JPH06197765A - Cholescystokinin b/gastrin receptor - Google Patents

Abstract

(57) [Abstract] [Structure]
The present invention relates to a human CCKB / gastrin receptor nucleotide sequence or polypeptide and a method for producing the same. [Effect] The availability of the human CCKB / gastrin receptor is useful for screening and evaluation of drugs acting on the human diseases such as gastric acid secretion inhibitors and anxiolytic agents.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a human CCKB / gastrin receptor used in an evaluation system necessary for developing a drug, particularly a cholecystokinin (CCK) B / gastrin receptor agonist.

[0002]

Cholecystokinin (CCK) is a neurogenic peptide existing in the central nerves and peripheral nerves such as the digestive system. On the other hand, gastrin is a peptide hormone secreted by G cells present in the gastric pylorus and has an action of inducing acid secretion in the gastric mucosa. Both peptides have a common 5 C-terminal amino acid sequence,
It has been shown to have an affinity for the same receptors present on the cell membrane. The physiological effects of these peptides are
Since it is induced by binding to the receptor, elucidation of the property of each receptor is important for elucidating the physiological phenomenon of each peptide, and thus the relation with the pathological condition.

CCK and gastrin receptors can be roughly classified into two types (CCKA receptor and CCKB / gastrin receptor) according to the difference in affinity for each peptide. The CCKA receptor has an affinity for CCK about 1000 times higher than that of gastrin, whereas the CCKB / gastrin receptor shows almost the same affinity for gastrin and CCK. The CCKA receptor is expressed in the digestive tract, particularly in the pancreas, and is considered to be involved in the secretion of digestive enzymes. On the other hand, C
The CKB / gastrin receptor is mainly expressed in the brain and gastric mucosa. The CCKB / gastrin receptor expressed in gastric mucosa controls gastric acid secretion and gastric mucosal proliferation by gastrin, and the centrally expressed receptor causes CCK-induced mental anxiety and panic symptoms. It is believed to be related. Therefore, inhibition of the binding between the CCKB / gastrin receptor and its ligand may lead to the improvement of central diseases such as gastrointestinal diseases such as gastric ulcer due to excessive gastric acid secretion and anxiety reactions.

[0004] Generally, a binding inhibition experiment between a ligand and a receptor is carried out using a ligand labeled with a radioactive element and an animal tissue that highly expresses the receptor. As the animal tissue to be used, in reality, available animal tissue other than human is selected. However, the nature of the receptor, even if it is classified into the same type, usually varies depending on the species from which it is derived. In other words, when searching for inhibitors of ligand / receptor binding targeting human diseases, human-derived receptors are required.

[0005]

Therefore, the present inventors have
As a result of earnest research using genetic engineering techniques, the present invention was completed by first isolating a gene encoding a human-derived CCKB / gastrin receptor and further determining the entire nucleotide sequence. That is, the object of the present invention is CC
It is to provide a gene encoding a KB / gastrin receptor or a polypeptide having a function similar to that of the receptor. Another object of the present invention is to provide a CCKB / gastrin receptor expressed using the gene or a polypeptide having a function similar to that of the receptor. A further object is to provide a vector incorporating the above gene, a cell transformed with the vector, and an antibody that binds to the CCKB / gastrin receptor. Other objects of the present invention will be apparent from the description.

[0006]

The gene provided by the present invention is the third of the sequences shown in SEQ ID NO: 1 in the sequence listing below.
The base sequence is from the 1st to the 1346th.
This gene encodes the human CCKB / gastrin receptor. The gene of the present invention also includes a partial sequence of the above sequence, which is capable of expressing a polypeptide having a function similar to that of the human CCKB / gastrin receptor. The human CCKB / gastrin receptor of the present invention is a polypeptide having the amino acid sequence shown by SEQ ID NO: 2.

Next, a method for producing the gene or polypeptide of the present invention will be described. First production method: First, human CCKB / gastrin receptor DN
M from a human cell or tissue capable of producing A
Extract RNA. Then, using this mRNA as a template, two types of primers sandwiching the receptor mRNA or a part of the mRNA region are used. The receptor cDNA or a part thereof can be obtained by performing reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR). Furthermore, by incorporating the obtained human CCKB / gastrin receptor cDNA or a part thereof into an appropriate expression vector, a host cell can be expressed and the receptor DNA can be produced.

The above manufacturing method will be described in detail below. 1) Extraction of mRNA The human CCKB / gastrin receptor DNA of the present invention is known as an mRNA that includes a human cell having the receptor-producing ability, such as a human brain cell or a human gastric mucosal cell, which encodes the receptor. Extract by the method of. Examples of the extraction method include the guanidine / thiocyanate / hot / phenol method, the guanidine / thiocyanate / guanidine / hydrochloric acid method, and the guanidine / thiocyanate cesium chloride method is preferable.

2) Purification of mRNA Purification of mRNA may be carried out by a conventional method. For example, the mRNA can be purified by adsorbing and eluting it on an oligo (dT) cellulose column. Furthermore, mRNA can be further fractionated by a sucrose density gradient centrifugation method or the like. Also,
Extracted mRNA is commercially available without extracting mRNA. To confirm that the above mRNA contains that encoding the human CCKB / gastrin receptor, m
RNA is translated to examine the affinity of the receptor for CCK or gastrin, or the change in intracellular second messenger (eg, IP ₃ production or increase in intracellular Ca ⁺⁺ concentration) upon gastrin or CCK stimulation, or It can be carried out by a method such as allowing a specific antibody to act on the receptor for detection.

For example, Xenopus la
evis) oocytes were injected with mRNA for translation (Gurdon, JB et al . (1972) Nature 233 , 177-182), or a translation reaction using rabbit reticulocyte system or wheat germ system was performed. (Schleif, RFand Wensink, PC (1
981): "Practical Methods in Molecular Biology", Spri
nger-Verlog, NY.).

3) Synthesis of first strand cDNA and said cDNA
PCR using purified mRNA as a template with random primer or oligo d
In the presence of T primer, reverse transcriptase reaction is performed to synthesize first strand cDNA. This synthesis can be performed by a conventional method. The obtained first-strand cDNA or a part of the region thereof was subjected to PCR using two kinds of primers sandwiched between
The target human CCKB / gastrin receptor DNA is amplified. The obtained DNA is fractionated by agarose gel electrophoresis or the like.

Second production method: The gene of the present invention can be produced by a conventional genetic engineering method in addition to the above-mentioned production method. First, single-stranded cDNA was synthesized using reverse transcriptase using the mRNA obtained in 2) above as a template.
Double-stranded cDNA is synthesized from the single-stranded cDNA. As the method, the Si nuclease method (Efstratiadis, A. et al .
(1976) Cell 7 .279-288), Land method (Land, H. et al . (198
1) Nucleic Acids Res. 9.2251-2266), O.Joon Yoo method (Yo
o, OJ et al . (1983) Proc.Natl.Acad.Sci.USA 79 , 1049-1
053), Okayama-Berg method (Okayama, H. and Berg, P. (1982) M
ol.Cell.Biol. 2 , 161-170) and the like.

Next, the recombinant plasmid obtained by the above-mentioned method is introduced into Escherichia coli, for example, DH5 α strain for transformation, and recombinants can be selected using tetracycline resistance or ampicillin resistance as an index. Transformation of a host cell can be performed, for example, when the host cell is E. coli.
Hanahan method (Hanahan, D. (1983) J.Mol.Biol. 166, 5
57-580), ie CaCl ₂ or MgCl ₂ or Rb
It can be carried out by a method of adding the recombinant DNA to a competent cell prepared by coexisting with Cl. As the vector, a phage vector such as lambda can be used in addition to the plasmid. As a method for selecting a strain having the desired human-derived CCKB / gastrin receptor DNA from the transformants obtained above, for example, the following various methods can be adopted.

Screening Method Using Synthetic Oligonucleotide Probes Oligonucleotides corresponding to all or part of the human-derived CCKB / gastrin receptor were synthesized (in this case, the nucleotide sequence derived using the frequency of codon usage or a possible nucleotide sequence It may be any of a plurality of nucleotide sequences in combination, and in the latter case, inosine can be included to reduce its type), and this can be used as a probe (labeled with ³² P or ³⁵ S) to transform the transformant. The DNA is hybridized with a denaturing and fixed nitrocellulose filter, and the obtained positive strain is searched for,
Select this.

Screening Method Using Probes Produced by Polymerase Chain Reaction Oligonucleotides of sense strand and antisense strand corresponding to a part of human-derived CCKB / gastrin receptor were synthesized, and these were combined to perform polymerase chain reaction (Saiki, Rk et al . (1988) Science 239 , 487-49.
1) is performed to amplify a DNA fragment encoding all or part of the desired CCKB / gastrin receptor. As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of cells producing human CCKB / gastrin receptor or genomic DNA can be used. A fragment of the DNA thus prepared is labeled with ³² P or ³⁵ S, and this is used as a probe to carry out colony hybridization or plaque hybridization to select a target clone.

Method for screening by producing human-derived CCKB / gastrin receptor in other animal cells A transformant is cultured, a gene is amplified, and the gene is transfected into an animal cell (in which case self-renewal is possible). A plasmid containing a transcription promoter region or a plasmid capable of being integrated into the chromosome of an animal cell may be used), and a protein encoded by the gene is produced on the cell surface. Then, the binding to gastrin or cholecystokinin is measured, the change in the second messenger in response to gastrin or cholecystokinin, or the antibody to human CCKB / gastrin receptor is used to detect the receptor. By doing so, a strain having a cDNA encoding the desired human-derived CCKB / gastrin receptor is selected from the original transformants.

Method for selecting using antibody to human-derived CCKB / gastrin receptor In advance, cDNA is incorporated into an expression vector to produce a protein on the surface of a transformant, and an antibody to human-derived CCKB / gastrin receptor and the antibody to the antibody A secondary antibody is used to detect a desired CCKB / gastrin receptor-producing strain, and a target strain is selected.

Method using selective hybridization translation system cDNA obtained from the transformant is blotted on a nitrocellulose filter or the like to hybridize mRNA from human CCKB / gastrin receptor cells, and then cDNA The mRNA bound to is dissociated and collected. The recovered mRNA is translated into a protein by a protein translation system, such as injection into Xenopus oocytes, or cell-free system such as rabbit reticulocyte lysate or wheat germ, and binding of the protein with gastrin or cholecystokinin. Or a human-derived CCKB / gastrin receptor antibody is used to select the strain of interest.

From the target transformant obtained, human-derived C
The method for collecting the DNA encoding the CKB / gastrin receptor is known in the art (Maniatis, T. et al . (1982): "Mol.
ecular Cloning-A Laboratory Manual "Cold Spring Har
bor Laboratory, NY). For example, it can be carried out by separating a fraction corresponding to plasmid DNA from cells and cutting out a cDNA region from the plasmid DNA.

As described above, for the sequencing of the obtained DNA, for example, the Maxam-Gilbert chemical modification method (Maxam, AMand
Gilbert, W. (1980): "Methods in Enzymology" 65 , 499-55
9) and dideoxynucleotide chain termination method using M13 phage (Messing, J. and Vieira, J (1982) Gene 19 , 269-27.
6) etc. The fragment containing the gene encoding the human-derived CCKB / gastrin receptor thus cloned can be transformed into another eukaryotic host cell by reintegration into an appropriate vector DNA. Furthermore, it is possible to express the gene in each host cell by introducing an appropriate promoter and a sequence involved in expression into these vectors.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like. Examples of vertebrate cells are COS cells (Gluzman, Y. (1981) Ce, which are monkey cells).
ll 23, 175-182) and the dihydrofolate reductase deficient strain of Chinese hamster ovary cells (CHO) (Urlau
b, G.and Chasin, LA (1980) Proc.Natl.Acad.Sci.USA 7
7 , 4216-4220) are often used, but the invention is not limited to these.

As an expression vector for vertebrate cells, one having a promoter located upstream of the gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. It may have an origin of replication. An example of the expression vector is pS having an early promoter of SV40.
V2dhfr (Subramani, S. et al . (1981) Mol.Cell.Biol. 1 , 85
4-864), etc., but not limited thereto.

Taking the case of using COS cells as a host cell, the expression vector has an SV40 origin of replication and is capable of autonomous growth in COS cells. Furthermore, a transcription promoter, a transcription termination signal and an RNA splice are used. It is possible to use a device having a part. The expression vector is the DEAE-dextran method (Luth
man, H.and Magnusson, G. (1983) Nucleic Acids Res. 11 , 1
295-1308), calcium phosphate-DNA coprecipitation method (Graham, F.
L. and van der Ed, AJ (1973) Virology 52 , 456-457) and electric pulse drilling (Neumann, E. et al . (1982) EMBO J.
1 , 841-845) and the like, and can be incorporated into COS cells, and thus desired transformed cells can be obtained. When CHO cells are used as the host cells, a vector capable of expressing the neo gene that functions as a G418 resistance marker, such as p
RSVneo (Sambrook, J. et al . (1989): "Molecular Cloning
-A Laboratory Manual "Cold Spring Harbor Laborator
y, NY) or pSV2-neo (Southern, PJandBerg, P. (1982) J.Mo
l.Appl.Genet. 1 , 327-341) etc. and selected by selecting G418-resistant colonies.
Transformed cells that stably produce the KB / gastrin receptor can be obtained.

The desired transformant obtained above can be cultured according to a conventional method, and the CCKB / gastrin receptor is produced in the cell or on the cell surface by the culture.
As the medium used for the culturing, various kinds of medium commonly used can be appropriately selected depending on the adopted host cell. For example, in the case of the above COS cells, RPMI-1640 medium, Dulbecco's modified Eagle minimum essential medium (DMEM), etc. A medium to which a serum component such as fetal bovine serum (FBS) is added can be used.

As described above, the CCKB / gastrin receptor produced intracellularly or extracellularly of the transformant can be obtained by various known separation procedures utilizing the physical properties and chemical properties of the receptor. It can be separated and purified from them. Specific examples of the method include treatment with an ordinary protein precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography ( Examples include various liquid chromatography such as HPLC), dialysis methods, and combinations thereof.

Thus, the substance obtained by the genetic engineering method using the DNA of the present invention is human-derived CCKB.
/ In order to express the gastrin receptor function, it does not necessarily have to have all of the amino acid sequences shown in SEQ ID NO: 2 in the Sequence Listing, for example, a partial sequence thereof, which is human-derived CCKB / As long as they exhibit gastrin receptor function, those amino acid sequences are also included in the polypeptide of the present invention. The present invention also includes a DNA encoding the receptor represented by SEQ ID NO: 1 in Sequence Listing. The C
The DNA encoding the CKB / gastrin receptor is the 3rd to 1st nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing.
A nucleotide sequence up to the 346th position is preferred.

Generally, eukaryotic genes are polymorphic (polymorp), as is known for interferon genes and the like.
hism) (for example, Nishi, T. et al . (198
5) J. Biochem. 97 , 153-159), this polymorphism may result in the replacement of one or more amino acids, or a change in the nucleotide sequence but no amino acid changes at all. There is also.

CCKB / gastrin receptor consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, one or more amino acid residues in one or more sites in the amino acid sequence are missing The inserted or substituted polypeptide may also have CCKB / gastrin binding activity. In the present invention, these polypeptides are called synonymous with CCKB / gastrin receptor.

For example, interleukin 2 (IL-2)
It has already been known that a protein obtained by converting a nucleotide sequence corresponding to cysteine of a gene into a nucleotide sequence corresponding to serine retains IL-2 activity (Wang, A. et al . (1984). Science 224 , 143-1433). Therefore, any such naturally occurring or artificially synthesized polypeptide CCKB / gastrin binding activity is included in the present invention.

The present invention also includes all DNAs having the same nucleotide sequence encoding these polypeptides. Such various DNAs of the present invention are
Based on the information on the human-derived CCKB / gastrin receptor, for example, the phosphite triester method (Hunkapil
Ler, M. et al . (1984) Nature 10 , 105-111) and the like, and can also be produced by chemical synthesis of nucleic acid.

The codon for the desired amino acid is known per se, and its selection may be arbitrary. For example, it can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used (Crantham, R. et al . (1981). ) Nucleic Acids Res. 9 r43
-r74). Furthermore, partial modification of the codons of these nucleotide sequences is carried out according to a conventional method using a site-specific mutagenesis (site specific mutagenesis) using a primer composed of a synthetic oligonucleotide encoding the desired modification.
mutagenesis) (Mark, DF et al . (1984) Proc.Natl.Aca
d.Sci.USA8 1 , 5662-5666) etc.

Although the genes and polypeptides of the present invention and methods for producing them have been described above, the present invention can also provide a monoclonal antibody of the receptor as a CCKB / gastrin receptor agonist. Hereinafter, a method for producing a monoclonal antibody of the receptor will be described. Human-derived C as a monoclonal antibody or fragment
Any one that binds to the CBK / gastrin receptor is included, but in particular, the monoclonal antibody and fragment F (a) prepared by sensitizing cells expressing the receptor
b ′) ₂ Fab ′ and Fab. Monoclonal antibodies are produced by culturing mouse hybridomas, or hybridomas selected by further cloning by a method such as limiting dilution, for example, by culturing a variant of cells with high antibody productivity in a medium or ascites of mice. .
The fragment F (ab ') ₂ Fab', Fab is a monoclonal antibody which is a proteolytic enzyme, preferably trypsin,
It can be obtained by digesting with an enzyme selected from the group consisting of papain and pepsin and performing appropriate purification.

Alternatively, it can be obtained by incorporating a part of a gene encoding a monoclonal antibody into an expression vector and introducing it into Escherichia coli for production. Mouse hybridoma was sensitized with cells expressing the receptor B
alb / c mouse splenocytes and mouse myeloma cells P3
X 63 Ag8 / U1 (P3U1) can be obtained by fusion with a conventional method, for example, the cell fusion method of Kueller and Milstein.

As a medium for culturing the above hybridoma, a minimal essential medium (Dalbec) modified by Dulbecco's modified Eagle.
co's modified minimum essential medium: DME
A medium in which fetal calf serum, L-glutamine, glucose, sodium pyruvate, 2-mercaptoethanol, and antibiotics (for example, penicillin G, streptomycin, gentamicin, etc.) is contained in M) is used. Cultivation of hybridomas of this expression is usually carried out in a gas phase of 5% carbon dioxide and 95% air at 37 ° C in a medium.
10 to 2 in the abdominal cavity of Balb / c mouse pretreated with 4,6,10,14-tetramethylpentadecane (trade name, Pristane, manufactured by Aldrich) for 4 days.
It is carried out in about 0 days to produce a purifiable amount of antibody. The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional method for protein isolation and purification. Examples of such methods include centrifugation, dialysis, salting out with ammonium sulfate, DEAE.
Column chromatography with cellulose, hydroxyapatite, protein A agarose and the like. The thus separated and purified antibody is digested by a proteolytic enzyme such as pepsin, papain, etc. by a conventional method, and subsequently, an active fragment such as F ( ab ') ₂
, Fab ′, Fab, Fv can be obtained.

[0035]

INDUSTRIAL APPLICABILITY The present invention makes it possible to obtain the human CCKB / gastrin receptor, and is useful for searching and evaluating drugs that act on the human disease, such as gastric acid secretion inhibitors and anxiolytics. Also, a monoclonal antibody of the receptor can be provided as a drug acting on the receptor.

It is also possible to prepare a CCKB / gastrin receptor mutein by modifying a part of the vector of the present invention. By examining the obtained mutant ligand binding activity, it is possible to examine which amino acid sequence of the receptor is important for binding. The information obtained is important for searching what kind of structure is required for the binding inhibitor. Furthermore, the receptor has not only a structure for binding to the ligand but also a structure for controlling the fluctuation of intracellular second messenger. The creation of muteins by vector modification will reveal not only the structure important for binding, but also the structure involved in second messenger control. This would provide important information for the design of inhibitors of cholecystokinin or gastrin whose mechanism of action is to regulate the fluctuation of second messengers.

Evaluation method of cholecystokinin (CCK) and gastrin agonist using CCKB / gastrin receptor The agonist is measured by measuring the affinity of CCK or gastrin for the receptor of the present invention in the presence of a test drug. Can be screened. The affinity of CCK or gastrin for the receptor of the present invention is the agonist labeled with a radioisotope (for example,
[ ¹²⁵ I] Asp ¹ -CCK8 or [ ¹²⁵ I] Tyr ¹² -ga
It can be determined by measuring the amount of strin I) bound to the receptor. The actual measurement of the binding amount is in accordance with Example 3.

[0038]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples.

Example 1 Isolation of Human CCKB / Gastrin Receptor cDNA Clone CCKB / Gastrin Receptor cDNA was already isolated from dog gastric mucosa (Kopin et al .: Proc.Natl.Acad.Sci.USA, 89,3).
605-3609, 1992), rat brain (Wank et al .: Proc.Natl.Aca
d.Sci.USA, 89,8691-8695,1992) and mastomys n
atalensis ECL tumor cells (Nakata et al .: Biochim.Bi
ophys.Res.Commun., 187,1151-1157,1992). The N- and C-termini of the proteins encoded by these cDNAs are well conserved.

Therefore, in order to obtain the full-length cDNA of human CCKB / gastrin receptor, we used nucleotides (sequences derived from dog gastric mucosa) encoding amino acid sequences at both ends as a primer (20-mer),
Using human brain-derived mRNA as a template, reverse transcri
It was decided to perform a ptase-polymerase chain reaction (RT-PCR). The details of the execution are shown below.

Human brain mRNA (Clonetech Labo. Inc.
) From 0.5 μg, AMV reverse transcriptase (40 un
First strand cDNA was synthesized by its; Boehringer).
The reverse transcripase reaction was carried out in a 20 μl reaction solution using the random primer p (dN) ₆ .
Using the obtained first strand cDNA (1 μl) as a template, 10
In 0 μl reaction solution, add Taq DNA polymerase (2.5 unit
s; Perkin-Elmer). Forward
As a primer, 5'-CCATGGAGCGTGCT
AAAGCTG-3 'as a reverse primer,
5'-CTCAGCCAGGCCCTAGCGTG-
3'was used at 100 pmol each. PCR, denaturation temperature 94 ℃ (1 min), annealing temperature 60 ℃
(2 min), polymerase reaction temperature 72 ° C (3 mi
The cycle consisting of n) was carried out for 30 cycles. When the obtained PCP product was fractionated by agarose gel electrophoresis, a DNA fragment of about 1300 bases was found. Cut out the gel containing this DNA fragment,
Purified by GENECLEAN (BIO 101 Inc.).

The purified DNA fragment was transformed into pCRI according to the procedure of TA Cloning Kit (Invitrogen).
It was inserted into the I vector and cloned. The nucleotide sequence of the RT-PCR product inserted into the pCRII vector is
Taq DyeDeoxy Terminator Cycle Sequencing method (Applie
d Biosystems, Inc.). The nucleotide sequence and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The homology with the amino acid sequence of the receptor cloned from canine gastric mucosa, rat brain and Mastomys natalensis ECL tumor cells was 88, 88 and 90%, respectively.

Example 2 Expression of Cloned Human CCKB / Gastrin Receptor in Heterologous Cells The isolated human CCKB / Gastrin receptor cDNA was incorporated into an expression vector and transduced into African green monkey kidney-derived fibroblast cell line COS-7. Then, the receptor was expressed. First, the human CCKB / gastrin receptor cDNA was excised from the pCRII vector by treatment with Hind III and Xba I restriction enzymes and inserted into the expression vector pCDM8 (Invitrogen). 5 μg of the obtained plasmid DNA was added to COS-7 (about 1.6 × 10 ⁶ cells) culture medium together with DEAE / dextrane (200 μg / ml). To improve the efficiency of transfection, DNA / DEAE
・ 2 hours after the addition of dextrane, 15% glycerol / HEPES-buffe
It was treated with red saline for 2 minutes. The cells are [ ¹²⁵ I] CCK
Up to 8 binding experiments 2 in DMEM with 10% fetal calf serum
The cells were cultured for a day (37 ° C, 5% CO ₂ -95% air).

Example 3 [ ¹²⁵ I] CCK8 Binding Experiment The affinity of the human CCKB / gastrin receptor expressed in COS-7 for agonist was [ ¹²⁵ I] CCK8.
It can be evaluated by measuring the binding amount of. COS-7 should be peeled off from the culture dish with a cell scraper to give 2 × 10 ⁵ cells / ml.
binding buffer (25 mM HEPES (pH 7.4) and 0.1% BS
A was suspended in Hank's balanced salt solution).
0.2 ml of this cell suspension was added to [ ¹²⁵ I] CCK80.
The reaction was started by adding 05 ml (final concentration 10-240 pM). To determine the amount of non-specific binding,
The reaction was performed in the presence of 1 μM unlabeled CCK8.

Shake at 37 ° C. for 60 minutes (100 times /
Min)), then add 0.75 ml of binding buffer,
The cells were precipitated by centrifugation (5000 x g, 1 min). After removing the supernatant, the radioactivity of the precipitated cells was measured by a gamma counter. The value obtained by subtracting the non-specific binding amount from the total binding amount is defined as the specific binding amount.
Scatchard analysis of ¹²⁵ I] CCK8 binding was performed. As a result, the Kd value was 0.12 nM and the Bmax value was 7.8 fmol / 10 ⁵ cells.

[Sequence list]

SEQ ID NO: 1 Sequence Length: 1347 Sequence Type: Nucleic Acid Topology: Linear Sequence CCATGGAGCT GCTAAAGCTG AACCGGAGCG TGCAGGGAAC CGGACCCGGG CCGGGGGCTT 60 CCCTGTGCCG CCCGGGGGCG CCTCTCCTCA ACAGCAGCAGC 180 AGCGCCCTG ACAGCAGACGCAG AGCGACGACCG ACGACAGACGCAG GTGGTCCTGG 240 GACTGAGCCG CCGCCTGAGG ACTGTCACCA ATGCCTTCCT CCTCTCACTG GCAGTCAGCG 300 ACCTCCTGCT GGCTGTGGCT TGCATGCCCT TCACCCTCCT GCCCAATCTC ATGGGCACAT 360 TCATCTTTGG CACCGTCATC TGCAAGGCGG TTTCCTACCT CATGGGGGTG TCTGTGAGTG 420 TGTCCACGCT AAGCCTCGTG GCCATCGCAC TGGAGCGGTA CAGCGCCATC TGCCGACCAC 480 TGCAGGCACG AGTGTGGCAG ACGCGCTCCC ACGCGGCTCG CGTGGTTGTA GCCACGTGGC 540 TGCTGTCCGG ACTACTCATG GTGCCCTACC CCGTGTACAC TGTCGTGCAA CCAGTGGGGC 600 CTCGTGTGCT GCAGTGCGTG CATCGCTGGC CCAGTGCGCG GGTCCGCCAG ACCTGGTCCG 660 TACTGCTGCT TCTGCTCTTG TTCTTCATCC CGGGTGTGGT TATGGCCGTG GCCTACGGGC 720 TTATCTCTCG CGAGCTCTAC TTAGGGCTTC GCT TTGACGG CGACAGTGAC AGCGACAGCC 780 AAAGCAGGGT CCGAAACCAA GGCGGGCTGC CAGGGGCTGT TCACCAGAAC GGGCGTTGCC 840 GGCCTGAGAC TGGCGCGGTT GGCGAAGACA GCGATGGCTG CTACGTGCAA CTTCCACGTT 900 CCCGGCCTGC CCTGGAGCTG ACGGCGCTGA CGGCTCCTGG GCCGGGATCC GGCTCCCGGC 960 CCACCCAGGC CAAGCTGCTG GCTAAGAAGC GCGTGGTGCG AATGTTGCTG GTGATCGTTG 1020 TGCTTTTTTT TCTGTGTTGG TTGCCAGTTT ATAGTGCCAA CACGTGGCGC GCCTTTGATG 1080 GCCCGGGTGC ACACCGAGCA CTCTCGGGTG CTCCTATCTC CTTCATTCAC TTGCTGAGCT 1140 ACGCCTCGGC CTGTGTCAAC CCCCTGGTCT ACTGCTTCAT GCACCGTCGC TTTCGCCAGG 1200 CCTGCCTGGA AACTTGCGCT CGCTGCTGCC CCCGGCCTCC ACGAGCTCGC CCCAGGGCTC 1260 TTCCCGATGA GGACCCTCCCCCTCTCTCCA TTGCTTCGCT GTCCAGGCTT AGCTACACCA 1320 CCATCAGCAC GCTAGGGCCT GGCTGAG 1347

SEQ ID NO: 2 Sequence length: 447 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro 15 Gly Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn 30 Ser Ser Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg 45 Gly Ala Gly Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu 60 Tyr Ala Val Ile Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile 75 Ile Val Val Leu Gly Leu Ser Arg Arg Leu Arg Thr Val Thr Asn 90 Ala Phe Leu Leu Ser Leu Ala Val Ser Asp Leu Leu Leu Ala Val 105 Ala Cys Met Pro Phe Thr Leu Leu Pro Asn Leu Met Gly Thr Phe 120 Ile Phe Gly Thr Val Ile Cys Lys Ala Val Ser Tyr Leu Met Gly 135 Val Ser Val Ser Val Ser Thr Leu Ser Leu Val Ala Ile Ala Leu 150 Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu Gln Ala Arg Val Trp 165 Gln Thr Arg Ser His Ala Ala Arg Val Val Val Ala Thr Trp Leu 180 Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr Thr Val Val 195 Gln Pro Val Gly Pro Arg Val Leu Gln Cys Val His Arg Trp Pro 210 Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Leu Leu 225 Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser 255 Asp Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro 270 Gly Ala Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala 285 Val Gly Glu Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser 300 Arg Pro Ala Leu Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly 315 Ser Gly Ser Arg Pro Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg 330 Val Val Arg Met Leu Leu Val Ile Val Val Leu Phe Phe Leu Cys 345 Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg Ala Phe Asp Gly 360 Pro Gly Ala His Arg Ala Leu Ser Gly Ala Pro Ile Ser Phe Ile 375 His Leu Leu Ser Tyr Ala Ser Ala Cys Val Asn Pro Leu Val Tyr 390 Cys Phe Met His Arg Arg Phe Arg Gln Ala Cys Leu Glu Thr Cys 405 Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg Pro Arg Ala Leu 420 Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser Leu Ser Arg 435 Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 447

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 5/10 C12P 21/02 C 8214-4B // (C12P 21/02 C12R 1:91)

Claims (11)

[Claims] 1. The third to 1346 described in SEQ ID NO: 1.
Gene containing the th base sequence and its partial base sequence 2. A vector incorporating the gene according to claim 1. 3. A cell transformed with the vector according to claim 2. 4. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and human cholecystokinin (CCK) B /
A polypeptide comprising a partial sequence of the amino acid having a gastrin receptor function 5. A method for expressing a polypeptide having the amino acid sequence according to claim 4 or a partial sequence thereof by culturing the cell according to claim 3. 6. A method for screening a human CCKB / gastrin receptor agonist using the amino acid sequence according to claim 4 or a partial sequence thereof. 7. The method according to claim 6, wherein the human CCKB / gastrin receptor agonist is an anti-ulcer drug. 8. The method of claim 6, wherein the human CCKB / gastrin receptor agonist is an anxiolytic. 9. An antibody that binds to the human CCKB / gastrin receptor. 10. An oligonucleotide capable of hybridizing to a gene having the nucleotide sequence of SEQ ID NO: 1. 11. The oligonucleotide according to claim 10, which can be used as a primer for PCR.