasmid pMSRαNBC26 neo was prepared by transforming competent cells of JM109 (Takara Shuzo). Plasmid pMSRαNBC26 neo carrying the DNA encoding the heart-derived protein of the present invention was transformed into Escherichia coli
i) Transformant: Escheric (Escheric)
hia coli) JM109 / pMSRαNBC26 neo was obtained.

Example 4 Introduction of Plasmid for Expression of Protein Derived from Human Heart into CHO-K1 Cells and Acquisition of Expressing Cells 10% Fetal Bovine Serum (Life Tech
nologies, Inc.), including the United States) Ham F12
CHO-K1 cells grown in a 150 ml tissue culture flask (Corning) using a medium (Nissui Pharmaceutical)
g / L Trypsin-0.2g / L EDTA (Life Technology (L
ife Technologies, Inc.), and the cells were removed by treatment with PBS (Life Technology (L)
ife Technologies, Inc.), centrifuged (1000 rpm, 5 minutes), and suspended in PBS. next,
DNA was introduced into the cells using Gene Pulser (manufactured by Bio-Rad) under the following conditions. That is, 8 × 10 ⁶ cells and 10 μg of a plasmid pMSRαNBC26 neo for expressing a novel human heart-derived protein were added to a 0.4 cm gap cuvette, and electroporation was performed under a voltage of 0.25 kV and a capacitance of 960 μF. Thereafter, the cells were transferred to Ham's F12 medium containing 10% fetal bovine serum, cultured for 24 hours, the cells were again detached and centrifuged, and then Geneticin (Life Technologies, Inc., USA) ) was suspended in ham F12 medium containing 10% fetal calf serum was added was such that 500 μg / ml, 10 ⁴ diluted to 96-well plates so that the cells / ml (Corning Costar (Corning Costar Corporation) Geneticin-resistant transformant was obtained by culturing in a carbon dioxide gas incubator at 37 ° C. Next, the obtained transformant was seeded on a 96-well white plate and cultured in a carbon dioxide gas incubator at 37 ° C. for 48 hours. A solution (140 mM tetramethylammonium chloride, 25 mM potassium bicarbonate, 0.8 mM dipotassium phosphate,
0.2mM monopotassium phosphate, 1mM calcium chloride, 1mM magnesium chloride, 10mM HEPES, pH7.4) After washing with 150μl, B solution containing 5μM BCECF-AM (Dojinsha) in the cells
(100 mM tetramethylammonium chloride, potassium bicarbonate, 0.8 mM dipotassium phosphate, 0.2 mM monopotassium phosphate, 1 mM calcium chloride, 1 mM magnesium chloride, 40
150 mM of ammonium chloride, 10 mM HEPES, pH 7.4) was added, and incubated at room temperature for 15 minutes. After aspirating the solution and washing with 150 μl of the A solution, the solution was aspirated and the A solution containing 1 mM amiloride (manufactured by Sigma) was removed.
After adding 50 μl, the fluctuation of intracellular pH was measured by a fluorescent drug screening system FDSS2000 (manufactured by Hamamatsu Photonics). C solution (115 mM sodium chloride, 25 mM potassium chloride, 25 mM sodium bicarbonate, 0.8 mM dipotassium phosphate, 0.2 mM monopotassium phosphate, 1 mM calcium chloride,
A hNBC2b-33 / CHO-K1 cell in which an increase in intracellular pH was observed after 200 μl of 1 mM magnesium chloride, 1 mM amiloride (Sigma), 10 mM HEPES pH7.4) was selected.

Example 5 Functional Analysis of Human Heart-Derived Protein by Intracellular pH Measurement 10% Fetal Bovine Serum (Life Tech
nologies, Inc.), including the United States) Ham F12
HNBC2b-33 / CHO-K1 cells cultured in a medium (manufactured by Nissui Pharmaceutical) and wild-type CHO-K1 cells as a control were inoculated into a 96-well white plate at 2 × 10 ⁴ cells / well, for 48 hours and 37 hours. The cells were cultured in a carbon dioxide gas incubator at ℃. A
Solution (140mM tetramethylammonium chloride, 25m
(M potassium bicarbonate, 0.8 mM dipotassium phosphate, 0.2 mM monopotassium phosphate, 1 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, pH 7.4)
B solution containing μM BCECF-AM (manufactured by Dojin) (100 mM tetramethylammonium chloride, potassium bicarbonate, 0.8 mM dipotassium phosphate, 0.2 mM monopotassium phosphate,
150 μl of 1 mM calcium chloride, 1 mM magnesium chloride, 40 mM ammonium chloride, 10 mM HEPES, pH 7.4) was added, and the mixture was incubated at room temperature for 15 minutes. Aspirate the solution, A
After washing with 150 μl of the solution, the solution was removed by suction, and 1 mM
50 μl of A solution containing amiloride (manufactured by Sigma) was added, and the change in intracellular pH was measured using a fluorescent drug screening system FDSS2000 (Hamamatsu Photonics). C solution (115 mM sodium chloride, 25 mM potassium chloride, 25 mM sodium hydrogen carbonate, 0.8 mM dipotassium phosphate, 0.2 mM monopotassium phosphate, 1 mM calcium chloride, 1 mM
Magnesium chloride, 1mM amiloride (Sigma), 1
After adding 200 μl of 0 mM HEPES (pH 7.4), the amount of increase in intracellular pH (the ratio of fluorescence values at 450 nm and 490 nm (450 nm / 490 nm)) that increased during 1 minute was calculated. further,
The amount of increase in intracellular pH when the anion exchanger inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (manufactured by Sigma) was added was measured. As a result, the hNBC2b-33 / CHO-K1 cells expressing the human heart-derived protein of the present invention were transformed into wild-type CHO-K1 cells after the addition of a C solution containing sodium ions and bicarbonate ions.
Significant compared to cells (significant difference test by t-test p <0.00
1) An increase in intracellular pH was observed. In addition, the addition of DIDS, an inhibitor of the anion transporter, allows intracellular
The increase in pH was suppressed. [Fig. 6]

[0038]

The protein of the present invention and the DNA of the present invention
The availability of antibodies and antisera, construction of an expression system for the protein of the present invention, Na ⁺ -HCO ₃ using the expression system ^- screening for development and drug candidate compounds in a system for measuring the activity of the co-transporter, Na Implement drug design based on the three-dimensional structure of the ⁺ -HCO ₃ ^- cotransporter protein, reagents for the preparation of probes and PCR primers in gene diagnosis, preparation of transgenic animals or drugs such as gene preventive and therapeutic agents Can be used.

[0039]

[Sequence Listing] [Sequence Listing] <110> Takeda Chemical Industries, Ltd. <120> Novel Protein and its DNA <130> A99283 <150> JP 10-362190 <151> 1998-12-21 <160> 8 <210 > 1 <211> 971 <212> PRT <213> Human <400> 1 Met Ser Leu Phe Ala Phe Cys Ser Gly Leu His His Phe Ser Phe Ile 1 5 10 15 Cys Phe Ile Ser Gly Asp Gly Leu Ser Ala Ser Arg His Ser Leu Arg 20 25 30 Thr Gly Leu Ser Ala Ser Asn Leu Ser Leu Arg Gly Glu Ser Pro Leu 35 40 45 Ser Leu Leu Leu Gly His Leu Leu Pro Ser Ser Arg Ala Gly Thr Pro 50 55 60 Ala Gly Ser Arg Cys Thr Thr Pro Val Pro Thr Pro Gln Asn Ser Pro 65 70 75 80 Pro Ser Ser Pro Ser Ile Ser Arg Leu Thr Ser Arg Ser Ser Gln Glu 85 90 95 Ser Gln Arg Gln Ala Pro Glu Leu Leu Val Ser Pro Ala Ser Asp Asp 100 105 110 Ile Pro Thr Val Val Ile His Pro Pro Glu Glu Asp Leu Glu Ala Ala 115 120 125 Leu Lys Gly Glu Glu Gln Lys Asn Glu Glu Asn Val Asp Leu Thr Pro 130 135 140 Gly Ile Leu Ala Ser Pro Gln Ser Ala Pro Gly Asn Leu Asp Asn Ser 145 150 155 160 Lys Ser Gly Glu Ile Lys Gly Asn Gly Ser Gly Gly Ser Arg Glu Asn 165 170 175 Ser Thr Val Asp Phe Ser Lys Val Asp Met Asn Phe Met Arg Lys Ile 180 185 190 Pro Thr Gly Ala Glu Ala Ser Asn Val Leu Val Gly Glu Val Asp Phe 195 200 205 Leu Glu Arg Pro Ile Ile Ala Phe Val Arg Leu Ala Pro Ala Val Leu 210 215 220 Leu Thr Gly Leu Thr Glu Val Pro Val Pro Thr Arg Phe Leu Phe Leu 225 230 235 240 Leu Leu Gly Pro Ala Gly Lys Ala Pro Gln Tyr His Glu Ile Gly Arg 245 250 255 Ser Ile Ala Thr Leu Met Thr Asp Glu Ile Phe His Asp Val Ala Tyr 260 265 270 Lys Ala Lys Asp Arg Asn Asp Leu Leu Ser Gly Ile Asp Glu Phe Leu 275 280 285 285 Asp Gln Val Thr Val Leu Pro Pro Gly Glu Trp Asp Pro Ser Ile Arg 290 295 300 Ile Glu Pro Pro Lys Ser Val Pro Ser Gln Glu Lys Arg Lys Ile Pro 305 310 315 320 Val Phe His Asn Gly Ser Thr Pro Thr Leu Gly Glu Thr Pro Lys Glu 325 330 335 Ala Ala His His Ala Gly Pro Glu Leu Gln Arg Thr Gly Arg Leu Phe 340 345 350 Gly Gly Leu Ile Leu Asp Ile Lys Arg Lys Ala Pro Phe Phe Phe Leu Ser 355 360 365 Asp Phe Lys Asp Ala Leu Ser Leu Gln Cys Leu Ala Ser Ile Leu Phe 370 375 380 Leu Tyr Cys Ala Cys Met Ser Pro Val Ile Thr Phe Gly Gly Leu Leu 385 390 395 400 Gly Glu Ala Thr Glu Gly Arg Ile Ser Ala Ile Glu Ser Leu Phe Gly 405 410 415 Ala Ser Leu Thr Gly Ile Ala Tyr Ser Leu Phe Ala Gly Gln Pro Leu 420 425 430 Thr Ile Leu Gly Ser Thr Gly Pro Val Leu Val Phe Glu Lys Ile Leu 435 440 445 Tyr Lys Phe Cys Arg Asp Tyr Gln Leu Ser Tyr Leu Ser Leu Arg Thr 450 455 460 Ser Ile Gly Leu Trp Thr Ser Phe Leu Cys Ile Val Leu Val Ala Thr 465 470 475 480 Asp Ala Ser Ser Leu Val Cys Tyr Ile Thr Arg Phe Thr Glu Glu Ala 485 490 495 Phe Ala Ala Leu Ile Cys Ile Ile Phe Ile Tyr Glu Ala Leu Glu Lys 500 505 510 Leu Phe Asp Leu Gly Glu Thr Tyr Ala Phe Asn Met His Asn Asn Leu 515 520 525 Asp Lys Leu Thr Ser Tyr Ser Cys Val Cys Thr Glu Pro Pro Asn Pro 530 535 540 Ser Asn Glu Thr Leu Ala Gln Trp Lys Lys Asp Asn Ile Thr Ala His 545 550 555 560 Asn Ile Ser Trp Arg Asn Leu Thr Val Ser Glu Cys Lys Lys Leu Arg 565 570 575 Gly Val Phe Leu Gly Ser Ala Cys Gly His His Gly Pro Tyr Ile Pro 580 585 590 Asp Val Leu Phe Trp Cys Val Ile Leu Phe Phe Thr Thr Phe Phe Leu 595 600 605 Ser Ser Phe Leu Lys Gln Phe Lys Thr Lys Arg Tyr Phe Pro Thr Lys 610 615 620 Val Arg Ser Thr Ile Ser Asp Phe Ala Val Phe Leu Thr Ile Val Ile 625 630 635 640 Met Val Thr Ile Asp Tyr Leu Val Gly Val Pro Ser Pro Lys Leu His 645 650 655 Val Pro Glu Lys Phe Glu Pro Thr His Pro Glu Arg Gly Trp Ile Ile 660 665 670 Ser Pro Leu Gly Asp Asn Pro Trp Trp Thr Leu Leu Ile Ala Ala Ile 675 680 685 Pro Ala Leu Leu Cys Thr Ile Leu Ile Phe Met Asp Gln Gln Ile Thr 690 695 700 Ala Val Ile Ile Asn Arg Lys Glu His Lys Leu Lys Lys Gly Ala Gly 705 710 715 715 Tyr His Leu Asp Leu Leu Met Val Gly Val Met Leu Gly Val Cys Ser 725 730 735 Val Met Gly Leu Pro Trp Phe Val Ala Ala Thr Val Leu Ser Ile Ser 740 745 750 His Val Asn Ser Leu Lys Val Glu Ser Glu Cys Ser Ala Pro Gly Glu 755 760 765 Gln Pro Lys Phe Leu Gly Ile Arg Glu Gln Arg Val Thr Gly Leu Met 770 775 780 Ile Phe Ile Leu Met Gly Leu Ser Val Phe Met Thr Ser Val Leu Lys 785 790 795 800 Phe Ile Pro Met Pro Val Leu Tyr Gly Val Phe Leu Tyr Met Gly Val 805 810 815 Ser Ser Leu Lys Gly Ile Gle Leu Phe Asp Arg Ile Lys Leu Phe Gly 820 825 830 Met Pro Ala Lys His Gln Pro Asp Leu Ile Tyr Leu Arg Tyr Val Pro 835 840 845 Leu Trp Lys Val His Ile Phe Thr Val Ile Gln Leu Thr Cys Leu Val 850 855 860 Leu Leu Trp Val Ile Lys Val Ser Ala Ala Ala Val Val Phe Pro Met 865 870 875 880 Met Val Leu Ala Leu Val Phe Val Arg Lys Leu Met Asp Leu Cys Phe 885 890 895 Thr Lys Arg Glu Leu Ser Trp Leu Asp Asp Leu Met Pro Glu Ser Lys 900 905 910 lys Lys Lys Glu Asp Asp Lys Lys Lys Lys Glu Lys Glu Glu Ala Glu 915 920 925 Arg Met Leu Gln Asp Asp Asp Asp Thr Val His Leu Pro Phe Glu Gly 930 935 940 Gly Ser Leu Leu Glu Ile Pro Val Lys Ala Leu Lys Tyr Ser Asn Ile 945 950 955 960 Phe Ser Ala Lys Val Ile Arg Gly His His Cys 965 970 <210> 2 <211> 3157 <212> DNA <213> Human <400> 2 CTTTTTAAAA ATGCCATGCT ATTGGTCATT CTAATTTCTA ACAGGATATT GCCCCCACCA 60 TCTGTCATTA ACAATTTTTT TTTATGT GTT CTGAGAACTA ATTACAGTTT TAACCAACTT 120 TTTAGTGACT TTCTAACATG TAAAAACCCG ATTGACATGC TAACCATCAC ATGACTTTGA 180 AATTTTGGGC CTTTGAAGTA TGCAATTTAT AGTGTAAATT GATTTTTTTT TAAATGTCTC 240 TATTTGCCTT CTGCAGTGGA CTTCATCATT TTTCATTTAT TTGTTTTATC TCAGGGGATG 300 GCCTTTCAGC CTCCCGCCAC TCTTTGCGAA CAGGTCTGTC TGCCTCAAAC CTTTCCTTGA 360 GAGGAGAATC ACCTTTATCT CTTCTTCTCG GTCATCTTCT TCCTTCTTCA AGAGCTGGAA 420 CCCCTGCAGG CTCAAGGTGT ACAACCCCAG TACCCACCCC TCAAAACAGT CCTCCTTCTA 480 GCCCTAGCAT CAGCCGCCTG ACCTCCAGAA GTTCCCAAGA GAGTCAGCGT CAGGCCCCAG 540 AACTACTGGT TTCACCTGCC AGTGATGATA TTCCCACAGT AGTAATTCAT CCGCCTGAGG 600 AAGACTTAGA AGCAGCGCTG AAAGGCGAGG AGCAGAAGAA TGAGGAAAAT GTTGACTTAA 660 CTCCAGGTAT TTTGGCCTCT CCCCAGTCTG CTCCTGGAAA CTTGGACAAT AGTAAAAGTG 720 GAGAAATTAA AGGTAATGGA AGTGGTGGAA GCAGAGAAAA TAGTACTGTT GACTTCAGCA 780 AGGTTGATAT GAATTTCATG AGAAAAATTC CTACGGGTGC TGAGGCATCC AACGTCCTGG 840 TGGGCGAAGT AGACTTTTTG GAAAGGCCAA TAATTGCATT TGTGAGACTG GCTCCTGCTG 900 TCCTCCTTAC AGGGTTGACT GAGGTCCCTG TTCCAACCAG GTTTT TGTTT TTGTTATTGG 960 GTCCAGCGGG CAAGGCACCA CAGTACCATG AAATTGGACG ATCAATAGCC ACTCTCATGA 1020 CAGATGAGAT TTTCCATGAT GTAGCTTATA AAGCAAAAGA CAGAAATGAC CTCTTATCTG 1080 GAATTGATGA ATTTTTAGAT CAAGTAACTG TCCTACCTCC AGGAGAGTGG GATCCTTCTA 1140 TACGCATAGA ACCACCAAAA AGTGTCCCTT CTCAGGAAAA GAGAAAGATT CCTGTGTTTC 1200 ACAATGGATC TACCCCCACA CTGGGTGAGA CTCCTAAAGA GGCCGCTCAT CATGCTGGGC 1260 CTGAGCTACA GAGGACTGGA CGGCTTTTTG GTGGTTTGAT ACTTGACATC AAAAGGAAAG 1320 CACCTTTTTT CTTGAGTGAC TTCAAGGATG CATTAAGCCT GCAGTGCCTG GCCTCGATTC 1380 TTTTCCTATA CTGTGCCTGT ATGTCTCCTG TAATCACTTT TGGAGGGCTG CTTGGAGAAG 1440 CTACAGAAGG CAGAATAAGT GCAATAGAGT CTCTTTTTGG AGCATCATTA ACTGGGATTG 1500 CCTATTCATT GTTTGCTGGG CAACCTCTAA CAATATTGGG GAGCACAGGT CCAGTTCTAG 1560 TGTTTGAAAA AATTTTATAT AAATTCTGCA GAGATTATCA ACTTTCTTAT CTGTCTTTAA 1620 GAACCAGTAT TGGTCTGTGG ACTTCTTTTT TGTGCATTGT TTTGGTTGCA ACAGATGCAA 1680 GCAGCCTTGT GTGTTATATT ACTCGATTTA CAGAAGAGGC TTTTGCAGCC CTTATTTGCA 1740 TCATATTCAT CTACGAGGCT TTGGAGAAGC TCTTTGATTT AGGAGAAACA T ATGCATTTA 1800 ATATGCACAA CAACTTAGAT AAACTGACCA GCTACTCATG TGTATGTACT GAACCTCCAA 1860 ACCCCAGCAA TGAAACTCTA GCACAATGGA AGAAAGATAA TATAACAGCA CACAATATTT 1920 CCTGGAGAAA TCTTACTGTT TCTGAATGTA AAAAACTTCG TGGTGTATTC TTGGGGTCAG 1980 CTTGTGGTCA TCATGGACCT TATATTCCAG ATGTGCTCTT TTGGTGTGTC ATCTTGTTTT 2040 TCACAACATT TTTTCTGTCT TCATTCCTCA AGCAATTTAA GACCAAGCGT TACTTTCCTA 2100 CCAAGGTGCG ATCGACAATC AGTGATTTTG CTGTATTTCT CACAATAGTA ATAATGGTTA 2160 CAATTGACTA CCTTGTAGGA GTTCCATCTC CTAAACTTCA TGTTCCTGAA AAATTTGAGC 2220 CTACTCATCC AGAGAGAGGG TGGATCATAA GCCCACTGGG AGATAATCCT TGGTGGACCT 2280 TATTAATAGC TGCTATTCCT GCTTTGCTTT GTACCATTCT CATCTTTATG GATCAACAAA 2340 TCACAGCTGT AATTATAAAC AGAAAGGAAC ACAAATTGAA GAAAGGAGCT GGCTATCACC 2400 TTGATTTGCT CATGGTTGGC GTTATGTTGG GAGTTTGCTC TGTCATGGGA CTTCCATGGT 2460 TTGTGGCTGC AACAGTGTTG TCAATAAGTC ATGTCAACAG CTTAAAAGTT GAATCTGAAT 2520 GTTCTGCTCC AGGGGAACAA CCCAAGTTTT TGGGAATTCG TGAACAGCGG GTTACAGGGC 2580 TAATGATTTT TATTCTAATG GGCCTCTCTG TGTTCATGAC TTCAGTCCTA AAGTTTA TTC 2640 CAATGCCTGT TCTGTATGGT GTTTTCCTTT ATATGGGAGT TTCCTCATTA AAAGGAATCC 2700 AGTTATTTGA CCGTATAAAA TTATTTGGAA TGCCTGCTAA GCATCAGCCT GATTTGATAT 2760 ACCTCCGTTA TGTGCCGCTC TGGAAGGTCC ATATTTTCAC AGTCATTCAG CTTACTTGTT 2820 TGGTCCTTTT ATGGGTGATA AAAGTTTCAG CTGCTGCAGT GGTTTTTCCC ATGATGGTTC 2880 TTGCATTAGT GTTTGTGCGC AAACTCATGG ACCTGTGTTT CACGAAGAGA GAACTTAGTT 2940 GGCTTGATGA TCTTATGCCA GAAAGTAAGA AAAAGAAAGA AGATGACAAA AAGAAAAAAG 3000 AGAAAGAGGA AGCTGAACGG ATGCTTCAAG ACGATGATGA TACTGTGCAC CTTCCATTTG 3060 AAGGGGGAAG TCTCTTGCAA ATTCCAGTCA AGGCCCTAAA ATATAGTAAC ATATTTTCAG 3120 CCAAGGTGAT CAGAGGGCAT CATTGCTAGA GTGCTGC 3157 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 3 AAGACAGAT TGACCTC > 24 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 CAAAAAGCCG TCCAGTCCTC TGTA 24 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> <400 > 5 TTGGGAATTC GTGAACAGCG GGTTACAGGG 30 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223 > <400> 6 TAGTAAGCTT GCAGCACTCT AGCAATGATG CCCTC 35 <210> 7 <211> 665 <212> DNA <213> Human <400> 7 CTTTTTAAAA ATGCCATGCT ATTGGTCATT CTAATTTCTA ACAGGATATT GCCCCCACCA 60 TCTGTCATTA ACAATTTTTT TAGTATCTACGTATCTATCTATCTATCTATCTATCATC 180 AATTTTGGGC CTTTGAAGTA TGCAATTTAT AGTGTAAATT GATTTTTTTT TAAATGTCTC 240 TATTTGCCTT CTGCAGTGGA CTTCATCATT TTTCATTTAT TTGTTTTATC TCAGGGGATG 300 GCCTTTCAGC CTCCCGCCAC TCTTTGCGAA CAGGTCTGTC TGCCTCAAAC CTTTCCTTGA 360 GAGGAGAATC ACCTTTATCT CTTCTTCTCG GTCATCTTCT TCCTTCTTCA AGAGCTGGAA 420 CCCCTGCAGG CTCAAGGTGT ACAACCCCAG TACCCACCCC TCAAAACAGT CCTCCTTCTA 480 GCCCTAGCAT CAGCCGCCTG ACCTCCAGAA GTTCCCAAGA GAGTCAGCGT CAGGCCCCAG 540 AACTACTGGT TTCACCTGCC AGTGATGATA TTCCCACAGT AGTAATTCAT CCGCCTGAGG 600 AAGACTTAGA AGCAGCGCTG AAAGGCGAGG AGCAGAAGAA TGAGGAAAAT GTTGACTTAA 660 CTCCA 665 <210> 8 <211> 507 <212> DNA <213> Human <400> 8 CCTTGGAGAC TAGAAAGAAA CTGCTAGATG GCTGTAACAC AGTTCATCCA TTTCCGTGAA 60 GAGA TCATGG GGAATATGTT CTTCATCATC ATCTTCAGTA CCAAGGATAA ACTGTGTTAC 120 AGAGATGGAG AAGAATATGA ATGGAAAGAA ACTGCTAGAT GGCTGAAATT TGAAGAGGAT 180 GTTGAAGATG GCGGTGACCG ATGGAGTAAA CCTTATGTGG CAACTCTCTC TTTGCACAGT 240 CTTTTTGAAC TAAGGAGTTG CATCCTCAAT GGAACAGTCA TGCTGGATAT GAGAGCAAGC 300 ACTCTAGATG AAATAGCAGA TATGGTATTA GACAACATGA TAGCTTCTGG CCAATTAGAC 360 GAGTCCATAC GAGAGAATGT CAGAGAAGCT CTTCTGAAGA GACATCATCA TCAGAATGAG 420 AAAAGATTCA CCAGTCGGAT TCCTCTTGTT CGATCTTTTG CAGATATAGG CAAGAAACAT 480 TCTGACCCTC ACTTGCTTGA AAGGAAT 507

[0040]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the DNA encoding the heart-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continued from FIG. 2).

FIG. 2 shows the nucleotide sequence of the DNA encoding the heart-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continued from FIG. 1 and continued from FIG. 3).

FIG. 3 shows the nucleotide sequence of the DNA encoding the heart-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continuation of FIG. 2).

FIG. 4 shows a hydrophobicity plot of the protein of the present invention derived from the amino acid sequence shown in FIGS. Portions 1 to 10 indicate hydrophobic domains.

FIG. 5: mR encoding the heart-derived protein of the present invention
The result of having investigated the expression level of NA in each human tissue by Northern hybridization is shown. Heart is the heart, Bra
in indicates the brain, Placenta indicates the placenta, Lung indicates the lungs, Liver indicates the liver, Skeletal Muscle indicates the skeletal muscle, and Kidney indicates the kidney. The number (kb) on the left indicates the size of the RNA molecular weight marker. A is the human heart-derived protein hNBC2 of the present invention.
b represents the expression level, and B represents the expression level of the human retina-derived Na ⁺ -HCO ₃ ^- cotransporter protein hNBC2.

FIG. 6 shows human heart-derived protein of the present invention expressed.
[Fig. 7] Fig. 7 shows the results of measuring the increase in intracellular pH after pre-pulsing ammonium ions to hNBC2b-33 / CHO-K1 cells and then adding a buffer containing sodium ions and bicarbonate ions. NBC2b-33 indicates hNBC2b-33 / CHO-K1 cells expressing the heart-derived protein of the present invention, and CHO-K1
Indicates wild-type CHO-K1 cells. The numbers on the left indicate the respective concentrations (final concentrations) of DIDS, an anion transport inhibitor.
0, 100 μM) shows the amount of increase in intracellular pH (ratio of fluorescence values at 450 nm / 490 nm) increased 10 minutes after the addition of a buffer containing sodium ions and bicarbonate ions when added. Data were expressed as mean ± standard error. Comparison between the two groups was performed by T test, and p <0.001 was considered significant.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61K 48/00 C07K 14/47 C07K 14/47 16/18 16/18 C12N 1/21 C12N 1/21 C12P 21/02 C 5/10 A01K 67/027 C12P 21/02 C12P 21/08 // A01K 67/027 G01N 33/15 Z C12P 21/08 33/53 D G01N 33/15 A61K 37/02 33/53 C12N 5/00 B (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/02 C12R 1:19)

Claims (16)

[Claims] 1. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 2. The protein according to claim 1, which has Na ⁺ -HCO ₃ ^- cotransporter activity, or a salt thereof. 3. A partial peptide of the protein according to claim 1, or an amide or ester thereof or a salt thereof. 4. A DNA containing a DNA having a base sequence encoding the protein according to claim 1. 5. The DNA according to claim 4, which has the nucleotide sequence represented by SEQ ID NO: 2. 6. A recombinant vector containing the DNA according to claim 5. (7) A transformant transformed with the recombinant vector according to (6). 8. A method for producing a protein or a salt thereof according to claim 1, wherein the transformant according to claim 7 is cultured, the protein according to claim 1 is produced, accumulated, and collected. . 9. An antibody against the protein according to claim 1 or a salt thereof, or the partial peptide according to claim 3 or an amide or ester thereof or a salt thereof. 10. The protein according to claim 1 or a salt thereof, or the partial peptide according to claim 3 or an amide or ester thereof or a salt thereof, wherein the protein or a salt thereof is used. 3. A method for screening a compound or a salt thereof which promotes or inhibits the Na ⁺ -HCO ₃ ^- cotransporter activity of the partial peptide according to 3, or an amide or ester thereof or a salt thereof. 11. The protein according to claim 1, comprising the protein or a salt thereof, or the partial peptide according to claim 3 or an amide or ester thereof or a salt thereof.
A screening kit for a compound or a salt thereof which promotes or inhibits the Na ⁺ -HCO ₃ ^- cotransporter activity of the partial peptide or amide or ester thereof or salt thereof described above. 12. A protein according to claim 1 or a salt thereof, or a partial peptide according to claim 3 or an amide thereof, which can be obtained by using the screening method according to claim 10 or the screening kit according to claim 11. ester or its salt Na ⁺ -HCO ₃ ^-
A compound or a salt thereof that promotes or inhibits co-transporter activity. 13. A protein according to claim 1 or a salt thereof, or a partial peptide according to claim 3 or an amide thereof or an amide thereof, which can be obtained by using the screening method according to claim 10 or the screening kit according to claim 11. ester or its salt Na ⁺ -HCO ₃ ^-
A medicament comprising a compound that promotes or inhibits co-transporter activity or a salt thereof. 14. A method for treating myocardial infarction and associated dysfunction, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and associated tissue damage, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and associated cerebral damage. 14. The medicament according to claim 13, which is a prophylactic / therapeutic agent. 15. Myocardial infarction and dysfunction associated therewith, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and associated tissue damage characterized by containing the antibody of claim 9, Diagnostic agent for cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and associated brain damage. 16. A method for quantifying the protein or its salt according to claim 1 or the partial peptide or its amide or ester or its salt according to claim 3, wherein the antibody according to claim 9 is used.