WO1997037018A1

WO1997037018A1 - Novel gene

Info

Publication number: WO1997037018A1
Application number: PCT/JP1997/000919
Authority: WO
Inventors: Yoshio Takada; Chihiro Kato
Original assignee: Asahi Kasei Kogyo Kabushiki Kaisha
Priority date: 1996-04-03
Filing date: 1997-03-19
Publication date: 1997-10-09
Also published as: JPH11299487A

Abstract

An isolated DNA involving at least a peptide coding region of a human-origin full-length DNA characterized by the restriction enzyme cleavage sites as shown in the restriction enzyme map of the figure; a novel gene which causes expression in accordance with the shear stress of vascular endothelial cells and thus enables the detection by means of the DNA whether or not the vascular endothelial cells are present in a normal physiological environment on which a shear stress is imposed, which has been unclear heretobefore; a DNA hybridizable with the above DNA; a DNA complementary to these DNAs; a recombinant DNA prepared by integrating these DNAs into a replicable vector; a transformant thereof; a substantially pure peptide encoded by the above DNA; an antibody capable of binding to the peptides; a method of detecting a substance capable of binding to the above peptides by utilizing a peptide encoded by the gene of the invention; and a method of detecting a substance which suppresses or inhibits the binding between the above peptides and the substance capable of binding thereto.

Description

Description New gene technology field

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. Also, 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. Further, 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. Conventional technology In recent years, research on the function of blood vessels has made remarkable progress, and various things about the function of blood vessels have been elucidated. Above all, vascular endothelial cells are known to play an extremely important role in blood vessels. Maintenance of action, regulation of expansion and contraction of blood vessel lumen, and the like. It has also been pointed out that the inability of these functions to work for some reason leads to various vascular diseases.

As one of the factors affecting the function of vascular endothelial cells as described above, restress is known as a physical factor (Frontiers of Medicare and Biological Engineering, 5, 245- 264 (1993)]. The shear stress is determined by the blood flow velocity and blood viscosity, and also by the diameter and shape of the blood vessel. It is known that arteriosclerosis often occurs in low-shear stress areas such as the inside of the side wall of a blood vessel, such as in the side wall of a blood vessel (Role of Blood Flow in At her ogenesis (Y. Yo shidaeta 1. As can be seen from the above, it has been pointed out that shear stress is important for the control of the function of vascular endothelial cells., Eds) Springer-Verlag, Tokyo (1988)].

However, studies on the relationship between stress and vascular endothelial cells are not always sufficient, and it is not clear how the function of vascular percutaneous cells is controlled in its physiological environment. Was.

How much shear stress is applied to individual cells Measurement of individual endothelial cell functions, and the mechanism of the onset of various vascular diseases caused by vascular endothelial cell dysfunction. Efficient analysis of expressed substances and search for related substances for clarification are possible.

In general, 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. . For example, human and human γ-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)]. Furthermore, recombinant rat protein C and rat plasma-derived protein C prolong the activated partial thromboplastin time using rat plasma in a dose-dependent manner. It has been reported that protein C has a weak elongation effect. CThrombos is and Haemos Iasis 54-61 (1994)]. In such a situation where species differences are expected, it is argued that obtaining human-specific proteins is extremely preferable for the future development of the human pharmaceutical field. do not have.

Therefore, when providing a method for detecting whether or not vascular endothelial cells are in a normal physiological environment where restress is applied, species differences and This means that the possibility of having a very large impact must be considered in advance. In other words, when performing the above-mentioned detection on vascular skin cells or human-derived vascular endothelial cells in a human body, the expression level of a specific DNA or protein must be measured by the method described below. Is most effective. However, DNA obtained from tissues or cells of other organisms may have a low homology in humans, and the amino acid sequence of the protein to be encoded may differ considerably. In some cases, quantitative or qualitative detection may not be possible. Considering such a possibility, it is desirable to detect using DNA isolated from humans.

As one of the methods to measure the environment where vascular endothelial cells are placed and the function of vascular endothelial cells, etc., and to clarify how the function of vascular endothelial cells is controlled in a physiological environment It has been desired to provide a method for detecting whether or not vascular endothelial cells are in a normal physiological environment to which shear stress is applied. Therefore, 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. Summary of the Invention

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). Furthermore, they found that 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. Furthermore, 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.

That is, 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 And 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. D

Show. Preferable examples of 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.

Accordingly, 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.

The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and the appended claims, with reference to the accompanying sequence listing and drawings. Brief description of the sequence listing

In the sequence listing:

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. BRIEF DESCRIPTION OF THE FIGURES

In the drawing:

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. Detailed description of the invention

According to the present invention, there is provided 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. Next, in order to facilitate understanding of the present invention, first, basic features and preferred embodiments of the present invention will be listed.

1. 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.

2. The peptide code region has the amino acid sequence of SEQ ID NO: 1. 2. The DNA according to the above item 1, which is a nucleotide sequence encoding the included peptide.

3. The DNA of the preceding item 1, wherein the peptide code region is the nucleotide sequence of SEQ ID NO: 2.

4. The DNA according to the above item 1, wherein the full-length human DNA derived from the restriction enzyme map is the nucleotide sequence of SEQ ID NO: 3.

5. A DNA complementary to the DNA described in any one of 1 to 4 above.

6. A replicable recombinant DNA comprising the DNA according to any one of 1 to 4 above incorporated into a replicable vector.

7. DNA that hybridizes to DNA containing the nucleotide sequence of SEQ ID NO: 2.

8. A complementary DNA to the DNA described in 7 above.

9. A replicable recombinant DNA obtained by incorporating the DNA according to 7 above into a replicable vector.

10. A microorganism or cell transformed with the replicable recombinant DNA according to 6 or 9 above.

1 1. A substantially pure peptide recalled by the DNA described in 1 or 7 above.

12. The peptide according to item 11, which comprises the amino acid sequence of SEQ ID NO: 1.

13. (a) Binding the DNA described in 1 or 7 above to a replicable expression vector and combining the DNA with the replicable expression vector Obtaining a replicable recombinant DNA containing

(b) transforming a microorganism or cell with the replicable recombinant DNA to form a transformant,

(c) selecting the transformant from the parent cell of the microorganism or cell,

(d) culturing the transformant, allowing the transformant to express the DNA,

(e) A peptide produced by a method comprising substantially isolating the peptide from a transformant in which the peptide has been cultured. 1 4. An antibody capable of binding to a peptide comprising at least the amino acid sequence of SEQ ID NO: 1.

15. 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.

16. 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.

Hereinafter, the present invention will be described specifically.

In the present invention, A in the nucleotide sequence indicates a deoxyadenyl residue, C indicates a deoxycytidyl residue, G indicates a deoxyguanyl residue, and 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, G1y Is glycine, His is histidine, I 1 e is isoleucine, Leu is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine, and Pro is prolin. Ser, serine, Thr is threonine, Trp is triptophan, Dyyr is tyrosine, and Va1 is norine.

Further, in the present invention, the term “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. For example, 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.

In the present invention, 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 . In addition, 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. You can choose, but usually 1, OOO dyn es Z cm ² or less, preferably 25 dynes Z cm ² or less. By changing the value of the restress applied to the cultured cells, 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.

(6X perfusion medium flow rate)

Shear stress = viscosity of perfusion medium X

(The width of the flow path of the culture device X the height of the flow path ² ) In other words, the shear stress value easily changes due to changes in the viscosity of the culture solution, the flow rate of the culture solution, and the shape of the flow path. In preparing a cDNA library, it is preferable that the average value of shear stress applied to cells be within the above range. 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. In a culture device where the width and height of the flow path are not the same, the stress applied to the cell differs depending on the surface to which the cell adheres, and the stress is different. In 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

This makes it possible to apply a uniform stress to all cells.

A known composition can be used as a culture solution used for culturing cells. For example, in the case of 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. Specific examples include E-GMUV medium (containing 2% fetal calf serum, manufactured by Kurabo Industries, Japan) or Ml99 medium supplemented with 20% fetal calf blood. In addition, ECGS (Endothelia cel l growth supp lemen 门, EGF [Epi derma l growt hf actor! Journal lof Cell Biol ,, 774-788 (1978)) or basic — Addition of cell growth factors such as FGF [Fibroblast growth actor; Journa 1 of Cell Biology, 7_7, 774-788 (1978)] increases cell growth. It can be better. It is also possible to increase the viscosity of the culture medium by adding dextran or the like, and to apply a high stress to the cultured cells.

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 銬 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.られる It is possible.

There is no particular limitation on the method for extracting mRNA from cells subjected to stress, but a method with less physical and chemical degradation of RNA is preferred. Specifically, for example, after crushing and lysing cells with a solution containing guanidine, total RNA obtained by ultracentrifugation using a cesium chloride density gradient is analyzed using an oligo (dT) column to obtain ρο1y (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. 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. For example, a method described in “Method for Genetic Gene Library” (edited by Hiroshi Nojima, Yodosha, Japan) In-primer. Using this linker primer, when the synthesized cDNA is digested with the restriction enzyme NotI, a NotI end is generated at the 3 'end downstream of the p01y (A) region of the cDNA. Insertion of the cDNA into the No NI site of the vector is facilitated. In this case, Not I does not cleave if a specific deoxycytidine residue in the recognition sequence is methylated, so even if there is a Not I cleavage site in the mRNA-derived cDNA sequence. Supply 5—Methyldeoxycytidine 15′-triphosphate instead of Deoxycytidine 15′-triphosphate during single-stranded cDNA synthesis to prevent digestion of the cDNA. This is preferred. Also, by creating different restriction enzyme ends at the 5 'end and 3' end of the cDNA, the cDNA can be inserted unidirectionally into the vector. Becomes possible. The method for preparing the restriction enzyme end is not limited, but a method using a restriction enzyme adapter is preferable, and specific examples include an Ec0RI adapter. By ligating the EcoRI adapter after cDNA synthesis, an EcoRI terminus is created at the 5 'end of the cDNA, and insertion into the EcoRI site of the vector is facilitated. In other words, cDNA having an EcoRI end at the 5 ′ end and a NoΐI end at the 3 ′ end is obtained, and the cDNA is unidirectionally inserted into the EcoRI and NotI sites of the vector. Becomes possible. 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! Has the promoter SV40ear1y that can be expressed in animal cells. 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. M

By using the prepared peptide to detect the activity related to the peptide, it is possible to search for an active substance and easily clone the gene encoding the substance. Can also. In other words, when a gene belonging to a family that is structurally homologous to the active substance is cloned, probe DNA, probe RNA, or PCR (polymerase) can be obtained from a known gene belonging to the family. (e cha in react ion) It is also possible to design a primer, etc., make the cDNA library into a 铸 shape, and efficiently and easily clone the gene that appears during restressing. .

Since 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. In addition, 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. In other words, it is possible to isolate the aforementioned related gene or the peptide encoded by the difference using the difference as an index. The cloning of genes whose expression level changes when a restress is applied includes Subtractive Hybrid dizat ion [Proceed ngs of the Nat iona l Academy of Sciences of the United St at es of America, 8> 2825-2829 (1991)) )] Etc. can be used. It is also possible to analyze the gene expression level of a known bioactive factor using this cDNA library, and it is also possible to analyze the involvement of the factor in the above-mentioned diseases and the like. It is.

c As a method for preparing the DNA of the present invention from a DNA library, 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). Specifically, for example, the DNA described in SEQ ID NOs: 4 and 5 can be used as a primer to amplify the gene by PCR. When the DNAs set forth in SEQ ID NOs: 4 and 5 are used as primers, 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 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. For example, specifically, 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. In addition, 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. Specifically, 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.

Probe the isolated DNA that hybridizes above As a result, a hybrid can be formed to detect a target DNA. 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瀵度1 0 0 μ 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 sufficient amount of fresh washing solution. Incubate twice at 50 ° C for 20 minutes. If the above-mentioned membrane is the one where DNA is fixed Although not particularly limited, usually those made from two-cell cellulose or nylon are used. 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. After the hybridization reaction, the presence or absence of rehybridization can be confirmed by the optimal detection method for each label. 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.

In the present invention, 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). In addition, 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. Specifically, 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. . The method of using complementary DNA or RNA as an antisense is not particularly limited.Specifically, for example, 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.

As described in detail in Examples of the present specification, when obtaining the HGIR gene of the present invention, 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) Was prepared and hybridized to mouse chromosomal DNA, but no positive signal was detected. As a result, this HGIR gene was determined to be characteristic of humans. After the completion of the HGIR nucleotide sequence determination, which was subsequently carried out, a homologous search was performed on a known protein database based on the determined amino acid sequence. High homology with the amino acid sequence encoded by the protein-coupled receptor !: Molecular Endocrinology, 1331-1338 (1991)] was confirmed. Although it was found after determining the nucleotide sequence of the HGIR gene of the present invention, the experimental results that no positive signal could be detected at all by the above hybridization were as follows. It was recognized that the result was a natural one, not from the error of the experiment, but from the nucleotide sequence of the mouse-derived G protein-coupled receptor gene. The sequences of the two are not completely identical, and from the above experimental results, it is appropriate to judge that they are different genes.

In any case, it is clear that 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. In other words, 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). On March 29, 1996 (Hara Deposit Date), Institute of Biotechnology, Institute of Industrial Science and Technology [Deposited at 1-3, 1-3-1, Higashi, Tsukuba, Ibaraki, Japan P EGIR ll and And PEGIR12-FLAG can be easily constructed using pGIR10 as a raw material according to the method described in Examples. Specifically, the non-translated region at the 5 'and 3' untranslated regions of the HGIR gene contained in pGIR10 was deleted by PCR using a synthetic PCR primer. The expression plasmid p EGIR l 1 was obtained by introducing a fragment of about 1.3 kbp containing the DNA region into an expression vector pcDNA3.1 (+) that works in animal cells. Can be constructed. In addition, using a PCR primer designed to add a nucleotide sequence encoding a marker peptide called FLAG peptide to the 3 ′ end of HGIR, the modified expression plasmid PEGIR 12 — Can build FLAG.

Further, it is preferable to introduce 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.

As the substantially pure peptide of the present invention, a peptide comprising the amino acid sequence encoded by the above-mentioned DNA or DNA fragment is preferable. In particular, a peptide containing the amino acid sequence of 1 to 45 of SEQ ID NO: 1 is mentioned as a preferable example.

In the present invention, 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. Furthermore, 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. Is an amino acid having a continuous length of at least 5 amino acids, more preferably at least 10 amino acids, particularly preferably at least 20 amino acids.

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. In addition, 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

Get A,

(d) culturing the transformant, allowing the transformant to express the DNA,

(e) a peptide produced by a method including substantially isolating the peptide from a transformant in which the peptide has been cultured. Although this method 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). The recombinant DNA in which HGIR is connected downstream of such a promoter is not particularly limited. For example, the above-mentioned DNA having pEGIRl1 includes PEGIR12-FLAG. By introducing the recombinant DNA into a host cell to express HGIR, the peptide to be coded can be easily obtained. The obtained peptide can be isolated and purified by a method such as ammonium sulfate fractionation or column chromatography.

When the amount of mRNA re-transcribed by the gene HGIR of the present invention was examined, it was found that the amount of mRNA was higher in the vascular endothelial cells subjected to the re-stress than in the vascular endothelial cells cultured in the static state. Furthermore, it was confirmed that the amount of mRNA changes according to the value of shear stress applied to vascular endothelial cells. Therefore, 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.

Also, since 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.For example, 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. 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.

By using 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 . In addition, 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.

It has been clarified in the study of the present inventors that 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. This result is considered to be consistent with the fact that the full-length HGIR clone was successfully used in the examples of the present application by using a human brain-derived cDNA library. . Further, by analyzing the distribution of mRNA in the brain in detail, it is possible to analyze the function of the peptide in more detail, but 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 μμπι, 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. In particular, these expression sites are said to be sites involved in emotion and memory among the central functions.In the end, 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.

That is, 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. As an indicator, 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.

As a typical example of the detection method of the present invention, 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.

Further, 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.

Furthermore, if a specific embodiment of the detection method of the present invention is shown, as a first embodiment, 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.

In a second embodiment, 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.

As described above, 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. In some cases, 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. For example, 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. Further, in addition to 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. In addition, even if the subject is considered to contain a substance that binds to the peptide and a substance that inhibits the binding to the peptide, an extract derived from a biological tissue or a cell, 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. For example, a method for detecting a phenomenon caused by the binding between the peptide and a substance to be detected as an index Or a change in the signal of a cell that occurs when a substance that binds to the peptide binds to the cell that expresses the peptide or a cell membrane fraction thereof and affects the function of the peptide. There is a method of detecting by using as an index. Binding to the peptide and the peptide The method for detecting the binding to such a substance is not particularly limited. For example, 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. Usually, in this method, it is preferable that the peptide, cell or cell membrane fraction is immobilized on a stationary phase. In this case, 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. As the detection method, an optimal method may be appropriately used depending on the type of the label.

On the other hand, when detecting a substance that binds to a cell or a cell membrane fraction expressing DNA encoding the peptide and affects the function of the peptide, it is necessary to detect a change in the signal. Thus, 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 されず 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. Specifically, 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.

As a specific example of the above detection method, a method for measuring the amount of inositol phosphate released from cells will be described below.

Prepare cells that express the peptide that HGIR encodes and adhere to the cells at the level of confluence, and prepare phosphate-buffered saline CPBS (-) (137 mM sodium chloride , 2.7 mM potassium chloride, 4.3 mM sodium hydrogen phosphate and

After washing the cells with 1.4 mM calcium dihydrogen phosphate)], l OC i Zml myo — [ ^2-3 H) inosito 1 and 10% 1 Incubate for 24 hours in a non-containing medium. After removing the culture solution, the cells are washed with PBS (1), and then cultured in an in siti 1-free culture solution containing 10 mM lithium chloride for 10 minutes. To the cells from which the culture solution has been removed, add an appropriate amount of a sample containing a substance capable of binding to the peptide and a culture solution containing no inosito 1 containing 1 OmM lithium chloride, and incubate for about 20 minutes. Remove the culture medium, immediately add 1 O mM ice-cold formic acid, and leave on ice for 20 minutes <

After neutralization by adding 1 OmM ammonia solution, the whole cell extract was equilibrated with a solution containing 5 mM sodium tetraborate and 6 OmM sodium formate. Ion exchange column AG 1-X 8 (manufactured by BI0-RAD, USA) to adsorb the released inositol phosphoric acid. The column was washed with 10 volumes of water over the column carrier, followed by a solution of 10 volumes of 5 mM sodium tetraborate and 6 OmM sodium formate. After washing, elute inositol phosphoric acid using a solution consisting of 1 M ammonium formate and 0.1 M formic acid. Add a liquid scintillation counter to a part of the eluate, mix well, and measure the radiation using a liquid scintillation counter.

In addition, since these measurement methods can perform quantitative analysis, it is possible to analyze the abundance of a substance capable of binding to the peptide and the level of its activity from a change in the measured value.

Further, 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. By the above, in the presence of the peptide and the substance capable of binding to the peptide, 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. 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.

The thus obtained substance capable of binding to the peptide or the substance capable of binding to the peptide and inhibiting the binding of the peptide has a physiological function of the peptide. Controlled Enables application to pharmaceuticals.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1

Human umbilical vein endothelial cells (HUVECs) and restressing and preparation of mRNA [po1y (A) ⁺ RNA]

(1) Culture of HUVEC

HU VEC (Endoc e1 1 — UV, manufactured by Kurabo Industries, Japan) used third passage cells. As the cell culture device, 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) on a 150 mm φ plastic dish (nunc, Denmark). , 1% gelatin

5 ml of a physiological saline solution (derived from Siskin, manufactured by Sigma, USA) was poured, and the mixture was allowed to stand at 4 ° C for a long time to obtain a gelatin-coated cover slip. Culture medium in a new 150 mm dish

. (E - GMUV, Japan, click Rabou Co., Ltd.) was allowed to stand Zerah Chinko door processing Kabasu Clip was washed with, 1 0 X 1 0 were planted ^four / cm ² and HUVEC in cormorants'll become. Set the cover slip on the parallel plate type shear stress culture device, and Perfusion culture was performed for 8 hours in a 37 ° C. incubator filled with carbon dioxide gas to apply a shear stress of 15 dynes / cm ² .

(2) Extraction of mRNA from cells

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 )], 0.7 ml of 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

(Manufactured by Sigma, USA)]. Using a 1 ml syringe equipped with a 21 G injection needle, the solution containing the cells was taken in and out about 10 times to completely disrupt the cells. This solution was transferred to a 1.5 ml micro tube and stored frozen at 180 ° C.

3.5 ml of 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. 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.

Gently remove the supernatant from the bottom to about 0.5 cm using a sterile pasteur pit, and cut about 1 cm from the bottom of the centrifuge tube using a sterilized pestle. did. While taking care not to allow the precipitated RNA fraction to flow out of the bottom of the centrifuge tube, it was overturned and allowed to stand on sterile paper, and the upper portion was completely removed. After standing at room temperature for 5 minutes, the precipitate was washed with 3601 TE-SDS solution (10 mM Tris-HCl buffer (PH 7.4), 5 mM EDTA and 1% sodium dodecyl sulfate). (SDS)] and transferred to a 1.5 ml microtube. 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 μl of sterilized purified water, and RNRN was collected again by ethanol precipitation.The precipitate was dissolved in 100 μl of sterilized purified water to obtain a total RNA solution. .

Separation and purification of mRNA [po 1 y (A) ⁺ RNA] from total RNA was performed using the mRNA purificat ion kit (Pharmacia, Sweden) according to the attached manual. Was. Example 2

Preparation of cNA library

The cDNA library was prepared in accordance with the linker primer method described in "Preparation of Gene Library" (edited by Hiroshi Nojima, Yodosha, Japan).

(1) Preparation of expression vector arm

100 / g pSI vector (Promega, USA) containing 50 U of restriction enzyme NotI (Takara Shuzo Co., Ltd., Japan) 200 Notlbuffer [10 mM Tris-hydrochloric acid buffer (pH 7.5), 150 mM sodium chloride, 7 mM magnesium chloride, 1 mM DTT, 0.01% bovine serum albumin (BSA) and 0.01% Triton X—100) After incubating at 37 ° C for 120 minutes, 20 U of NotI was further added, and the mixture was incubated at 37 ° C for 60 minutes to complete digestion. 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. // 言う口 / 言う言う). After adding an equal volume of the clot mouth form and mixing well, centrifuge at room temperature for 12 minutes at 12,000 rpm, and transfer the upper part to another 1.5 ml microcap tube. (Hereinafter, this operation is referred to as mouth-holm extraction.) After recovering the DNA by ethanol precipitation, the precipitate was washed with 75% ethanol. The obtained precipitate was treated with 200 μl of Bacterial alkaline phosphatase buffer containing 1 U of Bacterial alkaline phosphatase (E ^ _co1iC75, manufactured by Takara Shuzo, Japan) [50 mM Tris—dissolved in hydrochloric acid buffer (pH 8.0) and ImM magnesium chloride) and incubated at 37 ° C. for 30 minutes. After two rounds of phenol / black mouth extraction and black mouth extraction, DNA was recovered by ethanol precipitation, and the precipitate was washed with 75% ethanol. The resulting precipitate was reconstituted with 200 μl of H buffe containing 100 U of restriction enzyme Ec 0 RI (Takara Shuzo, Japan). r Dissolve in 50 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride, 1 mM DTT and 10 OmM sodium chloride, and add For 120 minutes. After extraction of phenol Z black mouth form and chlorofonolem, DNA was collected by ethanol precipitation to form a vector arm.

(2) Preparation of cDNA fragment

To 4 μl of 2 μg of the mRNA (0.5 μg / μ) obtained in Example 1, 3.5 μl of 5 mM Tris-HCl buffer (PH 7.5) was added. In addition, the mixture was kept at 65 ° C for 5 minutes and then placed on ice. This mRNA solution was prepared by adding 200 U of SUPERSCRIPT II reverse transcriptase (manufactured by GIBCO BRL, U.S.A.) to the 1.6 μ 法 linker primer described in Genetic Library J Preparation Method J (Japan). , Manufactured by Nippon Bio Services), 1 OmM DTT, 0.6 mM each of 2'-deoxyadenosine 15'-triphosphate (dATP), 2'-deoxyguanosine 15 and 13 lines Acid (dGTP) and thymidine—5′-triphosphoric acid (dTTP) (both from Pharmacia, Sweden), 0.3 mM of 5—methyldioxycytidine—5′—3 Phosphoric acid (Pharmacia, Sweden) and 20 U of RNase inhibitor

(Manufactured by Toyobo Co., Ltd., Japan) 25 μl cDNA first sirand buf fer [5 OmM Tris-HCl buffer (pH 8.3), 75 mM Chloride Rim And 3 mM magnesium chloride) at 37 ° C for 60 minutes, and then add 200 U of SUPERSCRIPT 1 I Reverse transcriptase was added and the mixture was incubated at 45 ° C. for 30 minutes to synthesize single-stranded cDNA.

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 μ d of dATP, dGTP and dTTP, and 375 μΜ 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. Transfer the upper 200 / il after extraction of phenolic chlorophenol and chlorophore to Millipore final letter (UFCP 3 TK50, manufactured by Millipore, USA), and place at room temperature. The mixture was centrifuged at 7,500 rpm for 10 minutes until the solution became almost zero. Add 100 μl of TE solution [1 OmM Tris-HCl buffer (PH 8.0) and ImM EDTA] to the filter cap, and add room temperature at 7,500 rpm. Centrifugation was performed for 10 minutes until the solution became almost zero. Again, the TE solution of 1001 was added to the filter, and centrifuged at room temperature at 7,500 rpm for 10 minutes until the solution almost disappeared. Then, add a TE solution with a concentration of 110 // 1 in a concentration of 110, wash the filter by pipetting, and The inverted solution was placed in a 1.5 ml microtube, and the linear stranded cDNA solution was recovered by centrifugation at 5,000 rpm for 10 seconds at room temperature. The operation is called purification by Millipore filter).

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 μ μ each of dATP, dCTP, dGTP and dTTP. 0 // 1 T4 DNA polymerase buf fer [5 OmM Tris-HCl buffer (pH 8.3), 5 OmM sodium chloride, 1 OmM magnesium chloride and [10 mM DTT] at 37 ° C for 30 minutes. After extraction of phenol-n-hol form and clo-form form, supernatant 10

0 μl was purified by Millipore filter and finally purified.

The blunt-ended cDNA solution dissolved in 20 μl of the TE solution was recovered.

4 μl of the resulting blunt-ended cDNA solution was mixed with 4 U of T4 DNA li gase (GIBC0 BRL, USA) and 0.35 § £ (: 0 1 ^ I adapter per [25 g of Eco RI — S maladaptor (No. 4502, manufactured by Takara Shuzo, Japan) and 14.2 pS mal 1 inker (No. 45085P, manufactured by Takara Shuzo, Japan) Was suspended in a 1: 1 molar ratio of 1 1 2 μl of 1 OmM Tris-HCl buffer (pH 7.5), lmM ED-D A and 10 mM magnesium chloride, 2 minutes at 5 ° C

Reaction was performed at 37 ° C for 10 minutes and at room temperature for 5 minutes. After kneeling, one hundred and twenty. 20 μl of Τ4 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. . Incubate the T4 DNA 1 gase by incubating at 70 ° C for 30 minutes, place on ice, and then continue with 24 U Not I and 27/1 Not I replenisher (2 7 8 mM sodium chloride, 8 mM magnesium chloride, 1.8 mM DTT, 0.018% BSA, and 0.018% Driton X—100). The total volume was 50 μl, and the mixture was incubated at 37 ° C for 90 minutes ₍ 5 μl 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.

(3) Ligation of vector and cDNA fragment and transformation of E. coli

The precipitate of the Not I digested cDNA fragment was dissolved in 30 μl of 14 DNA ligase buf fer containing 313.6 μ of vector arm and 4 U of Τ4 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 μμΟ, 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 μ〗 Ε 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 μl 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 Ω, voltage:

2. A voltage pulse was applied under the condition of OkV. Immediately after that

0.8 ml SOC medium (2% Bac 10 Tryptone (manufactured by Difco, USA), 0.5% Bac to

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. To which ampicillin was added as described above), and further cultured at 37 ° C for 16 hours with shaking. Add 3.5 m1 of dimethinoresolefoxide (DMSO, manufactured by Wako Pure Chemical Industries, Japan) per 50 m1 of bacterial solution — store at 80 ° C, cDNA library Bacterial solution.

Example 3 Cloning of partial fragment of novel gene HGIR

(1) Preparation of cDNA library template 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.

(2) Cloning of DNA width and width products using Degene ratePCR primer

2.5 U of 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 μg each) consisting of the sequence shown in

The sense primer mixture shown in 4 and SEQ ID NO: 5

) 100 μl 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.

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. Fifty ng of the recovered DNA fragment was After performing PCR amplification reaction under the same conditions, DNA was recovered by phenol Z chloroform extraction and ethanol precipitation, and the precipitate was washed with 75% ethanol. 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. After dissolving the precipitate in a 50 / u1 TE solution, adding 5 μl of 10-fold STE and 10 g of tRNA, and then using a Chroma Spin-100 spin column (CL0NTECH, USA) And small fragments were removed. After extraction of phenol / closed-hole form and precipitation with ethanol, the precipitate was washed and recovered with 75% ethanol to obtain a wide DNA fragment.

10 μg of plasmid vector pcDNAII (manufactured by InVitrogen, The Netherlands) containing 10 U of each restriction enzyme XhoI (manufactured by Takara Shuzo, Japan) and EcoRI. It was dissolved in Ο Ο μ 1 of Η buffer and incubated at 37 ° C for 2 hours to completely digest. Agarose gel electrophoresis was performed to cut out an agarose gel containing a DNA fragment of about 3.0 kbp detected by UV irradiation after staining with bromide thidium. Purification of the DNA fragment from the excised agarose gel is performed using EASYTRAP, a DNA powder for DNA recovery, and extraction of the DNA fragment by phenol / chloroform extraction and ethanol precipitation. pcDNAI I Xho I-EcoRl arm.

0.5 μl of pcDNAII in a 0.5 ml microtube

The Xho I-EcoRI arm and 0.4 μg of the amplified cDNA fragment were transferred and the total volume was adjusted to 10 μl with sterile purified water. To this was added 10 µl 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 μg / m1 ampicillin, 0.02%. X—Ga1 (5—Bromo 4—Chloro 3—Indolinore—Galactoside, Wako Pure Chemical Industries, Japan) and 50 mM IPTG (A LB agar medium containing 1.5% Bact 0 Agar in LB medium (Difco, U.S.A.) containing isopropyl-/ 3-D-thiogalactopyranoside (manufactured by Wako Pure Chemical Industries, Japan) The product was added to the mixture and sterilized by high-pressure steam and then spread on a petri dish and solidified.) The mixture was allowed to stand at 37 ° C for 16 hours to form a colony.

The single white color that appeared was shaken with 2 m 1 L B / Amp! [The cells were cultured, and the cells were collected by centrifugation at 1200 rpm for 3 minutes. 25 mM Tris-hydrochloric acid buffer containing 1 mg gZml of lysozyme (manufactured by Seikagaku Corporation, Japan)

(PH 8.0), 10 mM EDTA and 50 mM Darco 5 o

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 μl 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.

(3) Determination of clone base sequence

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). In a 0.5 ml micro tube, 9.5 l of the reaction stock solution, 0.8 pmol of 4, O jul / μ1 D7 promoter primer (manufactured by GIBCO BRL, USA) and 6.51 0.16 igμ 铸 sequence DNA for sequencing are added and mixed, and 50 μ1 mineral oil After overlaying, perform 25 cycles of PCR for 30 cycles at 96 ° C, 15 seconds at 55 ° C, and 4 minutes at 60 ° C for 25 cycles. Incubated for minutes. After the reaction, 800 1 of sterile purified water was added and stirred, and the aqueous layer after centrifugation was subjected to phenol / cloth form extraction three times. To the aqueous layer of 100 μl, add 10 μl of sodium triacetate (Ρ5.2) and ethanol to the aqueous layer, and stir the mixture at room temperature. The precipitate was collected by centrifugation at rpm for 15 minutes. After the precipitate was washed with 75% ethanol, it was allowed to stand under vacuum for 2 minutes and dried to obtain a sample for sequencing. The sequence sample was dissolved in formamide containing 41 1 OmM EDTA, denatured at 90 ° C for 2 minutes, cooled in ice, and subjected to a sequence. As a result, a nucleotide sequence consisting of a part of the gene HGIR having the sequence of SEQ ID NO: 2 was confirmed. Example 4

Cloning of full length gene HGIR

(1) Preparation of HGIR probe DNA

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.

Dissolve 1 μg of the prepared plasmid DNA in 100 μl of H buffer containing 10 U of each restriction enzyme Sphl (Takara Shuzo Co., Ltd.) and EcoRI at 37 ° C. For 2 hours to complete digestion. Agarose gel electrophoresis was performed to cut out an agarose gel containing approximately 500 bp of a DNA fragment detected by UV irradiation after bromide staining with chidium. Purification of the DNA fragment from the agarose gel was performed using a DNA collecting glass EASYTRAP, and the DNA fragment recovered by phenol / chloroform extraction and ethanol precipitation was used as the HGIR probe DNA.

Labeling of probe DNA [shed one ³² P] d CTP (1 1 ITB q / mmo 1, USA, E. I. Du pontde Nemours & and o., Inc., Inc.) using a Random Primer DNA Label ing Ki t (manufactured by Takara Shuzo Co., Ltd., Japan).

(2) Acquisition of positive clones by plaque hybridization

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. 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.

5-fold concentration of SSPE [0.9 M sodium chloride, 5 OmM sodium phosphate and 5 mM EDTA (pH 7.7)], 5-fold concentration of Enhardt's solution (each 0%) 1% BSA, ficoll and polyvinylpyrrolidone) and 0.5% SDS force Denatured salmon sperm DNA (salmon sperm DNA (manufactured by Sigma, USA) is dissolved in sterile purified water so that the final solution becomes 100 μg Zm1 in the resulting solution. The solution was drawn in and out using the attached syringe, and the DNA was cut into small pieces. After phenol / chloroform extraction and ethanol precipitation, dissolve in sterile purified water to a concentration of 1 Omg / m1, and incubate the required amount on ice at 100 ° C for 5 minutes. Was used after rapid cooling) to obtain a Brahy Hydration Solution. Put about 1 2 0 thousands bra over click menu assembler emissions which the DNA was fixed previously prepared hive Li Daizei sucrose Nba' grayed, main assembler down 1 cm ² per Li 0. 1 ml of flop Rehai Buri Dizei The solution was sealed with a ionic solution, and kept in a water bath at 65 ° C for 3 hours with shaking. This back grayed, The quenched labeled HGIR probe DNA on ice in addition to the earthenware pots by a final concentration of ^{1 X 1 0 6 c P m} / m 1 and seal after incubation for 5 minutes at 1 0 0 ° C Then, the mixture was kept in a water bath at 65 ° C for 24 hours with shaking. The membrane was removed and washed at room temperature for 10 minutes using Washing Solution 1 (2x SSPE and 0.1% SDS). Subsequently, after washing with washing solution 2 (1x SSPE and 0.1% SDS) at 65 ° C for 15 minutes, washing solution 3 (1 / 10x SSPE and 0.1% SSPE) was added at 65 ° C. Then, 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.

Next, 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. 3 3 1 3 7 2), and use a Beckman ultracentrifuge rotor (SW41Ti) at 18 ° C, 30 ° C. , And centrifuged at 00 rpm for 35 minutes. _{S is} buf f er of 4 0 0 μ 1 completely except the residue of the supernatant (1 0 0 g / ml of proteinase K, 0. 2 Μ chloride na Application Benefits um and 0.1% of SDS) to In addition, the reaction was carried out at 55 ° C for 90 minutes. Equivalent amount of water-saturated phenol solution [500 g of phenol (manufactured by Wako Pure Chemical Industries, Japan) and 0.5 g of 8-hydroxyquinoline

(Manufactured by Nacalai Tesque, Japan), and then added to 200 ml of purified water and shaken. The lower layer was used. After centrifugation at 00 rpm for 5 minutes, the upper part was transferred to another 1.5 ml micro tube. A phenol / cloth form extraction and ethanol precipitation were performed, and the positive clone was designated as DNA.

(3) Preparation of recombinant plasmid and determination of nucleotide sequence 1 μg of the prepared positive clone ADNA was dissolved in 50]} ^ buffer containing 1 U of restriction enzyme EcoRI, and incubated at 37 ° C for 2 hours to complete digestion. Agarose gel electrophoresis was performed to cut out an agarose gel containing a DNA fragment of about 1.5 kbp detected by UV irradiation after staining with bromide thidium. Purification of the DNA fragment from the agarose gel was performed using EASYTRAP, a glass powder for DNA recovery, and the DNA fragment recovered by phenol / chlorofonolem extraction and ethanol precipitation was used to remove the HGIR fragment (1). 5 kbp).

Dissolve 1 μg of Plasmid Vector PUC 118 (Takara Shuzo, Japan) in 50 μl of Η 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.

Transfer 0.2 g of pUCl18 EcoRI vector and 0.1 g of HGIR fragment (1.5 kbp) to a 0.5 ml microtube, and adjust to 5 / l with sterile purified water. Was. Then, 5 μl of solution I of DNA liat ion kitver.2 was added thereto, mixed well, and then reacted at 16 ° C for 1 hour. The reaction was stopped by incubating at 70 ° C for 30 minutes, and the DNA was recovered by phenol / chole-form extraction, ethanol precipitation, and then added to a 5 μl solution. The mixture was dissolved to obtain a mixture of ligne and ligne. Big Transformation of enterobacteria was performed using a competent cell, Escherichia coli 1i JM109 (Toyobo Co., Ltd., Japan). 100 μl of the competent cell dissolved in ice and 5 μl of the ligation mixture were mixed, and the mixture was allowed to stand still on ice for 30 minutes. After incubating at 42 ° C for 30 seconds, 0.9 ml SOC medium pre-warmed at 37 ° C was added and transferred to a 15 ml centrifuge tube. Shake for 1 hour at C! ! Cultured. This bacterial solution was added to 50 μl 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 μl After reacting at 37 ° C for 5 minutes, add 180 μl 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 μl 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

Analysis of homologous genes using mouse chromosome DNA

(1) Preparation of probe DNA fragment

100 μg 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

Subsequently, 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.

(2) Southern hybridization of mouse chromosomal DNA 5 / g mouse chromosomal DNA (manufactured by CL0NTECH, USA) was prepared by adding 5 U of restriction enzyme EcoRI to 1 OOj1 H buffer or 5 U Dissolved in 100/1 M buffer containing Hind III (Takara Shuzo Co., Ltd., Japan).

Incubation was performed at 37 ° C for 2 hours to complete digestion. After extraction of phenol / cloth holme and extraction of croat holme, respectively, DNA was collected at the ethanol precipitation.

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. Using the probe DNA fragment of about 270 bp prepared in (1) above, hybridization was performed on the membrane by the following method. That is, a final concentration of 100 μg / 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. Probe DNA fragment prep at this back grayed in 1 0 0 ° C, 5 minutes quenched above in ice after heat retention (1), Ni Let 's at a final concentration of ^{1 X 1 0 6 cpm / m} 1 In addition, it was sealed, and was further kept under shaking in a water bath at 42 ° C for 24 hours. The membrane was removed and washed with Washing Solution 1 at room temperature for 10 minutes. Subsequently, the substrate was washed at 50 with the washing solution 1 for 30 minutes, and further washed at 50 ° C. with the washing solution 3 for 30 minutes. The membrane was read and detected for radioactivity with a BAS 2000 imaging analyzer (Fuji Photo Film Co., Ltd.). As a result, no positive signal was detected from mouse chromosomal DNA. In other words, the HGIR gene obtained this time was considered to be unique to humans. Example 6

Determination of HGIR whole nucleotide sequence

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

M1 3 (— 21) and M1 3 reverse sequence primers as primers for React ion Dye Terminator After preparing a sequence sample, the nucleotide sequence was determined using a fluorescent sequencer manufactured by Applied Biosystems.

As a result, the nucleotide sequence of cDNA containing the HGIR gene consisting of the nucleotide sequence of SEQ ID NO: 3 was determined. Example 7

Analysis of mRNA amount by Northern hybridization method

In 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. 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. The mRNA was placed on a UV transilluminator with the transfer-facing side down and irradiated with UV at 254 nm for 5 minutes. 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 μg / τα \. In addition, 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. This back grayed, Ni Let 's become labeled HGIR probe DNA prepared in 1 0 0 ° Example 4 C in quenched with ice after 5 minutes incubated with a final concentration of ^{1 X 1 0 6 cpm / m} 1 In addition, it was sealed, and was further incubated while being shaken in a water bath at 42 ° C for 24 hours. The membrane was taken out and washed with a washing solution 1 at room temperature for 10 minutes. Subsequently, the substrate was washed for 30 minutes at 50 ° C. with the washing solution 1, and further washed at 50 ° C. for 30 minutes with the washing solution 3. After the hybridization, the radioactivity was read out and detected by a BAS 2000 imaging analyzer. As a result, as shown in Fig. 4, when using mRNA extracted from HUVECs subjected to stress-reactive culture, compared to the case using mRNA extracted from static culture HUVECs, it became clear. A kana signal was detected. In other words, it became clear that the amount of HGIR gene expression increased in vascular endothelial cells when shear stress was applied. Example 8

Preparation of anti-HGIR peptide antiserum

Among the amino acid sequences predicted from the nucleotide sequence of the HGIR gene determined in Example 6, 14 amino acids represented by 17 to 30 of SEQ ID NO: 1 were selected, and the peptide was selected. The rabbits were immunized, immunized and bleeding of egrets, and their titers were checked to prepare anti-HGIR peptide antiserum.

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. Example 9

Construction of HGIR expression plasmid

(1) Construction of 5 'and 3' untranslated region deletion expression plasmids

5 'untranslated region and 3' untranslated region of HGIR gene An expression plasmid from which the region was removed was constructed according to the following procedure.

First, 50 ng of 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). Dissolve in 100 μl of Taq DNA polymerase buffer containing the indicated antisense primer (manufactured by Nippon Bioservices, Japan), and overlay with Ι Ο Ο μ ミ mineral oil. 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.

The obtained PCR amplification product and 10 μg of Crowjung vector pBlueScriptll KS + (manufactured by STRATAGENE CLONING SYST EMS, USA) were each combined with 10 U of restriction enzyme Kpnl (manufactured by Takara Shuzo, Japan). And dissolved in 100 / zl of L buffer [10 mM Tris-HCl buffer (pH 7.5), 10 mM magnesium chloride and I mM DTT] at 37 ° C. After 2 hours of complete digestion, incubate in 保 Ο Ο μΙ H buffer containing 10 U of restriction enzyme EcoRI, and incubate at 37 ° C for 2 hours to complete digestion did. After adding 10 g of each tRNA, fractionation was carried out using a Chroma Spίη-400 spin column to remove small fragments. Phenol / c Mouth Mouth Holm extraction After the ethanol precipitation, the precipitate was washed with 75% ethanol to recover the DNA, and the HGIR (EcoRI-Kpnl) fragment and pBlueScr! pt II KS + EcoRI—Kpnl arm.

After ligation reaction of 0.5 pBlueScript II KS + EcoRI-Kpnl arm and 0.2 g of HGIR (EcoRI-Kpnl) fragment in a 20 μl 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 μl 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 μg / 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. However, the delay mitigation PstI (Takara Shuzo Co., Ltd., Japan) and BamHI were used as restriction enzymes for the preparation. As a result, it was confirmed that 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.

Subsequently, 5 g of PBSZHGIR was dissolved in 50 µl of Lbuffer containing 5 U of the restriction enzyme Kpnl, and completely digested by incubation at 37 ° C for 2 hours. FUNOOL Z-Cross mouth After extracting the form, precipitating ethanol, washing the precipitate with 75% ethanol and collecting DNA, 5 U T4 DNA po 1merase, 1 each The mixture was incubated at 37 ° C for 30 minutes in a T4 DNA polymerase buffer containing 25 µl of dATP, dCTP, dGTP and dTTP to prepare blunt-ended DNA. After extraction with phenol Z micro-mouthed form and ethanol precipitation, the precipitate was washed with 75% ethanol, and the recovered DNA was collected from a 50% plasmid containing 5 U of restriction enzyme EcoRI. It was dissolved in H buffer and completely digested by incubating at 37 ° C for 2 hours. Agarose gel electrophoresis is performed.Agarose gel containing about 1.3 kbp DNA fragment detected by UV irradiation after bromide staining with chidium is cut out, recovered and purified, and HGIR (EcoRI-BLUNT ) Fragments.

In addition, 10 ig of the expression vector pcDNA3.1 (+) for animal cells (produced by Inviogen, The Netherlands) was replaced with 10 U of each of the restriction enzymes EcoRI and EcoRV (Japan). Dissolve in Ο Ο Ο μ 国 H buffer (Takara Shuzo Co., Ltd.) at 37 ° C Completely digested for hours to keep warm. After adding 1 Q μg of tRNA, fractionation was performed using a Chroma Spin-400 spin column, and small fragments were removed. After extraction with phenol / closed-hole form and ethanol precipitation, the precipitate was washed with 75% ethanol to recover DNA, which was used as a pcDNA3.1 (+) EcoRI-EcoRV arm.

0.5 μg of pcDNA3.1 (+) EcRI-EcoRV arm and 0.2 μg of HGIR (EcoRI-BLUNT) fragment were subjected to ligation reaction at 20 μl and then the reaction mixture was used. E. coli was transformed. Transformation of Escherichia coli was carried out using a combi- nation cell, Escherichia coli 1JM109. 5 μl of the ligation reaction solution was added to 100 μl of the competent cell melted in ice, mixed, and then allowed to stand still on ice for 30 minutes. 42 After incubating at 30 ° C for 30 seconds, add 0.9 ml of SOC medium pre-incubated to 37 ° C, transfer to a 15m centrifuge tube, and heat to 37 ° C. For 1 hour. This bacterial solution was spread on an LB agar medium containing 50 g / ml of ampicillin, spread and allowed to stand at 37 ° C. for 16 hours to form colonies. The single colony that emerged was transplanted into 2 ml of LB / Amp, and cultivated overnight with shaking to extract lipoplasm DNA, thereby obtaining pEGIRIl. FIG. 5 shows the restriction enzyme cleavage site and functional map of the constructed expression plasmid pEGIR11.

(2) Construction of variant expression plasmid

In the same manner as (1), to facilitate the detection of the product An expression plasmid was prepared in which a marker peptide called FLAG peptide [BioTechnology, 61205-1210 (1988)] was attached to the carboxyl terminus of the HGIR peptide.

That is, 50 ng of PGIR10 was replaced with 2.5 U of Taq DNA polymerase and 0.1 lnmol of each of the PCR primers (sense primer shown in SEQ ID NO: 6 and antisense primer shown in SEQ ID NO: 8; The 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. After the reaction, the 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 に Ο Ο μ Ο 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 Ι Ο Ο μ H H buffer containing RI and completely digested by incubation at 37 ° C for 2 hours. After adding 10 μg of tRNA, small fragments were removed by fractionation using a Chroma Spin-400 spin column. After ethanol precipitation, the precipitate was washed with 75% ethanol and the DNA was recovered to obtain HGIR-FLAG (EcoR Kpnl) fragments. 0.5 μg (pBlueScript 1 I KS + EcoRI-Kpn I arm and 0.2 μg 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 μ1 and mix, and then stood still on ice for 30 minutes 42. Incubate for 30 seconds at 22 ° C, then incubate at 37 ° C in advance Add 0.9 ml of SOC medium, transfer to a 15 ml centrifuge tube, and culture with shaking for 1 hour at 37 ° C. Add 50 μg / ml ampicillin And spread on an LB agar medium containing 0.02% of 0-31 and 50-111] ^ and incubated at 37 ° C for 16 hours. The single white colony that appeared was 2 m 1 The cells were cultured with shaking using LB / Amp, and liposome DNA was extracted from the obtained cells to obtain pBSZHGIR-FLAG.The base sequence of the insert DNA was the same as in Example 6. Using the pBS / HGIR-FLAG as the material and the Dei-unit for various sensor lengths using Ki 1 o-Sequence De 1 eti on Kit However, 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 / χ1 L buffer containing 5 U of the restriction enzyme Kpnl, and kept at 37 ° C for 2 hours to completely digest. After extraction of phenol / close-up form and precipitation of ethanol, 75. /. After recovering the DNA by washing the precipitate in ethanol, the DNA was recovered in a T4 DNA polymerase buffer containing 5 U of T4 DNA polymerase, 125 M each of dATP, dCTP, dGTP and dTTP. The mixture was kept at 30 ° C for 30 minutes to prepare blunt-ended DNA. After extracting the phenolic mouth form, precipitating ethanol, and washing the precipitate with 75% ethanol, the recovered DNA was mixed with 50 μl of 5 U restriction enzyme Ec0RI. The cells were dissolved in H buffer (1) and completely digested at 37 ° C for 2 hours. HGIR-FLAG (EcoRI-BLUNT) fragment.

Next, 0.5 μg of pcDNA3.1 (+) EccRI-EcoRV arm and 0.2 μg of HGIR-FLAG (EcoRI-BLUNT) fragment were added to 20 μl after the ligation reaction. Then, Escherichia coli was transformed using the reaction solution. Transformation of Escherichia coli was carried out using a competent cell Escherichia c_o1i JM109. 5 μl of the ligation reaction solution was added to 100 μl of the competent cell melted in ice, mixed, and then allowed to stand on ice for 30 minutes. 42 After keeping the temperature at 22 ° C for 30 seconds, keep the 0.9 m1 The SOC medium was added, the mixture was transferred to a 15 ml centrifuge tube, and cultured with shaking at 37 t for 1 hour. This bacterial solution was spread on an LB agar medium containing 50 μg / m 1 of ampicillin, and allowed to stand still at 37 ° C. for 16 hours to form colonies. The emerged single colony was transplanted into 2 ml of LB / Amp, and cultured with shaking for a while to extract bacterial plasmid DNA, thereby obtaining PEGIR12-FLAG. Figure 5 shows the restriction enzyme cleavage site and functional map of the constructed expression plasmid pEGIRl2-FLAG.

Example 10

Confirmation of HGIR gene product

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).

(1) Introduction of expression plasmid into cells

The medium used is DMDM (Dainippon Pharmaceutical Co., Japan) containing 10% fetal serum (FCS), 50 IU / m1 ニ 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 μg of the expression plasmid was added to 41 LI POFECTAMINE

The samples were left standing at room temperature for 45 minutes at room temperature and in OPTI-MEM I Reduced Serum Mediurn (manufactured by GIBCO BRL, USA) of 2011. 800 μ \ (OPT I MEM I Reduced Serum Med iuni) was added to the reaction solution to prepare an introduction reaction solution. O

First, place the COS 1 cells on a 35 mm dish dish 2 X

1 0 ⁵ crowded seeded at a density of cells, at 5% CO _z to satisfy the 3 7 ° C Lee Nkyubeta one were cultured for 24 hours. After culturing the cells, wash the cells with OPTI-EI Reduced Serum Medium, add the transfer reaction solution described above, culture in the above incubator for 5 hours, and add 1 ml of medium containing 20% FCS. After further culturing for 18 hours in the above incubator, the introduced reaction solution was removed, and then 2 ml of a new medium was added, followed by further culturing for 3 days.

(2) Preparation of cell extract

On the third day after the introduction of plasmid, the medium was removed, and the cells were washed with ice-cold PBS (1). It was left on ice for 30 minutes. Next, the cells are detached using a silicon scraper, transferred to a 1.5 ml microtube, and centrifuged at 4 ° C at 3,000 rpm for 10 minutes.淸 excluded. Add 801 urine / Triton ZD TT solution (9 M urea, 2% Triton X—100 and 1% DTT) to the precipitate and add a Titec ultrasonic homogenizer US— Using a 5 S type, ultrasonic treatment was performed on the scale 2 for 1 second. Next, add 20 μl of an iDS solution (10% lithium dodecyl sulfate to which bromophenol blue (BPB) has been added to the extent that the color is obtained), and then add 1 μl.

About 5 μl of M-ris was added until the solution turned from yellow to blue. This was again subjected to ultrasonic treatment until the viscous property of the solution was completely eliminated, and the cell extract was obtained.

(3) Analysis of gene expression products by Western plotting

SDS - PAGE gels (Mul t igel4 / 20, Japan, the manufactured one of Gakusha) using a 1 lane per Li 1 X 1 0 ⁴ SDS according to the cell content of the cell extract exudates to the accompanying sample - PAGE Was performed. However, in this case, the same sample was subjected to electrophoresis using two gels for the purpose of performing the detection using two kinds of antibodies without performing the heat treatment of the sample. After completion of the electrophoresis, each gel was transferred to a Hybond-ECL (Amersham, UK) finalizer using a Bio-Rad mini-prototype cell. 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. Next, the plate is washed three times with a TBS solution containing 0.1% Tween 20 and then diluted with 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. Wash 3 times with TBS solution containing 0.1% Tween 20 After that, a secondary antibody solution diluted 100-fold with the above-mentioned antibody diluent [When using anti-HGIR antiserum as the primary antibody, HRP-labeled anti-Egret antibody (Amersham, UK)] When an anti-FLAGM 2 antibody was used as the primary antibody, an HRP-labeled anti-mouse antibody (manufactured by Amersham, UK) was used], and the mixture was reacted at room temperature with shaking for 1 hour. This was further washed three times with a TBS solution containing 0.1% Tween 20. After the last wash, soak in ECL Western Blotting Detection Reagent (Amersham, UK) for 1 minute, seal in a hybridization bag, and transfer to Hyno Inorem ECL. photosensitive be allowed a _c as a result, the extract of COS 1 cells derived from the introduction of PEGIR 1 2- FLAG, anti HGIR antiserum signal detected even when using any anti-FLAG antibody to primary antibody Was observed. Since the behavior of this signal was the same, it was confirmed that the gene expression product of the introduced expression plasmid was an HGIR-FLAG fusion peptide. On the other hand, in the extract derived from COS 1 cells into which pEGIR11 was introduced, a signal was observed only when anti-HGIR antiblood was used as the primary antibody. This signal behaves exactly the same as the fusion peptide described above, confirming that this is a peptide generated by HGIR. On the other hand, COS1 cells derived from pcDNA3.1 (+) vector transfected using as a negative control No signal was detected in any of the extracts from any of the antibodies.

According to the above procedure, the peptide encoded by the HGIR1 gene could be confirmed immunochemically. Example 1 1

Analysis of HGIR mRNA distribution in human tissue by Northern hybridization method

As 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 μg / m 1. DNA was added to prepare a prehybridization solution. Put the membranes with the above mRNA immobilized in the pre-hybridization bag, and add 0.1 ml of pre-hybridization solution per 1 cm ^{2 of the} membrane and seal. The mixture was kept in a water bath at 42 ° C. for 4 hours with shaking. 5 at 100 ° C in this bag After maintaining the temperature for 1 minute and quenching in ice, 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. For 24 hours in a water bath. The membrane was removed and washed with Washing Solution 1 at room temperature for 10 minutes. Subsequently, 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. After the hybridization, 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.

Industrial applicability

By using the peptide encoded by the novel gene HGIR of the present invention or its gene, 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.

Furthermore, it has been clarified that 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 listing

SEQ ID NO: 1

Array length: 4 2 3

Array type: peptide

Sequence type: amino acid

Array

Me t Va 1 Pro His Leu Leu Leu Leu Cys Leu Leu Pro Leu Va 1 Ai A 1 a

1 5 10 15

Th r Gl u Pro His Glu Gly y Ar g Ala Asp Gl u Gin Ser Ala Gl u Ala Ala

20 25 30

Leu Ala Va 1 Pro Asn Ala Set His Phe Phe Se i Trp Asn Asn Tyr Thr

35 40 45

Phe Set Asp Trp Gin Asn Phe Va 1 Gly Aig Atg Arg Tyr Gl Ala Glu

50 55 60

Ser Gin Asn Pro Thr Va 1 Lys Ala Leu Leu lie Va 1 Ala Tyr Sei Phe 65 70 75 80 lie I 1 e Val Phe Ser Leu Pbe Gly Asn Va 1 Leu Va 1 Cys His Va! Lie

85 90 95

Phe Lys Asn Gin Arg Met His Ser Ala Thr Ser Leu Phe lie Va 1 Asn

100 105 110

Leu Ala Val Ala Asp lie Met lie Thr Leu Leu Asn Thr Pro Phe Thr

115 120 125

Leu Val Aig Phe Val Asn Se i Thr Trp 11 e Phe Gly Lys Gly Met Cys

130 135 140

His Val Se 【Arg Phe Ala Gin Tyr Cys Ser Leu His Val Se i Ala Leu 145 150 155 160

Thr Leu Thr Ma [le Ala Va 1 Asp Arg His Gin Val lie Met His Pro >

Leu Pro Thr Ser Gin Leu Gin Ser Gly Lys Thr Asp Leu Sei Ser r Va 1

405 410 15

Glu Pro lie Va I Thi Met Sei

420 423

SEQ ID NO: 2

Array length: 1 2 6 9

Sequence type: nucleic acid

Sequence type: DN A

Array

ATGGTCCCTC ACCTCTTGCT GCTCTGTCTC CTCCCCTTGG TGCGAGCCAC CGAGCCCCAC 60 GAGGGCCGGG CCGACGAGCA GAGCGCGGAG GCGGCCCTGG CCGTGCCCAA TGCCTCGCAC 120 TTCTTCTCTT GGAACAACTA CACCTTCTCC GACTGGCAGA ACTTTGTGGGGGCAGGAGGC

TACGGCGCTG AGTCCCAGAA CCCCACGGTG AAAGCCCTGC TCATTGTGGC TTACTCCTTC 240

ATCATTGTCT TCTCACTCTT TGGCAACGTC CTGGTCTGTC ATGTCATCTT CAAGAACCAG 300

CGAATGCACT CGGCCACCAG CCTCTTCATC GTCAACCTGG CAGTTGCCGA CATAATGATC 360

ACGCTGCTCA ACACCCCCTT CACTTTGGTT CGCTTTGTGA ACAGCACATG GATATTTGGG 420

AACGGCATGT GCCATGTCAG CCGCTTTGCC CAGTACTGCT CACTGCACGT CTCAGCACTG 480

ACACTGACAG CCATTGCGGT GGATCGCCAC CAGGTCATCA TGCACCCCTT GAAACCCCGG 540

ATCTCAATCA CAAAGGGTGT CATCTACATC GCTGTCATCT GGACCATGGC TACGTTCTTT 600

TCACTCCCAC ATGCTATCTG CCAGAAATTA TTTACCTTCA AATACAGTGA GGACATTGTG 660

CGCTCCCTCT GCCTGCCAGA CTTCCCTGAG CCAGCTGACC TCTTCTGGAA GTACCTGGAC 720

TTGGCCACCT TCATCCTGCT CTACATCCTG CCCCTCCTCA TCATCTCTGT GGCCTACGCT 780

CGTGTGGCCA AGAAACTGTG GCTGTGTAAT ATGATTGGCG ATGTGACCAC AGAGCAGTAC 840

TTTGCCCTGC GGCGCAAAAA GAAGAAGACC ATCAAGATGT TGATGCTGGT GGTAGTCCTC 900

TTTGCCCTCT GCTGGTTCCC CCTCAACTCC TACGTCCTCC TCCTGTCCAG CAAGGTCATC 960

CGCACCAACA ATGCCCTCTA CTTTGCCTTC CACTGGTTTG CCATGAGCAG CACCTGCTAT 1020 AACCCCTTCA TATACTGCTG GCTGAACGAG AACTTCAGGA TTGAGCTAAA GGCATTACTG 1080

AGCATGTGTC AAAGACCTCC CAAGCCTCAG GAGGACAGGC CACCCTCCCC AGTTCCTTCC 1140

TTCAGGGTGG CCTGGACAGA GAAGAATGAT GGCCAGAGGG CTCCCCTTGC CAATAACCTC 1200

CTGCCCACCT CCCAACTCCA GTCTGGGAAG ACAGACCTGT CATCTGTGGA ACCCATTGTG 1260

ACGATGAGT 1269 SEQ ID NO: 3

Array length: 1 5 6 2

Sequence type: nucleic acid

Sequence type: D N A

Array

GTCCCGAGGA GCCAGGGAGC CCGGACGCGC GGAGCACCGC GCCGCCCGCC ACCAGCCCGC 60

GGTCGGCCTT GTATGCTGTG CGCCCCGCAC CCCGGCTGCG CGCTCCGATC CCAGCGGGAG 120

GGGACGCCCG GCCCGCAGGC TCCCAGGGGA GGGGTGGCTC CTGCAAAATG GTCCCTCACC 180

TCTTGCTGCT CTGTCTCCTC CCCTTGGTGC GAGCCACCGA GCCCCACGAG GGCCGGGCCG 240

ACGAGCAGAG CGCGGAGGCG GCCCTGGCCG TGCCCAATGC CTCGCACTTC TTCTCTTGGA 300

ACAACTACAC CTTCTCCGAC TGGCAGAACT TTGTGGGCAG GAGGCGCTAC GGCGCTGAGT 360

CCCAGAACCC CACGGTGAAA GCCCTGCTCA TTGTGGCTTA CTCCTTCATC ATTGTCTTCT 420

CACTCTTTGG CAACGTCCTG GTCTGTCATG TCATCTTCAA GAACCAGCGA ATGCACTCGG 480

CCACCAGCCT CTTCATCGTC AACCTGGCAG TTGCCGACAT AATGATCACG CTGCTCAACA 540

CCCCCTTCAC TTTGGTTCGC TTTGTGAACA GCACATGGAT ATTTGGGAAG GGCATGTGCC 600

ATGTCAGCCG CTTTCCCCAG TACTGCTCAC TGCACGTCTC AGCACTGACA CTGACAGCCA 660

TTGCGGTGGA TCGCCACCAG GTCATCATGC ACCCCTTGAA ACCCCGGATC TCAATCACAA 720

AGGGTCTCAT CTACATCGCT GTCATCTGGA CCATGGCTAC GTTCTTTTCA CTCCCACATG 780

CTATCTGCCA GAAATTATTT ACCTTCAAAT ACAGTGAGGA CATTGTGCGC TCCCTCTGCC 840

TGCCAGACTT CCCTGAGCCA GCTGACCTCT TCTGGAAGTA CCTGGACTTG GCCACCTTCA 900

TCCTCCTCTA CATCCTGCCC CTCCTCATCA TCTCTCTGGC CTACGCTCGT GTGGCCAAGA 960 AACTGTGGCT GTGTAATATG ATTGGCGATG TGACCACAGA GCAGTACTTT GCCCTGCGGC 1020

GCAAAAAGAA GAAGACCATC AAGATGTTGA TGCTGGTGGT AGTCCTCTTT GCCCTCTGCT 1080

GGTTCCCCCT CAACTGCTAC GTCCTCCTCC TGTCCAGCAA GGTCATCCGC ACCAACAATG 1140

CCCTCTACTT TGCCTTCCAC TGGTTTGCCA TGAGCAGCAC CTGCTATAAC CCCTTCATAT 1200

ACTGCTGGCT GAACGAGAAC TTCAGGATTG AGCTAAAGGC ATTACTGAGC ATGTGTCAAA 1260

GACCTCCCAA GCCTCAGGAG GACAGGCCAC CCTCCCCAGT TCCTTCCTTC AGGGTGGCCT 1320

GGACAGAGAA GAATGATGGC CAGAGGGCTC CCCTTGCCAA TAACCTCCTG CCCACCTCCC 1380

AACTCCAGTC TGGGAAGACA GACCTGTCAT CTGTGGAACC CATTGTGACG ATGAGTTAGA 1440

AGAGGTTGGG AAGAGGGAGT GGGAGGGGTC TGTCTCCACC TGAGGCAGGG AAAGAGAGCC 1500

TATTCTCACA CATGATCTTC AGAGTGCTGG AAACACACTC CTGCAGAAGC TGTAGGACTC 1560

TT 1562 SEQ ID NO: 4

Array length: 3 8

Sequence type: nucleic acid

Sequence type: D N A

Array

TAGTCGACWK YCT1ACIGYI MTIRSIRTIG AYMGITWY 38 SEQ ID NO: 5

Array length: 3 6

Sequence type: nucleic acid

Sequence type: D N A

Array

TGGAATTCTT RYTIAA AAI SCRTAIATIA VIGGRT 36 SEQ ID NO: 6

Array length: 3 6

Sequence type: nucleic acid

Sequence type: D N A

Array

GAATTCGGAT CCGGAGGGGT GGCTC CTGCA AAATGG 36 SEQ ID NO: 7

Array length: 2 9

Sequence type: nucleic acid

Sequence type: D N A

Array

GGTAC CTAAC TCATCGTCAC AATGGGTTC 29 SEQ ID NO: 8

Array length: 5 3

Sequence type: nucleic acid

Sequence type: D N A

Array

GGTACCTA CT TGTCATCCTC GTCCTTGTAG TCACTCATCG TCACAATGGG TTC 53

Claims

The scope of the claims

1. DNA comprising at least the peptide coding region of the full-length human DNA derived from the restriction enzyme cleavage site shown in the restriction map shown in FIG.

2. The DNA according to claim 1, wherein the peptide code region is a nucleotide sequence encoding a peptide including the amino acid sequence of SEQ ID NO: 1.

3. The DNA of claim 1, wherein the peptide code region is the nucleotide sequence of SEQ ID NO: 2.

4. The DNA according to claim 1, wherein the human-derived full-length DNA characterized by the restriction enzyme map is the nucleotide sequence of SEQ ID NO: 3.

5. A complementary DNA to the DNA according to any one of claims 1 to 4.

6. A replicable recombinant DNA comprising the DNA according to any one of claims 1 to 4 incorporated in a replicable vector.

7. DNA that can hybridize to DNA containing the nucleotide sequence of SEQ ID NO: 2.

8. A complementary DNA to the DNA of claim 7.

9. A replicable recombinant DNA comprising the DNA according to claim 7 incorporated into a replicable vector.

10. A microorganism or cell transformed with the replicable recombinant DNA according to claim 6 or 9.

11. A substantially pure peptide coded by the DNA of claim 1 or 7.

12. The peptide according to claim 11, comprising the amino acid sequence of SEQ ID NO: 1.

13. (a) binding the DNA according to claim 1 or 7 to a replicable expression vector, and preparing a replicable recombinant DNA containing the DNA and the replicable expression vector; Get

(d) culturing the transformant, allowing the transformant to express the DNA,

(e) A peptide produced by a method comprising substantially isolating the peptide from a transformant in which the peptide has been cultured.

14. An antibody that binds to a peptide comprising at least the amino acid sequence of SEQ ID NO: 1.

15. DNA containing at least the peptide coding region of human-derived full-length DNA characterized by the restriction enzyme cleavage site shown in the restriction enzyme map in Figure 1 and SEQ ID NO: 2 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 DNA containing the base sequence of 135 is bound to the peptide. The sample is brought into contact with a sample which is considered to contain such a substance, and the change that occurs in response to the binding of the peptide to the substance that binds to the peptide is used as an index, and A detection method characterized by detecting a substance that binds to a substance.

16. DNA containing at least the peptide coding region of the full-length human-derived DNA recharacterized by the restriction enzyme cleavage site shown in the restriction enzyme map of FIG. 35 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 DNA containing the base sequence of 5 and a substance capable of binding to the peptide Is brought into contact with a sample which is considered to contain a substance which inhibits the binding between the peptide and the substance which binds to the peptide, and then binds to the peptide and the substance which binds to the peptide. Changes that occur in response to the binding of these substances A detection method characterized by detecting, as an indicator, a substance that inhibits the binding between the peptide and a substance that binds to the peptide.