JP2000184890A - New protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which contains a specific amino acid sequence, consists of a protein having Na+-HCO3- cotransport activity and is useful in the search of therapeutic drugs for diseases caused by an abnormal intracellular ion environment and the gene diagnosis, treatment or the like due to its gene. SOLUTION: This protein is a new protein (salt) containing the amino acid sequence homologous or substantially homologous with the amino acid sequence represented by the formula, shows Na+-HCO3- cotransport activity, possesses and effect for regulating the intracellular ion environment to conditions suitable for survival and is useful in the measurement of Na+-HCO3- cotransport activity, the screening of compounds being considered for medicines, the drug design based on the steric structure of Na+-HCO3- cotransport protein, the gene diagnosis due to its gene and the utilization as a gene preventive - therapeutic agent. This protein is obtained by using a cDNA library arising from human brain as a template, cloning it by means of PCR using a synthetic nucleotide as a primer, integrating the resultant gene into an expression vector and introducing it in the host cell to express.

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
^{The +} -HCO ₃ ^- cotransporter plays an important role in maintaining cell homeostasis in normal times, but is involved in the occurrence of cytotoxicity in ischemia. Ischemia causes intracellular acidosis, and Na ⁺ -HCO ₃ ^- co-transporter and Na ⁺ -H ⁺
The exchanger works. As a result, the intracellular Na ⁺ concentration increases, and Ca ²⁺ overload into the cell occurs via the Na ⁺ -Ca ²⁺ exchanger. This overloaded Ca ²⁺ is thought to be one of the factors that result in cell damage [Basic Research in Cardiology, 91, 191- (1
996)]. The Na ⁺ -HCO ₃ ^- cotransporter gene from the kidney has been identified as a salamander [amphibian] [Nature, 387, 409- (199
7)], rat [American Journal of Physiology, 274,
F425- (1998)], human [The Journal of Biological Che
mistry, 272, 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,該該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 The purpose is to provide medicines and the like.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that Na specifically expressed in human-derived brain
⁺ -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;
(a) a protein or a salt thereof according to the above (1), which has a ⁺ -HCO ₃ ^- cotransporter activity; (3) a partial peptide of the protein according to the above (1) or a salt thereof;
Having a base sequence encoding the protein described in claim 1.
(5) a DNA according to the above (4) having the base sequence represented by SEQ ID NO: 2, (6) a recombinant vector containing the DNA according to the above (5), (7)
(8) culturing the transformant described in the above (7), the transformant transformed with the recombinant vector according to the above (6),
Producing the protein according to (1), accumulating the protein, collecting the protein, and collecting the protein; (9) a method for producing the protein or salt thereof according to (1); (9) the protein according to (1); )) An antibody against the partial peptide or a salt thereof; (10) the protein according to (1), wherein the protein according to (1) or the partial peptide or salt thereof according to (3) is used;
Na of the partial peptide or the salt thereof according to (3) above
⁺ -HCO ₃ ^- co test compound to transporter activity to promote or inhibit (11) above (1) proteins described, characterized by using a partial peptide or salt thereof according to (3), wherein A kit for screening a compound or a salt thereof which promotes or inhibits the Na ⁺ -HCO ₃ ^- cotransporter activity of the protein according to the above (1), the partial peptide according to the above (3) or a salt thereof. (12) The protein according to (1), the partial peptide according to (3) or the Na ^{+ of} a salt thereof obtained by using the screening method according to (10) or the screening kit according to (11).
-HCO ₃ ^- cotransporter active compound or its salt that promotes or inhibits, obtained using (13) (10) the screening method or the (11) the screening kit according to, wherein said (1), wherein Na ⁺ of the partial peptide according to the above (3) or a salt thereof
-HCO ₃ ^- about such pharmaceutical agent comprising the compound or its salt that promotes or inhibits the cotransporter activity.

More specifically, (14) the protein:
Amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:
An amino acid sequence in which one or more (preferably about 1 to 30, more preferably about 1 to 9, and still more preferably several) amino acids in the amino acid sequence represented by 1, have been deleted, SEQ ID NO: An amino acid sequence obtained by adding one or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several) amino acids to the amino acid sequence represented by 1, SEQ ID NO: 1 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10,
More preferably the (1, a protein containing the amino acids are amino acid sequence are substituted with other amino acids, or a combination thereof several)) above, wherein the Na ⁺ -HCO ₃ ^- cotransporter protein or The salt, (1
5) A comparison is made between the case where the test compound and the protein or the salt thereof described in (1) or the partial peptide or the salt thereof described in (2) above are brought into contact with the test compound. (10) The screening method according to the above (16), wherein Na ⁺ -HCO is compared with a case where a test compound is brought into contact with a cell containing the protein according to the above (1) or not. _A method for screening a compound or a salt thereof that alters the activity of the ₃ ^- cotransporter, (17) the protein according to (1), which is expressed on the cell membrane of the transformant by culturing the transformant according to (7). A compound or a salt thereof that alters the function of the protein according to the above (1), which is compared with the case where the test compound is brought into contact with the test compound A cleaning method, (18) a compound or a salt thereof that changes the activity of the protein according to (1), which is obtained by the screening method according to (15) to (17), (19) a method according to (15) to ( 17) Prevention or treatment of ischemic disease (particularly ischemic brain disease, etc.) characterized by containing the compound or a salt thereof that alters the activity of the protein of (1), which is obtained by the screening method of (1). Agent, (20)
The protein according to (1), wherein the antibody according to (9) is brought into contact with the protein according to (1), the partial peptide according to (2), or a salt thereof. And (21) a prophylactic or therapeutic agent for ischemic disease (particularly ischemic brain disease or the like) characterized by containing the antibody described in (8) above. provide.

The protein of the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (FIGS. 1 to 3). The protein of the present invention can be used, for example, in all cells (eg, spleen cells, nerve cells, glial cells, pancreatic β) of humans and warm-blooded animals (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.). Cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Or progenitor cells of these cells, stem cells or cancer cells, etc.), or any tissue in which those cells are present, for example, the brain, various parts 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.

[0007] The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 70% or more, preferably about 80% or more, and more preferably the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequences having about 90% or more, more preferably about 95% or more homology, and the like 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 identical" to the activity of the protein, for example, co-transporter activity (Na ⁺ -HCO ₃ ^- cotransporter activity), such as physiological properties,
Means substantially the same. Amino acid substitutions, deletions,
Additions or insertions often do not significantly alter the physiological or chemical properties of the polypeptide, in which case the substituted, deleted, added, or inserted proteins will Alternatively, it would be substantially identical to the one without the insertion. 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. Polar (neutral) amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine and the like. Examples of positively charged (basic) amino acids include arginine, lysine, histidine and the like. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0008] The term "substantially the same quality" means that their activities are qualitatively (physicochemically or pharmacologically) the same. Therefore, it is preferable that the activities are the same, but quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. The co-transporter activity can be measured according to a method known per se, for example, according to a screening method described later. In addition, the protein of the present invention includes an amino acid sequence in which one or more amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 1, and one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence with added amino acid, SEQ ID NO: 1
A protein containing an amino acid sequence in which one or more amino acids in the amino acid sequence represented by is replaced with another amino acid, or 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 in the above-mentioned protein 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 and acetyl). ), Gln formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, substituents on the side chains of amino acids in the molecule (eg, -OH, -CO
OH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, formyl group,
Protected by a C _1-6 acyl group such as a C _2-6 alkanoyl group such as acetyl, or a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto. 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.
Specifically, the partial peptide of the protein having the amino acid sequence represented by SEQ ID NO: 1 is an extracellular region (hydrophilic site) in the hydrophobic plot analysis shown in FIG. It is a peptide containing the part analyzed. 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. In addition, the partial peptide of the present invention is characterized in that
Or, two or more amino acids have been deleted, one or more amino acids have been added to the amino acid sequence, or one or more amino acids in the amino acid sequence have been substituted with another amino acid. You may. In the 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. Further, the partial peptide of the present invention also includes those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide having a sugar chain bonded thereto. . 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. As the salt of the protein of the present invention or its partial peptide, a physiologically acceptable acid addition salt is particularly preferable. 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 can be produced from the above-described human or mammalian cells or tissues by a purification method known per se, or a protein encoding the protein of the present invention described below.
It can also be produced by culturing a transformant containing NA. 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, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated. 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. Known methods for condensation and elimination of protecting groups include, for example, 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.

[0009] The DNA encoding the protein of the present invention may be any DNA as long as it contains 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 base sequence represented by SEQ ID NO: 2 include, for example,
A DNA containing a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the base sequence represented by SEQ ID NO: 2 is used. Hybridization is performed by a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook eta).
l, Cold Spring Harbor 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 to 40 mM, preferably about 19 to 20 mM.
In mM, the temperature is about 50-70 ° C, preferably about 60-6
The condition at 5 ° C. is shown. In particular, when the sodium concentration is about 19 mM
And a temperature of about 65 ° C. is most preferred. More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 includes SEQ ID NO: 2
DNA having the base sequence represented by The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the partial peptide 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. Reverse Transcription was performed directly using the mRNA fraction prepared from the cells and tissues described above.
scriptasePolymerase Chain Reaction (hereinafter referred to as RT-P
It can also be amplified by the CR method). Specifically, as the DNA encoding the partial peptide of the present invention, 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 represented by SEQ ID NO: 2 It has a nucleotide sequence that hybridizes under high stringency conditions and has substantially the same activity as the protein of the present invention (eg, Na ⁺ -HC
O ₃ ^- such as a DNA having a partial nucleotide sequence of the DNA encoding a protein having a co-transporter activity, etc.) is used. The DNA capable of hybridizing the base sequence represented by SEQ ID NO: 2 includes, for example, about 80% or more, preferably about 90% or more, and more preferably about 95% or more homology with the base sequence represented by SEQ ID NO: 2. For example, DNA containing a base sequence having 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, abbreviated as the protein of the present invention) include (1)
Synthetic DNA having partial nucleotide sequence of protein of the present invention
Amplification by PCR using primers, or (2) hybridization of DNA incorporated into an appropriate vector with a DNA fragment encoding a partial or entire region of the protein of the present invention or labeled with synthetic DNA Sorting.
Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Samb.
rook et al., Cold Spring Harbor Lab. Press, 1989)
And the like. Also,
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 using a known kit,
For example, by using Mutan ^™ -G (Takara Shuzo Co., Ltd.), Mutan ^™ -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 is at its 5 '
It has ATG as a translation initiation codon on the terminal side, and TAA and TGA as translation termination codons on the 3 ′ terminal side.
Alternatively, it may have a TAG. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA 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,
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, 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.

[0012] In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like, if desired, may be used. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes 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, virus is A
In the case of cNPV, a cell line derived from night larvae of moth (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells derived from Mamestra brassicae or
Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN; BmN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21
Cells (Vaughn, JL et al., In Viv
o), 13, 213-217, (1977)). As insects, for example, silkworm larvae and the like are used [Maeda et al., Nature, 315, 592 (198).
5)]. As animal cells, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf)
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, HEK293 cells, C127 cells, BALB3T3 cells, Sp-2 cells, and the like. Among these,
CHO cells, CHO (dhfr ⁻ ) cells, HEK293 cells and the like are preferred. In order to transform Escherichia, for example, Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69
Vol. 2110 (1972) and Gene, Vol. 17, 1
07 (1982). In order to transform Bacillus sp., For example, Molecular & General Genetics, 168, 11
1 (1979). In order to transform yeast, for example, Methods in Enzymology, 1
Vol. 94, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Natl. Aca)
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). Methods for introducing an expression vector into animal cells include, for example, the calcium phosphate method [Graham, FL and van der Eb, AJ Virology (Virology) 52, 456-467 (1973)], electroporation [Nuemann, E. et al. . Embo Journal (EMBO
J.) 1, 841-845 (1982)]. Thus, a transformant transformed with the expression vector containing the 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, cultivation was carried out while gradually increasing the MTX concentration, and the dhfr gene was selected by selecting a resistant strain.
DN encoding the protein of the present invention together with the gene
A can be amplified intracellularly to obtain a more highly expressed animal cell line.

The above transformant is cultured under conditions that allow expression of the DNA encoding the protein of the present invention, and the protein of the present invention is produced and accumulated to produce the protein of the present invention or a salt thereof. Can be.
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 the carbon source, for example, glucose, dextrin,
Nitrogen sources such as soluble starch and sucrose include, for example, ammonium salts, nitrates, corn steep liquor,
Inorganic or organic substances such as peptone, casein, meat extract, soybean meal, potato extract, and inorganic substances include, for example, 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
Journals of Experiments in Molecular Genetic
s), 431-433, Cold Spring Harbor Laborator
y, New York 1972]. Here, in order to make the promoter work efficiently if necessary, for example, 3β
-An agent such as indolylacrylic acid can be added. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 1
The reaction is carried out at a temperature of 5 to 43 ° C. for about 3 to 24 hours, and if necessary, ventilation and stirring may 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, as a medium,
For example, Burkholder minimal medium [Bo
Stian, KL et al., Proc. of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA),
77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Aca
USA, 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 in which the host is an insect cell or an insect, the medium used is 10% immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)).
Those to which additives such as bovine serum are appropriately added are used. The pH of the medium 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, about 5-
MEM medium containing 20% fetal bovine serum [Science (Se
ience), 122, 501 (1952)], DMEM medium [Virology, 8, 396 (195)
9)], RPMI 1640 medium [Journal of the American Medical Association (The Jo
unal of the American Medical Association) 199
Volume, 519 (1967)], 199 Medium [Proceeding of the Society for the Biological Medicine (Proceeding of the Society for the
Biological Medicine), 73, 1 (1950)]. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 72 hours,
Vent or agitate as needed. As described above,
The transformant can express the protein of the present invention. The protein of the present invention can be separated and purified from the culture by, for example, the following method. When the protein of the present invention is extracted from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude extract of the protein of the present invention by centrifugation or filtration is used as appropriate. 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,
Methods that mainly use differences in molecular weight, such as gel filtration and SDS-polyacrylamide gel electrophoresis, methods that use differences in charge, such as ion exchange chromatography, and specific novelty, such as affinity chromatography , A method using a difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, and a method using a difference in isoelectric point such as isoelectric focusing. When the thus obtained protein of the present invention is obtained in a free form, it can be converted to 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, By the method according to it,
It can be converted to a free form or other salts. The protein of the present invention produced by the recombinant, before or after purification by the action of an appropriate protein-modifying enzyme,
Optionally, modifications may be made, and the polypeptide may be partially removed. 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.

The antibody against the protein of the present invention, its partial peptide or a salt thereof can be used as the protein of the present invention,
Any of a polyclonal antibody and a monoclonal antibody may be used as long as the antibody can recognize the partial peptide or a salt thereof. Antibodies against the protein of the present invention, its partial peptide or a salt thereof (hereinafter abbreviated as the protein of the present invention) are produced by using the protein or the like of the present invention as an antigen in accordance with a known method for producing an antibody or antiserum. can do. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein or the like of the present invention is administered to a mammal at a site where the antibody can be produced by administration 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, and 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 measurement of the antibody titer in the antiserum can be performed, for example, by reacting the labeled protein of the present invention described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is performed in a known manner, for example,
The method of Koehler and Milstein [Natural
e), 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 /
And myeloma cells of warm-blooded animals such as 0, AP-a, etc., 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 to PEG6000) is 10 to 80%.
Cell fusion can be carried out efficiently by being added at about the concentration and incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes. 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 an antigen such as the protein of the present invention is directly or adsorbed together with a carrier, and Anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A, and the monoclonal antibody bound to the solid phase is detected. Method, 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, an enzyme, or the like, and detecting a monoclonal antibody bound to the solid phase And the like. The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, 1 to 20%, preferably 10 to 20%
% RPMI 1640 medium containing fetal calf serum, 1-1
GIT medium containing 0% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SF
M-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
° 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 gas. 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 Monoclonal antibody can be separated and purified in the same manner as normal polyclonal antibody separation and purification of immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis Method, adsorption and 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 the antibody and dissociate the bond. Specific Purification Method for Obtaining Antibody). [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, an immunizing antigen (antigen such as the protein 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 an antibody-containing substance against the antibody and the like 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. What 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 2 with respect to 1 hapten
A method of coupling at a rate of 0, preferably about 1 to 5 is used. Further, various coupling agents can be used for coupling the hapten and the carrier, but glutaraldehyde, carbodiimide, maleimide active ester,
An active ester reagent containing a thiol group or a dithioviridyl group 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. Administration is usually about 2
The procedure can be performed once every 6 weeks or about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the 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 polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody.

The protein of the present invention, its partial peptide or a salt thereof, and the DNA encoding them are
Obtaining antibodies and antisera, construction of an expression system for recombinant proteins, Na ⁺ -HCO ₃ using the expression system ^- Screening Construction and drug candidate compounds in the system for measuring the activity of the co-transporter, Na ⁺ - HCO ₃ ^- implementation of drug design based on the three-dimensional structure of the co-transporter, probes and PC in genetic diagnosis
It can be used as a reagent for preparing an R primer, a transgenic animal, or a medicament such as a gene preventive / 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. A protein, a partial peptide or a salt thereof of the present invention (hereinafter sometimes abbreviated as a protein of the present invention), a DNA encoding the protein of the present invention or a partial peptide thereof (hereinafter referred to as D of the present invention)
The use of antibodies expressing the protein of the present invention and cells of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below. . (1) Method for screening for a compound that alters the activity of the protein of the present invention Using the protein or the like of the present invention, or constructing an expression system for the protein or the like of the present invention, and using the expression system, Na ⁺ -HCO
₃ ^- The use of cotransporter 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 ) Or a salt thereof 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 characterized by comparing the case where the test compound is brought into contact with the protein of the present invention, its partial peptide or a salt thereof and the case where the test compound is not brought into contact. Typically, Na ⁺ −HC
O ₃ ^- provides a method of screening a compound or its salt that alters the cotransporter activity). More specifically, the present invention is characterized in that a protein of the present invention or a salt thereof or a partial peptide of the protein of the present invention or a salt thereof is compared with a case where the test compound is brought into contact with a test compound and a case where it is not brought into contact. The function of the protein of the present invention, which is characterized by comparing a case where a test compound is brought into contact with a cell containing the protein of the present invention and a case where a test compound is not contacted (specifically, ⁺ -HCO ₃ ^-
A method of screening for a compound or a salt thereof that alters (cotransporter activity), and contacting a test compound with the protein of the present invention expressed on the cell membrane of the transformant by culturing the transformant containing the DNA of the present invention. The function of the protein of the present invention, which is compared with the case where the protein is not allowed to be used and the case where the protein is not used (specifically, Na ⁺ -HC
O ₃ ^- provides a method of screening a compound or its salt that alters the cotransporter activity). Before the protein, etc. of the present invention is obtained, for example, Na ⁺ -HCO ₃ ^- cotransporter when screening activity altering compound, Na ⁺ -HCO ₃ such first rat ^- cells comprising co-transporter protein, Using the tissue to obtain candidate compounds (primary screening)
Was co-transporter alter the activity of confirming whether the compounds tested (secondary screening) is required ^- then the candidate compounds are indeed human Na ⁺ -HCO ₃ in. 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 eliminates the need for primary screening, Na ⁺ -HCO ₃ ^- a compound that alters the cotransporter activity can be efficiently screened. The specific description of the screening method of the present invention is as follows. First, the protein or the like of the present invention used in the screening method of the present invention may be any protein as long as it contains the aforementioned protein or the like of the present invention. Cells from animal organs are preferred. However, since human-derived organs are particularly difficult to obtain, cells used to express human-derived proteins and the like are suitable for screening. The above-described method is used for constructing a cell in which the protein or the like of the present invention is expressed, and it is preferable to construct the cell by expressing the DNA of the present invention in a mammalian cell or an insect cell. 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 DNA fragment is used as a nuclear polyhedrosis virus belonging to baculovirus using an insect as a host.
s; NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα
It is preferably incorporated downstream such as a promoter. The cells used for the expression may be any cells as long as the expression of the protein of the present invention can be confirmed. Preferably, cells having low Na ⁺ -HCO ₃ ^- cotransporter activity are used. The quantity and quality of the expressed Na ⁺ -HCO ₃ ^- cotransporter can be examined by a method known per se. For example, intracellular p
H Method of Measurement (The Journal of Biological Chemistry),
Vol. 272, pp. 19111-19114 (1997)), a method for measuring intracellular sodium ion concentration using a sodium ion indicator (Investigative Offthermology.
And Visual Science (Investigative Opht
halmology and Visual Science), Vol. 33, pp. 3068-3079 (1992)), a method for measuring the incorporation activity of labeled sodium into cells (Journal of Clinical Investigation (The Journal of Clinical Investigation)).
Investigation), Vol. 79, pp. 1276-1280 (1987)
Year)), the method of measuring the membrane potential (Nature
), Vol. 387, pp. 409-413 (1997)). More specifically, first, cells containing the protein or the like of the present invention are cultured in a multiwell plate or the like. After replacing with fresh medium or an appropriate buffer that is not toxic to cells, BCECF-AM (3'-O-acetyl-2 ', 7'
-biscarboxyethyl-4 or 5-carboxyfluorescein, diaceto
xymethyl ester) and Quene 1-AM (8-amino-2- (trans-2-
aminostyryl) -6-methoxyquinoline-N, N, N ', N'-tetraace
tic acid, tetraacetoxymethyl ester)
H Load indicator. Thereafter, the buffer is replaced with a buffer containing an ammonium salt such as ammonium chloride or ammonium sulfate, and after a certain period of incubation, the buffer is replaced with an appropriate buffer containing no ammonium chloride. Thereafter, screening can be performed by adding a test compound or the like and incubating for a certain period of time, adding a buffer containing sodium ions and bicarbonate ions, and measuring an increase in intracellular pH thereafter. As test compounds, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like are used. Or a known compound. Compounds that inhibit the activity of the protein or the like of the present invention include, for example,
Cerebral infarction, ischemic stroke, acute cerebral thrombosis, ischemic brain disease such as transient cerebral ischemic attack, cerebral bleeding due to cerebral hemorrhage and subarachnoid hemorrhage, myocardial infarction and accompanying dysfunction, unstable angina, It is useful as a safe and low-toxic drug for restenosis after PTCA, arrhythmia, heart failure, hypertension and accompanying tissue damage. 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 obtained in this way are safe and have low toxicity, for example, humans and mammals (eg, rats,
Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.)
Can be administered. 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 for the protein or the like of the present invention is orally administered for the purpose of treating ischemic brain disease, it is generally used In adults (as 60 kg), the inhibitor is used in an amount of about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, the target disease, and the like. For example, for the purpose of treating ischemic brain disease, an inhibitor for the protein or the like of the present invention may be used as an injection. Usually in adult form (60k
g), the inhibitor is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day.
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. 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, its partial peptide, or a salt thereof The antibody of the present invention can specifically recognize the protein of the present invention and the like. It can be used for quantification of proteins and the like, particularly for quantification by sandwich immunoassay. That is, the present invention provides, for example, (i) competitive reaction between an antibody of the present invention, a test solution and a labeled protein of the present invention, and the like. A method for quantifying the protein or the like of the present invention in a test solution, which comprises measuring the proportion of the protein or the like; (ii) the method of the present invention in which the test solution and the antibody of the present invention are insolubilized on a carrier; The present invention provides a method for quantifying the protein or the like of the present invention in a test solution, which comprises reacting an antibody simultaneously or continuously and measuring the activity of a labeling agent on an insolubilized carrier. In (ii) above,
Preferably, 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 and the like of the present invention can be measured using a monoclonal antibody against the protein and the like 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, and the F (a
b ′) ₂ , Fab ′, or Fab fraction may be used. The measurement method using an antibody against the protein or the like 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 the antigen in the liquid to be measured (eg, the amount of the protein of the present invention). Any method may be used as long as it is a method for detecting the amount of by chemical or physical means and calculating the amount from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use the 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. As the radioisotope, for example, [ ¹²⁵
I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like. 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 the insolubilization of the antigen or the antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme 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).
Thereafter, the amount of the protein of the present invention in the test liquid can be determined by measuring the activity of the labeling agent on the insolubilized carrier. Even if the primary reaction and the secondary reaction are performed in reverse order,
Further, they may be performed at the same time or at different 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 or the like 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 or the like 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. The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like. 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 reacted.
(B / F separation), the amount of labeling of either B or 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 to the antibody, or a solid phased antibody is used as the first antibody. And an immobilization method using an immobilized antibody as the second antibody. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess 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 measurement system for the protein of the present invention or a salt thereof 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, it is possible to refer to reviews, books, and the like [for example, "Radio Immunoassay" edited by Hiro Irie (published in Kodansha, 1974), "Radio Immunoassay" edited by Hiro Irie. (Kodansha, published in 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. (Medical Publishing, published in 1978), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. (2nd edition) (Medical Publishing, Showa 57) Ed., Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunoch)
emical Techniques (Part A)), ibid.Vol. 73 (Immunoch
emical Techniques (Part B)), ibid.Vol. 74 (Immunoch
emical Techniques (Part C)), ibid.Vol. 84 (Immunoche
mical Techniques (Part D: Selected Immunoassays)),
Ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoc
lonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodies))
(These are published by Academic Press Co., Ltd.). As described above, the protein of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.

(3) Neutralizing antibody Of the antibodies of the present invention, they bind to the extracellular region of the protein of the present invention and function as proteins (eg, Na ⁺ -HCO
Neutralizing antibodies capable of suppressing ₃ ^- cotransporter activity) can be used as therapeutic / prophylactic agents for cerebral ischemic diseases and the like. The antibody of the present invention can be administered to humans or warm-blooded animals as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary 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 suppress the function of the protein or DNA of the present invention in vivo. Therefore, it can be used as a therapeutic / prophylactic agent for cerebral ischemic disease and the like. When the above-mentioned antisense DNA is used as the above-mentioned therapeutic / prophylactic agent, the antisense DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector or the like. It can be administered to humans or warm-blooded animals according to the 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. An antisense DNA having a base sequence substantially complementary to a DNA encoding the protein of the present invention or a partial peptide thereof (hereinafter, may be abbreviated as the DNA of the present invention) is substantially the same as the DNA of the present invention. Any antisense DNA may be used as long as it has a nucleotide sequence complementary to that of DNA and has an action of suppressing expression of the DNA. 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%
% Or more, more preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the complementary sequence of the base sequence of the portion encoding the N-terminal portion of the protein of the present invention or its partial peptide (for example, the base sequence near the start codon) is approximately the same as the complementary strand. Antisense DNA having a homology of 80% or more, preferably about 90% or more, more preferably about 95% or more is suitable. These antisense DNAs have the known D
It can be manufactured using an NA 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 or the like of the present invention can be prepared using the DNA of the present invention. Non-human animals include mammals (for example, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) (hereinafter abbreviated as animals), and in particular, mice, rabbits and the like. Is preferred. When transferring the DNA of the present invention to a target animal, the DN
It is generally advantageous to use A 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 rabbit, a gene construct linked to downstream of various promoters which are highly homologous to the animal and can express the DNA of the present invention in animal cells, For example, D which produces the protein of the present invention at a high level by microinjection into fertilized rabbit eggs
NA transfer animals can be created. 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 brain 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 or the like 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 or the like of the present invention in all of its germinal and somatic cells. The progeny of this type of animal that has inherited the gene has the protein of the present invention in all of its germinal and somatic cells. After confirming that the DNA-transferred animal of the present invention stably retains the gene by breeding, it can be reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring have the DNA. Breeding can be subcultured. The animal into 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 transgenic animal of the present invention can also be used as a cell source for tissue culture. For example, DN in the tissue of the DNA transgenic mouse of the present invention
The protein or the like of the present invention can be analyzed by directly analyzing A or RNA, or by analyzing a tissue in which the protein of the present invention expressed by a gene is present. Cells of a tissue having the protein or the like of the present invention can be cultured by standard tissue culture techniques, and these can be used to study the function of cells from generally difficult-to-cultivate tissues such as, for example, those derived from brain and peripheral tissues. it can. In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. Further, 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 and amino acids are indicated by abbreviations, IUPAC-IUB
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given 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: threonine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lysine arginine arginine : 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 Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention. [SEQ ID NO: 2] This shows the base sequence of DNA encoding the Na ⁺ -HCO ₃ ^- cotransporter 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 Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention. [SEQ ID NO: 4] This shows the base sequence of a primer used for cloning DNA encoding the Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention. [SEQ ID NO: 5] This shows the base sequence of a primer used for cloning DNA encoding the Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention. [SEQ ID NO: 6] This shows the base sequence of the primer used for cloning the DNA encoding the Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention. [SEQ ID NO: 7] This shows the base sequence of the human brain-derived Na ⁺ -HCO 3 ^- cotransporter 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 kidney-derived Na ⁺ -HCO 3 ^- cotransporter hNBC1 DNA fragment used in Northern blotting in Example 2. [SEQ ID NO: 9] Na + -HCO 3 − derived from human retina used for Northern blotting in Example 2

The base sequence of the DNA fragment of the co-transporter hNBC2 is shown below. The transformant Escherichia coli JM109 / pMSRaNBC3 neo obtained in Example 1 described later.
Has been deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) on November 24, 1998 under the deposit number FER.
MBP-6586, deposited on November 5, 1998 with the Institute of Fermentation (IFO) under the deposit number IFO 1
No. 6216.

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS 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. Genetic manipulation using Escherichia coli is based on molecular cloning (Molecular clonin).
g) was followed.

【Example】

Example 1 derived from human brain Na ⁺ -HCO ₃ ^- co transporter protein cDNA Cloning and nucleotide sequence encoding the determined human brain-derived cDNA library (Clontech) as a template, synthetic nucleotide (SEQ ID NO: : 3, SEQ ID NO:
It was cloned by PCR using 4) as a primer. The PCR reaction was performed by the Hot Start method using Ampliwax PCR Gem 100 (Takara Shuzo). 10X P
yrobest Buffer (Takara Shuzo) 2 ml, 2.5 mM dNTP solution
4 ml and 1 mM each of a 10 mM primer solution were mixed with 12 ml of sterile distilled water. As the upper layer mixture, 1 ml of human brain-derived cDNA library (Clontech) solution, 10X Pyrobest Buf
fer (Takara Shuzo) 3ml, 2.5mM dNTP solution 4ml, Pyrobest
DNA polymerase (Takara Shuzo) 0.5ml, sterile distilled water 21.5
ml. Ampliwax PCR Gem
Add 100 and treat at 80 ° C for 5 minutes, 4 ° C for 5 minutes,
The upper layer mixture was added to prepare a PCR reaction solution. The reaction was carried out by repeating a cycle of 94 ° C. for 30 seconds and 68 ° C. for 5 minutes 45 times. The PCR reaction solution was diluted 50-fold with TE buffer and used as a template for the second PCR. In the second PCR, the synthetic oligonucleotides (SEQ ID NO: 3 and SEQ ID NO: 5) were used as primers for cloning the first half of the cDNA encoding the Na ⁺ -HCO ₃ ^- cotransporter protein from human brain, For partial cloning, synthetic oligonucleotides (SEQ ID NO: 6, SEQ ID NO: 4) were used as primers. 2.5 ml of PCR reaction solution previously prepared as a template, 10X P
5 ml of yrobest Buffer, 8 ml of 2.5 mM dNTP, 1 ml each of 10 mM primer solution 36 ml of sterile distilled water and 0.5 ml of Pyrobest were mixed to prepare a PCR reaction solution. The reaction conditions are 94 ° C for 30 seconds, 68
A 5 minute cycle at 45 ° C. was repeated 45 times. When the reaction solution was electrophoresed on a 1% agarose gel, a DNA fragment (about 1.9 kb) thought to encode the first half and a DNA fragment (about 1.6 kb) thought to encode the second half were specifically amplified. . The DNA fragment was recovered from the agarose gel according to a conventional method, and PCR Zero Blunt Kit
(Invitrogen) and subcloned into a plasmid vector. The plasmid having the cDNA encoding the first half was digested with restriction enzymes KpnI (Takara Shuzo) and PstI (Takara Shuzo) and electrophoresed on a 1% agarose gel, and a 1.9 kb DNA fragment was recovered. In addition, the plasmid having the cDNA encoding the latter half was replaced with the restriction enzyme PstI.
(Takara Shuzo) and NotI (Takara Shuzo) were digested and electrophoresed on a 1% agarose gel, and then a 1.6 kb DNA fragment was recovered. On the other hand, the SRα promoter derived from pTB1411 described in JP-A-5-076385 is digested with BglII (manufactured by Takara Shuzo), blunted, and then digested with EcoRI (manufactured by Takara Shuzo). (Takara Shuzo) to produce pCI-SRα. Next, this pCI-SRα was converted to ClaI
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 D
The ends were blunted by treatment with NA polymerase (manufactured by Takara Shuzo), the both were ligated with a DNA Ligation kit (manufactured by Takara Shuzo), and the plasmid pMSRαneo was obtained by transforming competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo). Plasmid pMS
Rαneo was digested with KpnI (Takara Shuzo) and NotI (Takara Shuzo) to obtain a 5.4 kb DNA fragment. The above 5.4 kb DNA fragment, the 1.95 kb DNA fragment encoding the first half of the previously prepared human brain-derived Na + -HCO3-cotransporter protein, and the 1.25 kb DNA fragment encoding the second half were mixed, and a DNA Ligation kit (Takara Shuzo) And transforming competent cells of Escherichia coli JM109 (Takara Shuzo) to obtain plasmid pMSRaNBC
3 neo was prepared. The nucleotide sequence of the cDNA was determined using a 377 DNA sequencer (PerkinElmer). Acquired
The cDNA had the base sequence represented by SEQ ID NO: 2. This cDNA fragment encoded a novel Na ⁺ -HCO ₃ ^- cotransporter protein consisting of 1044 amino acids represented by SEQ ID NO: 1. Brain-derived Na ⁺ -HCO ₃ of the present invention ^- cotransporter protein E. coli plasmid PMSRaNBC3 neo for holding the DNA encoding (Escherichia coli) JM109
Transformant: Escherichia coli
JM109 / pMSRaNBC3 neo was obtained.

[0028]

Example 2 Confirmation of Expression Specificity in Each Tissue (Northern Hybridization) The specificity analysis of a tissue expressing a gene by Northern hybridization was performed using a membrane filter of a human multiple tissue northern blot (Clontech). This membrane filter was prehybridized in Express Hybridization Buffer (Clontech) at 64 ° C. for 1 hour. On the other hand, a DNA fragment of the gene encoding the protein of the present invention represented by SEQ ID NO: 7 was used as a probe.
Labeling was performed using [a- ³² P] dCTP (Amersham) and Bca BEST labeling kit (Takara Shuzo). Hybridization was performed at 64 ° C. for 1 hour in an express hybridization buffer containing a labeled probe.
The filter was washed twice in 2X SSC, 0.05% SDS solution at room temperature, and further washed in 0.1X SSC, 0.1% SDS solution at 50 ° C.
Washed twice. Using BAS2000 (Fuji Film), an autoradiogram of the filter was taken to detect a band hybridized with the probe. As a result, a specific band was detected in the brain. [Figure 5-A] On the other hand, hNB, a known Na ⁺ -HCO ₃ ^- cotransporter protein
C1 used the DNA fragment represented by SEQ ID NO: 8 as a probe, and hNBC2 carried out hybridization using the DNA fragment represented by SEQ ID NO: 9 as a probe under the same conditions as described above. As a result, bands were detected for hNBC1 in the kidney and pancreas [FIG. 5-B], and for hNBC2, bands were detected in pancreas, kidney, skeletal muscle, lung, placenta, brain, and heart. [
Fig. 5-C]

[0029]

EXAMPLE 3 Human brain-derived Na ⁺ -HCO ₃ ^- cotransporter get 10% fetal bovine serum CHO-K1 Transfer and Expression cells into cells expressing plasmid (Life Technologies (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) at 0.5 g /
L trypsin-0.2g / L EDTA (Life Technology (Lif
e Technologies, Inc.) (country name: USA).
e Technologies, Inc., USA), centrifuged (1000 rpm, 5 minutes), and suspended in PBS. Next, DNA was introduced into the cells using Gene Pulser (Bio-Rad) under the following conditions. That is, 8 × 10 ⁶ cells and 10 mg of human brain-derived new Na ^{+ in} a 0.4 cm gap cuvette
-HCO ₃ ^- cotransporter expression plasmid PMSRaNBC3 neo addition, the voltage 0.25 kV, and electroporated under capacitance 960 mF. 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 the 500 mg / 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 the cells in a carbon dioxide 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 150ml,
B solution containing 5 mM BCECF-AM (Dojinsha) in 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 mM ammonium chloride, 10 m
(M HEPES, pH 7.4) was added and incubated at room temperature for 15 minutes. Aspirate the solution, wash with 150 ml of A solution, aspirate the solution, add 50 ml of A solution containing 1 mM amiloride (manufactured by Sigma), and use the fluorescent drug screening system FDSS2000 (Hamamatsu Photonics (Manufactured by the company). 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, 1 mM magnesium chloride, 1 mM amiloride (Sigma), 10 mM HEPES pH7. 4) 200ml
A strain showing an increase in intracellular pH after addition, hNBC3-10 /
CHOK1 cells were selected.

[0030]

Example 3 Na ⁺ -H derived from human brain by intracellular pH measurement
Functional analysis of CO3 ^- cotransporter protein 10% fetal bovine serum (Life Tech
nologies, Inc.), including the United States) Ham F12
HNBC3-10 / CHOK1 cells cultured in a culture medium (Nissui Pharmaceutical) and wild-type CHOK1 cells as a control were seeded at 2 × 10 ⁴ cells / well on a 96-well white plate for 48 hours.
The cells were cultured in a carbon dioxide gas incubator at 7 ° C. A solution
(140 mM tetramethylammonium chloride, 25 mM 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) After washing with 150 ml, 5 mM BCE was added to the cells.
B solution containing CF-AM (manufactured by Dojin) (100mM tetramethylammonium chloride, potassium bicarbonate, 0.8mM dipotassium phosphate, 0.2mM monopotassium phosphate, 1mM calcium chloride, 1mM magnesium chloride, 40mM ammonium chloride
150 ml of 10 mM HEPES (pH 7.4) was added and incubated at room temperature for 15 minutes. Aspirate the solution, wash with 150 ml of A solution, aspirate the solution, add 50 ml of A solution containing 1 mM amiloride (manufactured by Sigma), add
Fluorescent drug screening system FDSS for pH fluctuation
2000 (Hamamatsu Photonics). C solution (1
15 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, 1 mM magnesium chloride, 1 mM amiloride (Sigma), 10 mM HEPES pH
7.4) After the addition of 200 ml, the increase in intracellular pH increased per minute (the ratio of fluorescence values at 450 nm and 490 nm (4
50 nm / 490 nm)) was calculated. Furthermore, the anion exchanger inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2 '
-Disulfonic acid) (manufactured by Sigma) was measured for the increase in intracellular pH. As a result, hNBC3 expressing the Na ⁺ -HCO ₃ ^- cotransporter protein of the present invention
-10 / CHOK-1 cells showed a marked increase in intracellular pH compared to wild-type CHOK1 cells after the addition of C solution containing sodium ions and bicarbonate ions. Furthermore, the addition of DIDS, an inhibitor of anion transporters, suppressed the increase in intracellular pH in a concentration-dependent manner. [Fig. 6]

[0031]

Industrial Applicability The protein of the present invention, a partial peptide thereof or a salt thereof, and a DNA encoding them
The availability of antibodies and antisera, the construction of expression system for recombinant proteins, 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 ⁺ -HCO ₃ ^- implementation of drug design based on the three-dimensional structure of the co-transporter protein, a reagent for the creation of probes or PCR primers in gene diagnosis, is used as a medicament such as making or gene agent for the prophylaxis or treatment of transgenic animals be able to.

[0032]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 1044 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Pro Ala Ala Gly Ser Asn Glu Pro Asp Gly Val Leu Ser Tyr Gln 1 5 10 15 Arg Pro Asp Glu Glu Ala Val Val Asp Gln Gly Gly Thr Ser Thr Ile 20 25 30 Leu Asn Ile His Tyr Glu Lys Glu Glu Leu Glu Gly His Arg Thr Leu 35 40 45 Tyr Val Gly Val Arg Met Pro Leu Gly Arg Gln Ser His Arg His His 50 55 60 Arg Thr His Gly Gln Lys His Arg Arg Arg Gly Arg Gly Lys Gly Ala 65 70 75 80 Ser Gln Gly Glu Glu Gly Leu Glu Ala Leu Ala His Asp Thr Pro Ser 85 90 95 Gln Arg Val Gln Phe Ile Leu Gly Thr Glu Glu Asp Glu Glu His Val 100 105 110 Pro His Glu Leu Phe Thr Glu Leu Asp Glu Ile Cys Met Lys Glu Gly 115 120 125 Glu Asp Ala Glu Trp Lys Glu Thr Ala Arg Trp Leu Lys Phe Glu Glu 130 135 140 Asp Val Glu Asp Gly Gly Glu Arg Trp Ser Lys Pro Tyr Val Ala Thr 145 150 155 160 Leu Ser Leu His Ser Leu Phe Glu Leu Arg Ser Cys Leu Ile Asn Gly 165 170 175 Thr Val Leu Leu Asp Met His Ala Asn Ser Ile Glu Glu Ile Ser Asp 180 185 190 Leu Ile Leu Asp Gln Gln Glu Leu Ser Ser Asp Leu Asn Asp Ser Met 195 200 205 Arg Val Lys Val Arg Glu Ala Leu Leu Lys Lys His His His Gln Asn 210 215 220 Glu Lys Lys Arg Asn Asn Leu Ile Pro Ile Val Arg Ser Phe Ala Glu 225 230 235 240 Val Gly Lys Lys Gln Ser Asp Pro His Leu Met Asp Lys His Gly Gln 245 250 255 Thr Val Ser Pro Gln Ser Val Pro Thr Thr Asn Leu Glu Val Lys Asn 260 265 270 Gly Val Asn Cys Glu His Ser Pro Val Asp Leu Ser Lys Val Asp Leu 275 280 285 His Phe Met Lys Lys Ile Pro Thr Gly Ala Glu Ala Ser Asn Val Leu 290 295 300 Val Gly Glu Val Asp Ile Leu Asp Arg Pro Ile Val Ala Phe Val Arg 305 310 315 320 Leu Ser Pro Ala Val Leu Leu Ser Gly Leu Thr Glu Val Pro Ile Pro 325 330 335 Thr Arg Phe Leu Phe Ile Leu Leu Gly Pro Val Gly Lys Gly Gln Gln 340 345 350 Tyr His Glu Ile Gly Arg Ser Met Ala Thr Ile Met Thr Asp Glu Ile 355 360 365 Phe His Asp Val Ala Tyr Lys Ala Lys Glu Arg Asp Asp Leu Leu Ala 370 375 380 Gly Ile Asp Glu Phe Leu Asp Gln Val Thr Val Leu Pro Pro Gly Glu 385 390 395 400 Trp Asp Pro Ser Ile Arg Ile Glu Pro Pro Lys Asn Val Pro Ser Gln 405 410 415 Glu Lys Arg Lys Met Pro Gly Val Pro Asn Gly Asn Val Cys His Ile 420 425 430 Glu Gln Glu Pro His Gly Gly His Ser Gly Pro Glu Leu Gln Arg Thr 435 440 445 Gly Arg Leu Phe Gly Gly Leu Val Leu Asp Ile Lys Arg Lys Ala Pro 450 455 460 Trp Tyr Trp Ser Asp Tyr Arg Asp Ala Leu Ser Leu Gln Cys Leu Ala 465 470 475 480 Ser Phe Leu Phe Leu Tyr Cys Ala Cys Met Ser Pro Val Ile Thr Phe 485 490 495 Gly Gly Leu Leu Gly Glu Alu Thr Glu Gly Arg Ile Ser Ala Ile Glu 500 505 510 Ser Leu Phe Gly Ala Ser Met Thr Gly Ile Ala Tyr Ser Leu Phe Ala 515 520 525 Gly Gln Ala Leu Thr Ile Leu Gly Ser Thr Gly Pro Val Leu Val Phe 530 535 540 Glu Lys Ile Leu Val Lys Phe Cys Lys Asp Tyr Ala Leu Ser Tyr Leu 545 550 555 560 Ser Leu Arg Ala Cys Ile Gly Leu Trp Thr Ala Phe Leu Cys Ile Val 565 570 575 Leu Val Ala Thr Asp Ala Ser Ser Leu Val Cys Tyr Ile Thr Arg Phe 580 585 590 Thr Glu Glu Ala Phe Ala Ser Le u Ile Cys Ile Ile Phe Ile Tyr Glu 595 600 605 Ala Ile Glu Lys Leu Ile His Leu Ala Glu Thr Tyr Pro Ile His Met 610 615 620 His Ser Gln Leu Asp His Leu Ser Leu Tyr Tyr Cys Arg Cys Thr Leu 625 630 635 640 Pro Glu Asn Pro Asn Asn His Thr Leu Gln Tyr Trp Lys Asp His Asn 645 650 655 Ile Val Thr Ala Glu Val His Trp Ala Asn Leu Thr Val Ser Glu Cys 660 665 670 Gln Glu Met His Gly Glu Phe Met Gly Ser Ala Cys Gly His His Gly 675 680 685 Pro Tyr Thr Pro Asp Val Leu Phe Trp Ser Cys Ile Leu Phe Phe Thr 690 695 700 Thr Phe Ile Leu Ser Ser Thr Leu Lys Thr Phe Lys Thr Ser Arg Tyr 705 710 710 715 720 Phe Pro Thr Arg Val Arg Ser Met Val Ser Asp Phe Ala Val Phe Leu 725 730 735 Thr Ile Phe Thr Met Val Ile Ile Asp Phe Leu Ile Gly Val Pro Ser 740 745 750 Pro Lys Leu Gln Val Pro Ser Val Phe Lys Pro Thr Arg Asp Asp Arg 755 760 765 Gly Trp Ile Ile Asn Pro Ile Gly Pro Asn Pro Trp Trp Thr Val Ile 770 775 780 Ala Ala Ile Ile Pro Ala Leu Leu Cys Thr Ile Leu Ile Phe Met Asp 785 790 795 800 Gln Gln Ile Thr Ala Val Ile I le Asn Arg Lys Glu His Lys Leu Lys 805 810 815 Lys Gly Cys Gly Tyr His Leu Asp Leu Leu Met Val Ala Ile Met Leu 820 825 830 Gly Val Cys Ser Ile Met Gly Leu Pro Trp Phe Val Ala Ala Thr Val 835 840 845 Leu Ser Ile Thr His Val Asn Ser Leu Lys Leu Glu Ser Glu Cys Ser 850 855 860 Ala Pro Gly Glu Gln Pro Lys Phe Leu Gly Ile Arg Glu Gln Arg Val 865 870 875 880 Thr Gly Leu Met Ile Phe Val Leu Met Gly Cys Ser Val Phe Met Thr 885 890 895 Ala Ile Leu Lys Phe Ile Pro Met Pro Val Leu Tyr Gly Val Phe Leu 900 905 910 Tyr Met Gly Val Ser Ser Leu Gln Gly Ile Gln Phe Phe Asp Arg Leu 915 920 925 Lys Leu Phe Gly Met Pro Ala Lys His Gln Pro Asp Phe Ile Tyr Leu 930 935 940 Arg His Val Pro Leu Arg Lys Val His Leu Phe Thr Leu Ile Gln Leu 945 950 955 960 Thr Cys Leu Val Leu Leu Trp Val Ile Lys Ala Ser Pro Ala Ala Ile 965 970 975 Val Phe Pro Met Met Val Leu Ala Leu Val Phe Val Arg Lys Val Met 980 985 990 Asp Leu Cys Phe Ser Lys Arg Glu Leu Ser Trp Leu Asp Asp Leu Met 995 1000 1005 Pro Glu Ser Lys Lys Lys Lys Le u Asp Asp Ala Lys Lys Lys Ala Lys 1010 1015 1020 Glu Glu Glu Val Ile Val Leu Ala Pro Thr Val Tyr Leu Gly Ala Ser 1025 1030 1035 1040 Asn Tyr Arg Thr 1044

[SEQ ID NO: 2] Sequence length: 3561 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA sequence TAGTTCGGCT CCGCCATGCC GGCCGCCGGG AGTAACGAGC CGGACGGCGT CCTCAGCTAT 60 CAGAGACCAG ATGAAGAAGC TGTGATGATCAGCAGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA AAGAAGAGCT GGAAGGTCAC AGAACTCTGT ATGTGGGAGT TCGGATGCCG 180 CTTGGCCGGC AGAGCCATCG GCATCACCGC ACTCATGGCC AGAAGCACCG GAGACGAGGG 240 CGGGGCAAAG GAGCCAGCCA GGGGGAGGAA GGCCTGGAAG CCCTGGCCCA CGACACACCA 300 TCTCAGCGTG TTCAGTTCAT TCTTGGCACC GAGGAAGATG AAGAGCATGT GCCTCATGAG 360 CTGTTTACAG AGCTGGATGA GATCTGTATG AAAGAGGGAG AAGATGCTGA GTGGAAGGAA 420 ACAGCCAGGT GGCTGAAGTT TGAAGAAGAT GTTGAAGATG GGGGAGAACG CTGGAGCAAG 480 CCTTATGTGG CAACCCTTTC ATTGCACAGC CTGTTTGAGC TAAGGAGCTG CCTTATTAAT 540 GGAACAGTCC TCCTGGATAT GCATGCAAAT AGCATAGAAG AAATTTCAGA CCTGATCCTG 600 GATCAGCAAG AACTGTCCAG TGACCTGAAT GACAGCATGA GGGTTAAAGT GCGGGAAGCC 660 CTTCTCAAAA AGCATCATCA TCAGAATGAA AAGAAGAGAA ACAACCTCAT TCCCATTGTT 720 CG CTCCTTTG CTGAGGTTGG CAAGAAGCAG TCTGATCCTC ATTTGATGGA TAAACATGGT 780 CAAACCGTGT CTCCTCAGTC TGTTCCAACT ACAAATCTTG AAGTAAAAAA TGGAGTGAAT 840 TGTGAACATA GTCCTGTGGA TTTAAGCAAG GTAGACCTTC ATTTCATGAA AAAAATTCCT 900 ACTGGGGCCG AGGCCTCCAA TGTCCTGGTT GGAGAGGTGG ATATTTTGGA CCGTCCCATT 960 GTTGCCTTTG TGAGGCTGTC TCCAGCTGTT CTTCTCTCAG GCCTAACAGA AGTGCCAATC 1020 CCAACAAGAT TTTTGTTTAT CTTATTGGGT CCAGTAGGGA AAGGTCAGCA GTACCATGAG 1080 ATTGGCAGAT CCATGGCCAC CATCATGACA GATGAGATTT TTCATGACGT AGCATATAAG 1140 GCAAAAGAGC GAGATGATCT CCTGGCGGGG ATTGATGAGT TCCTAGACCA GGTGACGGTG 1200 CTCCCTCCAG GAGAGTGGGA TCCCTCCATT AGAATTGAGC CACCCAAAAA TGTCCCTTCC 1260 CAGGAGAAAA GGAAAATGCC TGGAGTTCCA AATGGAAATG TTTGCCACAT AGAACAGGAA 1320 CCACATGGGG GTCACAGTGG GCCAGAACTT CAGCGCACTG GGCGGCTATT TGGGGGCTTG 1380 GTGCTGGACA TCAAGCGGAA GGCCCCCTGG TACTGGAGCG ACTACCGAGA TGCACTCAGC 1440 TTACAGTGTT TGGCTTCCTT TCTGTTCCTG TACTGTGCCT GCATGTCACC TGTCATCACC 1500 TTTGGGGGAC TGCTTGGAGA AGCCACTGAG GGACGCATAA GTGCAATTGA ATCCTTGTTT 1560 GGAGCTTCCA T GACTGGGAT TGCTTATTCC TTGTTTGCGG GACAGGCTCT CACCATCCTG 1620 GGAAGTACTG GACCAGTGCT TGTGTTTGAA AAGATTTTGG TCAAATTCTG CAAAGACTAT 1680 GCTCTTTCAT ACCTCTCCCT GCGAGCTTGT ATTGGACTGT GGACCGCTTT CCTGTGTATT 1740 GTCCTTGTGG CAACTGATGC CAGTTCCCTT GTCTGCTACA TTACCCGTTT CACTGAAGAA 1800 GCATTTGCCT CCCTAATTTG CATTATTTTC ATCTATGAAG CAATAGAAAA ACTGATTCAC 1860 CTGGCAGAGA CCTACCCCAT CCACATGCAC AGCCAGCTGG ACCACCTTAG CCTCTATTAC 1920 TGCAGGTGTA CTCTGCCAGA GAATCCAAAC AATCACACCC TCCAGTACTG GAAGGACCAC 1980 AACATCGTGA CAGCAGAAGT CCACTGGGCT AACCTGACTG TCAGTGAATG CCAGGAGATG 2040 CATGGAGAGT TCATGGGATC TGCGTGCGGC CATCATGGAC CCTACACTCC TGATGTCCTC 2100 TTTTGGTCCT GTATTCTCTT TTTCACCACC TTCATCCTCT CAAGCACCTT AAAGACGTTT 2160 AAGACGAGCC GTTATTTCCC AACCAGAGTA CGCTCCATGG TGAGTGACTT TGCTGTTTTC 2220 CTCACTATCT TCACAATGGT GATTATTGAT TTTTTGATTG GAGTCCCATC ACCAAAGCTT 2280 CAAGTTCCCA GTGTGTTCAA GCCAACAAGG GATGATCGCG GATGGATTAT TAATCCCATT 2340 GGCCCCAATC CCTGGTGGAC TGTGATAGCT GCAATTATCC CAGCTCTTCT CTGTACTATC 2400 TTGATATTCA TGGATC AGCA GATCACAGCC GTCATTATTA ACAGGAAGGA ACATAAGCTC 2460 AAGAAAGGCT GTGGCTACCA CCTGGACCTA CTGATGGTGG CCATCATGCT GGGTGTCTGC 2520 TCCATCATGG GCCTGCCCTG GTTTGTAGCT GCAACTGTCT TGTCCATCAC ACATGTGAAC 2580 AGCCTCAAGC TAGAATCTGA ATGCTCTGCT CCTGGAGAAC AGCCCAAGTT CCTGGGCATC 2640 CGAGAACAGA GAGTGACAGG CCTTATGATC TTTGTGCTGA TGGGCTGCTC AGTCTTCATG 2700 ACGGCTATCT TAAAGTTTAT TCCAATGCCA GTACTCTACG GAGTTTTCCT TTACATGGGA 2760 GTTTCTTCAC TACAGGGAAT TCAGTTCTTT GATCGTCTAA AGCTCTTTGG GATGCCCGCA 2820 AAGCACCAGC CAGATTTCAT CTACCTGCGG CATGTGCCGC TGCGCAAAGT GCACCTCTTC 2880 ACCCTCATCC AGTTGACCTG TCTCGTCCTG CTCTGGGTCA TCAAGGCATC TCCAGCTGCC 2940 ATTGTTTTCC CAATGATGGT TTTGGCCTTG GTCTTTGTCA GGAAAGTCAT GGATCTCTGT 3000 TTCTCTAAGC GAGAGCTGAG CTGGCTAGAT GATCTCATGC CTGAAAGCAA AAAGAAGAAG 3060 TTGGATGATG CCAAAAAGAA GGCCAAGGAG GAAGAGGTCA TAGTCCTTGC ACCAACTGTA 3120 TACCTGGGGG CCTCAAATTA CAGAACATAG GAAGGGTCAT GTGAAAAGTC AGCATGTCTG 3180 GAATCCCGAG GGTTATATTT AGGAGCTGGG AAGATTACCC CCAAAGATGT TCTCAGCTAA 3240 AGAATGGATT AGGGATTCTT G CTTCTGTCT GGTCTTAATT TTTGGGTTTG ACAACCACTT 3300 ATTTTTTCCT TTGGTTACAA TCTACTCACC AGGCTCATAC CTACAATGTG AACATACAGT 3360 ATGCCCTTAT TAGCAGATTC AATGGCTCAC ATTCTTTCAA AAGGTCTAAT TTGACAAATA 3420 CATAAGACCC ATTATTTCCT AGAATGGTTG GAATATATCT AATTGCAAAT GGTGCTGTGG 3480 TTGGCACCAT GCAAAGATAA CTTGCATAGG ACTTTCTGTC TTTTTTCATT TCCCTTCAGC 3540 ACTTGGCATC TTGTCATCTA C 3561

[0033]

[SEQ ID NO: 3] Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GGGGGGTACC TAGTTCGGCT CCGCCATGC 29

[0034]

[SEQ ID NO: 4] Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGGGGCGGCC GCGTAGATGA CAAGATGCCA AGTGC 35

[0035]

[SEQ ID NO: 5] Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ACACCTGCAG TAATAGAGGC TAAGGTGG 28

[0036]

[SEQ ID NO: 6] Sequence length: 28 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCCTCTATTA CTGCAGGTGT ACTCTGCC 28

[0037]

[SEQ ID NO: 7] Sequence length: 302 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATTCACCTGG CAGAGACCTA CCCCATCCAC ATGCACAGCC AGCTGGACCA CCTTAGCCTC 60 TATTACTGCA GGTGTACTCT GCCAGAGAAT CCAAACAGACACT GACGTCAACTCACACT TCGTGACAGC AGAAGTCCAC TGGGCTAACC TGACTGTCAG TGAATGCCAG 180 GAGATGCATG GAGAGTTCAT GGGATCTGCG TGCGGCCATC ATGGACCCTA CACTCCTGAT 240 GTCCTCTTTT GGTCCTGTAT TCTCTTTTTC ACCACCTTCA TCCTCTCAAG CACCTTAAAG 300 AC 302

[0038]

[SEQ ID NO: 8] Sequence length: 314 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence CAAGCTTGCA GATTACTACC CCATCAACTC CAA
CTTCAAAA GTGGGCTACA ACACTCTCTT 60 TTCCTGTACC TGTGTGCCAC CTGACCCAGC TAA
TATCTCA ATATCTAATG ACACCACACT 120 GGCCCCAGAG TATTTGCCAA CTATGTCTTC TAC
TGACATG TACCATATA CTACCTTTGA 180 CTGGGGCATTT TTGTCGAAGA AGGAGTGTTC AAA
ATACGGA GGAAACCTTG TCGGGAACAA 240 CTGTAATTTT GTTCCTGATA TCACCACTCAT GTC
TTTTATC CTCTTCTTGG GAACCTACAC 300 CTCTTCCATG GCTC
314

[0039]

[SEQ ID NO: 9] Sequence length: 292 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence TTTAGGAGAA ACATATGCAT TTAATATGCA CAACAACTTA GATAAACTGA CCAGCTACTC 60 ATGTGTATGT ACTGAACCTC CAAACCCCAG CAATGAAGATCATAGAGAT 120 GCACACAATA TTTCCTGGAG AAATCTTACT GTTTCTGAAT GTAAAAAACT 180 TCGTGGTGTA TTCTTGGGGT CAGCTTGTGG TCATCATGGA CCTTATATTC CAGATGTGCT 240 CTTTTGGTGT GTCATCTTGT TTTTCACAAC ATTTTTTCTG TCTTCATTCC TC 292

[0040]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the DNA encoding the brain-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 brain-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 brain-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 brain-derived protein of the present invention prepared based on the amino acid sequences shown in FIGS. Portions 1 to 10 indicate hydrophobic domains.

FIG. 5: mRN encoding the brain-derived protein of the present invention
The result of examining the expression level of A in each human tissue by Northern hybridization is shown. Heart is the heart, Brain
Is the brain, Placenta is the placenta, Lung is the lungs, Liver is the liver, Skeletal Muscle is the skeletal muscle, and Kidney is the kidney. The number (kb) on the left indicates the size of the RNA molecular weight marker. A is the expression level of the protein of the present invention, B is the human kidney-derived Na ⁺ -HCO ₃ ^- cotransporter protein hNBC1
The amount of expression, C is Na ⁺ -HCO ₃ from human retina ^- showing the expression level of the protein HNBC2.

FIG. 6 shows hNBC3-expressing the brain-derived protein of the present invention.
10 shows the results of measuring the increase in intracellular pH after 10 / CHOK1 cells were subjected to ammonium ion prepulse and then a buffer containing sodium ions and bicarbonate ions was added. NBC3-10 brain-derived Na ⁺ -HCO ₃ of the present invention ^- indicates hNBC3-10 / CHOK1 cells expressing the co-transporter protein,
CHOK1 indicates wild-type CHOK1 cells. The numbers on the left are
The intracellular pH (450nm / 450nm / It shows the amount of increase in the ratio of fluorescence at 490 nm).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C07K 14/47 C07K 14/47 C12N 1/21 C12N 1/21 9/99 9/99 C12P 21/02 C12P 21/02 C 21/08 21/08 G01N 33/566 G01N 33/566 33/577 33/577 B // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) F term ( Reference) 4B024 AA01 CA04 DA02 GA27 HA17 4B064 AG02 AG27 CA02 CA10 CA19 CA20 CC24 DA03 DA13 4B065 AA26X AA91X AA93Y AB01 BA02 CA24 CA25 CA44 CA46 4C084 AA07 AA17 BA01 BA22 BA44 CA53 DA39 NA06 NA07 ZA021 ZA361 ZA361 ZA361 ZA361 ZA361 FA74

Claims (13)

[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 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, the partial peptide according to claim 3, or a salt thereof. 10. The protein according to claim 1, the partial peptide according to claim 3 or a salt thereof, wherein the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof is used. ⁺ -HCO ₃ ^- method of screening a compound or its salt that promotes or inhibits the cotransporter activity. 11. The protein according to claim 1, the partial peptide according to claim 3, or a salt thereof, wherein the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof is used. ⁺ -HCO ₃ ^- cotransporter activity that promotes or inhibits kit for screening a compound or a salt thereof. 12. The protein according to claim 1, obtained by using the screening method according to claim 10 or the screening kit according to claim 11, the partial peptide according to claim 3, or a Na ⁺ -HCO salt thereof. ₃ ^- the compound or its salt that promotes or inhibits the cotransporter activity. 13. A protein according to claim 1, a partial peptide according to claim 3, or a Na ⁺ -HCO salt thereof, which is obtained by using the screening method according to claim 10 or the screening kit according to claim 11. _A medicament comprising a compound that promotes or inhibits ₃ ^- cotransporter activity or a salt thereof.