JP5999542B2 - Method for detecting molecules having ligand-like activity - Google Patents

Description

  The present invention relates to a novel detection technique for ligand-like activity such as AGEs or OxLDL, and a detection technique for lifestyle-related disease risk factors.

  Lectin-like oxidized low density lipoprotein (LDL) receptor 1 (Lectin-like Oxidized LDL receptor: hereinafter referred to as LOX-1) is an oxidized LDL (hereinafter referred to as LOX-1) that causes atherogenesis. A unique scavenger receptor for denatured LDL (also referred to as OxLDL) and was first identified in 1997 in cultured bovine aortic endothelial cells. Although LOX-1 is functionally similar to other scavenger receptors, it has a unique structure that is structurally different.

  Bovine LOX-1 (bLOX-1) is a glycoprotein consisting of 273 amino acid residues belonging to the C-type lectin family and having a molecular weight of about 50 kDa, the N-terminus is in the cytoplasm, and the C-terminus is outside the cell membrane. A single-penetration type II membrane protein. Human LOX-1 (hLOX-1) is also a type II membrane protein of about 30 kDa consisting of 273 amino acid residues belonging to the C-type lectin family (molecular weight of about 40 kDa due to addition of sugar chains at positions 139 and 183). This receptor is structurally composed of the following four domains: an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, a neck domain, and a C-type lectin-like domain (hereinafter referred to as CTLD). . This CTLD is highly conserved among species, particularly at the position of 6 cysteine residues, and is a functional domain for recognizing LOX-1 ligands. Six cysteine residues in this CTLD are involved in the intramolecular disulfide bond of hLOX-1. In addition to this conserved CTLD, neck domains in hLOX-1 and other known species have high sequence identity. According to Xie et al. (Non-patent Document 1: Protein Expression and Purification 32: 68-74 (2003)), CTLD is a minimal domain sufficient to bind denatured LDL, even if CTLD is glycosylated. It has been shown that denatured LDL can be recognized and bound without it.

According to previous studies, LOX-1 is expressed not only in vascular endothelial cells but also in macrophages and activated vascular smooth muscle cells, and various structurally unrelated macromolecules (modified LDL, bacteria, aging) Erythrocytes, cells undergoing apoptosis, and platelets), and plays an important role in various biological phenomena such as biological defense mechanisms and inflammatory mechanisms, and its expression is under various conditions, Conditions such as hyperlipidemia, diabetes, hyperglycemia, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia-reperfusion injury, vascular balloon injury; and oxidized LDL, angiotensin II, endothelin, TNF-α, advanced glycation products (AGE), TGF-β, 8- iso - prostaglandin _{F 2.alpha,} by stimuli such as shear stress, are adjusted Bets are known (Non-Patent Document 2:. Folia Pharmacol.Jpn 127,103-107 (2006)).

  Regarding the measurement of denatured LDL, it is expected to be used in various fields such as early diagnosis of diseases, evaluation of preventive effects of functional foods, evaluation of treatment effects by improving lifestyle and medication. So far, monoclonal antibodies have been used to measure denatured LDL, which is a risk factor for arteriosclerosis in human plasma. In particular, denatured LDL has a definite molecular modification structure, and the meaningful molecular species is not clear. For this reason, there are many problems in detection using monoclonal antibodies.

  Late Glycation End Products (hereinafter referred to as AGE in the present specification) are involved in the development and development of diabetic vascular disorders known as vascular complications that are the primary cause of impaired quality of life for diabetic patients. ing. Eye, nerve, and kidney disorders due to vascular complications are called diabetic retinopathy, neuropathy, and nephropathy (a total of three major complications), which are characteristic pathologies for diabetic patients.

  A receptor for recognizing AGE (Receptor for AGE: hereinafter referred to as RAGE) was identified from bovine lung in 1992 and belongs to the immunoglobulin superfamily that binds to AGE, and is a type I membrane with a molecular weight of about 35 kDa. It is a protein (complete RAGE subjected to sugar chain modification has a molecular weight of 55 kDa). The extracellular domain of RAGE has a structure in which a domain having three immunoglobulin fold structures of one V-type immunoglobulin domain and then two C-type immunoglobulin domains (C1 region and C2 region) are combined. . RAGE also contains a single-pass membrane domain and a 43 amino acid intracellular domain. RAGE interacts with various classes of ligands (AGE, S100 / calgranulin, amphoterin and amyloid-β peptides (and β-sheet fibril class)). The V domain is an essential site for ligand binding, and the intracellular domain has been shown to be essential for RAGE-mediated intracellular signal transduction (Non-patent Document 3: Circ Res. 2003; 93: 1159-1169). ).

  RAGE is expressed only at low levels in normal tissues and the vascular system. However, this receptor is upregulated where the ligand accumulates. For example, in diabetic blood vessels, typical ligands for RAGE include the following AGE structures: (carboxymethyl) lysine-protein adduct (major AGE present in vivo), carboxyethyl-lysine (CEL) ) Protein adducts, pentosidine-adducts (major AGE crosslinkers found in diabetic tissues associated with collagen and basement membrane destabilization), pyralin, imidazolone, methylglyoxal (precursors for the formation of other ranges of AGEs) ), Croslin, fluorolink, propyridine, argpyrimidine, vesperlysine, glyoxal-derived lysine dimer, deoxyglucosamine-derived lysine dimer, and the like. RAGE expression is increased in endothelial cells, smooth muscle cells, and infiltrating mononuclear phagocytes in the diabetic vasculature. AGE-RAGE interactions change cellular properties important in vascular homeostasis. For example, after RAGE binds to AGE, endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, and their permeability to macromolecules. In mononuclear phagocytes, RAGE activates the expression of cytokines and growth factors and induces cell migration in response to soluble AGE, whereas chemotaxis occurs with a fixed ligand (Non-Patent Document 4: J Clin. Invest. 108: 949-955 (2001)).

  The present inventors have applied for a method for detecting LOX-1 mutant-surfactant complex and RAGE mutant-surfactant complex, and lifestyle-related disease risk factors using these. (Patent Document 7).

JP 2002-320489 A JP 2002-181820 A JP 2003-36499 A JP 2003-238404 A JP 2003-125786 A JP 2002-17353 A JP 2009-108037 A

Protein Expression and Privacy 32: 68-74 (2003) Folia Pharmacol. Jpn. 127, 103-107 (2006) Circ Res. 2003; 93: 1159-1169. J. et al. Clin. Invest. 108: 949-955 (2001)

  In Patent Document 7 submitted by the present inventors, since a specific molecule is used, it is necessary to rely on the reaction specificity, and it is impossible to comprehensively detect OxLDL-like molecules and RAGE molecules. It was difficult to overcome this. Therefore, it is considered that the possibility of false negatives cannot be minimized even in the detection of lesions. Therefore, it is an object to overcome this.

  Therefore, in the present invention, the present inventors, as a method for detecting or quantifying a molecule exhibiting OxLDL-like activity or a method for detecting or quantifying a molecule exhibiting AGEs-like activity, RAGE143 immobilized on a solid phase or a variant thereof or a solid phase. Exhaustive detection or quantification was made possible by utilizing the step of contacting the sample with labeled AGE or labeled OxLDL in the presence of CTLD14 or a variant thereof immobilized on the phase.

Accordingly, the present invention provides the following.
(1) A method for detecting or quantifying a molecule exhibiting AGE-like activity,
(A) A sample to be detected or quantified is contacted with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, or labeled in the presence of an AGE molecule immobilized on a solid phase. Contacting the RAGE molecule;
And (B) detecting the binding of the RAGE molecule to the AGE molecule, wherein the presence or level of inhibition of the binding indicates the presence or level of a molecule exhibiting AGEs-like activity in the sample. The method of inclusion.
(2) A preliminary detection method for diabetic nephropathy,
(A) A sample from a subject for detection of diabetic nephropathy is contacted with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, or an AGE molecule immobilized on a solid phase Contacting with a labeled RAGE molecule in the presence of
And (B) detecting the binding of the RAGE molecule to the AGE molecule, wherein the presence or absence of inhibition of the binding indicates that the subject will or will have future diabetic nephropathy A method comprising the steps.
(3) A preliminary detection method for diabetic nephropathy in diabetes,
(A) A sample from a subject suffering from diabetes that is subject to detection of diabetic nephropathy is contacted with an immobilized AGE molecule in the presence of a RAGE molecule immobilized on a solid phase or immobilized on a solid phase Contacting the labeled RAGE molecule in the presence of the labeled AGE molecule;
And (B) detecting the binding of the RAGE molecule to the AGE molecule, wherein the presence or absence of inhibition of the binding indicates that the subject will or will have future diabetic nephropathy A method comprising the steps.
(4) A method for detecting diabetes or diabetic nephropathy,
(A) A sample from a subject for detection of diabetic nephropathy is contacted with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, or an AGE molecule immobilized on a solid phase Contacting with a labeled RAGE molecule in the presence of
And (B) detecting binding of the RAGE molecule or ligand of the AGE or variant thereof to the immobilized AGE or variant thereof, wherein the presence or level of inhibition of the binding is determined by the subject A method comprising the step of indicating that the patient is suffering from diabetes or diabetic nephropathy.
(5) The method according to any one of Items 1 to 4, wherein the RAGE molecule is RAGE143, RAGE1, RAGE223, RAGE226, or a variant thereof.
(6) The AGE molecule may be Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE), or modifications thereof 6. The method according to any one of items 1 to 5, wherein the method is a body.
(7) A method for detecting or quantifying a molecule exhibiting OxLDL-like activity,
(A) The sample to be detected or quantified is brought into contact with the labeled denatured LDL in the presence of CTLD molecules immobilized on the solid phase, or labeled in the presence of denatured LDL immobilized on the solid phase. Contacting the CTLD molecule;
And (B) detecting the binding of the CTLD molecule to the modified LDL, wherein the presence or level of inhibition of the binding comprises the presence or level of a molecule exhibiting OxLDL-like activity, Method.
(8) The method according to item 7, wherein the CTLD molecule is CTLD14, LOX-1 full length, LOX-1 extracellular region full length, CTLD, or a variant thereof.
(9) The modified LDL is oxidized LDL (OxLDL), malondialdehyde-modified LDL (MDA-LDL), acrolein modified LDL, nonenal modified LDL, crotonaldehyde (CRA) modified LDL, 4-hydroxynonenal (HNE) modified LDL Item 9. The method according to Item 7 or 8, which is a hexanoyl (HEL) -modified LDL, a small particle LDL, a saccharified LDL, an acetylated LDL, or a variant thereof.
(10) A kit for detecting or quantifying a molecule exhibiting AGE-like activity,
(A) a solid phase having a fixed RAGE molecule and a labeled AGE molecule, or a solid phase having a fixed AGE molecule and a labeled RAGE molecule; and (B) detecting binding of the RAGE molecule to the AGE molecule. A kit comprising means, wherein the presence or level of inhibition of the binding indicates the presence or level of molecules exhibiting AGEs-like activity.
(11) A kit for preliminary diagnosis of diabetic nephropathy,
(A) a solid phase having a immobilized RAGE molecule and a labeled AGE molecule, or a solid phase having a fixed AGE molecule and a labeled RAGE molecule; and (B) means for detecting binding of the RAGE molecule to the AGE Wherein the presence or level of inhibition of the binding indicates that the subject is or will likely suffer from diabetic nephropathy in the future.
(12) A kit for preliminary diagnosis of diabetic nephropathy in diabetes,
(A) a solid phase having an immobilized RAGE molecule and a labeled AGE molecule, or a solid phase having an immobilized AGE molecule and a labeled RAGE molecule; and (B) means for detecting binding of the molecule to the AGE. Prepared,
The presence or level of inhibition of the binding indicates that the subject is or will likely suffer from diabetic nephropathy in the future.
(13) A kit for diagnosis of diabetes or diabetic nephropathy,
(A) a solid phase having an immobilized RAGE molecule and a labeled AGE molecule, or a solid phase having an immobilized AGE molecule and a labeled RAGE molecule; and (B) means for detecting binding of the molecule to the AGE. Prepared,
The presence or level of inhibition of the binding indicates that the subject is suffering from diabetes or diabetic nephropathy.
(14) The kit according to any one of Items 10 to 13, wherein the RAGE molecule is RAGE143, RAGE1, RAGE223, RAGE226, or a modified form thereof.
(15) The AGE molecule may be Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE) or a modification thereof The kit according to any one of Items 10 to 14, which is a body.
(16) A kit for detecting or quantifying a molecule exhibiting OxLDL-like activity,
(A) a solid phase having immobilized CTLD molecules and labeled denatured LDL, or a solid phase having immobilized denatured LDL and labeled CTLD molecules; and (B) detecting binding of the CTLD molecules to the denatured LDL. A kit comprising means, wherein the presence or level of inhibition of binding indicates the presence or level of a molecule exhibiting OxLDL-like activity.
(17) The kit according to item 16, wherein the CTLD molecule is CTLD14, LOX-1 full length, LOX-1 extracellular region full length, CTLD or a variant thereof.
(18) The modified LDL is oxidized LDL (OxLDL), malondialdehyde-modified LDL (MDA-LDL), acrolein-modified LDL, nonenal-modified LDL, crotonaldehyde (CRA) -modified LDL, 4-hydroxynonenal (HNE) -modified LDL Item 18. The kit according to item 16 or 17, which is a hexanoyl (HEL) -modified LDL, a small particle LDL, a saccharified LDL, an acetylated LDL, or a variant thereof.
(19) A preliminary diagnostic marker for diabetic nephropathy comprising a molecule exhibiting AGE-like activity.
(20) A preliminary diagnostic marker for diabetic nephropathy in diabetes, comprising a molecule exhibiting AGE-like activity.
(21) A diagnostic marker for diabetes or diabetic nephropathy, comprising a molecule exhibiting AGE-like activity.
(22) Use of AGEs-like activity in a subject to serve as a preliminary diagnosis index of diabetic nephropathy, wherein the AGEs-like activity is higher than a standard value. Use to indicate that it is likely or likely to have diabetic nephropathy in the future.
(23) Use of AGEs-like activity in a subject to provide an index for preliminary diagnosis of diabetic nephropathy in diabetes, wherein the AGEs-like activity is higher than a standard value. Uses to indicate that the body will or will have future diabetic nephropathy.
(24) Use of AGEs-like activity in a subject for use as an index for diagnosis of diabetes or diabetic nephropathy, wherein the AGEs-like activity is higher than a standard value when the subject is diabetic Or use to indicate that you are suffering from diabetic nephropathy.

  Still further embodiments and advantages of the invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as needed.

  It can be said that the convenience is remarkably improved by the assay using the CTLD molecule of the present invention. The conventional method required three separate reactions after sample addition, but the reaction is only one step of sample addition, and no powerful drugs such as strong acids are used as in the conventional method. It can be said that a safe assay was provided.

  In addition, the assay using CTLD molecules exhibited a significant effect on the comprehensiveness. That is, the molecule has been extended to a molecule that has a structure different from that of denatured LDL and has been shown to be recognized by CTLD14 (Hsp: heat shock protein that is a molecular chaperone, although the function is not clear). In addition, it became possible to detect molecules that could not be captured by the conventional method, and the coverage was improved. In addition, although the binding is weak, AGEs can also be recognized as a ligand for CTLD14. In the conventional technique, it was necessary to prepare a completely different antibody (and low specificity) and perform the reaction separately. In the technique of the present invention, it can be detected as a molecule exhibiting a ligand-like activity simultaneously with denatured LDL. became.

  It has been found that the assay using the RAGE molecule of the present invention also has a remarkable effect. Although RAGE also recognizes denatured LDL and hemoglobin A1C (HbA1c) used for blood glucose level management, the structure differs from AGEs (late glycation reaction products) such as R-AGE. However, simultaneous detection is difficult with the conventional method. According to the method of the present invention, it has become possible to simultaneously detect as molecules showing ligand-like activity. In addition, since low molecular ligands such as Lys-AGE can be detected at the same time, it can be said that the comprehensiveness of the assay using the RAGE molecule of the present invention is improved.

  Since the AGE-like molecule rises after the blood glucose level rises, and then the amount of drinking water increases, an increase in the amount of drinking water is one index of diabetic nephropathy, so it can be said that “predictive diagnosis” is possible.

FIG. 1 shows that quercetin was added at a dose of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay using a 96-well plate with immobilized RAGE. , Relative binding activity (%, quercetin-free addition is taken as 100%). FIG. 2 shows that using 96-well plates with immobilized RAGE, OxLDL was modeled as a LOX-1 inhibitor at 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / Relative binding activity (%, with no quercetin added as 100%) when added at assay dose. FIG. 3 shows that using 96-well plates with immobilized RAGE, G-AGE as a model for LOX-1 inhibitor 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and Relative binding activity (%, with no quercetin added as 100%) when added at a dose of 5 μg / assay. FIG. 4 shows that 96-well plates with immobilized RAGE were used to model R-AGE as a model for LOX-1 inhibitors at 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and Relative binding activity (%, with no quercetin added as 100%) when added at a dose of 5 μg / assay. FIG. 5 shows a control experiment conducted using R-AGE labeled with the water-soluble dye Alexa546 as a fluorescently labeled model ligand. The left phase of FIG. 5 shows the phase difference image of the cells used, the center of FIG. 5 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 5 shows the cell image with the ligand bound. The white dots on the right side of FIG. FIG. 6 shows a photograph in the case of adding quercetin in an experiment conducted in the same order as in FIG. 6 shows the phase difference image of the used cell, the center of FIG. 6 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 6 shows a cell image in which quercetin is added in addition to the ligand. Indicates. As shown in the right of FIG. 6, it was confirmed by high-sensitivity observation in the cells that the binding of the ligand was inhibited by the addition of quercetin. FIG. 7 shows a photograph in the case of adding R-AGE in the same order as FIG. The left phase of FIG. 7 shows the phase difference image of the cells used, the center of FIG. 7 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 7 added R-AGE in addition to the ligand. A cell image is shown. As shown in FIG. 7 right, a fluorescence image showing inhibition of ligand binding by addition of R-AGE is shown. FIG. 8 shows that quercetin was added at a dose of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay using a 96-well plate immobilized with LOX-1. The relative binding activity (%, quercetin-free addition is defined as 100%). FIG. 9 shows that using LOX-1 immobilized 96-well plates, OxLDL was added at doses of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay. The relative binding activity (%, quercetin-free addition is defined as 100%). FIG. 10 shows that using LOD-1 immobilized 96-well plates, AcLDL was added at doses of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay. The relative binding activity (%, quercetin-free addition is defined as 100%). FIG. 11 shows a 96-well plate with immobilized OxLDL when OxLDL was added at doses of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay. , Relative binding activity (%, quercetin-free addition is taken as 100%). FIG. 12 shows that quercetin was added at a dose of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay using a 96-well plate immobilized with OxLDL. , Relative binding activity (%, quercetin-free addition is taken as 100%). FIG. 13 shows quercetin at 0 μg / assay (100% control), 0.5 μg / assay, using a 96-well plate immobilized with CTLD14 as a CTLD molecule, and an example of labeling OxLDL as an example of denatured LDL. Relative binding activity (%, with no quercetin added as 100%) when added at doses of 5 μg / assay and 5 μg / assay. FIG. 14 shows a control experiment using cells in which LOX-1 was forcibly expressed as a fusion protein with a fluorescent protein and OxLDL as a control ligand. FIG. 14 shows the phase contrast image of the cell used on the left, FIG. 14 shows the fluorescence of LOX-1 (forced expression as a fusion protein with a fluorescent protein) expressing cell in the center of FIG. Show. A white dot on the right side of FIG. 14 is a bound ligand. FIG. 15 shows a photograph when quercetin is added in the same order as in FIG. FIG. 15 shows the phase contrast image of the cells used on the left, FIG. 15 shows the fluorescence of the cells expressing LOX-1 (forced expression as a fusion protein with a fluorescent protein) in the center, and FIG. 15 shows the addition of quercetin in addition to the ligand. A cell image is shown. As shown in the right of FIG. 15, it was confirmed by high-sensitivity observation in cells that the binding of ligand was inhibited by the addition of quercetin. FIG. 16 shows the measurement results of serum lipid concentrations in mice fed with a high cholesterol / Clinton-Sibrusky rodent diet or a normal diet in Example 7. The result of having measured the total cholesterol amount, the HDL cholesterol amount, and the LDL cholesterol amount by a colorimetric method (Wako Pure Chemicals, Osaka, Japan) is shown. In the high-cholesterol diet group, the total cholesterol level was significantly higher than that in the control group (FIG. 16-A), and among the cholesterol, there was no significant difference in the HDL cholesterol level (FIG. 16-B, control group (69. 7 ± 3.4 mg / dl) vs. high cholesterol diet group (65.4 ± 2.4 mg / dl)), LDL cholesterol level was control group (47.5 ± 4.0 mg / dl) vs . It was confirmed that it was significantly higher than the high cholesterol diet group (112.9 ± 4.9 mg / dl) (FIG. 16-C), and it was confirmed that hyperlipidemia was developed in the high cholesterol diet group. It was. From this, it was shown that the anti-cholesterol diet mouse used in this example can be used as a hyperlipidemia model mouse. * Indicates statistical significance relative to the normal diet (control diet) (P <0.05). FIG. 17 shows the results of measuring the binding inhibitory activity of serum derived from a hyperlipidemia model mouse (high cholesterol diet group) and a healthy group mouse (control) to DiI-labeled AcLDL on an LOX-1-immobilized plate. Sera from hyperlipidemic model mice showed 30% inhibitory activity, but sera from healthy groups had only 12% inhibition and were statistically significant at a significant level (P <0.05). Inhibitory activity was observed (it was shown that there were molecules showing ligand-like activity). Regarding LDL, LDL derived from a hyperlipidemic model mouse showed 13% inhibitory activity, but LDL derived from a healthy group was only 3% inhibited, and was statistically significant (P <0.05). Scientifically significant inhibitory activity was observed. * Indicates statistical significance relative to the normal diet (control diet) (P <0.05). FIG. 18 shows 4-hydroxy-2-nonenal, which is a structure characteristic of oxidized LDL (a molecule showing ligand activity) generated by oxidation of LDL in hyperlipidemia model mice and LDL derived from healthy mice. This is a result of colorimetric determination of the formation of (HNE) by the reactivity with the anti-HNE monoclonal antibody. Although LDL derived from healthy mice hardly responds (OD≈0), LDL derived from hyperlipidemic model mice shows significant reactivity (OD = 0.3), and a significant level (P <0.05) A statistically significant difference was observed. This indicates that LDL derived from hyperlipidemia model mice has a statistically significant molecule showing LOX-1 ligand-like activity, and the inhibition shown in FIG. Together with the activity results, it has been shown that the techniques of this application can efficiently detect molecules that exhibit ligand-like activity. * Indicates statistical significance relative to the normal diet (control diet) (P <0.05). FIG. 19 is a graph showing a correlation between a blood glucose level measured by a blood glucose meter and a glucose concentration in serum measured by a glucose measurement kit. FIG. 20 shows the results of measuring the glucose concentration in the serum of healthy mice and diabetes model mice in which diabetes was induced by administration of streptozotocin. FIG. 21 is a diagram showing a correlation between serum glucose concentration and creatine concentration for each individual. FIG. 22 is a diagram showing the blood creatine concentration of a healthy mouse and a diabetes model mouse in which diabetes was induced by administration of streptozotocin. In the group in which the molecule showing the ligand-like activity of RAGE of the present invention was detected, it was confirmed at a later date that the blood creatinine concentration as an index of the onset of nephropathy increased. FIG. 23 shows the “Alexa546-labeled R-AGE binding inhibitory activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “blood glucose level or blood glucose concentration” Is a graph on the 0th day among the graphs showing the correlation for each individual measured over time. FIG. 24 shows the “Alexa546-labeled R-AGE binding inhibitory activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “blood glucose level or blood glucose concentration” Is a graph on the 16th day among the graphs showing the correlation for each individual measured over time. FIG. 25 shows the “Alexa546-labeled R-AGE binding inhibition activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “blood glucose level or blood glucose concentration” Is a graph on the 22nd day among the graphs showing the correlation of each individual measured over time. FIG. 26 shows the “Alexa546-labeled R-AGE binding inhibitory activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “blood glucose level or blood glucose concentration” Is a graph on the 57th day among the graphs showing the correlation of each individual measured over time. FIG. 27 shows the “Alexa546-labeled R-AGE binding inhibition activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “blood glucose level or blood glucose concentration” Is a graph on the 71st day among the graphs showing the correlation for each individual measured over time. FIG. 28 shows the “Alexa546-labeled R-AGE binding inhibitory activity to RAGE-immobilized plate” and “the amount of creatinine in serum” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz). It is the graph of the 71st day which measured with time and showed the correlation for every individual.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, the term “receptor” is a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands. Here, this complexation has a biological structure. Receptors can be completely outside the cell (extracellular receptors), inside the cell membrane (but directing the receptor portion to the extracellular environment and cytosol), or completely inside the cell (intracellular Of the receptor). They can also function independently of cells. Receptors in the cell membrane allow cells to communicate (eg, signal transduction) with space outside their boundaries and to function in the transport of molecules and ions to the inside and outside of the cell. As used herein, a receptor may be a full length receptor or a fragment of a receptor.

  As used herein, the term “lectin-like oxidized low density lipoprotein (LDL) receptor 1 (Lectin-like Oxidized LDL receptor)” is also referred to as LOX-1, and is shown in (1) SEQ ID NO: 4. (2) an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions in the amino acid sequence shown in SEQ ID NO: 4, and a natural LOX-1 (3) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and exhibiting the activity of natural LOX-1; (4) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 4 And a polypeptide exhibiting the activity of natural LOX-1; (5) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 3; (6) against the nucleic acid sequence represented by SEQ ID NO: 3 A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes with a complementary nucleic acid sequence under stringent conditions and exhibiting the activity of natural LOX-1; (7) the nucleic acid represented by SEQ ID NO: 3 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having one or several nucleotide substitutions, additions and / or deletions in the sequence and exhibiting the activity of native LOX-1; (8) SEQ ID NO: 3 above Comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity to the nucleic acid sequence shown in A polypeptide exhibiting the activity of native LOX-1; and (9) an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 3 above, and native LOX It is one of the polypeptides that show the activity of -1. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art. Further, as LOX-1, feeding such as human LOX-1 (also referred to as hLOX-1), bovine LOX-1 (also referred to as b-LOX1), porcine LOX-1, mouse LOX-1, and rabbit LOX-1 Examples include, but are not limited to, animal LOX-1. bLOX-1 is a glycoprotein having a molecular weight of about 50 kDa consisting of 273 amino acid residues belonging to the C-type lectin family, the N-terminal is in the cytoplasm, and the C-terminal is a cell membrane once-penetrating type II. It is a type membrane protein. hLOX-1 is also an approximately 30 kDa type II membrane protein consisting of 273 amino acid residues belonging to the C-type lectin family. LOX-1 is structurally composed of the following four domains: an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, a neck domain, and a C-type lectin-like domain.

  As used herein, the term “C-type lectin-like domain” is also referred to as “CTLD” and has homology with the sugar chain recognition sites of members belonging to the C-type lectin family. CTLD is very well conserved among these members, among species, and the position of the 6 cysteine residues is completely conserved. CTLD is a functional domain for recognizing the ligand of LOX-1, and six cysteine residues among them are involved in three intramolecular disulfide bonds of hLOX-1. Therefore, in the mutant LOX-1-surfactant complex of the present invention, cysteines at positions 144, 155, 172, 243, 256, 264 are retained in the amino acid sequence of SEQ ID NO: 4. Is preferred. Similarly, in the CTLD-surfactant complex of the present invention, amino acids corresponding to cysteines at positions 144, 155, 172, 243, 256, and 264 in the amino acid sequence of SEQ ID NO: 4 are retained. Preferably it is. In addition to this conserved CTLD, neck domains in hLOX-1 and other known species have high sequence identity. In addition, cysteine at position 140 in hLOX-1 is involved in an intermolecular disulfide bond and forms a dimer of hLOX-1. However, since the cysteine at position 140 is not essential for recognition of denatured LDL, it is not always necessary to retain the cysteine at position 140 when expressed, and it may be mutated. Furthermore, in hLOX-1, sugar chains are added at N at position 183 and N at position 139. The molecular weight of hLOX-1 to which a sugar chain is added is 50 kDa. Although hLOX-1 is usually glycosylated, it can recognize and bind to denatured LDL, even though it is not glycosylated, in the same way as normal glycosylated hLOX-1. The CTLD is a minimal domain necessary and sufficient to bind denatured LDL. The 4 C-terminal residues (LRaq) of LOX-1 are essential for ligand recognition and uptake, and the 7 C-terminal residues (KANLRAQ) are essential for hLOX-1 folding and transport. hLOX-1 W150, R208, R229, R231, R248 and the like are essential amino acids for ligand recognition and uptake. LOX-1 has also been reported to be cleaved from the cell membrane, released as a soluble form, and also present in the blood of healthy individuals.

As used herein, the term "CTLD-like polypeptide", "CTLD", "PR (P rotease- R esistant (protease resistance)) - CTLD ', C-terminal, the" CTLD14 "(CTLD + neck domain Polypeptide having 14 amino acids on the side), “PR-CTLD14”, “CTLD + neck” (polypeptide having CTLD + neck domain), or “PR-CTLD + neck” all polypeptides or variants thereof Include.

  As used herein, the term “CTLD molecule” is understood to include CTLD-like polypeptides as well as any complexes thereof. Therefore, it is understood that CTLD molecules include LOX-1 full length, LOX-1 extracellular region full length (S61 to Q273), CTLD14 (129-143), CTLD (143-273), and the like.

  As used herein, “complex” means any construct comprising two or more parts. For example, when one part is a polypeptide, the other part may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.). . In the present specification, two or more parts constituting the complex may be bonded by a covalent bond, or bonded by other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). May be. When two or more parts are polypeptides, they can also be referred to as chimeric polypeptides. Therefore, in this specification, the “complex” includes a molecule formed by linking a plurality of molecules such as a polypeptide, a polynucleotide, a lipid, a sugar, and a small molecule. Examples of such a complex include, but are not limited to, glycolipids and glycopeptides. As used herein, a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a variant or fragment thereof, and a nucleic acid molecule that encodes each variant or fragment as long as it has biological activity with respect to LOX-1. Can be used. Complexes containing such nucleic acid molecules can also be used.

  As used herein, the terms “CTLD” and “PR-CTLD” typically include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (2) SEQ ID NO: 2 above A polypeptide comprising an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions in the amino acid sequence represented by (3); (3) other than positions 90 and 107 in the amino acid sequence represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence comprising one or several substitutions, additions and / or deletions at the amino acid position and exhibiting the activity of native LOX-1; (4) at least an amino acid sequence represented by SEQ ID NO: 2 above A polypeptide comprising an amino acid sequence having 90% sequence identity; (5) at least 80% with the amino acid sequence shown in SEQ ID NO: 2 above A polypeptide comprising an amino acid sequence having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 1; and (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 1. A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) a nucleic acid sequence complementary to the nucleic acid sequence set forth in SEQ ID NO: 1 and under stringent conditions An amino acid sequence encoded by a nucleic acid molecule that hybridizes with the amino acid at positions 90 and 107 in the encoded amino acid sequence, retains the corresponding amino acid in SEQ ID NO: 2 and (9) a nucleic acid sequence represented by SEQ ID NO: 1 A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or several substitutions, additions and / or deletions and having the activity of native LOX-1; (10) shown in SEQ ID NO: 1 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence; (11) by a nucleic acid sequence having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 1 above Indicated by one of the polypeptides comprising the encoded amino acid sequence. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “CTLD14” and “PR-CTLD14” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6; (2) the amino acid sequence shown in SEQ ID NO: 6 above. A polypeptide comprising an amino acid sequence comprising a substitution, addition and / or deletion of one or several amino acids in (3); or 1 or 2 at amino acid positions other than positions 104 and 121 in the amino acid sequence shown in SEQ ID NO: 6 above A polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions and exhibiting the activity of native LOX-1; (4) at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 above (5) at least 80% of the amino acid sequence shown in SEQ ID NO: 6 A polypeptide comprising an amino acid sequence having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 5; (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 5 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 5 and under stringent conditions Wherein the amino acids at positions 104 and 121 in the encoded amino acid sequence retain the corresponding amino acids in SEQ ID NO: 6 and A polypeptide comprising an amino acid sequence exhibiting activity; (9) the nucleic acid sequence represented by SEQ ID NO: 5 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or several substitutions, additions and / or deletions in and exhibiting the activity of native LOX-1; (10) shown in SEQ ID NO: 5 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence; (11) by a nucleic acid sequence having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 5 above Indicated by one of the polypeptides comprising the encoded amino acid sequence. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “CTLD + Neck” and “PR-CTLD + Neck” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8; (2) shown in SEQ ID NO: 8 above. A polypeptide comprising an amino acid sequence comprising a substitution, addition and / or deletion of one or several amino acids in the amino acid sequence; (3) in amino acid positions other than positions 172 and 189 in the amino acid sequence shown in SEQ ID NO: 8 above; A polypeptide comprising an amino acid sequence comprising one or several substitutions, additions and / or deletions and exhibiting the activity of native LOX-1; (4) at least 90% of the amino acid sequence shown in SEQ ID NO: 8 above A polypeptide comprising an amino acid sequence having sequence identity; (5) at least the amino acid sequence represented by SEQ ID NO: 8 A polypeptide comprising an amino acid sequence having 0% sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule represented by SEQ ID NO: 7; (7) a nucleic acid sequence represented by SEQ ID NO: 7 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule which hybridizes under stringent conditions with a complementary nucleic acid sequence; (8) a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 7 and stringent An amino acid sequence encoded by a nucleic acid molecule that hybridizes under various conditions, wherein the amino acids at positions 172 and 189 in the encoded amino acid sequence retain the corresponding amino acids in SEQ ID NO: 6 and are naturally occurring A polypeptide comprising an amino acid sequence exhibiting LOX-1 activity; (9) shown in SEQ ID NO: 7 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or several substitutions, additions and / or deletions in said nucleic acid sequence and exhibiting the activity of native LOX-1; (10) SEQ ID NO: 7 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in (11); (11) having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 7 above Indicated by one of the polypeptides comprising the amino acid sequence encoded by the nucleic acid sequence. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  The CTLD-like polypeptide may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of natural LOX-1 is retained.

  Also in the CTLD-like polypeptide-surfactant complex shown above, cysteine residues are involved in intramolecular disulfide bonds. Therefore, in the CTLD-like polypeptide of the present invention, 144 of the amino acid sequence of SEQ ID NO: 4 is used. It is preferred that cysteines corresponding to positions 155, 172, 243, 256, 264 are retained.

  As used herein, the term “ligand” is a binding partner for a specific receptor or family of receptors. The ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological means.

As used herein, “denatured LDL” refers to contact of LDL with active oxygen, oxidative enzymes, Fe ^3+, etc. in the body, or cell-dependent chemical changes caused by vascular endothelial cells, macrophages, etc. Any LDL variant with various molecular modifications that occur. As modified LDL present in a living body, typically, oxidized LDL (referred to as OxLDL in the present specification, for example, fully oxidized LDL (also referred to as F-OxLDL in the present specification) and partially oxidized LDL (present In the specification, it is also referred to as M-OxLDL), malondialdehyde-modified LDL (MDA-LDL), acrolein-modified LDL, nonenal-modified LDL, crotonaldehyde (CRA) -modified LDL, 4-hydroxynonenal (HNE) -modified LDL , Hexanoyl (HEL) -modified LDL, small particle LDL (LDL having a diameter of 255 nm or less), saccharified LDL, acetylated LDL, and the like. When oxidized LDL shows abnormal values, arteriosclerosis, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular disorder (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), Diseases such as aortic aneurysm, renal infarction, and hyperlipidemia are expected, but are not limited to these (see “Today's clinical test 2007-2008” publisher, Nankodo, Inc.). In a generally used test method, MDA-LDL (normal range: 10 to 80 L) and phosphatidylcholine oxide (normal range: 8.4 U / mL to 17.6 U / mL) are used as reference substances. As used herein, “a molecule exhibiting OxLDL-like activity” refers to a molecule having at least one of the above-mentioned OxLDL activities (referred to herein as “OxLDL-like activity”). Examples of such OxLDL-like activity include, but are not limited to, LOX-1 binding activity (ligand activity).

  As used herein, the term “advanced glycation end products” is also referred to as AGE and is a diabetic blood vessel known as a vascular complication that impairs the quality of life of diabetic patients. Involved in the onset and progression of disabilities. Reducing sugars represented by glucose react non-enzymatically with amino groups of proteins and amino acids to form glycation products such as Schiff bases or Amadori rearrangement compounds. The reaction so far is reversible and is called the first reaction. Thereafter, a late saccharification reaction product is formed through complicated and irreversible reactions such as condensation, cleavage, and cross-linking. Such a series of reactions is called glycation. AGE is also a general term for structures generated through such a process. Examples of the AGE structure present in the living body include, but are not limited to, carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, pyralin, imidazoline, methylglyoxal, and croslin. A product obtained by glycation of albumin, immunoglobulin, ovalbumin or the like present in plasma is also AGE, and is widely used in experimental systems as AGE. Furthermore, in the in vitro experimental system, BSA (bovine serum albumin) subjected to saccharification treatment, for example, R-AGE (BSA saccharified with ribose), F-AGE (BSA treated with fructose); G- AGE (BSA saccharified with glucose) and the like are also widely used. Hemoglobin A1c used as an index for blood glucose control is an Amadori transfer compound, but is included in AGE. Arbitrary proteins can also be converted to AGE. For example, CML albumin and CEL albumin included in AGE are both AGEs in which albumin is glycated. Such an AGE production reaction can occur in the living body either in the circulating blood, in the extracellular matrix, or in the cell. For example, AGEs present in the blood vessels of diabetic patients include: those that are fluorescent and have a cross-linked structure (such as pentosidine and croslin) and those that do not fluoresce or cross-link (such as carboxymethyllysine, pyralin, methylglyoxal (MG) -imidazolone) ). When AGE shows an abnormal value, diseases such as microangiopathy (nephropathy, retinopathy, neurosis, etc.) and macrovascular disorder (ischemic heart disease, cerebrovascular disease, obstructive arteriosclerosis) are expected. In the test method generally used, as reference substances, pyralin (normal range: less than 23 pmol / mL in plasma), pentosidine (normal range: 0.00915 to 0.0431 μg / mL in plasma (when measured by ELISA)) (See “Today's clinical test 2007-2008” publisher, Nanedo Co., Ltd.).

  As used herein, “AGE molecule” refers to any molecule contained in the AGE. For example, as AGE, Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE) or a modified form thereof These complexes can be mentioned, but are not limited thereto.

  In the present specification, the “molecule exhibiting AGE-like activity” refers to a molecule having at least one of the above-mentioned AGE activities (referred to herein as “AGEs-like activity”). Examples of such AGE-like activity include, but are not limited to, binding activity (ligand activity) to RAGE.

  As used herein, the term “AGE receptor (Receptor for AGE)” is also referred to as RAGE, and (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10; (2) SEQ ID NO: 10 above A polypeptide comprising an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions in the amino acid sequence represented by (2) and exhibiting the activity of natural RAGE; (3) the amino acid represented by SEQ ID NO: A polypeptide comprising an amino acid sequence having at least 90% sequence identity with the sequence and exhibiting the activity of natural RAGE; (4) an amino acid having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 10 above A polypeptide comprising a sequence and exhibiting the activity of native RAGE; (5) shown in SEQ ID NO: 9 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule; (6) an amino acid encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 9 above. A polypeptide comprising a sequence and exhibiting the activity of native RAGE; (7) encoded by a nucleic acid molecule having one or several nucleotide substitutions, additions and / or deletions in the nucleic acid sequence shown in SEQ ID NO: 9 above (8) an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 9; And a polypeptide exhibiting the activity of natural RAGE; and (9) shown in SEQ ID NO: 9 above Comprises an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with acid sequence, and polypeptides displaying the activity of natural RAGE, it is one of. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art. RAGE is also a type I membrane protein with a molecular weight of about 35 kDa, which was identified from bovine lung in 1992 and belongs to the immunoglobulin superfamily that binds to AGE (complete RAGE with sugar chain modification has a molecular weight of 55 kDa). . The extracellular domain of RAGE has a structure in which one V-type immunoglobulin domain and then two C-type immunoglobulin domains (C1 region and C2 region) are combined with a domain having three immunoglobulin fold structures. Yes. RAGE also contains a single-pass membrane domain and a 43 amino acid cytoplasmic domain. RAGE interacts with various classes of ligands (AGE, S100 / calgranulin, amphoterin and amyloid-β peptide). The V domain is an essential site for ligand binding, and the cytoplasmic domain is essential for RAGE-mediated intracellular signaling. Since RAGE also has a disulfide bond within each domain, the mutant RAGE-surfactant complex of the present invention has positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 10. It is preferred to retain a cysteine residue corresponding to the position. RAGE is expressed only at low levels in normal tissues and the vascular system. However, this receptor is upregulated where the ligand accumulates. RAGE expression is increased in endothelial cells, smooth muscle cells, pericytes, renal mesangial cells and infiltrating mononuclear phagocytes in the diabetic vasculature. RAGE expression is also increasing in pathological sites such as arteriosclerotic lesions where AGE is accumulated. AGE-RAGE interactions change cellular properties important in vascular homeostasis. For example, after RAGE binds to AGE, vascular endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, and their permeability to macromolecules. In mononuclear phagocytes, RAGE activates the expression of cytokines and growth factors and induces cell migration in response to soluble AGEs, whereas chemotaxis occurs with fixed ligands.

  As used herein, the term “RAGE-like polypeptide” refers to “RAGE8”, “mRAGE8”, “RAGE1”, “mRAGE1”, “RAGE2”, “mRAGE2”, “RAGE3”, “mRAGE3”. , “RAGE4”, “mRAGE4”, “RAGE7”, “mRAGE7”, “RAGE143”, “mRAGE143”, “RAGE223”, “mRAGE223”, “RAGE226” and “mRAGE226” or their variants Includes the body.

  As used herein, the term “RAGE molecule” is understood to include any complex thereof as well as RAGE-like polypeptides. Therefore, it is understood that RAGE molecules include RAGE-like polypeptides, such as RAGE (full length), RAGE extracellular region (positions 22-332 of SEQ ID NO: 10), RAGE143, RAGE223, RAGE226, and the like. .

  As used herein, the terms “RAGE8” and “mRAGE8” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12; (2) 1 in the amino acid sequence shown in SEQ ID NO: 12 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) 1 or several amino acids at amino acid positions other than positions 92 and 95 in the amino acid sequence shown in SEQ ID NO: 12 above A polypeptide comprising an amino acid sequence containing amino acid substitutions, additions and / or deletions and exhibiting the activity of natural RAGE; (4) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 above (5) at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 12 above (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 11; (7) a nucleic acid complementary to the nucleic acid sequence represented by SEQ ID NO: 11 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes with the sequence under stringent conditions; (8) one or several substitutions, additions and / or deletions in the nucleic acid sequence shown in SEQ ID NO: 11 above; (9) a polypeptide encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 11 above. A peptide comprising amino acids at positions 92 and 95 in the encoded amino acid sequence; A polypeptide which retains the corresponding amino acid in SEQ ID NO: 12 and exhibits the activity of native RAGE; (10) a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 11 above And (11) one of the polypeptides comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 11 above. Indicated by one. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE1” and “mRAGE1” include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14; (2) 1 in the amino acid sequence shown in SEQ ID NO: 14 above. Or a polypeptide comprising an amino acid sequence containing several substitutions, additions and / or deletions; (3) amino acid positions other than positions 92, 95, 206 and 250 in the amino acid sequence shown in SEQ ID NO: 14 A polypeptide comprising an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions, and exhibiting the activity of natural RAGE; (4) at least 90% of the amino acid sequence represented by SEQ ID NO: 14 above A polypeptide comprising a variant having the sequence identity of: (5) less than the amino acid sequence shown in SEQ ID NO: 14 above A polypeptide comprising a variant having a sequence homology of 80%; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 13; (7) the nucleic acid sequence represented by SEQ ID NO: 13 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a complementary nucleic acid sequence; (8) one or several substitutions in the nucleic acid sequence shown in SEQ ID NO: 13 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having addition and / or deletion; (9) hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 13 above Amino acid sequence encoded by a nucleic acid molecule, wherein the amino acid sequence is at position 92 A polypeptide that retains the corresponding amino acid in SEQ ID NO: 14 and exhibits the activity of native RAGE; (10) at least a nucleic acid sequence shown in SEQ ID NO: 13 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having 90% sequence identity; and (11) encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 13 above. Indicated by one of the polypeptides comprising the amino acid sequence. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE2” and “mRAGE2” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16; (2) 1 in the amino acid sequence shown in SEQ ID NO: 16 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 16, 1 or 2 at amino acid positions other than positions 92, 95 and 206 A polypeptide comprising an amino acid sequence comprising several amino acid substitutions, additions and / or deletions and exhibiting the activity of native RAGE; (4) at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 16 above (5) at least 80% of the amino acid sequence shown in SEQ ID NO: 16 A polypeptide comprising a variant having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 15; (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 15 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) one or several substitutions, additions and / or in the nucleic acid sequence shown in SEQ ID NO: 15 above Or a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having a deletion; (9) encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 15 above. A polypeptide having a position of 92 and 95 in the encoded amino acid sequence. And the amino acid sequence at positions 206 retains the corresponding amino acid in SEQ ID NO: 16 and exhibits the activity of natural RAGE; (10) at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 15 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having sex; and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 15 above. Indicated by one of the peptides. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE3” and “mRAGE3” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 18; (2) 1 in the amino acid sequence shown in SEQ ID NO: 18 above. Or a polypeptide comprising an amino acid sequence containing several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 18, 1 or 2 at amino acid positions other than positions 92, 95 and 206 A polypeptide comprising an amino acid sequence comprising several amino acid substitutions, additions and / or deletions and exhibiting the activity of native RAGE; (374) at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 18 above (5) an amino acid sequence represented by SEQ ID NO: 18 and at least 8 A polypeptide comprising a variant having% sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 17; (7) against the nucleic acid sequence represented by SEQ ID NO: 17 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a complementary nucleic acid sequence; (8) one or several substitutions or additions in the nucleic acid sequence shown in SEQ ID NO: 17 above And / or a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having a deletion; (9) a nucleic acid molecule that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 17 under stringent conditions A polypeptide encoded by the amino acid sequence at positions 92 and 95 in the encoded amino acid sequence And the amino acid sequence at positions 206 retains the corresponding amino acid in SEQ ID NO: 18 and exhibits the activity of native RAGE; (10) at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 17 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having sex; and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence shown in SEQ ID NO: 17 above. Indicated by one of the peptides. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE4” and “mRAGE4” are (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20; (2) 1 in the amino acid sequence shown in SEQ ID NO: 20 above. Or a polypeptide comprising an amino acid sequence containing several substitutions, additions and / or deletions; (3) amino acid positions other than positions 14, 17, 128 and 172 in the amino acid sequence shown in SEQ ID NO: 20 above. A polypeptide comprising an amino acid sequence comprising one or several amino acid substitutions, additions and / or deletions, and exhibiting the activity of native RAGE; (4) at least 90% of the amino acid sequence represented by SEQ ID NO: 20 above A polypeptide comprising a variant having the sequence identity of: (5) less than the amino acid sequence shown in SEQ ID NO: 20 above (6) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule represented by SEQ ID NO: 19; (7) a nucleic acid sequence represented by SEQ ID NO: 19 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to said nucleotide sequence; (8) one or several substitutions in the nucleic acid sequence shown in SEQ ID NO: 19 above A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having additions and / or deletions; (9) hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 19 above A polypeptide encoded by a nucleic acid molecule, wherein the polypeptide is at position 14 in the encoded amino acid sequence. A polypeptide that retains the corresponding amino acid in SEQ ID NO: 20 and shows the activity of native RAGE, wherein the amino acid sequence at positions 17, 128, and 172; (10) at least the nucleic acid sequence shown in SEQ ID NO: 19 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having 90% sequence identity; and (11) encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 19 above. Indicated by one of the polypeptides comprising the amino acid sequence. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE7” and “mRAGE7” include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22; (2) 1 in the amino acid sequence shown in SEQ ID NO: 22 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 22, one or several amino acids at amino acid positions other than positions 92 and 95 A polypeptide comprising an amino acid sequence containing amino acid substitutions, additions and / or deletions and exhibiting the activity of natural RAGE; (4) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 above A polypeptide comprising a variant; (5) at least 80% sequence homology with the amino acid sequence set forth in SEQ ID NO: 22 above. (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 21; (7) a nucleic acid complementary to the nucleic acid sequence represented by SEQ ID NO: 21 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes with the sequence under stringent conditions; (8) one or several substitutions, additions and / or deletions in the nucleic acid sequence shown in SEQ ID NO: 21 above; (9) a polypeptide encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 21 above. A peptide comprising amino acids at positions 92 and 95 in the encoded amino acid sequence; A polypeptide which retains the corresponding amino acid in SEQ ID NO: 22 and exhibits the activity of native RAGE; (10) a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 21 above A polypeptide comprising an amino acid sequence encoded by: and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 21 above Indicated by one. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE143” and “mRAGE143” include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 24; (2) 1 in the amino acid sequence shown in SEQ ID NO: 24 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 24, one or several amino acids at amino acid positions other than positions 92 and 95 A polypeptide comprising an amino acid sequence containing amino acid substitutions, additions and / or deletions and exhibiting the activity of native RAGE; (4) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24 above A polypeptide comprising a variant; (5) at least 80% of the amino acid sequence shown in SEQ ID NO: 24 above A polypeptide comprising a variant having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 23; (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 23 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) one or several substitutions, additions and / or in the nucleic acid sequence shown in SEQ ID NO: 23 above Or a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having a deletion; (9) encoded by a nucleic acid molecule that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 23 under stringent conditions A polypeptide wherein the positions 92 and 95 in the encoded amino acid sequence A polypeptide that retains the corresponding amino acid in SEQ ID NO: 24 and exhibits natural RAGE activity; (10) a nucleic acid having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 23 above A polypeptide comprising an amino acid sequence encoded by the molecule; and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 23 above Indicated by one of the following. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  In the present invention, it may be preferable to use RAGE143. Although not wishing to be bound by theory, RAGE143 has the same recognition activity as RAGE1 and is excellent in molecular stability. RAGE1, RAGE223, RAGE226, etc. can also be used. However, since decomposition is likely to occur during storage and reactivity may change during the experiment, it is used using any method that does not allow degradation to proceed. Preferably, RAGE143 may be preferred for long-term assays. It is understood that similar results can be obtained not only with RAGE 143 but also with RAGE1, RAGE223, RAGE226, and the like.

  As used herein, the terms “RAGE223” and “mRAGE223” include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 26; (2) 1 in the amino acid sequence shown in SEQ ID NO: 26 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 26, one or several amino acids at amino acid positions other than positions 92 and 95 A polypeptide comprising an amino acid sequence containing amino acid substitutions, additions and / or deletions and exhibiting the activity of natural RAGE; (4) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 26 above (5) at least 80% of the amino acid sequence shown in SEQ ID NO: 26 A polypeptide comprising a variant having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 25; (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 25 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) one or several substitutions, additions and / or in the nucleic acid sequence shown in SEQ ID NO: 25 above Or a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having a deletion; (9) encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 25 above. A polypeptide wherein the positions 92 and 95 in the encoded amino acid sequence A polypeptide that retains the corresponding amino acid in SEQ ID NO: 26 and exhibits natural RAGE activity; (10) a nucleic acid having at least 90% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 25 above A polypeptide comprising an amino acid sequence encoded by the molecule; and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 25 above. Indicated by one of the following. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  As used herein, the terms “RAGE226” and “mRAGE226” include (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 28; (2) 1 in the amino acid sequence shown in SEQ ID NO: 28 above. Or a polypeptide comprising an amino acid sequence comprising several substitutions, additions and / or deletions; (3) in the amino acid sequence shown in SEQ ID NO: 28, one or several amino acids at amino acid positions other than positions 92 and 95 A polypeptide comprising an amino acid sequence comprising amino acid substitutions, additions and / or deletions and exhibiting the activity of native RAGE; (4) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 28 above (5) at least 80% of the amino acid sequence shown in SEQ ID NO: 28 A polypeptide comprising a variant having sequence homology; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule represented by SEQ ID NO: 27; (7) complementary to the nucleic acid sequence represented by SEQ ID NO: 27 A polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a specific nucleic acid sequence; (8) one or several substitutions, additions and / or in the nucleic acid sequence shown in SEQ ID NO: 27 above Or a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having a deletion; (9) encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 27 above. A polypeptide wherein the positions 92 and 95 in the encoded amino acid sequence A polypeptide that retains the corresponding amino acid in SEQ ID NO: 28 and exhibits the activity of native RAGE; (10) a nucleic acid having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 27 above A polypeptide comprising an amino acid sequence encoded by the molecule; and (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 27 above. Indicated by one of the following. The above-mentioned identity or homology is calculated using BLAST (NCBI BLAST 2.2.9 (issued 2004.5.12)) as a sequence analysis tool and using default parameters. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of those in the art.

  The RAGE-like polypeptide may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of the natural RAGE is retained.

  Also in the above RAGE-like polypeptide, since it is important to form an intramolecular disulfide bond, it corresponds to positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 10. Cysteine is preferably retained.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is done.

  In the present specification, the “amino acid” may be natural or non-natural as long as it satisfies the object of the present invention.

  As used herein, “amino acid derivative” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art. As used herein, it is understood that amino acid derivatives and amino acid analogs can be used alternatively as long as they can provide the same biological function as an amino acid.

  As used herein, “natural amino acid” means an L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  As used herein, “amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool. The identity search can be performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12). In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, a “corresponding” amino acid has, or is predicted to have, in a certain protein molecule or polypeptide molecule, the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison. An amino acid. For example, in LOX-1, the amino acids are at positions 232 and 249, and in RAGE, the amino acids are at positions 114, 117, 228, and 272.

  In the present specification, the “corresponding” gene refers to a gene having, or expected to have, the same action as that of a predetermined gene in a species as a reference for comparison in a certain species. When there are a plurality of genes having the same, those having the same origin evolutionarily. Thus, a gene corresponding to a gene (eg, LOX-1) can be an ortholog of that gene. Therefore, genes corresponding to human genes can be found in other animals (mouse, rat, pig, rabbit, guinea pig, cow, sheep, etc.). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal is the sequence of that animal (eg, mouse, rat, pig, rabbit, guinea pig, cow, sheep, etc.) using the sequence of the gene serving as the reference for the corresponding gene as a query sequence. It can be found by searching the database.

  As used herein, “heterologous” refers to nucleotide or amino acid sequences that are different or non-corresponding sequences, or sequences from different species. For example, the human nucleotide or amino acid sequence is heterologous to the mouse nucleotide or amino acid sequence, and the human LOX-1 nucleic acid or amino acid sequence is heterologous to the human albumin nucleotide or amino acid sequence. It is.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.

  As used herein, “biological function” refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. Examples of the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance. Examples of the present invention include, but are not limited to, a function that LOX-1 recognizes denatured LDL and a function that RAGE recognizes AGE. As used herein, a biological function can be exerted by “biological activity”. As used herein, “biological activity” refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule. When two factors interact, their biological activity depends on the binding between the two molecules and the resulting biological change, eg, when one molecule is precipitated with an antibody, the other When molecules also coprecipitate, the two molecules are considered bound. Therefore, seeing such coprecipitation is one method of judgment. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.

  Thus, “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other Similar measures of function are mentioned.

  In this specification, the term “interaction” refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included. Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, “contacting” means bringing a compound into physical proximity, either directly or indirectly, to a polypeptide or polynucleotide of the present invention. . The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse α-hemoglobin genes are orthologs, but the human α- and β-hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, orthologs may also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, site-specific base substitution methods using synthetic oligonucleotides (specification) Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other modifications may also be made by methods usually used in the field of molecular biology. it can. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, the binding site of a ligand molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in ligand binding capacity). It is well known. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, “substitution, addition and / or deletion” of a polypeptide or polynucleotide refers to an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, relative to the original polypeptide or polynucleotide. , Replacing, adding, or removing. Such substitution, addition and / or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. These changes in the reference nucleic acid molecule or polypeptide can occur at the 5 ′ end or 3 ′ end of the nucleic acid molecule as long as the desired function (eg, AGE recognition ability, etc.) is retained, or It can occur at the amino-terminal or carboxy-terminal site of the amino acid sequence representing this polypeptide, or can occur anywhere between those terminal sites and is interspersed individually between residues in the reference sequence. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, AGE recognition ability, etc.) intended in a variant having the substitution, addition or deletion. As long as it is). For example, such a number can be one or several, and preferably within 20%, within 15%, within 10%, within 5%, or less than 150, less than 100, less than 100, It can be 50 or less, 25 or less, and the like.

  As used herein, the term “tag sequence” refers to a substance for sorting molecules by a specific recognition mechanism such as a receptor-ligand, more specifically, to bind a specific substance. A substance (for example, having a relationship such as biotin-avidin or biotin-streptavidin) that serves as a binding partner. Thus, for example, a specific substance to which a tag sequence is bound can be selected by contacting the substrate to which the binding partner of the tag sequence is bound. Such tag sequences are well known in the art. Representative tag sequences include, but are not limited to, myc tag, His tag, HA, Avi tag and the like.

  As used herein, the term “detection agent” broadly refers to any agent capable of detecting a target substance (eg, denatured LDL, AGE, etc.).

  As used herein, the term “solid phase” refers to a material used interchangeably herein with “substrate” and “substrate” from which the device of the present invention is constructed. A planar support to which molecules such as antibodies can be immobilized. In the present invention, when detecting using the principle of surface plasmon resonance, the solid phase is preferably a glass substrate base material having a metal thin film containing gold, silver or aluminum on one side. In the present invention, when detecting using the principle of the crystal oscillator microbalance, a frequency conversion element (for example, a crystal oscillator or a surface acoustic wave element) is used as a solid phase, and the receptor is directly coupled. One side of the quartz plate is coated with silicone, and the other side is provided with a gold electrode as a solid phase. In the present invention, when a mechanism such as an enzyme-linked immunosorbent assay (ELISA) is used, a microtiter plate is generally used as the solid phase (substrate). The material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials. Suitable substrates include beads, gold particles, plates (eg, microtiter plates), test tubes, chips, magnetic particles, membranes, fibers, glass slides, metal films, filters, tubes, balls, diamond-like carbon coated stainless steel. However, it is not limited to these.

  As such a material for use as a solid phase and a substrate, any material capable of forming a solid surface can be used, for example, glass, silica, silicone, ceramic, silicon dioxide, plastic, metal (also alloys) Natural) and synthetic polymers (including, but not limited to) polystyrene, cellulose, amylose, chitosan, dextran, and nylon. The substrate may be formed from a plurality of layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. In the present invention, a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, and a PVDF membrane can also be used. When analyzing a high-density material, it is preferable to use a material having hardness such as glass. A preferable material for the substrate varies depending on various parameters such as a measuring instrument, and those skilled in the art can appropriately select an appropriate material from the various materials described above.

  In this specification, “chip” refers to a micro integrated circuit that has various functions and is a part of a system. In the present specification, the solid phase on which the receptor is immobilized is referred to as a receptor chip and / or a receptor microchip.

  As used herein, the term “dry form” means a state in which the water content is substantially reduced in a detection agent, device, diagnostic agent or the like containing the components used in the present invention. Say. In general, the “dry state” can be easily achieved by using techniques well known in the art such as air drying and drying under reduced pressure.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecμlar Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecule Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecule Biology: A Compendium of Methods from Current Protocols in Molecule Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecule Biology: A Compendium of Methods from Current Protocols in Molecule Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecule Biology: A Compendium of Methods from Current Protocols in Molecule Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecule Biology: A Compendium of Methods from Current Protocols in Molecule Biology, Wiley, and annual updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  The polypeptide having the amino acid sequence of SEQ ID NO: 2 and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

  As used herein, the term “label” refers to a molecule that is attached to a substance that is known to bind to that substance in order to detect that substance. Examples of labels include, but are not limited to, fluorescent labels, chemiluminescent labels, bioluminescent labels, and the like.

  As used herein, the term “refolding” is used to unravel the abnormal structure of a polypeptide that has lost its original function due to abnormal folding, while preventing reaggregation with a surfactant. This refers to refolding to the original correct structure of the polypeptide by utilizing the inclusion ability of cycloamylose.

As used herein, the term “surfactant” refers to a substance that, when dissolved in a liquid, significantly reduces the surface tension of the solution. A surfactant forms a micelle colloid when it exceeds the sea micelle concentration in the solution. Since the surfactant has a hydrophilic group and a lipophilic group, it is an amphiphilic compound, and forms a stable layer by directional coordination at the two-phase interface. Surfactants include anionic surfactants such as long chain fatty acid salts and sodium dodecyl sulfate (SDS), cationic surfactants such as cetyltrimethylammonium bromide, amphoteric surfactants, Triton ^TM series and It is divided into nonionic surfactants such as the Tween ^TM series. Suitable surfactants include, but are not limited to, polyoxyethylene surfactants and ionic surfactants.

(Description of Preferred Embodiment)
The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment. It should be understood that those skilled in the art can easily make modifications, changes and the like within the scope of the present invention with reference to the following preferred embodiments.

(Method for detecting or quantifying molecules exhibiting AGE-like activity)
In one aspect, the present invention provides a method for detecting or quantifying a molecule exhibiting AGE-like activity, wherein (A) a sample to be detected or quantified is labeled in the presence of a RAGE molecule immobilized on a solid phase. Contacting with a labeled RAGE molecule in the presence of an AGE molecule immobilized on a solid phase, or (B) detecting the binding of the RAGE molecule to the AGE molecule. Thus, the presence or level of inhibition of binding provides a method comprising the step of indicating the presence or level of a molecule exhibiting AGEs-like activity in the sample.

  In this method, either a receptor molecule RAGE molecule or its ligand AGE molecule is immobilized on a solid phase, and a sample to be detected or quantified in the presence of the other is assayed. The effect of the increase in the sensitivity which was not able to be obtained, and the increase in the comprehensiveness of the screening of the molecule | numerator which shows AGEs-like activity was acquired.

  In the embodiment of the present invention, any solid phase can be used as long as a molecule to be immobilized, for example, a RAGE molecule or an AGE molecule can be immobilized. In one embodiment, when using a mechanism such as an enzyme linked immunosorbent assay (ELISA), a microtiter plate is generally used as the solid phase (substrate). The material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials. Examples of the material include polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile. Use organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. Can do. The immobilization method to the solid phase can be carried out by any known method in the art. For example, preferably, a bonding reaction via a silanol group (SiOH) on a solid surface, a hydrophobic bond by a matrix of a material and an ionic bond by a modified functional group, a bonding reaction via an aromatic ring contained in the matrix Can be used. After fixation on the solid phase, the solid phase may be dried or used in a wet state. It becomes easy to preserve by drying.

  In the present invention, any technique known in the art can be used as a technique for bringing the sample of the present invention into contact with an AGE molecule or a RAGE molecule. For example, both of them are mixed in a solution, or then incubated. However, it is not limited to these methods.

  In the present invention, as a technique for detecting the binding of the RAGE molecule to the AGE molecule, any technique known in the art can be used. For example, when any molecule is labeled (for example, fluorescent labeling) It will be appreciated that any technique for detecting the label can be utilized. Here, the presence / absence or level of inhibition of the binding indicates the presence or level of a molecule exhibiting AGE-like activity in the sample.

  In the present invention, the quantification of the binding of the RAGE molecule to the AGE molecule can be realized by any method for quantifying in the above detection. Such quantification may be relative (level) or displayed as an absolute amount. For example, as such a technique, for known amounts of AGE molecules and RAGE molecules, the intensity of the label to be detected is plotted, a calibration curve is created, and extrapolated from the sample detection data. It is not limited.

  In one embodiment, the RAGE molecule used in the present invention can be RAGE143, RAGE1, RAGE223, RAGE226 or a variant thereof. In one preferred embodiment, RAGE 143 is used. Although it is because stability is long with respect to long-term storage, it is not limited to this.

  In one embodiment, the AGE molecule used in the present invention is Lys-AGE (glutaraldehyde modified lysine modified AGE), glucose modified AGE (G-AGE), ribose modified AGE (R-AGE), fructose modified AGE (F -AGE) or variants thereof.

(Preliminary detection method for diabetic nephropathy)
In another aspect, the present invention provides a preliminary detection method (or a prediction method or a preliminary diagnosis method) for diabetic nephropathy, wherein (A) the subject is a target for detection of diabetic nephropathy. Contacting the sample with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, or contacting a labeled RAGE molecule in the presence of an AGE molecule immobilized on a solid phase; and B) detecting the binding of the RAGE molecule to the AGE molecule, wherein the presence or level of inhibition of the binding indicates that the subject will or will have future diabetic nephropathy A method comprising: Thus, it has not been shown in the past that RAGE molecules or AGE molecules have been used to show that a subject will or will have diabetic nephropathy in the future, and significant progress has been made in the diagnostic field. It can be said that

  Here, in the preliminary detection method (or prediction method or preliminary diagnosis method) of diabetic nephropathy of the present invention, detection of AGE molecules and RAGE molecules, contact, binding, calculation of the level of binding, etc. It will be appreciated that any of the embodiments described in Methods for detecting or quantifying molecules exhibiting AGE-like activity may be utilized.

  In the present specification, the “subject” refers to an organism (for example, a human) that is a target of the diagnosis or detection method of the present invention.

  As used herein, “sample” refers to any substance obtained from a subject or the like, and includes, for example, body fluids (blood, saliva, urine, tears, etc.). Preferably blood, urine or tears are used.

  A method for determining whether or not a subject will suffer from diabetic nephropathy in the future based on the presence or absence or the level of inhibition of binding is as follows. Samples are prepared from this regardless of the condition of the subject. When a labeled AGE molecule sample is reacted at 37 ° C. for 1 hour in the presence of RAGE, the activity of inhibiting the binding of the labeled AGE molecule is quantified. The amount of labeled AGE binding when a buffer solution is added instead of the sample (when the label is a fluorescent label, the fluorescence intensity measured by a fluorometer) is 100%, and the greater the degree of inhibition of binding, the more AGE-like It is determined that the abundance of molecules showing activity is high and the possibility of being affected is high.

(Preliminary detection method of diabetic nephropathy in diabetes)
In another aspect, the present invention relates to a preliminary detection method (or a prediction method or a preliminary diagnosis method) of diabetic nephropathy in diabetes, and (A) Diabetes targeted for detection of diabetic nephropathy A sample from a subject suffering from is contacted with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, or a labeled RAGE molecule in the presence of an AGE molecule immobilized on a solid phase. And (B) detecting the binding of the RAGE molecule to the AGE molecule, wherein the presence or absence of inhibition of the binding is determined by whether the subject will suffer from diabetic nephropathy in the future or A method is provided that includes a step that indicates a possibility. Thus, it has not been shown in the past that a subject suffering from diabetes using RAGE molecule or AGE molecule has or will show the possibility of having diabetic nephropathy in the future. It can be said that it shows remarkable progress.

  Here, in the preliminary detection method (or prediction method or preliminary diagnosis method) of diabetic nephropathy in diabetes of the present invention, detection of AGE molecule and RAGE molecule, contact, binding, calculation of binding level, etc. It is understood that any of the embodiments described in (Methods for detecting or quantifying molecules exhibiting AGE-like activity) can be utilized.

  A method for determining whether or not a subject suffering from diabetes will suffer from or is likely to suffer from diabetic nephropathy based on the presence or absence or the level of binding inhibition is as follows. Samples are prepared from subjects with diabetes. When a labeled AGE molecule sample is reacted at 37 ° C. for 1 hour in the presence of RAGE, the activity of inhibiting the binding of the labeled AGE molecule is quantified. The amount of labeled AGE binding when a buffer solution is added instead of the sample (when the label is a fluorescent label, the fluorescence intensity measured by a fluorometer) is 100%, and the greater the degree of inhibition of binding, the more AGE-like It is determined that the abundance of molecules showing activity is high and the possibility of being affected is high.

(Detection method of diabetes or diabetic nephropathy)
In another aspect, the present invention relates to a detection method (or diagnostic method) for diabetes or diabetic nephropathy, in which (A) a sample from a subject to be detected for diabetic nephropathy is used as a solid phase. Contacting with the labeled AGE molecule in the presence of the RAGE molecule immobilized on, or contacting with the labeled RAGE molecule in the presence of the AGE molecule immobilized on the solid phase, and (B) the immobilized Detecting the binding of the RAGE molecule or a ligand of the AGE or a variant thereof to an AGE or variant thereof, wherein the presence or level of inhibition of the binding is determined by the subject having diabetes or diabetic nephropathy A method is provided comprising the step of indicating that the patient is afflicted. Thus, it has been shown that RAGE molecules or AGE molecules can be used to diagnose having diabetes or diabetic nephropathy, and has made remarkable progress in the diagnostic field. It can be said to be a thing.

  Here, in the method for detecting diabetes or diabetic nephropathy of the present invention, AGE molecules and RAGE molecules, contact, detection of binding, calculation of the level of binding, etc. (method of detecting or quantifying molecules exhibiting AGEs-like activity) It is understood that any of the embodiments described in can be utilized.

  A technique for determining that a subject is suffering from diabetes or diabetic nephropathy based on the presence or absence or level of inhibition of binding is as follows. Samples are prepared from a subject suspected of having or suffering from diabetes or diabetic nephropathy. When a labeled AGE molecule sample is reacted at 37 ° C. for 1 hour in the presence of RAGE, the activity of inhibiting the binding of the labeled AGE molecule is quantified. The amount of labeled AGE binding when a buffer solution is added instead of the sample (when the label is a fluorescent label, the fluorescence intensity measured by a fluorometer) is 100%, and the greater the degree of inhibition of binding, the more AGE-like It is determined that the abundance of molecules showing activity is high and the possibility of being affected is high.

(Method for detecting or quantifying molecules exhibiting OxLDL-like activity)
In one aspect, the present invention provides a method for detecting or quantifying a molecule exhibiting OxLDL-like activity, wherein (A) a sample to be detected or quantified is labeled in the presence of a CTLD molecule immobilized on a solid phase. Contacting with the denatured LDL or contacting with the labeled CTLD molecule in the presence of the denatured LDL immobilized on the solid phase, and (B) detecting the binding of the CTLD molecule to the denatured LDL. Thus, the presence or level of inhibition of binding provides a method comprising the step of indicating the presence or level of a molecule exhibiting OxLDL-like activity.

  In this method, either a CTLD molecule as a receptor molecule or a denatured LDL as a ligand thereof is immobilized on a solid phase, and a sample to be detected or quantified is assayed in the presence of the other. The effect of the increase in the sensitivity which was not able to be obtained, and the increase in the comprehensiveness of the screening of the molecule | numerator which shows OxLDL-like activity was acquired.

  In the embodiment of the present invention, any solid phase can be used as long as a molecule to be immobilized, for example, a CTLD molecule or a modified LDL can be immobilized. In one embodiment, when using a mechanism such as an enzyme linked immunosorbent assay (ELISA), a microtiter plate is generally used as the solid phase (substrate). The material of the substrate can be any solid, either covalently or noncovalently, that has the property of binding to the biomolecules used in the present invention or that can be derivatized to have such properties. Materials. Examples of the material include polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile. Use organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. Can do.

  The immobilization method to the solid phase can be carried out by any known method in the art. For example, it is preferable to use a bond by a silanol group (SiOH) on a solid surface, a hydrophobic bond by a matrix of a material and an ionic bond by a modified functional group, or a bond through an aromatic ring contained in the matrix. it can. After fixation on the solid phase, the solid phase may be dried or used in a wet state. It becomes easy to preserve by drying.

  In the present invention, as a method of bringing the sample of the present invention into contact with a CTLD molecule or denatured LDL, any method known in the art can be used. For example, both of them are mixed in a solution, or then incubated. However, it is not limited to these methods.

  In the present invention, as a technique for detecting the binding of denatured LDL to a CTLD molecule, any technique known in the art can be used. For example, when any molecule is labeled (for example, a fluorescent label), It will be appreciated that any technique for detecting the label can be utilized. Here, the presence / absence or level of inhibition of the binding indicates the presence or level of a molecule exhibiting OxLDL-like activity in the sample.

  In the present invention, the quantification of the binding of denatured LDL to CTLD molecules can be realized by any method of quantifying in the above detection. Such quantification may be relative (level) or displayed as an absolute amount. For example, as such a technique, for known amounts of CTLD molecules and denatured LDL, the intensity of the label to be detected is plotted, a calibration curve is created, and extrapolated from the sample detection data. It is not limited.

  In one embodiment, the CTLD molecule used in the present invention may be CTLD14, LOX-1 full length, LOX-1 full length extracellular region or a variant thereof. Preferably, CTLD14 can be used. Although it is because stability is long with respect to long-term storage, it is not limited to this.

  In one embodiment, the modified LDL used in the present invention is oxidized LDL (OxLDL), malondialdehyded LDL (MDA-LDL), acrolein modified LDL, nonenal modified LDL, crotonaldehyde (CRA) modified LDL, 4- It may be hydroxynonenal (HNE) modified LDL, hexanoyl (HEL) modified LDL, small particle LDL, saccharified LDL, acetylated LDL or a variant thereof.

(Kit for detection or quantification of molecules exhibiting AGE-like activity)
In one aspect, the present invention provides kits for the detection or quantification of molecules that exhibit AGEs-like activity. The kit comprises (A) a solid phase and labeled AGE molecule having an immobilized RAGE molecule, or a solid phase and labeled RAGE molecule having an immobilized AGE molecule; and (B) the RAGE molecule to the AGE molecule. Means for detecting binding are provided. In this kit, the presence or absence or level of inhibition of the binding indicates the presence or level of molecules exhibiting AGEs-like activity. The RAGE molecule, solid phase, AGE molecule, label, contact, binding detection technique or means that can be used in the kit of the present invention, the calculation of the level of binding, etc. (the method for detecting or quantifying molecules exhibiting AGE-like activity) It will be understood that any of the embodiments described in FIG.

(Kit or marker for detection or diagnosis of diabetes or diabetic nephropathy)
In another aspect, the present invention provides a kit for preliminary detection or diagnosis (or prediction) of diabetic nephropathy. Wherein the kit comprises (A) a solid phase and labeled AGE molecule having an immobilized RAGE molecule, or a solid phase and labeled RAGE molecule having an immobilized AGE molecule; and (B) the RAGE molecule to the AGE The presence or level of inhibition of the binding indicates that the subject is or will be suffering from diabetic nephropathy in the future. The RAGE molecule, solid phase, AGE molecule, label, contact, binding detection technique or means that can be used in the kit of the present invention, the calculation of the level of binding, etc. (detection or quantification method of molecules exhibiting AGEs-like activity) It is understood that any of the embodiments described in (Methods for detecting diabetes or diabetic nephropathy) can be utilized.

  In another aspect, the present invention provides a preliminary diagnostic marker for diabetic nephropathy comprising a molecule that exhibits AGE-like activity. Alternatively, the present invention is the use of AGEs-like activity in a subject to serve as a preliminary diagnostic indicator of diabetic nephropathy, wherein the AGEs-like activity is higher than a standard value, Uses are provided that indicate that a subject will or will likely suffer from diabetic nephropathy in the future. Here, when the diagnostic marker of the present invention is measured, the fact that the AGE-like activity is higher than the standard value indicates that the subject will suffer from or may have diabetic nephropathy in the future. Molecules that exhibit AGEs-like activity or AGEs-like activity can be measured using a technique using the AGE molecule or RAGE molecule of the present invention. AGEs-like activity, molecules exhibiting AGEs-like activity that can be used in the diagnostic markers or uses of the present invention, and RAGE molecules, solid phases, AGE molecules, labels, contacts, binding detection techniques or means, etc. As for the calculation of the level of the above, it is possible to use any of the embodiments described in (Method for detecting or quantifying a molecule exhibiting AGE-like activity), (Method for detecting diabetes or diabetic nephropathy) and other columns. Understood.

  In another aspect, the present invention provides a kit for preliminary detection or diagnosis (or prediction) of diabetic nephropathy in diabetes. The kit includes (A) a solid phase and labeled AGE molecule having an immobilized RAGE molecule, or a solid phase and labeled RAGE molecule having an immobilized AGE molecule; and (B) binding of the molecule to the AGE. Means for detecting are provided. Here, the presence or level of inhibition of the binding indicates that the subject is or will likely suffer from diabetic nephropathy in the future. The RAGE molecule, solid phase, AGE molecule, label, contact, binding detection technique or means, etc. that can be used in this kit of the present invention include the calculation of the level of binding, etc. (detection or quantification of molecules showing AGEs-like activity) It is understood that any embodiment described in (Method), (Method of Detecting Diabetes or Diabetic Nephropathy) can be utilized.

  In another aspect, the present invention provides a preliminary diagnostic marker for diabetic nephropathy in diabetes comprising a molecule that exhibits AGE-like activity. Alternatively, the present invention is the use of AGEs-like activity in a subject to serve as a preliminary diagnostic indicator of diabetic nephropathy in diabetes, wherein the AGEs-like activity is higher than a standard value. The use is provided wherein the subject is or will likely suffer from diabetic nephropathy in the future. Here, when the diagnostic marker of the present invention is measured, the fact that the AGE-like activity is higher than the standard value indicates that the subject will suffer from or may have diabetic nephropathy in the future. Molecules that exhibit AGEs-like activity or AGEs-like activity can be measured using a technique using the AGE molecule or RAGE molecule of the present invention. AGEs-like activity, molecules exhibiting AGEs-like activity that can be used in the diagnostic markers or uses of the present invention, and RAGE molecules, solid phases, AGE molecules, labels, contacts, binding detection techniques or means, etc. As for the calculation of the level of the above, any of the embodiments described in (Method of detecting or quantifying a molecule exhibiting AGE-like activity), (Method of detecting diabetes or diabetic nephropathy in diabetes) and other columns may be used. It is understood.

  In another aspect, the present invention provides a kit for the detection or diagnosis of diabetes or diabetic nephropathy. Wherein the kit comprises (A) a solid phase and labeled AGE molecule having an immobilized RAGE molecule, or a solid phase and labeled RAGE molecule having an immobilized AGE molecule; and (B) the molecule of said molecule against the AGE. Means for detecting binding are provided. Here, the presence or level of inhibition of the binding indicates that the subject is suffering from diabetes or diabetic nephropathy. The RAGE molecule, solid phase, AGE molecule, label, contact, binding detection technique or means, etc. that can be used in this kit of the present invention include the calculation of the level of binding, etc. (detection or quantification of molecules showing AGEs-like activity) It is understood that any embodiment described in (Method), (Method of Detecting Diabetes or Diabetic Nephropathy) can be utilized.

In another aspect, the present invention provides a diagnostic marker for diabetes or diabetic nephropathy comprising a molecule that exhibits AGEs-like activity. Alternatively, the present invention relates to the use of AGEs-like activity in a subject to serve as an index for diagnosis of diabetes or diabetic nephropathy, wherein the AGEs-like activity is higher than a standard value. Uses are provided that indicate that the body is suffering from diabetes or diabetic nephropathy. Here, when the diagnostic marker of the present invention is measured, the fact that the AGEs-like activity is higher than the standard value indicates that the subject suffers from diabetes or diabetic nephropathy. Molecules that exhibit AGEs-like activity or AGEs-like activity can be measured using a technique using the AGE molecule or RAGE molecule of the present invention. AGEs-like activity, molecules exhibiting AGEs-like activity that can be used in the diagnostic markers or uses of the present invention, and RAGE molecules, solid phases, AGE molecules, labels, contacts, binding detection techniques or means, etc. As for the calculation of the level of the above, it is possible to use any of the embodiments described in (Method for detecting or quantifying a molecule exhibiting AGE-like activity), (Method for detecting diabetes or diabetic nephropathy) and other columns. Understood.

(Kit for detection or quantification of molecules exhibiting OxLDL-like activity)
In another aspect, the present invention provides kits for the detection or quantification of molecules that exhibit OxLDL-like activity. The kit comprises (A) a solid phase with labeled CTLD molecule and labeled denatured LDL, or a solid phase with labeled denatured LDL and labeled CTLD molecule; and (B) the CTLD molecule against the denatured LDL. Means for detecting binding are provided. Here, the presence or absence or level of inhibition of the binding indicates the presence or level of a molecule exhibiting OxLDL-like activity. The modified LDL, CTLD molecule, solid phase, label, contact, binding detection technique or means, etc. that can be used in this kit of the present invention include the calculation of the level of binding, etc. (detection or quantification of molecules exhibiting OxLDL-like activity) It is understood that any embodiment described in (Method) can be utilized.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  The handling of the animals used in the following examples complied with the standards defined by the Food Research Institute. In addition, although a manufacture example is described below, it is understood that non-patent document 7 can be further referred to for detailed information.

(Production Example 1: Production of CTLD-like polypeptide)
A nucleic acid encoding a CTLD-like polypeptide (G232A / G249A; also referred to as PR-CTLD in this example) having mutations introduced at two cleavage sites was produced according to a standard method. By introducing this nucleic acid into a mammalian expression vector pECFP (Clontech, USA) fused with cyan fluorescent protein (CFP) according to a standard method, a plasmid containing PR-CTLD-encoding nucleic acid fused with CFP at the N-terminus is obtained. It was constructed. CHO cells were cultured in F12 medium supplemented with 10% FCS at 37 ° C. in a humidified atmosphere of 5% CO ₂ . 400 μl of CHO cells suspended at a density of 1 × 10 ⁴ cells / ml were seeded on a cover glass 24 hours before transfection. Cells were transfected with LipofectAMINE reagent according to the manufacturer's protocol, incubated for 48 hours and transiently expressed in CHO cells. Thereafter, the cells were observed with a fluorescence microscope, and CTLD-like polypeptide-expressing cells were confirmed by CFP fluorescence. Further, the modified LDL as a ligand is labeled with 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine (DiD; Molec μlar Probes, Eugene, OR) as a fluorescent dye. When this modified LDL was added to CTLD-like polypeptide-expressing cells, the ligand recognition ability and uptake ability could be confirmed.

  From these results, PR-CTLD expressing cells showed a clear ligand uptake ability, and it was shown that the amino acid substitution performed for conferring protease resistance had no effect on the ability to recognize denatured LDL. .

  Furthermore, when the protease sensitivity of this PR-CTLD was examined, degradation occurred even when 0.02 mg of PR-CTLD was treated for 18 hours with 2u (1u is a concentration capable of degrading 1 mg of the target protein) thrombin. It was confirmed that there was no protease, and a protease-resistant CTLD-like polypeptide (PR-CTLD) was successfully produced.

(Production Example 2: Generation of tag sequence-added CTLD-like polypeptide)
In addition to the CTLD-like polypeptide (PR-CTLD) produced in Production Example 1, the protein PR-CTLD14 (including 14 amino acids in the neck region) having a different length in which a protease resistance mutation similar to that of the CTLD-like polypeptide is further introduced. , Amino acids 129 to 273 of LOX-1). The amino acid sequence of PR-CTLD14 includes Cys involved in intermolecular S—S bond. Although there is recognition ability without forming an intermolecular S—S bond, it is necessary to take a molecular density of a certain level or higher when expressing a function on a cell (Xie et al., DNA and Cell Biology 23 (2 ): 111-117 (2004) and Matsunaga et al., Experimental Cell Research 313: 1203-1214 (2007)). Accordingly, PR-CTLD14 capable of forming an intermolecular SS bond as a structure that can contribute to high-density integration can also be used to construct an assay system for detecting denatured LDL.

When utilizing the expressed protease-resistant CTLD-like polypeptides (PR-CTLD and PR-CTLD14) in a detection / evaluation system, it is desirable that effective modifications are applied. Therefore, two types of tags (one sequence AviTag (GLNDIFEAQKIEWHE (SEQ ID NO: 29)) that undergoes biotinylation in E. coli and the other are Streptag (AWRHPQFGGG) that is a sequence directly recognized by streptavidin. (SEQ ID NO: 30)) or Streptag II (WSHPQFEK (SEQ ID NO: 31)) was added.The binding of biotin and streptavidin was the strongest by non-covalent bond (KD = 10 ⁻¹⁵ M), immobilization via streptavidin, etc. However, in order to biotinylate, it takes time and effort to add biotin to the medium and to co-express biotin ligase. (KD = 10 ⁻⁷ M), tag added on gene sequence and colon A StreptagII (WSHPQFEK (SEQ ID NO: 31))-added protein capable of binding to streptavidin when expressed in bacteria was also generated, and in each case, a nucleic acid sequence encoding a tag sequence was introduced into the 5 ′ side of the pET system vector. The plasmids for expression (pET N-Avi, pET N-St II) PR-CTLD and PR-CTLD14 were introduced into each of these expression vectors according to a standard method to generate an expression construct. For conversion, a BirA gene encoding biotin ligase was introduced into a pAC vector that can be maintained in the same microbial cell as the pET vector to prepare an expression plasmid (BirA / pAC).

  In both PR-CTLD and PR-CTLD14, formation of an intramolecular S—S bond is essential for structural stabilization and function. Therefore, Origami B (DE3) (Novagen, USA) was used as an expression host. Origami B (DE3) is a thioredoxin reductase and glutathione reductase mutant and has the property of promoting S—S bond formation of proteins expressed in the cytoplasm. When AviTag was added to a protease resistant CTLD-like polypeptide, it was simultaneously transformed with a vector containing a nucleic acid sequence encoding AviTag and the target protein and an expression vector containing a BirA gene. The resulting transformed host cells were added with kanamycin (15 μg / ml), ampicillin (50 μg / ml), tetracycline (12.5 μg / ml), chloramphenicol (34 μg / ml) (all at the final concentration). The cells were cultured at 37 ° C. in LB medium. When transformed with an expression vector containing StreptagII-encoding nucleic acid, it was cultured at 37 ° C. in LB medium supplemented with kanamycin (15 μg / ml), ampicillin (50 μg / ml), and tetracycline (12.5 μg / ml). Expression of PR-CTLD and PR-CTLD14 was induced by adding IPTG.

  As a preliminary experiment, temperature conditions and induction time for expressing PR-CTLD and PR-CTLD14 as soluble polypeptides were examined. The temperature conditions were set to (1) 37 ° C, (2) 25 ° C and (3) 20 ° C. The induction time was set to (A) 2 hours, (B) 4 hours, (C) 8 hours, (D) 18 hours, and (E) 24 hours. First, these host cells were induced at 37 ° C., and the molecular weight of the induced protein was measured. As a result, induction of the protein having the expected molecular weight was confirmed, but 100% was recovered in the insoluble fraction. In view of this result, when the induction temperature was lowered to 25 ° C. without changing the induction time, expression as a soluble polypeptide was confirmed slightly. Further, the induction time was lowered to 20 ° C. and the induction time was examined. In order to recover the soluble polypeptide, the temperature condition (3) and the induction time (D) 18 hours to (E) 24 It was concluded that induction of time, particularly 20-24 hours, was appropriate.

  The above transformed cells were pre-cultured at 37 ° C. for 12 hours. In the main culture, this pre-cultured solution was added to each of the LB media so as to be diluted 20-fold, and swirled at 25 ° C. Based on the conditions obtained above, when the optical density (A600) of the culture solution reached about 0.5, the culture temperature was lowered to 20 ° C. and at the same time, 1 mM (final concentration) IPTG was added. Protein expression was induced. When the AviTag-added protein was produced, 50 μM (final concentration) of biotin was added simultaneously with the start of induction, and the target protein to be expressed was simultaneously biotinylated. Twenty to 24 hours after the start of induction, the cells were pelleted by centrifugation at 12,000 rpm × 5 minutes, the supernatant was discarded, and washed with an appropriate amount of TBS.

  The bacterial cells suspended in TBS were crushed with an ultrasonic crusher, and the supernatant recovered by centrifugation at 15,000 rpm × 30 minutes was used as the soluble fraction. More than 90% of the expressed target protein remained in the insoluble fraction. Since the His protein for purification was added to the C-terminal side, the target protein was purified by eluting with imidazole after binding to Ni-agarose. AviTag added for detection can also be used for purification. In this case, it was adsorbed on streptavidin Mutein matrix (Roche) and then eluted with biotin. In the case of StrepTag II, it was adsorbed on Strep-Tactin Sepharose (IBA, US) and then eluted with dessiobiotin. For both PR-CTLD and PR-CTLD14, the yield per liter was less than 0.5 mg as shown in Table 1 below.

(Production Example 3: RAGE-like molecule)
A sRAGE1-like polypeptide (R114Q / V117A / R228N / M272I; also referred to as mRAGE1 in this example) having a mutation introduced at four cleavage sites was produced according to a standard method. By introducing this nucleic acid into a mammalian expression vector pECFP (Clontech, USA) fused with cyan fluorescent protein (CFP) or pTarget (Promega) according to a standard method, an mRAGE1-encoding nucleic acid fused with CFP at the N-terminus is obtained. The containing plasmid was constructed. CHO cells were cultured in F12 medium supplemented with 10% FCS at 37 ° C. in a humidified atmosphere of 5% CO ₂ . 400 μl of CHO cells suspended at a density of 1 × 10 ⁴ cells / ml were seeded on coverslips 24 hours before transfection. Cells were transfected with LipofectAMINE reagent according to the manufacturer's protocol, incubated for 48 hours and transiently expressed in CHO cells. Then, when this cell was observed with the fluorescence microscope, the mRAGE1 expression cell was confirmed by the fluorescence of CFP. Further, BSA (R-AGE, see Production Example 8 for the production method) saccharified with ribose as a ligand is labeled with Alexa633 (Molecula Probe) as a fluorescent dye, and this labeled glycated BSA (AGE molecule) is labeled mRAGE1. When added to the expression cells, the ability to recognize and take up the ligand was confirmed.

  mRAGE1-expressing cells showed clear ligand uptake ability, and it was shown that amino acid substitution aimed at increasing protease resistance did not affect ligand recognition ability.

  Furthermore, when the protease sensitivity was examined, it was shown that the resistance was higher than that of the natural type, and the RAGE molecule with increased protease resistance was successfully produced.

(Production Example 4: Determination of minimum region essential for ligand recognition ability of RAGE)
In this example, miniRAGE was manufactured.

Based on the sequence shown in GenBank accession number AB036432 as miniRAGE, the following primers:
Forward primer (common to RAGE1, RAGE2, RAGE3, RAGE7, RAGE8, RAGE9, RAGE143, RAGE223 and RAGE226)
5′-CTACATATGGCTCAAAAACATCACAGC-3 ′ (SEQ ID NO: 32)
For reverse primer RAGE1
5′-TTACTCGAGAGCCTGCAGTTGGCCC-3 ′ (SEQ ID NO: 33)
・ About RAGE2
5′-TTACTTCGAGACCAGACACGGGGCTG-3 ′ (SEQ ID NO: 34)
・ About RAGE3
5′-TTACTTCGAGAAGCTACTGCTCACC-3 ′ (SEQ ID NO: 35)
・ About RAGE7
For 5′-TTACTTCGAGAAAACACCAGCCGTGAGT-3 ′ (SEQ ID NO: 36) RAGE8,
For 5′-TTACTCGAGAAATCTGGATAGACGG-3 ′ (SEQ ID NO: 37) RAGE9,
5′-TTACTTCGAGAACTTGGTTCCCTTTTCC-3 ′ (SEQ ID NO: 38)
-For RAGE143, 5'-TTACTCGAGTCCCCCACCTTATTGGGG-3 '(SEQ ID NO: 41),-For RAGE223,
5′-TTACTCGAGCTGTGCGCAAGGCCCG-3 ′ (SEQ ID NO: 42)
・ About RAGE226
5′-TTACTCGAGACTGGATGGGGGCTGTG-3 ′ (SEQ ID NO: 43)
For RAGE4, forward primer 5′-TCGCATATGGCAATGAACAGGAATGG-3 ′ (SEQ ID NO: 39) reverse primer 5′-TTACTCGAGAGCCTGCAGGTTGGCCCC-3 ′ (SEQ ID NO: 40)
DNA encoding RAGE2 (amino acid sequence 23 to 250 of natural RAGE), DNA encoding RAGE3 (amino acid sequence 23 to 230 of natural RAGE), RAGE4 (amino acid sequence 101 to natural RAGE) 332), a DNA encoding RAGE7 (amino acid sequence 23 to 137 of natural RAGE), a DNA encoding RAGE8 (amino acid sequence 23 to 120 of natural RAGE), and RAGE9 (natural RAGE). DNA encoding amino acid sequence 23 to 112), DNA encoding RAGE143 (amino acid sequence 23 to 143 of natural RAGE), DNA encoding RAGE223 (amino acid sequence 23 to 223 of natural RAGE), and RAGE226 (Natural RAGE To obtain a DNA encoding the amino acid sequence 23 to 226 position).

  These miniRAGE-encoding DNAs were amplified by PCR using specific primers to which a restriction enzyme recognition sequence was added, and then TA cloning was performed using pCR2.1 (Invitrogen) as a cloning vector to confirm the nucleotide sequence.

(2) Generation of tag sequence-added RAGE-like polypeptide RAGE-like polypeptides (sRAGE1, mRAGE1, RAGE2-RAGE8, RAGE143, RAGE223, and RAGE226) generated in the above production example are effective when used in a detection / evaluation system. It is desirable that such modification is applied. Therefore, two types of tags (one sequence AviTag (GLNDIFEAQKIEWHE (SEQ ID NO: 29)) that undergoes biotinylation in E. coli and the other are Streptag (AWRHPQFGGG) that is a sequence directly recognized by streptavidin. (SEQ ID NO: 30) or WSHPQFEK (SEQ ID NO: 31)) The binding of biotin and streptavidin is the strongest non-covalent bond (KD = 10 ⁻¹⁵ M), and development to immobilization via streptavidin, etc. However, in order to biotinylate, it takes time and effort to add biotin to the medium and to co-express biotin ligase, etc. Therefore, the binding strength is inferior (KD = ^10-7 M), adding a tag on the gene sequence and expressing it in E. coli A StreptagII (WSHPQFEK (SEQ ID NO: 31))-added protein capable of binding to toavidin was also generated, and in each case, a nucleic acid sequence encoding a tag sequence was introduced into the 3 ′ side of the pET-based vector to obtain an expression plasmid ( pET C-Avi, pET C-StII) According to a conventional method, mRAGE and miniRAGE were introduced into these expression vectors to generate an expression construct.For biotinylation of AviTag addition protein, the same bacteria as the pET system vector BirA / pAC was prepared by introducing a BirA gene encoding biotin ligase into a pAC vector that can be maintained in the body.

  In RAGE, as with CTLD, the formation of an SS bond is essential for structural stabilization and function. Therefore, OrigamiB (DE3) (Novagen, USA) was used as an expression host. Origami B (DE3) is a thioredoxin reductase and glutathione reductase mutant and has the property of promoting S—S bond formation of proteins expressed in the cytoplasm. When AviTag is added to a RAGE-like polypeptide (sRAGE1, mRAGE1, RAGE2-RAGE8, RAGE143, RAGE223, and RAGE226 are collectively referred to as “RAGE-like polypeptide”), a vector containing a nucleic acid sequence encoding AviTag and the polypeptide And an expression vector containing the BirA gene. The resulting transformed host cells were added with kanamycin (15 μg / ml), ampicillin (50 μg / ml), tetracycline (12.5 μg / ml), chloramphenicol (34 μg / ml) (all at the final concentration). The cells were cultured at 37 ° C. in LB medium. When transformed with an expression vector containing StreptagII-encoding nucleic acid, it was cultured at 37 ° C. in LB medium supplemented with kanamycin (15 μg / ml), ampicillin (50 μg / ml), and tetracycline (12.5 μg / ml). RAGE-like polypeptide expression was induced by adding IPTG.

  As a preliminary experiment, the temperature conditions and induction time for expressing a RAGE-like polypeptide as a soluble polypeptide were examined. The temperature conditions were set to (1) 37 ° C, (2) 25 ° C and (3) 20 ° C. The induction time was set to (A) 18 hours, (B) 20 hours, and (C) 24 hours. First, when these host cells were induced at 37 ° C. and the molecular weight of the induced protein was measured, most of them were recovered in the insoluble fraction. When the culture temperature was lowered to 25 ° C., 90% or more was expressed as a soluble polypeptide in the case of sRAGE, but only about 20% was expressed as a soluble polypeptide in the case of mRAGE1. For each miniRAGE, a tendency was observed that the solubilization rate decreased as the deficient portion became longer. RAGE8 and RAGE9 were almost insoluble even when the culture temperature was lowered, and it was difficult to express them as soluble polypeptides. In view of the above results, when the induction time was examined while maintaining the culture temperature at 25 ° C., it was found that the induction time (A) of 18 hours was appropriate.

  The above transformed cells were pre-cultured at 37 ° C. for 12 hours. In the main culture, this pre-cultured solution was added to each of the LB media so as to be diluted 20-fold, and swirled at 25 ° C. Based on the conditions obtained above, when the optical density (A600) of the culture reached about 0.5, 1 mM (final concentration) IPTG was added to induce expression of the target protein. When the AviTag-added protein was produced, 50 μM (final concentration) of biotin was added simultaneously with the start of induction, and the target protein to be expressed was simultaneously biotinylated. 18 hours after the start of induction, the cells were pelleted by centrifugation at 12,000 rpm × 5 minutes, the supernatant was discarded, and washed with an appropriate amount of TBS.

  The bacterial cells suspended in TBS were crushed with an ultrasonic crusher, and the supernatant recovered by centrifugation at 15,000 rpm × 30 minutes was used as the soluble fraction. Since the His protein for purification was added to the C-terminal side, the target protein was purified by eluting with imidazole after binding to Ni-agarose. AviTag added for detection can also be used for purification. In this case, it was adsorbed on streptavidin Mutein matrix (Roche) and then eluted with biotin. In the case of StrepTag II, it was adsorbed on Strep-Tactin Sepharose (IBA, US) and then eluted with dessiobiotin.

(3) Determination of minimum region essential for ligand recognition ability of RAGE The expression efficiency of each MiniRAGE eluted as described above, the ratio expressed as a soluble polypeptide, the yield, etc. are as shown in Table 2 below. .

  From the above results, it was found that the minimum region essential for the ligand recognition ability of RAGE is RAGE8 (amino acid sequence 23 to 120 of natural RAGE).

(Production Example 5: Preparation method of LOX-1 extracellular region)
In this production example, a preparation example of the LOX-1 extracellular region is shown. CTLD14 and CTLD were produced in the same manner.

  The expression host was BL21 (DE3), and after expressing nearly 100% as inclusion bodies, it was prepared by the following two methods.

1) Refolding with cycloamylose (see Non-Patent Document 7)
BL21 (DE3) transformed simultaneously with an expression construct and a vector containing the BirA gene, or BL21 (DE3) transformed with an expression construct was ampicillin (50 μg / ml), chloramphenicol (34 μg / ml, expression) It was cultured at 37 ° C. in LB medium containing (not added in the case of construct alone) (both final concentrations). When the turbidity (A600) of the culture reached about 0.5, 1 mM (final concentration) IPTG was added to induce expression of the target protein. When the target protein was biotinylated, 50 μM (final concentration) of biotin was added simultaneously with the start of induction, and biotinylation was performed simultaneously. Four hours after the start of induction, the cells were collected by centrifugation at 12,000 rpm × 5 minutes, and washed with TBS.

  Subsequently, the bacterial cells suspended in TBS were crushed with an ultrasonic crusher, and the precipitate fraction of the cell lysate was washed with TBS to obtain inclusion bodies. The inclusion bodies were treated with 6M guanidine hydrochloride solution containing DTT at a final concentration of 40 mM for 1 hour at room temperature, followed by addition of 70 volumes of 0.1% CTAB (containing 2 mM DL-cystine) / TBS solution, After reacting at room temperature for 1 hour, a 3% CA solution was added to a final concentration of 0.6%, and the reaction was further allowed to proceed at room temperature for 1 hour. The reaction solution was centrifuged at 15,000 rpm for 5 minutes, and the resulting supernatant was used as a refolding solution. Purification was by Ni-agarose purification using a His tag.

2) Refolding and purification by dilution dialysis (see Vohra RS et al. (2007) Protein Expr. Purif. 52: 415-421)
1) The cells obtained in the same manner were suspended in a lysis buffer (1 mg / ml lysozyme containing 10 mM Tris, pH 7.8, protease inhibitor cocktail), reacted at 4 ° C. for 30 minutes, Centrifuge for 15 minutes at 10,000 g. The obtained precipitate (inclusion body) was refolded according to the method of Vorah et al.

Add 100 mg of inclusion body to 2.2. It was suspended in ml of buffer A (Buffer A) (10 mM Tris-HCl, pH 7.8, 6M guanidine hydrochloride, 100 mM NaH ₂ PO ₄ ) and reacted at 4 ° C. for 2 hours. The supernatant obtained by centrifugation at 100,000 × g for 30 minutes is used as a solubilized inclusion body solution, bound to COSMOGEl His-Accept (Nacalai) using His tag, and an imidazole concentration gradient of 10-100 mM by FPLC (GE Healthcare) Was eluted with. The protein peak fraction was dialyzed against 50 mM Tris (pH 8.0) containing 6 M guanidine hydrochloride. After adding 100 mM DTT at a final concentration to the dialysis solution, the protein concentration was adjusted to 1 mg / ml. Subsequently, dialysis was repeated for 14 hours each with a buffer solution in which the guanidine hydrochloride concentration was gradually reduced to obtain a refolded purified protein.

(Production Example 6: RAGE preparation method)
RAGE-like polypeptides (RAGE-related proteins) corresponding to all RAGE molecules were prepared by the following two methods as described in this Production Example.

1) Method of preparing and refolding as an inclusion body The same method as in Production Example 5 was used. Since the expression construct is different from that in Production Example 5, only the antibiotics added at the time of culturing are changed to ampicillin (50 μg / ml), tetracycline (12.5 μg / ml), and chloramphenicol (34 μg / ml). Used.

2) Technique for expressing as a soluble protein (see Non-Patent Document 7)
Based on the information described in Production Example 4, Origami B (DE3), which was simultaneously transformed with the expression construct and the vector containing the BirA gene, was treated with ampicillin (50 μg / ml), tetracycline (12.5 μg / ml), chloram The cells were cultured at 25 ° C. in LB medium containing phenicol (34 μg / ml) (both final concentrations). When the turbidity (A600) of the culture solution reached about 0.5, 1 mM (final concentration) IPTG and 50 μM (final concentration) biotin were added, and biotinylation was performed simultaneously with the induction of the target protein. 18 hours after the start of induction, the cells were collected by centrifugation at 12,000 rpm × 5 minutes, and washed with TBS. The bacterial cells were suspended in TBS, crushed with an ultrasonic crusher, and centrifuged at 15,000 rpm × 30 minutes, and the collected supernatant was used as a soluble fraction. The target protein was bound to Ni-agarose by a C-terminal His tag and then eluted with imidazole. The peak fraction containing the target protein is dialyzed with TBS, and after binding to Streptavidin Mutein matrix (Roche) with biotin added to the N-terminal, the target protein obtained by elution with biotin is purified. RAGE.

(Production Example 7: Preparation of oxidized LDL)
In this production example, oxidized LDL was prepared (see Non-Patent Document 7).

In preparation, everything possible was sterilized and operated as aseptically as possible. LDL prepared from human plasma (EDTA was removed by dialysis) was adjusted to 1 mg / ml with PBS (−), and CuSO ₄ was added to a final concentration of 5 μM. The solution was then reacted at 37 ° C. for 20 hours, and 1 mM EDTA (final concentration) was added to stop the oxidation reaction. This solution was dialyzed against Tris-EDTA (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% EDTA), and after sterile filtration, 0.02% NaN ₃ (final concentration) was added to oxidize. An LDL solution was obtained.

(Production Example 8: Method for preparing glycated bovine serum albumin (BSA) which is an AGE molecule)
In this production example, various glycated BSAs were prepared as AGE molecules (see Non-Patent Document 7).

  Glycated BSA was prepared as follows. All that was possible during the preparation was sterilized and manipulated as aseptically as possible.

  Each sugar (4.51 g of fructose, 4.51 g of fructose, and 3.75 g of ribose) was weighed using a spatula washed with ethanol and placed in a cap-capped plastic tube that had been γ-sterilized. 20 ml of sterile 1M phosphate buffer (pH 7.4) was aseptically added to each tube, and gently mixed by inversion to mix the solution. Finally, 21 ml of endotoxin-free distilled water was added, and the mixture was aseptically filtered through a 0.22 μm filter, and then allowed to react at 37 ° C., protected from light for 12 weeks. Once a week, 80 μl of the sugar-BSA preparation was removed under aseptic conditions, the pH was confirmed, and the pH of the reaction solution was adjusted to pH 7.4 with a 10N NaOH solution (endotoxin free). After 12 weeks, dialysis was performed with sterile PBS to completely remove sugar, and the resulting reaction solution was saccharified BSA (depending on the reacted sugar, ribose-BSA, glucose-BSA, fructose-BSA) (in this specification, These are referred to as ribose-modified AGE (also referred to as ribose AGE or R-AGE), glucose-modified AGE (also referred to as glucose AGE or G-AGE), and fructose-modified AGE (also referred to as fructose-AGE or F-AGE), respectively. And aseptically stored at 4 ° C.

Example 1
In this example, using a 96-well plate on which the receptor RAGE was immobilized, ribose-modified AGE (R-AGE) was labeled as a ligand to confirm whether the assay of the present invention was functional.

(1) Measurement of fluorescent plate reader A 96-well plate (manufactured by Nuku) on which a receptor (RAGE produced in Production Example 6) was immobilized was prepared.

  The 96-well plate was blocked overnight at 4 ° C. using protein-free blotting buffer (Therm Scientific).

  Molecules for activity measurement (quercetin made by Sigma, OxLDL, G-AGE produced as in the above production example (prepared as in Production Example 8), R-AGE (prepared as in Production Example 8)) Was added at 50 μl / well at doses of 0 μg / assay (100% control), 0.05 μg / assay, 0.5 μg / assay and 5 μg / assay. Reaction by adding fluorescently labeled ligand (water-soluble dye Alexa546 (Moleclar Probes) labeled R-AGE (prepared as in Production Example 8)) in a volume of 50 μl / well followed by incubation at 37 ° C. for 60 minutes. Made progress. Subsequently, washing with a buffer solution (Tris buffered saline (TBS) (Tris / Tris-HCl 10 mM; NaCl 150 mM) was performed 5 minutes × 2 times, 10 minutes × 3 times, and 100 μl of buffer solution (TBS) was added. Measurement was performed with a fluorescent plate reader (Wallac ARVO SX, manufactured by Perkin Elmer).

  As a control, a similar experiment was performed using ribose-modified AGE (R-AGE) (prepared in Production Example 8) labeled with a water-soluble dye Alexa546 (Molecular Probes) as a fluorescently labeled model ligand. Quercetin (Quercetin; manufactured by Sigma) is a flavonoid that has been reported to be effective in preventing the onset of diabetes, and was used as a model for RAGE inhibitors.

(result)
The results are shown in Table 3 below, and the plots are shown in FIGS. 1 to 4 (quercetin, OxLDL, G-AGE and R-AGE, respectively).

  FIG. 5 shows a control experiment conducted using ribose-modified AGE (R-AGE) labeled with the water-soluble dye Alexa546 as a fluorescently labeled model ligand. The left phase of FIG. 5 shows the phase difference image of the cells used, the center of FIG. 5 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 5 shows the cell image with the ligand bound. The white dots on the right side of FIG.

  FIG. 6 shows a photograph in the case of adding quercetin in the same order as FIG. 6 shows the phase difference image of the used cell, the center of FIG. 6 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 6 shows a cell image in which quercetin is added in addition to the ligand. Indicates. As shown in the right of FIG. 6, it was confirmed by high-sensitivity observation in the cells that the binding of the ligand was inhibited by the addition of quercetin.

  FIG. 7 shows a photograph in the case of adding R-AGE in the same order as in FIG. The left phase of FIG. 7 shows the phase difference image of the cells used, the center of FIG. 7 shows the fluorescence of cells expressing RAGE (forced expression as a fusion protein with a fluorescent protein), and the right side of FIG. 7 added R-AGE in addition to the ligand. A cell image is shown. As shown in FIG. 7 right, a fluorescence image showing inhibition of ligand binding by addition of R-AGE is shown.

(Quantitative assay)
Next, using a glucose-modified AGE (G-AGE) (produced as described in Production Example 8) immobilization plate, Lys-AGE (glyceraldehyde-modified lysine: low molecular AGE model) (0 0.09 g of lysine was prepared by dissolving 0.45 g of DL glyceraldehyde in 50 ml of 0.2 M phosphate buffer (pH 7.4) and reacting at 37 ° C. for 1 week under light-shielded conditions.) Quantitative assays were performed using ribose-modified AGE (R-AGE) (prepared as described in Preparative Example 8) in amounts of 0, 0.25 μg / assay and 2.5 μg / assay, respectively. .

  The results are shown in Table 4.

(Example 2)
In this example, whether or not the assay of the present invention functions was confirmed using R-AGE or G-AGE immobilized 96-well plates as AGE molecules.

  As an AGE molecule, glucose-modified AGE (G-AGE) (produced as described in Production Example 8) or ribose-modified AGE (R-AGE) (produced as described in Production Example 8) A 96-well plate (manufactured by Nuku) was prepared.

  The 96-well plate was blocked overnight at 4 ° C. using protein-free blotting buffer (Therm Scientific).

  Molecules for activity measurement (Lys-AGE (glutaraldehyde-modified lysine-modified AGE: low molecular weight AGE model)) (0.09 g of lysine, 0.45 g of DL glyceraldehyde, 50 ml of 0.2 M phosphate buffer (pH 7.4) And glucose-modified AGE (G-AGE) or ribose-modified AGE (R-AGE) at 50 μl / well, 0 μg / assay. (100% control), 0.25 μg / assay and 2.5 μg / assay were added.

  The reaction was allowed to proceed by adding biotin-labeled RAGE 143 (prepared in Production Example 6) in a volume of 50 μl / well followed by incubation at 37 ° C. for 60 minutes. Next, washing with a buffer solution (TBS) was performed 5 minutes × 2 times, 10 minutes × 3 times. Horseradish peroxidase (HRP) -labeled streptavidin was added to the wells (100 μl / well) and reacted at room temperature for 60 minutes. Thereafter, it was washed with a buffer solution (TBS). Thereafter, 100 μl / well of chromogenic substrate 3,3 ′, 5,5′-tetramethylbenzidine (TMB) was added. This was allowed to react at room temperature for approximately 10 minutes, and the reaction was stopped by adding 100 μl / well of 1N hydrochloric acid (HCl). Thereafter, the absorbance at 450 nm was measured.

  The results are shown in Table 5 below. In any case, it is understood that the binding is quantitatively inhibited.

(Example 3)
In this example, the assay was performed using a model in which a LOX-1 receptor was immobilized and a model that had been competed with the ligand, and acetylated LDL labeled with a lipid-soluble dye DiI as a fluorescent-labeled model ligand. I confirmed that it works.

  A 96-well plate (manufactured by NUNK) on which LOX-1 receptor (prepared in the same manner as in Production Example 5) was immobilized was prepared.

  This 96-well plate was blocked overnight at 4 ° C. using blocking reagent N102 (manufactured by NOF Corporation).

  Molecules to be measured for activity (quercetin from Sigma, OxLDL and AcLDL produced as in the above production example) in a volume of 50 μl / well (final amounts used were 0, 0.05 μg / assay, 0.5 μg / assay) 5 μg / assay). Quercetin (Quercetin; manufactured by Sigma) is a flavonoid that has been reported to suppress cell damage caused by OxLDL, and was used as a model for LOX-1 inhibitors.

  Acetylated LDL (acetylated LDL (manufactured by Molec μlar Probe)) labeled with a fat-soluble dye DiI which is a fluorescently labeled ligand is dialyzed with PBS (−), and after sterile filtration, 10 μl of DiI / DMSO solution per 1 ml (1 mg / ml) (300 mg / ml) was added aseptically and allowed to react for 18 hours under light-shielded conditions at 37 ° C. After 0.0834 g of KBr was added and dissolved per ml of the reaction solution, 105,000 g at 12 ° C. The sample was subjected to ultracentrifugation for 20 hours, the uppermost layer fraction was collected, and DiI-labeled AcLDL was added at a volume of 50 μl / well, and the reaction was allowed to proceed by incubating at 37 ° C. for 60 minutes. It was. Next, washing with a buffer solution (TBS) was performed 5 minutes × 2 times, 10 minutes × 3 times. 100 μl of buffer solution (TBS) was added and measurement was performed with a fluorescent plate reader (Wallac ARVO SX, manufactured by Perkin Elmer).

  The results are shown in Table 6. The plots are shown in FIGS. 8-10 (quercetin, OxLDL, AcLDL, respectively). 8 to 10, in each of the drawings, the vertical axis represents relative binding activity (%, the value of absorbance at the addition amount of 0 is 100%). From the left, relative binding activities are shown using 0, 0.05 μg / assay, 0.5 μg / assay, and 5 μg / assay, respectively.

Example 4
In this example, it was confirmed whether OxLDL was immobilized as an example of denatured LDL and CTLD14 was labeled as a CTLD molecule to confirm whether the present invention functions.

  A 96-well plate (Nunk) having OxLDL immobilized thereon was prepared.

  This 96-well plate was blocked overnight at 4 ° C. using blocking reagent N102 (manufactured by NOF Corporation).

A molecule for activity measurement (OxLDL produced in Production Example 5, quercetin manufactured by Sigma) in a volume of 50 μl / well (final amount used was 0, 0.05 μg / assay, 0.5 μm).
g / assay, 5 μg / assay. ).

  Biotin-labeled CTLD14 (prepared in Production Example 6) was added in a volume of 50 μl / well, and then the reaction was allowed to proceed by incubation at 37 ° C. for 60 minutes.

  Next, washing with a buffer solution (TBS) was performed 5 minutes × 2 times, 10 minutes × 3 times.

  Horseradish peroxidase (HRP) -labeled streptavidin was added to the wells (100 μl / well) and allowed to react at room temperature for 60 minutes. Thereafter, it was washed with a buffer solution (TBS). Thereafter, 100 μl / well of chromogenic substrate 3,3 ′, 5,5′-tetramethylbenzidine (TMB) was added. This was allowed to react at room temperature for approximately 10 minutes, and the reaction was stopped by adding 100 μl / well of 1N hydrochloric acid (HCl). Thereafter, the absorbance at 450 nm was measured.

  The results are shown in Table 7 below, and the plots are shown in FIGS. 11 to 12 (OxLDL and quercetin, respectively). In any case, it is understood that the binding is quantitatively inhibited.

(Example 5)
In this example, it was confirmed whether CTLD14 was immobilized as a CTLD molecule and OxLDL was labeled as an example of denatured LDL to confirm that the present invention functions.

  A 96-well plate (15 μg / ml, 50 μl / well) (manufactured by Nuku) on which CTLD14 prepared as in Production Example 5 was immobilized was prepared.

  This 96-well plate was blocked overnight at 4 ° C. using blocking reagent N102 (manufactured by NOF Corporation).

  Quercetin, which is a molecule for measuring activity, and rutin (manufactured by Sigma), a glycoside of quercetin, were added at a dose of 50 μl / well.

  The reaction was then allowed to proceed by adding OxLDL at a dose of 50 μl / well followed by incubation at 37 ° C. for 60 minutes. Next, washing with a buffer solution (TBS) was performed 5 minutes × 2 times, 10 minutes × 3 times.

  Horseradish peroxidase (HRP) -labeled anti-ApoB was added to the well at 100 μl / well and allowed to react at 37 ° C. for 60 minutes. Thereafter, it was washed with a buffer solution (TBS) (5 minutes × 2, 10 minutes × 3). After washing with a buffer again, 100 μl / well of chromogenic substrate 3,3 ′, 5,5′-tetramethylbenzidine (TMB) was added. This was allowed to react at room temperature for approximately 10 minutes, and the reaction was stopped by adding 100 μl / well of 1N hydrochloric acid (HCl). Thereafter, the absorbance at 450 nm was measured.

  The results are shown in Table 8 below, and the plot for quercetin is shown in FIG. It is understood that quercetin is quantitatively inhibited from binding while its glycoside rutin is not.

  FIG. 14 shows a control experiment using cells in which LOX-1 was forcibly expressed as a fusion protein with a fluorescent protein and OxLDL as a control ligand. FIG. 14 shows the phase contrast image of the cell used on the left, FIG. 14 shows the fluorescence of LOX-1 (forced expression as a fusion protein with a fluorescent protein) expressing cell in the center of FIG. Show. A white dot on the right side of FIG. 14 is a bound ligand.

  FIG. 15 shows a photograph when quercetin is added in the same order as in FIG. FIG. 15 shows the phase contrast image of the cells used on the left, FIG. 15 shows the fluorescence of the cells expressing LOX-1 (forced expression as a fusion protein with a fluorescent protein) in the center, and FIG. 15 shows the addition of quercetin in addition to the ligand. A cell image is shown. As shown in the right of FIG. 15, it was confirmed by high-sensitivity observation in cells that the binding of ligand was inhibited by the addition of quercetin.

  In the case of rutin, inhibition of binding on the cells was not detected, which was consistent with the results on the microplate.

(Example 6: Diagnosis or predictive diagnosis of diabetes or diabetic nephropathy)
In this example, it was confirmed whether the assay of the present invention can be used to diagnose or predict diabetes or diabetic nephropathy.

  In this example, a model animal for diabetes or diabetic nephropathy is prepared, blood is collected from the model animal, and values such as blood glucose level and other parameters for diabetes and diabetic nephropathy (for example, one of the symptoms of nephropathy). And the AGEs-like activity developed in the present invention were measured and correlated to determine whether diabetes or diabetic nephropathy can be diagnosed or predicted. Details are as follows.

  First, as a model animal for diabetes or diabetic nephropathy, a model to which streptozotocin (STZ) was administered was prepared (Am J Physiol Renal Physiol (2006) 290, F214-F222, J Endocrinology (1999) 162, 189-195). . Blood was collected at an interval of about one week with the administration day as zero day, and blood glucose level, increased water consumption, which is one of the symptoms of nephropathy, and AGEs-like activity were measured.

  AGE activity was measured by the same method as in Example 2.

(result)
The measurement results of blood glucose level and AGE activity are shown in Tables 9 to 11 below. The measured value (%) was displayed with the measured value when the molecule showing AGEs activity was zero as 100%. Therefore, the displayed AGE-like activity is evaluated by the inhibition rate (decrease in measured value%).

A summary of the above results is shown in Table 12 below.

  Therefore, an increase in blood glucose level, which is one of the indicators of diabetes, was observed on the 16th day. At this point, neither an increase in AGEs-like activity (ie, a decrease in the inhibition rate (measured value%)) nor nephropathy symptoms (including an increase in water consumption) was observed. On the 22nd day when an increase in blood glucose level, which is one of the indicators of diabetes, was observed, an increase in AGE-like activity began to be observed. No symptoms of nephropathy (including increased water consumption) were observed at the 22nd day.

  From around day 26, an increase in water consumption, one of the symptoms of nephropathy, began to be observed.

  Thus, it is understood that an increase in AGE-like activity can be used to diagnose diabetes and can be used for preliminary diagnosis and diagnosis of diabetic nephropathy.

(Example 7: Inhibition of ligand binding to LOX-1 (CTLD14) using LDL)
In this example, it is demonstrated that the binding inhibition of the ligand to LOX-1 (CTLD14) can be confirmed for the AcLDL used in the above examples. It is also demonstrated that confirmation can be obtained for LDL, not serum.

(Method)
Mice: C57BL / 6 male mice were purchased from Charles River Laboratory Japan (Yokohama, Japan) at the age of 4 weeks, and sold to SPF (Specific Pathogen Free) animal facilities within the National Institute of Agricultural Research and Food Research Institute. And those that reached 5 weeks of age were used. Six mice were given AIN-93M (oriental yeast) as a control group, and 16 mice were fed a high cholesterol Clinton-Siblesky rodent diet (15.8% fat, 1.25% cholesterol) as a high cholesterol diet. And 0.5% sodium cholate (D12109C, Research Diet Inc., New Brunswkc, NJ, USA) for 16 weeks. Guidelines for animal experiments, etc. of the National Research Institute for Agricultural and Food Sciences, and the National Institute for Agricultural Sciences for Food and Food Technology, based on the `` Standards for Use and Storage and Pain Reduction '' To the animal experiment committee of the research institute Are those that have undergone screening was approved that.

(Serum lipid measurement)
Mice were sacrificed by whole blood collection under isoflurane light anesthesia. The total cholesterol level (FIG. 16A), HDL cholesterol level (FIG. 16B), and LDL cholesterol level (FIG. 16C) of each mouse were measured by a colorimetric method (Wako Pure Chemicals, Osaka, Japan). LDL was separated from the serum of a high cholesterol diet and normal diet mice by continuous ultracentrifugation, and then dialyzed against PBS containing 0.03 mM EDTA.

(Competitive binding assay using microplate)
50 μl of refolded CTLD14 solution (containing 15 μg / ml of CTLD14) was added to each well of a 96-well plate and allowed to react for 14 hours at 4 ° C. After removing the protein solution, the plate was washed with 200 μl of TBS, and subsequently 200 μl of blocking solution LN102 (NOF Corporation) was added, followed by reaction at 4 ° C. for 14 hours to complete blocking. 50 μl of sample (serum diluted with TBS, or LDL) was added to each well, followed by addition of 50 μl of DiI-labeled DiD-AcLDL and allowed to react at 37 ° C. for 2 hours. After removing the reaction solution and washing each well 5 times with 200 μl of TBS, 100 μl of TBS was added, and fluorescence intensity (excitation wavelength: 520 nm) was measured with a fluorescence plate reader (Wallac Arvo SX, Perkin Elmer, Waltham MA, USA). , Fluorescence wavelength: 550 nm). As a result, the value obtained from three independent measurements was expressed as a relative value (%) with the fluorescence intensity obtained from a well without a sample as 100% (FIG. 17, A: serum, B: LDL). .

(Enzyme-linked immunosorbent assay)
100 μl of LDL (1 to 100 μg / ml protein amount) prepared from a normal diet or a high cholesterol diet group was added to each well of a 96-well plate and reacted at 4 ° C. for 14 hours. After removing the LDL solution, each well was washed with 200 μl of TBS. Subsequently, 200 μl of blocking solution LN102 (NOF Corporation) was added and reacted at 37 ° C. for 2 hours for blocking. Anti-HNE-histidine monoclonal antibody (Nippon Yushi, Tokyo, Japan) 100 μl (containing 0.5 μg / ml antibody) was added to each well and reacted at 37 ° C. for 2 hours. After removing the antibody solution, the plate was washed 5 times with TBS, 100 μl of horseradish peroxidase (HRP) -labeled anti-mouse antibody (Bio Rad) was added, reacted at 37 ° C. for 1 hour, and then washed 5 times with TBS. . 100 μl of the substrate TMB solution was added to each well, the color was developed at room temperature, the reaction was stopped by adding 100 μl of 1N HCl, and then the absorbance at 450 nm was measured with a microplate reader (Wallac Arvo SX, Perkin Elmer). The results are shown as an average of three independent experiments (FIG. 18, B-2).

(Example 8: Serum (plasma) glucose level as an indicator of diabetes progression)
As shown in Example 6, an increase in blood glucose level as an index for the onset of diabetes was already observed on the 16th day, and an increase in AGE-like activity was observed on the 22nd day. However, no symptoms of nephropathy (such as increased water consumption) were observed, indicating that AGEs-like activity increased before nephropathy symptoms. Thereafter, the patient's progress was observed (the amount of water consumed increased slightly from around day 26), and blood was collected on day 71. The concentration of creatinine, a nephropathy marker, was measured to confirm that nephropathy had developed. did. At the same time, the glucose concentration in the serum was also measured by a precise measurement method. Here, the main focus of blood collection on day 71 is measurement of the amount of creatinine, and it is described in Example 6 that the blood glucose concentration, which is an index of the onset of diabetes, has been significantly higher than before.

  In this example, up to Example 6, the glucose concentration was simply measured with a blood glucose meter without directly quantifying it, but as a final confirmation, it was strictly measured with a glucose measurement kit. The strictness of the method of the present invention was confirmed.

  More specifically, in this example, whole blood was collected on day 71 and glucose CII-Test Wako was used to accurately measure the glucose concentration in the serum, and the calibration curve was determined by the manufacturer's instructions. Based on the above, measurements were made.

(Mouse used)
The mouse used until Example 6 (Stz-treated BALB / c mouse) was used.

(Measurement method and results)
The coloring reagent was prepared by dissolving one bottle of coloring agent (for 150 ml) in a buffer solution (150 ml). 2 μl each of serum and standard solution (blank is distilled water) was placed in a 96-well microplate (measured in 3 wells for the same sample), and then 300 μl of a coloring reagent was added to each well and mixed well. After 5 minutes at 37 ° C., the absorbance at 505 nm and 600 nm was measured, and the difference between the two was taken as the measured value. A calibration curve was created from the standard solution, and the glucose concentration in the sample was determined from the calibration curve. In FIG. 19, the results of plotting these measurement results on the horizontal axis and the measurement values obtained by a conventional blood glucose meter on the vertical axis are shown. In this figure, the correlation between the blood glucose level and the glucose measurement kit is shown. Also, when the glucose concentration in serum is accurately measured, the results in the control group and the streptozotocin administration group are shown in FIG.

  From these results, a calibration curve with standard serum was prepared, and when the glucose concentration in the serum of each mouse was measured, there was a correlation between the measured value by the glucose kit and the measured value by the blood glucose meter (FIG. 19), Even when the glucose concentration in the serum was accurately measured, a significant difference was naturally recognized between the control group and the streptozotocin-administered group (FIG. 20), but simple measurement using a blood glucose meter that had quantified changes over time. But it was confirmed that there was no problem.

(Example 9: Creatinine amount in serum (plasma) as an indicator of the progression of nephropathy)
In this example, AGEs-like activity was already detected on the 22nd day, but thereafter, for the purpose of clarifying whether nephropathy was reached, serum creatinine concentration was measured at the time of whole blood collection. The measurement was performed using a lab assay creatinine ^TM (Jaffe method; Wako Pure Chemical Industries). At the time of measurement, a standard curve with standard serum was prepared based on the manufacturer's instructions, and the creatinine concentration of the sample was determined.

(Mouse used)
The mouse used until Example 6 (Stz-treated BALB / c mouse) was used.

(Measurement method and results)
A 50 μl sample was mixed with 300 μl of a protein removal reagent, allowed to stand at room temperature for 10 minutes, and then centrifuged at 2,500 rpm for 10 minutes. A deproteinized sample and 100 μl of a standard solution for preparing a calibration curve were added to a 96-well microplate (3 wells for the same sample). As a blank (zero standard solution), 100 μl of distilled water was added to the well. Subsequently, 50 μl of picric acid was added, and further 50 μl of 0.75 mol / l sodium hydroxide solution was added, mixed well, and allowed to stand at 25-30 ° C. for 20 minutes. Thereafter, the absorbance at 520 nm was measured within 30 minutes. FIG. 21 shows the correlation between serum glucose concentration and creatine concentration for each individual. In the control group, the glucose concentration is 200 mg / dl or less and the creatine concentration is in the range of 0.4 to 0.7 mg / dl, whereas in the Stz group, the glucose concentration exceeds 400 mg / dl. Among the individuals with high concentrations, there were many individuals in which the amount of creatinine also showed a value of 0.7 mg / dl or more (FIG. 21). Furthermore, the average value of the creatine concentration in the control group and the Stz group was significantly higher in the Stz group (FIG. 22). From this, it is considered that nephropathy also developed following the onset of diabetes in the Stz group. However, on the 22nd day when AGE-like activity was detected, an increase in the amount of drinking water was measured as the simplest index for the onset of nephropathy, and the onset of nephropathy could not be clearly captured.

  Therefore, AGEs-like activity was already detected on the 22nd day, but it was subsequently demonstrated that nephropathy was reached.

(Example 10: Summary of time-series data)
In addition to the previous examples, in addition to the previous data (day 0, day 16 and day 22), blood glucose level or blood glucose concentration, samples of day 57 and day 71 were the same. The data collected and collected in time series are shown in FIG. 23 to FIG. 27 (the data presented in this example is therefore partially duplicated in the examples 1 to 9). To do.)

  Specifically, FIGS. 23 to 27 show “Alexa546-labeled R-AGE binding inhibitory activity to RAGE-immobilized plate” of serum derived from control mice and mice in which diabetes was induced by administration of streptozotocin (Stz), and “ The blood glucose level or blood glucose concentration "was measured over time, and was a graph showing the correlation for each individual, on the 0th day (Fig. 23), the 16th day (Fig. 24), and the 22nd day (Fig. 25). ), 57th day (FIG. 26), and 71st day (FIG. 27). The AGEs-like activity in the figure is a value expressed with a concentration at which the binding of Alexa546-labeled R-AGE is 100% inhibited as 100, and a concentration when there is no inhibition at all. The higher this value, the more the AGEs-like activity. Indicates that the content of the indicated molecule is high. The 0th day (see FIG. 23) is the start date of Stz administration. On the 16th day (FIG. 24), an increase in blood glucose level was observed in most individuals in the Stz-administered group, and it began to show a significantly higher blood glucose level than in the control group, and diabetes was already induced. Conceivable. On the other hand, an increase in AGEs-like activity was detected in several individuals, but the correlation between individuals showing a significant increase in blood glucose level and individuals showing an increase in AGEs-like activity was not clear. On the 22nd day (FIG. 25), the difference in blood glucose level between the Stz group and the control group becomes significant, the number of individuals having an AGEs-like activity value of 20 or more increases, and in individuals with high blood glucose levels, the AGEs-like activity is also high. AGEs-like activity has been detected in the onset group such as there is a tendency. It is considered that molecules exhibiting AGEs-like activity have been generated with the onset of diabetes. At this stage, no signs of diabetic nephropathy such as increased water consumption are observed, and it is considered that molecules showing AGE-like activity are captured before signs of nephropathy appear.

  In the serum collected on day 57 ((FIG. 26)), a high glucose concentration in the serum was detected in the Stz group, and AGEs-like activity was generally high, but the correlation with the glucose concentration was not high. Furthermore, the AGEs-like activity of the control group was also increased, and this blood collection result did not clearly show that molecules exhibiting AGEs-like activity were markedly accumulated as diabetes progressed.

  The Stz group on November 25, which is the 71st day, showed a high glucose concentration and a significant difference from the control group. Furthermore, AGEs-like activity was higher than that of the control group, and there were many individuals with high glucose concentration and AGEs-like activity. FIG. 28 shows the amount of creatinine in the serum on day 71, and the correlation with AGEs-like activity was observed. In contrast to the control group, the Stz group had many individuals with higher creatinine concentration and AGEs-like activity. Observed. Since creatinine is a marker of nephropathy, it can be said that nephropathy has been confirmed to develop as diabetes progresses in the Stz group. In addition, AGEs-like activity has already been detected from the 22nd day, and the result on the 57th day shows that the control value shows an abnormal value. It can be detected at an early stage, and it can be said that measurement of AGE-like activity can be a marker for detecting nephropathy onset at an early stage.

  As described above, changes in serum glucose concentration and AGEs-like activity in control mice and Stz mice were traced over time. Also, from day 0 to day 22, it was observed that there were a large number of individuals whose blood glucose level and AGE-like activity both increased in the Stz group, and that tendency was also observed on day 71. At the stage of the 22nd day, an increase in the amount of drinking water, which is a simple measure for the onset of nephropathy, was not observed, but at this point, an increase in AGE-like activity was already observed. Furthermore, on the 71st day, blood creatinine amount, which is an index of nephropathy onset, was also measured, but it was also confirmed that there were many individuals with high AGE activity and creatinine concentration in the Stz group.

  From the above, AGEs-like activity can be used for the diagnosis of diabetes or diabetic nephropathy, and is a preliminary diagnosis (prediction) of diabetic nephropathy, a preliminary diagnosis of diabetic nephropathy in diabetes ( Prediction) is also possible and proved to be useful as a diagnostic marker.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  CTLD-like polypeptide-surfactant binding complex and RAGE-like polypeptide-surfactant binding complex are useful for early diagnosis of diabetes, arteriosclerosis, diabetic vasculopathy, diabetic nephropathy, etc. It can be used in various fields such as evaluation, life improvement and evaluation of treatment effects by medication.

SEQ ID NO: 1: Nucleic acid sequence encoding PR-CTLD or CTLD SEQ ID NO: 2: Amino acid sequence of PR-CTLD or CTLD SEQ ID NO: 3: Nucleic acid sequence encoding PR-Lox-1 or Lox-1 SEQ ID NO: 4: PR- Lox-1 or Lox-1 amino acid sequence SEQ ID NO: 5: Nucleic acid sequence encoding PR-CTLD14 or CTLD14 SEQ ID NO: 6: PR-CTLD14 or CTLD14 amino acid sequence SEQ ID NO: 7: Lox-1 CTLD + neck or PR-CTLD + Nucleic acid sequence encoding SEQ ID NO: 8: Lox-1 CTLD + Neck or PR-CTLD + Neck amino acid sequence SEQ ID NO: 9: Nucleic acid sequence encoding RAGE or mRAGE SEQ ID NO: 10: RAGE or mRAGE amino acid sequence SEQ ID NO: 11 RAGE Or nucleic acid sequence encoding mRAGE8 SEQ ID NO: 12: amino acid sequence of RAGE8 or mRAGE8 SEQ ID NO: 13: nucleic acid sequence encoding RAGE1 or mRAGE1 SEQ ID NO: 14: amino acid sequence of RAGE1 or mRAGE1 SEQ ID NO: 15: nucleic acid encoding RAGE2 or mRAGE2 SEQ ID NO: 16: amino acid sequence of RAGE2 or mRAGE2 SEQ ID NO: 17: nucleic acid sequence encoding RAGE3 or mRAGE3 SEQ ID NO: 18: amino acid sequence of RAGE3 or mRAGE3 SEQ ID NO: 19: nucleic acid sequence encoding RAGE4 or mRAGE4 SEQ ID NO: 20: RAGE4 Or the amino acid sequence of mRAGE4 SEQ ID NO: 21: nucleic acid sequence encoding RAGE7 or mRAGE7 SEQ ID NO: 22: RAGE7 or mRA Amino acid sequence of E7 SEQ ID NO: 23: Nucleic acid sequence encoding RAGE143 or mRAGE143 SEQ ID NO: 24: Amino acid sequence of RAGE143 or mRAGE143 SEQ ID NO: 25: Nucleic acid sequence encoding RAGE223 or mRAGE223 SEQ ID NO: 26: Amino acid sequence SEQ ID NO: RAGE223 or mRAGE223 27: Nucleic acid sequence encoding RAGE226 or mRAGE226 SEQ ID NO: 28: Amino acid sequence of RAGE226 or mRAGE226 SEQ ID NO: 29: AviTag sequence GLNDIFEAQKIEWHE
SEQ ID NO: 30: Strepttag sequence AWRHPQFGG
Sequence number 31: StrepttagII arrangement | sequence WSHPQFEK
SEQ ID NO: 32: forward primer (common to RAGE1, RAGE2, RAGE3, RAGE7, RAGE8, RAGE9, RAGE143, RAGE223, and RAGE226) 5′-CTACATATGGCTCAAAACATCACAGC-3 ′
SEQ ID NO: 33: RAGE1 reverse primer 5′-TTACTCGAGAGCCTGCAGTTGGCCC-3 ′
SEQ ID NO: 34: RAGE2 reverse primer 5′-TTACTTCGAGACCAGACACGGGGCTG-3 ′
SEQ ID NO: 35: RAGE3 reverse primer 5′-TTACTTCGAGAAGCTACTGCTCCCACC-3 ′
SEQ ID NO: 36: RAGE7 reverse primer 5′-TTACTTCGAGAAACACCAGCCGTGAGT-3 ′
SEQ ID NO: 37: RAGE8 reverse primer 5′-TTACTCGAGAAATCTGGTAGACACGG-3 ′
SEQ ID NO: 38: RAGE9 reverse primer 5′-TTACTCGAGACTTGGTCTCCTTTC-3 ′
SEQ ID NO: 39: RAGE4 forward primer 5′-TCGCATATGGCAATGAACAGGAATGG-3 ′
SEQ ID NO: 40: RAGE4 reverse primer 5′-TTACTCGAGAGCCTGCAGTTGGCCC-3 ′
SEQ ID NO: 41: RAGE143 reverse primer 5′-TTACTCGAGTCCCCCACTTTATTGGGG-3 ′
SEQ ID NO: 42: AGE223 reverse primer 5′-TTACTCGAGCTGTCGCACAGCGCCG-3 ′
SEQ ID NO: 43: RAGE226 reverse primer 5′-TTACTCGAGACTGATGGGGGCTGTGC-3 ′

Claims (8)

A method for detecting or quantifying AGE molecules comprising:
(A) contacting a sample to be detected or quantified with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, wherein the RAGE molecule is RAGE143, RAGE1, RAGE223, RAGE226 or A process that is a variant thereof,
And (B) a step of detecting binding of the RAGE molecule for the AGE molecule, presence or level of inhibition of said binding indicates the presence or the level of presence of AGE content element in said sample, comprising the step, Method. A method for detecting diabetes or diabetic nephropathy,
(A) contacting a sample from a subject to be detected for diabetic nephropathy with a labeled AGE molecule in the presence of a RAGE molecule immobilized on a solid phase, the RAGE molecule comprising RAGE143 , RAGE1, RAGE223, RAGE226 or a variant thereof,
And (B) said a fixed detecting binding of said labeled AGE molecule to R AGE molecule, high Ikoto as compared levels of inhibition of said binding is normal individuals, said subject is diabetic Or a method comprising the step of indicating that the patient is suffering from diabetic nephropathy. The method according to claim 1, wherein the RAGE molecule is RAGE143. The labeled AGE molecules are labeled Lys-AGE (glutaraldehyde modified lysine modified AGE), labeled glucose modified AGE (G-AGE), labeled ribose modified AGE (R-AGE), labeled fructose modified AGE (F The method according to any one of claims 1 to 3, which is -AGE) or a modification thereof. A kit for the detection or quantification of AGE minute child,
(A) a solid phase having an immobilized RAGE molecule and a labeled AGE molecule; and (B) means for detecting the label of the labeled AGE molecule bound to the immobilized RAGE molecule, from the intensity of the label , binding of the RAGE molecule is detected for AGE molecule, comprising means, the RAGE molecule, RAGE143, RAGE1, RAGE223, a RAGE226 or a variant thereof, presence or level of inhibition of said binding, AGE content child A kit that indicates the presence or level of presence. A kit for the diagnosis of diabetes or diabetic nephropathy,
(A) a solid phase having an immobilized RAGE molecule and a labeled AGE molecule; and (B) means for detecting the label of the labeled AGE molecule bound to the immobilized RAGE molecule, from the intensity of the label Means for detecting the binding of the RAGE molecule to the AGE molecule, wherein the RAGE molecule is RAGE143, RAGE1, RAGE223, RAGE226 or a variant thereof, and the level of inhibition of the binding is higher than that in a normal individual. Indicates that the subject is suffering from diabetes or diabetic nephropathy. The kit according to any one of claims 5 to 6, wherein the RAGE molecule is RAGE143. The labeled AGE molecules are labeled Lys-AGE (glutaraldehyde modified lysine modified AGE), labeled glucose modified AGE (G-AGE), labeled ribose modified AGE (R-AGE), labeled fructose modified AGE (F The kit according to any one of claims 5 to 7, which is -AGE) or a modification thereof.