WO2006035702A1

WO2006035702A1 - C-peptide specific binding molecule and use thereof

Info

Publication number: WO2006035702A1
Application number: PCT/JP2005/017615
Authority: WO
Inventors: Keiji Naruse; Jiro Nakamura; Keiko Naruse
Original assignee: National University Corporation Nagoya University
Priority date: 2004-09-28
Filing date: 2005-09-26
Publication date: 2006-04-06
Also published as: JPWO2006035702A1

Abstract

A molecule capable of specific binding to C-peptide, and a method of using the same. There is provided a C-peptide binding agent comprising newly identified two types of C-peptide specific binding molecules (SEQ ID NO: 1 and SEQ ID NO: 3). Further, there is provided a method of estimating the action and state of C-peptide with the use of the amount of relevant proteins as an indicator. Still further, there is provided a method of screening a compound capable of regulating the action and state of C-peptide with the use of these proteins.

Description

Specification

C-peptide specific binding molecules and uses thereof

Technical field

[0001] The present invention relates to a C peptide-specific binding molecule and use thereof. Specifically, it relates to a C peptide binding agent using a C peptide-specific binding molecule, a method for evaluating the action state of C peptide, a method for screening a compound that regulates the action state of C peptide, and the like.

Background art

[0002] Insulin preparations are indispensable for the treatment of diabetic patients. Currently, insulin drugs with various action times such as ultra-fast, fast-acting, intermediate, and ultra-long-acting types have been developed. It contributes to the treatment. However, especially in type 1 diabetes, where insulin is depleted, the onset and progress of diabetic complications has a major impact on the quality of life and prognosis of patients. It has also been pointed out that C-peptide resistance may exist in type 2 diabetes as well as insulin resistance.

The C peptide is a 31-amino acid peptide that is produced when proinsulin is broken down into insulin in the splenic Langenorehans island, and has long been thought to have no physiological activity. In recent years, it has been proved that C-peptide has physiological activities such as vasodilatory action and neuroprotective action, and its action attracts attention. However, the mechanism of action of the C-peptide was unclear.

[0003] Non-Patent Document 1: Biochem Cell Biol. 1990 Dec; 68 (12): 1428- 32.

Non-Patent Document 2: Acta Physiol Hung. 1995; 83 (4): 333-42.

Non-Patent Document 3: Diabetes Metab Res Rev. 2003 Sep- Oct; 19 (5): 375-85.

Non-Patent Document 4: Comment in: Diabetes Metab Res Rev. 2003 Sep- Oct; 19 (5): 345- 7. Non-Patent Document 5: Int J Exp Diabetes Res. 2002 Oct- Dec; 3 (4): 241- Five.

Non-Patent Document 6: Nitric Oxide. 2003 Sep; 9 (2): 95-102.

Non-Patent Document 7: J. Biol. Chem. 2003, 278 (23) 20915-24

Disclosure of the invention

Problems to be solved by the invention [0004] If the mechanism of action of C peptide is clarified, experimental evidence will be obtained when C peptide is used as a drug, and specific drug discovery targeting C peptide or its binding molecule will be possible. Will open up the way to clinical application of C-peptide.

In order to elucidate the mechanism of action of C-peptide, it is essential to first identify the C-peptide receptor molecule, that is, the molecule that specifically binds to C-peptide (C-peptide specific binding molecule). Most important.

On the other hand, C peptide-specific binding molecules are indispensable for C peptides to exert their actions, so the expression state (expression level, localization, etc.) of C peptide-specific molecules is the action of C peptides. It directly affects the state (action amount, etc.). Therefore, the action state of the c peptide can be regulated by controlling the expression state of the c peptide specific binding molecule. This means that C peptide-specific binding molecules are targets for developing drugs for diseases involving C peptides. On the other hand, the state of expression of c-peptide-specific binding molecules Since it is possible to indirectly evaluate the action state of c-peptides, the pathology of diseases involving C-peptides using C-peptide-specific binding molecules It is also possible to grasp the risk and evaluate the risk of morbidity. That is, application of the C peptide-specific binding molecule to the diagnostic field is also expected.

As described above, in addition to being a key molecule for elucidating the mechanism of action of C-peptide, C-peptide-specific binding molecules are themselves target molecules in drug discovery and diagnostic fields.

. The first object of the present invention is to provide a c-peptide-specific binding molecule having such a high utility value. It is also intended to provide medical use or diagnostic use of C peptide specific molecules.

Means for solving the problem

[0005] In order to achieve the above object, the present inventors tried to identify a C peptide-specific binding molecule. As a result, we have succeeded in identifying two types of proteins that have specific binding properties to C-peptide by conducting various studies with a focus on setting extraction conditions from cells. The present invention has been completed based on the above findings and provides the following configuration.

The first aspect of the present invention is the above-described protein (C peptide-specific binding tag) that has been successfully identified. C peptide-binding agent using a protein. The C peptide binding agent of the present invention comprises a first protein having the amino acid sequence of SEQ ID NO: 1, a second protein having the amino acid sequence of SEQ ID NO: 3, and a protein substantially homologous to the first protein or the second protein. Contains one or more proteins selected from the group of power and strength. The C peptide binding agent can be used to regulate the action state of the C peptide. It can also be used for C peptide detection, measurement or quantification, C peptide separation and purification.

[0006] Another aspect of the present invention relates to a method for evaluating the action state of a C peptide by detecting the protein (C peptide-specific binding protein or a protein substantially homologous thereto). In the evaluation method of the present invention, the first protein having the amino acid sequence of SEQ ID NO: 1, the second protein having the amino acid sequence of SEQ ID NO: 3, and the first protein or the second protein substantially. A step of detecting in the sample one or more proteins selected from the group consisting of homologous proteins. Detection (measurement) Use the results to evaluate the action state of C-peptide. Based on the action state of the C peptide, it is possible to diagnose a disease involving the C peptide. In another embodiment of this aspect, the nucleic acid in the sample is the detection target. That is, a nucleic acid encoding the first protein having the amino acid sequence of SEQ ID NO: 1, a nucleic acid encoding the second protein having the amino acid sequence of SEQ ID NO: 3, and the first protein or the second protein. A step of detecting in a sample a nucleic acid that encodes a homologous protein and one or more nucleic acids selected from a powerful group, based on the detection results! Evaluate the action state of the C peptide in the sample. Alternatively, a polymorphism of the gene encoding the first protein having the amino acid sequence of SEQ ID NO: 1, a polymorphism of the gene encoding the second protein having the amino acid sequence of SEQ ID NO: 3, and the first protein or A step of analyzing in a sample one or more polymorphisms selected from the group consisting of a polymorphism of a gene encoding a protein substantially homologous to the second protein, and based on the analysis results Evaluate the C peptide activity in the sample.

[0007] Still another aspect of the present invention relates to a screening method using the above protein (C peptide-specific binding protein or a protein substantially homologous thereto). That is, in this aspect, a means for finding a compound capable of regulating the action state of C peptide is proposed. Provided. In the screening method of the present invention, the step of bringing the C peptide into contact with the protein is carried out in the presence (test group) and absence (control group) of the test compound. Then, the change in the binding state (binding amount, binding force, etc.) between the C peptide and the above protein due to the presence of the test compound is examined. According to such a procedure, a compound that changes the binding state between the C peptide and its specific binding molecule, that is, a compound that can regulate the action state of the c peptide can be selected. Selected compounds will be potential candidates for drugs for diseases involving C-peptides, or useful materials in developing such drugs. In another embodiment of this aspect, a cell-based screening method is provided. In this embodiment, after the step of culturing cells for a predetermined time in the presence (test group) and absence (control group) of the test compound, the expression level of the protein in the cells after culturing is determined. taking measurement. Then, by comparing the expression level of the test group with the expression level of the control group, it is evaluated whether or not the test compound has the ability to regulate the action state of the C peptide (and if so) .

[0008] Yet another aspect of the present invention relates to a compound capable of regulating the action state of a C peptide. Specifically, as one aspect, there is provided an antibody that has a specific binding property to the C peptide or the protein and inhibits the binding between the C peptide and the protein. The antibody can be used, for example, for the binding assay between the C peptide and the protein by utilizing its specific binding property. Further, since the antibody has an activity of suppressing or inhibiting the action of the C peptide, it can be used as a drug for diseases caused by high expression of the C peptide.

[0009] The present invention further provides a method for regulating the action state of a C peptide in a target cell. In one embodiment of this aspect, the step of artificially introducing the protein (C peptide-specific binding protein or a protein substantially homologous thereto) into the target cell is performed, thereby increasing the amount of action of the C peptide. Is prompted. In another embodiment, a step of introducing into the target cell a compound that inhibits the binding of the C peptide to the protein is carried out, thereby promoting a reduction in the amount of action of the C peptide. In yet another embodiment, the expression of the protein in the target cell is inhibited by using an antisense method, RNA interference, ribozyme, etc., and the amount of action of the C peptide is reduced. According to the method of this aspect Thus, it is possible to examine the effect of an increase or decrease in the action amount of c-peptide on target cells. In addition, if the cells constituting the living body are targeted cells, the method of the present invention can be used to treat a subject suffering from a disease caused by high or low expression of C peptide.

[0010] The present invention further provides a kit for simply carrying out the method of the present invention.

Brief Description of Drawings

[0011] FIG. 1 shows the results of electrophoresis (cell staining) of a cell extract prepared from human aortic smooth muscle cells. Left lane: migration result of sample solution prepared in the above procedure (C peptide (+)), right lane: NHS-activated Sepharose 4 Fast Flow without C peptide binding is used instead of C peptide binding column The rest are migration results (C peptide (-)) of a control sample prepared in the same procedure. In the lower row, the band positions are indicated by lines (1, 2).

[FIG. 2] FIG. 2 shows the results of a competition experiment with C peptide (gel after staining). A: Control 1 (C peptide (-) column), B: Control 2 (C peptide (+) column, no competitive inhibition), C: Sample lane (C peptide (+) column, competitive inhibition).

FIG. 3 shows the results of PCR using HSPA5 or HSPA9B-specific primers using cDNA prepared from human vascular endothelial cells (HUVEC), human fetal kidney cells (HEK), and HeLa cells as a cage.

FIG. 4 is a result of analyzing a sample prepared by E. coli transformed with HSPA5 expression vector or HSPA9B expression vector by Western plot. (1) is the lane to which the precipitate fraction obtained by centrifugation after E. coli solubilization was applied, (2) is the lane to which the supernatant fraction obtained by centrifugation after E. coli solubilization was applied, (3) Is the lane where the supernatant obtained by centrifugation after E. coli solubilization was purified with Glutachione-Sepharose 4B.

FIG. 5 shows the results of analysis of a sample prepared by HEK cell power transformation with an HSPA5 expression vector or HSPA9B expression vector, by Western plotting. The left column (1) is the lane to which the cell disruption solution was applied, and (2) is the lane to which the Co-beads extract was applied. (1) in the right column is the lane to which the cell lysate was applied, and (2) is the supernatant after centrifuging the cell lysate. (3) is the lane to which the Co-beads extract was applied.

FIG. 6 is a fluorescent immunostaining image of HEK cells transformed with an HSPA5 expression vector or HSPA9B expression vector. Upper row is control, middle row is HEK cell transformed with HSPA5 expression vector, and lower row is HEK cell transformed with HSPA9B expression vector.

[Fig. 7] Experimental protocol for binding experiment of HSPA5 and C peptide.

FIG. 8 shows the result of binding experiment (Western blot) between HSPA5 and C peptide. (1) is a cell lysate prepared from cells transformed with the HSPA 5 expression vector, (2) is a cell lysate prepared by transformation with an empty vector, and (3) is transformed with an HSPA5 expression vector. Cell force The component bound to the C peptide beads in the supernatant fraction of the prepared cell lysate, (4) was obtained by reacting non-peptide-bound beads (No peptide beads) instead of C peptide beads Sample, (5) is the component bound to the C peptide beads in the supernatant fraction of the cell disruption solution prepared from cells transformed with the empty vector, and (6) is the cell force transformed with the empty vector. The components bound to the peptide-unbound beads in the supernatant fraction of the cell lysate prepared. (7) is the cell force transformed with the HSPA5 expression vector. The supernatant fraction of the prepared cell lysate is first reacted with the C peptide. Reaction with C peptide beads In some cases, the component bound to the C peptide, (8) is the cell force transformed with the empty vector. The supernatant fraction of the prepared cell lysate is first reacted with the C peptide and then with the C peptide beads. Ingredients bound to C peptide.

FIG. 9 is an immunostained image of HUVEC cells stimulated with C peptide. The right column is a stained image obtained by reacting anti-HSPA5 antibody and FITC anti-goat antibody. The left column is the control.

[Fig. 10] Protocol of HSP knockdown experiment.

[Fig. 11] Results of HSP knockdown experiment. No.1 (sense strand introduction, without C peptide stimulation), No.2 (sense strand introduction, with C peptide stimulation), No.3 (antisense strand introduction, without C peptide stimulation), No.4 (antisense strand) Introduction, C peptide stimulation).

BEST MODE FOR CARRYING OUT THE INVENTION

(C peptide binding agent)

The first aspect of the present invention relates to a C peptide binder. Here, the “C peptide” is produced when proinsulin is broken down into insulin in the splenic islets of Langeron. It is a peptide consisting of mino acid (SEQ ID NO: 5). C-peptide has been used as a diagnostic marker for diabetes. Recently, it has been reported that the C peptide itself has a specific physiological activity.

The C peptide binding agent of the present invention includes a protein identified as a C peptide specific binding molecule, or a protein substantially homologous thereto (hereinafter collectively referred to as “the present protein”). As shown in Examples described later, the present inventors succeeded in obtaining two types of proteins as C peptide-specific binding molecules. As a result of amino acid sequence analysis, each of these proteins is also referred to as a 70 kDa protein 9B precursor (hereinafter also referred to as “HSPA9B”. The amino acid sequence of the protein is represented by SEQ ID NO: 1, and the base sequence encoding the protein is represented by SEQ ID NO: 2, respectively. And heat shock 70 kDa protein 5 (hereinafter also referred to as “HSPA5”! /, And the amino acid sequence of the protein is shown in SEQ ID NO: 3 and the base sequence encoding it is shown in SEQ ID NO: 4). Was identified. In the present specification, the former is sometimes referred to as a first protein and the latter as a second protein.

Here, the term “substantially homologous” used in the present specification for the two types of proteins to be compared is used in the application utilizing the binding property to the C peptide! It means a state in which the two types of proteins possess the binding ability to the C peptide to the extent possible. Regarding “substantial homology” here, the region not involved in binding to the C peptide is less important. Therefore, substantial homology can be recognized even if the region not involved in binding to the C peptide is altered (deletion or substitution of some or all amino acids, addition of other amino acids, etc.). Even when other molecules or substances are added, substantial homology may be maintained before and after the addition. On the other hand, if the two types of proteins to be compared differ in terms of their constituent amino acids and molecular weight, there may be cases where substantial homology can be recognized between them. However, in general, if amino acid sequences have high homology, they are often homologous in function. When the binding to one C-peptide is 100%, the other is at least 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably about 100%. %, Substantial homology can be observed between the two proteins. For example, as a protein substantially homologous to HSPA9B, a protein having a sequence obtained by modifying a part of the amino acid sequence of SEQ ID NO: 1 and having a specific binding property to a C peptide can be mentioned. Similarly, as a protein substantially homologous to HSPA5, a protein having a sequence obtained by modifying a part of the amino acid sequence of SEQ ID NO: 3 and having a specific binding property to a C peptide can be exemplified. The modification of the amino acid sequence herein means that the amino acid sequence is changed by deletion or substitution of one to several amino acids constituting the amino acid sequence, or addition or insertion of one to several amino acids, or a combination thereof. This modification can be performed as long as the property of specific binding to C peptide is highly maintained. “Highly retained” as used herein means 50% or more after modification, preferably 70% or more, and more preferably, when the specific binding to C peptide before modification is defined as 100%. Is 80% or more, more preferably 90% or more, and most preferably about 100%.

As long as such a high degree of retention is recognized, the position of the amino acid sequence change is not particularly limited. Further, a change may occur at a plurality of positions (may be consecutive positions). The number of amino acids required for modification is, for example, a number corresponding to within 10% of all amino acids constituting the amino acid sequence before modification, preferably a number corresponding to within 5% of all amino acids, more preferably within 1% of all amino acids. It is a number corresponding to. However, in some cases, significant modifications beyond this range, such as addition of amino acids to the 5 'end or 3' end, may be permitted.

[0013] Other examples of proteins substantially homologous to HSPA9B (or HSPA5) include naturally occurring mutant HSPA9B (or HSPA5). Yet another example is HSPA9B (or HSPA5) of a species other than human. Examples of the “non-human species” here include non-human primates (monkeys, chimpanzees, etc.), mice, rats, usagis, horses, horses, hidges, nu, cats, and the like. Such a C peptide binding agent containing HSPA9B (or HSPA5) derived from a non-human species can be suitably applied (in vivo or ex vivo) to a species other than human. Alternatively, it can be suitably used for in vitro experiments.

[0014] The C peptide binding agent of the present invention comprises at least one of the present proteins.

In other words, the C peptide binding agent of the present invention contains two or more of the present proteins. Typically, the C peptide binding agent of the present invention comprises HSPA9B or HSPA5 alone or both. The protein constituting the C-peptide binding agent of the present invention is modified such as methylation or daricosylation! /, Or even! /.

[0015] The C peptide-binding agent of the present invention can be used to control the action state of a C peptide. For example, if the C peptide binding agent of the present invention is administered to a living body, it can be expected that the C peptide binds to it and that the action of the C peptide is exerted via the C peptide binding agent. Therefore, if applied to an individual exhibiting a pathological condition as a result of the reduced action of the C peptide, the pathological condition can be improved.

On the other hand, the peptide binding agent of the present invention can also be used for detection, measurement or quantification of C peptide. When the peptide binding agent of the present invention is used for such detection of C peptide, it is preferable to label the peptide binding agent of the present invention with a detectable labeling substance. However, if another substance capable of detecting the peptide binding agent of the present invention is used in combination, such a labeling agent becomes unnecessary.

The detection or the like here may be performed in a living body or in a cell or tissue separated from a living body! / 生体.

[0016] The C peptide binding agent of the present invention may be used for separation and purification of C peptides. For example, a C peptide affinity column can be constructed by binding the C peptide binding agent of the present invention to a support and filling it in a suitable container. Such a column can be used to detect and sort powerful C-peptides such as biological samples.

[0017] The present protein constituting the C peptide binding agent of the present invention (HSPA9B, HSPA5, or a protein homologous to any of them) is, for example, an animal (eg, human, monkey, It can be obtained from cells or tissues of chimpanzees, etc.) by using means such as extraction and purification (for details of the extraction method, see the examples below). The protein can also be prepared using genetic engineering techniques. That is, it can be prepared by introducing a DNA encoding the target protein into an appropriate host cell and recovering the protein expressed in the transformant. The collected protein is appropriately purified according to the purpose. When prepared as a recombinant protein, various modifications are possible. For example, DNA encoding the protein of interest and other suitable By carrying out transformation using a vector retaining a suitable DNA, it is possible to obtain a recombinant protein in which the target protein is not a peptide encoded by the other DNA, and the protein is linked. Other DNA can be incorporated in advance in the vector. By such modification, it is possible to extract the recombinant protein, simplify the purification, or add a biological function.

[0018] (Detection / evaluation / diagnosis method using C peptide-specific binding molecule)

Another aspect of the present invention relates to a method for evaluating the working state of a C peptide by detecting the above-mentioned protein. As used herein, the term “action state of C peptide” refers to the amount of action of C peptide exerted through the protein, and depends on the amount of the protein present. In this specification, the term “evaluation” is used interchangeably with determination and discrimination.

[0019] In the method of the present invention, a step of detecting the amount of the present protein (preferably HSPA9B or HSPA5) present in a sample is performed. The detection results are used to evaluate the action state of the C peptide in the sample. In the present invention, for example, cells, tissues, blood, etc. collected from the living body of the subject (subject), cultured cells, established cells, etc. can be used.

[0020] The amount of the protein can be detected (measured) using, for example, a compound that specifically binds to the protein. The detection method (or measurement method) is not limited to this, but is preferably an immunological method. In an immunological technique, an antibody (specifically, an anti-HSPA9B antibody or an anti-HSPA5 antibody) against the above-described protein is used, and the present protein is detected using the binding property (binding amount) of the antibody as an index. The term “antibody” used herein is a polyclonal antibody, monoclonal antibody, chimeric antibody, single chain antibody, CDR grafted antibody, humanized antibody, or a fragment thereof (however, it has a specific binding ability to HSPA9B etc. Etc.). The antibody in the present invention can be prepared by using an immunological technique, a phage display method, a ribosome display method, and the like.

The immunological staining method enables rapid and sensitive detection. Therefore, the burden on the subject (patient) is reduced when a small amount of sample is used. Also, the operation is simple. Examples of detection methods include ELISA, radioimmunoassay. And qualitative or quantitative methods such as FACS, immunoprecipitation, and immunoblotting.

The polyclonal antibody can be prepared by the following procedure. Prepare an antigen (HSPA9B, etc.) and use it to immunize animals such as mice, rats, rabbits, goats, etc. As an antigen, HSPA9B or a homologous protein thereof, HSPA5 or a homologous protein thereof, or any one of them can be used. When an effective immune-inducing action cannot be expected due to the low molecular weight, it is preferable to use an antigen bound with a carrier protein. As the carrier protein, KLM (Keyhole Light Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin), etc. are used. In addition, for the binding of carrier protein, the calpositimide method, dartalaldehyde method, diazo condensation method, MBS (maleimido benzoyl succinimide) method and the like can be used.

Repeat immunization as necessary, and collect blood when the antibody titer rises sufficiently, and obtain serum by centrifugation. The antiserum obtained is purified by the utility. In this way, a polyclonal antibody can be obtained. On the other hand, monoclonal antibodies can be prepared by the following procedure. First, immunization is performed in the same procedure as above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hyperidoma. Subsequently, after this hybridoma is monoclonalized, a clone producing an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing hyperpridoma to a desired number or more and then transplanting it into the abdominal cavity of an animal (for example, a mouse) and growing it in ascites to purify the ascites. For the purification of the culture medium or the ascites, affinity chromatography using protein G, protein A or the like is preferably used. In addition, affinity chromatography with an antigen immobilized thereon can also be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination. Regarding the antibody production method, Kohler and Milstein (1975) Nature 256: 495-497; Brown et al. (1981) J. Immunol. 127: 539— 46; Brown et al. (1980) J. Biol. Chem. 255: 4980-83; Yeh et al. (1976) Proc. Natl. A cad. Sci. USA 76: 2927-31; Yeh et al. (1982 ) Int. J. Cancer 29: 269-75; Kozbor et al. (1983) Immunol. Today 4:72; Kenneth, RH in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); Lerner, EA (1981) Yale J. Biol. Med. 54: 387-402; Gefter, ML et al. (1977) Somatic Cell Genet. 3: 231-36, etc. .

[0022] Various documents regarding phage display methods, such as Huse et al. (1989) Science 246: 1275-1281; McCafferty et al. (1990) Nature 348: 552-554; Fuchs et al. (1991) B io / Technology 9: 1370—1372; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88: 797 8-7982; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133—4137; Gram et al. (1 992) Proc. Natl. Acad. Sci. USA 89: 3576-3580; Hay et al. (1992) Hum. Antibod. Hy bridomas 3: 81-85; Griffiths et al. (1993) EMBO J 12 PCT International Publication No. WO 90/02809; PCT International Publication No. WO 92/20791; PCT International Publication No. WO 92/15679; PCT International Publication No. WO 92/09690, etc. it can. Moreover, kits for preparing and screening phage display libraries are commercially available, and can be suitably used.

[0023] By using a labeled antibody, the above detection or the like can be easily performed. Examples of antibody labeling include fluorescent dyes such as fluorescein, rhodamine, Texas red, and Oregon green, horseradish peroxidase, microperoxidase, alkaline phosphatase, 13 D-galactosidase, and other enzymes, luminol, and acrylidine. Chemical or bioluminescent compounds such as dyes, radioactive isotopes such as ³² P, ¹³¹ ι, and ¹²⁵ ι, and piotin can be used.

[0024] The amount of the protein can also be determined using a nucleic acid encoding the present protein (preferably a nucleic acid encoding HSPA9B or HSPA5) as opposed to detecting the protein itself. Specifically, for example, the amount of mRNA encoding this protein is detected. In addition, genomic DNA can be targeted for detection when the number of copies of the gene encoding the protein varies with the amount of the protein. For example, if an increase in the gene copy number of this protein is found as a result of detection, It can be predicted that the expression level of the quality increases.

Methods for measuring the amount of a specific nucleic acid are known in the art. For example, the Southern hybridization method, the Northern hybridization method, the in situ hybridization method, RT-PCT The law etc. can be used.

Another embodiment of the present invention relates to a method for evaluating the action state of a C peptide in a subject using a polymorphism of a gene encoding the protein, and preferably a polymorphism of a gene encoding the protein in the subject (preferably Includes a step of analyzing a polymorphism of a gene encoding HSPA9B or HSPA5.

In general, there are individual differences in the DNA sequence that constitutes a gene. This individual difference is called genetic polymorphism. Genetic polymorphisms are those in which one base is replaced by another base (SNP (single nucleotide polymorphism)), and usually one to several tens of bases are deleted or inserted (inserted Z deletion type polymorphism). Type), those having a different number of repeats in a repetitive sequence of 2 to several basic forces (VNTR (variable number of tandem repeat) and microsatellite polymorphism), and the like are known. These gene polymorphisms may regulate the expression state of the gene, or change the amino acid in the protein encoded by the gene and affect its function. Therefore, by analyzing the polymorphism of a certain gene, the potential function of the protein encoded by the gene can be evaluated. Thus, by examining the polymorphism of the gene encoding this protein, the expression state and Z or action state of this protein can be predicted. The prediction results obtained in this way can be used to understand and evaluate the action state of C-peptide. Specifically, for example, as a result of analyzing the gene polymorphism of the protein in a certain target, the protein is expressed when it is considered that the protein is highly expressed or when the interaction with the C peptide is performed well. When it is considered that the C peptide is likely to exert its action, it can be determined that the subject is at high risk of suffering from a disease caused by an increase in the action of the C peptide. It can also be determined that high effects can be obtained when C peptide is administered to the subject. The information obtained in this way is used when making a treatment plan for medical treatment (treatment or prevention) using C peptide (including the case of stopping the administration of C peptide and changing to other treatment means). Useful. [0026] The evaluation results regarding the action state of the C peptide obtained by the above methods are used to diagnose a disease characterized by an abnormality in the action amount of the C peptide (hereinafter also referred to as "target disease of the present invention"). Available to: Accordingly, the present invention also provides the following diagnostic method.

One aspect of the diagnostic method of the present invention is a method for diagnosing a disease characterized by an abnormal amount of action of a C peptide, wherein the first protein having the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 are used. A sample collected from a subject (subject) of at least one protein selected from the group consisting of a second protein having a protein, a protein substantially homologous to the first protein or the second protein, and a force And detecting the C peptide in the sample using the detection result. Another aspect of the present invention is a method for diagnosing a disease characterized by an abnormal amount of action of C peptide, comprising a nucleic acid encoding a first protein having the amino acid sequence of SEQ ID NO: 1, and SEQ ID NO: One or more nucleic acids selected from the group consisting of a nucleic acid encoding a second protein having an amino acid sequence of 3 and a nucleic acid encoding a protein substantially homologous to the first protein or the second protein, A step of detecting in a sample collected from a subject (subject), and a step of using the detection result to evaluate the working state of the C peptide in the sample.

Still another embodiment of the present invention is a method for diagnosing a disease characterized by an abnormal amount of action of a C peptide, comprising a polymorphism of a gene encoding the first protein having the amino acid sequence of SEQ ID NO: 1, A polymorphism of a gene encoding a second protein having the amino acid sequence of SEQ ID NO: 3, and a polymorphism of a gene encoding a protein substantially homologous to the first protein or the second protein; Analyzing one or more polymorphisms selected from the group consisting of: a sample collected from the sample; and a step of evaluating the action state of the C peptide in the sample using the analysis result; including.

[0027] As used herein, "a disease characterized by an abnormal amount of action of C peptide" refers to a disease accompanied by high or low expression of C peptide as at least one characteristic. Therefore, not only diseases directly caused by high or low expression of C-peptide, but also diseases caused by other causes result in high or low expression of C-peptide. If included, it is included in the disease. Here, HSPA5 (also known as glucose regulated protein 78kDa (G RP78)), one of the proteins, has been reported to be associated with diabetes (Biochem Cell Biol. 19 90 Dec; 68 ( 12): 1428-32 .; Acta Physiol Hung. 1995; 83 (4): 333-42.). HSPA5 has also been confirmed to inhibit apoptosis (J. Biol. Chem. 2003, 278 (23) 20915-24). On the other hand, it has been reported that C peptide also has an inhibitory effect on apoptosis (Diabetes Metab Res Rev. 2003 Sep- Oct; 19 (5): 375- 85.; Comment in: Diabetes Metab Res Rev. 2003 Sep- Oct; 19 (5): 345— 7.; Int J Exp Diabetes Res. 2002 Oct— Dec; 3 (4): 241— 5.) o Regarding the C peptide, it further increased intracellular calcium and activated eNOS. It has been reported that there is a vasodilatory effect by wandering (Nitric Oxide. 2003 Sep; 9 (2): 95-10 2.) In addition, as a result of our research, (1) insulin and Similarly, it promotes phosphorylation by p42 / p44 MAP kinase, (2) inhibits phosphorylation by Akt, unlike insulin, and (3) promotes Rho translocation to Rho Through these actions, it became clear that the C peptide was involved in the function of vascular smooth muscle cells.

Considering the above facts, the amount of action of C-peptide is effective in diseases caused by apoptosis and vasodilatory effects, such as diabetic complications (macrovascular and microvascular disorders) and metabolic syndrome (Metabolic syndrome). It is considered relevant. Therefore, the method of the present invention is expected to be an effective means for diagnosing these diseases. In addition, it is possible to treat these diseases (improving symptoms) by suppressing this especially when the action of C-peptide is abnormally high, or by increasing it when the action of C-peptide is abnormally low! Or it is expected that it can be prevented.

Here, the action amount of the C peptide depends primarily on the expression amount of the C peptide. That is, if the expression level of C peptide is abnormal, the action amount of C peptide is usually abnormal. In view of this, the term “disease characterized by an abnormal amount of C peptide action” is used interchangeably with the term “disease characterized by an abnormal expression level of C peptide”. Shall be. Accordingly, diseases characterized by abnormal expression levels of C peptide also fall under the disease targeted by the present invention.

In this specification, the term “disease” means an abnormal condition such as a disease, illness, or disease state. Used interchangeably with terminology.

[0029] The action amount of the C peptide provides useful information regarding the target disease of the present invention. That is, since there is a relationship between the amount of action of C peptide and the target disease of the present invention, by examining the amount of action of C peptide in the target (human etc.), It is possible to grasp or evaluate the presence or absence of disease, the degree of risk of being affected, and Z or disease state. It is also possible to monitor changes (improvement or deterioration) of the disease state by measuring changes in the action amount of C peptide over time.

As described above, the C peptide has been shown to have an anti-apoptotic action, a vasodilatory action, a neuroprotective action, and the like. Therefore, the expression state of this protein can also be used for evaluating or evaluating the degree of these actions of the C peptide. That is, the present protein is effective not only as a determination index for diseases caused as a result of these actions, but also as a means for directly grasping these actions.

[0030] (Method for screening compound that modulates action state of C peptide)

Yet another aspect of the present invention relates to a screening method using the present protein. In the screening method of the present invention, the step (contact step) of contacting the present protein (preferably HSPA9B or HSPA5) and the peptide in the presence (test group) and absence (control group) of the test compound, respectively. To be implemented. Specifically, for example, the protein is brought into contact with the C peptide immobilized on an insoluble support such as a plate, a membrane, or a bead in the reaction solution. In the test group, such contact is performed with the test compound present in the reaction solution (ie, with the test compound added), while in the control group, the test compound is not present in the reaction solution. Similar contact is performed. Next, after a predetermined time has elapsed, the nonspecific binding component is removed by washing with an appropriate solution. Subsequently, a complex of the C peptide and the present protein is detected, and the detection amount is compared between the test group and the control group (detection amount comparison step). The detection of the complex can be performed by, for example, an immunological technique using an antibody having a specific binding property to the present protein.

[0031] The reaction solution used for contacting the present protein with the C peptide is not particularly limited, and a known or commercially available buffer solution, physiological saline, or the like can be used. The reaction conditions (composition and pH of the reaction solution, reaction temperature, reaction time, etc.) are as follows. It can be easily set based on experiments using quality as an index. For example, phosphate buffer, citrate buffer, Tris-HCl buffer, Tris-acetate buffer, etc. can be used as the anti-application solution, and the pH thereof is, for example, pH 6.0 to pH 8.0, preferably pH 6.5. Set to ~ pH 7.5. The reaction temperature can be, for example, 4 ° C to 45 ° C, preferably 4 ° C to 40 ° C. The reaction time can be set, for example, in the range of 1 minute to 24 hours (specifically, for example, the reaction is performed overnight).

[0032] Instead of contacting the present protein with the C peptide immobilized on the insoluble support, the present protein may be immobilized on the insoluble support and then contacted with the C peptide. In this case, the amount of the complex can be determined by detecting the C peptide bound to the present protein on the solid phase.

[0033] Using the result of the detection amount comparison step, the change in the binding mode (binding amount, binding force, etc.) between the C peptide and the present protein due to the presence of the test compound is examined. Specifically, for example, if the amount of binding between the C peptide and this protein decreases due to the presence of the test compound, it is determined that the test compound has an action to inhibit or suppress the binding between these two molecules. Can do. On the other hand, if the amount of binding between the C peptide and this protein increases due to the presence of the test compound, it is judged that the test compound has the effect of promoting or enhancing the binding between these two molecules. be able to. In this manner, a compound that changes the binding property between the C peptide and the present protein, that is, a compound that can regulate the action state of the C peptide can be selected. The selected compounds are effective for medical treatment of diseases involving C peptide (ie, diseases characterized by abnormal action of C peptide). For example, a compound that has been shown to inhibit or inhibit the binding of the C peptide and the protein is expected to be used to suppress the action of the C peptide. Therefore, the compound is expected to be applied for the treatment and prevention of diseases caused by high expression of C peptide. On the other hand, compounds that have been shown to promote or enhance the binding of C peptide and the present protein can be used to enhance the action of C peptide. Therefore, the compound is expected to be used for the treatment and prevention of diseases caused by low expression of c-peptide.

As described above, the compound selected by the screening method of the present invention is C peptide. It is a promising candidate for drugs for diseases in which it is involved, or a useful material in developing such drugs.

[0034] In another aspect of the invention, a cell-based screening method is provided. In this embodiment, it is examined whether the intracellular expression level of the present protein (preferably HSPA9B or HSPA5) changes due to the presence of the test compound. For example, a step of culturing a cell for a predetermined time in the presence of a test compound (cultivation step), a step of measuring the expression level of the protein in the cultured cell (expression level measurement step), The step of comparing the expression level of this protein with the expression level of the protein in the cell after similarly culturing the cells in the absence of the test compound (expression level comparison step) For example, first prepare a cell group (test group) cultured in the presence of the test compound and a cell group (control group) consisting of the same type of cells and cultured in the absence of the test compound. Incubate under the same conditions except for the presence or absence. After incubation, the intracellular expression level of this protein is measured for each group. Finally, the measurement results are compared. When the intracellular expression level of the protein in the absence of the test compound is known in advance, the test compound may be evaluated using this as the intracellular expression level of the protein in the control group. In this case, culturing in the absence of the test compound can be omitted.

[0035] The intracellular expression level can be measured, for example, by directly detecting the protein using an immunological technique using an antibody having a specific binding property to the protein. Alternatively, mRNA encoding the present protein may be detected. In this case, the expression level of the protein can be ascertained using the amount of mRNA encoding the protein as an index. For detection of mRNA, conventional methods such as various hybridization methods using a specific probe (eg, Southern blotting) can be employed.

[0036] Based on the comparison results between the test group and the control group, whether or not the presence of the test compound affects the expression level of the protein in the cell is determined, and if so, the degree thereof. As a result, if there is a change in the intracellular expression level of the protein due to the presence of the test compound, it can be determined that the test compound has an effect of regulating the expression level of the protein. Here, as a result of the study by the present inventors, this protein is a C peptide in the living body. It was found that it is a binding substance for tide and a key molecule that exerts the action of c-peptide. In other words, the action state (action amount) of c-peptide in the living body depends on the expression level (and utility) of this protein. From this, it can be said that a test compound having an action of regulating the expression level of this protein can regulate the action state of the C peptide in the living body. As described above, compounds capable of regulating the action state of C peptide can also be selected by a cell-based screening method.

[0037] In the screening method of the present invention, the test group and the control group may be composed of the same cells (cell group). For example, after culturing cells in the absence of the test compound and measuring the expression level of this protein (measurement in the control group), the test compound is added to the culture medium, and further cultured for a predetermined time. After completion of the culture, measure the intracellular expression level of this protein (measurement of the test group).

[0038] As long as the condition that the protein can be expressed is satisfied, the type of cell used is not limited. For example, established cells such as HeLa cells, COS cells, and CHO cells can be used. These cells are also readily available for cell banking, such as ATCC. In addition, cells separated from a living body can be used directly. For example, cells collected from humans, monkeys, horses, horses, mice, rats, rabbits, chickens and the like can be used in the screening method of the present invention.

Furthermore, it must be separated from the living body on the condition that it is a non-human animal cell (for example, mouse, rat, rabbit, chicken, etc.), and the state (that is, the state that constitutes the living body) is detailed. Blasts may be used. In this case, for example, the test compound is orally or parenterally administered to an individual, and after a predetermined time has elapsed, the amount of the protein in a specific cell (for example, vascular smooth muscle cell, nervous system cell, etc.) is measured. . Then, compare the measured amount with the amount of the protein in the case where the protein is not administered!

[0039] A cell group in which a network is formed between cells other than dispersed cells (for example, cells forming a specific tissue) can be used for screening. Further, the screening method of the present invention may be performed using two or more different types of cells in combination.

[0040] The number of cells to be used is not particularly limited, and can be determined in consideration of the detected amount of the present protein, experimental equipment, and the like. For example, 1 to 10 ^5, preferably 10 to 10 ^4, more preferably 10 ² ~ 10 ³ cells can be used.

[0041] (Compound that modulates the action state of C-peptide)

Yet another aspect of the present invention relates to a compound capable of modulating the action state of a C peptide. Specifically, as one aspect, there is provided an antibody that specifically binds to a C peptide or a protein having the amino acid sequence of SEQ ID NO: 1 and inhibits the binding between the C peptide and the protein. In another embodiment, there is provided an antibody having a specific binding property to a C peptide or a protein having the amino acid sequence of SEQ ID NO: 3 and inhibiting the binding between the C peptide and the protein. These antibodies can be used, for example, for the binding assay between the C peptide and the present protein by utilizing its specific binding property. In addition, since the antibody has an activity of inhibiting the action of C peptide, it can be used as a drug for diseases caused by high expression of C peptide. As the antibody production method, the method described in the detection method column of the present invention can be employed.

[0042] (Method of adjusting action state of C peptide)

Yet another aspect of the present invention relates to a method for regulating the action state of a C peptide in a target cell. In one embodiment of this aspect, the step of artificially introducing the protein (preferably HSPA 9B or HSPA5) into the target cell is performed. As a result, the abundance of the protein, which is a specific binding molecule of C peptide, in the target cell increases, and the action amount of C peptide is enhanced accordingly. Therefore, it is possible to examine the influence of an increase in the action amount of C peptide on target cells. In other words, a suitable experimental system is provided for investigating the physiological effects of C-peptide. Here, if the target cell is a cell constituting a living body (including a cell to be transplanted into the living body later), the amount of action of the C peptide in the living body can be increased. Therefore, the method of the present invention can be used to treat a subject suffering from a disease caused by low expression of C peptide.

In another embodiment of the present invention, the step of administering to the target cell a compound that inhibits the binding between the C peptide and the protein is performed. As a result, the action amount of the C peptide in the target cell can be reduced. Therefore, it is possible to examine the influence of a decrease in the action amount of C peptide on the target cells. In other words, also in this embodiment, a suitable experimental system is provided for examining the physiological action of C peptide. Where If the target cell is a cell constituting a living body (including cells that are transplanted into the living body later), the amount of action of the C peptide in the living body can be reduced. Therefore, the method of the present invention can be used to treat a subject suffering from a disease caused by high expression of C peptide.

[0043] In still another embodiment of the present invention, there is provided a method for regulating the action state of a C peptide by inhibiting or suppressing the expression of the protein (preferably HSPA9B or HSPA5) in a target cell. Inhibition or suppression of the expression of this protein can be achieved by antisense methods, RNA interference, or by using ribozymes.

[0044] In the case of performing expression inhibition by an antisense method, for example, an antisense construct that generates RNA complementary to a unique part of mRNA encoding this protein when transcribed in a target cell is used. Is done. Such an antisense 'construct is introduced into a target cell, for example, in the form of an expression plasmid. On the other hand, an oligonucleotide 'probe that hybridizes with the mRNAZ or genomic DNA sequence encoding this protein and inhibits its expression when introduced into the target cell as an antisense' construct can also be employed. . Such oligonucleotide probes are preferably those that are resistant to exonucleases and endogenous nucleases such as Z or endonucleases.

When a DNA molecule is used as an antisense nucleic acid, an oligodeoxyribonucleotide derived from a region containing a translation initiation site (for example, a region of −10 to +10) of mRNA encoding the present protein is preferable.

[0045] The complementarity between the antisense nucleic acid and the target nucleic acid is preferably strict, but there may be a few mismatches. The ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. In general, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large. One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.

[0046] The antisense nucleic acid may be DNA, RNA, or a chimeric mixture thereof, or a derivative or modified form thereof. Moreover, it may be single-stranded or double-stranded. Base part, sugar part, Alternatively, the stability of the antisense nucleic acid, the hybridization ability, etc. can be improved by modifying the phosphate skeleton. In addition, antisense nucleic acids can promote cell membrane transport (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6 556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; see PCT Publication No. W088 / 09810, published December 15, 1988) or substances that increase affinity for specific cells.

The antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (198 8), Proc. Natl. Acad. Sci. USA 85: 7448 -You can refer to 7451 etc.

[0047] In order to enhance the action of the antisense nucleic acid in the target cell, a strong promoter such as pol II or pol III can be used. That is, if a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter. Expression of the antisense nucleic acid can be performed by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells). For example, SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), rous sarcoma virus 3 ′ end region promoter (Yamamoto et al., 1980, Cell 22: 787-797), herpes zoster thymidine Promoters such as 'kinase' promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.

[0048] In one embodiment of the present invention, the expression of the protein is inhibited by RNA interference (RNAi). RN Ai is a sequence-specific post-transcriptional gene repression process that can occur in eukaryotic cells. In RNA interference, a double-stranded RNA (dsRNA) having a sequence corresponding to the sequence of the target mRNA is used. Mammalian cells are known to have two pathways affected by dsRNA (sequence-specific pathways and sequence non-specific pathways)! In sequence-specific pathways, relatively long dsRNAs are split into short interfering RNAs (siRNAs). This siRNA is about 2 which forms about 19 nucleotide siRNA each with an overhang at the 3 'end. Has a 1 nucleotide sense and antisense strand. On the other hand, a sequence non-specific pathway is thought to be triggered by any dsRNA related to the sequence if it is longer than a predetermined length. In this pathway, dsRNA is involved in the synthesis of two enzymes: PKR, which becomes active and phosphorylates the translation initiation factor el F2 to stop all protein synthesis, and RNAase L activation molecule 2 ', 5 'Oligoadenylate synthase is activated. In the method of the present invention, it is preferable to use dsRNA shorter than about 30 base pairs in order to minimize the progression of this non-specific pathway (Hunter et al. (1975) J Biol Chem 250: 409- 17; Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 25 4: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8. See).

RNAi has been confirmed to be an effective means for reducing gene expression in various cell types (eg, HeLa cells, NIH / 3T3 cells, COS cells, 293 cells, etc.). In general, the expression can be inhibited more effectively than the antisense method.

[0049] The dsRNA used for RNAi can be prepared in vitro or in vivo by chemical synthesis or using an appropriate expression vector. The latter method is particularly effective for preparing relatively long dsRNA. In designing dsRNA, a sequence unique to the target nucleic acid (continuous sequence) is usually used. A program and algorithm for selecting an appropriate target sequence have been developed.

[0050] In another embodiment of the present invention, the expression of the protein is inhibited by a ribozyme. A force capable of destroying mRNA encoding the protein using a ribozyme that cleaves mRNA with a site-specific recognition sequence. Preferably, a hammerhead ribozyme is used. For the construction method of the non-header ribozyme, for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.

As in the antisense method, ribozymes may be constructed using modified oligonucleotides, for example, for the purpose of improving stability and targeting ability. To generate an effective amount of a ribozyme in a target cell, for example, a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter (eg, pol II or pol III) can be used. preferable.

[0051] (Kit used in the method of the present invention) Each of the above-described methods of the present invention (a detection method using the present protein as a detection target, a screening method using the present protein, etc.) may be carried out using a kited reagent or the like. Another aspect of the present invention provides kits used for such purposes. For example, in the case of a kit used for a detection method using the present protein as a detection target, a reagent (first reagent) having specific binding property to the present protein is included as a component (component) thereof. The first reagent is preferably an antibody or antibody fragment. Further, if a detectable substance labeled with a label is used as the first reagent, direct detection (measurement) is possible using the amount of the labeling substance as an index. On the other hand, if a reagent that specifically binds to the reagent (second reagent) with a labeling substance (for example, a labeled antibody secondary antibody) is used, the reagent is bound via the first reagent. Second Reagent Power Using the amount of label obtained as an index, it is possible to indirectly detect the binding amount of the first reagent. In addition to the reagents described above, the kit of the present invention can contain a diluent, a reaction solution, a reaction vessel, and the like necessary for the reaction using the first reagent and the like.

On the other hand, in the case of a kit used in the screening method of the present invention, typically, as a component (component) thereof, a first reagent containing C peptide and a second reagent containing the present protein (for example, HSPA9B or HSPA5) are used. 2 reagents. The C peptide or the protein may be bound to an insoluble support. If the first reagent or the second reagent is labeled in advance, the kit can directly detect the binding amount between the C peptide and this protein. On the other hand, a labeled compound (for example, an antibody) having a specific binding property to C peptide or the present protein may be included in the kit. When a powerful kit is used, the amount of binding between the C peptide and the protein is indirectly detected using the amount of the labeled compound (label amount) as an index.

The kit of the present invention usually includes instructions for use.

Example 1

[0052] <Separation of protein having specific binding property to C-peptide> Purification and identification>

1. Preparation of C peptide bond column

About lml NHS-activated Sepharose 4 Fast Flow (Amersham Bioscience, 71-50

00-14) was bound with 2 mg of C peptide according to a predetermined method provided by the manufacturer.

[0053] 2. Separation and purification of C peptide-specific binding protein In the following procedure, separation and purification of a protein having specific binding property to c-peptide was attempted.

(1) Human aortic smooth muscle cells cultured in DMEM, 5% CO, 37 ° C with 10% FCS

2

m Collected from 10 dishes (number of cells: about 1 X 10 ⁸ ).

(2) The recovered cells were suspended in Lysis buffer 1.5 ml: 25 mM Tris-HCl (pH 7.5), 1% NP40, inhibitor (Complete Mini, Roche, 1 836 153) and left at 4 ° C for 30 min. . This treatment solubilized the cells.

(3) After the above treatment, the mixture was centrifuged at 15,000 X g and lOmin, and the supernatant was collected (cell extract, approximately 1 ml).

(4) The cell extract was reacted with the C peptide binding column (lml) prepared in 1. at room temperature.

(5) After completion of the reaction, washing was performed with Lysis buffer (lml) (3 times) to remove components that non-specifically bound to the C peptide binding column.

(6) The binding component was also eluted with 1 ml of 0.1 M glycine (pH 3.0) in the C peptide binding column force after the washing treatment (once). The eluate was immediately neutralized with glycine (pH 3.0).

(7) The eluate after neutralization was concentrated to about 100 1 (about 10-fold concentration) using polyethylene glycol (sample solution).

(8) The sample solution obtained by the above procedure was separated by SDS-PAGE and then subjected to silver staining.

[0054] The stained gel is shown in the upper part of FIG. Left lane: electrophoresis result of sample solution prepared in the above procedure (C peptide (+)), right lane: NHS-activated Sepharose 4 Fast Flow without C peptide binding is used instead of C peptide binding column. Except for is the migration result (C peptide (-)) of the control sample prepared by the same procedure. In the lower part of Fig. 1, the band positions are indicated by lines (1, 2). As is clear from FIG. 1, two bands are observed in the left lane (C peptide (+)). That is, two types of proteins that specifically bind to the C peptide binding column were obtained. From the mobility, the molecular weights of these proteins were estimated to be about 70 kDa and about 68 kDa, respectively.

[0055] 3. Identification of C peptide-specific binding proteins

Next, we attempted to identify two proteins that were successfully separated. First, the same hand as above After preparing the sample solution in order ((1) to)), separation by SDS-PAGE and transfer to PVDF membrane were performed, and MALDI-TOF / MS analysis (PMF analysis) was performed. As a result, the protein observed as an approximately 70 kDa band by SDS-PAGE is the heat shock 70 kDa protein 9B precursor (ACCESSION: NP—004125, DEFINITION: heat shock 70 kDa protein 9B precursor; heat shock 70kD protein 9; stress—70 protein, mitochondrial; 75 kDa glucose regu lated protein; peptide— binding protein 74; mortalin, perinuclear; p66— mortalin; heat shock 70kD protein 9B (mortalin— 2) [Homo sapiens], Entrez Protein, NCBI, http: // www .ncbi.nlm.nih.gov /). On the other hand, the protein observed as an approximately 68kDa band on SDS-PAGE is heat shock 70kDa protein 5 (ACCESSION: NP.005338, DEFINITION: heat shock 70kDa protein 5 (glucose- regulated protein, 78kDa); He at-shock 70kD protein. -5 (glucose-regulated protein, 78kD); heat shock 70kD prote in 5 (glucose-regulated protein, 78kD) [Homo sapiens], Entrez Protein, NCBI, http: //www.ncbi.nlm.nih.gov/) It was half IJ to be.

Example 2

In order to confirm the binding ability of the two identified proteins to the C-peptide, a competition experiment was performed according to the following procedure.

2

m Collected from 10 dishes (number of cells: about 1 X 10 ⁸ ).

(4) The cell extract and C peptide binding column (see 1. in Example 1, 1 ml) were reacted at room temperature in the presence of C peptide. As a control 1 (C peptide (-) column), the cell extract was reacted under the same conditions as NHS-activated Sepharose 4 Fast Flow to which C peptide was not bound. As control 2 (no competitive inhibition), cell extract and C peptide binding column Were reacted in the same manner in the absence of c-peptide.

(5) After the reaction was completed, the cells were washed with Lysis buffer (lml) (3 times).

(6) Elution with 1 ml of 0.1 M glycine (pH 3.0) (once). The eluate was immediately neutralized with glycine (pH 3.0).

[0057] Fig. 2 shows the gel after staining. A: Control 1 (C peptide (-) column), B: Control 2 (C peptide (+) column, no competitive inhibition), C: Sample lane (C peptide (+) column, competitive inhibition). As is clear from FIG. 2, two types of bands (about 70 kDa and about 68 kDa) observed around 70 kDa disappeared by competitive inhibition of C peptide. This indicates that an excess of free C-peptide competitively inhibited the binding of the two proteins to the column. In other words, it can be said that the two types of proteins that have been purified by the C peptide binding column have specific binding properties to the C peptide.

Example 3

[0058] <Expression experiment of identified protein and verification of function>

1. Confirmation of HSPA5 and HSPA9B in human-derived cells

Human vascular endothelial cells (HUVEC), human fetal kidney cells (HEK), and HeLa cell force were extracted from total RNA, converted to cDNA, and PCR was performed using HSPA5 or HSPA9B specific primers. The reaction solution after PCR was subjected to electrophoresis. Fig. 3 shows the results of electrophoresis. The upper part of Fig. 3 shows the amplification product with the HSPA5-specific primer, and the lower part shows the amplification product with the HSP A9B-specific primer. The molds used for PCR are as follows. (L) HUVEC cDNA, (2) HUVEC Total RNA, (3) HEK cDNA, (4) HEK Total RNA, (5) HeLa cDNA, (6) HeLa Total RNA, (7) None .

As is clear from FIG. 3, it was confirmed that HSPA5 and HSPA9B were expressed in HUVEC cells, HEK cells, and HeLa cells!

[0059] 2. Closing HSPA5 and HSPA9B First, HSPA5 and HSPA9B were cloned from HEK cells by PCR. Next, in order to utilize the Gatew a _y System (Invitrogen) to prepare an entry clone incorporating Kuroyungu the cDNA. Using the resulting entry clone, vectors for E. coli expression and mammalian expression were prepared. Make sure that the GST-tag is added to the C-terminal side of the cDNA clone for E. coli expression, and the His-tag and GFP-tag are attached to the C-terminal side of the cDNA clone for mammalian expression. did.

[0060] 3. Expression in E. coli

Escherichia coli was transformed with the E. coli expression vector obtained in 2. (HSPA5 expression vector, HSPA9B expression vector). After inducing expression with IPTG, the GST fusion protein was purified from the cell disruption solution and subjected to Western plotting. The results of the Western plot are shown in Fig. 4. The left lanes ((1) to (3)) are obtained for the samples that also obtained E. coli power transformed with the HSPA5 expression vector, and the right lanes ((1) to (3)) are transformed with the HSPA9B expression vector. The results are for samples that also have converted E. coli potency. In addition, (1) is a lane to which a precipitate fraction obtained by centrifugation after E. coli solubilization was applied, (2) is a lane to which a supernatant fraction obtained by centrifugation after E. coli solubilization was applied, ( 3) is the lane where the supernatant obtained by centrifugation after E. coli solubilization was purified with Glutachione-Sepharose 4B.

As can be seen from Fig. 4, a band is observed at a position below the expected position (arrow). For HSPA9B, a decrease in the amount extracted with GSP beads is observed. From these results, it was speculated that the target proteins (HSPA5 and HSPA9B) were expressed in the full length state!

[0061] 4. Expression in mammalian cells

(1) Confirmation of expression by Western plot

HEK cells were transformed with the mammalian expression vector obtained in 2. (HSPA5 expression vector, HSPA9B expression vector). Effectene was used for transformation. His-tag fusion protein was extracted from the cell lysate using Cobeads and subjected to Western blotting. Figure 5 shows the results of the Western plot. In both left and right columns, sample lanes (control) obtained from HEK cells transformed with empty vectors in order from the left, formed with HSPA5 expression vector Sample lane (HSPA5) obtained from transformed HEK cells, and sample lane (HSPA9B) from which HEK cells transformed with the HSPA9B expression vector were also obtained. In the left column, (1) is the lane to which the cell lysate was applied, and (2) is the lane to which the Co-beads extract was applied. Similarly, in the right column, (1) is the lane to which the cell lysate was applied, (2) was the lane to which the supernatant after centrifuging the cell lysate was applied, and (3) was applied to the Co-beads extract. Lane.

As shown in Fig. 5, in the lane where the sample extracted with Co beads was applied, a band was observed at the expected position, indicating that both HSPA5 and HSPA9B were expressed in full length.

[0062] (2) Confirmation of expression by fluorescent immunostaining

First, HEK cells were seeded in a 35 mm dish. Subsequently, HEK cells were transformed with the mammalian expression vector (HSPA5 expression vector, HSPA9B expression vector) obtained in 2. (HSPA5-His (0.4ug), HSPA9B-His (0.4ug)). Effectene was used for transformation. The transformed cells were collected and seeded in a 1 × 1 glass chamber. Fluorescent immunostaining was performed using Anti-V5 (Alexa 488 mouse) and Hoechst. The staining results are shown in FIG. The upper part of Fig. 6 shows the control panel (the result of staining HEK cells transformed with the empty vector). The middle row shows the results of staining of HEK cells transformed with the HSPA5 expression vector, and fluorescence is observed. Similarly, the lower panel shows the result of staining of HEK cells transformed with the HSPA9B expression vector, and fluorescence is observed. As is clear from these results, HSPA5-His and HS PA9B-His were successfully expressed in mammalian cells.

[0063] 5. Binding experiment with C peptide

The C-peptide binding property of HSPA5 was verified by Western blot. Samples were prepared according to the protocol shown in Fig. 7 and subjected to Western blotting. The results of Western blot are shown in Fig. 8. Samples applied to each lane are as follows. That is, (3) is the cell force transformed with the HSPA5 expression vector, the component bound to the C peptide beads in the supernatant fraction of the prepared cell lysate, and (4) is the peptide unbound bead instead of the C peptide beads. It is a sample obtained by reacting Also, (1) is a cell lysate prepared with cell force transformed with HSPA5 expression vector, and (2) is a cell force prepared with empty vector. (5) Cell force transformed with the empty vector. (5) Components bound to the C peptide beads in the supernatant of the prepared cell lysate. (6) Cells transformed with the empty vector. The component bound to the peptide-unbound beads in the supernatant fraction of the prepared cell lysate, (7) is the cell force transformed with the HSPA5 expression vector The supernatant fraction of the prepared cell lysate is first C After reacting with the peptide, the component bound to the C peptide when reacted with the C peptide beads, (8) is the cell force transformed with the empty vector. This component is bound to C peptide when reacted with C peptide beads after reacting with peptide.

As is clear from FIG. 8, a clear band is observed in the lane (3). A faint band is also observed in the lane (4). Bands are not recognized in other lanes. From these results, it was confirmed that HS PA5 that was forcibly expressed bound to the C peptide, although non-specific binding was slightly observed.

[0064] 6. Confirmation of expression on cell membrane surface

HUVEC cells are seeded in a 1 x 1 glass chamber, stimulated with C peptide (0.3nM, lOmin) and TG (triglyceride luM, 5hr), then anti-HSPA5 antibody (using FITC anti-goat antibody as secondary antibody) Was used for immunostaining and fluorescence was observed. The results of immunostaining are shown in FIG. The right column in Fig. 9 is a stained image obtained by reacting anti-HSPA5 antibody and FITC anti-goat antibody. On the other hand, the left column shows the staining results when only the FITC anti-goat antibody is reacted (control). As is clear from FIG. 9, it was suggested that HSPA5 was present on the membrane surface.

[0065] 7. Response to C-peptide during HSP knockdown

ERK activity was analyzed as an index of cellular response to C peptide stimulation. If HSP is involved in this reaction, HSP knockdown will suppress ERK activity.

The outline of the experimental procedure is as follows (details are shown in Fig. 10). First, a sense strand and an antisense strand composed of a morpholino oligo for HSPA5 were introduced into HUVEC cells by Endo-Porter. C peptide stimulation was performed after a predetermined time, and ERK activity was evaluated by Western blotting using anti-phosphorylated ERK antibody.

The results are shown in FIG. In HUVEC cells into which sense strand has been introduced, ER is induced by C peptide stimulation. Although activation of K was observed (Νο.2 in Fig. 11), ERK activity was significantly suppressed in HUVEC cells into which the antisense strand had been introduced (Νο.4 in Fig. 11). This result suggests the existence of HSP-mediated pathway as one of the mechanisms of ERK activity induced by C peptide stimulation.

Industrial applicability

[0066] According to the present invention, a C peptide-specific binding molecule is clarified, and its application in the field of medical diagnosis is achieved. It also provides a useful tool for elucidating the mechanism of action of C-peptide.

The C peptide-specific binding molecule in the present invention is expected to be a particularly effective therapeutic target when it has not only insulin resistance but also C peptide resistance. In addition, for type 1 diabetes with which the C-peptide dies out, agents that control the activation of the molecule, such as the antigen of the molecule, exert an anti-apoptotic action or a vasodilatory action. It may be possible to suppress the development of diabetic complications (macrovascular and microangiopathy).

At present, in the category of diabetes related to peptides, the therapeutic effect of neuropathy by vasodilatory action and neuroprotective action is attracting special attention. For these, it is highly possible that the C peptide-specific binding molecule in the present invention is involved. Therefore, it is indispensable not only for elucidating the mechanism of action of C-peptide, but also for the molecule itself to become a future drug discovery target.

The present invention is not limited to the description of the embodiment and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

The contents of papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

Claims

The scope of the claims

[1] The first protein having the amino acid sequence of SEQ ID NO: 1, the second protein having the amino acid sequence of SEQ ID NO: 3, and the protein substantially homologous to the first protein or the second protein are also powerful. A C-peptide binding agent comprising one or more proteins selected from the group.

[2] A method for evaluating the action state of a C peptide in a sample, comprising the following steps: a first protein having the amino acid sequence of SEQ ID NO: 1, a second protein having the amino acid sequence of SEQ ID NO: 3, and the above Detecting in a sample one or more proteins selected from the group consisting of the first protein or a protein substantially homologous to the second protein and also having a force.

[3] A method for evaluating the action state of a C peptide in a sample, comprising the following steps: a nucleic acid encoding a first protein having the amino acid sequence of SEQ ID NO: 1 and a second protein having the amino acid sequence of SEQ ID NO: 3 Detecting in a sample one or more nucleic acids selected from the group consisting of a nucleic acid encoding a protein, a nucleic acid encoding a protein substantially homologous to the first protein or the second protein, and a force group.

[4] A method for evaluating the action state of a C peptide in a sample comprising the following steps: a polymorphism of a gene encoding the first protein having the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 A polymorphism of a gene that encodes the second protein, and a polymorphism of a gene that encodes the first protein or a protein substantially homologous to the second protein, and one or more selected from the group The step of analyzing polymorphisms in a sample.

[5] A method for screening for a compound that modulates the action state of C-peptide, comprising the following steps:

A first protein having the amino acid sequence of SEQ ID NO: 1, a second protein having the amino acid sequence of SEQ ID NO: 3, and the first protein or a protein substantially homologous to the second protein, selected from the group that has power Contacting one or more of the proteins and C peptide in the presence (test group) and absence (control group) of the test compound, respectively; c removing components that are non-specifically bound to the peptide;

Detecting a complex of a C peptide and said protein; and

Comparing the detection result of the test group with the detection result of the control group.

[6] A method for screening for a compound that modulates the action state of C-peptide, comprising the following steps:

Culturing the cells for a predetermined time in the presence (test group) and absence (control group) of the test compound;

A first protein having the amino acid sequence of SEQ ID NO: 1, a second protein having the amino acid sequence of SEQ ID NO: 3, and the first protein or a protein substantially homologous to the second protein, selected from the group that has power Measuring the expression level of the one or more proteins in the cells after culturing;

Comparing the expression level of the test group with the expression level of the control group.

[7] An antibody having a specific binding property to a C peptide or a protein having the amino acid sequence of SEQ ID NO: 1 and inhibiting binding between the C peptide and the protein.

[8] An antibody having a specific binding property to a C peptide or a protein having the amino acid sequence of SEQ ID NO: 3 and inhibiting the binding between the C peptide and the protein.

[9] A first protein having the amino acid sequence of SEQ ID NO: 1, a second protein having the amino acid sequence of SEQ ID NO: 3, and a protein substantially homologous to the first protein or the second protein. A method for modulating the action state of a C peptide in a target cell, comprising introducing one or more proteins selected from the group into the target cell.

[10] A target cell comprising the step of introducing into the target cell a C peptide and a compound that inhibits the binding of the first protein having the amino acid sequence of SEQ ID NO: 1 or the second protein having the amino acid sequence of SEQ ID NO: 3 A method for regulating the action state of C peptide in the protein.

[11] The first protein having the amino acid sequence of SEQ ID NO: 1, the second protein having the amino acid sequence of SEQ ID NO: 3, and the protein substantially homologous to the first protein or the second protein are also powerful. A method of modulating the action state of a C peptide in a target cell, comprising inhibiting the expression of one or more proteins selected from the group in the target cell.

[12] The method according to claim 11, wherein the step force is performed by using an antisense method, RNA interference, or a ribozyme.

[13] For evaluating the action state of C peptide or acting amount of C peptide, which contains a reagent having specific binding property to the first protein having the amino acid sequence of SEQ ID NO: 1 or the second protein having the amino acid sequence of SEQ ID NO: 3. For diagnosing diseases characterized by abnormalities

14. The kit according to claim 13, wherein the disease is diabetes or metabolic syndrome.

[15] The kit according to claim 13 or 14, wherein the reagent comprises an antibody having a specific binding property to the first protein or the second protein.