JP2020134368A - Assay system for detecting AGEs - Google Patents

Abstract

To provide an assay system for detecting irritative glycation end products (AGEs) in a sample.SOLUTION: One aspect of the present invention relates to a kit for detecting irritative glycation end products (AGEs) in a sample obtained from a subject. The kit includes a substrate to which AGEs for capturing are fixed and an advanced glycation end products receptor (sRAGE) containing an amino-acid sequence at least 90% identical with the arrangement number 3. The kit can detect different types of irritative AGEs in the sample exhaustively.SELECTED DRAWING: None

Description

The present invention relates to a detection agent, kit or method for detecting advanced glycation end products (AGEs).

Advanced glycation end products (AGEs) are glycation reactions in the body and glycation of proteins produced during the processing of foods. Some AGEs induce dysfunction in the body and diabetic angiopathy (nephropathy, retinopathy). , Neuropathy, etc.) and triggers age-related diseases. However, AGEs are a general term for various structures, and it is difficult to measure AGEs that trigger age-related diseases. Furthermore, although many agricultural, forestry and fishery products have been reported to have an effect of suppressing AGEs production, a technique for detecting and evaluating AGEs that is meaningful to living organisms has not been established. Therefore, it is difficult to provide objective information on the prevention of age-related diseases by food, and it is required to establish an effective evaluation method. On the other hand, RAGE (receptor for AGEs) is known as a receptor for recognizing AGEs. RAGE recognizes AGEs of various structures, and AGEs are recognized by RAGE, which leads to the onset. Therefore, AGEs recognized by RAGE are considered to be meaningful AGEs for living organisms.

Under such circumstances, a method has been developed in which an antibody against a typical structure of AGEs (carboxymethyl lysine or the like) is prepared and a part of AGEs is detected by an enzyme antibody reaction method or the like. Among AGEs, a method for detecting and quantifying some molecules whose structure is clear by HPLC, MS, or the like has been developed. For fluorescent AGEs (such as pentosidine), a method for detecting by fluorescence has been developed. The present inventors have previously developed a system that stably expresses sRAGE that recognizes AGEs (Patent Document 1).

International Publication No. 2016/051808

The present inventors have created and provided a method for comprehensively detecting AGEs having a wide variety of structures.

More specifically, as a result of diligent efforts, the present inventors comprehensively detect AGEs in a sample by competing the binding of sRAGE to AGEs in the sample with a capture (or competing) substance. Newly found that is possible. Further, according to the present invention, it is possible to detect AGEs (stimulant AGEs) that are more related to the disease.

Therefore, the present invention provides the following items.
(Item 1)
A kit for detecting irritating advanced glycation end products (AGEs) in a sample.
A substrate on which capture AGEs are fixed,
A kit comprising an advanced glycation end product receptor (sRAGE) comprising an amino acid sequence having at least about 90% identity with SEQ ID NO: 3.
(Item 2)
The kit according to item 1, wherein the capture AGEs are glucose (G) -AGEs or fructose (F) -AGEs.
(Item 3)
The kit according to item 2, wherein the capture AGEs are glucose (G) -AGEs.
(Item 4)
The kit according to any one of items 1 to 3, wherein the AGEs for capture are immobilized on the substrate using a carbonate-bicarbonate buffer solution.
(Item 5)
The kit of item 4, wherein the carbonate-bicarbonate buffer has a pH value of about 9.2 to about 10.6.
(Item 6)
The kit of item 5, wherein the carbonate-bicarbonate buffer has a pH value of about 9.6.
(Item 7)
The stimulating AGEs are glyoxal (GO) -AGEs, glycolaldehyde (Glycol) -AGEs, glyceraldehyde (Glycer) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. Any of items 1 to 6, including AGEs, acetaldehyde (AA) -AGEs, ribose-AGEs, xylose-AGEs, arabinose-AGEs, fructose (F) -AGEs, sorbitol-AGEs, galactose-AGEs or any combination thereof. The kit described in item 1.
(Item 8)
The stimulating AGEs are glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. 7. The kit of item 7, comprising AGEs, acetaldehyde (AA) -AGEs, or any combination thereof.
(Item 9)
The kit according to item 8, wherein the irritating AGE comprises glyceraldehyde-AGEs, glycolaldehyde-AGEs or any combination thereof.
(Item 10)
The capture AGEs are bovine serum albumin, human serum albumin, ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin, fibrinogen, and high-density lipoprotein. (HDL), immunoglobulins, low-density lipoprotein (LDL), insulin, keratin, osteocalcin, alcohol dehydrogenase, aldose reductase, catepsin B, spuroxide dysmidase, myosin ATPase, erythrocyte spectroins, or AGEs of myerin, The kit according to any one of items 1 to 9.
(Item 11)
The kit according to item 10, wherein the capture AGEs are albumin AGEs.
(Item 12)
The kit according to any one of items 1 to 11, wherein the sRAGE is biotinylated.
(Item 13)
The kit according to any one of items 1 to 12, further comprising a detection agent that specifically binds to the sRAGE.
(Item 14)
The kit according to item 13, wherein the detection agent is streptavidin.
(Item 15)
The kit according to any one of items 1 to 14, wherein the kit can detect the stimulating AGEs in a sample containing the stimulating AGEs of about 100 ng / ml or more.
(Item 16)
The kit according to item 15, wherein the kit can detect the stimulating AGEs in a sample containing the stimulating AGEs of about 10 ng / ml or more.
(Item 17)
A kit for detecting irritating advanced glycation end products (AGEs) in a sample.
Competitive AGEs and
A kit comprising a substrate on which an advanced glycation end product receptor (sRAGE) containing an amino acid sequence having at least about 90% identity with SEQ ID NO: 3 is immobilized.
(Item 18)
The kit according to item 17, wherein the competing AGEs are glucose (G) -AGEs or fructose (F) -AGEs.
(Item 19)
The kit according to item 18, wherein the competing AGEs are glucose (G) -AGEs.
(Item 20)
The stimulating AGEs are glyoxal (GO) -AGEs, glycolaldehyde (Glycol) -AGEs, glyceraldehyde (Glycer) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. Any of items 17-19, including AGEs, acetaldehyde (AA) -AGEs, ribose-AGEs, xylose-AGEs, arabinose-AGEs, fructose (F) -AGEs, sorbitol-AGEs, galactose-AGEs or any combination thereof. The kit described in item 1.
(Item 21)
The stimulating AGEs are glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. 20. The kit of item 20, comprising AGEs, acetaldehyde (AA) -AGEs, or any combination thereof.
(Item 22)
21. The kit of item 21, wherein the irritating AGE comprises Glycer-AGEs, Glycol-AGEs or any combination thereof.
(Item 23)
The competing AGEs are albumin, ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin, fibrinogen, high density lipoprotein (HDL), immunoglobulins. , Low Density Lipoprotein (LDL), Insulin, Keratin, Osteocalcin, Alcohol Dehydrogenase, Ardos Reductase, Catepsin B, Speroxide Dismudase, Myosin AMPase, Erythrocytorin, or Myerin AGEs, Items 17-22. The kit described in item 1.
(Item 24)
The kit according to any one of items 17 to 23, further comprising a detection agent that specifically binds to the competitive AGEs.
(Item 25)
The kit according to item 24, wherein the detection agent is an antibody against the competitive AGEs.
(Item 26)
A substrate for detecting irritating advanced glycation end products (AGEs) in a sample, wherein the capture AGEs are immobilized.
(Item 27)
The substrate according to item 26, wherein the trapping AGEs are glucose (G) -AGEs or fructose (F) -AGEs.
(Item 28)
The substrate according to item 27, wherein the trapping AGEs are glucose (G) -AGEs.
(Item 29)
The substrate according to any one of items 26 to 28, wherein the trapping AGEs are immobilized on the substrate using a carbonate-bicarbonate buffer solution.
(Item 30)
29. The substrate of item 29, wherein the carbonate-bicarbonate buffer has a pH value of about 9.2 to about 10.6.
(Item 31)
The substrate of item 30, wherein the carbonate-bicarbonate buffer has a pH value of about 9.6.

In the present invention, it is intended that the above one or more features may be provided in addition to the specified combinations. Further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

According to the present invention, it is possible to comprehensively detect AGEs in a sample, particularly AGEs (stimulant AGEs) associated with a disease. Therefore, the present invention provides a kit and method for detecting irritant AGEs useful for diagnosing a disease, and a substrate that can be used for the kit and method.

FIG. 1 shows the generation pathways of various AGEs. FIG. 2 shows the results of SDS-PAGE of sRAGE expressed in the posterior silk gland and the results of Western blotting. The figure on the left is the result of protein staining with kumasi brilliant blue, and the figure on the right is the result of visualizing sRAGE with an anti-RAGE antibody after transfer to a nitrocellulose membrane. In each lane, lane 1 is a molecular weight marker; lanes 2 to 8 are silk gland extracts (middle + rear), and lanes 9-11 are cocoon extracts. Of the silk gland extracts (lanes 2-8), lane 2 is a non-genetically modified silkworm; lanes 3-5 are the expression results by the USA-GAL4 expression system, and lanes 6-8 are the expression results by the TALEN activator expression. The result. Further, when described in detail for each lane, lanes 3 and 6 are silk gland extracts of the silkworm expressing the middle silk gland; lanes 4 and 7 are the silk gland extract of the silkworm expressing the posterior silk gland; lane 5 , 8 are the results of running the extract when the silk gland of the rear silkworm expressing transgenic silkworm was extracted with 10 times the amount of the extract of lane 4. Regarding the cocoon extract, lane 9 is a cocoon extract of non-genetically modified silkworm, lane 10 is a cocoon extract of transgenic silkworm expressed in the posterior silk gland by the UAS-GAL4 expression system, and lane 11 is It is the result of the cocoon extract of the recombinant silkworm expressed in the TALEN activator expression system. FIG. 3 shows the results of visualization of the biotinylated protein with HRP-labeled streptavidin after transcription to a nitrocellulose membrane to confirm the biotinylation of the sRAGE expressed in FIG. FIG. 4 shows an outline of the assay system in Example 1. FIG. 5 shows a graph of the detection results of typical stimulant AGEs (GO, MGO, Glycer, Glycol-treated OVA). The vertical axis shows the OD value, and the horizontal axis shows the concentration of stimulating AGEs in the sample. As capture AGEs, G-BSA and F-BSA were used. FIG. 6 shows the measurement results of AGEs in a sample containing human plasma. The vertical axis shows the OD value, and the horizontal axis shows the dilution rate of plasma. w / o means no plasma addition. FIG. 7 shows the detection results of typical stimulant AGEs (GO, Glycer, MGO, Glycol-treated OVA) in the presence of 20-fold diluted Vietnamese serum. The horizontal axis represents the AGEs concentration (ng / ml), and the vertical axis represents the absorbance (A450). FIG. 8 shows the results of detection of stimulating AGEs in the assay system when capture AGEs are fixed in wells. The horizontal axis shows the AGEs concentration (ng / ml), and the vertical axis shows the relative value (%) when the absorbance (A450) without the addition of stimulating AGEs is 100. 8A-8D show the results of glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs and methylglyoxal (MGO) -AGEs, respectively. The upper part of each figure shows the result in the sample in which each stimulating AGEs was added to PBS (-), and the lower part shows the result in the sample in which the stimulating AGEs were added to human serum diluted 20 times. FIG. 8 shows the results of detection of stimulating AGEs in the assay system when capture AGEs are fixed in wells. The horizontal axis shows the AGEs concentration (ng / ml), and the vertical axis shows the relative value (%) when the absorbance (A450) without the addition of stimulating AGEs is 100. 8A-8D show the results of glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs and methylglyoxal (MGO) -AGEs, respectively. The upper part of each figure shows the result in the sample in which each stimulating AGEs was added to PBS (-), and the lower part shows the result in the sample in which the stimulating AGEs were added to human serum diluted 20 times. FIG. 8 shows the results of detection of stimulating AGEs in the assay system when capture AGEs are fixed in wells. The horizontal axis shows the AGEs concentration (ng / ml), and the vertical axis shows the relative value (%) when the absorbance (A450) without the addition of stimulating AGEs is 100. 8A-8D show the results of glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs and methylglyoxal (MGO) -AGEs, respectively. The upper part of each figure shows the result in the sample in which each stimulating AGEs was added to PBS (-), and the lower part shows the result in the sample in which the stimulating AGEs were added to human serum diluted 20 times. FIG. 8 shows the results of detection of stimulating AGEs in the assay system when capture AGEs are fixed in wells. The horizontal axis shows the AGEs concentration (ng / ml), and the vertical axis shows the relative value (%) when the absorbance (A450) without the addition of stimulating AGEs is 100. 8A-8D show the results of glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs and methylglyoxal (MGO) -AGEs, respectively. The upper part of each figure shows the result in the sample in which each stimulating AGEs was added to PBS (-), and the lower part shows the result in the sample in which the stimulating AGEs were added to human serum diluted 20 times. FIG. 9 shows the measurement results of AGEs in a sample containing mouse serum or plasma. The vertical axis shows the OD value, and the horizontal axis shows the dilution rate of serum or plasma. w / o means no addition of serum or plasma. FIG. 9 shows the measurement results of AGEs in a sample containing mouse serum or plasma. The vertical axis shows the OD value, and the horizontal axis shows the dilution rate of serum or plasma. w / o means no addition of serum or plasma. FIG. 10 shows the results of detection of irritant AGEs in food. The vertical axis shows the relative value (%) when the OD value without the sample is set to 100, and the horizontal axis shows the ratio of the samples in the measurement system. FIG. 11 shows a calibration curve of Glycer-BSA prepared in an assay system when capture AGEs were immobilized on a substrate at pH 9.6 and pH 7.4. FIG. 12 shows a calibration curve of Glycer-BSA prepared when the concentration of sRAGE used in the assay system was 0.03 μg / ml and 0.1 μg / ml. FIG. 13 shows a calibration curve of Glycer-BSA prepared when the concentrations of the immobilized capture AGEs were 0.1 μg / ml, 0.3 μg / ml, and 0.9 μg / ml. FIG. 14 shows an outline of the assay system in Example 7. A method similar to a competitive / sandwich ELISA, which is a non-limiting embodiment of the present invention, is described. FIG. 15 shows a calibration curve prepared using a glyceraldehyde-treated BSA. From the OD value (horizontal axis) of the measured sample, the AGEs concentration converted into the glyceraldehyde-treated BSA concentration (μg / ml) (vertical axis) can be obtained. FIG. 16A shows the results of measuring the concentration of AGEs in the serum of experimental animals (mice). The vertical axis shows the OD value, and the horizontal axis shows the number of weeks. Male mice aged 7, 18, 34, 57 weeks were used, and female mice aged 7, 14, 36, 54 weeks were used. , The measurement results in the youth, middle age, and old age groups are shown. FIG. 16B shows the results of measuring the concentration of AGEs in the plasma of an experimental animal (mouse). The vertical axis shows the OD value, and the horizontal axis shows the number of weeks. For male mice, 7,18,34,57-week-old animals were used, and for female mice, 7,14,36,54-week-old animals were used, and the measurement results in the juvenile, middle-aged, and aged groups are shown. FIG. 17 shows the detection results of stimulant AGEs contained in peptides extracted from calcined pork. FIG. 18A shows the stimulating AGEs concentration generated in BSA prepared by reacting various reducing sugars and carbonyls with BSA. The stimulant AGEs concentration was measured by the method shown in FIG. The stimulating AGEs concentration is shown by the Glycer-AGEs equivalent converted from the measured value (OD value) to the Glycer-AGEs concentration by the calibration curve in the upper row (see the figure below). FIG. 18B shows the results showing the activity as stimulating AGEs when each glycated BSA shown in FIG. 18A was treated with trypsin to form a glycated peptide in terms of Glycer-AGEs equivalent.

Hereinafter, the present invention will be described. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, this specification (including definitions) takes precedence.

(Definition of terms)
The following is a list of definitions of terms specifically used herein.

As used herein, "about" means ± 10% of the value shown.

As used herein, the terms "advanced glycation end products" or "advanced glycation end products" (both in English, Advanced Glycation End Products) are also referred to as AGEs, which are glycation products of proteins and have various structures. It is a general term for the body. It occurs in the process of processing food and is important for improving the taste, but it is also produced in the living body, and a part of it induces dysfunction in the living body and triggers age-related diseases. It is also known to be involved in the onset and progression of diabetic angiopathy known as vascular complications, which is the main cause of impairing the quality of life of diabetic patients. Damage to the eyes, nerves, and kidneys due to vascular complications is called diabetic retinopathy, neurosis, and nephropathy (total of the three major complications), and is a pathological condition characteristic of diabetic patients. Reducing sugars such as 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 up to this point is reversible and is called the early reaction. After that, advanced glycation end products are formed through complicated and irreversible reactions such as condensation, cleavage, and crosslink formation. Such a series of reactions is called a saccharification reaction. AGEs are also a general term for structures produced through such a process. Examples of the AGEs structure present in the living body include, but are not limited to, carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pentosidine, pyrarin, imidazoline, methylglyoxal, and crosulin. The product in which albumin, immunoglobulin, ovalbumin and the like present in plasma undergo the above-mentioned glycation is also AGE, and is widely used in experimental systems as AGE. Furthermore, in the in vitro experimental system, BSA (bovine serum albumin) that has been glycated, for example, R-AGE (BSA that has been glycated with ribose), F-AGE (BSA that has been treated with fructose); G- AGE (BSA saccharified with glucose) and the like are also widely used. Hemoglobin A1c, which is used as an index of glycemic control, is an Amadori rearrangement compound, but is included in AGEs. Also, any protein can be converted to AGEs. For example, CML albumin and CEL albumin included in AGEs are both AGEs in which albumin is glycated. Such an AGEs production reaction can occur in the circulating blood, extracellular matrix, or intracellular in vivo. For example, AGEs present in the blood vessels of diabetic patients include: fluorescent and cross-linked structures (pentosidine, crosulin, etc.) and non-fluorescent and non-cross-linked (carboxymethyl lysine, pyrarin, methylglyoxal (MG) -imidazolone, etc.) ) Can be roughly divided into two. When AGEs show abnormal values, diseases such as microangiopathy (nephropathy, retinopathy, neuropathy, etc.) and macroangiopathy (ischemic heart disease, cerebrovascular disease, arteriosclerosis obliterans, etc.) are expected. In commonly used test methods, the reference substances are pyrarin (normal range: less than 23 pmol / mL in plasma) and pentocidin (normal range: 0.00915-0.0431 μg / mL in plasma (as measured by ELISA)). Etc. are used (see "Today's Clinical Examination 2007-2008" Publisher Nanedo Co., Ltd.).

In the present specification, "stimulated advanced glycation end products" or "stimulated AGEs (stimulated AGEs)" refer to AGEs that are highly related to diseases and have a property of strongly binding to sRAGE. .. Previously, it was thought that glycation by glucose in blood was the main cause, but it has begun to be suggested that glycation by glucose takes a long time and glucose glycation AGEs are less irritating to the living body. Excess glucose is metabolized in the polyol metabolism system to produce glyceraldehyde (Glycer), and the oxidation reaction produces glyoxal (GO), glycolaldehyde (Glycol), etc., which are highly reactive and can be produced in a short time. In addition to producing AGEs, it has been reported that the glycated product is highly biostimulant (Fig. 1).

As used herein, "capture (or competing) AGEs" are AGEs that compete with the binding of stimulating AGEs to be measured to sRAGE in the competitive assay system of the present invention, and are more than AGEs to be measured. Also refers to those with low binding ability to sRAGE. For example, since glucose (G) -AGEs have a lower binding ability to sRAGE than any AGEs, any stimulating AGEs having a higher binding ability to sRAGE than glucose (G) -AGEs can be detected. In this case, it is effective when it is desired to comprehensively detect irritating AGEs in a sample. Moreover, since fructose (F) -AGEs have a higher binding potential for sRAGE than G-AGEs, it is possible to detect stimulating AGEs having a higher binding potential for sRAGE than F-AGEs, excluding G-AGEs. is there. As described above, as the capture AGEs, the capture AGEs can be appropriately selected according to the type of the AGEs to be measured. Typical examples of capture AGEs include glucose (G) -AGEs, fructose (F) -AGEs, galactose-AGEs, and the like, and more specific examples include bovine serum albumin and human serum albumin. , Ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin, fibrinogen, high density lipoprotein (HDL), immunoglobulin, low density lipoprotein (LDL) ), Insulin, keratin, osteocalcin, alcohol dehydrogenase, aldose reductase, catepsin B, spuroxide dysmidase, myosin ATTase, erythrocyte spectroline, or AGEs of myelin, but not limited to these.

As used herein, the term "immobilization" means that a substance such as AGEs is immobile with respect to another substance of interest (for example, a substrate (base material, solid phase)). Immobilization is achieved by a variety of means, including the use of carbonate-bicarbonate buffers, the use of plates coated with a substance that binds to tags (eg biotin) (eg avidin), and the like. Not limited.

As used herein, "complex" means any construct that includes two or more parts. For example, if one portion is a polypeptide, the other portion may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.). .. In the present specification, two or more portions constituting the complex may be bonded by covalent bonds or other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). It may have been done. When two or more parts are a polypeptide, it can also be referred to as a chimeric polypeptide. Therefore, in the present 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.

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

As used herein, the term "AGEs molecule" refers to any molecule contained in the above-mentioned AGE. For example, AGEs include Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE), or a variant thereof. Complexes thereof can be mentioned, but are not limited to them.

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

As used herein, the term "Receptor for AGE" is also referred to as RAGE and is (1) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 8; (2) SEQ ID NO: 8 above. A polypeptide containing an amino acid sequence containing one or several amino acid substitutions, additions and / or deletions in the amino acid sequence shown in the above, and exhibiting the activity of natural RAGE; (3) The amino acid shown in SEQ ID NO: 8 above. A polypeptide containing 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: 8 above. Polypeptide containing a sequence and exhibiting the activity of natural RAGE; (5) Polypeptide containing an amino acid sequence encoded by the amino acid molecule shown in SEQ ID NO: 7; (6) In the nucleic acid sequence shown in SEQ ID NO: 7 above. A polypeptide containing an amino acid sequence encoded by a nucleic acid molecule that hybridizes with a complementary nucleic acid sequence and a nucleic acid molecule that hybridizes under stringent conditions, and exhibits the activity of natural RAGE; (7) The nucleic acid shown in SEQ ID NO: 7 above. A polypeptide containing 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 RAGE; (8) shown in SEQ ID NO: 7 above. A polypeptide containing an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence and exhibiting the activity of native RAGE; and (9) at least the nucleic acid sequence shown in SEQ ID NO: 7 above. It is one of the polypeptides containing an amino acid sequence encoded by a nucleic acid molecule having 80% sequence homology and exhibiting the activity of natural RAGE. The above identity or homology is calculated using the default parameters using BLAST (NCBI's BLAST 2.2.9 (issued on 2004.5.12)), which is a tool for sequence analysis. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of one of ordinary skill in the art. RAGE is also a type I membrane protein with a molecular weight of approximately 35 kDa, which was identified in 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 RAGEs has a structure in which one V-type immunoglobulin domain is followed by two C-type immunoglobulin domains (C1 region and C2 region), which have three immunoglobulin fold structures. There is. RAGE also contains a transmembrane domain and a 43 amino acid cytoplasmic domain. RAGE interacts with various classes of ligands (AGEs, 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 is at positions 38, 99, 144, 208 and 259 and 301 in the amino acid sequence of SEQ ID NO: 3. It is preferable to retain the cysteine residue corresponding to the position. RAGE is expressed only at low levels in normal tissues and vascular systems. 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. In addition, the expression of RAGE is also increased in pathological sites such as atherosclerotic lesions where AGEs are accumulated. AGEs-RAGE interactions alter important cellular properties in vascular homeostasis. For example, after RAGE binds to AGEs, vascular endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, as well as their permeability to macromolecules. In mononuclear phagocytes, RAGE activates cytokine and growth factor expression and induces cell migration in response to soluble AGEs, whereas haptotaxis occurs with fixed ligands.

As used herein, the term "RAGE ligand recognition region" or "sRAGE (Soluble Receptor For Advanced Glycation End products)" is used interchangeably and refers to a region recognized by a RAGE ligand. Specifically, the sRAGE or RAGE ligand recognition region refers to all or part of the extracellular region of RAGE. The sRAGE is typically composed of, but not limited to, positions 22-332 of SEQ ID NO: 3 or SEQ ID NO: 8.

As used herein, the term "RAGE-like polypeptide" means "RAGE8", "mRAGE8", "RAGE1", "mRAGE1", "RAGE2", "mRAGE2", "RAGE3", "mRAGE3". , "RAGE4", "mRAGE4", "RAGE7", "mRAGE7", "RAGE143", "mRAGE143", "RAGE223", "mRAGE223", "RAGE226" and "mRAGE226" or their variants Including the body. These explanations are disclosed in Japanese Patent Application Laid-Open No. 2013-209330 and the like, and the contents thereof are appropriately incorporated herein by reference.

As used herein, the term "RAGE molecule" is understood to include RAGE-like polypeptides as well as any complex thereof. Therefore, it is understood that the RAGE molecule includes RAGE-like polypeptides and the like, for example, RAGE (full length), RAGE extracellular region (position 22-332 of SEQ ID NO: 8), RAGE143, RAGE223, RAGE226 and the like. .. The RAGE molecule also includes a RAGE (mini RAGE) lacking an entire domain or a part of a domain among the three domains constituting the RAGE. The mini RAGE also includes a mini RAGE of a RAGE-like polypeptide.

In the present specification, the molecule containing the "RAGE ligand recognition region" is a "RAGE molecule" other than the full-length RAGE (including the "RAGE-like polypeptide"), for example, "RAGE8", "mRAGE8", "RAGE1". , "MRAGE1", "RAGE2", "mRAGE2", "RAGE3", "mRAGE3", "RAGE4", "mRAGE4", "RAGE7", "mRAGE7", "RAGE143", "mRAGE143", "RAGE223", Examples thereof include "mRAGE223", "RAGE226" and "mRAGE226", and RAGE extracellular region (position 22-332 of SEQ ID NO: 8).

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.

Since it is important to form an intramolecular disulfide bond also in the above RAGE-like polypeptide, it corresponds to the 38th, 99th, 144th, 208th, 259th and 301st positions of the amino acid sequence of SEQ ID NO: 8. Cysteine is preferably retained.

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

In the present specification, "antibody" is broadly defined as a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and a functional fragment thereof (for example, F (ab') ₂ and the like. Fab fragments), as well as conjugates or functional equivalents produced by other recombination (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies). , Single chain antibody (scFV), diabody, sc (Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc). Further, such an antibody may be covalently linked or recombinantly fused to an enzyme such as alkaline phosphatase, horseradish peroxidase, galactosidase, or the like. Further, such an antibody may be covalently linked or recombinantly fused to an enzyme such as alkaline phosphatase, horseradish peroxidase, galactosidase, or the like. When used in a narrow sense, an antibody refers to a full-length antibody (eg, polyclonal antibody, monoclonal antibody, etc.), and the others may be referred to as a variant or an antigen-binding fragment. The antibody used in the present invention may bind to its target, and its origin, type, shape, etc. are not limited. Specifically, it can be produced based on known antibodies such as non-human animal antibodies (for example, mouse antibody, rat antibody, camel antibody), human antibody, chimeric antibody, and humanized antibody. In the present invention, single chain antibodies are used. The binding of the antibody to the target is preferably a discriminative or specific binding. The variant of the antibody may be a combination of the antibody and various molecules such as polyethylene glycol. Variants of the antibody can be obtained by chemically modifying the antibody using known techniques.

As used herein, the term "single chain antibody" is also referred to as "scFv (single chain Fv)", and the variable region regions ( _VH and _VL ) of the heavy and light chains of the antibody are linked with appropriate linker peptides. Corresponds to the one. A single-chain antibody protein can be expressed by constructing such a construct at the gene level and introducing it into Escherichia coli using a protein expression vector.

As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to its purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, and so on. Amino acids such as 15, 20, 25, 30, 40, 50, and above are mentioned, and lengths represented by integers not specifically listed here (eg, 11) are also suitable as lower limits. possible. Further, in the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and are specifically listed here. A length represented by a non-integer (eg, 11) may also be a suitable lower bound. In the present specification, the length of a polypeptide and a polynucleotide can be expressed by the number of amino acids or nucleic acids, respectively, as described above, but the above number is not absolute and is an upper limit as long as it has the same function. Alternatively, the above-mentioned number as the lower limit is intended to include several above and below the number (or, for example, 10% above and below). The length of a fragment useful herein can be determined by whether at least one of the functions of the full-length protein that is the basis of the fragment is retained.

As used herein, the term "homology" of a gene refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes are homologous can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When comparing two gene sequences directly, the DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%. , 95%, 96%, 97%, 98% or 99%, the genes are homologous.

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

In the present specification, comparison of amino acid sequence and base sequence similarity, identity and homology is calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.2.9 (issued on 2004.5.12). The value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value.

As used herein, the term "modified" refers to a substance that has been partially modified from the original polypeptide or substance such as a polynucleotide. Such variants include substitution variants, addition variants, deletion variants, truncated variants, allelic variants and the like. An allele is a genetic variant that belongs to the same locus and is distinguished from each other. Therefore, the "allele variant" refers to a variant having an allele relationship with a certain gene. A "species homolog or homolog" is, within a species, homologous (preferably 60% or more, more preferably 80% or more,) at the amino acid or nucleotide level with a gene. Those having 85% or more, 90% or more, 95% or more homology). The method for obtaining such species homologues is clear from the description herein. The "ortholog" is also referred to as an orthologous gene and refers to a gene derived from speciation from a common ancestor with two genes. For example, in the hemoglobin gene family with multiple gene structures, human and mouse α-hemoglobin genes are orthologs, while human α-hemoglobin and β-hemoglobin genes are paralogs (genes generated by gene duplication). .. Since orthologs are useful for estimating the molecular phylogenetic tree, orthologs may also be useful in the present invention.

As used herein, the term "conservative (modified) variant" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, it is essentially the same. Refers to an array. Such base sequence modification methods include cleavage with restriction enzymes, ligation by treatment with DNA polymerase, Klenow fragment, DNA ligase, etc., and site-specific base substitution method using synthetic oligonucleotides (specification). Site-directed mutation method; MarkZollerand Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other methods usually used in the field of molecular biology can also be used for modification. Due to the degeneracy of the genetic code, many 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 alanine is identified by a codon, that codon can be changed to any of the corresponding corresponding codons described, without changing the encoded polypeptide. Such nucleic acid variation is a "silent modification (mutation)", which is a type of conservatively modified mutation. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent mutations 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) to produce a functionally identical molecule. It is understood that it can be modified. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is implicitly included in each of the sequences described. Preferably, such modifications can be made to avoid substitution of the amino acid cysteine, which has a profound effect on the higher order structure of the polypeptide.

One amino acid can be replaced by another amino acid in a protein structure, such as the binding site of a ligand molecule, without a significant reduction or loss of the ability to interact. It is the ability and nature of a protein to interact that defines the biological function of a protein. Thus, substitutions of a particular amino acid can be made at the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after substitution. Thus, various modifications can be made in the peptides disclosed herein or in the corresponding DNA encoding the peptides, without any apparent loss of biological utility.

Such nucleic acids can be obtained by well-known PCR methods and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.

The hydrophobicity index of amino acids can be taken into account when designing modifications as described above. The importance of the hydrophobic amino acid index in imparting interactive biological functions in proteins is generally recognized in the art (Kyte.J and Doolittle, LFJ. 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 that 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); Aspartic acid (-3.5); Leucine (-3.9) And arginine (-4.5)).

It is possible in the art to replace one amino acid with another amino acid having a similar hydrophobicity index and to produce a protein that still has similar biological functions (eg, a protein equivalent in ligand binding potential). It is well known. In such amino acid substitutions, 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 substitutions of amino acids based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity index is assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); aspartic acid (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline ( -0.5 ± 1); alanin (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.5) -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be replaced by another that has a similar hydrophilicity index and can still give a bioequivalent. In such amino acid substitutions, 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 original amino acid and the amino acid to be replaced are similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and aspartic acid; and valine, leucine, and isoleucine, Etc., but are not limited to these.

In addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made herein to make functionally equivalent polypeptides. Amino acid substitution refers to substituting one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids of the original peptide. The addition of amino acids means the addition of one or more amino acids, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids to the original peptide chain. Amino acid deletion refers to the deletion of one or more, for example, 1-10, preferably 1-5, more preferably 1-3 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 substituted or added may be a natural amino acid, an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.

As used herein, the term "substitution, addition and / or deletion" of a polypeptide or polynucleotide means that the original polypeptide or polynucleotide is an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively. , To be replaced, to be added, or to be removed. Techniques for such substitutions, additions and / or deletions are well known in the art, and examples of such techniques include site-specific mutagenesis techniques. These changes in the reference nucleic acid molecule or polypeptide can occur at the 5'end or 3'end of this nucleic acid molecule, as long as the desired function (eg, RAGE recognition ability) is retained. It can occur at the amino-terminal or carboxy-terminal sites of the amino acid sequences that represent this polypeptide, or can occur anywhere between those terminal sites and are individually interspersed among the 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 a number may be increased as long as the desired function is retained in the variant having the substitution, addition or deletion. be able to. For example, such numbers can be one or several, and preferably within 20%, within 15%, within 10%, within 5%, or below 150, below 100, of the total length. It can be 50 or less, 25 or less, and so on.

As used herein, the term "tag sequence" is used to bind a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a specific substance. A substance that acts as a binding partner of (for example, having a relationship such as biotin-avidin and biotin-streptavidin). Therefore, for example, a specific substance to which the tag sequence is bound can be selected by contacting the base material to which the binding partner of the tag sequence is bound. Such tag sequences are well known in the art. Typical tag sequences include, but are not limited to, myc tags, His tags, HA, Avi tags, and the like.

As used herein, the term "detecting agent" broadly refers to any factor (agent) capable of detecting a substance or state of interest (eg, AGEs).

As used herein, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural, or may be a modified amino acid. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally 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). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done.

As used herein, the "amino acid" may be natural or non-natural as long as it satisfies the object of the present invention. As used herein, the term "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. It is understood herein that amino acid derivatives and amino acid analogs can be used as alternatives as long as they can provide the same biological functions as amino acids. As used herein, the term "natural amino acid" means the 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, aspartic acid, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornitine. , And lysine. Unless otherwise specified, all amino acids referred to herein are L-form, but forms using D-form amino acids are also within the scope of the present invention. As used herein, the term "unnatural amino acid" means an amino acid that is not normally found in proteins. Examples of unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, D- or L-form of homoarginine and D-phenylalanine. As used herein, the term "amino acid analog" refers to a molecule that is not an amino acid but has similar physical properties and / or functions to an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

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

As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". The "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably. Specifically, such an oligonucleotide includes, for example, 2'-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphate diester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphate diester bond in an oligonucleotide. Is an oligonucleotide derivative converted to N3'-P5'phosphoroamidate bond, an oligonucleotide derivative in which ribose and phosphate diester bond in the oligonucleotide are converted into peptide nucleic acid bond, and uracil in the oligonucleotide is C- 5 Oligonucleotide derivatives substituted with propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced with C-5 propynylcytosine, oligonucleotide An oligonucleotide derivative in which cytosine in a nucleotide is replaced with phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in DNA is replaced with 2'-O-propyl ribos, and an oligonucleotide derivative in which ribose in an oligonucleotide is 2 Examples include oligonucleotide derivatives substituted with'-methoxyethoxyribose. Unless otherwise indicated, certain nucleic acid sequences are also conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as are the explicitly indicated sequences. Is intended to include. Specifically, the degenerate codon substituent creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91- 98 (1994)).

As used herein, the "nucleotide" may be natural or non-natural. The "nucleotide derivative" or "nucleotide analog" refers to one that is different from the naturally occurring nucleotide but has the same function as the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2'-O-methylribonucleotide, peptide-type nucleic acid (PNA). Not done.

As used herein, the term "complex molecule" refers to a molecule formed by linking a plurality of types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such a complex molecule include, but are not limited to, glycolipids, glycolipides, and the like. In the present specification, a polypeptide having an amino acid exemplified by SEQ ID NO: 3 or the like or a variant or fragment thereof, as long as it has biological activity involved in diagnosis, each variant or fragment is used. The encoding nucleic acid molecule can also be used. In addition, a complex molecule containing such a nucleic acid molecule can also be used.

As used herein, "nucleic acid" is also used interchangeably with genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. Specific nucleic acid sequences also include "splice variants". Similarly, a particular protein encoded by a nucleic acid implicitly includes any protein encoded by a splice variant of that nucleic acid. As the name suggests, "splice variants" are the product of alternative splicing of genes. After transcription, the first nucleic acid transcript can be spliced so that different (different) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but involves alternative splicing of exons. Another polypeptide derived from the same nucleic acid by over-read transcription is also included in this definition. Any product of the splicing reaction, including recombinant splice products, is included in this definition.

As used herein, the term "gene" refers to a factor that defines a genetic trait. It is usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that influences its expression is called a regulatory gene. As used herein, "gene" may refer to "polynucleotide", "oligonucleotide" and "nucleic acid" and / or "protein" "polypeptide", "oligopeptide" and "peptide".

As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a polynucleotide selected from the polynucleotides of the present invention as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, using a filter on which DNA derived from colonies or plaques is immobilized, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl, and then the concentration is 0.1 to 2 times. Means a polynucleotide that can be identified by washing the filter under 65 ° C. conditions using an SSC (saline-sodium citrate) solution (the composition of the 1-fold SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed by Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A PRac1tical Approach, Second Edition, Oxford, etc. Here, sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions. Therefore, the polypeptide used in the present invention (eg, RAGE, etc.) is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule encoding the polypeptide specifically described in the present invention. Polypeptides to be produced are also included.

As used herein, the term "hybridizable polynucleotide" refers to a polynucleotide that can hybridize to another polynucleotide under the above-mentioned hybridization conditions. Specifically, as a polynucleotide capable of hybridizing, a polynucleotide having at least 60% or more homology with the base sequence of DNA encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 3 or the like, preferably 80%. Polynucleotides having the above homology, more preferably polynucleotides having 95% or more homology can be mentioned.

Factors that affect the stability of the DNA double strand include the composition, length and degree of base pair mismatch of the base. Hybridization conditions can be adjusted by one of ordinary skill in the art, applying these variables and allowing different sequence-related DNAs to form hybrids. The melting temperature of a perfectly matched DNA double strand can be estimated by the following formula.
Tm (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) +0.41 (% G + C) -600 / N-0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization solution or wash solution, and% G + C is the (guanine +) in the hybrid. Cytosine) The percentage of base. For incompletely matched hybrids, the melting temperature is reduced by about 1 ° C. for each 1% mismatch.

As used herein, "refolding" refers to the inclusion of cycloamylose while unraveling the abnormal structure of a polypeptide that has lost its original function due to its abnormal folding and preventing reaggregation with a surfactant. It refers to refolding to the original correct structure of the polypeptide by utilizing the contact ability or by dilution dialysis under the control of the oxidation-reduction potential.

As used herein, the term "purification column" refers to a solid or semi-solid support that has an affinity for or can bind to a molecule such as a protein (eg, an antibody), such as metal chelated agarose. Columns and the like can be mentioned. Each molecule can be separated by the difference in affinity or binding force of each molecule. Appropriate supports will be selected by those skilled in the art depending on the properties of the molecule to be purified. For example, in the TALON column, a protein to which a His tag or the like is added can be efficiently purified.

As used herein, the term "TALON" refers to a metal chelate affinity resin using Co.

As used herein, "phosphate buffered saline (PBS) is an aqueous solution of pH 7 to 8 containing NaCl, KCl, Na ₂ HPO ₄ , and KH ₂ PO _4. The concentration and pH of each component are , Can be suitably adjusted according to the application. In the present specification, "PBS (+)" means containing calcium ion and magnesium ion, and "PBS (-)" means calcium ion. And magnesium ion-free, but herein, "PBS" shall mean "PBS (-)" unless explicitly stated. In the present specification, Dulbecco's PBS (−) can be typically used. The composition of Dulbecco's PBS (−) is NaCl 8 g, KCl 0.2 g, Na ₂ HPO ₄ 1.15 g, KH ₂ PO ₄ 0.2 g / L, (pH 7.4).

As used herein, the term "cellulose ester membrane tube" refers to a dialysis tube produced by extruding a polymer mixture of cellulose acetate.

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 complex has a biological structure. Receptors are either completely outside the cell (extracellular receptors), inside the cell membrane (but directing parts of the receptors to the outside environment and cytosol), or completely inside the cell (intracellular). Can be present in the receptor). They can also function independently of the cell. Receptors in the cell membrane allow cells to communicate (eg, signal transduce) with space outside their boundaries and to function in the transport of molecules and ions inside and outside the cell. As used herein, the receptor may be the full length of the receptor or a fragment of the receptor.

As used herein, the term "antigen-antibody reaction" is used in the broadest sense used in the art, and particularly refers to a reaction based on a specific binding between an antigen and an antibody. Reagents and methods for detecting and quantifying antigens in a sample by using the immunoblotting (Western blot) format as the detection system are also provided.

As used herein, the term "binding" 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. The physical interaction (binding) can be direct or indirect, the indirect being mediated by 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 is not accompanied by other substantial chemical intermediates.

As used herein, the term "specifically interacting" with a second substance or factor means that the first substance or factor interacts with the second substance or factor. It means interacting with a substance or factor other than a substance or factor (particularly, another substance or factor present in a sample containing a second substance or factor) with a higher affinity. Specific interactions with a substance or factor include transcription factors when both nucleic acids and proteins are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions. Examples include, but are not limited to, protein-lipid interactions and nucleic acid-lipid interactions, such as the reaction of the transcription factor with the binding site. Therefore, when a substance or factor is both nucleic acids, the first substance or factor "specifically interacts" with the second substance or factor means that the first substance or factor is the second substance. Alternatively, it includes having at least a partial complementarity to the factor. Also, for example, when both substances or factors are proteins, the first substance or factor "specifically interacts" with the second substance or factor means, for example, an interaction by an antigen-antibody reaction, a receptor. Interactions by -ligand reaction, enzyme-substrate interaction, etc. are included, but are not limited thereto. When two substances or factors include proteins and nucleic acids, the "specific interaction" of the first substance or factor with the second substance or factor is the subject of the transcription factor and its transcription factor. Includes interactions with the binding region of the nucleic acid molecule. Therefore, as used herein, a "factor that specifically interacts" with a biological factor such as a polynucleotide or a polypeptide is defined as having an affinity for the polynucleotide or a biological factor such as the polypeptide. Representatively equal or higher, or preferably significantly (eg, statistically significant), than the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. ) Including high ones. Such affinity can be measured, for example, by hybridization assay, binding assay and the like.

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

When a mechanism such as enzyme-linked immunosorbent assay (ELISA) is used in the present invention, a microtiter plate is generally used as the solid phase (base material or substrate). As used herein, the term "solid phase" is used interchangeably herein with "board" and "base" to refer to the material on which the device of the invention is constructed. A planar support on which molecules such as antibodies can be immobilized. The material of the substrate is any solid, either covalently or non-covalently, that has the property of binding to the biomolecule used in the present invention or can be derivatized to have such property. Materials can be mentioned. Suitable substrates include beads, gold particles, plates (eg, microtiter plates), test tubes, chips, magnetic particles, membranes, fibers, glass slides, metal thin films, filters, tubes, balls, diamond-like carbon-coated stainless steel. However, it is not limited to these.

As such materials for use as solids and substrates, any material capable of forming a solid surface can be used, for example glass, silica, silicone, ceramics, silicon dioxide, plastics, metals (alloys also Included), natural and synthetic polymers (eg, polystyrene, cellulose, amylose, chitosan, dextran, and nylon), including, but not limited to. The substrate may be formed from multiple 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. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinylacetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, 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 and other organic materials can be used. In the present invention, a film used for blotting, such as a nylon film, a nitrocellulose film, and a PVDF film, can also be used. When analyzing a high-density material, it is preferable to use a hard material such as glass as a material. The material preferable as the substrate varies depending on various parameters such as measuring equipment, and those skilled in the art can appropriately select an appropriate material from the various materials as described above.

"Sandwich ELISA" refers to a particular form of ELISA in which a molecule capable of specifically binding to an antibody or antigen (eg, RAGE) is bound to a solid material and served in a sample containing the antigen, followed by an unbound antigen. The surface of the solid material is washed to remove the antibody, after which a labeled antibody (eg, an enzyme-linked labeled antibody) is bound to the bound antigen (if present) of the antibody-antigen-antibody. A form that forms a sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease and β-galactosidase. The enzyme-linked antibody reacts with the substrate to produce a measurable color reaction product. Instead of labeled antibodies, unlabeled antibodies can also be used. In this case, it can be detected by binding the labeled secondary antibody to the unlabeled antibody.

In the present specification, "silkworm" means silkworm (silkworm) in the usual meaning, and is considered to be a kind of insect belonging to the order Lepidoptera (Lepidoptera) and Bombyx mori. The official Japanese name is Bombyx mori (scientific name: Bombyx mori), which is the name of this larva, but generally also refers to this species in general. It feeds on mulberry and produces silk to make pupal cocoons. Silkworms, also known as silkworms, are not wild insects. The ancestor of the silkworm is thought to be the Bombyx mandarina, which inhabits East Asia. Silkworms and bombyx mandarins are academically distinct species, but these hybrids are fertile. In the present specification, the silkworm includes a mulberry. As used herein, the term "organism that imparts sugar chains similar to silkworm" refers to an organism that has the ability to add sugar chains similar to silkworm, such as a gene encoding an enzyme that adds sugar chains similar to silkworm. Can include transgenic organisms and the like.

In the present specification, the "silk gland" is a pair of left and right organs existing in the body of a mature silkworm, and a large amount of protein (amino acid) ingested from mulberry leaves is combined with two types of silk proteins (fibroin and sericin). Means an organ that changes to. The silk glands are paired on the left and right, and secrete liquid silk, which is the raw material for eyebrows. The silk gland is divided into three parts: a posterior silk gland, a middle silk gland, and an anterior silk gland. In the present invention, any silk gland can be used for synthesis, but in consideration of handling after synthesis, a posterior silk gland and a middle silk gland are usually used, and a middle silk gland is preferably used. It is not limited. In addition, it can be expressed throughout the body and recovered from the whole body, or it can be recovered from the eyebrows after forming the eyebrows. The posterior silk gland is an elongated part at the rearmost part, and when stretched, it is approximately. It will be 20 cm. Here, the fibroin protein, which is the center of the eyebrows, is synthesized later.

The central silk gland is a thick part bent in an S shape in the central part, and when extended, it becomes about 6 cm. The fibroin protein sent from the posterior silk gland is concentrated and stored, and shaped into fibers. It also secretes another silk protein, sericin. When spitting out eyebrows, it acts as an adhesive that holds fibroin proteins together.

The anterior silk gland is a thin tube that connects to the spout, which is about 4 cm in length, and becomes thinner toward the end. Molecules of liquid fibroin protein are stretched and aligned in one direction and aggregate with each other to further remove water. At the tip of the tube, it merges with another pair of tubes and is spit out from the spout to become a single eyebrows.

Silkworms stop eating mulberries at the end of their fifth instar (mature silkworms). The body of a mature silkworm is filled with a pair of organs (silk glands) that store a liquid (liquid silk) like starch syrup, which is the raw material for eyebrows. The silk gland is connected to the spout at the mouth of the silkworm through a thin spout tube. Liquid silk is stretched and hardened by passing through a thin spit tube to become eyebrows. Furthermore, the eyebrows are pulled out from the silk glands one after another by a series of movements in which the thread spit out from the spit tube by the larva is attached to a nearby object, the head and chest are moved in a figure eight shape, and the thread is pulled. It is.

As used herein, the term "caiko-type sugar chain" refers to a sugar chain structure peculiar to sugar proteins produced in silkworm, and is typically trimannosyl core (itself), oligomannose-type sugar chain, and complex-type sugar chain. , Or its hybrid type. In the present invention, silkworm-type glycoprotein is produced using the central silk gland. Therefore, unless otherwise specified, the “silkworm-type sugar chain” refers to a unique sugar chain type produced in the central silk gland. Say. As such a silkworm-type sugar chain, for example, two N-acetylglucosamine (GlcNAc) bound to asparagine (Asn) are bound, and then three molecules of mannose (Man) are bound (referred to as trimannosyl core, described below. It has a structure branched from the core (represented by Eq. (1)), and various sugar chains are further bound to it.

As used herein, a "purified" biological factor (eg, nucleic acid or protein) is one in which at least a portion of the factors naturally associated with the biological factor have been removed. Therefore, the purity of the biological factor in the purified biological factor is usually higher (ie, enriched) than in the state in which the biological factor is normally present.

The term "purified" as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. It means that the same type of biological factor is present.

As used herein, an amino acid or nucleic acid "corresponding" has, or has, in a polypeptide molecule or a polynucleotide molecule, the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide to be compared. Amino acids or nucleotides that are expected to be predicted, especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and make similar contributions to catalytic activity. For example, if it is an antisense molecule, it can be a similar part in the ortholog corresponding to a particular part of the antisense molecule. The corresponding amino acids are specified to be, for example, cysteineized, glutathioneized, SS bond formed, oxidized (eg, methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Can be an amino acid. Alternatively, the corresponding amino acid can be the amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids may be regions or domains over a range. Thus, such cases are referred to herein as "corresponding" regions or domains.

As used herein, a "corresponding" gene (eg, a polypeptide molecule or a polynucleotide molecule) has, or is expected to have, in some species, similar action to a given gene in the species of reference for comparison. A gene (for example, a polypeptide molecule or a polynucleotide molecule) to be produced, and when a plurality of genes having such an action exist, those having the same evolutionary origin. Therefore, the gene corresponding to a gene can be the ortholog of that gene. Thus, mouse and rat RAGEs (or soluble forms of sRAGE) can each find a corresponding RAGE (sRAGE or soluble form of sRAGE) in humans. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an animal (eg, a mouse) may be a reference gene for the corresponding gene (eg, RAGE or a soluble form of sRAGE), which may use the sequence of the animal as a query sequence. For example, it can be found by searching the sequence database of humans and rats).

As used herein, "heterologous" refers to a nucleotide or amino acid sequence that is a different sequence, an uncorresponding sequence, or a sequence from a different species. For example, a human nucleotide or amino acid sequence is heterologous to a mouse nucleotide or amino acid sequence, and a human RAGE nucleic acid or amino acid sequence is heterologous to a human albumin nucleotide or amino acid sequence. ..

As used herein, the term "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 include, but are not limited to, specific antibody production, enzymatic activity, and resistance imparting. In the present invention, for example, the function of RAGE to recognize a marker such as hemopexin can be mentioned, but the present invention is not limited thereto. As used herein, biological function can be exerted by "biological activity". As used herein, the term "biological activity" refers to the activity that a certain factor (for example, polynucleotide, protein, etc.) can have in vivo, and exerts various functions (for example, transcription promoting activity). Activities include, for example, the activity of activating or inactivating another molecule by interacting with one molecule. When two factors interact, their biological activity is the binding between the two molecules and the resulting biological changes, eg, when one molecule is precipitated with an antibody to the other. When the molecules also co-deposit, the two molecules are considered to be bound. Therefore, seeing such coprecipitation is one of the judgment methods. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. In another example, when a factor is a ligand, the ligand involves 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). Various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or, for example, the amount of upstream or downstream protein or other after some stimulation or event. A scale of similar function is mentioned.

As used herein, the term "marker (substance)" refers to whether or not the patient is suffering from a certain condition (disease related to AGEs such as diabetes, diabetic nephropathy, diabetic retinopathy, diabetic complications such as diabetic neuropathy). Or a substance that serves as a marker for tracking whether or not there is a risk. Examples of such markers include genes, gene products, metabolites, enzymes and the like. In the present invention, diseases related to AGEs such as diabetic angiopathy, non-alcoholic steatosis, Alzheimer's dementia, diabetes, hypertension, hypertension, hypertensive nephrosclerosis, arteriosclerosis, ischemia-reperfusion injury, Diabetes, preliminary detection, prediction or pre-diagnosis of a condition such as after vascular balloon injury is a drug, agent, factor or means specific to the marker associated with the condition, or a composition, kit or system containing them. It can be realized by using. Further, in the present invention, diagnosis, preliminary detection, prediction or pre-diagnosis of a certain condition (for example, a disease such as diabetic complications such as diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy), ( Assessment of therapeutic efficacy (such as dialysis) can also be achieved using agents, agents, factors or means specific to the marker associated with the condition, or compositions, kits or systems containing them.

As used herein, the term "gene product" refers to a protein or mRNA encoded by a gene. Here, it has been found that gene products that are not directly related to glucose metabolism (ie, proteins that are not directly related to glucose metabolism such as insulin) can be used as indicators of diabetes.

As used herein, the term "subject" refers to an organism (eg, human) that is the subject of the diagnosis or detection of the present invention.

As used herein, the term "sample" refers to any substance obtained from a subject or the like, and includes, for example, body fluids (blood, saliva, urine, tears, cerebrospinal fluid, etc.). Although blood, urine, and tears are preferably used and the specificity varies depending on the marker, those skilled in the art can appropriately select a preferable sample based on the description herein.

As used herein, "drug", "drug" or "factor" (both of which correspond to agents in English) are used interchangeably as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly. Saccharides, oligosaccharides, lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.)) , These complex molecules include, but are not limited to. Factors specific to a polynucleotide typically include polynucleotides that are complementary to the sequence of the polynucleotide with certain sequence homology (eg, 70% or more sequence identity). Examples include, but are not limited to, polypeptides such as transcription factors that bind to the promoter region. Factors specific for a polypeptide are typically an antibody or derivative thereof or an analog thereof (eg, a single chain antibody) specifically directed to the polypeptide, which the polypeptide accepts. Specific ligands or receptors when they are bodies or ligands, and substrates when their polypeptides are enzymes are included, but are not limited thereto.

As used herein, the term "interaction" refers to two substances, that is, a force (for example, an intermolecular force (Van der Waals force), a hydrogen bond, a hydrophobic interaction) between one substance and the other substance. Etc.). Usually, the two interacting substances are in an associative or bound state.

Therefore, as used herein, a "factor that specifically interacts" with a biological factor such as a polynucleotide or a polypeptide is defined as having an affinity for the polynucleotide or a biological factor such as the polypeptide. Representatively equal or higher, or preferably significantly (eg, statistically significant), than the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. ) Including high ones. Such affinity can be measured, for example, by hybridization assay, binding assay and the like.

As used herein, the term "specifically interacting" with a second substance or factor means that the first substance or factor interacts with the second substance or factor. It means interacting with a substance or factor other than a substance or factor (particularly, another substance or factor present in a sample containing a second substance or factor) with a higher affinity. Specific interactions for a substance or factor include transcription factors when both nucleic acids and proteins are involved, such as ligand-receptor reactions, hybridizations in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions. Examples include, but are not limited to, protein-lipid interactions and nucleic acid-lipid interactions, such as the reaction of the transcription factor with the binding site. Therefore, when a substance or factor is both nucleic acids, the first substance or factor "specifically interacts" with the second substance or factor means that the first substance or factor is the second substance. Alternatively, it includes having at least a partial complementarity to the factor. Also, for example, when both substances or factors are proteins, the first substance or factor "specifically interacts" with the second substance or factor means, for example, an interaction by an antigen-antibody reaction, a receptor. Interactions by -ligand reaction, enzyme-substrate interaction, etc. are included, but are not limited thereto. When two substances or factors include proteins and nucleic acids, the "specific interaction" of the first substance or factor with the second substance or factor is the subject of the transcription factor and its transcription factor. Includes interactions with the binding region of the nucleic acid molecule.

In the present specification, the "means" means a tool that can be an arbitrary tool for achieving a certain purpose. In particular, in the present specification, the "means for selectively recognizing" means a certain object as another. A means that can be recognized differently.

As used herein, the term "binding" 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. The physical interaction (binding) can be direct or indirect, the indirect being mediated by 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 is not accompanied by other substantial chemical intermediates.

As used herein, "contacting" means physics of a compound, either directly or indirectly, with respect to a polypeptide or polynucleotide capable of functioning as a marker, ligant, etc. of the invention. It means to bring them closer to each other. Polypeptides or polynucleotides can be present in many buffers, salts, solutions and the like. Contact includes placing the compound in, for example, a beaker, a microtiter plate, a cell culture flask or a microarray (eg, a gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.

As used herein, the term "label" refers to an entity (eg, substance, energy, electromagnetic wave, etc.) for identifying a molecule or substance of interest from others. Examples of such a labeling method include an RI (radioisotope) method, a fluorescence method, a biotin method, and a chemiluminescence method. When a plurality of markers or factors or means for capturing the markers of the present invention are labeled by the fluorescence method, the markers are labeled with fluorescent substances having different fluorescence maximum wavelengths. The difference in the maximum wavelength of fluorescence emission is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but when labeling AGEs, Alexa Fluor is desirable as the fluorescent substance. Alexa Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that supports a wide range of fluorescent wavelengths. Compared to other fluorescent dyes of the corresponding wavelength, it is extremely It is stable, bright, and has low pH sensitivity. Examples of the combination of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa555 and Alexa633, a combination of Alexa488 and Alexa555, and the like. When labeling a nucleic acid, any one that can bind to the base portion can be used, but cyanine dyes (for example, Cy3, Cy5, etc. of CyDye ^TM series), Rhodamine 6G reagent, N-acetoxy-N2- It is preferable to use acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and Rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of Rhodamine 6G reagent and fluorescein, and the like. In the present invention, such a label can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art and those skilled in the art can appropriately implement such methods depending on the label and the subject of interest.

As used herein, the term "diagnosis" refers to identifying various parameters related to a disease, disorder, condition, etc. in a subject and determining the current state or future of such a disease, disorder, condition. By using the methods, devices, and systems of the present invention, conditions within the body can be investigated and such information can be used to formulate a disease, disorder, condition, treatment to be administered or preventive action in a subject. Alternatively, various parameters such as a method can be selected. In the present specification, in a narrow sense, "diagnosis" means diagnosing the current situation, but in a broad sense, it includes "predictive diagnosis", "pre-diagnosis" and the like. Early diagnosis is sometimes called "early diagnosis".

In particular, in the present specification, "predictive diagnosis" or "pre-diagnosis" is used interchangeably with a molecule capable of recognizing sRAGE, AGEs, etc., and diabetic, diabetic nephropathy, diabetic nephropathy. , When referring to diabetic complications such as diabetic neuropathy, it means to detect the pre-symptomatic stage of diabetic complications such as diabetes, diabetic nephropathy, diabetic nephropathy, diabetic neuropathy, etc. It includes determining the risk of developing the disease in the future and determining the risk of developing diabetes for the purpose of preventing diabetic complications such as diabetes, diabetic nephropathy, diabetic nephropathy, and diabetic neuropathy. By using the methods, kits, compositions, detection agents, diagnostic agents, systems, etc. of the present invention, the condition in the body can be investigated in advance, and such information can be used for diseases, disorders, and conditions in the subject. Various parameters can be selected, such as the procedure or method for treatment or prevention to be administered. In the present specification, "predictive diagnosis" or "pre-diagnosis" is used in part with the concept of "early diagnosis" because it also includes diagnosis at a stage that cannot be diagnosed by other conventional methods.

As a general rule, the diagnostic method of the present invention is industrially useful because it can be used from the body and can be carried out without the hands of a medical worker such as a doctor. In the present specification, in particular, "predictive diagnosis, pre-diagnosis or diagnosis" may be referred to as "support" in order to clarify that it can be carried out without the hands of medical personnel such as doctors.

As used herein, the term "detecting agent" broadly refers to any factor capable of detecting a substance of interest (eg, modified LDL, AGEs, etc.).

As used herein, the term "diagnostic agent" broadly refers to any factor capable of diagnosing a condition of interest (eg, disease).

As used herein, the term "treatment" refers to a disease or disorder that, in the event of such a condition, prevents the exacerbation of such disease or disorder, preferably maintains the status quo, more preferably alleviates, and further. Preferably, it means to withdraw.

As used herein, the term "prevention" refers to preventing a disease or disorder from becoming such a condition before it becomes such a condition. By performing the predictive diagnosis or pre-diagnosis of the present invention, diseases related to AGEs such as diabetic angiopathy, non-alcoholic steatohepatitis, Alzheimer's dementia, etc. are prevented or measures for prevention are taken. be able to. By performing the predictive diagnosis or pre-diagnosis of the present invention, diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy can be prevented, or measures for prevention can be taken. ..

(Explanation of Preferred Embodiment)
Although the description of the preferred embodiment is described below, it should be understood that this embodiment is an example of the present invention and the scope of the present invention is not limited to such preferred embodiment. It should be understood that those skilled in the art can also easily make modifications, changes, etc. within the scope of the present invention with reference to the following preferred embodiments. For these embodiments, those skilled in the art can appropriately combine any of the embodiments.

<A assay system 1: A system in which capture AGEs are immobilized on a substrate>
The present inventors have found that the reactivity of AGEs with sRAGE differs depending on the type of AGEs. Based on this finding, the inventors have selected less reactive AGEs (captured AGEs (also referred to as competing AGEs)) and more reactive AGEs (stimulant AGEs) in the sample. By competitively binding to sRAGE, an assay system capable of detecting stimulating AGEs in a sample was constructed. The more reactive stimulant AGEs bind strongly to sRAGE and the amount of binding of AGEs for capture is reduced, so that the signal (for example, color development) from AGEs for capture or from sRAGE bound to AGEs for capture is reduced. However, it is possible to calculate the amount of stimulating AGEs in the sample from the degree of this decrease. Examples of the signal include colorimetricity, fluorescence, chemiluminescence, radioisotope and the like. The amount of AGEs can be calculated by preparing a calibration curve in advance and converting the absorbance (OD value) into a concentration.

Therefore, in one aspect, the present invention is a kit for detecting irritating advanced glycation end products (AGEs) in a sample, wherein the kit includes a substrate on which capture AGEs are immobilized and SEQ ID NO: 3. A kit is provided that comprises an advanced glycation end product receptor (sRAGE) containing an amino acid sequence having at least about 90% identity. As shown in FIG. 14, by immobilizing the capture AGEs on the substrate and detecting the labeled (for example, biotinylated) sRAGE, the binding between the capture AGEs and the sRAGE can be detected. This system is advantageous because it is not necessary to use a primary antibody against competing AGEs used in a system in which sRAGE is immobilized on a substrate as shown in FIG. 4 and a secondary antibody that recognizes the primary antibody. However, there is no big difference in detection ability, and it can be used in the same way. For the system in which sRAGE is immobilized on the substrate, refer to <assay system: system in which sRAGE is immobilized on the substrate>.

Further, when the capture AGEs are immobilized on the substrate, the site recognized by sRAGE does not appear on the surface, and there is a concern that the binding may be inhibited. However, unexpectedly, the AGEs immobilized on the substrate The binding between sRAGE and sRAGE was not inhibited.

Any AGEs can be used as the capture AGEs. For example, fructose (F) -AGEs have a higher binding ability to sRAGE than G-AGEs. Therefore, when F-AGEs are used as capture AGEs, they have a higher binding ability to sRAGE than F-AGEs, excluding G-AGEs. Is able to detect highly irritating AGEs. In this way, capture AGEs can be appropriately selected according to the measurement target. In a system that comprehensively detects stimulating AGEs, the capture AGEs are preferably glucose (G) -AGEs or fructose (F) -AGEs, and more preferably glucose (G) -AGEs.

Immobilization of capture AGEs on a substrate can be performed based on techniques well known in the art, but a carbonate-bicarbonate buffer (sodium carbonate-sodium bicarbonate buffer) is used on the substrate. It is preferably immobilized. Carbonate-bicarbonate buffers that can be used for immobilization have a pH in the range of about 9.2 to about 10.6, eg, about 9.2 to about 9.6, about 9.2 to about. 9.8, about 9.2 to about 10.0, about 9.2 to about 10.2, about 9.2 to about 10.4, about 9.4 to about 9.6, about 9.4 to about 9.8, about 9.4 to about 10.0, about 9.4 to about 10.2, about 9.4 to about 10.4, about 9.6 to about 9.8, about 9.6 to about It has a pH in the range of 10.0, about 9.6 to about 10.2, or about 9.6 to about 10.4, preferably in the range of about 9.4 to about 9.8. And more preferably can have a pH of about 9.6.

Examples of the stimulating AGEs include glyoxal (GO) -AGEs, glycolaldehyde (Glycol) -AGEs, glyceraldehyde (Glycer) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, and methylglyoxal (MGO)-. AGEs, acetaldehyde (AA) -AGEs, ribose-AGEs, xylose-AGEs, arabinose-AGEs, fructose (F) -AGEs, sorbitol-AGEs, galactose-AGEs or any combination thereof can be used, but not limited to these. In particular, in the assay system of the present invention, glyceraldehyde (Glycer) -AGEs and glycolaldehyde (Glycol) -AGEs can be detected with high sensitivity.

In some embodiments, the capture AGEs are bovine serum albumin, human serum albumin, ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin. , Fibrinogen, High Density Lipoprotein (HDL), Immunoglobin, Low Density Lipoprotein (LDL), Insulin, Keratin, Osteocalcin, Alcohol Dehydrogenase, Ardos Reductase, Catepsin B, Speroxide Dismudase, Myosin AMPase, Erythrocyte Spectrin, Or it can be myelin AGEs. In a preferred embodiment, the capture AGEs are albumin AGEs.

In the embodiment in which the capture AGEs are immobilized on the substrate, the kit of the present invention may further contain a detection agent for detecting sRAGE bound to the capture AGEs. In some embodiments, the sRAGE may be biotinylated. Examples of the detection agent include streptavidin, anti-sRAGE antibody and the like. In other embodiments, the sRAGE may be tagged with a myc tag, a flag tag, an HA tag, or the like, and can be detected using a detection agent such as an anti-myc antibody, an anti-flag antibody, or an anti-HA antibody. ..

The assay system of the present invention can detect stimulating AGEs in a very wide range of concentrations and is above about 100 ng / ml, for example in the concentration range of about 100 ng / ml to about 100,000 ng / ml, especially about 10 ng / ml. As described above, for example, it is possible to detect stimulating AGEs in a sample in a concentration range of about 10 ng / ml to about 10,000 ng / ml. A high-concentration sample may be diluted before measurement, and the upper limit of the measurement concentration range is not limited to the above.

The sRAGE that can be used in the kit of the present invention may have the amino acid sequence set forth in SEQ ID NO: 3, but is at least about 80%, at least about 85%, with SEQ ID NO: 3 as long as it has the ability to recognize AGEs. , At least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% may have amino acid sequences that are identical. In some embodiments, sRAGE can be expressed by expression systems well known in the art, such as E. coli, yeast, insect cells, animal cells and the like. In a preferred embodiment, the kit of the present invention may comprise a silkworm-type sRAGE using a silkworm expression system. By using the silkworm expression system, silkworm-type sugar chain modification is performed, and sRAGE with high stability can be produced. The method for producing the silkworm type sRAGE will be described in detail in <sRAGE (silkworm type) and its production method>. As a method for stabilizing sRAGE in addition to stabilization by silkworm-type sugar chain modification, a method well known in the art such as adding a stabilizer such as sugar or glycerol can be used.

<assay system 2: system in which sRAGE is immobilized on a substrate>
In another aspect, the invention also provides a system in which sRAGE is immobilized on a substrate. More specifically, the present invention is a kit for detecting irritating advanced glycation end products (AGEs) in a sample, the kit containing competing AGEs and SEQ ID NO: 3 at least about 90%. A kit is provided that includes a substrate on which an advanced glycation end product receptor (sRAGE) containing an amino acid sequence having the same identity is immobilized. In this embodiment, detection is performed using a detection agent specific for competing AGEs bound to sRAGE. In some embodiments, the kit of the present invention may further comprise a detector that specifically binds to competing AGEs. The detection agent can be appropriately selected according to the type of AGEs used. For example, when bovine serum albumin (BSA) is used as competitive AGEs, an anti-albumin antibody can be used as a detection agent. As various other embodiments, the above-mentioned <assay system 1: a system in which capture AGEs are immobilized on a substrate> and the other embodiments described above can be appropriately used.

<Substrate for detecting stimulant AGEs>
In a further aspect, the invention provides a substrate for detecting irritating advanced glycation end products (AGEs) in a sample, wherein the capture AGEs are immobilized. As various embodiments relating to immobilization, the above-mentioned <assay system 1: a system in which capture AGEs are immobilized on a substrate> and other embodiments described above can be appropriately used.

<Method of detecting irritating AGEs>
In a further aspect, the invention provides a method of detecting irritating advanced glycation end products (AGEs) in a sample. In the embodiment using the assay system 1, the method contacts a sample expected to contain stimulating AGEs with the sRAGE described herein on a substrate on which capture AGEs are immobilized. It may include a step and a step of adding a detection agent for sRAGE. The method may further include the step of measuring the absorbance and the step of converting the absorbance into the concentration of irritating AGEs in the sample. Conversion of stimulating AGEs to concentrations can be performed using a calibration curve. As various other embodiments, the above-mentioned <assay system 1: a system in which capture AGEs are immobilized on a substrate> and the other embodiments described above can be appropriately used.

In one embodiment, as the solid phase used in the detection method of the present invention, any molecule to be immobilized, for example, any molecule as long as AGEs or sRAGE can be immobilized can be used. In one embodiment, when using a mechanism such as enzyme-linked immunosorbent assay (ELISA), a microtiter plate is generally used as the solid phase (base material). The material of the substrate is any solid, either covalently or non-covalently, that has the property of binding to the biomolecule used in the present invention or can be derivatized to have such property. Materials can be mentioned. Examples of the material include polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, and polyacrylonitrile. , 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 and other organic materials. Can be done.

As used herein, the term "solid phase" is used interchangeably herein with "board" and "base" to refer to the material on which the device of the invention is constructed. A planar support on which molecules such as AGEs or sRAGE can be immobilized. When detecting using the principle of surface plasmon resonance in the present invention, the solid phase is preferably a base material of a glass substrate having a metal thin film containing gold, silver or aluminum on one side. When a mechanism such as enzyme-linked immunosorbent assay (ELISA) is used in the present invention, a microtiter plate is generally used as the solid phase (base material). The material of the substrate is any solid, either covalently or non-covalently, that has the property of binding to the biomolecule used in the present invention or can be derivatized to have such property. Materials can be mentioned. Suitable substrates include beads, gold nanoparticles, semiconductor nanoparticles (eg, CdTe nanoparticles, CdSe nanoparticles, GaN nanoparticles, ZnS nanoparticles, InP nanoparticles, etc.), silica nanoparticles, polystyrene nanoparticles, acrylic nanoparticles. Particles, latex nanoparticles, carbon nanoparticles, plates (eg microtiter plates), test tubes, chips, magnetic particles, films, fibers, slide glass, metal thin films, filters, tubes, balls, diamond-like carbon-coated stainless steel, etc. However, it is not limited to these.

As such materials for use as solids and substrates, any material capable of forming a solid surface can be used, for example glass, silica, silicone, ceramics, silicon dioxide, plastics, metals (alloys also Included), natural and synthetic polymers (eg, polystyrene, cellulose, amylose, chitosan, dextran, and nylon), including, but not limited to. The substrate may be formed from multiple 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. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinylacetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, 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 and other organic materials can be used. In the present invention, a film used for blotting, such as a nylon film, a nitrocellulose film, and a PVDF film, can also be used. When analyzing a high-density material, it is preferable to use a hard material such as glass as a material. The material preferable as the substrate varies depending on various parameters such as measuring equipment, and those skilled in the art can appropriately select an appropriate material from the various materials as described above.

As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression can be achieved using suitable methods, including, for example, mRNA measurement and immunological measurement methods. Examples of the molecular biological measurement method include Northern blotting, dot blotting, PCR method and the like. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blotting method, and an immunohistochemical staining method. Moreover, as a quantitative method, the ELISA method, the RIA method and the like are exemplified. It can also be performed by a gene analysis method using an array (for example, DNA array, protein array). DNA arrays are widely outlined in (Shujunsha ed., Cell Engineering Separate Volume "DNA Microarrays and Latest PCR Methods"). For protein arrays, see NatGenet. It is described in detail in 2002 Dec; 32 Suppl: 526-32. Examples of the gene expression analysis method include, but are not limited to, RT-PCR, RACE method, SCSP method, immunoprecipitation method, two-hybrid system, in vitro translation, and the like, in addition to the above. Such further analytical methods are described, for example, in the Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, edited by Yusuke Nakamura Yodosha (2002), and all of these descriptions are incorporated herein by reference. Will be done.

As used herein, the term "search" refers to finding another nucleobase sequence having a specific function and / or property by utilizing one nucleobase sequence electronically, biologically, or by some other method. Say. For electronic searches, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444- 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. -453 (1970)), but is not limited thereto. Biological searches include stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays (microarray assays) in which genomic DNA is attached to a glass plate, PCR and in situ hybridization, and the like. Not limited to. As used herein, it is intended that the genes used in the present invention should also include the corresponding genes identified by such electronic and biological searches.

As used herein, the term "kit" refers to a unit that is usually divided into two or more compartments and is provided with a portion to be provided (eg, antibody, label, etc.). The form of this kit is preferred when it is intended to provide a composition that should not be provided mixed and is preferably mixed and used immediately prior to use. Such kits preferably include the parts provided (eg, instructions or instructions describing how the reagents should be treated. As used herein, kits are reagent kits. When used as an antibody, the kit usually includes instructions and the like that describe how to use the antibody.

As used herein, the "instruction" describes the method of using the present invention to a doctor or another user. This instruction sheet contains words instructing the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like. In addition, the instruction sheet may include words indicating that the skeletal muscle is administered (for example, by injection) as the administration site. This instruction is prepared in accordance with the format prescribed by the regulatory agency of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.) and approved by the regulatory agency. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, which is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail). But can be provided.

As used herein, the term "in vivo" or "in vivo" refers to the inside of a living body. In a particular context, "in vivo" refers to the location where a substance of interest should be placed.

As used herein, the term "in vitro" refers to a condition in which a part of a living body is removed or released "in vitro" (for example, in vitro) for various research purposes. A term that contrasts with in vivo.

As used herein, the term "exovibo" refers to a series of actions that are performed outside the body but are intended to be returned to the body afterwards for a certain procedure.

The procedure for prescribing the in vitro diagnostic agent of the present invention as a medicine or the like is known in the art, and is described in, for example, the Japanese Pharmacopoeia, the United States Pharmacopeia, and the Pharmacopoeia of other countries. Therefore, one of ordinary skill in the art can determine the amount to be used without undue experimentation, as described herein.

<SRAGE (silkworm type) and its manufacturing method>
In one aspect, the present invention provides a reconstituted advanced glycation end product acceptor (sRAGE) containing a silkworm-type sugar chain and containing the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.

In a preferred embodiment, the silkworm-type sugar chain in the present invention includes a trimannosyl core, a complex-type sugar chain, an oligomannose-type sugar chain, or a hybrid-type sugar chain.

In one embodiment, the silkworm-type sugar chain is a trimannosyl core (from asparagine residue to GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man) -α1,3-Man). In addition to the structure, each molecule contains a sugar chain in which 0 to 2 molecules of GlcNAc and 0 to 4 molecules of Man 0 to 4 are bound. In another embodiment, the silkworm-type sugar chain of RAGE of the present invention is trimannosyl core (from asparagine residue GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man) -α1, In addition to the structure of 3-Man), each 2 molecules contain a sugar chain in which 0 to 4 molecules of GlcNAc and 0 to 8 of Man 0 to 8 molecules are bound. In another embodiment, the silkworm-type sugar chain of sRAGE includes, but is not limited to, sugar chains having the structures (1) to (8) ([Chemical formula 1] to [Chemical formula 8]).

In one embodiment, the composition ratio of sugar chains of silkworm type sRAGE is 90% or more for oligomannose type and less than 5% for complex type and hybrid type. The sugar composition of silkworm-type sRAGE is (Man) ₅ (GlcNAc) ₂ from 46% to 56%, (Man) ₇ (GlcNAc) ₂ from 23% to 33%, and (Man) ₆ (GlcNAc). ) ₂ is 7% to 15%, (Man) ₃ (GlcNAc) ₂ is 1% to 5%, (Man) ₃ (GlcNAc) ₃ is 2% to 8%, and (Man) ) ₄ (GlcNAc) ₃ is 1% to 5%. In a preferred embodiment, the sugar composition is (Man) ₅ (GlcNAc) ₂ from 48% to 54%, (Man) ₇ (GlcNAc) ₂ from 25% to 31%, and (Man) ₆ (Man) ₆ ( GlcNAc) ₂ is 9% to 13%, (Man) ₃ (GlcNAc) ₂ is 2% to 4%, (Man) ₃ (GlcNAc) ₃ is 3% to 6%, and ( Man) ₄ (GlcNAc) ₃ is 2% to 4%. In a more preferred embodiment, the sugar composition is (Man) ₅ (GlcNAc) ₂ is 51.4%, (Man) ₇ (GlcNAc) ₂ is 27.5%, and (Man) ₆ (GlcNAc). ) ₂ is 10.8%, (Man) ₃ (GlcNAc) ₂ is 2.8%, (Man) ₃ (GlcNAc) ₃ is 4.6%, and (Man) ₄ ( GlcNAc) ₃ is 2.5%. For these detailed information, reference is made to FIG. 54 of WO2016 / 051808, which is incorporated herein by reference.

The sRAGE that has been subjected to the addition of the sugar chain or a sugar chain in which a further sugar is added to the sugar chain has excellent stability and has the ability to recognize AGEs having various structures.

In one embodiment, the silkworm-type sugar chain binds to asparagine at position 3 and / or asparagine at position 59 of SEQ ID NO: 3.

In another embodiment, the sRAGE of the present invention is biotinylated. Biotinylation enables highly sensitive measurement results in bioassays and high-density accumulation in a certain direction on beads or membranes via streptavidin.

In one embodiment, the sRAGE of the present invention can be used as a composition for detecting advanced glycation end products (AGEs). Therefore, the present invention provides a composition for detecting AGEs, which comprises sRAGE containing the silkworm-type sugar chain of the present invention.

Although we do not want to be bound by theory, by using the sRAGE of the present invention, it is possible to raise the AGEs to be analyzed from about 10% of the conventional level to a level corresponding to almost all, and more accurate diagnosis. It has a remarkable effect in that it has become possible to do. In addition, a wide range of AGEs can be detected by the conventional detection method using reconstructed RAGE (sRAGE), but sRAGE has problems in practical use such as fragmentation in about one and a half months and loss of cognitive ability. It is also remarkable that this can be overcome by using the sRAGE of the present invention. The improved sRAGE of the present invention has undergone glycosylation, is a very stable molecule, and has maintained its cognitive activity for a long period of time when stored at 4 ° C. for about 1 year or more. In addition, the improved sRAGE of the present invention made it possible to concentrate a small amount of AGEs and detect AGEs having various structures.

Therefore, in another aspect, the present invention is also a method for producing sRAGE, in which A) a nucleic acid molecule encoding sRAGE is expressively incorporated into a silkworm or an organism that imparts a sugar chain similar to that of silkworm; B. ) Provide a method comprising the step of arranging the silkworm or an organism that imparts a sugar chain similar to the silkworm under the conditions under which the gene is expressed to express the sRAGE; and C) the step of obtaining the sRAGE.

Although the present invention is produced in silkworm, any organism may be used as long as it can impart silkworm-type sugar chains, and any organism that imparts sugar chains similar to silkworm can be used. It is understood that it can be done.

In one embodiment, the expression takes place in the silkworm or the central silk gland of an organism that imparts sugar chains similar to the silkworm.

In a preferred embodiment of the present invention, step A) is achieved by microinjecting an expression vector containing the nucleic acid molecule encoding sRAGE. Of course, other than microinjection, it is understood that the expression vector can be introduced as described herein or by any other known method.

In one embodiment, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or a variant thereof.

In one embodiment, the method for producing a protein of the present invention comprises a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silkworm gland, and any protein whose expression is directly or indirectly regulated by the promoter. An organism that imparts a sugar chain similar to that of a transgenic silkworm or silkworm having the encoding nucleic acid sequence, and that expresses the arbitrary protein in the silk gland or secretes the sugar chain into the cocoon thread. The step of producing the imparting organism; and (b) the step of recovering the arbitrary protein from the organism imparting the same sugar chain as the produced transgenic silkworm silkworm may be achieved.

In one exemplary embodiment, the step of producing a silkworm or an organism that imparts a sugar chain similar to the silkworm of the present invention begins with a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silkworm gland, and It produces silkworm eggs having a nucleic acid sequence encoding any protein whose expression is directly or indirectly regulated by the promoter. Next, from the silkworms produced from the produced silkworm eggs or the organisms that impart sugar chains similar to silkworms, the transgenic silkworms that express any protein or the organisms that impart sugar chains similar to silkworms are selected. Can be realized by.

In one exemplary embodiment, transgenic silkworms or organisms that impart sugar chains similar to silkworms can be selected, for example, using selectable markers. As the selectable marker in the present invention, a marker generally used by those skilled in the art, for example, a fluorescent protein such as CFP, GFP, YFP, DsRed, etc. can be used. By using these markers, transgenic silkworms can be detected only by observing with a stereoscopic fluorescence microscope. Moreover, since the fluorescent colors are different, a plurality of markers can be used at the same time.

In yet another embodiment, as a method for recovering an arbitrary protein from a produced transgenic silkworm or an organism that imparts a sugar chain similar to that of silkworm, for example, a sugar chain similar to that of transgenic silkworm or silkworm is imparted. There is a method of recovering an arbitrary protein from a silkworm spit out by a living organism. As a recovery method, a method well known to those skilled in the art, for example, a method of dissolving a cocoon with 60% LiSCN and dialyzing with 20 mM Tris and 5 urea (Inoue, S., Tsuda, H., Tanaka, H. , Magoshi, Yand Mizuno (2001) Sericologia 4,157-163.) Can be used. Further, as another protein recovery method, for example, a method using a surfactant, a method of dissolving in an aqueous solution, or the like is possible.

In addition, a silkworm egg or a sugar chain similar to silkworm having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silk gland and a nucleic acid sequence encoding an arbitrary protein indirectly controlled by the promoter. Examples of the eggs of the organism to be imparted include (i) a nucleic acid sequence in which a nucleic acid sequence encoding a transcriptional regulatory factor is functionally bound downstream of a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silk gland. And (ii) downstream of the target promoter of the transcriptional regulator, a silkworm egg having a nucleic acid sequence to which a nucleic acid sequence encoding an arbitrary protein is operably linked or an egg of an organism that imparts a sugar chain similar to that of a silkworm Can be mentioned.

A sugar chain similar to that of a silkworm egg or silkworm having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silk gland and a nucleic acid sequence encoding an arbitrary protein whose expression is directly controlled by the promoter is imparted. As the egg of an organism, for example, a silkworm egg having a nucleic acid sequence in which a nucleic acid sequence encoding an arbitrary protein is operably linked downstream of a promoter of a nucleic acid sequence encoding a protein specifically expressed in the central silk gland or Examples thereof include eggs of organisms that impart sugar chains similar to those of silkworms. In such a silkworm egg, a nucleic acid sequence in which a nucleic acid sequence encoding an arbitrary protein is operably linked is introduced into the egg downstream of the promoter of the nucleic acid sequence encoding a protein specifically expressed in the central silk gland. Can be manufactured with.

As used herein, the term "operably linked" means that the protein is linked so as to realize the expression of the protein of interest, and typically, the promoter is linked by binding of a transcriptional regulator to the promoter. It means that the promoter and the nucleic acid sequence are bound so that the expression of the nucleic acid sequence existing downstream of the promoter is induced. Examples of the combination of the transcriptional regulator and the target sequence include GAL4 and UAS, TetR and TRE, and the like. By using GAL4 and UAS, or TetR and TRE, the expression site, timing, and amount of the target gene can be accurately controlled, and can be easily expressed in many tissues. Moreover, even if the gene to be expressed is a lethal gene, a strain can be created. The nucleic acid sequence used in the present invention can be prepared by a method such as hybridization technique, polymerase chain reaction (PCR) technique, site-directed mutagenesis method, or DNA synthesis. Whether or not the prepared nucleic acid sequence has promoter activity can be examined by those skilled in the art by a well-known reporter assay or the like using a reporter gene. Reporter genes that can be used are described elsewhere herein and any known one can be used.

The glycoprotein of the present invention is preferably a protein that does not cause irreversible denaturation in silk thread. Further, as such a protein, a protein having no secretory signal from the silk gland cell to the silk gland lumen can be exemplified.

The introduction of DNA into silkworm eggs or eggs of organisms that impart sugar chains similar to silkworms is, for example, a method of injecting a transposon as a vector into early developmental eggs (Tamura, T., Thibert, C., Royer, C). ., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., It can be done according to Prudhomme, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84).

Also, for example, the above DNA between the transposon inverted terminal repeats (Handler AM, McCombs SD, Fraser MJ, Saul SH. (1998) Proc. Natl. Acad. Sci. USA 95 (13): 7520-5). A vector having a nucleic acid sequence encoding a transposon transferase (helper vector) can be introduced into a silkworm egg or an egg of an organism that imparts a sugar chain similar to that of a silkworm. Helper vectors include pHA3PIG (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84). It is not limited.

As the transposon in the present invention, piggyBac is preferable, but the transposon is not limited to this, and marina, minos, etc. can also be used (Shimizu, K., Kamba, M., Sonobe, H). ., Kanda, T., Klinakis, AG, Savakis, C. and Tamura, T. (2000) Insect Mol. Biol., 9, 277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).

In the present invention, it is also possible to produce transgenic silkworms or organisms that impart sugar chains similar to silkworms by using a baculovirus vector (Yamao, M., N. Katayama, H. Nakazawa, M. Yamakawa, Y. Hayashi et al., 1999, Genes Dev 13: 511-516).

Further, the silkworm in the present invention or an organism that imparts a sugar chain similar to that of silkworm is not particularly limited, but for mass production of the target glycoprotein, a protein constituting silk thread such as fibroin protein is used. It is preferable to use a silkworm or an organism that imparts a sugar chain similar to that of silkworm, in which the production of proteins constituting silkworms is suppressed by mutation of a coding gene region (including a coding region, a promoter region, and an untranslated region). .. Examples of the silkworm or the organism that imparts the same sugar chain as the silkworm include the silkworm or the silkworm of a mutant strain in which the production of the protein constituting the silkworm is suppressed by the mutation of the gene region encoding the protein constituting the silkworm. Organisms that impart similar sugar chains, preferably silkworms of the bare silkworm line in which the production of proteins constituting silk thread is suppressed by the mutation, or organisms that impart sugar chains similar to silkworms, more preferably Nd-s ^D. However, the production of silkworm proteins is suppressed regardless of whether the cause of the suppression of silkworm protein production is artificial or depends on mutations that occur in nature. Any silkworm or an organism that imparts a sugar chain similar to that of the silkworm may be used. Such silkworms or organisms that impart sugar chains similar to silkworms are silkworms well known to those skilled in the art as sericin silkworms. The use of sericin silkworms further facilitates the purification of proteins synthesized from any gene introduced into the chromosome.

Further, as the silkworm or the organism that imparts the same sugar chain as the silkworm in the present invention, not only the silkworm having the property of producing non-diapausing eggs but also the silkworm having the property of producing dormant eggs (for example, in a practical variety). Arugunma, 200, Harumine, Kanetsuki, Kinshu, Kanewa, etc.) can also be used. Here, the dormant egg means an egg in which embryonic development is temporarily stopped after spawning, and the non-sleeping egg means an egg in which embryonic development does not stop after spawning and the larvae hatch.

When a silkworm having the property of laying a dormant egg is used, a non-sleeping egg is laid and DNA is introduced into the non-sleeping egg. As a method of laying a non-sleeping egg, for example, in Gunma, a method of culturing a dormant egg at 15 ° C. to 21 ° C. to allow an adult worm generated from the dormant egg to lay a non-sleeping egg, preferably a dormant egg. Is cultivated at 16 ° C to 20 ° C to allow the adult worms produced from the dormant eggs to lay non-sleeping eggs, more preferably the dormant eggs are cultivated at 18 ° C. A method of laying a dormant egg, most preferably a method of culturing a dormant egg at 18 ° C. to raise larvae generated from the dormant egg in full light and causing a grown adult to lay a non-sleeping egg. Can be done. Further, in 200, a method of culturing a dormant egg at 15 ° C. to 21 ° C. to allow an adult worm generated from the dormant egg to lay a non-sleeping egg, preferably a dormant egg is cultivated at 16 ° C. to 20 ° C. A method of laying a non-sleeping egg on an adult laid from the dormant egg, more preferably a method of culturing the dormant egg at 18 ° C. to cause an adult worm born from the dormant egg to lay a non-sleeping egg, or dormant. A method in which larvae born from eggs are bred in full light and grown adults lay non-sleeping eggs, most preferably by culturing the dormant eggs at 25 ° C., the larvae born from the dormant eggs are bred in full light. However, there is a method of causing a grown adult to lay a non-sleeping egg. Eggs can be cultured, for example, by placing them in an incubator at 18 ° C to 25 ° C or in a room at a constant temperature, and larvae can be bred in a breeding room at 20 ° C to 29 ° C using artificial feed. ..

In the present invention, the day length condition means a daily light-dark cycle during egg culture or larval breeding. Such conditions include a bright condition and a dark condition, and in particular, the full-bright condition means a 24-hour bright condition without darkness. The day length condition can be changed according to the variety. The above-mentioned dormant egg of the present invention can be cultured by those skilled in the art according to a general method for culturing silkworm eggs. For example, culturing is carried out according to the method described in "Ministry of Education (1978) Silkworm Species Manufacturing. Pp193, Jikkyo Shuppansha, Tokyo". Further, the breeding of silkworm larvae in the present invention can be carried out by a method well known to those skilled in the art. For example, breeding is carried out according to the method described in "Ministry of Education (1978) Silkworm Species Manufacturing. Pp193, Jikkyo Shuppansha, Tokyo".

In the present invention, whether or not the laid egg is a non-sleeping egg can be determined by the color of the egg. It is generally known that dormant eggs are colored dark brown and non-diapause eggs are yellowish white. Therefore, in the present invention, it is determined that the laid egg is a non-sleeping egg because it is not dark brown, more preferably yellowish white.

In another aspect, the invention provides a silkworm or an organism that imparts a sugar chain similar to a silkworm, in which a nucleic acid molecule encoding sRAGE is expressively incorporated.

In an embodiment of this aspect, the nucleic acid molecule used in the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 2 or a variant thereof.

In one embodiment, the silkworm of the present invention or an organism imparting a sugar chain similar to the silkworm is a promoter of a nucleic acid sequence encoding sRAGE specifically expressed in the central silkworm gland, and directly or indirectly by the promoter. A transgenic silkworm or silkworm that imparts a sugar chain similar to that of a transgenic silkworm or silkworm having a nucleic acid sequence encoding an arbitrary protein whose expression is regulated, and that expresses the sRAGE in the silkworm gland or secretes it into the cocoon thread. Provide an organism that imparts a sugar chain similar to that of Silkworm. The state of the transgenic silkworm of the present invention or an organism that imparts a sugar chain similar to that of silkworm is not particularly limited, and may be, for example, an egg state. By using the transgenic silkworm of the present invention or an organism that imparts a sugar chain similar to that of silkworm, sRAGE can be produced in large quantities.

The transgenic silkworm of the present invention or an organism that imparts a sugar chain similar to silkworm is preferably (i) operably linked downstream of the promoter of the nucleic acid sequence encoding sRAGE specifically expressed in the central silk gland. A transgenic silkworm or a sugar chain similar to silkworm having a nucleic acid sequence encoding a transcriptional regulatory factor to be produced and (ii) a nucleic acid sequence encoding sRAGE operably linked downstream of the target promoter of the transcriptional regulatory factor. Similar to transgenic silkworms or silkworms that have a nucleic acid sequence operably linked to the sRAGE-encoding nucleic acid sequence downstream of the promoter of the sRAGE-encoding nucleic acid sequence that is specifically expressed in the central silk gland. It is an organism that imparts the sugar chain of.

In other embodiments, the sRAGE can be extracted from the posterior silk glands (which produce fibroin instead of sericin) or eyebrows. In addition, in one other embodiment, the invention provides silk glands or cocoons of the transgenic silkworms of the invention or organisms that impart sugar chains similar to silkworms (those that produce silk glands or cocoons). Such silk glands or cocoons are useful as silk glands or cocoons containing a large amount of sRAGE.

The present invention also provides nucleic acid sequences or nucleic acid molecules (eg, DNA) for use in the methods of the invention. Such nucleic acid sequences or nucleic acid molecules include (a) a nucleic acid sequence encoding a transcriptional regulatory factor operably linked downstream of the promoter of the nucleic acid sequence encoding sericin, and (b) a target of the transcriptional regulatory factor. Nucleic acid sequence encoding any protein operably linked downstream of the promoter, (c) Nucleic acid sequence operably linked downstream of the sRAGE-encoding nucleic acid sequence of the nucleic acid sequence encoding sericin , Etc., and may be provided as a kit composed of a combination of these. The present invention also provides a vector in which the nucleic acid sequences (a) to (c) are inserted between the inverted terminal repeat sequences of the transposon. Furthermore, a kit containing the vector and a vector (helper vector) having a nucleic acid sequence encoding a transposon transferase is provided.

<Structure of sugar chain>
There are two types of sugar chains, a sugar chain that binds to asparagine (called an N-glycosidic bond sugar chain) and a sugar chain that binds to serine, threonine, etc. (called a 0-glycosidic bond sugar chain), depending on the sugar chain binding mode. It is roughly divided. The above-mentioned N-glycosidic bond sugar chains have various structures [Biochemical Experimental Method 23-Glycoprotein Sugar Chain Research Method (Glycoprotein Sugar Chain Research Center), edited by Reiko Takahashi (1989)], but in each case. It is preferable to have a common core structure as shown below. However, this point is the same even when the above-mentioned glycoprotein is not an antibody.

The silkworm type is as follows (Iizuka et al. FEBS Journal 276 (2009) 5806-5820). That is, in the above-mentioned production method, the above-mentioned silkworm-type glycoprotein has a sugar chain structure represented by the following chemical formula (1) (also referred to as trimannosyl core).

It is preferable to contain a glycoprotein having an N-glycosidic bond sugar chain (trimannosyl core) containing. Although not bound by theory, the above-mentioned sugar chain structure is a common feature of silkworm-type sugar chains, and as shown in Examples and the like, it has excellent stability and modified LDL or AGEs. This is because it is presumed that this sugar chain plays a certain role because it has been found that can be detected widely and sensitively. In the present specification, the sugar chain (1) is also referred to as N4-1 or sugar chain 000.1.

In the above structure, the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end. Examples of the binding of fucose to N-acetylglucosamine at the reducing end include α1,3 binding and α1,6 binding.

The N-glycosidic bond sugar chain has a high mannose type (oligomannose type) in which only mannose is bound to the non-reducing end of the core structure, and galactose-N-acetylglucosamine (hereinafter, Gal-GlcNAc) on the non-reducing end side of the core structure. It has one or more branches in parallel, and further has a structure such as sialic acid and bisecting N-acetylglucosamine on the non-reducing terminal side of Gal-GlcNAc (also referred to as a complex type). Both are synonymous.), Hybrid type having both high mannose type (oligomannose type) and complex type (complex type) branches on the non-reducing end side of the core structure.

In the present embodiment, the above-mentioned glycoprotein has a sugar chain structure represented by the following chemical formulas (2), (3) or (4).

This type is "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man-β1,2-GlcNAc) -α1,3-Man-β1,2-GlcNAc" in terms of asparagine residues. It is also described as. In the present specification, the sugar chain (2) is also referred to as a sugar chain 200.1.

(This type is also described as "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man-β1,2-GlcNAc) -α1,3-Man" in terms of asparagine residues, and is complex. It may also be classified into a type.) In the present specification, the sugar chain (3) is also referred to as a sugar chain 100.1.

(This type is also described as "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man) -α1,3-Man-β1,2-GlcNAc" in terms of asparagine residues, and is complex. It may be classified into types.)
It is preferable to include a glycoprotein having an N-glycosidic bond sugar chain containing. This is because it is known that the silkworm-type sugar chain contains the above-mentioned complex type sugar chain. In the present specification, the sugar chain (4) is also referred to as N4-2 or sugar chain 100.2.

Then, in the present invention, (2) is typically recognized. Among the above-mentioned glycoproteins, a glycoprotein having an N-glycosidic bond sugar chain containing a sugar chain structure represented by the chemical formula (3) or (4) may be contained.

Whether the proportion of glycoprotein having an N-glycosidic bond sugar chain containing the sugar chain structure represented by the chemical formula (2), (3) or (4) is 10 mol% or more, 20 mol% or more, or 30 mol% or more. For example, the N-glycosidic bond sugar chain of the glycoprotein is characterized by being imparted by the silkworm type sugar chain as compared with the N-glycoside bond sugar chain of the glycoprotein produced by general cells that do not produce the silkworm type. Because it will be strengthened.

(Oligomannose type)

This structure is "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,3-Man) -α1,6-Man (-α1,3-Man) -α1,6--" from the viewpoint of asparagine residues. Also described as "Man". In the present specification, the sugar chain (5) is also referred to as N3 or sugar chain M5.1. In the present invention, in addition to (2), this (5) is also typically recognized.

This structure is "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,3-Man-α1,2-Man) -α1,6-Man (-α1,3-Man) when viewed from the asparagine residue. ) -Α1,6-Man ”. In the present specification, the sugar chain (6) is also referred to as N2 or sugar chain M6.1. In the present invention, in addition to (2), this (6) is also typically recognized.

Of course, it is understood that in addition to these oligomannose types, those to which additional sugars are added can also be included as the sugar chain of the present invention.

(Sugar chain of sRAGE)
Examples of the sugar chain type of sRAGE include the following in addition to the above (1) to (6) ([Chemical formula 1] to [Chemical formula 6]).

(This type is "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man-α1,3-Man) -α1,3-Man-β1,2-GlcNAc" in terms of asparagine residues. ”, And may be classified as a hybrid type.) In the present specification, the sugar chain (7) is also referred to as N4-3 or sugar chain H4.11.

(This structure is "GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,3-Man-α1,2-Man) -α1,6-Man (-α1,3-Man)" when viewed from the asparagine residue. It is also described as "Man) -α1,6-Man-α1,2-Man" and may be classified as an oligomannose type.) In the present specification, the sugar chain (8) is N1 or sugar chain M7.2. Is also displayed.

The sugar chain structural analysis of sRAGE revealed that about 90% or more of them are oligomannose type sugar chains and contain several% of complex type and hybrid type sugar chains. Furthermore, it has an excellent effect that stability is improved by adding sugar chains.

As the sugar chain type of sRAGE that can be included in the present invention, FIGS. 20 and 54 of WO2016 / 051808, which are incorporated herein by reference, can be referred to.

In one embodiment, the silkworm-type sugar chain of RAGE of the present invention is trimannosyl core (from asparagine residue GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man) -α1, In addition to the structure of 3-Man), each molecule contains a sugar chain in which 0 to 2 molecules of GlcNAc and 0 to 4 of Man 0 to 4 molecules are bound. In another embodiment, the silkworm-type sugar chain of RAGE of the present invention is trimannosyl core (from asparagine residue GlcNAc-β1,4-GlcNAc-β1,4-Man (-α1,6-Man) -α1, In addition to the structure of 3-Man), each 2 molecules contain a sugar chain in which 0 to 4 molecules of GlcNAc and 0 to 8 of Man 0 to 8 molecules are bound.

In the sRAGE of the present invention, there are two sites that can receive sugar chain addition, and the sugar chain may be added to only one of the two sites or to both of them. In a preferred embodiment, sugar chains are added to both of the sites that can receive sugar chain addition at two sites. When sugar chains are added to both of the two places, the added sugar chains may be the same sugar chain or may be different. Examples of the sugar chain to be added include sugar chains having the structures (1) to (8) ([Chemical formula 1] to [Chemical formula 8]).

As described above, silkworm-type sugar chains are classified into oligomannose-type, complex-type, and hybrid-type. The sugar chain structure of sRAGE is 87% to 97% oligomannose type, 2% to 8% complex type, and 1% to 5% hybrid type. Preferably, 90% to 94% is oligomannose type, 3% to 6% is complex type, and 2% to 4% is hybrid type. Most preferably, 92.5% is oligomannose type, 4.6% is complex type and 2.5% is hybrid type.

In one embodiment, the sugar composition of silkworm-type sRAGE is (Man) ₅ (GlcNAc) ₂ (WO2016 / 051808 N3 in FIG. 54, which is incorporated herein by reference), ranging from 46% to 56% ( Man) ₇ (GlcNAc) ₂ is 23% to 33%, (Man) ₆ (GlcNAc) ₂ is 7% to 15%, and (Man) ₃ (GlcNAc) ₂ is 1% to 5%. (Man) ₃ (GlcNAc) ₃ is 2% to 8%, and (Man) ₄ (GlcNAc) ₃ is 1% to 5%. Preferably, the sugar composition is (Man) ₅ (GlcNAc) ₂ from 48% to 54%, (Man) ₇ (GlcNAc) ₂ from 25% to 31%, and (Man) ₆ (GlcNAc). ₂ is 9% to 13%, (Man) ₃ (GlcNAc) ₂ is 2% to 4%, (Man) ₃ (GlcNAc) ₃ is 3% to 6%, (Man). ₄ (GlcNAc) ₃ is 2% to 4%. Most preferably, the sugar composition is (Man) ₅ (GlcNAc) ₂ at 51.4%, (Man) ₇ (GlcNAc) ₂ at 27.5%, and (Man) ₆ (GlcNAc) ₂ Is 10.8%, (Man) ₃ (GlcNAc) ₂ is 2.8%, (Man) ₃ (GlcNAc) ₃ is 4.6%, and (Man) ₄ (GlcNAc). ₃ is 2.5%. For a detailed description of these, reference can be made to FIG. 54 of WO 2016/051808, which is incorporated herein by reference.

There are not many types of sugars that make up sugar chains that are bound to glycoproteins, and the most common ones are glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, There are about 7 to 8 types such as xylose. The structural modes are also limited to some extent, and slight structural differences among them are identified and precisely recognized to control various biological phenomena.

In the present specification, "fucose" means ordinary fucose, which is 6-deoxy-galactose, which is a kind of deoxy sugar, and has a chemical formula of C ₆ H ₁₂ O ₅ , molecular weight 164.16, and melting point 163. It is classified into hexose and monosaccharide at ° C and specific rotation of -76 °. Naturally, the L type is in the form of L-fucoside, which is widely present in animals and plants. In mammals and plants, it is found on N-linked sugar chains on the cell surface.

As used herein, "N-acetylglucosamine" means ordinary N-acetylglucosamine (N-acetyl-D-glucosamine, GlcNAc, NAG), which is a glucose-derived monosaccharide and is a few biochemical substances. It is an important substance for the chemical mechanism. Chemically this substance is an amide between glucosamine and acetic acid. N-acetylglucosamine is a component of glycosaminoglycan (mucopolysaccharide) such as glycoprotein and hyaluronic acid in mammals. N-acetylglucosamine forms the skeleton of an N-linked glycoprotein in which oligosaccharide chains centered on mannose are bound to asparagine (chitobiose structure), and is a major constituent sugar of sugar chains having a more complicated structure.

As used herein, the term "galactose" means ordinary galactose, and has the same molecular formula and molecular weight as glucose, C ₆ H ₁₂ O ₆ and 180. The configuration is 2-position (second from the top in Fischer projection), -OH at 5-position is in the same direction, 3-position and 4-position are in opposite directions, and D-galactose has the same orientation as 5-position D-glyceraldehyde. There is. It is a 4-epimer of glucose. Most of them are D-galactose in nature.

As used herein, "mannose" means ordinary mannose, which is a type of monosaccharide classified as aldohexose, and has the same molecular formula and molecular weight as glucose, C ₆ H ₁₂ O ₆ , 180. is there. The configuration is the 2-position (the second from the top in the Fischer projection), the -OH at the 3-position is in the same direction, the 4-position and the 5-position are in opposite directions, and the D-mannose has the same orientation as the 5-position D-glyceraldehyde. There is. It is a 2-epimer of glucose. Most of them are D-mannose in nature.

<Analysis method of sugar chains>
Analysis of Neutral Sugar / Amino Sugar Composition A sugar chain is typically composed of neutral sugars such as galactose, mannose and fucose, amino sugars such as N-acetylglucosamine, and acidic sugars such as sialic acid. In the composition analysis of sugar chains, neutral sugars or amino sugars can be released by acid hydrolysis of the sugar chains with trifluoroacetic acid or the like, and the composition ratio thereof can be analyzed. As a specific method, a method using a sugar composition analyzer (BioLC) manufactured by Dionex can be mentioned. BioLC is an HPAEC-PAD (high performance anion-exchange chromatography-pulsed aperture metric detection) method [J. Liq. Chromatogr. , 6, 1577 (1983)] to analyze the sugar composition.

The composition ratio can also be analyzed by a fluorescent labeling method using 2-aminopyridine. Specifically, a known method [Agric.Biol. Chem. , 55 (1), 283-284 (1991)], the sample is fluorescently labeled by 2-aminopyridylation, and the composition ratio can be calculated by HPLC analysis.

Sugar chain structural analysis The structural analysis of sugar chains is performed by the two-dimensional sugar chain mapping method [Anal. Biochem. , 171, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Sugar Chain Research Method (Glycoprotein Publishing Center), edited by Reiko Takahashi (1989)]. In the two-dimensional sugar chain mapping method, for example, the retention time or elution position of the sugar chain by reverse phase chromatography is plotted on the X-axis, and the retention time or elution position of the sugar chain by normal phase chromatography is plotted on the Y-axis. , A method of estimating the sugar chain structure by comparing with those results of known sugar chains.

Specifically, the glycoprotein is decomposed with hydrazine to release the sugar chain from the glycoprotein, and the sugar chain is fluorescently labeled with 2-aminopyridine (hereinafter abbreviated as PA) [J. Biochem. , 95, 197 (1984)], the sugar chain is separated from the excess PA-forming reagent by gel filtration, and reverse phase chromatography is performed. Next, normal phase chromatography is performed on each peak of the separated sugar chains. Based on these results, plot on a two-dimensional sugar chain map, sugar chain standard (manufactured by TakaRa), literature [Anal. Biochem. , 171, 73 (1988)], and the sugar chain structure can be estimated. Further, mass spectrometry such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain mapping method.

<Glycoprotein production method>
In this embodiment, glycoproteins are expressed in silk gland cells of insects classified into Lepidoptera such as silkworms.

At that time, the above-mentioned insect is preferably silkworm. This is because, in the case of silkworms, the breeding method has been established in the sericulture industry, and the ability to eject cocoons from the silk glands is excellent, so that the above-mentioned glycoprotein can be efficiently obtained in large quantities.

Silkworms typically produce 0.3-0.5 g of cocoons per head, most of which are silk proteins such as fibroin synthesized in the posterior silk glands and sericin synthesized in the middle silk glands. In this way, silkworms are organisms with excellent protein synthesis ability, and by utilizing this ability, recombinant proteins for analysis and evaluation can be produced in large quantities and at low cost. ..

Further, the above-mentioned silk gland cells are preferably middle silk gland cells. This is because sericin is synthesized in the central silk gland, so if the above-mentioned glycoprotein is expressed in sericin, the recombinant protein is localized in the sericin layer that exists around fibroin and is relatively soluble in water. Therefore, the recombinant protein can be efficiently expressed in the central silk gland, and the recombinant protein contained in the cocoon can be easily extracted without modifying its three-dimensional structure.

That is, in the method for producing glycoprotein in the present embodiment, the step of expressing glycoprotein is genetically modified so as to express the glycoprotein of the present invention, and the silk thread gland of the above-mentioned insect (for example, silkworm) individual. It is preferable to include a step of producing a silkworm containing the glycoprotein of the present invention. Here, usually, the production of a cocoon containing the above-mentioned glycoprotein in the silk gland of an individual insect means that the above-mentioned glycoprotein is typically expressed in the silk gland cell of an insect.

The step of extracting the target glycoprotein may include a step of discharging the cocoon containing the glycoprotein from the above-mentioned silk thread gland and a step of extracting the above-mentioned glycoprotein from the above-mentioned discharged cocoon. preferable. Because fibroin or sericin is synthesized in the silk gland, if the above-mentioned glycoprotein is expressed in fibroin or sericin, it is efficient in the silk gland to localize the recombinant protein in the fibroin layer or the sericin layer. This is because the recombinant protein can be expressed well and the recombinant protein contained in the cocoon can be extracted.

In one embodiment, the step of expressing the above-mentioned glycoprotein is such that in the above-mentioned individual insect, the gene encoding the above-mentioned glycoprotein is provided so that it can be expressed downstream of the sericin promoter in the genome of the above-mentioned insect. It is preferable to include a step of using an individual and a step of producing a cocoon containing the above-mentioned glycoprotein in the sericin portion as the above-mentioned cocoon. Then, in this case, the step of extracting the above-mentioned glycoprotein is a step of immersing the above-mentioned cocoon in the extract and extracting the above-mentioned glycoprotein from the above-mentioned sericin portion of the above-mentioned cocoon into the above-mentioned extract. Is preferably included. This is because the silk thread that constitutes the cocoon has a structure in which a sericin layer exists around the fibroin layer that exists in the center. The silk glands that synthesize silk proteins are functionally and morphologically divided into posterior silk glands, middle silk glands, and anterior silk glands, while the sericin that constitutes the sericin layer is synthesized in the middle silk glands. , The fibroin that constitutes the fibroin layer is synthesized in the posterior silk gland. When the tissue expressing the glycoprotein is the central silk gland, the glycoprotein is secreted into the sericin layer of the silk thread. On the other hand, when the tissue expressing the glycoprotein is the posterior silk gland, the glycoprotein is secreted into the fibroin layer of the silk thread. Then, in order to prepare a transgenic silkworm that secretes a glycoprotein into the sericin layer, in order to express the glycoprotein in the central silk gland in the vector, in addition to the structural gene of the glycoprotein, for example, of each structural gene An promoter that induces gene expression in central sericin cells may be assembled upstream. Examples of promoters that induce gene expression in central silk gland cells include promoters of the sericin 1 gene or the sericin 2 gene. The middle or posterior silk glands are commonly used synthetic pathways and can be used in the present invention as well. Alternatively, it may be expressed systemically. When expressed in silk glands, there are a method of extracting the middle (and posterior) silk glands and extracting them, and a method of extracting them from the cocoon. In addition, when expressed in the whole body, there is a method of grinding the whole body or squeezing the body fluid.

By using such an expression vector, a transgenic silkworm that secretes glycoprotein into the sericin layer can be produced by a single gene transfer operation. On the other hand, transgenic silkworms that secrete glycoproteins into the sericin layer can also be produced by multiple gene transfer operations.

However, it is not intended to exclude the method of producing transgenic silkworms that secrete glycoproteins in the fibroin layer. In order to express glycoproteins in the posterior silk gland, a fibroin layer is used using a vector in which, in addition to the structural genes of the glycoprotein, a promoter that induces gene expression in the posterior silk gland cells is incorporated upstream of each structural gene. A transgenic silkworm that secretes protein may be produced. Examples of promoters that induce gene expression in posterior silk gland cells include promoters of genes such as fibroin L chain, fibroin H chain, and fibroin hexamarin.

Similar to the case of producing a transgenic silkworm that secretes glycoprotein in the sericin layer, in the case of producing a transgenic silkworm that secretes glycoprotein in the fibroin layer, the above-mentioned expression vector set is used to generate multiple genes. Transgenic silkworms may be produced by the introduction operation.

These expression vectors have a function for introducing a gene into the silkworm chromosome. For example, if a partial sequence of an insect-derived DNA-type transposon is integrated, the gene can be introduced into the silkworm chromosome. Specifically, it is a plasmid vector having a pair of inverted repeat sequences existing at the end of a DNA-type transposon, and a gene sequence inserted into a chromosome in a region sandwiched between the pair of inverted repeat sequences, that is, It is an expression vector in which a gene and a promoter of a glycoprotein are integrated. Known insect-derived DNA-type transposons include piggyBac, mariner (Insect Mol. Biol. 9, 145-155, 2000), and Minos (Insect Mol. Biol. 9, 277-281, 2000). Among them, the sequence derived from piggyBac is most frequently used. In order to produce transgenic silkworms, this plasmid is microinjected into silkworm eggs together with piggyBac's transposase expression vector (helper plasmid). This helper plasmid is a recombinant plasmid vector that lacks one or both of the inverted repeat sequences of piggyBac and incorporates substantially only the transposase gene region of piggyBac. In the helper plasmid, as the promoter for expressing transposase, the endogenous transposase promoter may be used as it is, or the silkworm actin promoter, the Drosophila HSP70 promoter, or the like may be used. To facilitate screening of next-generation silkworms, the marker gene can be simultaneously incorporated into a vector incorporating the polynucleotide to be inserted.

In the present specification, the promoter can be bound to the nucleic acid sequence so that the expression of the nucleic acid sequence of the structural gene existing downstream of the promoter is induced by the binding of the transcriptional regulator to the promoter. Examples of the combination of the transcriptional regulatory factor and the target sequence include GAL4 and UAS, TetR and TRE, and the like. By using GAL4 and UAS, or TetR and TRE, the expression site, timing, and amount of the target gene can be accurately controlled, and can be easily expressed in many tissues. Moreover, even if the gene to be expressed is a lethal gene, a strain can be created.

In one embodiment, the glycoprotein produced by the method of the present invention is not limited as long as it is produced by the method of the present invention, and the signal sequence (eg, fibroin H signal peptide), enteroquinase recognition site (eg, DDDDK). (SEQ ID NO: 1)), biotinylated site (eg, BioEase tag), or tag sequence (eg, BioEase tag, His tag, etc.) may or may not be present. That is, the glycoprotein produced by the method for producing a glycoprotein of the present invention includes both a glycoprotein having a signal sequence and a glycoprotein not having a signal sequence.

Examples of silkworm eggs having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland of the present invention and a nucleic acid sequence encoding a glycoprotein whose expression is directly controlled by the promoter are specific to the silk gland. Downstream of the promoter of the nucleic acid sequence encoding the protein expressed in, there is a silkworm egg having a nucleic acid sequence operably linked to the nucleic acid sequence encoding the sugar protein of interest. In such a silkworm egg, a nucleic acid sequence in which the nucleic acid sequence encoding the target sugar protein is operably linked is introduced into the silkworm egg downstream of the promoter of the nucleic acid sequence encoding the protein specifically expressed in the silkworm gland. It can be manufactured by doing.

Further, as a silkworm egg having a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland of the present invention and a nucleic acid sequence encoding a recombinant glycoprotein whose expression is indirectly controlled by the promoter, for example, (I) A nucleic acid sequence in which a nucleic acid sequence encoding a transcriptional regulatory factor is functionally bound downstream of a promoter of a nucleic acid sequence encoding a protein specifically expressed in the silk gland, and (ii) a target promoter of the transcriptional regulatory factor. Downstream of, there is a silkworm egg having a nucleic acid sequence in which a nucleic acid sequence encoding a recombinant antibody is operably linked. In the present invention, the nucleic acid sequence encoding the recombinant antibody whose expression is directly or indirectly controlled by the promoter of the nucleic acid sequence encoding the protein specifically expressed in the silk gland promotes the secretion of the antibody and the amount recovered. It is preferable to have a signal sequence in order to increase the number of proteins. Specific embodiments of the signal sequence are described elsewhere herein and any known signal sequence can be used.

As one exemplary production method, larvae (F0 generation) hatched from silkworm eggs injected with a small amount of vector are bred. All the obtained F0 generation silkworms are crossed with wild-type silkworms or between F0 silkworms, and transgenic silkworms are selected from the next generation (F1 generation) silkworms. Transgenic silkworms are selected by, for example, PCR method or Southern blotting method. In addition, when a marker gene is incorporated, it can be selected by using its phenotypic trait. For example, when a fluorescent protein gene such as GFP is used as a marker gene, it can be carried out by irradiating F1 generation silkworm eggs and larvae with excitation light and detecting the fluorescence emitted by the fluorescent protein. Transgenic silkworms can be produced by the above methods.

When the glycoprotein of interest is recovered from the cocoon of the transgenic silkworm, it is recovered from the sericin layer when the glycoprotein is localized in the sericin layer, and from the fibroin layer when it is contained in the fibroin layer. Recovery of glycoproteins from the sericin layer can be carried out particularly easily. Since the sericin constituting the sericin layer is hydrophilic, the recombinant protein localized in this layer can be extracted without using a solution that denatures the protein. The extract for extracting the target glycoprotein from the sericin layer is not particularly limited as long as the glycoprotein can be extracted. For example, it may be a neutral salt solution, or it may be a solution containing a surfactant and other reagents for efficient extraction. In order to extract glycoprotein from the cocoon using these extracts, for example, a method such as immersing the fragmented cocoon in the extract and stirring it can be used. Further, the cocoon may be pulverized before extraction, or mechanical treatment such as ultrasonic treatment may be performed at the time of extraction. In addition, the silkworm silkworm gland cells described above may be appropriately transformed to express a transferase (for example, β-galactose transferase, N-acetylglucosamine transferase, etc.) so that further glycosylation is carried out. ..

In practicing the present invention, a reporter gene may be incorporated to confirm expression. Such a reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, and a β-glucuronidase gene (GUS) commonly used in those skilled in the art are used. ) And the GFP gene and the like. The expression level of the reporter gene can be measured by a method known to those skilled in the art, depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting the acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, it is a fluorescent compound by detecting the color development of the dye compound due to the catalytic action of the gene expression product, and when it is a luciferase gene, it is a fluorescent compound due to the catalytic action of the gene expression product. By detecting the fluorescence of Fluorescence, and in the case of β-chronidase gene (GUS), luminescence of Fluoron (ICN) and 5-bromo-4-chloro-3-indrill by the catalytic action of the gene expression product. The expression level of the reporter gene can be measured by detecting the color development of -β-glucuronide (X-Gluc) and, in the case of the GFP gene, by detecting the fluorescence by the GFP protein.

The method for producing a recombinant glycoprotein of the present invention includes a step of recovering a target glycoprotein synthesized in the silkworm body. The synthesized glycoprotein of interest is secreted into the middle or posterior silk glands in an active state without being insolubilized. Therefore, the glycoprotein of interest can be recovered from the central or posterior silk glands. As a method of recovering the target sugar protein from the middle silk gland or the posterior silk gland, for example, the silkworm in the spitting stage is dissected, and the middle silk gland or the posterior silk gland is excised in 20 mM Tris-HCl pH 7.4. However, the target sugar protein in the silk gland can be recovered by scratching the silk gland with a tweezers or a scalpel.

The glycoprotein of the object of the present invention can also be recovered from, for example, a cocoon spit out by a transgenic silkworm. As a recovery method, a method well known to those skilled in the art, for example, a method of dissolving a cocoon with 60% LiSCN and dialyzing with 20 mM Tris and 5 M urea (Inoue, S., Tsuda, H., Tanaka, H., Magoshi, Yand Mizuno (2001) Sericologia 4,157-163.) Can be used. Further, as another protein recovery method, for example, a method using a surfactant, a method of dissolving in an aqueous solution, or the like is possible.

The silkworm in the present invention is not particularly limited, but for mass production of the target sugar protein, a nucleic acid sequence region (coding region, promoter region, non-) that encodes a protein constituting silk thread such as fibroin protein. It is preferable to use silkworms whose production of proteins constituting silk threads is suppressed by mutations (including translation regions). As such silkworms, silkworms of a mutant strain in which the production of the proteins constituting the silk thread is suppressed by the mutation of the nucleic acid sequence region encoding the proteins constituting the silk thread, preferably the production of the proteins constituting the silk thread by the mutation. The silkworm of the bare silkworm line, which is suppressed, is more preferably Nd-s ^D, but the cause of the suppression of protein production of silk thread depends on whether it is artificial or not and the mutation that occurs in nature. Regardless of whether or not the silkworm is used, any silkworm whose production of proteins constituting silk thread is suppressed may be used.

One aspect of such a silkworm is a silkworm known to those skilled in the art as a sericin silkworm. The use of sericin silkworm enables mass production of the target glycoprotein in the central silk gland, and facilitates purification of the protein synthesized from the nucleic acid sequence encoding the recombinant antibody introduced into the chromosome. It is also preferable to use sericin silkworm in terms of production amount when producing a recombinant antibody in the posterior silk gland.

The silkworms in the present invention include silkworms having the property of producing non-sleeping eggs and silkworms having the property of producing dormant eggs (for example, the practical varieties Gunma, 200, Harumine, Kanetsuki, Kinshu, etc. Kanewa etc.) can be used. Here, the dormant egg means an egg in which embryonic development is temporarily stopped after spawning, and the non-sleeping egg means an egg in which embryonic development does not stop after spawning and the larvae hatch. When a silkworm having the property of laying a dormant egg is used, a non-sleeping egg is laid and DNA is introduced into the non-sleeping egg. As a method of laying a non-sleeping egg, for example, in Gunma, a method of culturing a dormant egg at 15 ° C. to 21 ° C. to allow an adult worm generated from the dormant egg to lay a non-sleeping egg, preferably a dormant egg. Is cultivated at 16 ° C to 20 ° C to allow the adult worms produced from the dormant eggs to lay non-sleeping eggs, more preferably the dormant eggs are cultivated at 18 ° C. A method of laying a dormant egg, most preferably a method of culturing a dormant egg at 18 ° C. to raise larvae generated from the dormant egg in full light and causing a grown adult to lay a non-sleeping egg. Can be done. Further, in 200, a method of culturing a dormant egg at 15 ° C. to 21 ° C. to allow an adult worm generated from the dormant egg to lay a non-sleeping egg, preferably a dormant egg is cultivated at 16 ° C. to 20 ° C. A method of laying a non-sleeping egg on an adult laid from the dormant egg, more preferably a method of culturing the dormant egg at 18 ° C. to lay a non-sleeping egg on the adult worm born from the dormant egg, or dormant. A method in which larvae born from eggs are bred in full light and grown adults lay non-sleeping eggs, most preferably by culturing the dormant eggs at 25 ° C., the larvae born from the dormant eggs are bred in full light. However, there is a method of causing a grown adult to lay a non-sleeping egg.

Eggs can be cultured, for example, by placing them in an incubator at 18 ° C to 25 ° C or in a room at a constant temperature, and larvae can be bred in a breeding room at 20 ° C to 29 ° C using artificial feed. ..

The above-mentioned dormant egg of the present invention can be cultured by those skilled in the art according to a general method for culturing silkworm eggs. For example, culturing is carried out according to the method described in "Ministry of Education (1978) Silkworm Species Manufacturing. Pp193, Jikkyo Shuppansha, Tokyo". Further, the breeding of silkworm larvae in the present invention can be carried out by a method well known to those skilled in the art. For example, breeding is carried out according to the method described in "Ministry of Education (1978) Silkworm Species Manufacturing. Pp193, Jikkyo Shuppansha, Tokyo".

In the present invention, whether or not the laid egg is a non-sleeping egg can be determined by the color of the egg. It is generally known that dormant eggs are colored dark brown and non-diapause eggs are yellowish white. Therefore, in the present invention, it is determined that the laid egg is a non-sleeping egg based on the fact that the egg is not dark brown, more preferably yellowish white.

Hereinafter, specific examples of the method for introducing a nucleic acid molecule into a silkworm egg will be described, but the method for introducing a nucleic acid molecule into a silkworm egg in the present invention is not limited to this method. For example, it is possible to introduce a nucleic acid molecule directly into an egg using a tube for injecting the nucleic acid molecule into a silkworm egg, but in a preferred embodiment, the eggshell is physically or chemically punctured in advance. Nucleic acid molecules are introduced through the holes. At this time, a tube for injecting a nucleic acid molecule can be inserted into the egg through the hole so that the insertion angle is substantially perpendicular to the ventral side surface of the egg.

In the present invention, as a method of physically making a hole in the eggshell, for example, a method of making a hole using a needle, a minute laser, or the like can be mentioned. A hole can be preferably made in the eggshell by a method using a needle. The material, strength, and the like of the needle are not particularly limited as long as the needle can make a hole in the eggshell of the silkworm. The needle in the present invention usually refers to a rod-shaped needle having a sharp tip, but the shape is not limited to this, and the overall shape is not particularly limited as long as it can make a hole in the eggshell. For example, a pointed pyramid-shaped material or a pointed triangular pyramid-shaped material is also included in the "needle" of the present invention. In the present invention, a tungsten needle can be preferably used. The thickness (diameter) of the needle of the present invention may be as long as it can make a hole through which the capillary, which will be described later, can pass, and is usually 2 to 20 μm, preferably 5 to 10 μm. On the other hand, as a method of chemically making a hole in the eggshell, for example, a method of making a hole using a chemical (hypochlorous acid or the like) or the like can be mentioned.

In the present invention, the position for making a hole is not particularly limited as long as the insertion angle with respect to the ventral side surface of the egg can be made substantially vertical when a tube for injecting a nucleic acid molecule is inserted through the hole, but it is preferable. Is the ventral side surface or the opposite side, more preferably the ventral side surface, and even more preferably the central portion of the ventral side surface of the egg from the slightly posterior end.

In the present invention, "almost vertical" means 70 ° to 120 °, preferably 80 ° to 100 °. In the present invention, the "position to become a germ cell in the future" is usually a position close to the egg surface on the ventral side of the egg (usually a position 0.01 mm to 0.05 mm from the egg surface), and is preferable. , The position near the egg surface in the center of the ventral side of the egg and slightly from the posterior pole.

The tube for injecting nucleic acid molecules of the present invention is not particularly limited in the material, strength, inner diameter, etc. of the tube, but when a hole is physically or chemically made in the eggshell before inserting the tube for injecting nucleic acid molecules. Is preferably a thickness (outer diameter) that allows the hole to pass through the hole. Examples of the tube for injecting nucleic acid molecules of the present invention include glass capillaries and the like.

In the method for introducing a nucleic acid molecule of the present invention, as a preferred embodiment, a hole is physically or chemically formed in the above-mentioned silkworm egg, and a tube for inserting the nucleic acid molecule is inserted at an insertion angle with respect to the ventral side surface of the egg. The step of inserting the nucleic acid molecule into the egg through the hole so as to be substantially vertical is performed using a manipulator in which the needle and the tube for injecting the nucleic acid molecule are integrated. In a preferred embodiment, the invention is typically practiced using a device that includes the manipulator as one of its components.

Such devices include an anatomical microscope, a lighting device, a movable stage, a coarse-moving manipulator fixed to the microscope with metal fittings, a micromanipulator attached to this manipulator, and air pressure for injecting nucleic acid molecules. It consists of an injector that adjusts. The pressure used for the injector is supplied from a nitrogen cylinder, and the pressure switch can be turned on by a foot switch. Injections are given to eggs fixed on a substrate such as a glass slide, and the position of the eggs is determined by a mobile stage. The glass capillary of the micromanipulator is operated by an operation unit connected by four tubes. The actual procedure is to position the tungsten needle with respect to the egg with a coarse-moving manipulator and move the egg horizontally with the lever on the stage to make a hole. Subsequently, the lever of the operation unit of the micromanipulator is operated to guide the tip of the glass capillary to the position of the hole, and the capillary is inserted into the egg again by the lever of the stage. In this case, the glass capillary needs to be inserted perpendicular to the ventral side of the egg. Turn on the foot switch, inject nucleic acid molecules, and operate the lever to remove the capillary from the egg. Close the holes with instant adhesive, etc., and protect them with an incubator at a constant temperature and humidity. The device used in the present invention preferably includes the device described in Japanese Patent No. 1654050 or a device obtained by improving the device.

Further, in one embodiment of the present invention, the silkworm egg used for introducing the nucleic acid molecule may be fixed to the substrate. As the substrate of the present invention, for example, a slide glass, a plastic plate, or the like can be used, but the substrate is not particularly limited thereto. In the above aspect of the present invention, it is desirable to align and fix the eggs in order to accurately inject the nucleic acid molecule into a position in the silkworm egg that will become a germ cell in the future. Further, in the above aspect, the number of silkworm eggs fixed to the substrate is not particularly limited. When a plurality of silkworm eggs are used, the direction of fixing the silkworm eggs to the substrate is preferably a direction in which the dorsoventral direction is constant. The above-mentioned silkworm eggs of the present invention are fixed to a substrate, for example, by spawning eggs on a commercially available mount (rose seed mount) coated with water-based glue in advance, adding water to the mount to peel off the eggs, and then in a wet state. Eggs are aligned on a substrate and air-dried. Eggs are preferably fixed on a slide glass in the same direction. The egg can also be fixed to the base by using double-sided tape, an adhesive or the like.

Whether or not a nucleic acid molecule has been introduced into a silkworm egg is determined, for example, by re-extracting the injected nucleic acid molecule from the egg and measuring it (Nagaraju, J., Kanda, T., Yukuhiro, K., Chavancy, G., Tamura, T. and Couble, P. (1996) Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. Zool., 31, 589-598) and injected nucleic acid molecules How to see the expression of silkworms in eggs (Tamura, T., Kanda, T., Takiya, S., Okano, K. and Maekawa, H. (1990). Transient expression of chimeric CAT genes injected into early embryos of the It can be confirmed by domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65, 401-410).

In one aspect, the invention can build a system to realize the methods of the invention.

As used herein, the term "system" refers to any system for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and generally consists of one or more components, and if there are a plurality of components, those. The elements of are acting and related to each other, and refer to a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole. The system can be in any form such as a device, composition, diagnostic agent. Thus, the system is, for example, a diagnostic agent that is an in vitro drug, from a large-scale system with a measuring device, to a system with chromatography, a kit utilizing an immune response, a composition containing an antibody (ie, a monoclonal antibody of a marker). ) Etc. are understood to be included.

In one embodiment, the invention comprises sRAGE for detecting AGEs in a sample derived from a subject, the subject has AGEs-related diseases such as diabetes, diabetic nephropathy, diabetic nephropathy, and the like. Provided is a system or composition for pre-diagnosing or diagnosing whether or not diabetic complications such as diabetic nephropathy). It can be understood that these compositions or systems can use any factor or means as long as the AGEs can be identified. Therefore, it is understood that not only the factors or means specifically described herein, but any equivalent factor or means known in the art can be used.

In one embodiment, the factor used in the present invention is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, organic small molecules and complex molecules thereof, preferably the factor is a protein or complex. It is a molecule (eg, glycoprotein, lipid protein, etc.). Preferably, the factor is an antibody (eg, a polyclonal antibody or a monoclonal antibody). Such factors are preferably labeled or labelable. This is because it is easier to diagnose.

In a preferred embodiment of the invention, the means used are a mass analyzer, a nuclear magnetic resonance measuring instrument, an X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology. Target means (eg, Western blotting, ELISA, RIA), biochemical means (eg, pI electrophoresis, Southern blotting, two-dimensional electrophoresis), electrophoresis equipment, chemical analysis equipment, fluorescence two-dimensional differential electrophoresis (eg, pI electrophoresis, Southern blotting, two-dimensional electrophoresis). It is selected from the group consisting of 2DE-DIGE), isotope labeling method (ICAT), tandem affinity purification method (TAP method), physical means, laser microdissection and combinations thereof.

In a preferred embodiment of the invention, the system of the invention further comprises a standard of AGEs. Such standards are preferably used to confirm whether the AGEs detecting means are functioning normally.

In a preferred embodiment, the invention may further comprise means for purifying the sample of interest. Examples of such purification means include chromatography and the like. Purification can be used in preferred embodiments because it can improve diagnostic accuracy, but this is not essential.

In the present invention, the subject comprises a mammal, and in one embodiment, the subject comprises a rodent. Such rodents (eg, rats, mice, etc.) are preferred because model animals, especially diabetic or diabetic nephropathy model animals (eg, streptozotocin (Stz) mice, etc.) have been produced. In another preferred embodiment, the subject comprises a human.

In one embodiment, the factors or means used in the present invention have the ability to quantify the AGEs of the present invention. Such quantification should be a means or factor that allows the calibration curve to be drawn properly when a standard curve is drawn. Preferably, for example, antibody, mass spectrometry, chromatography analysis and the like can be mentioned. Therefore, in certain embodiments, the system of the present invention further comprises a quantification means for quantifying AGEs.

In one embodiment, the quantification means includes a determination means for comparing the standard curve with the measurement result to determine whether the AGEs are within the normal value range. Such a determination means can be realized by using a computer. In the detection or diagnosis of the present invention, as the method for measuring the concentration of AGEs, a method generally used for quantification of proteins can be used as it is as long as the method can specifically measure the concentration of AGEs. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

One of the preferred embodiments in the detection or diagnosis of the present invention is to capture AGEs on a carrier and measure the concentration of the captured AGEs. That is, a substance having an affinity for AGEs is immobilized on the carrier, and AGEs are captured on the carrier via the substance having the affinity. According to this embodiment, the influence of contaminants contained in the sample can be reduced, and the concentration of AGEs can be measured with higher sensitivity and accuracy. Examples of the detection method include known techniques such as EIA (enzyme immunoassay), RIA (radioimmunoassay), and ELISA (enzyme-linked immunosorbent assay).

When an immunoassay is used as a method for measuring AGEs in the present embodiment, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using an antibody immobilized on a carrier as a primary antibody can be easily constructed. For example, two types of antibodies specific for AGEs or sRAGE and having different epitopes can be prepared, one can be immobilized on a carrier as a primary antibody, and the other can be enzymatically labeled as a secondary antibody to construct a sandwich EIA system. it can. In addition, it is possible to construct an immunoassay system by a binding inhibition method or a competitive method. Furthermore, when a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentrations of a plurality of markers can be measured at the same time, and rapid measurement is possible. Therefore, the present invention can provide a reagent in which sRAGE is immobilized on a substrate. Compositions for detecting AGEs can also be provided using such reagents.

On the other hand, when mass spectrometry is used as the method for measuring AGEs in the present embodiment, AGEs can be captured on the carrier by ionic bonding or hydrophobic interaction in addition to the antibody. Ionic bonds and hydrophobic interactions are not as specific as bioaffinity such as antigens and antibodies, and substances other than AGEs are also captured, but according to mass spectrometry, they are quantified by a mass spectrometer spectrum that reflects the molecular weight. No problem. In particular, using a protein chip using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter, "SELDI-TOF"). -MS ") can be performed to measure the concentration of AGEs more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, especially weak cation exchange, can be used. A substrate and a metal ion substrate are preferably used.

<Manufacturing of biotinylated protein>
(Silkworm co-expressing biotin ligase)
In one aspect of the present invention, there is provided a silkworm or an organism that imparts a sugar chain similar to silkworm, in which a nucleic acid molecule encoding a target protein and biotin ligase are co-expressably incorporated.

Since the nucleic acid sequence encoding the target protein, for example, sRAGE, contains a biotinylated tag sequence, co-expression of biotin ligase causes the target protein to undergo biotinification. In one embodiment, the biotin ligase is BirA (SEQ ID NO: 10). The tag sequence to be biotinylated is BioEase. tag, Avi. Examples include, but are not limited to, tag, any sequence that can undergo biotinylation. These silkworms can efficiently produce biotinylated sRAGE by oral administration of biotin. Further, according to the sugar chain structural analysis of the obtained sRAGE, oligomannose-type sugar chains mainly composed of (Man) ₅ (GlcNAc) ₂ accounted for 90% or more, and several% of complex-type and hybrid-type sugar chains. Clarified to include. Furthermore, the addition of sugar chains improves stability (stable for 1 year or more), and immobilization that maintains directionality via biotin makes it possible to detect AGEs with various structures.

(Production of biotinylated protein by silkworm co-expressing biotin ligase)
In another aspect of the present invention, which is a method for producing a biotinylated protein, A) a step of co-expressing biotin ligase and a nucleic acid molecule encoding the protein into a silkworm or an organism that imparts a sugar chain similar to that of silkworm. B) The step of arranging the silkworm or an organism imparting a sugar chain similar to the silkworm under the conditions in which the nucleic acid molecule is expressed to express the biotin ligase and the protein; and C) administering biotin to the organism. , Provide a method comprising the step of obtaining the biotinylated protein. The method for producing a silkworm-type biotinylated protein of the present invention can express a target protein (for example, sRAGE, etc.) in a large amount within a few days in a biotinylated state with high biotinlation efficiency using a soluble protein as a soluble protein. It has an excellent effect of being. Furthermore, the glycosylated protein expressed using a silkworm or an organism that imparts a sugar chain similar to that of the silkworm is superior in pH stability to a protein using an Escherichia coli expression system in which sugar chain addition is not performed, and is depleted. Since the rate of aggregation by salt treatment is low and various buffer solutions can be applied, the range of utilization is wide. Further, according to the sugar chain structural analysis of the obtained sRAGE, oligomannose-type sugar chains mainly composed of (Man) ₅ (GlcNAc) ₂ accounted for 90% or more, and several% of complex-type and hybrid-type sugar chains. Clarified to include. Furthermore, the addition of sugar chains improves stability (stable for 1 year or more), and immobilization that maintains directionality via biotin makes it possible to detect AGEs with various structures.

The administration of biotin to silkworms is preferably oral administration of a diet containing biotin, but it can also be administered by a method such as injection.

In a preferred embodiment of the invention, step A) is accomplished by microinjecting an expression vector containing a nucleic acid molecule encoding biotin ligase and a protein. Of course, other than microinjection, it is understood that the expression vector can be introduced as described herein or by any other known method.

(Silkworm biotinylated sRAGE)
In another aspect, the invention provides a highly efficient biotinylated sRAGE. Due to the high efficiency of biotinification, it is possible to fix the AGEs on a substrate or the like that maintains the directionality via biotin, and it is possible to detect AGEs having various structures. In one embodiment, the biotinylation efficiency of the sRAGE of the present invention is about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, or about 60% or more. In a preferred embodiment, the biotinylation efficiency of the sRAGE of the present invention is about 60% or more. According to the sugar chain structural analysis of the obtained sRAGE, oligomannose-type sugar chains mainly composed of (Man) ₅ (GlcNAc) ₂ account for 90% or more, and contain several% of complex-type and hybrid-type sugar chains. It revealed that. Furthermore, the addition of sugar chains improves stability (stable for 1 year or more), and immobilization that maintains directionality via biotin makes it possible to detect AGEs with various structures.

In one embodiment, the sRAGE of the present invention is subjected to silkworm-type glycosylation. In a particular embodiment of the invention, the sRAGE of the invention has the amino acid sequence of SEQ ID NO: 3 in which there are two sites that can undergo glycosylation. The sugar chains added to these glycosylation sites may be the same or different. When the sugar chains are different, the added sugar chains can be any combination of the sugar chains (1) to (8) ([Chemical formula 1] to [Chemical formula 8]).

<Production of single chain antibody by silkworm>
In one embodiment, the protein produced by the present invention is susceptible to the addition of silkworm-type sugar chains. Examples of the sugar chain to be added include the above-mentioned trimannosyl core, complex type, oligomannose type and hybrid type, and preferably the above-mentioned structures (1) to (8) ([Chemical formula 1] to [Chemical formula 8]). It is a sugar chain having any of.

(General technology)
The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. et al. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. et al. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. et al. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. et al. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. et al. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. et al. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. et al. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual Biology; J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Volume Experimental Medicine "Gene transfer & Expression Analysis Experimental Method", Yodosha, 1997, etc. Is used as a reference.

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

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

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

(Production Example 1: Production of biotinylated sRAGE in the central silk gland by transgenic silkworm)
Briefly, the sRAGE vector was microinjected into silkworm eggs to obtain RAGE-expressing silkworms. Furthermore, by crossing with a line capable of expressing in the central silk gland, a recombinant silkworm expressing sRAGE in the central silk gland (easy to extract and high in purity) was produced. Furthermore, biotinylated sRAGE is produced by feeding a recombinant silkworm co-expressing biotin ligase with a diet containing biotin. A method for producing sRAGE by recombinant silkworm is also described in detail in WO 2016/051808, which is incorporated herein by reference.

(Materials and methods)
(Lineage production, expression, etc.)
UAS (Upstream Activation Sequence) sequence of pBac [Ser-UAS / 3xP3EGFP] (Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 19, 473 (2010)) A UAS vector was constructed by inserting a fragment encoding a signal peptide of the fibroin H chain amplified by PCR and a fragment encoding sRAGE downstream of. A genetically modified silkworm was created using this expression vector. The w1-pnd strains of white eyes, white eggs, and non-diapause strains maintained by the National Institute of Agrobiological Sciences were used as host strains. Lines expressing GAL4 in the central silk glands of the obtained transgenic silkworms (Ken-ichiro Tatematsu, Isao Kobayashi, Keiro Uchino, Hideki Sezutsu, Tetsuya Iizuka, Naoyuki Yonemura, Toshiki Tamura, Transgenic Research, 19, 473 (2010)) ) UAS (Upstream Activation Sequence). Among the obtained next-generation silkworms, individuals having both a GAL4 construct and a UAS construct were selected by a selection marker. The larvae on the 5th and 6th days were dissected, and the central silk gland was removed. The protein was extracted by shaking with 1 mL of PBS + 1% Triton X-100 extract at 4 ° C. for 2 hours.

(purification)
A solution obtained by removing sericin from the central silk gland extract by freeze-thaw treatment is bound to a TALON resin (metal chelate affinity chromatography resin using Co) equilibrated with PBS to gradually increase the imidazole concentration in PBS. The sRAGE was eluted with. A 50 mM to 500 mM imidazole-eluting fraction was collected and dialyzed against PBS (−). Further, the fraction was bound to Mutein Matrix equilibrated with PBS, and the fraction eluted with PBS containing 1.5 mM to 3 mM biotin was dialyzed against PBS to purify the biotinylated sRAGE.

(Array)
The sequence of sRAGE produced in this example is shown in SEQ ID NO: 3. The structure of the N-linked sugar chain of the silkworm central silk gland-expressing protein is as follows, and sRAGE is specifically expressed in the silkworm central silk gland. Glycoprotein sugar chains expressed in the central silk gland have almost no bouch mannose-type (with fucose) sugar chains or high-mannose-type sugar chains found outside the central silk glands, and are complex-type sugar chains, hybrid-type sugar chains, or , Oligomannose type sugar chain.

(Production Example 2: Production of sRAGE in the posterior silk gland by transgenic silkworm)
In this production example, sRAGE was expressed in the posterior silk gland. The production of sRAGE from both the posterior silk gland and the posterior / middle silk gland was carried out according to Production Example 1, but by increasing the amount of buffer used for extraction per silk gland 5-10 times. An increase in the amount of extraction was confirmed.

(result)
The results are shown in FIGS. 2 and 3. As shown in FIG. 2, it was shown that sRAGE was fully expressed in the posterior silk glands. It was also shown that sRAGE was biotinylated, as shown in FIG.

(Example 1: Detection of stimulating AGEs)
In this example, typical stimulant AGEs (GO, MGO, Glycer, Glycol-treated OVA) were detected. The outline of the assay system in Example 1 is shown in FIG.

(Materials and methods)
In the assay system used in Example 1, sRAGE is immobilized on the wells. The competitive AGEs and the stimulating AGEs in the sample bind to the competitively immobilized sRAGE, and the larger the stimulating AGEs, the smaller the binding amount of the competitive AGEs. Color is developed using a detection reagent specific to competing AGEs, and the lower the absorbance (the lower the competing AGEs), the more irritating AGEs in the sample. The concentration of stimulating AGEs in the sample can be determined by preparing a calibration curve in advance and converting the absorbance into a concentration.

・ Immobilization (50 μl / well)
The sRAGE (B. mori sRAGE) (5 μl / well / TBS) produced in the above production example was immobilized at 4 ° C. overnight. Since sRAGE is inactivated when left at the pH on the alkaline side for a long time, PBS (-) was used as a buffer for immobilization. Then, it was washed 3 times with TBST (200 μl / well).

-Blocking (250 μl / well)
Blocking was performed at room temperature for 2 hours using a Protein free blocking buffer (Thermo Fisher scientific). Then, it was washed 3 times with TBST (200 μl / well).

Competitive binding Samples and competitive AGEs were added to the wells (100 μl / well) and incubated for 2 hours at room temperature. The sample contains various reducing sugar-treated OVAs (final concentration 1-1500 ng / ml PBS (−)). For competing AGEs, fructose or glucose glycated BSA (final concentration 100 ng / ml PBS (−)) was used. Then, it was washed 5 times with TBST (200 μl / well).

Primary antibodies against competing AGEs (100 μl / well) were added and incubated for 1 hour at room temperature. As the primary antibody, anti-BSA (mouse monoclonal) (diluted 1: 6000 with Pierce blocking buffer) was used. Then, it was washed 5 times with TBST (200 μl / well). Further, an HRP-labeled secondary antibody (100 μl / well) was added and incubated at room temperature for 1 hour. Then, it was washed 5 times with TBST (200 μl / well).

Finally, TMB (50 μl / well), which is a substrate of HRP, was added and incubated at 25 ° C. for 10 minutes to develop color, and the reaction was stopped by 1N HCl (50 μl / well). 450 nm O.D. D. The value was measured.

(result)
The results are shown in FIG. Due to the competitive method, the larger the amount of stimulating AGEs, the smaller the amount of competitive AGEs bound to sRAGE and the lower the OD value. Due to competition with G-BSA, it was confirmed that any AGEs could be sufficiently detected at a concentration of 100 ng / ml or more (Glycer and Glycol-treated AGEs could be detected even at a concentration of about 10 ng / ml in the past. From the report, it seems that a measurement range of 500 to 3000 ng / ml is required for the detection of AGEs in human samples (Hiroaki Muramoto, Toshio Muto, Masayoshi Takeuchi, Journal of Diagnosis 46 (5), 467-473; and Takeuch et al., PLoS One. 2015; 10 (3): e0118652.), It was revealed that the assay system of the present invention can detect sensitively at a very low concentration.

(Example 2: Confirmation of the effect of the components in the sample on the assay system)
In this example, it was confirmed whether components other than AGEs in plasma or serum did not affect the ability of the assay system. In this example, the assay system of Example 1 was used. In addition, diluted plasma or serum was used as a sample.

(result)
As shown in FIG. 6, it was revealed that the OD value was almost constant at a dilution rate of 1/20 to 1/1000, and there was almost no difference in detection ability from the plasma-free sample. Similar results were obtained with the use of serum. As described above, the assay system of the present invention can detect AGEs with high sensitivity even in a biological sample.

(Example 3: Detection of typical irritating AGEs in the presence of serum)
In this example, the measurement range / detection sensitivity of typical stimulant AGEs was confirmed in the presence of 20-fold diluted Vietnamese serum. The detection was carried out in the same manner as in Example 1. Samples were prepared by dissolving each reducing sugar-treated OVA (GO, Glycer, MGO, Glycol-treated OVA) in human serum diluted 20-fold with PBS (−) (100 μl / well).

(result)
The results are shown in FIG. The concentration of each AGEs on the horizontal axis of FIG. 7 indicates the final concentration in the assay system. It has been shown that the assay system of the present invention can also be used for the detection of AGEs in human serum.

(Example 4: Construction of an assay system in which AGEs for capture are immobilized)
In Example 1, detection was performed in a system in which sRAGE was immobilized in a well, but in this example, it was examined whether detection could be performed in an assay system in which AGEs for capture were immobilized in a well.

(Materials and methods)
・ Immobilization (100 μl / well)
Capture AGEs (G-BSA or F-BSA, 0.1 μg / ml) diluted with PGS (-) were immobilized at 4 ° C. overnight and TBS-0.05% Tween 20 (TBST) (200 μl / well). Was washed 3 times.

-Blocking (250 μl / well)
Blocking was performed at 37 ° C. for 2 hours using a Protein free blocking buffer (Thermo Fisher scientific). Then, it was washed 3 times with TBST (200 μl / well).

Competitive binding sample (100 μl / well) and biotinylated B. Mori sRAGE (0.05 μg / ml / 20-fold diluted serum) was added to the wells and incubated overnight at 4 ° C. The sample contains various AGEs (20-fold diluted serum). Then, it was washed 5 times with TBST (200 μl / well).

HRP-conjugated streptavidin (50 μl / well) diluted with Pierce blocking buffer was added, incubated for 30 minutes at room temperature, and washed 5 times with TBST (200 μl / well). TMB (50 μl / well), which is a substrate for HRP, was added and incubated at room temperature for 10 minutes to develop color, and the reaction was stopped by 1N HCl (50 μl / well). 450 nm O.D. D. The value was measured.

(result)
It was shown that stimulating AGEs can be detected even in a system in which capture AGEs are immobilized in wells, as in Example 1 (FIGS. 8A to 8D).

(Example 5: Confirmation of the effect of mouse serum / plasma on the assay system)
In this example, the effect of experimental animal (mouse) serum / plasma on the assay system was confirmed.

(Materials and methods)
As experimental animals, 7-week-old mice (male, female) were used. A heparinized syringe was used to divide the blood into two parts after cardiac blood collection. Half of the plasma was prepared using a plasma separator, and the rest was allowed to stand for 30 minutes to coagulate the blood and then centrifuged to prepare serum. The prepared plasma and serum were stored at -80 ° C until use (samples do not freeze and thaw repeatedly).

・ Immobilization (100 μl / well)
Capture AGEs (G-BSA or F-BSA, 0.1 μg / ml) diluted with PGS (-) were immobilized at 4 ° C. overnight and TBS-0.05% Tween 20 (TBST) (200 μl / well). Was washed 3 times.

-Blocking (250 μl / well)
Blocking was performed at 37 ° C. for 2 hours using a Protein free blocking buffer (Thermo Fisher scientific). Then, it was washed 3 times with TBST (200 μl / well).

Competitive binding sample (100 μl / well) and biotinylated B. Mori sRAGE (0.05 μg / ml / 20-fold diluted serum) was added to the wells and incubated overnight at 4 ° C. Samples are mouse serum or plasma diluted with PBS (−). Then, it was washed 5 times with TBST (200 μl / well).

HRP-conjugated streptavidin (50 μl / well) diluted with Pierce blocking buffer was added, incubated for 30 minutes at room temperature, and washed 5 times with TBST (200 μl / well). TMB (50 μl / well), which is a substrate for HRP, was added and incubated at room temperature for 10 minutes to develop color, and the reaction was stopped by 1N HCl (50 μl / well). 450 nm O.D. D. The value was measured.

(result)
When serum was used, a slight inhibitory effect on detection was observed at 200-fold dilution, but it was shown that stimulant AGEs in serum can be detected in consideration of this inhibitory effect (Fig. 9A). ). On the other hand, when plasma was used, significant inhibition was observed even at a 200-fold dilution, so a 200-fold or higher dilution was also tested (FIG. 9B). It was shown that when the dilution ratio is 1000 times or more, it is possible to detect irritating AGEs in plasma in consideration of the inhibitory effect.

(Example 6: Detection of milk and lactic acid fermented beverages)
In this example, detection was performed on milk in which AGEs are expected to be produced and lactic acid fermented beverages A and B which are processed products thereof. The assay system used conforms to Example 4. The sample was biotinylated B. Includes mori sRAGE (0.05 μg / ml / 20-fold diluted serum) and milk or lactic acid fermented beverages diluted with PBS (−).

(result)
It was suggested that AGEs in animal-derived foods can be detected (Fig. 10). It has long been known as the Maillard reaction that AGEs are generated in the process of cooking and processing foods. AGEs in foods were regarded as added value such as flavor, but in recent years, it has been pointed out that some of AGEs in foods cause age-related diseases, so the flavor does not change and is added. There is a need for the development of processing methods that reduce only AGEs that cause aging diseases, and the utilization of foodstuffs that have the effect of reducing AGEs. It was shown that the assay system of the present invention can be applied to the detection of irritating AGEs in foods at that time and the evaluation of anti-glycation ability of agricultural, forestry and fishery products which are expected to have anti-glycation ability. ..

(Example 7: Optimization of assay system conditions)
In this example, the conditions of the assay system were optimized for better reproducibility.

The amount of protein to be immobilized and the stability after immobilization are considered to be important factors that determine the sensitivity and dynamic range of the assay. It is said that the treatment at the pH on the alkaline side improves the amount of protein immobilization and is less likely to cause peeling after immobilization, regardless of the material of all commonly used substrates (polystyrene, etc.). In the case of immobilizing a relatively stable protein such as an antibody, a carbonate buffer (pH 9.2 to 10.6) is widely used. The most important thing is that the immobilized protein is stable without problems such as exfoliation of the immobilized protein in the process of detection work. On the other hand, it is not always good if the amount of immobilization is large, and it is necessary to determine the optimum amount of immobilization (method) by examining the protein concentration at the time of the immobilization reaction.

Unlike antibodies, it was unclear whether AGEs had a stable structure against alkaline treatment and whether they functioned without losing their activity after alkaline treatment. Therefore, in Example 4, it was confirmed that they did not affect the structure / function of AGEs. The solidification reaction was carried out by diluting with PBS (-). It was sufficiently tolerable to detect irritating AGEs, but it was predicted that the AGEs immobilized on the substrate were not constant (the amount of immobilization was different, the AGEs were exfoliated during the detection process). .. Therefore, when the solidification conditions were examined, solidification was strongly performed under alkaline conditions (for example, pH 9.6), peeling during the detection work process was less likely to occur, and the mixture was exposed to alkaline conditions for about one night. However, it was confirmed that the AGEs immobilized by washing with PBS (-) after the immobilization maintained the structure (function). In this example, the immobilization reaction was carried out in sodium carbonate buffer (pH 9.6). When sodium carbonate buffer (pH 9.6) was used, G-BSA was firmly immobilized and better reproducibility was shown. Further, as shown in FIG. 11, when the fixation at pH 9.6 is performed, the correlation coefficient “R square” of the approximate curve of the calibration curve is higher than that when the fixation is performed at pH 7.4. It approached 1 and it was confirmed that the fitting was improved. As described above, when immobilization at pH 9.6 was performed, a more suitable calibration curve was obtained to obtain the measured value.

Further, when the concentration of biotinylated sRAGE is 0.1 μg / ml (final concentration in the reaction solution), the optimum competitive reaction with the immobilized G-BSA can be performed, and the reproducibility is improved. Was shown (Fig. 12). Further, when the amount of AGEs for capture was 0.9 μg / ml, a calibration curve more suitable for obtaining the measured value was obtained (FIG. 13). Those skilled in the art can appropriately adjust the concentrations of sRAGE and AGEs for capture in the assay system, and are not limited to the above concentrations.

(Materials and methods)
The outline of the assay system in this example is shown in FIG.

-Coating (100 μl / well)
0.9 μg / ml of capture AGEs (G-BSA or F-BSA) diluted in carbonate buffer (pH 9.6) were immobilized at 4 ° C. overnight. Washed 3 times with 200 μl / well of TBS-0.05% Tween 20 (TBST).

-Blocking (250 μl / well)
Blocking was performed at 37 ° C. for 2 hours using a Protein free blocking buffer (Thermo Fisher scientific). Then, it was washed 3 times with TBST (200 μl / well).

Competitive binding samples (100 μl / well) were added to the wells and incubated overnight at 4 ° C. The sample was biotinylated B. Includes mori sRAGE (0.1 μg / ml / PBS) and mouse serum or glycer-BSA. Then, it was washed 5 times with TBST (200 μl / well).

HRP-conjugated streptavidin (50 μl / well) diluted with Pierce blocking buffer was added, incubated for 30 minutes at room temperature, and washed 5 times with TBST (200 μl / well). TMB (50 μl / well), which is a substrate for HRP, was added and incubated at room temperature for 10 minutes to develop color, and the reaction was stopped by 1N HCl (50 μl / well). 450 nm O.D. D. The value was measured.

The assay system was used to track AGEs concentrations in the sera of laboratory animals (mice). In this example, the glyceraldehyde-BSA concentration equivalent (μg / ml), which is considered to be particularly related to age-related diseases, is displayed (FIG. 15). A calibration curve was prepared using glyceraldehyde-treated BSA, and the concentration of stimulant AGEs in the sample was converted into the glyceraldehyde-treated BSA concentration and shown, so that the stimulant AGEs concentration in each sample (μg / ml) was shown. ). Regarding human serum, it was confirmed that the measured value can be obtained in the linear part of the calibration curve by diluting 15 to 50 times (data was obtained by 2-step dilution because it depends on the healthy subject and the progress of the disease). ..

(result)
As shown in FIGS. 16A and 16B, it was revealed that the concentrations of AGEs in mouse serum and plasma can be measured in the juvenile, middle-aged, and aged groups. Furthermore, it was suggested that it is possible to track fluctuations over time.

(Example 8: Measurement of AGEs in foods derived from animals and plants, cooked foods)
Using the assay system of Example 7, it was verified whether AGEs in animal, plant-derived foods, and cooked foods could be measured.

(Materials and methods)
(1) Firing and sampling of pork Pork shoulder loin (6 sheets each) was divided into two groups, a non-firing group (3 sheets) and a calcined group (3 sheets), and a quantitative test of stimulating AGEs was carried out. Prior to the preparation of the baking sample, a frying pan (made by Heiwa Fraise, COM-9784) was placed on an IH heater (manufactured by Panasonic, KZ-PH33), and a radiation thermometer (made by Horiba, IT-550) was placed at a height of about 50 cm above the frying pan. I fixed it in the position. As a result, after confirming that the surface temperature of the frying pan was stable at a constant temperature (about 195 ° C.), three pieces of pork were sequentially baked. At that time, the sample was allowed to stand in the center of the frying pan and the front surface was fired for 3 minutes, the back surface was fired for 3 minutes, and then the front surface and the back surface were fired again for 1 minute each. The firing was unglazed, and the frying pan was covered with a lid to prevent drying. In addition, when a total of 6 firing group samples were fired intermittently, the oil remaining in the frying pan during sample replacement was removed with kitchen paper.

Next, meat pieces were collected from 9 places per sample in the fired group and the non-fired group using a leather punch (φ = 12 mm). The collected meat pieces were mixed and then shredded with a knife to homogenize.

(2) Extraction of Peptide from Pork Peptide was extracted from the homogenized sample by the following procedure. First, about 1 g of the homogenized sample was accurately weighed, and 9 mL of an 8 M urea solution (dissolved in physiological saline) was added per 1 g. After allowing to stand at room temperature for 1 hour, the sample suspension was disrupted by running a homogenizer (IKA Ultra-Turrax, DT-20) at 9000 rpm for 2 minutes twice. After separating 100 μL from the crushed sample, add 900 μL of physiological saline to reduce the urea concentration, then add 10 μL of 20 mg / mL trypsin (Wako Pure Drug), and stir with a rotator at 37 ° C. overnight. It was reacted. After completion of the reaction, the sample was allowed to stand at 95 ° C. for 5 minutes to inactivate trypsin, and centrifugation (15000 rpm, 15 minutes, room temperature) was performed to remove the residue as a precipitate fraction. On the other hand, 850 μL of the supernatant fraction containing the peptide was collected and cleaned by passing it through a filter (ADVANTEC, DISMIC-13, pore size 0.2 μm). Physiological saline was added to the sample thus obtained to make a total volume of 1.2 mL, of which 1 mL was subjected to HPLC (Shimadzu Corporation). After adsorbing on a reverse phase HPLC column (InertSustein C18 column, GL Science) equilibrated with ultrapure water / 0.2% formic acid, the mobile phase is replaced with 100% acetonitrile / 0.2% formic acid. The eluted fraction was fractionated. The eluted fraction was dissolved in 200 μL of ultrapure water after centrifugation, and the concentration was tested using a BCA protein quantification kit (Takara Bio, T9300A). As a result, several mg / mL peptide was obtained from each sample.

The obtained peptide was added to the AGEs detection system according to Example 7, and the AGEs produced by calcination were measured.

(result)
As shown in FIG. 17, by converting to Glycer-BSA equivalent, it can be confirmed that there is no significant difference in the amount of AGEs in the meat by simple baking without addition of sauce or the like. Similarly, stimulant AGEs could be measured in liquid samples such as green juice and milk. In the case of unglazed unglazed, it was not confirmed that cooking produced irritating AGEs that cause age-related diseases.

(Example 9: Measurement of BSA saccharified with various reducing sugars)
In this example, BSA saccharified with various reducing sugars (glucose, fructose, galactose, maltose, lactose, xylose, ribose, glycolaldehyde, glyoxal, methylglyoxal) was measured.

4 μg / mL BSA-AGEs and 0.1 μg / ml sRAGE were competed to quantify sRAGE bound to glucose-BSA immobilized in wells. The measurement was performed according to the assay system of Example 7. In addition, the stimulant AGEs when each glycated BSA was treated with trypsin to form a glycated peptide were also quantified. The trypsin treatment was performed to confirm whether or not the whole structure of the protein is essential for the recognition of RAGE, and whether or not the peptide modified by the reducing sugar also has the RAGE binding potential (stimulation to the living body).

(result)
The results are shown in FIGS. 18A and 18B. Depending on the type of reducing sugar used in the saccharification treatment, AGEs having different reactivity with sRAGE are generated, and the assay system of Example 7 can indicate AGEs saccharified with various reducing sugars in Glycer-BSA equivalent. It was confirmed that. It was also confirmed that the trypsin-treated glycated peptide can be measured. From the results, the binding force of sRAGE to AGEs saccharified with various reducing sugars is approximately as follows: glucose ≒ maltose ≒ lactose <fructose <galactose <ribose ≒ glyoxal <xylose ≒ glycolaldehyde ≒ methyl glyoxal.

(Note)
As described above, the present invention has been illustrated using the preferred embodiment of the present invention, but the present invention should not be construed as being limited to this embodiment. It is understood that the present invention should be construed only by the claims. It will be understood by those skilled in the art that from the description of specific preferred embodiments of the present invention, an equivalent range can be implemented based on the description of the present invention and common general technical knowledge. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood.

The present invention is useful in the field of diagnosing diseases related to AGEs, or in the field of predictive diagnosis, or in the food industry.

SEQ ID NO: 1: Enterocinase recognition site (DDDDK)
SEQ ID NO: 2: Nucleic acid sequence of sRAGE used in the present invention SEQ ID NO: 3: Amino nucleic acid sequence of sRAGE used in the present invention SEQ ID NO: 4: Biotinylated amino acid sequence "GLNDIFEAQKIEWHE"
SEQ ID NO: 5: Nucleic acid sequence of <Biotin-RAGE> (including fibroin H chain intron, signal peptide, BioEASE-tag & linker & FLAG and RAGE)
SEQ ID NO: 6: <Biotin-RAGE> amino acid sequence SEQ ID NO: 7: RAGE nucleic acid sequence SEQ ID NO: 8: RAGE amino acid sequence SEQ ID NO: 9: biotin ligase (BirA) nucleic acid sequence SEQ ID NO: 10: biotin ligase (BirA) Amino acid sequence

Claims (31)

A kit for detecting irritating advanced glycation end products (AGEs) in a sample.
A substrate on which capture AGEs are fixed,
A kit comprising an advanced glycation end product receptor (sRAGE) comprising an amino acid sequence having at least about 90% identity with SEQ ID NO: 3. The kit according to claim 1, wherein the capture AGEs are glucose (G) -AGEs or fructose (F) -AGEs. The kit according to claim 2, wherein the capture AGEs are glucose (G) -AGEs. The kit according to any one of claims 1 to 3, wherein the AGEs for capture are immobilized on the substrate using a carbonate-bicarbonate buffer solution. The kit according to claim 4, wherein the carbonate-bicarbonate buffer has a pH value of about 9.2 to about 10.6. The kit of claim 5, wherein the carbonate-bicarbonate buffer has a pH value of about 9.6. The stimulating AGEs are glyoxal (GO) -AGEs, glycolaldehyde (Glycol) -AGEs, glyceraldehyde (Glycer) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. AGEs, acetaldehyde (AA) -AGEs, ribose-AGEs, xylose-AGEs, arabinose-AGEs, fructose (F) -AGEs, sorbitol-AGEs, galactose-AGEs or any combination thereof, among claims 1-6. The kit described in any one item. The stimulating AGEs are glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. The kit according to claim 7, which comprises AGEs, acetaldehyde (AA) -AGEs, or any combination thereof. The kit according to claim 8, wherein the irritating AGE comprises glyceraldehyde-AGEs, glycolaldehyde-AGEs or any combination thereof. The capture AGEs include bovine serum albumin, human serum albumin, ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin, fibrinogen, and high-density lipoprotein. (HDL), immunoglobulins, low-density lipoprotein (LDL), insulin, keratin, osteocalcin, alcohol dehydrogenase, aldose reductase, catepsin B, spuroxide dissmudase, myosin ATPase, erythrocyte spectroins, or AGEs of myerin, The kit according to any one of claims 1 to 9. The kit according to claim 10, wherein the capture AGEs are albumin AGEs. The kit according to any one of claims 1 to 11, wherein the sRAGE is biotinylated. The kit according to any one of claims 1 to 12, further comprising a detection agent that specifically binds to the sRAGE. The kit according to claim 13, wherein the detection agent is streptavidin. The kit according to any one of claims 1 to 14, wherein the kit can detect the stimulating AGEs in a sample containing the stimulating AGEs of about 100 ng / ml or more. The kit according to claim 15, wherein the kit can detect the stimulating AGEs in a sample containing the stimulating AGEs of about 10 ng / ml or more. A kit for detecting irritating advanced glycation end products (AGEs) in a sample.
Competitive AGEs and
A kit comprising a substrate on which an advanced glycation end product receptor (sRAGE) containing an amino acid sequence having at least about 90% identity with SEQ ID NO: 3 is immobilized. The kit according to claim 17, wherein the competing AGEs are glucose (G) -AGEs or fructose (F) -AGEs. The kit according to claim 18, wherein the competing AGEs are glucose (G) -AGEs. The stimulating AGEs are glyoxal (GO) -AGEs, glycolaldehyde (Glycol) -AGEs, glyceraldehyde (Glycer) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. 17-19 of claims 17-19, comprising AGEs, acetaldehyde (AA) -AGEs, ribose-AGEs, xylose-AGEs, arabinose-AGEs, fructose (F) -AGEs, sorbitol-AGEs, galactose-AGEs or any combination thereof. The kit described in any one item. The stimulating AGEs are glyoxal (GO) -AGEs, glyceraldehyde (Glycer) -AGEs, glycolaldehyde (Glycol) -AGEs, 3-deoxyglucosone (3-DG) -AGEs, methylglyoxal (MGO)-. The kit according to claim 20, which comprises AGEs, acetaldehyde (AA) -AGEs, or any combination thereof. 21. The kit of claim 21, wherein the irritating AGE comprises glyceraldehyde-AGEs, glycolaldehyde-AGEs or any combination thereof. The competing AGEs include albumin, ovoalbumin, collagen, crystallin, tuberin, lysoteam, RNase, carmodulin, hemoglobin, muscle fiber protein, antithrombin III, ferritin, fibrin, fibrinogen, high density lipoprotein (HDL), immunoglobulins. , Low Density Lipoprotein (LDL), Insulin, Keratin, Osteocalcin, Alcohol Dehydrogenase, Ardos Reductase, Catepsin B, Sparoxide Dismudase, Myosin AMPase, Erythrocytorin, or Myerin AGEs, claims 17-22. The kit described in any one item. The kit according to any one of claims 17 to 23, further comprising a detection agent that specifically binds to the competitive AGEs. The kit according to claim 24, wherein the detection agent is an antibody against the competitive AGEs. A substrate for detecting irritating advanced glycation end products (AGEs) in a sample, wherein the capture AGEs are immobilized. The substrate according to claim 26, wherein the trapping AGEs are glucose (G) -AGEs or fructose (F) -AGEs. The substrate according to claim 27, wherein the trapping AGEs are glucose (G) -AGEs. The substrate according to any one of claims 26 to 28, wherein the AGEs for capture are immobilized on the substrate using a carbonate-bicarbonate buffer solution. 29. The substrate of claim 29, wherein the carbonate-bicarbonate buffer has a pH value of about 9.2 to about 10.6. The substrate according to claim 30, wherein the carbonate-bicarbonate buffer has a pH value of about 9.6.