WO1997037018A1 - Nouveau gene - Google Patents

Nouveau gene Download PDF

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Publication number
WO1997037018A1
WO1997037018A1 PCT/JP1997/000919 JP9700919W WO9737018A1 WO 1997037018 A1 WO1997037018 A1 WO 1997037018A1 JP 9700919 W JP9700919 W JP 9700919W WO 9737018 A1 WO9737018 A1 WO 9737018A1
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Prior art keywords
dna
peptide
substance
binding
seq
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PCT/JP1997/000919
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English (en)
Japanese (ja)
Inventor
Yoshio Takada
Chihiro Kato
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Asahi Kasei Kogyo Kabushiki Kaisha
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Publication of WO1997037018A1 publication Critical patent/WO1997037018A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel gene. More specifically, the present invention relates to DNA of a novel gene expressed in response to shear stress of vascular endothelial cells.
  • the use of the DNA of the present invention makes it possible to detect whether or not vascular endothelial cells are in a normal physiological environment where shear stress is applied, which has not been clarified conventionally. This makes it possible to elucidate the mechanism of vascular endothelial cell dysfunction.
  • the present invention relates to a DNA capable of hybridizing to the partial sequence of the above DNA; a DNA complementary to the DNA; a recombinant DNA obtained by incorporating the DNA into a replicable vector;
  • the present invention relates to a microorganism or a cell transformed with a body DNA; a peptide encoded by the DNA; and an antibody capable of binding to the peptide.
  • the present invention relates to a method for detecting a substance capable of binding to the peptide encoded by the DNA or a substance inhibiting the binding between the peptide and the substance capable of binding to the peptide. By using the detection method of the present invention, it is possible to detect a substance that develops as a drug for central nervous system diseases.
  • proteins such as physiologically active substances and receptors have different sequences depending on their biological species. Therefore, even if each organism has a related protein, it is expected that there is a difference in reactivity and effect when the protein derived from one species is given to a different species.
  • human and human ⁇ -IFN strongly bind to RNA and inhibit the activity of RNase A dimer isolated from male sperm, whereas mouse IFN has its inhibitory ability. Is reported to be missing [Biochemical Journal, March 7, 123-127 (1 995)].
  • recombinant rat protein C and rat plasma-derived protein C prolong the activated partial thromboplastin time using rat plasma in a dose-dependent manner.
  • an object of the present invention is to clarify how the function of vascular endothelial cells is controlled in a physiological environment, and more specifically, to reduce the stress of vascular endothelial cells. To detect the presence or absence of a physiological environment.
  • the present inventors have conducted intensive studies to solve the above problems, We succeeded in isolating a DNA fragment of a novel gene from a cDNA library obtained by using mRNA extracted from vascular endothelial cells subjected to stress.
  • the DNA fragment was used to isolate and obtain a DNA containing the entire length of a novel gene (hereinafter, often referred to as the HGIR gene).
  • HGIR gene a DNA containing the entire length of a novel gene.
  • the expression level of this gene was not affected by vascular endothelial cells and changed in response to restress. It was confirmed that it was possible to detect whether or not it was in a normal physiological environment.
  • the presence of the peptide encoded by the gene was confirmed in a cell extract obtained by introducing an expressible recombinant DNA into which the above gene was incorporated into animal cells. Furthermore, when the biodistribution of the mRNA of the gene of the present invention was examined, it was found that the mRNA had a high distribution in the brain, and the present invention was completed.
  • the present invention relates to an isolated DNA containing at least a peptide coding region of a human-derived full-length DNA characterized by a restriction enzyme cleavage site shown in the restriction enzyme map of FIG.
  • the transformant a substantially pure peptide encoded by the DNA, an antibody capable of binding to the peptide, and a peptide encoded by the DNA
  • a method for detecting a substance that inhibits or suppresses the binding between the peptide that is recovered by the DNA and the substance that binds to the peptide. is there.
  • the shaded area in Fig. 1 indicates the peptide code area.
  • the peptide coding region include a base sequence coding for the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1). More specifically, a preferred example is a nucleotide sequence of 1-1269 of the peptide code region. Further, it is preferable that the DNA characterized by the restriction enzyme cleavage site shown in the restriction enzyme map in FIG. 1 is 1 to 1562 of SEQ ID NO: 3. A preferred example of the peptide encoded by the DNA is the amino acid sequence of amino acid sequence of SEQ ID NO.
  • a main object of the present invention is to provide a DNA capable of detecting whether or not vascular endothelial cells are in a normal physiological environment to which restress is applied.
  • Another object of the present invention is to provide a peptide obtained and an antibody capable of binding to the peptide.
  • Another object of the present invention is to provide a method for detecting a substance capable of binding to the peptide encoded by the DNA, and a method for binding the peptide encoded by the DNA to the peptide.
  • An object of the present invention is to provide a method for detecting a substance that inhibits or suppresses binding to such a substance.
  • SEQ ID NO: 1 is an amino acid sequence of a peptide consisting of 4 23 amino acids
  • SEQ ID NO: 2 is a nucleotide sequence of a peptide code region consisting of 1269 bases;
  • SEQ ID NO: 3 is an example of the nucleotide sequence of full-length DNA represented by the restriction map in FIG. 1;
  • SEQ ID NOs: 4 and 5 were obtained using the PCR primers used to obtain the base sequences of 51 1 to 102 1 of SEQ ID NO: 2;
  • SEQ ID NOs: 6, 7, and 8 are PCR primers used for construction of the HGIR expression plasmid.
  • FIG. 1 shows a restriction enzyme map containing the DNA coding region of the DNA of the present invention.
  • the hatched region in the restriction enzyme map shows the peptide code region, the left side shows the amino terminal of the peptide, and the right side shows the carboxyl terminal.
  • FIG. 2 is a diagram showing the structure of a parallel plate type stress-reduction culture apparatus.
  • Figure 3 shows recombinant plasmids PGIR10 and PGIR2. It is a figure which shows a restriction enzyme cleavage site of 0 and a functional map. The blacked-out portion indicates the HGIR gene. The arrow corresponds to the peptide coding region, and the direction of the arrow indicates the 5 'end to the 3' end.
  • FIG. 4 shows the results of detecting the amount of mRNA corresponding to HGIR in vascular endothelial cells by Northern hybridization.
  • FIG. 5 is a diagram showing restriction enzyme cleavage sites and functional maps of the recombinant plasmids pEGRI11 and pEGIR12-FLAG.
  • the blacked-out portion indicates the HGIR gene.
  • the arrow corresponds to the peptide code region, and the direction of the arrow indicates the 5 'end to the 3' end.
  • a DNA comprising at least a full-length human DNA derived from a restriction enzyme cleavage site shown in the restriction map of FIG. 1 and comprising at least a peptide coding region.
  • DNA comprising at least the peptide coding region of the full-length human DNA derived from the restriction site shown in the restriction map shown in FIG.
  • the peptide code region has the amino acid sequence of SEQ ID NO: 1.
  • a replicable recombinant DNA comprising the DNA according to any one of 1 to 4 above incorporated into a replicable vector.
  • a replicable recombinant DNA obtained by incorporating the DNA according to 7 above into a replicable vector.
  • DNA containing at least the peptide coding region of human full-length DNA characterized by the restriction site shown in the restriction map in Figure 1, and SEQ ID NO: 1
  • At least one peptide selected from the group consisting of DNAs that can hybridize to DNA containing the nucleotide sequence of 135 is ligated to the peptide.
  • the sample is brought into contact with a sample that is considered to contain such a substance, and the change that occurs in response to the binding of the peptide to a substance that binds to the peptide is used as an index, and the A method for detecting a substance that binds to a substance.
  • At least a peptide of human-derived full-length DNA characterized by the restriction sites shown in the restriction map in Figure 1.
  • At least one kind of DNA selected from the group consisting of DNAs that hybridize to DNA containing the coding region and DNA containing the nucleotide sequence of SEQ ID NO: 2
  • Contacting a peptide that binds and a substance that binds to the peptide with an analyte that is thought to contain a substance that inhibits the binding of the peptide to a substance that binds to the peptide At least the binding between the peptide and the substance that binds to the peptide, using as an index the change that occurs in response to the binding of the peptide to the substance that binds to the peptide.
  • a detection method characterized by detecting a substance that inhibits lipase.
  • a in the nucleotide sequence indicates a deoxyadenyl residue
  • C indicates a deoxycytidyl residue
  • G indicates a deoxyguanyl residue
  • T indicates a thymidyl residue.
  • a la is alanine
  • Arg is arginine
  • a sn is asparagine
  • Asp is aspartic acid
  • Cys is cystine
  • G1n is glutamine
  • G1u is glutamic acid
  • His is histidine
  • I 1 e is isoleucine
  • Leu is leucine
  • Lys is lysine
  • Met is methionine
  • Phe is phenylalanine
  • Pro is prolin.
  • Ser, serine, Thr is threonine
  • Trp is triptophan
  • Dyyr is tyrosine
  • Va1 is norine.
  • peptide includes those generally understood by those skilled in the art as peptides, oligopeptides, polypeptides, proteins and the like.
  • the DNA of the present invention is an isolated DNA containing at least a peptide coding region of full-length DNA characterized by a restriction enzyme cleavage site shown in the restriction enzyme map of FIG. Genes containing this peptide coding region are collectively referred to as “HGIR”).
  • a preferred example of the DNA of the present invention is a DNA in which the above-mentioned peptide coding region encodes a peptide including the amino acid sequence of 1442 of SEQ ID NO: 1. Is mentioned. Specifically, the amino acid sequence of SEQ ID NO: 1 An example is a DNA in which the nucleotide sequence encoding the peptide containing the sequence is the nucleotide sequence of SEQ ID NO: 2.
  • the peptide containing the amino acid sequence of SEQ ID NO: 1 only needs to be a sequence having the characteristics thereof.Therefore, substitution or deletion of one or more bases in the nucleotide sequence encoding the above-mentioned peptide is sufficient. Or there may be additions.
  • a fusion protein can be obtained by adding a base sequence encoding a known peptide. 1205--1210 (1988)]. This fusion protein is preferable because it can be detected with an antibody against the FLAG peptide or the like.
  • the nucleotide sequence encoding the amino acid sequence of 1 to 16 of SEQ ID NO: 1 may encode a signal peptide. However, a peptide from which this site has been removed is also disclosed as a preferred example.
  • DNA encoding various amino acid residues or polypeptide residues upstream or downstream of the gene encoding the peptide containing the amino acid sequence of SEQ ID NO: 1 May be included. Further, as a preferable example of the full-length DNA recharacterized by the restriction enzyme cleavage site shown in the above restriction enzyme map, a DNA having the nucleotide sequence of SEQ ID NO: 3 including the nucleotide sequence of SEQ ID NO: 2 can be mentioned. These are preferably human-derived DNAs having genetic information that can be obtained from humans.
  • the present invention provides a method for substantially encoding the above various DNAs. And pure peptides.
  • Preferable is a peptide containing the amino acid sequence of 1-423 of SEQ ID NO: 1.
  • Cells used for preparing the DNA of the present invention are not particularly limited, but cells that respond to stress are preferably used, and particularly adherent cells, such as vascular endothelial cells, and preferably human vascular endothelium. Cells, particularly preferably human umbilical vein endothelial cells (HUVEC).
  • the vascular endothelial cells can be easily separated from the human band according to the method described in Cell, 1, 329 (1988) or Human Cell, 1, 188 (1988). It is also possible to obtain and use the separated secondary culture cells.
  • the number of passages of the vascular endothelial cells is not particularly limited as long as it retains the properties as vascular endothelial cells, but is preferably within 10 passages. Cells can be used alone or in combination of two or more.
  • perfusion culture is performed to load restress without the cells.
  • the shear stress applied to the cultured cells is not particularly limited as long as the expression is such that the expression of the gene produced by the cells can be changed, but it is generally preferable that the shear stress be 0.1 Idyne Z cm 2 or more, and it is preferable. Is at least 1.5 dynes Z cm 2, and more preferably at least 15 dynes / cm 2 .
  • the upper limit is not particularly limited as long as it is not strong enough to destroy cells or float without adhering to the adhesive surface of the culture device, and also in consideration of dynamic economic efficiency.
  • the endothelial cells can be cultured under the same restress as the restress received by the arterial and venous endothelial cells in the living body. It can be.
  • the value of shear stress can be obtained by the following method.
  • the rotating disk type is the culture device that can apply stress. : Bio rheology, 461 (1988)), parallel plate type (Bio Iechnology and Bioengineer) 1021 (1985)] and a microcarrier type [Japanese Patent Application Laid-Open No. 4-179494] are preferred, but a parallel plate type is preferred.
  • the stress applied to the cell differs depending on the surface to which the cell adheres, and the stress is different.
  • the culture device it is possible to uniformly apply shear stress to cells by selecting the adhesive surface. For example, cells may be adhered to only one side, or cells may be adhered to either one side and the opposite side, but not to the side. 1
  • a known composition can be used as a culture solution used for culturing cells.
  • vascular endothelial cells it is preferable to use a cell culture medium to which 0 to 20% of the blood of an animal such as a mouse is added.
  • E-GMUV medium containing 2% fetal calf serum, manufactured by Kurabo Industries, Japan
  • Ml99 medium supplemented with 20% fetal calf blood.
  • ECGS Endothelia cel l growth supp lemen ⁇
  • EGF [Epi derma l growt hf actor]
  • the culture conditions of vascular endothelial cells are not specified, but for example, culture is performed under the following conditions.
  • the vascular endothelial cells are adhered like a cobblestone to the cell culture surface of the culture device, and the culture conditions are set.
  • the culture temperature is not particularly limited as long as it is a temperature at which the cells can be cultured, but is preferably 37, and more preferably in an incubator filled with 5% carbon dioxide gas.
  • New The number of cells is particularly limited as long as the mRNA required for the preparation of type ⁇ cDNA can be extracted. It is not limited, but the number is such that it is performed in normal culture, preferably 1 ⁇ 10 4 or more, particularly preferably 1 ⁇ 10 6 or more.
  • the culturing time is not particularly limited as long as the expression state is clearly changed as compared with the static culture and the survival state of the cells is good, but it is preferably from 6 hours to 48 hours. ⁇ ⁇ ⁇ It is possible.
  • RNA obtained by ultracentrifugation using a cesium chloride density gradient is analyzed using an oligo (dT) column to obtain ⁇ 1y (A ) + Purification method as RNA.
  • the method of synthesizing cDNA by converting the obtained mRNA into type I is not particularly limited.For example, a primer that anneals to the po1y (A) region of mRNA is used, and the method is performed using reverse transcriptase.
  • a method for synthesizing the main-strand cDNA is exemplified.
  • the reverse transcriptase to be used at this time is not particularly limited as long as it is an enzyme capable of synthesizing a cDNA having the same length as the type III mRNA. ).
  • the obtained single-stranded cDNA can also be obtained as double-stranded cDNA using E. coli DNA polymerase I.
  • cDNA can be used as it is as type I, but after vector insertion, it is introduced into host cells. It is preferable to use the NA library.
  • a cDNA library By using a cDNA library, it is possible to maintain and maintain the cDNA, and to always amplify and supply the cDNA by culturing the transfected cells. Become.
  • the method for preparing the cDNA library is not particularly limited, and examples thereof include a linker primer method.
  • the primer used in the cDNA synthesis in this case is not particularly limited, but digestion of the cDNA synthesized using a specific restriction enzyme generates a pairing end with the vector's cloung site. It is preferable to use a method that facilitates the insertion of cDNA into a vector.
  • cDNA having an EcoRI end at the 5 ′ end and a No ⁇ I end at the 3 ′ end is obtained, and the cDNA is unidirectionally inserted into the EcoRI and NotI sites of the vector.
  • the vector holding the cDNA is not particularly limited as long as it can stably hold each cDNA, and examples thereof include plasmid vectors.
  • Preferable is Plasmid Vector PSI (promega, USA).
  • a vector arm obtained by digesting pSI with restriction enzymes EcoRI and NotI, and a cDNA having EcoRI and NotI terminus were ligated and introduced into E. coli.
  • a cDNA library that holds many types of cDNAs can be obtained efficiently. Plus middector ps!
  • CDNA is inserted in one direction from the 5 'side downstream of this promoter to create a recombinant plasmid, and the resulting recombinant plasmid is introduced into animal cells.
  • the expression of cDNA is enabled, and the protein encoded by the cDNA can be directly expressed and obtained.
  • the cDNA library is introduced into animal cells as described above.
  • this cDNA library reflects the characteristics of vascular endothelial cells that have been subjected to shear stress, it can be used for diseases that occur at low stress in blood vessels in vivo, such as arteriosclerosis. It greatly contributes to the study of bioactive factors and other related genes involved in the pathogenesis. Furthermore, the possibility of isolating the above-mentioned factors and genes using this cDNA library and the development of pharmaceuticals that control those factors and genes may be considered.
  • this cDNA library contains other cells, specifically a cDNA library prepared using mRNA extracted from vascular skin cells to which no shear stress is applied, and the like. The type of gene and its content are different.
  • a family gene cloning method using a Degenerate PCR primer [Proceedings of the National Academy of This can be performed with reference to Sciences of the United States of America, 16, 1603-1607 (1989), Sciences, UA, 569-572 (1989).
  • the DNA described in SEQ ID NOs: 4 and 5 can be used as a primer to amplify the gene by PCR.
  • the DNA prepared after amplification is a partial fragment of the gene of the present invention, and the DNA fragment sandwiched between the primers (ie, SEQ ID NO: 2 5 1 1 1 1 1 1 0 2 1 base sequence) is obtained. It is preferable that the fragment obtained by amplification is cloned on a vector. Furthermore, to clone the entire gene, for example, all or a part of the previously cloned DNA fragment, the base sequence of SEQ ID NO: 2 or 3, or a fragment thereof is used as the probe DNA. And obtaining a positive clone from the cDNA library by the hybridization method.
  • the cDNA library used at this time is not particularly limited as long as it contains the DNA of the present invention.
  • Human Brain 5, -Stretch Plus cDNA Library (Clontech, USA) Manufactured).
  • the hybridization method may be appropriately selected and carried out according to one feature of the cDNA library to be used, and a plaque hybridization method is preferable.
  • an amplification method by PCR using, as primers, DNAs having the respective sequences at the 5 ′ end and the 3 ′ end in the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 disclosed by the present application. May be prepared.
  • the DNA of the present invention can be obtained very easily by using the below-mentioned Escherichia coli M109 / pGlR 10 strain (FERM BP-5836) prepared by the present inventors as a raw material.
  • the above-described isolated DNA of the present invention is an isolated DNA containing at least a peptide coding region of full-length DNA characterized by a restriction enzyme cleavage site shown in the restriction enzyme map of FIG. DNA. Further, as the isolated DNA of the present invention, a DNA containing a partial sequence of the DNA is mentioned. this part The sequence is a continuous sequence of a length that can be used to characterize the gene HGIR by a method such as hybridization or annealing, and is a base sequence shown in the sequence listing or a salt thereof. It is a sequence containing a fragment of the base sequence complementary to the base sequence.
  • a sequence having a continuous sequence of at least 15 bases in the base sequence encoding the amino acid sequence of 1 to 45 of SEQ ID NO: 1 is preferred. Or a contiguous sequence of at least 20 bases, preferably at least 30 bases. In this fragment, more preferred embodiments include at least 10 amino acids in the amino acid sequence from 1 to 45 of SEQ ID NO: 1 (more preferably, 15 or more, particularly preferred). Or more than 20 or more than 30) continuous amino acid sequences, or a DNA fragment containing the nucleotide sequence. More specifically, the base sequence encoding the above partial amino acid sequence includes the base sequence of 11-135 of SEQ ID NO: 2.
  • the method of hybridization is not particularly limited for the isolated DNA that hybridizes to the DNA fragment having the nucleotide sequence of SEQ ID NO: 2, but is not limited to dot blot hybridization. It is possible to use a reduction, Southern blot hybridization, black hybridization, or colony hybridization.
  • the method is not particularly limited, and examples thereof include the above-mentioned hybridization method. More specifically, probe is detected after keeping the target DNA-immobilized membrane and labeled probe DNA at a fixed temperature for a certain period of time in a hybridization solution. A method for detecting a target DNA in the above method.
  • the conditions for the hybridization are not particularly limited, but, for example, the hybridization can be performed under the following conditions.
  • Pre-hybridization solution (5 ⁇ strength SSPE (0.9 M sodium chloride, 50 mM sodium phosphate and 50 mM sodium phosphate) 5 mM EDTA (pH 7.7) ⁇ , 5-fold concentration of Enhardt's solution (0.1% each: 8SA, alcohol and polyvinylpyrrolidone) and 0.5% SDS after 3 hours incubation at 5 0 ° C to liquid at a final ⁇ 1 0 0 ⁇ g / m 1 become by cormorants those plus denatured salmon sperm DNA] in denatured 32 P-labeled probe DNA to a final concentration 1 X 1 0 6 cpm / m 1 was added to the power sale by a, is et to 1 2 hours coercive heated at 5 0 ° C, the washing solution a sufficient amount of the main assembler down after thermal insulation [2X 3 3 C (0.3 M sodium chloride and 0.3 M sodium citrate) and 0.1% SDS) at room temperature, and then use a
  • the membrane is the one where DNA is fixed
  • the labeling of the probe DNA is not particularly limited, and a non-RI label such as 32 P, 33 P or 35 S, such as the strength of RI labeling, biotin, and digoxigenin can be used.
  • the high predication solution is not particularly limited, but it can be prepared using honolemamide, SSPE, SSC, Denhardt's solution, SDS, salmon sperm DNA and the like.
  • a complementary DNA to the above DNA can be obtained by a well-known base pairing rule (A and T are paired, and C and G are paired).
  • a complementary RNA to the above DNA and its complementary DNA can also be obtained by the well-known base pairing rule (A and U are paired, and C and G are paired).
  • DNA or RNA complementary to the above DNA or a fragment thereof is generally used as antisense.
  • the complementary DNA or RNA to the DNA of SEQ ID NO: 3 or the DNA fragment thereof can be used as a molecule that regulates the expression of the peptide encoded by HGIR. .
  • HGIR antisense is added to cultured cells or experimental animals expressing HGIR, The changes caused by the suppression of HGIR expression can be used to analyze the function of the peptide encoded by the HGI scale. Furthermore, administering antisense to humans for the purpose of suppressing the synthesis of the peptide is useful for treating diseases related to abnormal physiological functions of the peptide. .
  • the method of administering this antisense is not particularly limited.For example, a method of synthesizing an antisense and dissolving it in an appropriate solvent and then administering it directly, or incorporating a fragment containing an antisense sequence into an appropriate vector, It is also possible to use a method in which recombinant DNA is administered and designed so as to synthesize antisense in cells or in vivo.
  • the present inventors have conducted research on a novel gene that is expressed in response to shear stress of vascular skin cells, and at the same time, have also studied for a human-specific gene.
  • Proteins such as bioactive substances and receptors usually have different sequences depending on the species, and even if each organism has a related protein, the protein derived from one species is given to a different species. It is expected that there will be differences in reactivity and effects. Therefore, it is unquestionable that obtaining a human-specific protein would be extremely favorable for the future development of human medicine.
  • cDNA containing the full length of the HGIR gene of the present invention (that is, 1 to 15 6 of SEQ ID NO: 3) 2 base sequence).
  • Approximately 270 bp fragment upstream of this cDNA DNA fragment consisting of a piece ie, a DNA fragment containing the nucleotide sequence of SEQ ID NO: 3 obtained by digesting pGIR10 with restriction enzymes EcoRI and EaeI in a conventional manner
  • a piece ie, a DNA fragment containing the nucleotide sequence of SEQ ID NO: 3 obtained by digesting pGIR10 with restriction enzymes EcoRI and EaeI in a conventional manner
  • the HGIR gene of the present invention which is derived from a human and is different from the conventionally known mouse-derived G protein-coupled receptor gene, is a novel gene that has not been obtained so far. It was clear. Furthermore, the present invention is a recombinant DNA, comprising the DNA of any of the above-mentioned present invention.
  • the plasmid used for preparing the recombinant DNA in the present invention is not particularly limited, but a commonly used plasmid can be used.
  • Specific examples of the recombinant plasmid of the present invention include pGIR10, pGIR20, pEGIR11, and pEGIR12-FLAG.
  • PGIR10 and PGIR20 are recombinant plasmids obtained by inserting a DNA fragment of about 1.5 kbp containing the gene HGIR of the present invention into the EcoRI site of the cloning vector pUCl18. Yes, and each DNA fragment is inserted in the opposite direction.
  • FIG. 3 shows restriction enzyme cleavage sites and functional maps of pGIR10 and PGIR20.
  • the arrows shown in FIG. 3 for the DNA containing HGIR indicate about 1.2 kbp of the HGIR protein coding region in the direction from the N-terminal side to the C-terminal side of the protein.
  • the HGIR-containing DNA fragment has a restriction enzyme cleavage site shown in FIG.
  • the transformant obtained by introducing the recombinant plasmid PGIR10 is Escherichia co1i (Escherichia coli) JM109 ZPGIR10 strain (FERMBP-5836).
  • the modified expression plasmid PEGIR 12 Can build FLAG.
  • the recombinant DNA of the present invention into a known host. That is, a microorganism or cell transformed with the recombinant DNA of the present invention.
  • a peptide comprising the amino acid sequence encoded by the above-mentioned DNA or DNA fragment is preferable.
  • a peptide containing the amino acid sequence of 1 to 45 of SEQ ID NO: 1 is mentioned as a preferable example.
  • the peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided with various amino acid residues or polypeptide residues upstream or downstream of the amino acid sequence of SEQ ID NO: 1. It may contain a group. Specifically, in the amino acid sequence of SEQ ID NO: 1 It may have one or more amino acid residues upstream of the non-terminal Met, and examples of such amino acid residues include signal-like peptides. Can be Similarly, one or more amino acid residues may be further provided downstream of Ser on the carboxyl terminal side.
  • the peptide of the present invention is preferably a peptide having the amino acid sequence shown in SEQ ID NO: 1 or a sequence that retains the characteristics of the peptide as a partial fragment thereof, and is preferable.
  • the method for obtaining the peptide of the present invention is not particularly limited. Specifically, for example, a method for preparing a synthetic peptide based on information on the amino acid sequence or a method for encoding a gene encoding the peptide. A method of synthesizing a peptide by introducing it into a host cell may be mentioned.
  • the method for preparing the synthetic peptide is not particularly limited. Examples of the method include sequentially condensing an amino acid with a protected amino group with an amino acid with a protected carboxy group, and a method for preparing an oligopeptide. For example, there is a method of cutting the target.
  • a peptide-encoding gene can be introduced into a host cell to produce the peptide.
  • the peptides obtained by this method are: (a) the above-mentioned D of the present invention;
  • NA is bound to a replicable expression vector, and the replicable recombinant DN containing the DNA and the replicable expression vector
  • a peptide produced by a method including substantially isolating the peptide from a transformant in which the peptide has been cultured is not particularly limited, a gene encoding the peptide of interest, specifically, for example, the nucleotide sequence of SEQ ID NO: 2 It is preferable to obtain the peptide by introducing it into a host cell and expressing it using a recombinant DNA obtained by ligating the DNA containing the DNA downstream of a promoter that works in the host cell.
  • Microorganisms or cells as host cells are preferably non-limiting animal cells. Specifically, COS-7 cells or COS-1 cells derived from African kidney monkey kidneys are preferred. CHO-K1 cells derived from Chinese hamster ovary and the like.
  • the promoter is not particularly limited as long as it works efficiently in the host cell, and examples thereof include a promoter derived from the IE (immediate ear) gene of human cytomegalovirus (CMV).
  • CMV human cytomegalovirus
  • the recombinant DNA in which HGIR is connected downstream of such a promoter is not particularly limited.
  • the above-mentioned DNA having pEGIRl1 includes PEGIR12-FLAG.
  • vascular endothelial cells By measuring the amount of mRNA transcribed by HGIR, it is possible to indirectly measure the restress value without affecting the endothelial cells.
  • the method for analyzing the amount of mRNA is not particularly limited. For example, Northern hybridization using a probe DNA, a probe RNA, a PCR primer or the like created from the cDNA sequence of HGIR shown in SEQ ID NO: 3 Analysis methods such as in situ hybridization and reverse transcription PCR are used.
  • an increase in the amount of mRNA transcribed into the gene means an increase in the amount of the peptide encoded by the gene
  • the amount of the peptide encoded by HGIR present in vascular endothelial cells By measuring the stress, it is also possible to indirectly measure the restress value without acting on the endothelial cells.
  • the method of confirming the peptide encoded by HGIR in the cells or tissues is not particularly limited.
  • the extract obtained from the cells or tissues of interest is subjected to Western blotting, and detected using an antibody. Methods, methods for directly examining peptides on the cell membrane surface by immunostaining, and methods for analyzing by flow cytometry.
  • the method for obtaining the antibody of the present invention is not particularly limited. It is mentioned as.
  • a peptide for producing an antibody a peptide comprising the amino acid sequence of SEQ ID NO: 1 is used. Alternatively, some fragments thereof can be mentioned, and these peptides can be synthesized by the above-mentioned method.
  • the length of the peptide is not particularly limited as long as it can be characterized as a peptide encoded by HGIR, but is preferably, for example, 10 amino acids or more, and more preferably 20 amino acids. And the like.
  • This peptide can be used as is, or with KLH (keyho 1 e-1 impet hemocyanin), BSA (preferred blood phenol), and ivy carrier protein, and optionally with an adjuvant. Then, by collecting the serum, it is possible to obtain an antihemoprotein containing an antibody (polyclonal antibody) that recognizes the peptide encoded by HGIR. It is also possible to purify and use the antibody instead of the antiserum. Examples of animals to be inoculated include sheep, magpies, goats, goats, mice, rats, and the like. New It is also possible to obtain a recombinant monoclonal antibody by a known method for producing hybridoma cells. In this case, mouse-derived cells are preferred.
  • the method for measuring the gene of the present invention as a measurement target, for example, the above-described method for measuring the amount of mRNA or the amount of peptide, a disease occurring in a low-stress site of a blood vessel in a living body, specifically, an artery Detect areas where sclerosis or the like is likely to occur, and take preventive measures to prevent the onset of sclerosis, or to perform early treatment by controlling vascular endothelial cell function in those areas .
  • HGIR By estimating the function of this endothelial cell from the expression state of it, the involvement of a physiologically active substance in the state where shear stress is applied, or searching for new molecules related to stress, the disease onset It also leads to the elucidation of the mechanism of the work.
  • the peptide encoded by the gene HGIR of the present invention exists on the cell membrane as a receptor. Furthermore, the biodistribution of the mRNA of the gene HGIR of the present invention, that is, the tissues or cells expressing the mRNA, were examined using methods such as Northern hybridization and insitu hybridization. In this regard, for example, according to the analysis of mRNA distribution of HGIR in human tissues by Northern High Bridging, the gene of the present invention shows a high distribution in the brain. It became clear. In other words, the peptides encoded by HGIR are considered to work physiologically in the brain.
  • HGIR clone was successfully used in the examples of the present application by using a human brain-derived cDNA library.
  • the purpose is to use the insitu hybridization method. Is useful. In this method, a tissue derived from a living body is sliced to a thickness of about 1 ⁇ , fixed on a slide glass, and then a radiolabel or enzyme capable of detecting HGIR mRNA.
  • the probe DNA or RNA labeled with a label or the like is hybridized in an appropriate buffer, detected using the optimal method for the type of label, and the HGIR mRNA is present. It allows for detailed analysis of sites and cells. According to this analysis, HGIR mRNA is expressed at specific sites in the brain, including the hippocampus, thalamus, hypothalamus, papillary body, outer olfactory nucleus, ventromedial thalamus, and dorsal tegmental nucleus This is allowed. In other words, the peptides encoded by this HGIR are considered to have functions related to the central nervous system at these sites.
  • these expression sites are said to be sites involved in emotion and memory among the central functions.
  • this peptide has a physiological function as a receptor for a neurotransmitter in the central nervous system. It is thought that they have physiological functions related to emotion or memory. Therefore, the above-mentioned receptor encoded by HGIR is a ligand that is a substance that binds to the receptor and causes an action, that is, a substance that acts to enhance the function of the receptor, or suppresses or inhibits the function of the receptor. It can be used to detect inhibitors and can be applied to pharmaceuticals for central nervous disorders, as well as disorders of emotion or memory.
  • the present invention includes at least the peptide coding region of the above-mentioned peptide, that is, a full-length human-derived DNA characterized by the restriction site shown in the restriction map of FIG.
  • At least one kind of DNA encoded by at least one kind of DNA selected from the group consisting of DNAs that can hybridize to the DNA containing the base sequence of SEQ ID NO: 2 Used to contact a subject suspected of containing a substance capable of binding to the peptide and to examine changes occurring in response to the binding of the peptide to a substance capable of binding to the peptide.
  • a detection method characterized by detecting a substance that binds to the peptide, or the aforementioned peptide and a substance that binds to the peptide are referred to as the peptide.
  • the sample is brought into contact with a sample which is considered to contain a substance which inhibits the binding to the peptide, and the peptide and the substance which bind to the peptide are brought into contact with each other. Binding to the peptide and the peptide using the change that occurs in response to the binding as an indicator
  • This detection method is characterized by detecting substances that inhibit the binding of such substances.
  • the above-mentioned peptide of the present invention is brought into contact with a substance in a subject which is considered to contain a substance capable of binding to the peptide.
  • This is a detection method for detecting a substance using a change occurring in response to the substance as an index.
  • another typical example of the detection method of the present invention is a method of combining the peptide with a substance capable of binding to the peptide in the presence of the substance capable of binding to the peptide.
  • Bringing a test sample suspected of containing a substance that inhibits binding into contact with the peptide and the peptide Detection using a change that occurs in response to a substance that binds to the peptide as an indicator to detect a substance that suppresses or inhibits the binding of the peptide to the substance that binds to the peptide Is the way.
  • a substance that binds to the peptide, or the peptide and the peptide may bind to the peptide.
  • a screening method for selecting a substance that inhibits binding to a substance can be cited. According to this screening method, it will be possible to find new and useful drugs for central nervous system diseases.
  • the amount of a substance that binds to the peptide or a substance that inhibits the binding between the peptide and a substance that binds to the peptide is measured.
  • the measurement method is mentioned. It is a measurement of the abundance, and if exactly a quantitative, in some cases simply determines the presence or absence of the substance, Ri by the and this use of good c This measurement method be any, in the central It will provide a useful measurement tool for the study of drugs for neurological diseases.
  • the HGIR gene or its DNA fragment it is possible to use the HGIR gene or its DNA fragment to obtain a substantially pure peptide of the present invention encoded by the DNA.
  • the peptides can be obtained by peptide synthesis using a condensation reaction of amino acids.
  • the peptide of the present invention is expressed on a cell membrane.
  • a peptide in an impregnated form that is, a cell itself expressing the peptide on the cell membrane, a fraction of the cell membrane only of the above cells, or a peptide purified by a known method, etc. Therefore, various peptides can be used based on the specific purpose of the detection method of the present invention.
  • the subject used for screening for a substance capable of binding to the peptide is not particularly limited.
  • an extract or culture derived from a tissue or a cell derived from a living body considered to include a physiological ligand The above can be used. In this case, those derived from humans are preferred.
  • physiological ligands it is possible to search for a substance that binds to the peptide by using a synthetic compound or a microorganism for culture.
  • an extract derived from a biological tissue or a cell can be used.
  • Culture supernatants such as culture supernatants of synthetic compounds and microorganisms, can be used.
  • the method for detecting a substance that binds to the peptide is not particularly limited.
  • Binding to the peptide and the peptide The method for detecting the binding to such a substance is not particularly limited.
  • a substance capable of binding to the peptide is labeled in advance, and the peptide and the peptide are expressed.
  • the unbound substance is added to the cell or its cell membrane fraction, and the unbound substance is removed by washing, and the change in the amount of the label bound to the peptide, cell or cell membrane fraction can be detected.
  • the peptide, cell or cell membrane fraction is immobilized on a stationary phase.
  • the type of label is not particularly limited, and a radioactive label, a fluorescent label, a chemiluminescent label, an antigen label, an enzyme label, or the like can be used.
  • an optimal method may be appropriately used depending on the type of the label.
  • the presence or absence and the strength of the peptide on the physiological function can be measured, and a measurement system using these as an index can be constructed.
  • the signal is transmitted to the whole cell through a change in a signal transmitting substance in the cell.
  • the method for detecting the signal of the cell ⁇ ⁇ ⁇ is not particularly limited, and may be any method as long as it is a method for detecting a signal change in a cell caused by a substance that binds to the peptide via the peptide.
  • the concentration of calcimuion there are methods for detecting the concentration of calcimuion, the concentration of cAMP, the amount of inositol phosphoric acid released, the activity of phospholipase C, and the like.
  • Each inspection The extraction method is not particularly limited.For example, in the case of measuring the concentration of calcium, the method of measuring the fluorescence intensity using the fluorescent dye fura 2 which is sensitive to calcium or the method of measuring the calcium-binding photoprotein ethanol. An example is a detection method using phosphorus.
  • a system for detecting the binding between the peptide and a substance capable of binding to the peptide or a system for detecting a change in intracellular signal caused by the substance capable of binding to the peptide may be used.
  • the substance containing the substance capable of binding to the peptide and the substance inhibiting the binding of the peptide is contained. Addition of a possible analyte, and comparing the measured values of the group without the analyte and the group with the analyte indicate that the binding between the peptide and the substance that binds to the peptide is inhibited. You can search for substances, measure their abundances, and analyze their activity.
  • the substance that binds to the peptide to be added As the substance that binds to the peptide to be added, the aforementioned substance that binds to the peptide is used. In addition, a substance that binds to the peptide and a substance that inhibits binding to the peptide are included.
  • the analytes that may be considered to be possessed are not particularly limited, but may be extracts and cultures of tissues and cells derived from living organisms, culture supernatants of synthetic compounds and microorganisms, and the methods described above. And antibodies against the obtained peptide.
  • Example 1
  • HU VEC Endoc e1 1 — UV, manufactured by Kurabo Industries, Japan
  • a large parallel plate type restress load culture device described in Japanese Patent Application Laid-Open No. 7-274949, shown in FIG. 2, was used.
  • Place a glass cover slip PYREX, 94 x 52 x 1.0 mm, Corning, USA
  • a 150 mm ⁇ plastic dish unc, Denmark.
  • Cells subjected to shear stress are removed from the device together with the coverslip, and phosphate-buffered saline that has been kept at 37 ° C in advance.
  • CPBS (I) 137 mM sodium chloride, 2.7 mM calcium chloride, 4.3 mM sodium hydrogen phosphate, and 1.4 mM sodium dihydrogen phosphate
  • guanidine solution [4 M guanidine thiocyanate (Fluka Chenie AG, Switzerland), 20 mM sodium acetate (pH 5.2). ), 0.1 mM dithiothreitol (DTT, manufactured by Sigma, USA) and 0.5% N-lauuroyl sarcocinnadium
  • a cesium chloride solution [0.1 M ethylenediamine] is placed in a polyaromatic ultracentrifuge tube (No. 33131372, manufactured by Beckman, USA) that has been autoclaved.
  • Cesium chloride (manufactured by G1BC0 BRL, U.S.A.) was dissolved in tetraacetic acid (EDTA) solution (PH 8.0) to a concentration of 5.7 M, and 0.2% pyrocarbonate was dissolved.
  • EDTA tetraacetic acid
  • PH 8.0 tetraacetic acid
  • Add JETIL (DEPC, manufactured by Wako Pure Chemical Industries, Japan), stir vigorously, leave for 10 minutes, and sterilize by autoclaving at 120 ° C for 20 minutes.
  • the guanidine solution was added to the X 1 0 7 pieces of the cell disruption solution was gently layered what was 8. Align in a 2 ml total volume. Using an ultracentrifugal rotor (SW41Ti, manufactured by Beckman, USA), the mixture was centrifuged at 180 ° C and 150,000 Xg for 20 hours.
  • SW41Ti ultracentrifugal rotor
  • RNA solution To the RNA solution, add an equal volume of a mixture of black-mouthed form / 1-butanol (4: 1), stir vigorously, and centrifuge at room temperature for 15 minutes at 15, OOO rpm [TMA-2 rotor One (made by TOMY, Japan), rotor radius: 62.9 mm. Unless otherwise specified, the same rotor was used for the following centrifugation)]) and the aqueous layer was transferred to another 1.5 ml microtube. 1/10 amount of 3 M sodium acetate and 2.5 times amount of ethanol The mixture was stirred at ⁇ 80 ° C for 30 minutes, centrifuged at 4 ° C and 150,000 rpm for 20 minutes, and the precipitate was collected. The operation is called ethanol precipitation). The precipitate was dissolved in 360 ⁇ l of sterilized purified water, and RNRN was collected again by ethanol precipitation.The precipitate was dissolved in 100 ⁇ l of sterilized purified water to obtain a total RNA solution. .
  • the cDNA library was prepared in accordance with the linker primer method described in "Preparation of Gene Library” (edited by Hiroshi Nojima, Yodosha, Japan).
  • Equal volume of phenolic Z-neck mouth Holm mixture [500 ml of fluorohonolem and 500 g of phenol (manufactured by Wako Pure Chemical Industries, Japan) and lg 8 — Mix well with hydroxyquinoline (Nacalai Tesque, Japan) and add 200 ml of purified water to use the lower layer.) After stirring, the mixture was centrifuged at room temperature at 1200 rpm for 5 minutes, and the upper part was transferred to another 1.5 ml microtube. // ⁇ ⁇ ⁇ / ⁇ ⁇ ⁇ ⁇ ).
  • the synthesized single-stranded cDNA was converted to 2 U of RNase H (manufactured by Pharmacia, Sweden) and 50 U of ⁇ col i DNA po 1 ym erase I (manufactured by Fanolemasia, Sweden) ), Each of 225 ⁇ d of dATP, dGTP and dTTP, and 375 ⁇ of 2′-deoxycytidine-15′-triphosphate (dCTP, manufactured by Pharmacia, Sweden), In a cDNA second strand buf fer [25 mM Tris-HCl buffer (pH 8.3), 100 mM potassium chloride and 5 mM magnesium chloride] containing 5 mM DTT, 16 The linear stranded cDNA was synthesized by keeping the mixture at 150 ° C for 150 minutes.
  • the obtained double-stranded cDNA solution was prepared by adding 10 U containing 5 U of T4 DNA polymerase (manufactured by Takara Shuzo Co., Ltd., Japan) and 125 ⁇ ⁇ each of dATP, dCTP, dGTP and dTTP.
  • the blunt-ended cDNA solution dissolved in 20 ⁇ l of the TE solution was recovered.
  • Reaction was performed at 37 ° C for 10 minutes and at room temperature for 5 minutes. After kneeling, one hundred and twenty. 20 ⁇ l of ⁇ 4 DNA 1 i gase buf fer [50 mM Tris-HCl buffer (pH 7.6), 10 mM magnesium chloride, I mM adenosine 1-5'-triphosphoric acid, ImM DTT and 5% Polyethylene Render Recall 800) at 8 ° C and the adapters were ligated. .
  • the total volume was 50 ⁇ l, and the mixture was incubated at 37 ° C for 90 minutes ( 5 ⁇ l of a 10-fold concentration of STE solution [100 mM Tris-HCl buffer (pH 8.0)) 1 M After adding sodium chloride, 10 mM ED-chondration A] and 10 g of tRNA, fractionation was performed using Chroma Spin-400 Spin Column (CL0NTECH, USA) to fractionate small fragments. After ethanol precipitation, the precipitate was washed and recovered with 75% ethanol to obtain a cDNA fragment.
  • the precipitate of the Not I digested cDNA fragment was dissolved in 30 ⁇ l of 14 DNA ligase buf fer containing 313.6 ⁇ of vector arm and 4 U of ⁇ 4 DNA ligase, and 12 ° C. Incubate at 70 ° C for 30 minutes, inactivated at 40 ° C to inactivate T4 DNA ligase, and After adding E solution to make 10 ⁇ , the supernatant after phenol / chloroform extraction, and the supernatant after the extraction of macropore form were purified by Millipore filter. Finally, the DNA was recovered in a 30 ⁇ ⁇ solution, and used as a ligation mixture.
  • E. coli was transformed by the electroporation method using GENEPULSER (manufactured by BI0-RAD, USA), and the host Escherichia coli was transformed according to the method described in "Gene library preparation method".
  • iaco 1 i DH11S strain (GIBCO BRL, USA) was used as a competent cell. 3 ⁇ l of the ligation mixture was added to 501 concomitant cells melted in ice, and mixed without foaming. Transfer the bacterial solution into a cuvette (distance between electrodes: 0.1 cm, manufactured by BI0-RAD, USA) that has been cooled in ice in advance, and set it on a chamber slide. Impedance: 25 iu F, resistance: 200 ⁇ , voltage:
  • Yea st Ex tr act (manufactured by Difco, USA), 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium sulfate, 10 mM magnesium chloride And 20 mM glucose], mixed by pipetting, and transferred to a 15 ml centrifuge tube.
  • the cuvette was rinsed with 0.2 ml of SOC medium, combined with the bacterial solution, and cultured with shaking at 37 ° C for 1 hour.
  • Bacterial solution 500 ml of LB / Amp medium [LB medium (1% Bacto Trypt. Ne, 0.5% Bacto Yeast Extract and 1% sodium chloride) will be 50 / ig Zml.
  • DMSO dimethinoresolefoxide
  • Plasmid DNA for cDNA library template was prepared using QIAGEN P1 asmid Max i Kit (Q1AGEN, Germany). I did it. Using 150 ml of the DNA library bacterial solution as a starting material, the plasmid DNA was extracted and purified according to the attached manual, and finally dissolved in a 1 ml TE solution.
  • Taq DNA polymerase (Takara Shuzo Co., Ltd., Japan) prepared in a 0.5 ml microtube, 0.2 mM each of dATP, dCTP, dGTP and dTTP, and A set of primers (SEQ ID NO: 1 ⁇ g each) consisting of the sequence shown in
  • PCR amplification reaction 100 ⁇ l of Taq DNA polymerase buffer [10 mM Tris-HCl buffer (both manufactured by Nippon Bio Services, Japan) pH 8.3), 50 mM potassium chloride and 1.5 mM magnesium chloride] 2 g of the plasmid DNA for the cDNA library template prepared in (1) above. And overlay it with 100/1 mineral oil (Sigma, USA) and incubate at 95 ° C for 2 minutes using a PCR device (DNA Thermal Cycler, Takara Shuzo, Japan). After that, 40 cycles of an amplification reaction (hereinafter referred to as PCR amplification reaction) were performed in which 1 cycle was performed at 95 ° C, 2 minutes at 42 ° C, and 3 minutes at 72 ° C. Thereafter, the mixture was kept at 72 ° C for 7 minutes and at 4 ° C for 5 minutes.
  • PCR amplification reaction 40 cycles of an amplification reaction
  • Agarose gel electrophoresis was performed according to the method described in Molecule Clarinng, A Laboratory Manua 1 Second Edition (1989).
  • the amplifying reaction solution of 101 was subjected to agarose gel electrophoresis, and an agarose gel containing a DNA fragment of about 500 to 800 bp detected by UV irradiation after staining with bromide thidium was cut out.
  • Purification of the DNA fragment from the agarose gel was performed using EASYTRAP (a product of Takara Shuzo Co., Ltd., Japan). After the recovered DNA solution was extracted with phenol / closed-hole form, the DNA fragments were recovered by ethanol precipitation.
  • This precipitate is dissolved in 200 U of H buffer containing 50 U of each restriction enzyme Sa1I (Takara Shuzo, Japan) and EcoRI, and incubated at 37 ° C for 2 hours. Digested completely. After extraction with phenol / chloroform and ethanol precipitation, the precipitate was washed with 75% ethanol.
  • the Xho I-EcoRI arm and 0.4 ⁇ g of the amplified cDNA fragment were transferred and the total volume was adjusted to 10 ⁇ l with sterile purified water. To this was added 10 ⁇ l of solution I of DNA ligation kit ver. 2 (Takara Shuzo Co., Ltd., Japan), mixed well, and reacted at 16 for 1 hour. The reaction was stopped by keeping the temperature at 70 ° C. for 30 minutes, and the DNA was recovered by phenol-novel mouth-orifice extraction and ethanol precipitation. Transformation of Escherichia coli was performed by the electroporation method described in Example 2, and the transformed bacterial solution was added to 50 ⁇ g / m1 ampicillin, 0.02%.
  • Lysozyme solution consisting of water, and suspended in 3001, and incubated at 37 for 5 minutes, and then add 180 l of 0.3 N sodium hydroxide solution containing 2% SDS. The mixture was stirred vigorously to lyse the E. coli. After incubating at 65 ° C for 20 minutes and leaving it to stand at room temperature, add a mixture of 120 ⁇ 1 phenol-chloroform to the lysate, and mix vigorously. Was extracted. To the aqueous layer after centrifugation, add 1-10 volumes of 3 M sodium acetate and an equal volume of isopropanol, and allow to stand at room temperature for 5 minutes.
  • the precipitate was collected by centrifugation at 1500, rpm for 5 minutes.
  • the precipitate was dissolved in 50 ⁇ l of a TE solution containing lOgZml of RNaseA (Sigma, USA), and kept at 37 ° C for 3 hours to remove contaminating RNA.
  • 3-2. Add 20% poly-carbonate solution containing 2.5 M sodium chloride-600 solution, cool in ice for 15 minutes, then 4 ° C, 15, OOO rpm The precipitate was recovered by centrifugation for 20 minutes at, and the precipitate was washed with 75% ethanol to obtain type I DNA for sequencing.
  • the nucleotide sequence of the inserted cDNA fragment was determined using a fluorescent sequencer manufactured by Applied Biosystems.
  • the sequence sampzole was prepared using PRISM, Ready Reaction Dye Terminator Cycling Sequencing Kit (manufactured by Applied Biosystems, USA).
  • E. coli colonies containing plasmid DNA containing a partial fragment of HGIR obtained in The cells were transplanted to the mp, and cultured at 37 ° C with shaking. Plasmid DNA was prepared using QIAGEN Plasmid Maxi Kit.
  • the cDNA library used was the Human Brain 5'-Stretch Plus cDNA Library (HL3002a, CLONTECH, USA). E. coli Escherichia coli C600Hf1 strain was placed on LB medium containing 1 OmM magnesium sulfate and 0.2% maltose for 16 hours. The cells were cultured with shaking for use as a host bacterial solution. c After the DNA library is added to the host bacterial solution and infected, spread on LB agar medium containing 1 OmM magnesium sulfate, and allowed to stand at 37 ° C for 8 hours to obtain about 1.2 million cells. A single plaque was formed.
  • Hybond-N + membrane (Amersham, UK) was gently placed on the plaque-formed agar, and allowed to stand for about 1 minute. Gently peel off the membrane and place the surface in contact with the black on the filter paper soaked in a denaturing solution (1.5 M sodium chloride and 0.5 M sodium hydroxide). For 7 minutes. Similarly, the membrane was transferred onto filter paper containing a neutralizing solution [1.5 M sodium chloride and 0.5 M tris-HCl buffer (pH 7.5)]. After standing for 3 minutes, this operation was repeated using fresh filter paper soaked with a neutralized solution, and then the membrane was washed with double concentration SSC (0.3 M sodium chloride and The cells were washed with 0.03 M sodium citrate) to remove cell debris.
  • a denaturing solution 1.5 M sodium chloride and 0.5 M sodium hydroxide
  • the membrane was transferred onto filter paper containing a neutralizing solution [1.5 M sodium chloride and 0.5 M tris-HCl buffer (pH 7.5)]. After standing for 3 minutes, this operation was repeated using
  • plaque side Place the plaque side up on a dry filter paper and wrap the air-dried membrane with Saran Wrap (made by Asahi Kasei Corporation, Japan), and place the plaque side down on UV transillumination It was placed on a noun and irradiated with UV at 254 nm for 5 minutes.
  • the plaque-forming medium was stored at 4 ° C.
  • the plate was washed with 0.1% SDS) for 15 minutes.
  • the membranes were wrapped one by one in a salang wrap, fixed to a cassette, and baked on an X-ray film at 180 ° C using one piece of paper. After 6 hours, develop the X-ray film and The positive signal was detected and stored at 4 ° C to separate positive clones from the medium.
  • I phage DNA was extracted from the positive clones by the following method. Positive clones were punched out using a sterile pasteur pit, and the host strain of 200 ju1 Escherichia co1i C600Hf1 strain previously dispensed into a 50 ml centrifuge tube Suspended in liquid. After incubating at 37 ° C for 15 minutes, 12 ml of LB medium containing 10 mM magnesium sulfate was added, and cultured with shaking at 37 ° C for 16 hours. The bacterial solution was transferred to a 15-ml centrifuge tube, and centrifuged at 7,000 rpm for 10 minutes to remove the residue. Transfer the above 11 ml to an ultracentrifuge tube (No.
  • Plasmid Vector PUC 118 (Takara Shuzo, Japan) in 50 ⁇ l of ⁇ buffer containing 1 U of restriction enzyme Ec0RI, and heat to 37 ° C. And digested completely for 2 hours. The DNA recovered by phenol black-mouthed form extraction and ethanol precipitation was used as pUCl18EcoRI vector.
  • This bacterial solution was added to 50 ⁇ l of ampicillin, 0.02% of 0 31 and 50 was spread on an LB agar medium containing 1 to 10 and incubated at 37 ° C for 16 hours to form colonies. From the single white colony that appeared, the plasmid DNA was extracted using the same method as in Example 3, and the HGIR fragment was extracted.
  • Recombinant plasmids PGIR10 and PGIR20 (1.5 kbp) inserted in the reverse direction were obtained. That is, the single white colony that had emerged was cultured with 2 ml of LBZAmp for 10 minutes with shaking, and the cells were recovered by centrifugation at 12,200 rpm for 3 minutes. Lyse the cells with a lysozyme solution [25 mM Tris-HCl buffer (pH 8.0) containing lmg Zml of lysozyme, 1 OmM EDTA and 5 OmM glucose] 300 ⁇ l After reacting at 37 ° C for 5 minutes, add 180 ⁇ l of 0.3 N sodium hydroxide solution containing 2% SDS, and vigorously stir to remove E.
  • coli. Lysed After incubating at 65 ° C for 20 minutes, leave at room temperature for 120 ⁇ l of cooled lysate.
  • the phenolic hologram was extracted using a phenolic hologram mixture. To the aqueous layer after centrifugation, add 1/10 volume of 3 M sodium acetate and an equal volume of isopropanol, and allow to stand at room temperature for 5 minutes, then at 1500 rpm for 5 minutes. The precipitated plasmid DNA was recovered by centrifugation of the DNA.
  • Figure 3 shows the restriction enzyme map and function map of these plasmids.
  • the transformant carrying PGIR10 was named Escherichia coli JM109 / pGIR10 strain (FERMBP-58336) and deposited with the National Institute of Bioscience and Human Technology.
  • E. coli colonies carrying PGIR10 or PGIR20 were each transplanted to 150 ml of LB / Amp, incubated at 37 ° C with shaking, and the plasmid DNA was transferred to Q1AGEN PI. It was prepared using asmi d Maxi Kit.
  • Example 5 Example 5
  • 100 ⁇ g of recombinant plasmid pGIR10 was replaced with 100 M buffer [1] containing 10 U of each of the restriction enzymes EcoRI and Eael (Takara Shuzo Co., Ltd., Japan). 37 mM dissolved in 0 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 1 mM DTT and 50 mM sodium chloride. Incubate for 2 hours at C to digest completely. bo
  • agarose gel electrophoresis was performed, and an agarose gel containing a DNA fragment of about 270 bP detected by UV irradiation after staining with bromide thidium was cut out.
  • Purification of DNA fragments from agarose gel was performed using a DNA recovery glass. The DNA fragment was recovered using EASYTRAP, extracted with phenol phenol, and extracted with ethanol. The resulting approximately 2 7 0 bp DNA fragment, [a - 32 P] using d CTP labeled with Random Pr imer DNA Label ing Ki t , and the probe DNA fragment.
  • the obtained restriction enzyme-digested DNA was subjected to agarose gel electrophoresis, and the gel after the electrophoresis was subjected to 200 ml of a 0.5 N sodium hydroxide solution and a 1.5 M sodium chloride solution. In room temperature for 20 minutes. The gel was transferred to a vacuum transfer device (BC-600, manufactured by Biocraft, Japan) and used as a transfer buffer 0.4. N sodium hydroxide solution The DNA was aspirated at 7 cm ZHg for 40 minutes and the DNA was transferred to a Hybond-N + membrane. The membrane was gently peeled off, washed gently for 30 seconds in double concentration SSC, transferred to filter paper and air-dried for about 1 hour.
  • a vacuum transfer device BC-600, manufactured by Biocraft, Japan
  • hybridization was performed on the membrane by the following method. That is, a final concentration of 100 ⁇ g / m in 50% formamide (Wako Pure Chemical Industries, Japan), 5 times concentration SSPE, 5 times concentration Enhardt's solution and 2% SDS solution. Denatured salmon sperm DNA was added to obtain a prehybridization solution so as to obtain m1. Put main Npura down of the previously prepared high Buri da Izei to Nba' grayed, main assembler down 1 cm 2 per Li 0. By adding 1 ml Burehai Bed Li Dizei to down liquid sheet to one le, 4 2 ° The mixture was kept in a water bath for 4 hours.
  • Recombinant brassmid p GIR10 and PGIR20 are used as materials, and a variety of insert lengths are provided using the Deletion Kit for Kilo-Sequence (Takara Shuzo Co., Ltd., Japan). Were used to determine the DNA sequence including the full length of the HGIR gene. That is, 10 g of pGIR10 or PGIR20 is dissolved in an H buffer containing 20 U of restriction enzymes SphI and BamHI (Takara Shuzo Co., Ltd., Japan) at 37 ° C. For 2 hours to complete digestion. DNA attached and blunt-ended by Exonuc lease I II, ung Bean Nucl ease and Klenow Fragment were carried out. After circularizing the plasmid, Escherichia coli J109 strain was transformed to obtain clones containing DNA fragments of various lengths. 3 The same method as (3), ie, PRISM, Ready
  • Example 1 2 g of mRNA extracted from stress-reduced stress culture HUVEC and static culture HU VEC were used for molecular loning, A Laboratory Manual, Second Edition (1989). At, electrophoresis using a formaldehyde gel was performed. The gel after electrophoresis was allowed to stand at room temperature for 20 minutes in 200 ml of a 0.05 N sodium hydroxide solution. The gel was transferred to a Bacilleum transfer buffer apparatus, and a 0.05 N sodium hydroxide solution was used as the transfer buffer at 7 cm ZHg. Aspirate for 40 minutes and transfer mRNA to Hybond-N + membrane.
  • Example 5 High predication was performed in the same manner as described above. That is.
  • Denatured salmon sperm DNA is added to a solution consisting of 50% formamide, 5 times concentration SSPE, 5 times concentration of Denhardt's solution and 2% SDS to a final concentration of 100 ⁇ g / ⁇ ⁇ .
  • a pre-high bridging solution was used. Put main assembler down with a fixed m RNA prepared above to a high pre Daizi sucrose Nba' grayed, sealed by the addition of main Nburan lcm 2 per Li 0. 1 ml of Bureno I Bed Li da I THEY try down liquid, 42 The mixture was kept in a water bath at 22 ° C with shaking for 4 hours.
  • Peptide synthesis was performed by the Fmoc solid-phase synthesis method (New Chemistry Laboratory Course 1 ⁇ Protein VI synthesis and expression, published by Tokyo Kagaku Dojin), and the carrier protein KLH was equivalent to 2 mg of synthetic peptide. (keyhole-1 impet hemocyanin, manufactured by PIERCE, USA) conjugated by the maleimide method was used as an antigen.
  • 0.5 mg of the antigen was subcutaneously administered to the back of about 2.1 kg of a heron, and the same amount of the antigen was similarly administered on 21 days, 42 days and 63 days thereafter. Seventy-three days after the first administration, the blood of Perilla was collected by anesthesia blood collection under anesthesia, and the serum was separated and used as antiserum.
  • PGIR10 PGIR10 was added to 2.5 U (D Taq DNA poIymerase and each 0.5 lmmo1 PCR primer (sense primer shown in SEQ ID NO: 6 and SEQ ID NO: 7).
  • the PCR amplification reaction was performed for 30 cycles, with 1 cycle at 1 °, 2 minutes at 68 ° C, and 3 minutes at 72 ° C. After extraction of the form with ethanol and precipitation with ethanol, the precipitate was washed with 75% ethanol to recover DNA.
  • E. coli After ligation reaction of 0.5 pBlueScript II KS + EcoRI-Kpnl arm and 0.2 g of HGIR (EcoRI-Kpnl) fragment in a 20 ⁇ l reaction system, E. coli was transformed using the reaction solution. Converted.
  • Transformation of Escherichia coli was carried out using a combi- nation cell Escherichia coli J109.
  • the ligation mixture of 51 was added to 100 ⁇ l of the competent cell melted in ice, mixed, and allowed to stand still on ice for 30 minutes. 42 After incubating at 22 ° C for 30 seconds, add 0.9 ml of SOC medium, which was previously kept at 37 ° C, and transfer to a 15 ml centrifuge tube. At:! The cells were cultured with shaking for a long time.
  • the bacterial solution was added to 50 ⁇ g / m 1 of ampicillin 0.02% of 0 31 and 50 Then, the cells were spread on an agar medium containing 1 to 10 and incubated at 37 ° C for 16 hours to form colonies. The single white colony that emerged was cultured with shaking using 2 ml of LB ZAmp, and libramide DNA was extracted from the obtained cells to obtain pBSZH GIR.
  • the base sequence of the insert DNA was determined by the same method as in Example 6, using pBS ZHG IR as the material and the Deletion Kit for Kilo-Sequence, and using various Deletion Kits for the insert length. We decided to create and obtain a new Miuantant.
  • the delay mitigation PstI (Takara Shuzo Co., Ltd., Japan) and BamHI were used as restriction enzymes for the preparation.
  • the nucleotide sequence of the HGIR peptide code region contained in the insert was identical to the nucleotide sequence of SEQ ID NO: 2 and had no mutation.
  • E. coli was transformed. Transformation of Escherichia coli was carried out using a combi- nation cell, Escherichia coli 1JM109. 5 ⁇ l of the ligation reaction solution was added to 100 ⁇ l of the competent cell melted in ice, mixed, and then allowed to stand still on ice for 30 minutes.
  • FIG. 5 shows the restriction enzyme cleavage site and functional map of the constructed expression plasmid pEGIR11.
  • PCR amplification reaction was carried out using a PCR apparatus in 100% of Taq DNA poIymerase buf fer containing Japan (manufactured by Nippon Bioservices, Japan) using a PCR device for 30 cycles. However, the conditions of the amplification reaction at this time were 1 minute at 95 ° C, 2 minutes at 68 ° C, and 3 minutes at 72 ° C.
  • reaction solution was extracted with phenol and / or chloroform, and ethanol was precipitated. The precipitate was washed with 75% ethanol and the DNA was recovered. Dissolve in ⁇ ⁇ ⁇ ⁇ ⁇ L buffer containing 0 U restriction enzyme K pn I and incubate completely at 37 ° C for 2 hours, then continue with 10 U restriction enzyme Eco It was dissolved in ⁇ ⁇ ⁇ ⁇ H H buffer containing RI and completely digested by incubation at 37 ° C for 2 hours. After adding 10 ⁇ g of tRNA, small fragments were removed by fractionation using a Chroma Spin-400 spin column.
  • HGIR-FLAG (EcoR Kpnl) fragments 0.5 ⁇ g (pBlueScript 1 I KS + EcoRI-Kpn I arm and 0.2 ⁇ g of HGIR-FLAG (EcoR I-Kpnl) fragment were added to each other after ligation in 201.
  • Escherichia coli was transformed using the reaction solution.Escherichia coli was transformed using a competent cell Escherichia £ 01 i JM109. Add the ligation mixture of ⁇ 1 and mix, and then stood still on ice for 30 minutes 42.
  • the restriction enzymes used in the preparation of the delay mitogen were PstI and BamHI, and as a result, the HGIR peptide contained in the insert was used. It was confirmed that the nucleotide sequence of the coded region was the nucleotide sequence of SEQ ID NO: 2 and there was no change. Subsequently, 5 g of pBS / HGIR-FLAG was dissolved in 50 / ⁇ 1 L buffer containing 5 U of the restriction enzyme Kpnl, and kept at 37 ° C for 2 hours to completely digest.
  • the HGIR and its modified expression plasmid prepared in Example 9 were introduced into COS-1 cells (obtained from Dainippon Pharmaceuticals, Japan) using LI POFECTAMINE Reagent (GIBCO BRL, USA). The gene product was analyzed by Western blotting using anti-HGIR antiserum and anti-FLAGM2 antibody (KODAK, USA).
  • the medium used is DMDM (Dainippon Pharmaceutical Co., Japan) containing 10% fetal serum (FCS), 50 IU / m1 ⁇ nisin, and 50 g Zml streptmycin. did.
  • the expression plasmids (PEGIR11 and PEGIR12-FLAG) used for the transfection were transferred to a 150 ml LB / Amp colony of E. coli colonies carrying each plasmid. It was prepared from the cells obtained by culturing the cells at 7 ° C using QIAGEN Plasm id Max Kit. In addition, the same operation was performed for pcDNA3.1 (+) vector containing no HGIR as a negative control. Each 2 ⁇ g of the expression plasmid was added to 41 LI POFECTAMINE
  • the two filters thus prepared were combined with a blocking solution [5% skim milk (Difco, USA) and a TBS solution containing 0.1% Tween 20 (20 mM). Tris-HCl buffer (pH 7.6) and 1337 mM sodium chloride)] and reacted at room temperature with shaking for 1 hour.
  • a blocking solution [5% skim milk (Difco, USA) and a TBS solution containing 0.1% Tween 20 (20 mM). Tris-HCl buffer (pH 7.6) and 1337 mM sodium chloride)] and reacted at room temperature with shaking for 1 hour.
  • TBS solution containing 0.1% Tween 20
  • an antibody diluent TBS solution containing 2% skim milk and 0.1% Tween 20
  • Immerse it in a primary antibody solution one for anti-HGIR anti-blood solution and one for anti-FLAGM 2 antibody solution obtained by diluting it 1: 1000 and react for 1 hour while shaking at room temperature. I let it.
  • a membrane on which mRNA extracted from each human tissue was immobilized Human Multi-Tissue Template No. B lot (manufactured by CLONTECH, USA) was used.
  • the mRNA immobilized on this member is derived from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and spleen tissues.
  • This membrane was treated in the same manner as in Example 5, and mRNA immobilized on the membrane was analyzed by the Northern Hybridization method. That is, denatured salmon sperm was added to a solution containing 50% formamide, 5 times concentration SSPE, 5 times concentration Denhardt's solution and 2% SDS so that the final concentration was 100 ⁇ g / m 1.
  • DNA was added to prepare a prehybridization solution.
  • the mixture was kept in a water bath at 42 ° C. for 4 hours with shaking. 5 at 100 ° C in this bag
  • add the labeled HGIR probe DNA prepared in Example 4 to a final concentration of 1 ⁇ 10 6 cpm / m 1, seal, and further cool at 42 ° C.
  • the membrane was removed and washed with Washing Solution 1 at room temperature for 10 minutes.
  • the substrate was washed at 50 ° C. for 30 minutes using the cleaning solution 1, and further washed at 50 ° C. for 30 minutes using the cleaning solution 3.
  • the radioactivity was read and detected using a BAS 2000 imaging analyzer. As a result, a clear signal was detected in the lane in which mRNA extracted from human brain was fixed. In other words, it was revealed that HGIR is highly expressed in the brain in human-derived tissues.
  • the stress that does not control the function of vascular endothelial cells and is restressed in individual cells which was not clear until now, It is possible to easily measure the degree of load by replacing the expression level with the expression level, and it is possible to analyze in detail the function of individual dermal cells. Analyzing the relationship between the function of vascular endothelial cells and shear stress in more detail, analyzing the expressed substances efficiently to elucidate the mechanisms of onset of various vascular diseases consisting of vascular endothelial cell dysfunction, and It will also be possible to search for related substances.
  • the gene of the present invention is highly expressed in human brain tissue, and it was also found that the gene may have a physiological function as a receptor for a neurotransmitter. . Therefore, using the peptide coded by the gene of the present invention, a substance capable of binding to the above-mentioned peptide and a binding between the above-mentioned peptide and a substance capable of binding to the peptide are used. It is possible to detect a substance that suppresses or inhibits, for example, a substance obtained by the detection method of the present invention develops as a drug for a central nervous disease.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • TCCTCCTCTA CATCCTGCCC CTCCTCATCA TCTCTCTGGC CTACGCTCGT GTGGCCAAGA 960 AACTGTGGCT GTGTAATATG ATTGGCGATG TGACCACAGA GCAGTACTTT GCCCTGCGGC 1020
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

L'invention se rapporte à un ADN isolé comprenant au moins une région codant un peptide d'un ADN entier humain et qui se caractérise par des sites de clivage d'enzyme de restriction tels que présentés sur la carte enzymatique de restriction de la figure; à un nouveau gène qui s'exprime en fonction de la force de cisaillement des cellules endothéliales vasculaires, ce qui permet de détecter, au moyen de l'ADN, si ces cellules sont ou non présentes dans un environnement physiologique normal dans lequel on applique des forces de cisaillement, ce qui n'était pas clair jusqu'ici; à un ADN pouvant s'hybrider avec l'ADN précité; à un ADN complémentaire à ces ADN; à un ADN de recombinaison préparé par intégration de ces ADN dans un vecteur réplicable; à son transformant; à un peptide pratiquement pur codé par l'ADN précité; à un anticorps capable de se fixer aux peptides; à un procédé de détection d'une substance capable de se fixer aux peptides précités au moyen d'un peptide codé par le gène de l'invention; et à un procédé de détection d'une substance qui supprime ou inhibe la liaison entre les peptides précités et la substance capable de s'y fixer.
PCT/JP1997/000919 1996-04-03 1997-03-19 Nouveau gene WO1997037018A1 (fr)

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JP8081304A JPH11299487A (ja) 1996-04-03 1996-04-03 新規遺伝子
JP8/81304 1996-04-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0877083A1 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Récepteur humain (HCEPT09), connecté à la protéine G
EP0789076A3 (fr) * 1996-02-07 1999-04-14 Takeda Chemical Industries, Ltd. Protéines récepteurs couplés à la protéine G, leur production et utilisation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS646219A (en) * 1987-01-08 1989-01-10 Asahi Chemical Ind Peptide having promoting action on activation of protein c by thrombin
JPH03503757A (ja) * 1987-06-12 1991-08-22 ノボ―ノルディスク　アクティーゼルスカブ タンパク質およびその誘導体
WO1994008040A1 (fr) * 1992-09-25 1994-04-14 Synaptic Pharmaceutical Corporation Adn codant des recepteurs adrenergiques alpha 1 humains et leurs utilisations
JPH07274994A (ja) * 1994-04-08 1995-10-24 Asahi Chem Ind Co Ltd トロンボモジュリンの製造法
WO1995028157A1 (fr) * 1994-04-13 1995-10-26 Synaptic Pharmaceutical Corporation UTILISATION DE COMPOSES SPECIFIQUES DES RECEPTEURS α1C-ADRENERGIQUES POUR LE TRAITEMENT DE L'ADENOME PROSTATIQUE

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS646219A (en) * 1987-01-08 1989-01-10 Asahi Chemical Ind Peptide having promoting action on activation of protein c by thrombin
JPH03503757A (ja) * 1987-06-12 1991-08-22 ノボ―ノルディスク　アクティーゼルスカブ タンパク質およびその誘導体
WO1994008040A1 (fr) * 1992-09-25 1994-04-14 Synaptic Pharmaceutical Corporation Adn codant des recepteurs adrenergiques alpha 1 humains et leurs utilisations
JPH07274994A (ja) * 1994-04-08 1995-10-24 Asahi Chem Ind Co Ltd トロンボモジュリンの製造法
WO1995028157A1 (fr) * 1994-04-13 1995-10-26 Synaptic Pharmaceutical Corporation UTILISATION DE COMPOSES SPECIFIQUES DES RECEPTEURS α1C-ADRENERGIQUES POUR LE TRAITEMENT DE L'ADENOME PROSTATIQUE

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0789076A3 (fr) * 1996-02-07 1999-04-14 Takeda Chemical Industries, Ltd. Protéines récepteurs couplés à la protéine G, leur production et utilisation
EP0877083A1 (fr) * 1997-05-07 1998-11-11 Smithkline Beecham Corporation Récepteur humain (HCEPT09), connecté à la protéine G

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