JP4451158B2 - Transcriptional regulatory cis elements, transcriptional regulators that specifically bind to them, and uses thereof - Google Patents

Description

  The present invention relates to a novel transcriptional regulatory cis element in the SREBP-1c promoter, a novel transcriptional regulatory factor capable of binding thereto, a method for screening a prophylactic / therapeutic substance for metabolic disorders using them, which is related to genetic susceptibility to metabolic disorders, The present invention relates to a non-human recombinant animal in which a mutated promoter sequence that is less susceptible to metabolic disorders, as well as the SREBP-1c promoter or the gene for the above-mentioned transcriptional regulatory factor has been modified.

  The prevalence of type 2 diabetes has increased over the past decades. One important background of this phenomenon is the recent increase in populations in a state called metabolic syndrome consisting of several metabolic abnormalities such as hyperlipidemia, visceral obesity, impaired glucose tolerance, and hyperinsulinemia. That is. Metabolic syndrome can be induced in people with an unidentified genetic background by environmental factors such as a high-calorie diet and lack of exercise.

  High fructose diets induce metabolic disturbances in rats that are similar to metabolic syndromes, so rats with high fructose diets have been used as an established animal model of metabolic syndrome. Nagai et al. (Non-patent Document 1) induced the expression of sterol regulatory factor binding protein-1 (SREBP-1), a transcription factor that is a key for the expression of lipid synthase group in the liver, in rats fed with high fructose diet. However, it is disclosed that expression of peroxisome proliferator-responsive receptor α (PPARα), a ligand-responsive nuclear receptor that regulates the expression of enzymes involved in fatty acid oxidation, is down-regulated. These changes in transcription factor expression may play a central role in the development of metabolic disturbances in rats fed a high fructose diet.

  Furthermore, Nagata et al. (Non-Patent Document 2) show that there are strain differences in obesity and lipid metabolism abnormalities caused by a high fructose diet in mice, and that this is related to differences in the expression of SREBP-1c in the liver. Is disclosed. That is, while CBA mice gain weight and serum lipids due to high fructose diet, DBA / 2 mice show no change, and such metabolic abnormalities are positively correlated with the expression level of liver SREBP-1c mRNA. It was.

However, the mechanism of how fructose induces the expression of the SREBP-1 gene has not yet been elucidated, and the substance of genetic predisposition that tends to cause metabolic abnormalities due to a high fructose diet has not been confirmed.
Nagai et al., “Am J Physiol Endocrinol Metab”, (USA), 2002, Vol. 282, No. 5, p. E1180-1190 Nagata et al., "Diabetes mellitus", Japanese Diabetes Society, April 15, 2002, volume 45, augmentation number 2, p. S247

  The object of the present invention is to elucidate the mechanism of induction of SREBP-1c gene expression by fructose in mammals and to identify factors that regulate genetic susceptibility to dietary metabolic disorders, thereby effectively preventing metabolic disorders. • To provide a therapeutic agent.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that the SREBP-1c gene in CBA mice that cause lipid metabolism abnormalities due to a high fructose diet and DBA mice that hardly exhibit metabolic abnormalities. It was found that there is a difference in one base in the promoter region. Furthermore, as a result of gel shift assay using a nucleic acid probe having a nucleotide sequence containing the mutation site of these two groups of mice, the presence of two transcriptional regulatory factors capable of specifically binding only to the former mouse-derived nucleotide sequence was confirmed. Revealed. As a result of determining the amino acid sequences of these transcriptional regulatory factors by TOF-MS analysis, it was found that they are known Nonamer Binding Proteion (NBP), RNA binding motif protein, and X chromosome retrogene (RBMX) -like proteins. The expression of these transcriptional regulators markedly increased after meals, especially after feeding on a high fructose diet, but was hardly expressed on an empty stomach and correlated well with the expression of the SREBP-1c gene.
As a result of analyzing the promoter of a known human SREBP-1c gene, the present inventors have confirmed that a sequence homologous to the nucleotide sequence including the above-mentioned mutation site in CBA mice exists, and in humans as well as mice. We found that a polymorphism of may exist.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A nucleic acid comprising the same or substantially the same base sequence as part of the base sequence, comprising guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1,
[2] A nucleic acid having the following characteristics (1) and (2):
(1) 1 or 2 or more bases are substituted in the same or substantially the same base sequence as the part of the base sequence including the base sequence represented by SEQ ID NO: 1 or the guanine represented by base number 112, Contains a deleted, inserted or added nucleotide sequence
(2) A transcriptional regulatory factor capable of binding to a guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1 and a base sequence consisting of bases adjacent thereto cannot bind [3] represented by SEQ ID NO: 1. The nucleic acid according to [2] above, wherein the guanine represented by base number 112 in the base sequence is substituted with another base;
[4] The nucleic acid according to the above [3], wherein the other base is adenine,
[5] A part of the base sequence including the guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1 in the SREBP-1c promoter or a base sequence corresponding thereto is detected. , A method for diagnosing genetic susceptibility to metabolic disorders in test animals,
[6] The method according to [5] above, wherein the metabolic disorder is a sugar / lipid metabolic disorder,
[7] A screening method for a prophylactic / therapeutic substance for metabolic disorders, characterized by using the following (a) and the following (b) and / or (c):
(a) a DNA having the same or substantially the same base sequence as part of the base sequence, comprising the base sequence represented by SEQ ID NO: 1 or the guanine represented by base number 112
(b) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(c) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5, or a partial peptide thereof, or a salt thereof [8] The metabolic disorder is a sugar / lipid metabolic disorder [7] Described method,
[9] The method according to [7] above, wherein the inhibition of binding between the (a) and the (b) and / or (c) in the presence of a test substance is detected.
[10] A feature is that an animal cell containing a gene under the control of the promoter containing (a) is loaded with sugar, and the expression of the gene in the presence and absence of the test substance is compared. The method according to [7] above,
[11] The method according to [10] above, wherein the animal cell has the ability to produce (b) and / or (c),
[12] The method according to [10] above, wherein the animal cell is a hepatocyte.
[13] The method according to [10] above, wherein the sugar is fructose,
[14] It is under the control of a promoter containing DNA having the same or substantially the same base sequence as a part of the base sequence, including guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1. A method according to the above [10], wherein the animal containing the gene is loaded with sugar and the expression of the gene in the liver is compared under administration and non-administration of the test substance,
[15] The method according to [14] above, wherein the sugar is fructose,
[16] A prophylactic / therapeutic agent for metabolic disorders comprising a substance that suppresses the production or activity of the following (a) and / or (b):
(a) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(b) a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5, or a partial peptide thereof, or a salt thereof [17] The above [16], wherein the metabolic disorder is a sugar / lipid metabolic disorder Listed agents,
[18] The agent according to [16] above, wherein the activity inhibitor is an antibody against (a) and / or an antibody against (b),
[19] The above, wherein the activity-suppressing substance is DNA having the same or substantially the same base sequence as part of the base sequence, including guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1. [16] The agent according to
[20] The agent according to claim 16, wherein the production inhibitor is (c) and / or (d) below:
(c) a nucleic acid containing a base sequence complementary to the base sequence encoding (a) or a part thereof
(d) Nucleic acid containing a base sequence complementary to the base sequence encoding (b) or a part thereof [21] Feeding an effective amount of a substance that suppresses the production or activity of (a) and / or (b) below A method for preventing or treating metabolic disorders, including administration to animals,
(a) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(b) a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 5, or a partial peptide thereof, or a salt thereof [22] For the production of a prophylactic / therapeutic agent for metabolic disorders, use of a substance that suppresses production or activity of (a) and / or (b),
(a) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(b) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or a partial peptide thereof or a salt thereof [23] a protein having the following characteristics (1) and (2) or A peptide or a salt thereof,
(1) In a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3 or a partial peptide thereof or a salt thereof, one or more amino acids are substituted, deleted, inserted or added Containing the determined amino acid sequence
(2) A promoter containing the guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1 and binding to the same or substantially the same base sequence as a part of the base sequence, [24] a protein or peptide having the following characteristics (1) and (2) or a salt thereof,
(1) In a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 5, or a partial peptide thereof or a salt thereof, one or more amino acids are substituted, deleted, inserted or added Containing the determined amino acid sequence
(2) A promoter containing the guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1 and binding to the same or substantially the same base sequence as a part of the base sequence, [25] The prophylactic / therapeutic agent for metabolic disorders comprising the protein or peptide or salt thereof according to [23] above and / or the protein or peptide or salt thereof according to [24] above,
[26] A diagnostic agent for metabolic disorders comprising the following (a) and / or (b):
(a) an antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3 or a partial peptide thereof or a salt thereof
(b) an antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 5 or a partial peptide thereof or a salt thereof [27] contains the following (a) and / or (b) A diagnostic agent for metabolic disorders,
(a) a nucleic acid containing a base sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a part thereof
(b) Nucleic acid containing a base sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or a part thereof [28] A base sequence represented by SEQ ID NO: 1 A non-human transgenic animal into which a gene under the control of a promoter containing DNA having the same or substantially the same base sequence as the part of the base sequence, including guanine represented by middle base number 112,
[29] Features of (1) below:
(1) The following (a) and / or (b) includes a promoter that cannot bind
(a) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(b) An endogenous SREBP-1c gene having a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or a partial peptide thereof or a salt thereof is characterized by the following (2):
(2) It is under the control of a promoter containing DNA having the same or substantially the same base sequence as a part of the base sequence, including guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1. The non-human transgenic animal according to the above [28], which is substituted with a SREBP-1c gene having
[30] A non-human transgenic animal introduced with a gene under the control of a promoter having the following characteristics (1) and (2):
(1) In the base sequence represented by SEQ ID NO: 1 and including the guanine represented by base number 112, in the same or substantially the same base sequence as that part of the base sequence, one or more bases are substituted, Contains DNA having a deleted, inserted or added nucleotide sequence
(2) (a) and / or (b) below cannot be combined
(a) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 3, or a partial peptide thereof, or a salt thereof
(b) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 5, or a partial peptide thereof or a salt thereof [31] the same as or substantially the same as the base sequence shown in SEQ ID NO: 1 The endogenous SREBP-1c gene comprising a promoter having the same nucleotide sequence in the above is replaced with the SREBP-1c gene under the control of the promoter having the characteristics (1) and (2) above. Non-human transgenic animals,
[32] A non-human transgenic animal into which the following (a) and / or (b) is introduced,
(a) a DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a partial peptide thereof
(b) DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 or a partial peptide thereof
And [33] a non-human animal in which the following (a) and / or (b) is inactivated,
(a) DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3
(b) DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5
I will provide a.

  A novel transcriptional regulatory cis element in the SREBP-1c promoter found in the present invention includes the cis element because it interacts with a transcriptional regulatory factor that specifically binds to it and promotes the expression of SREBP-1c. A nucleic acid having a base sequence can be combined with the transcription regulator to screen for a compound that regulates the expression of the SREBP-1c gene, and thus a candidate compound for a prophylactic / therapeutic agent for metabolic disorders. Furthermore, the nucleic acid can detect the presence or absence of a mutation in the cis element in the SREBP-1c promoter, thus diagnosing the genetic susceptibility of mammals to dietary metabolic disorders.

The present invention relates to a novel transcriptional regulatory cis element (hereinafter referred to as “fructose responsive element; FRE”) that can promote transcription of downstream genes in response to feeding (sugar loading), particularly high fructose diet (fructose loading). ) "And nucleic acids containing the cis element.
The fructose responsive element (FRE) of the present invention is a guanine (hereinafter referred to as “G ₁₁₂ ”) represented by the base number 112 in the base sequence of the mouse-derived SREBP-1c promoter represented by SEQ ID NO: 1, such as CBA and C3H. partial nucleotide sequence comprising also) be abbreviated as, preferably partial nucleotide sequence consists of about 5 to about 30 bases, and more particularly the G ₁₁₂ part 5 downstream 3 'upstream 0-20 bases and G _112' The base sequence is the same or substantially the same as a partial base sequence having a total length of about 5 to about 30 bases consisting of 0 to 20 bases on the side.
The “substantially identical base sequence” means 1) a base sequence in which one or more bases (preferably 1 to several bases) except G ₁₁₂ are replaced with other bases in the above partial base sequence, 2 1) a base sequence in which one or more bases (preferably 1 to several bases) are deleted, 3) a base sequence in which 1 or 2 bases (preferably 1 to several bases) are inserted, and combinations thereof A nucleotide sequence that can promote transcription of downstream genes in response to food intake (sugar load), particularly high fructose diet (fructose load). Transcription-promoting activity is determined by the binding assay with transcription factors described below, or the sugar (eg, fructose) of a reporter gene (eg, luciferase, Green Fluorescent Protein (GFP), etc.) under the control of the promoter containing the nucleotide sequence to be examined. It can be assayed by detecting an increase in expression due to loading. As the “substantially identical base sequence”, for example, mammals other than mice (eg, humans, rats, rabbits, guinea pigs, hamsters, cows, horses, sheep, monkeys, dogs, cats, etc.) such as partial nucleotide sequence comprising a nucleotide corresponding to G ₁₁₂ of SREBP-1c gene promoter derived from strains or individuals postprandial) fructose diet show metabolic abnormalities trends such as serum lipids increase may preferably be mentioned. Specifically, for example, in the base sequence of the human-derived SREBP-1c promoter represented by SEQ ID NO: 13, a partial base sequence containing guanine represented by base number 39, preferably a partial base consisting of about 5 to about 30 bases The sequence, more specifically, a partial base sequence having a total length of about 5 to about 30 bases composed of the guanine and its 5 ′ upstream 0-20 bases and 3 ′ downstream 0-20 bases.

The nucleic acid containing the FRE of the present invention is a nucleic acid having the base sequence of the FRE of the present invention or a base sequence to which one or more bases are added 5 ′ upstream and / or 3 ′ downstream of the FRE. Any of them may be used (except for a nucleic acid containing the entire base sequence represented by SEQ ID NO: 1). The length of the base sequence to be added is not particularly limited. For example, the SREBP-1c promoter sequence (eg, SEQ ID NO: 6) further upstream than the 5 ′ upstream sequence of G ₁₁₂ of the base sequence represented by SEQ ID NO: 1 In which the base sequence represented by base numbers 1 to 637 and the like are included.
The nucleic acid may be DNA, RNA, or DNA / RNA chimera, and is appropriately selected according to its use (eg, expression promoter, diagnostic probe, therapeutic decoy nucleotide, etc.). However, DNA is preferable. The nucleic acid may be single-stranded or double-stranded, and in the case of double-stranded, it may be a hybrid of a DNA strand and an RNA strand. The nucleic acid may be a physiologically acceptable salt with an acid or a base, and for example, a physiologically acceptable acid addition salt is preferred. Such salts include, among others, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The nucleic acid (preferably DNA) containing the FRE of the present invention is a human or other mammal (eg, mouse, rat, rabbit, guinea pig, hamster, cow, horse, sheep, monkey, dog, cat etc.), preferably Cells or individuals that show a tendency of metabolic abnormalities such as serum lipid elevation after eating (especially after eating a high fructose diet), particularly preferably cells derived from CBA, C3H mice [eg, hepatocytes, spleen cells, neurons, glia Cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, Natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells Or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells exist [eg, brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus, Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine) ), Blood vessels, heart, thymus, spleen, salivary gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, cartilage, joint, skeletal muscle, etc.] from the known SREBP-1c gene promoter sequence (For example, Amemiya-Kudo et al., Journal of Biological Chemistry (J. Biol. Chem.), 2000, Vol. 275, No. 40, p. 31078-31085; GenBank registration) No. AB046200) is used as a probe to clone the genomic DNA containing the promoter region, cleaved into a DNA fragment containing the desired partial promoter sequence using a DNA-degrading enzyme, for example, an appropriate restriction enzyme, and separated by gel electrophoresis. It can be prepared by collecting the desired band and purifying the DNA. Alternatively, the SREBP-1c promoter containing the FRE of the present invention is obtained by PCR using the above crude cell extract or genomic DNA isolated therefrom as a template and a primer synthesized on the basis of a known SREBP-1c gene promoter sequence. Partial sequences can also be amplified and isolated.

Nucleic acids containing FRE of the present invention, based on the nucleotide sequence of SEQ ID NO: 1, a nucleic acid having substantially the same nucleotide sequence and its partial nucleotide sequence or base sequence containing G _112, It can also be obtained by chemical synthesis using a commercially available DNA / RNA automatic synthesizer.
In the case of chemical synthesis, the nucleic acid is an N-glycoside of a purine or pyrimidine base, other types of polynucleotides other than deoxyribonucleotides and ribonucleotides, or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetics). Sequence-specific nucleic acid polymers) or other polymers that contain special linkages, where the polymers contain nucleotides with configurations that allow base pairing and base attachment as found in DNA and RNA Or the like. They have known modifications, such as those with labels known in the art, capped, methylated, one or more natural nucleotides substituted with analogs, Intramolecular nucleotide modifications, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphoro Having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg monosaccharides, etc.) Possessing an intercurrent compound (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg α-anomeric type) Nucleic acid etc.). Here, the “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleotide may also have a modified sugar moiety, such as one or more hydroxyl groups substituted with halogens, aliphatic groups (eg, C _1-6 alkyl groups), or ethers, amines, etc. It may be converted to a functional group of Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
Such modified nucleic acids are useful for enhancing in vivo stability and improving cell permeability, for example, when the nucleic acids are used as therapeutic decoy nucleotides.

  When the nucleic acid containing the FRE of the present invention is used as a promoter for gene expression, a base sequence imparting basal promoter activity such as a TATA box is added downstream of the FRE. Furthermore, other transcription control cis sequences (eg, CAAT box, GC box, etc.) can be arranged at appropriate positions.

The present invention also provides a mutant SREBP-1c promoter having a mutation in the fructose responsive element of the present invention, and a transcriptional regulatory factor that can bind to the element (that is, NBP and / or RBMX-like protein described below) cannot be bound. Alternatively, a partial polynucleotide thereof containing the mutation site is provided. That is, the mutant FRE of the present invention or the mutant SREBP-1c promoter containing the mutant FRE includes a base sequence represented by SEQ ID NO: 1 or a part of the base sequence containing guanine (G ₁₁₂ ) represented by the base number 112 A base represented by SEQ ID NO: 1 containing a base sequence in which one or more bases (preferably 1 to several bases) are substituted, deleted, inserted or added in the same or substantially the same base sequence it is a nucleic acid, wherein the transcriptional regulatory factor capable of binding to a nucleotide sequence consisting of nucleotide adjacent to G ₁₁₂ and it in the sequence can not bind. Here, “substantially the same base sequence” has the same meaning as described above. The “adjacent base” may be either 5 ′ upstream side or 3 ′ downstream side of G ₁₁₂ , or may be both. Nucleotide sequence consisting of G ₁₁₂ and a base adjacent thereto is preferably from about 5 to about 30 bases, and more particularly the G ₁₁₂ Part 5 downstream 0-20 bases 3 'of the upstream 0-20 bases and G _112' Is a base sequence consisting of about 5 to about 30 bases in total length.
The mutant FRE and the mutant SREBP-1c promoter containing the mutant FRE can be used as a probe for detecting the mutation in the SREBP-1c promoter of an animal individual, and the mutant SREBP-1c promoter is a high fructose diet. It is useful as a transgene for preparing a resistant transgenic animal model.

Preferably, the mutant FRE of the present invention or the mutant SREBP-1c promoter containing the same is the above-described nucleic acid in which G ₁₁₂ is substituted with another base in the base sequence represented by SEQ ID NO: 1, particularly preferably, A nucleic acid as described above wherein G ₁₁₂ is replaced with adenine.

  The mutant FRE of the present invention or a mutant SREBP-1c promoter containing the mutant FRE is a human or other mammal having the mutant promoter (eg, mouse, rat, rabbit, guinea pig, hamster, cow, horse, sheep, monkey, dog, Cats), for example, cells or individuals that do not show metabolic abnormalities such as elevated serum lipids after eating (especially after eating a high fructose diet), particularly preferably cells derived from DBA, C57BL mice [eg, hepatocytes, spleen cells , Nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg macrophages, T Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, Bone cells, bone cells, osteoblasts, osteoclasts, mammary cells, or stromal cells, or precursor cells, stem cells or cancer cells of these cells] or any tissue in which these cells are present [eg, brain, brain (Eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gallbladder, bone marrow , Adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, salivary gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, cartilage, joint, skeletal muscle From the genomic DNA extracted from the known SREBP-1c gene promoter sequence (for example, Amemiya-Kudo et al., J. Biol. Chem., 2000, Vol. 275, Vol. 40 , P. 31078-31085; GenBank accession number: AB046200) as a probe, genomic DNA containing the promoter region is cloned, and a desired (partial) promoter sequence is obtained using a DNA-degrading enzyme such as an appropriate restriction enzyme. It can be prepared by cleaving the contained DNA fragment and separating it by gel electrophoresis, then recovering the desired band and purifying the DNA. Alternatively, it can also be isolated by PCR using a primer synthesized based on the known SREBP-1c gene promoter sequence using the crude cell extract or genomic DNA isolated therefrom as a template.

  In addition, the mutant FRE of the present invention or the mutant SREBP-1c promoter containing the mutant FRE has a known SREBP-1c gene promoter as a template, and has one or more bases (preferably 1 to several bases) in the FRE base sequence of the present invention. ) Can be obtained by site-directed mutagenesis by PCR using an oligonucleotide having a nucleotide sequence substituted, deleted, inserted or added as one primer. The fact that the obtained mutant FRE or the mutant SREBP-1c promoter containing the mutant FRE does not promote transcription of downstream genes in response to feeding (sugar load), particularly high fructose diet (fructose load), is described below. Or by examining changes in expression of a reporter gene (eg, luciferase, Green Fluorescent Protein (GFP), etc.) under the control of the mutant promoter due to sugar (eg, fructose) loading. .

  Alternatively, the mutant FRE of the present invention or the mutant SREBP-1c promoter containing the mutant FRE base sequence in the known SREBP-1c promoter has one or more bases (preferably 1 to several bases) substituted or missing. The lost, inserted or added base sequence can also be obtained by chemical synthesis using a commercially available DNA / RNA automatic synthesizer in the same manner as described above.

The present invention also provides a method for diagnosing genetic susceptibility to metabolic disorders in a subject animal (eg, a human or other mammal) by detecting a mutation in a fructose response element in the SREBP-1c promoter. That is, the method, SEQ ID NO: part of the base sequence containing a base sequence G _112, represented by 1 (i.e., CBA mouse, etc. FRE from) or the corresponding nucleotide sequence (i.e., the other of the present invention FRE or mutant FRE of the present invention) is detected.
Examples of the metabolic disorders include metabolic disorders caused by meals (especially high fructose diet), such as sugar / lipid metabolic disorders (eg, hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity, glucose tolerance) Abnormalities, fasting blood glucose disorders, hyperinsulinemia, hypertension, albuminuria, etc.).
As a method for detecting a mutation in FRE, any known SNP detection method can be used. As the detection method, for example, genomic DNA extracted from cells of a test animal is used as a sample, and the above FRE of the present invention or a nucleic acid containing the same, or a mutant FRE of the present invention or a nucleic acid containing the same as a probe. For example, according to the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)), hybridization is carried out while precisely controlling stringency, and only a sequence completely complementary to the probe is used. Or a mixed probe in which one of the FRE of the present invention or a nucleic acid containing the same and the mutant FRE of the present invention or a nucleic acid containing the same is labeled and the other is unlabeled, and gradually from the denaturation temperature. Hybridization is performed while lowering the reaction temperature, and a sequence that is completely complementary to one probe is hybridized in advance. And a method of preventing cross-reaction with the probe having a pitch.
Detection of mutations in FRE may be carried out by a known SNP detection method using PCR, such as PCR-SSCP method, allele-specific PCR, PCR-SSOP method, DGGE method, RNase protection method, PCR-RFLP method, etc. it can. For example, in the case of the PCR-SSCP method, the SREBP-1c promoter partial sequence 5 ′ upstream from the FRE of the present invention is a sense primer, and the SREBP-1c promoter complementary chain partial sequence 3 ′ downstream from the FRE is an antisense primer. Perform PCR using the cell extract of the test animal or genomic DNA purified from it as a template (label one of the primers or substrate nucleotides), and then convert the resulting amplified fragment into a single strand, followed by a non-denaturing gel The primary structure polymorphism can be detected from the difference in mobility when subjected to electrophoresis.

The present invention also provides two transcriptional regulators (hereinafter also referred to as “transcriptional regulators of the present invention”) that can bind to the FRE of the present invention. The transcriptional regulatory factor has an activity of specifically binding to the FRE of the present invention to promote transcription of a gene located downstream thereof, and has a DNA binding property that it cannot bind to the mutant FRE of the present invention. .
Specifically, the transcriptional regulatory factor of the present invention is (1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 3, and (2) represented by SEQ ID NO: 5. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 is specific for a conserved 9-mer sequence existing near the recombination site in the rearrangement of immunoglobulin or T cell receptor gene. It is a known protein called Nonamer Binding Protein (NBP) that was identified as a protein that binds to (Gene Dev., 3: 1801-1813, 1989). On the other hand, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 is one of the proteins belonging to the nuclear RNA-binding protein family having an RNA-binding motif, and is used in humans and mice. Is a protein encoded by a gene similar to an RNA binding motif protein, X chromosome retrogene (RBMX) gene (Nature Genet., 22: 223-224, 1999), which is known to exist on the X chromosome. Hereinafter, the former may be referred to as “NBP of the present invention” and the latter may be referred to as “RBMX-like protein of the present invention”.
The transcriptional regulatory factor of the present invention is a mammalian cell (eg, human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.). Cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells ( Examples, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, Osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells, etc.) or any tissue in which those cells are present, eg, brain, brain regions (eg Olfactory bulb, amygdala, basal cerebrum, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle Protein derived from lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testes, ovary, placenta, uterus, bone, joint, skeletal muscle It may be a protein synthesized by chemical synthesis or a cell-free translation system. Alternatively, it may be a recombinant protein produced from a transformant introduced with a polynucleotide having a base sequence encoding the amino acid sequence.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 is about 70% or more, preferably about 80% or more, more preferably about 90% or more, with the amino acid sequence represented by SEQ ID NO: 3. Particularly preferred are amino acid sequences having homology of about 95% or more, most preferably about 98% or more. Similarly, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 is about 70% or more, preferably about 80% or more, more preferably about 90%, with the amino acid sequence represented by SEQ ID NO: 5. %, Particularly preferably about 95% or more, and most preferably about 98% or more of amino acid sequences having homology.
Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 (or 5) are substantially the same as the amino acid sequence represented by SEQ ID NO: 3 (or 5). And a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 3 (or 5).

The substantially homogeneous activity includes, for example, the binding activity to the FRE sequence of the present invention and the transcriptional control activity of a gene under the control of a promoter containing the sequence. Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, for example, it is preferable that the transcriptional control activity is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). The quantitative factors such as the degree of protein and the molecular weight of the protein may be different.
The transcriptional control activity can be measured using a known method such as Northern analysis or gel shift assay for the target gene. Alternatively, the activity of the transcriptional regulatory factor of the present invention can be evaluated by a method using its intracellular localization, for example, by examining the degree of transfer from the cytoplasm to the nucleus.

The NBP of the present invention (or the RBMX-like protein of the present invention) is, for example, 1) 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 3 (or 5) About 1 to 2, preferably about 1 to 10, more preferably several (1 to 5) amino acids, 2) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 3 (or 5) An amino acid sequence added with the above (preferably about 1-30, preferably about 1-10, more preferably several (1-5)) amino acids, 3) represented by SEQ ID NO: 3 (or 5) An amino acid sequence in which 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5)) amino acids are inserted into the amino acid sequence; Represented by the number 3 (or 5) An amino acid sequence in which one or two or more (preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence are substituted with other amino acids. Or 5) a protein containing an amino acid sequence obtained by combining them is also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the transcriptional control activity is maintained.
The NBP of the present invention is preferably a protein having the amino acid sequence represented by SEQ ID NO: 3, ie, mouse NBP or a homolog thereof in other mammals. The RBMX-like protein of the present invention is preferably a protein having the amino acid sequence represented by SEQ ID NO: 5, ie, a mouse RBMX-like protein or a homologue thereof in other mammals.

The protein in the present specification has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. In the transcriptional regulatory factor of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C _6-12 aryl groups such as α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl and phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl; ₁₄ aralkyl group; pivaloyloxymethyl group is used.
When the transcriptional regulatory factor has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the transcriptional regulatory factor of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, the transcriptional regulatory factor of the present invention, amino acid residues at the N-terminus: amino protecting group (eg methionine residue) (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6} protected with an acyl group, N-terminal glutamine residue generated by cleavage in vivo is pyroglutamine oxidized, and a substituent on the side chain of an amino acid in the molecule (eg, —OH, — SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein having a sugar chain bound thereto.

The present invention also provides a partial peptide of the transcriptional regulatory factor of the present invention (hereinafter sometimes simply referred to as “the partial peptide of the present invention”). The partial peptide may be any peptide as long as it is a peptide having the partial amino acid sequence of the transcriptional regulatory factor of the present invention described above and has substantially the same activity as the transcriptional regulatory factor, For example, it includes a DNA binding domain and a transcriptional regulatory (activation) domain of the transcriptional regulatory factor. Here, “substantially the same quality of activity” means DNA (FRE of the present invention) binding activity and transcription promotion activity of a gene under the control of the FRE.
In addition, some peptides having a partial amino acid sequence of the transcriptional regulatory factor of the present invention have DNA (FRE of the present invention) binding activity, but do not have transcriptional promoting activity of genes under the control of the FRE ( For example, it includes a DNA-binding domain of the transcriptional regulatory factor but not a transcriptional regulatory (activation) domain. However, such a peptide can bind to the FRE sequence of the present invention in the SREBP-1c promoter and block the transcriptional activation of the SREBP-1c gene by the transcriptional regulatory factor of the present invention. In particular, it is useful as a prophylactic / therapeutic agent for disorders of sugar / lipid metabolism.

In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Examples of R in the ester include the same as those described above for the transcriptional regulatory factor of the present invention. When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Further, in the partial peptide of the present invention, as in the transcription regulator of the present invention described above, the amino group of the N-terminal methionine residue is protected with a protective group, and the N-terminal side is cleaved in vivo. Also included are those in which the glutamine residue is pyroglutamine oxidized, those in which the substituents on the side chain of the amino acid in the molecule are protected with an appropriate protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound It is.

  The transcriptional regulatory factor or partial peptide thereof of the present invention may be a free form or a salt form. Examples of the salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The transcriptional regulatory factor of the present invention can be produced from the aforementioned mammalian cells or tissues by a known protein purification method. Specifically, the transcriptional regulatory factor of the present invention is obtained by homogenizing mammalian tissues or cells and separating and purifying the nuclear fraction by chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. Can be manufactured.

Specifically, the transcriptional regulatory factor of the present invention is a human or other mammal (eg, mouse, rat, rabbit, guinea pig, hamster, cow, horse, pig, sheep, monkey, dog, cat, etc.), preferably A DNA having the FRE base sequence of the present invention is brought into contact with a cell nuclear extract derived from a strain or an individual showing a tendency of increased SREBP-1c expression or metabolic disorder due to a meal (sugar load), particularly a high fructose diet (fructose load). The protein can be obtained by recovering (separating and purifying) the protein bound to the DNA. The animal cell for obtaining the nuclear extract is not particularly limited as long as it expresses the target transcriptional regulatory factor. From the above-described various cells or the cells related to the preparation of the nucleic acid containing the FRE of the present invention. Examples of such tissues include hepatocytes, more preferably hepatocytes loaded with sugar, and particularly preferably hepatocytes loaded with fructose. Such hepatocytes may be those obtained by adding sugar (eg, fructose) to a cell line established from cells derived from liver or a culture medium of primary cultured cells and incubating for a certain period of time, or diet (eg, : Liver tissue excised from an animal body fed a high fructose diet).
Preparation of a nuclear extract from cells / tissues can be performed by a conventional method. For example, cells or tissues can be treated with a suitable buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, HEPES buffer, etc .; protein buffer such as urea and guanidine hydrochloride, Triton X-100 (It may contain a surfactant such as ^TM ), and the cells are disrupted by sonication, lysozyme treatment and / or freeze-thawing, and then the precipitate obtained by centrifugation or filtration is removed with, for example, a hypertonic solution. For example, a method of obtaining a crude extract of nucleoprotein by treating and centrifuging and collecting the supernatant is used.
Means for bringing the nuclear extract into contact with the DNA containing the FRE base sequence of the present invention is not particularly limited. For example, an affinity column in which the DNA is immobilized on a suitable insoluble carrier (eg, agarose, cellulose, sepharose, etc.) The target transcriptional regulatory factor and FRE are bound to the column through a nuclear extract, and then eluted using a concentration gradient of NaCl, KCl, etc., and the protein-containing fraction eluted at a high salt concentration is FRE It can be recovered as a fraction containing a transcriptional regulatory factor that can specifically bind to.
Isolation and purification of the transcriptional regulatory factor of the present invention contained in the fraction thus obtained can be performed according to a method known per se. As such a method, a method using solubility such as salting out or solvent precipitation method; mainly using a difference in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

The transcriptional regulatory factor of the present invention or a partial peptide thereof was subjected to complete degradation (acid or alkaline degradation) of the purified transcriptional regulatory factor to determine the amino acid composition, and further used chemical substances such as sequence-specific peptidases and bromocyan. The amino acid sequence of the partial peptide obtained by limited degradation is determined using a known technique such as Edman degradation, and the total amino acid sequence is determined by combining these information, and then the known peptide is based on the amino acid sequence. It can also be produced according to synthetic methods.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. It is possible to produce a target transcriptional regulator by condensing a partial peptide or amino acid that can constitute the transcriptional regulatory factor of the present invention and the remaining part, and removing the protective group when the product has a protective group. it can.
Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following 1) to 5).
1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
5) Supervised by Yajima Haruaki, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten

The transcriptional regulatory factor thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the transcriptional regulatory factor obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when a protein is obtained as a salt Alternatively, the salt can be converted to a free form or other salt by a known method or a method analogous thereto.

  For the synthesis of the transcriptional regulatory factor of the present invention or a partial peptide thereof, a commercially available protein synthesis resin can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid appropriately protected with an α-amino group and a side chain functional group is formed on the resin in accordance with the per se known various condensation methods according to the sequence of the target transcriptional regulator or its partial peptide. Allow to condense. At the end of the reaction, the protein (peptide) is cut out from the resin, and at the same time, various protecting groups are removed, and the intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein (peptide) or its amide. .

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. Can then be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about −20 ° C. to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the elimination of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protecting group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

  Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as -butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

As another method for obtaining an amide form of a protein (peptide), for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide chain is extended to the desired chain length on the amino group side. And a protein (peptide) from which only the protecting group of the α-amino group at the N-terminus of the peptide chain is removed and a protein (peptide) from which only the protecting group of the carboxyl group at the C-terminal has been removed are produced. ) In a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein (peptide) obtained by the condensation, all the protecting groups are removed by the above-described method to obtain the desired crude protein (peptide). This crude protein (peptide) can be purified by using various known purification means, and the main fraction can be freeze-dried to obtain an amide form of the desired protein (peptide).
To obtain an ester of a protein (peptide), for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired amide of the protein (peptide) is obtained. An ester of a protein (peptide) can be obtained.

  The partial peptide of the present invention can also be produced by cleaving the transcriptional regulatory factor of the present invention with an appropriate peptidase.

  Furthermore, the transcriptional regulatory factor of the present invention or a partial peptide thereof can be obtained by culturing a transformant containing the DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide thereof, It can also be produced by separating and purifying the partial peptide. Alternatively, the transcriptional regulatory factor of the present invention or a partial peptide thereof can be translated in vitro using an RNA corresponding to the DNA as a template using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc. Can also be synthesized. Alternatively, the DNA can be synthesized using a cell-free transcription / translation system further containing RNA polymerase as a template.

  Examples of the DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide thereof include genomic DNA, genomic DNA library, human or other mammals (for example, cattle, monkeys, horses, pigs, sheep, goats, dogs, cats, Any cell (eg, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts) in guinea pigs, rats, mice, rabbits, hamsters, etc. Cells, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, Synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells and stem cells of these cells Or cancer cells) or any tissue in which these cells exist, such as the brain, brain regions (eg, olfactory bulb, buccal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, Subthalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid , Gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, testicle, testis, ovary, placenta , CDNA derived from the uterus, bone, joint, skeletal muscle, etc. (especially each part of the brain or brain), cDNA library derived from the cells / tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as “RT-PCR method”) using a total RNA or mRNA fraction prepared from the cells / tissues described above.

Examples of the DNA encoding the NBP of the present invention include a DNA containing the base sequence represented by SEQ ID NO: 2 or a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 2 under highly stringent conditions. And a DNA encoding a protein having substantially the same activity (eg, transcription control activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 3 described above.
As the DNA encoding the RBMX-like protein of the present invention, for example, the DNA containing the base sequence represented by SEQ ID NO: 4 or the base sequence represented by SEQ ID NO: 4 is hybridized under highly stringent conditions. Examples thereof include DNA encoding a protein containing a base sequence and having substantially the same activity (eg, transcription control activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 5.
The DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 (or 4) under highly stringent conditions is, for example, preferably about 50% or more of the base sequence represented by SEQ ID NO: 2 (or 4). Is a DNA containing a base sequence having a homology of about 60% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more.

Hybridization should be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is preferable.
The DNA encoding the NBP of the present invention is preferably a DNA containing the base sequence represented by SEQ ID NO: 2. The DNA encoding the RBMX-like protein of the present invention is preferably a DNA containing the base sequence represented by SEQ ID NO: 4.

  The DNA encoding the partial peptide of the NBP of the present invention (or the RBMX-like protein of the present invention) encodes the same or substantially the same amino acid sequence as a part of the amino acid sequence represented by SEQ ID NO: 3 (or 5) A DNA that encodes a peptide that has substantially the same activity (eg, transcription control activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 3 (or 5) described above It can be anything. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can be directly amplified by RT-PCR using mRNA fraction prepared from the cells / tissues described above.

Specifically, examples of the DNA encoding the partial peptide of the NBP of the present invention (or the RBMX-like protein of the present invention) include (1) a DNA having the base sequence represented by SEQ ID NO: 2 (or 4). A DNA having a partial base sequence, or (2) having a base sequence that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 2 (or 4) under highly stringent conditions, and is encoded by the DNA For example, DNA encoding a peptide having substantially the same activity (eg, transcription control activity) as a protein containing an amino acid sequence is used.
The DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 (or 4) under highly stringent conditions is, for example, about 60% or more, preferably about 70% or more, more preferably about 80%, with the base sequence. % Or more, and most preferably, a polynucleotide containing a nucleotide sequence having about 90% or more identity is used.

  The DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide thereof may be a free form or a salt form. Examples of the salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide thereof is amplified by the PCR method using a synthetic DNA primer having a part of the base sequence encoding the factor or the partial peptide, or incorporated into an appropriate expression vector. The DNA can be cloned by hybridization with a DNA fragment encoding a part or all of the protein of the present invention or a labeled synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

Preferably, the DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide thereof is a reporter gene under the control of a host cell promoter containing DNA having the fructose response element sequence of the present invention, and the control of the host cell promoter. It can be obtained by introducing the underlying animal-derived cDNA library into the host cell and isolating the cDNA introduced into the cell that highly expresses the reporter gene. As the promoter for the host cell, any promoter that can exhibit promoter activity in the host cell to be used can be used, but preferably a promoter that can function in yeast cells, for example, PHO5 promoter, PGK promoter, GAP A promoter, an ADH promoter, or the like is used. Introduction of the FRE sequence of the present invention into such a promoter (construction of a chimeric promoter) can be carried out by combining per se known genetic engineering techniques. Any known reporter gene may be used, and examples thereof include, but are not limited to, a luciferase gene, a GFP gene, an alkaline phosphatase gene, a peroxidase gene, and a β-galactosidase gene.
Animal-derived cDNA libraries include any mammal expressing the transcription factor of interest (eg, human, mouse, rat, guinea pig, hamster, rabbit, sheep, cow, horse, pig, dog, cat, monkey, etc. ) Cells or tissues, but preferably from a strain or individual showing a tendency for increased SREBP-1c expression or metabolic disorder due to diet (sugar load), particularly high fructose diet (fructose load), More preferred is a cDNA library derived from the hepatocytes of the strain or individual, more preferably derived from hepatocytes loaded with sugar, and most preferably derived from hepatocytes loaded with fructose. Each cDNA constituting the library can be cloned downstream of a host cell promoter suitable for the host cell to be used using a genetic engineering technique known per se. As the promoter, a promoter capable of functioning in the same yeast cell as described above is preferably used.
As an introduction vector carrying the above expression cassette, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19) , PSH15); bacteriophage such as λ phage, etc., which contain other enhancer, poly A addition signal, selection marker, SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), etc., if desired Can be used. Selectable markers include, for example, dihydrofolate reductase gene [methotrexate (MTX) resistance], ampicillin resistance gene, neomycin resistance gene (G418 resistance), etc. and auxotrophic (leucine requirement, tryptophan requirement, etc.) mutations And the like.
As the host, for example, yeast, insect cells, mammalian cells and the like are used, and yeast cells are preferable in the sense of avoiding the background of transcriptional regulators inherent in the host. Specifically, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris 71)
Transformation can be performed according to a known method depending on the type of host. For example, in the case of yeast cells, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of Science. It can be transformed according to the method described in The USA (Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
The culture of the transformant can be performed according to a known method depending on the type of the host. For example, as a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimal medium [Bostian, KL et al., Proceedings of the National Academy of Sciences. Of the USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the -National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.

  After the transformant was cultured for a certain period of time, the expression of the reporter gene was examined and introduced into the transformant whose expression level was significantly increased compared to the control cell (host cell into which only the expression vector containing the reporter gene was introduced). By cloning the cDNA by a conventional method, it is possible to obtain the DNA encoding the transcriptional regulatory factor of the present invention or a partial peptide that retains the DNA binding properties and transcription promoting activity.

The nucleotide sequence of the obtained DNA was determined using a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc. Conversion can be carried out according to a method known per se such as duplex method, Kunkel method or the like, or a method analogous thereto.

  The cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

The DNA expression vector encoding the transcriptional regulatory factor of the present invention is obtained, for example, by cutting out the target DNA fragment from the DNA encoding the transcriptional regulatory factor of the present invention and ligating the DNA fragment downstream of the promoter in an appropriate expression vector. Can be manufactured.
As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); Bacteriophages; animal viruses such as retrovirus, vaccinia virus, baculovirus; pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferable.
When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene. (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a medium not containing thymidine.
If necessary, a signal sequence suitable for the host may be added to the N-terminal side of the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc .; When the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc .; When the host is yeast When the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, and the like are used, respectively.

A transformant containing the “DNA encoding the transcriptional regulatory factor of the present invention” obtained as described above is produced by transforming a host with an expression vector containing the DNA according to a known method. be able to.
Here, examples of the expression vector include those described above.
As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.
Examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. , 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], and the like.
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Used.

Examples of insect cells include, for the virus AcNPV, cabbage armyworm larvae derived cell line (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from mid-intestine of Trichoplusia ni, High Five ^TM cell derived from an egg of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

Transformation can be performed according to a known method depending on the type of host.
Examples of the genus Escherichia include, for example, Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene ( Gene), Vol. 17, 107 (1982) and the like.
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
Yeast is, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences of USA ( Proc. Natl. Acad. Sci. USA), 75, 1929 (1978), and the like.
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells are, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).

The culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc .; As the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5-8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
The transformant whose host is Escherichia is usually cultured at about 15 to 43 ° C. for about 3 to 24 hours. If necessary, aeration or agitation may be performed.
The transformant whose host is a Bacillus genus is usually cultured at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, aeration or agitation may be performed.
As a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or an insect, for example, 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) What added the additive suitably is used. The pH of the medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. The pH of the medium is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, the transcriptional regulatory factor of the present invention can be produced intracellularly (in the nucleus or cytoplasm) or extracellularly of the transformant.

The transcriptional regulatory factor of the present invention or a partial peptide thereof can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when the transcriptional regulatory factor of the present invention or the partial peptide of the present invention is extracted from the cultured cell or cytoplasm of the cell, the cell or cell collected from the culture by a known method is suspended in an appropriate buffer solution, and For example, a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by sonication, lysozyme treatment, and / or freeze-thawing, or the like is appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 ^™ . On the other hand, when extracting the transcriptional regulatory factor of the present invention or the partial peptide of the present invention from the nuclear fraction, the precipitate obtained by the above centrifugation or filtration is treated with, for example, a hypertonic solution, and the supernatant is recovered by centrifugation. Thus, a method of obtaining a crude extract of nucleoprotein is used.
Isolation and purification of the transcriptional regulatory factor of the present invention or the partial peptide of the present invention contained in the thus obtained soluble fraction or nuclear extract can be performed according to a method known per se. As such methods, methods utilizing the solubility such as salting out and solvent precipitation method; mainly utilizing differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography, a method utilizing a specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography, etc. A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

When the protein or peptide thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
The protein or peptide produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the transcriptional regulatory factor of the present invention or the partial peptide of the present invention thus obtained can be confirmed by an enzyme immunoassay or Western blotting using a specific antibody.

Furthermore, as described above, the transcriptional regulatory factor of the present invention or the partial peptide of the present invention is a rabbit reticulocyte lysate using the above-mentioned transcriptional regulatory factor of the present invention or a DNA encoding the partial peptide or RNA corresponding thereto as a template, It can also be synthesized by in vitro translation using a cell-free protein (transcription /) translation system consisting of wheat germ lysate, E. coli lysate and the like. The cell-free protein (transcription /) translation system can be a commercially available one, or a method known per se, such as Prat JM et al., Transcription and Tranlation, 179-209, Hames BD & Higgins SJ It can also be prepared according to the method described in eds., IRL Press, Oxford (1984). Examples of commercially available cell lysates include E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche), etc. derived from E. coli, and Rabbit Reticulocyte Lysate System derived from rabbit reticulocytes. Examples of those derived from wheat germ (Promega) include PROTEIOS ™ (TOYOBO). Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, the method described in Johnston FB et al., Nature, 179, 160-161 (1957) or Erickson AH et al., Meth. Enzymol., 96, 38-50 (1996), etc. Can be used.
As a system or apparatus for protein synthesis, a batch method (Pratt, JM et al. (1984) mentioned above) or a continuous cell-free protein synthesis system (Spirin AS, which continuously supplies amino acids, energy sources, etc.) to the reaction system. et al., Science, 242, 1162-1164 (1988)), dialysis (Kikawa et al., 21st Japan Molecular Biology Society, WID6), or multi-layer method (PROTEIOS ^TM Wheat germ cell-free protein synthesis core kit) Letter: manufactured by TOYOBO). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging the synthesized product and degradation product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.

The present invention also includes a fructose responsive element of the present invention or a DNA containing the same (which may include the entire base sequence represented by SEQ ID NO: 1; hereinafter, also referred to as “the DNA of the present invention”), and the element A metabolic disorder characterized by using a transcriptional regulatory factor of the present invention or a partial peptide thereof (hereinafter sometimes simply referred to as “transcriptional regulatory factor of the present invention”) that can bind to Examples: prevention and treatment of high TG, hyper LDL-C, hypo HDL-C, obesity, impaired glucose tolerance, fasting glycemic disorder, hyperinsulinemia, hypertension, albuminuria, etc.) A screening method is provided. As a specific embodiment of the screening method, for example,
1) a method for detecting binding inhibition between the DNA of the present invention and the transcriptional regulatory factor of the present invention in the presence of a test substance;
2) A method for comparing the expression of the gene in an animal cell containing a gene under the control of a promoter containing the DNA of the present invention in the presence and absence of the test substance. In the above method 2), there are cases where the measurement sensitivity can be improved by loading sugar on animal cells.

When the binding between the DNA of the present invention and the transcriptional regulatory factor of the present invention is used as an indicator, for example, the labeled DNA of the present invention and the transcriptional regulatory factor of the present invention (NBP (partial of the present invention), eg, ³² P, digoxigenin) And a RBMX-like protein of the present invention (including a partial peptide, or both of them) in the presence of a test substance, The reaction solution can be subjected to non-denaturing gel electrophoresis to detect the disappearance of the band corresponding to the DNA-transcription regulator complex or the decrease in signal intensity. Here, the transcriptional regulatory factor of the present invention may be used in an isolated and purified form, or may be used in the form of a nuclear extract of cells expressing the transcriptional regulatory factor. Such cells include, for example, cells derived from humans or other mammalian individuals whose expression of SREBP-1c is increased by sugar (eg, fructose) loading, preferably hepatocytes, more preferably hepatocytes loaded with sugar. More preferably, hepatocytes loaded with fructose, especially hepatocytes derived from CBA or C3H mice loaded with fructose are mentioned. Isolation of the nuclear extract from the cells can be performed according to the method described above.
Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
For example, when the signal intensity of the band corresponding to the DNA-transcription regulator complex decreases in the presence of the test substance by about 20% or more, preferably 30% or more, more preferably about 50% or more, the test substance Can be selected as a substance that inhibits the DNA binding activity of the transcriptional regulatory factor of the present invention.

When the expression of a gene under the control of a promoter containing the DNA of the present invention is used as an index, any promoter that can function in animal cells can be used as the promoter, for example, SRα promoter, SV40 promoter, LTR A promoter, a CMV (cytomegalovirus) promoter, an HSV-TK promoter, or the like is used. The DNA of the present invention can be inserted into an appropriate position in the promoter using a genetic engineering technique known per se. Alternatively, the SREBP-1c promoter containing the FRE of the present invention may be used as the “promoter containing the DNA of the present invention”.
The gene under the control of the promoter containing the DNA of the present invention is not particularly limited as long as its expression level can be easily measured, but is preferably a reporter such as luciferase, GFP, alkaline phosphatase, peroxidase, β-galactosidase, etc. Gene. In addition, the SREBP-1c gene containing the SREBP-1c promoter containing the FRE of the present invention can also be used as “a gene under the control of a promoter containing the DNA of the present invention”. In this case, a cell or tissue derived from a mammal (eg, CBA, C3H mouse, etc.) having the SREBP-1c gene or the individual animal (excluding humans) is “under the control of the promoter containing the DNA of the present invention. It can be used as an “animal cell containing a gene in”. When using a reporter gene as a gene under the control of a promoter containing the DNA of the present invention, a reporter gene linked to the downstream of the promoter containing the DNA of the present invention using a known genetic engineering technique, Suitable transfer vectors, for example, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); λ It can be inserted into a vector such as a bacteriophage such as a phage and introduced into a host animal cell. The introduction vector may contain other enhancer, poly A addition signal, selection marker, SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like, if desired. Examples of the selection marker include dihydrofolate reductase gene [methotrexate (MTX) resistance], ampicillin resistance gene, neomycin resistance gene (G418 resistance) and the like.
The animal cell is not particularly limited as long as it is a cell that can express the transcriptional regulatory factor of the present invention (preferably a cell that can express the factor in response to a glucose load). For example, monkey cell COS-7, Vero , Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster ovary cells (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3 Various cell lines such as human FL cells can also be used, but preferably hepatocytes, particularly preferably hepatocytes derived from CBA and C3H mice. These animal cells are described in, for example, “Cell Engineering Supplement 8 New Cell Engineering Experimental Protocol”, 263-267 (1995) (published by Shujunsha), “Virology”, Vol. 52, 456 (1973). Can be transformed according to the method described in 1. above.
When sugar is loaded into animal cells, the loaded sugar is not particularly limited as long as it is a carbohydrate that serves as an energy source. Monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and lactose, starch, glycogen And the like, or a mixture thereof, and the like, preferably fructose and a mixture of fructose and other sugars. Glucose loading is performed by adding sugar to the culture medium. When a non-human animal individual having a SREBP-1c gene containing the SREBP-1c promoter containing the FRE of the present invention is used, the animal is raised. It can be performed by ingesting a diet such as a commonly used diet or a per se known high fructose diet.
Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
Sugar is loaded in the presence and absence of the test substance, and in a suitable medium (eg, minimum essential medium, Dulbecco's modified Eagle medium, Ham medium, F12 medium, RPMI 1680 medium, William E medium, etc.) for a certain period of time. After culturing the cells, the expression of the gene under the control of the promoter containing the DNA of the present invention is compared under both conditions. When using the above non-human animal individuals, the test substance is administered orally or parenterally (eg, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracutaneously, etc.) and then fed for a certain period of time. Later, an appropriate biological sample (eg, hepatocytes, blood, etc.) may be collected from the animal, and the expression of the SREBP-1c gene may be detected and compared with an individual who has not been administered the test substance. The expression of the SREBP-1c gene can be detected and quantified by an immunoassay method such as ELISA using an anti-SREBP-1c antibody prepared by a conventional method, or an RT-PCR method.
As a result, for example, a test substance that inhibits gene expression by about 20% or more, preferably 30% or more, more preferably about 50% or more is selected as a substance that inhibits the transcription promoting activity of the transcriptional regulatory factor of the present invention. be able to.
Alternatively, using animal cells that can be used in the above-described method, the intracellular localization of the transcriptional regulatory factor of the present invention, for example, the degree of transfer of the factor from the cytoplasm to the nucleus in the animal cell By comparing in the presence and absence, a prophylactic / therapeutic substance for metabolic disorders (particularly, sugar / lipid metabolic disorders) can be screened. More specifically, for example, by immunostaining the cells with a fluorescently labeled antibody against the transcriptional regulatory factor of the present invention, the transfer of the factor from the cytoplasm to the nucleus can be monitored. Alternatively, by using a transformant capable of expressing the transcriptional regulatory factor of the present invention as a fusion protein with a fluorescent protein such as GFP, the transfer of the factor from the cytoplasm to the nucleus can be directly monitored (for example, Biochem. Biophys. Res. Commun., 278: 659-664 (2000)).

The “inhibitor of the transcriptional regulatory factor of the present invention” obtained by the screening method described above is that the gene of the mammal having the SREBP-1c gene containing the FRE of the present invention in the promoter region by diet (particularly a high fructose diet). Since the increase in expression can be suppressed, metabolic disorders involving abnormal expression of the gene, particularly sugar / lipid metabolic disorders (eg, hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity) It is useful as a prophylactic / therapeutic substance for impaired glucose tolerance, fasting blood glucose disorder, hyperinsulinemia, hypertension, albuminuria, etc.).
Therefore, inhibitors of the transcriptional regulatory factor of the present invention (these substances are peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.) And a salt may be formed, and specific examples of the salt include those similar to the salts of the transcriptional regulatory factor of the present invention described above). It can be used as a prophylactic / therapeutic agent for metabolic disorders after mixing with a possible carrier to obtain a pharmaceutical composition. Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations , Solubilizing agents, suspending agents, isotonic agents, buffers, soothing agents and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.

Preferable examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate and the like can be mentioned.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, light anhydrous silicic acid, low substituted hydroxypropylcellulose and the like.
Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
Preferable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
Preferable examples of the tonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
Preferable examples of the soothing agent include benzyl alcohol.
Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbate.
Suitable examples of the colorant include water-soluble edible tar dyes (for example, edible dyes such as edible red Nos. 2 and 3, edible yellows 4 and 5, edible blue Nos. 1 and 2, and water-insoluble lake dyes ( Examples thereof include aluminum salts of the water-soluble edible tar pigments, natural pigments (eg, β-carotene, chlorophyll, bengara, etc.).
Preferable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.

Examples of the dosage form of the pharmaceutical composition include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions and suspensions; and injections (eg, subcutaneous injection). Preparations, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (eg, nasal preparations, transdermal preparations, ointments, etc.), suppositories (eg, rectal suppositories, vaginal suppositories) Etc.), pellets, drops, sustained-release preparations (eg, sustained-release microcapsules, etc.) and the like, and these can be safely administered orally or parenterally.
The pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. Below, the specific manufacturing method of a formulation is explained in full detail. The content of the inhibitor of the transcriptional regulatory factor of the present invention in the pharmaceutical composition varies depending on the dosage form, the dose of the compound, etc., but is, for example, about 0.1 to 100% by weight.

  For example, oral preparations include active ingredients, excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, hatched starch, gum arabic). , Carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.) or a lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc., and then compression molded, and if necessary, taste masking, enteric or For the purpose of durability, it is produced by coating with a coating base by a method known per se.

Examples of the coating base include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.
As the sugar coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.
Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Acrylic polymers such as Rohm Pharma Co.], methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; Examples include natural products such as shellac.
Examples of sustained-release film coating bases include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co., Ltd.], ethyl acrylate / methyl methacrylate copolymer suspension Acrylic polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma) are listed.
Two or more of the coating bases described above may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.

  For injections, the active ingredient is a dispersant (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservative (eg, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, Phenol, etc.), isotonic agents (eg, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.), etc. and aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) or oily solvents (eg, , Olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like. At this time, additives such as a solubilizing agent (eg, sodium salicylate, sodium acetate, etc.), a stabilizer (eg, human serum albumin, etc.), a soothing agent (eg, benzyl alcohol, etc.) may be used as desired. The injection solution is usually filled in a suitable ampoule.

Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.). It can be administered orally or parenterally.
The dose of the preventive / therapeutic agent for metabolic disorders of the present invention varies depending on the target disease, administration subject, administration route, etc. For example, in an adult patient suffering from hyperTG (60 kg body weight), On the other hand, the inhibitor of the transcriptional regulatory factor of the present invention which is an active ingredient is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

  The present invention may be either or both of the transcriptional regulatory factor of the present invention (NBP of the present invention (including a partial peptide) and the RBMX-like protein of the present invention (including a partial peptide). Metabolic disorders comprising a substance that suppresses the production or activity (hereinafter also referred to as “production inhibitory substance” or “activity inhibitory substance”), particularly sugar / lipid metabolic disorders (eg, high TG, Provided is a prophylactic / therapeutic agent for LDL-C blood, low HDL-C blood, obesity, impaired glucose tolerance, fasting glycemic disorder, hyperinsulinemia, hypertension, albuminuria and the like.

The preventive / therapeutic agent for metabolic disorders containing the “production inhibitory substance” or “activity inhibitory substance” may be the “production inhibitory substance” or “activity inhibitory substance” itself. It is preferable that it is a pharmaceutical composition obtained by mixing with an acceptable carrier. Here, examples of the pharmacologically acceptable carrier include those similar to the case of the “inhibitor of the transcriptional regulatory factor of the present invention” obtained by the screening method described above.
The pharmaceutical composition can be produced in the same manner as in the case of the “inhibitor of the transcriptional regulatory factor of the present invention” described above.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.). It can be administered orally or parenterally.
The dose of the metabolic disorder preventive / therapeutic agent containing “production inhibitor” or “activity inhibitor” varies depending on the target disease, administration subject, administration route, etc., but for example, suffers from hyperTGemia In an adult patient (body weight 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 0.1 to 100 mg as an “production inhibitor” or “activity inhibitor” as an active ingredient per day. About 1.0 to 20 mg.

  The activity suppressing substance may be any substance as long as it can suppress the activity of the transcriptional regulatory factor of the present invention, that is, the transcription promoting activity of the gene under the control of the promoter containing the fructose responsive element of the present invention. A substance that binds to the transcriptional regulatory factor of the invention and inhibits the binding of the factor to the FRE sequence of the present invention (eg, an antibody against the transcriptional regulatory factor of the present invention, a nucleotide sequence to which the transcriptional regulatory factor of the present invention can bind) And a substance that can promote degradation, metabolism or inactivation of the transcriptional regulatory factor of the present invention (eg, protease, protein modifying enzyme, etc.).

Preferably, the activity inhibitory substance includes an antibody against the transcriptional regulatory factor of the present invention or a partial peptide thereof or a salt thereof. An antibody against the transcriptional regulatory factor of the present invention or a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as “the antibody of the present invention”) uses the transcriptional regulatory factor (including the partial peptide or salt) as an antigen, It can be produced according to a method known per se for producing antibodies or antisera. A monoclonal antibody or a polyclonal antibody against the transcriptional regulatory factor of the present invention can be produced, for example, as follows.
[Production of monoclonal antibodies]
(A) Production of Monoclonal Antibody-Producing Cells The transcriptional regulatory factor of the present invention (NBP of the present invention or RBMX-like protein of the present invention) is administered to a mammal at a site where antibody production is possible by administration to itself or a carrier And administered with diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
For example, an individual with an antibody titer selected from a mammal immunized with an antigen, such as a mouse, is collected 2 to 5 days after the final immunization, and spleen or lymph nodes are collected, and antibody-producing cells contained therein are allogeneic or Monoclonal antibody-producing hybridomas can be prepared by fusing with myeloma cells from different animals. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein (Nature, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

Examples of myeloma cells include mammalian myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
A monoclonal antibody-producing hybridoma can be prepared, for example, by adding a hybridoma culture supernatant to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then labeling with a radioactive substance or enzyme (anti-immunoglobulin antibody ( When the cells used for cell fusion are mice, anti-mouse immunoglobulin antibodies are used) or protein A is added to detect monoclonal antibodies bound to the solid phase; solid antibodies adsorbed with anti-immunoglobulin antibodies or protein A Screening can be performed by adding a hybridoma culture supernatant to a phase, adding a protein labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to a solid phase.
The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Selection of the monoclonal antibody can be usually performed in an animal cell medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). Any medium can be used for the selection and breeding of the monoclonal antibody as long as the hybridoma can grow. Examples of such a medium include RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, and GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.). Or a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
The monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G ] Can be separated and purified according to the above.

[Preparation of polyclonal antibody]
The polyclonal antibody against the transcriptional regulatory factor of the present invention can be produced according to a method known per se. For example, an immune antigen (protein antigen) itself or a complex of it and a carrier protein is prepared, and a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and the antibody-containing product of the present invention is collected from the immunized animal. Thus, the antibody can be produced by separating and purifying the antibody.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that an antibody can be efficiently produced against a hapten immunized by crosslinking to a carrier. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 to about 1 by weight with respect to hapten 1. A method of coupling at a rate of 5 is used.
For coupling of the hapten and the carrier, various condensing agents such as glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and active ester reagent containing a dithiobilidyl group are used.
The condensation product is administered to a mammal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks and about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably from blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

Another preferred embodiment of the activity inhibitory substance is the fructose response element of the present invention or a nucleic acid (preferably DNA) containing the same. Here, the FRE of the present invention or a nucleic acid containing the same is a decoy nucleotide for DNA to which the transcriptional regulatory factor of the present invention binds. The nucleic acid can be produced according to the method described above.
Since the nucleic acid has low toxicity and can suppress the function of the transcriptional regulatory factor of the present invention in vivo (that is, transcription promoting activity such as SREBP-1c gene), abnormal expression of SREBP-1c gene is involved. It can be used as an agent for preventing or treating metabolic disorders. The nucleic acid is formulated in the same manner as in the case of the inhibitor of the transcriptional regulatory factor of the present invention obtained by the above screening method, and a mammal (eg, human, mouse, rat, rabbit, sheep, pig, cow, horse, cat) , Dogs, monkeys, chimpanzees, etc.) orally or parenterally.
The nucleic acid can also be administered to the mammal after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, or the like.
The nucleic acid may be administered to the mammal by a catheter such as a gene gun or a hydrogel catheter, and after aerosolization, it can also be locally administered into the trachea as an inhalant.
The dose of the preventive / therapeutic agent for metabolic disorders comprising the FRE of the present invention or a nucleic acid containing the same varies depending on the target disease, administration subject, administration route, etc., but for example, suffers from hyperTGemia. In an adult patient (body weight 60 kg), the amount of the active ingredient nucleic acid per day is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

  The production regulator of the transcriptional regulatory factor of the present invention preferably includes a nucleic acid containing a base sequence complementary to the base sequence encoding the transcriptional regulatory factor of the present invention or a part thereof. The nucleic acid containing a base sequence complementary to the base sequence encoding the transcriptional regulatory factor of the present invention or a part thereof (hereinafter sometimes abbreviated as “antisense nucleic acid of the present invention”) is the transcription of the present invention. It has a nucleotide sequence that is completely complementary or substantially complementary to the nucleotide sequence encoding the regulatory factor (NBP of the present invention or RBMX-like protein of the present invention), or a part of the complementary nucleotide sequence. And what is necessary is just to have the effect | action which suppresses the translation of this protein from RNA which codes the transcriptional regulatory factor of this invention. The “substantially complementary base sequence” includes a base sequence encoding the transcriptional regulatory factor of the present invention and a base sequence capable of hybridizing under physiological conditions of a cell expressing the protein, more specifically, About 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% between the complementary strand of the base sequence encoding the transcriptional regulatory factor of the present invention or a partial base sequence thereof. Examples thereof include base sequences having the above homology.

  The antisense nucleic acid of the present invention can be designed and synthesized based on the nucleotide sequence information of the nucleic acid encoding the transcriptional regulatory factor of the present invention that has been cloned or determined. Such a nucleic acid can inhibit replication or expression of a gene encoding the transcriptional regulatory factor of the present invention. That is, the antisense nucleic acid of the present invention can hybridize with RNA transcribed from the gene encoding the transcriptional regulatory factor of the present invention, and inhibits synthesis (processing) or function (translation into protein) of mRNA. be able to.

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation of the transcriptional regulatory factor of the present invention is inhibited. The entire sequence or partial sequence of RNA encoding the transcriptional regulatory factor may be a short sequence of about 15 bases and a long sequence of mRNA or the initial transcript. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, the 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region of the gene encoding the transcriptional regulatory factor of the present invention ORF translation initiation codon, 3 ′ end untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop can be selected as target regions, but any region within the gene can be selected as a target. For example, it is also preferable to use the intron portion of the gene as a target region.
Furthermore, the antisense nucleic acid of the present invention not only hybridizes with mRNA or initial transcription product encoding the transcriptional regulatory factor of the present invention to inhibit translation into protein, but also has the transcription of the present invention which is double-stranded DNA. It may bind to a gene encoding a regulatory factor to form a triplex, thereby inhibiting RNA transcription.

  Antisense nucleic acids are deoxyribonucleotides containing 2-deoxy-D-ribose, ribonucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides Other polymers with backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a pair of bases as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a ring or a base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and also unmodified polynucleotides (or unmodified oligonucleotides), and more publicly known Modified, such as those with labels known in the art, capped, methylated, substituted one or more natural nucleotides with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, and toxins) , Antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelates Compounds containing compounds (for example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. As used herein, “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

  Antisense nucleic acids are RNA, DNA, or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic. Many such modifications are known in the art, such as J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed ., Antisense Research and Applications, CRC Press, 1993, etc.

  Antisense nucleic acids may be altered or contain modified sugars, bases, bonds, donated in specialized forms such as liposomes, microspheres, applied by gene therapy, or added Could be given in form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

A ribozyme capable of specifically cleaving mRNA or gene initial transcription product encoding the transcriptional regulatory factor of the present invention within the coding region (including intron part in the case of the initial transcription product) is also an antisense nucleic acid of the present invention. Can be included. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleavage activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA since it only uses RNA as a substrate. When the mRNA encoding the transcriptional regulatory factor of the present invention has a double-stranded structure by itself, a target sequence can be obtained by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. Can be made into a single strand [ Proc. Natl. Acad. Sci. USA , 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [ Nucleic Acids Res. , 29 (13): 2780-2788 (2001)].

Small interfering RNA (siRNA) complementary to mRNA encoding the transcriptional regulatory factor of the present invention or a partial sequence in the coding region of the gene initial transcription product (including intron in the case of the initial transcription product) Can also be included in the antisense nucleic acid of the invention. When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. Recently, it has been confirmed that this phenomenon also occurs in mammalian cells [ Nature , 411 (6836): 494-498 (2001)], and has attracted attention as an alternative to ribozyme.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence information encoding the transcriptional regulatory factor of the present invention, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman, etc.) can be used to synthesize a sequence complementary to this. For siRNA, a sense strand and an antisense strand are respectively synthesized by a DNA / RNA automatic synthesizer, denatured in an appropriate annealing buffer at, for example, about 90 to about 95 ° C. for about 1 minute, and then about 30 to It can be prepared by annealing at about 70 ° C. for about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase.

When the “production inhibitor” is the antisense nucleic acid of the present invention, the antisense nucleic acid is inserted into a suitable vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. It can also be administered to mammals as a prophylactic / therapeutic agent.
The antisense nucleic acid may be administered by a catheter such as a gene gun or a hydrogel catheter, and after aerosolization, it can also be locally administered into the trachea as an inhalant.

  Furthermore, the antisense nucleic acid of the present invention can also be used as a diagnostic nucleic acid probe for examining the presence and expression status of the nucleic acid encoding the transcriptional regulatory factor of the present invention in tissues and cells.

  The present invention also relates to a protein or peptide containing an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the transcriptional regulatory factor of the present invention or a partial peptide thereof, and the fructose of the present invention Provided is a protein or peptide characterized by having the ability to bind to a response element but not activating a promoter containing the FRE sequence. “Do not activate a promoter” means that transcriptional activation of a gene under the control of the promoter cannot be promoted. The protein or peptide may be any protein as long as it has the above-mentioned properties. For example, by substitution, deletion, insertion or addition of an amino acid in the transcriptional regulatory (activation) domain of the NBP or RBMX-like protein of the present invention, Examples include mutant proteins or peptides lacking transcription promoting activity.

  The above mutant protein or peptide binds to the SREBP-1c promoter containing the FRE sequence of the present invention, thereby binding the normal transcriptional regulatory factor of the present invention to the FRE sequence and activating transcription of the SREBP-1c gene. Metabolic disorders associated with abnormally increased SREBP-1c gene expression, particularly sugar / lipid disorders (eg, hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity, It is useful for the prevention and treatment of impaired glucose tolerance, fasting blood glucose disorder, hyperinsulinemia, hypertension, albuminuria, etc. Accordingly, the present invention provides a preventive / therapeutic agent for metabolic disorders, particularly sugar / lipid metabolic disorders, comprising the mutant protein or peptide.

The preventive / therapeutic agent for metabolic disorders comprising the above mutant protein or peptide may be the mutant protein or peptide itself, but is a pharmaceutical obtained by mixing it with a pharmacologically acceptable carrier. A composition is preferred. Here, examples of the pharmacologically acceptable carrier include those similar to the case of the “inhibitor of the transcriptional regulatory factor of the present invention” obtained by the screening method described above.
The pharmaceutical composition can be produced in the same manner as in the case of the “inhibitor of the transcriptional regulatory factor of the present invention” described above.
Since the preparation thus obtained is safe and has low toxicity, for example, for mammals (eg, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) And can be administered orally or parenterally.
The dose of the preventive / therapeutic agent for metabolic disorders of the present invention varies depending on the target disease, administration subject, administration route, etc. For example, in an adult patient suffering from hyperTG (60 kg body weight), The effective amount of the mutated protein or peptide as an active ingredient is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

  The present invention also provides a metabolic disorder comprising a nucleic acid having a base sequence encoding the transcriptional regulatory factor of the present invention or a part thereof, particularly a sugar / lipid disorder (eg, hyperTGemia, high LDL- C blood, low HDL-C blood, obesity, impaired glucose tolerance, fasting blood glucose disorder, hyperinsulinemia, hypertension, albuminuria and the like. For example, a mammal (eg, human, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog by using a nucleic acid having a base sequence encoding the transcriptional regulatory factor of the present invention as a probe. , Monkeys, chimpanzees, etc.) can detect abnormalities (gene abnormalities) of the DNA or mRNA encoding the transcriptional regulatory factor of the present invention, for example, damage, mutation or decreased expression of the DNA or mRNA, It is useful as a gene diagnostic agent for increasing DNA or mRNA or overexpression.

The above gene diagnosis can be performed by, for example, known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy, Of Sciences of USA (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, 2766-2770 (1989)).
For example, when excessive expression is detected by Northern hybridization or when a DNA mutation is detected by the PCR-SSCP method, for example, there is a possibility of suffering from a sugar / lipid metabolic disorder such as hyperTGemia. Can be diagnosed as high.

The present invention also provides a metabolic disorder comprising the above-described antibody of the present invention, particularly a sugar / lipid metabolic disorder (eg, hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity, glucose tolerance). Dysfunction, fasting blood glucose disorder, hyperinsulinemia, hypertension, albuminuria, etc.).
That is, the present invention
(I) Labeled transcription of the present invention bound to the antibody by competitively reacting the antibody of the present invention with a test solution and a labeled transcriptional regulatory factor of the present invention (including a partial peptide) A method of diagnosing a metabolic disorder, particularly a sugar / lipid metabolic disorder, characterized by quantifying the transcriptional regulatory factor of the present invention or a salt thereof in a test solution by measuring a ratio of the regulatory factor, and (ii) a subject The test solution is tested by measuring the activity of the labeling agent on the insolubilized carrier after reacting the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention simultaneously or successively. Provided is a method for diagnosing a metabolic disorder, particularly a sugar / lipid metabolic disorder, characterized by quantifying the transcriptional regulatory factor of the present invention or a salt thereof in fluid.

In the quantification of (ii) above, when one antibody is an antibody that recognizes the N-terminal part of the transcriptional regulatory factor of the present invention, the other antibody is another part of the transcriptional regulatory factor of the present invention, such as the C-terminal part. It is desirable that the antibody recognizes.
In addition to quantification of the factor using a monoclonal antibody against the transcriptional regulatory factor of the present invention, detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. Furthermore, a single chain antibody (scFv) in which the variable regions of the heavy chain and the light chain are linked by a linker can also be used.

  The quantification of the transcriptional regulatory factor of the present invention or a salt thereof using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the solution to be measured is determined. Any measurement method may be used as long as it is a measurement method that is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can also be used for binding of an antibody or antigen to a labeling agent.

  For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize and immobilize proteins or the like may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the amount of the transcriptional regulatory factor of the present invention or a salt thereof in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.

  In the method for measuring the transcriptional regulatory factor of the present invention or a salt thereof by the sandwich method described above, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is different from the site where the transcriptional regulatory factor of the present invention binds. Is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the transcriptional regulatory factor of the present invention, the antibody used in the primary reaction is: Preferably, an antibody that recognizes other than the C end, for example, the N end is used.

The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble one is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.
In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the transcriptional regulatory factor of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation method in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (Academic Press Issue) and the like can be referred to.
As described above, by using the antibody of the present invention, the transcriptional regulatory factor of the present invention or a salt thereof can be quantified with high sensitivity.

  In the above quantification method using the antibody of the present invention, a biopsy sample (eg, kidney cell, pancreatic cell, etc.) of a test animal is used as a test sample, and the concentration of the transcriptional regulatory factor of the present invention or a salt thereof in the test sample is determined. By quantifying, when an overexpression of the factor is detected, it can be diagnosed that there is a high possibility of suffering from a sugar / lipid metabolism disorder such as hyperTG.

The present invention also provides a non-human transgenic animal into which a gene under the control of a promoter containing the fructose response element (FRE) of the present invention has been introduced. The “promoter comprising the FRE of the present invention” is a promoter for animal cells similar to that described in the screening method of the present invention, and the FRE of the present invention linked to an appropriate position using a genetic engineering technique. Etc. are exemplified. Alternatively, the SREBP-1c promoter itself containing the FRE of the present invention can also be used as the “promoter containing the FRE of the present invention”.
As the gene under the control of the promoter, a reporter gene similar to that described in the screening method of the present invention is preferably exemplified, but the SREBP-1c gene itself containing the FRE of the present invention in the promoter region is referred to as “ It can also be used as “a gene under the control of a promoter containing the FRE of the present invention”.
As used herein, the term “transgenic animal” means that a gene under the control of a promoter containing the FRE of the present invention is permanently present in the host animal cell, and the gene is present in the host chromosome. Although it may be integrated above or may exist stably as an extrachromosomal gene, preferably the gene is retained in an integrated state on the host chromosome.

  A non-human transgenic animal into which a gene under the control of a promoter containing the fructose responsive element of the present invention (hereinafter referred to as “the FRE-Tg animal of the present invention”) is a fertilized egg of a non-human mammal or a non-human mammal. Fertilized eggs, sperm or progenitor cells thereof (primordial germ cells, oocyte cells, oocytes, egg cells, spermatogonia, spermatocytes, sperm cells, etc.), preferably in the early stage of embryo development of fertilized eggs (further Preferably, before the 8-cell stage), calcium phosphate coprecipitation method, electroporation method, lipofection method, aggregation method, microinjection method, gene gun (particle gun) method, DEAE-dextran method, etc. It is created by introducing the gene of interest by the gene transfer method. In addition, by the gene introduction method, a target gene can be introduced into a somatic cell, tissue, organ, etc. of a non-human mammal and used for cell culture, tissue culture, and the like. Transgenic animals can also be produced by fusing (or germline) cells with known cell fusion methods. Alternatively, as in the case of producing a knockout animal, the gene of interest was introduced into embryonic stem cells (ES cells) of a non-human mammal using the above gene transfer method, and the gene was stably integrated in advance. After selecting the clone, the ES cell is injected into the blastocyst or the ES cell mass and the 8-cell stage embryo are aggregated to produce a chimeric animal, and the one having the transgene transferred to the germ line is selected. It is also possible to obtain transgenic animals.

  In addition, a part of the living organism of the transgenic animal produced in this way (for example, 1) cells, tissues, organs, etc. that stably hold the transgene, 2) cells or tissues derived therefrom are cultured and necessary Can be used for the same purpose as “the FRE-Tg animal of the present invention” as “a part of the living body of the FRE-Tg animal of the present invention”. Examples of organs that are part of the living body of the FRE-Tg animal of the present invention include liver, heart, kidney, adrenal gland, blood vessel, gastrointestinal tract, and brain, and tissue and cells include tissue pieces and cells derived from the organ. Preferably exemplified.

The “non-human mammal” that can be the subject of the present invention is not particularly limited as long as it is a mammal other than a human for which a transgenic system has been established. For example, cows, pigs, sheep, goats, rabbits, dogs, cats, Examples include guinea pigs, hamsters, rats, and mice. Preferred are mice, rats, rabbits, dogs, cats, guinea pigs, hamsters, etc., among which rodents that have relatively short ontogeny and biological cycle and are easy to reproduce from the viewpoint of producing disease model animals are more preferred. Mice (eg, C57BL / 6 strain, DBA / 2 strain, etc. as pure strains, B6C3F ₁ strain, BDF ₁ strain, B6D2F ₁ strain, BALB / c strain, ICR strain, etc.) as hybrids, and rats (eg, Wistar, SD, etc.) ) Is preferred.
In addition to mammals, birds such as chickens can be used for the same purpose as the “non-human mammal” targeted in the present invention.

  While the structural gene in the transgene is preferably in an intron-free form (ie, cDNA), the 5 'and 3' end sequences of the intron are common to most eukaryotic genes, so another embodiment In, a form containing an intron (ie, genomic DNA) can also be preferably used.

  Examples of vectors carrying a transgene include plasmids derived from E. coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus, etc. Is used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, or plasmids derived from yeast are preferably used, and plasmids derived from Escherichia coli are particularly preferable.

  It is preferable to have a sequence (also called a polyadenylation (polyA) signal or terminator) that terminates the transcription of the target mRNA in the transgenic animal downstream of the transgene. Efficient transgene expression can be achieved using terminator sequences from various mammalian or avian genes. Preferably, a simian virus SV40 terminator or the like is used. In addition, for the purpose of further expressing the gene of interest, a splicing signal of each gene, an enhancer region, and a part of the intron of the eukaryotic gene are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region (5′UTR). Alternatively, it can be linked 3 ′ downstream (3′UTR) of the translation region depending on the purpose.

  In addition, when a transgenic animal is produced using ES cells, the above vector can be used as a selectable marker gene (eg, neomycin resistance gene, hygromycin resistance gene, etc.) for selecting a clone in which the transgene is stably integrated. It is preferable to further contain a drug resistance gene). In addition, if the transgene is intended to be integrated into a specific site on the host chromosome by homologous recombination (ie, creating a knock-in animal), the vector described above may contain a target site to eliminate random insertions. It is preferable that a herpes simplex virus-derived thymidine kinase gene or a diphtheria toxin gene is further included as a negative selection marker gene outside the homologous DNA sequence. These embodiments will be described in detail later.

  The above promoter, structural gene DNA, terminator, etc. can express the transgene in the correct arrangement in the above vector, that is, in a transgenic animal by a normal genetic engineering technique using an appropriate restriction enzyme and DNA ligase. Can be inserted in a simple arrangement.

  In a preferred embodiment, an expression vector containing a gene under the control of a promoter containing the FRE of the present invention obtained as described above is introduced into an early embryo of a target non-human mammal by a microinjection method. The

The early embryo of the target non-human mammal is obtained by collecting in-vivo fertilized eggs obtained by mating males and females of the same kind of non-human mammal, or by collecting eggs and sperm collected from male and female of the same kind of non-human mammal respectively. It can be obtained by fertilization.
Non-human mammals age and breeding conditions are different from each other by the animal species used, for example, a mouse (preferably an inbred mouse such as C57BL / 6J (B6), etc. F ₁ of B6 and another inbred strain) Are preferably about 4 to about 6 weeks of age for females and about 2 to about 8 months of age for males, and about 1 hour under light conditions (eg, 7:00 to 19:00) for about 12 hours. Those raised for a week are preferred.
In vivo fertilization may be by natural mating, but in order to regulate the sexual cycle and obtain a large number of early embryos from one individual, after inducing superovulation by administering gonadotropin to a female non-human mammal, A method of mating with a non-human mammal is preferred. Examples of methods for inducing ovulation in female non-human mammals include follicle stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG), and then luteinizing hormone (human chorionic gonadotropin, generally abbreviated as hCG). Is preferably administered by, for example, intraperitoneal injection, but the preferred dose and interval of administration of hormones differ depending on the type of non-human mammal. For example, when the non-human mammal is a mouse (preferably an inbred mouse such as C57BL / 6J (B6), F _{1 of} B6 and other inbred line, etc.), usually after administration of follicle stimulating hormone, 48 hours later, a method of obtaining a fertilized egg by administering luteinizing hormone and immediately mating with male mice is preferred. The dose of follicle stimulating hormone is about 20 to about 50 IU / individual, preferably about 30 IU / individual, luteinization. The dosage of the hormone is about 0 to about 10 IU / individual, preferably about 5 IU / individual.
After a certain period of time, the abdominal cavity of a female non-human mammal whose mating has been confirmed by inspection of the vaginal plug, etc. is opened, the fertilized egg is removed from the oviduct, and an embryo culture medium (eg, M16 medium, modified Whitten medium, BWW medium, M2 medium, WM-HEPES medium, BWW-HEPES medium, etc.) are washed to remove cumulus cells, and the cells are cultured until DNA microinjection under 5% carbon dioxide / 95% air by the microdrop culture method or the like. When microinjection is not performed immediately, the collected fertilized eggs can be stored frozen by a slow method or an ultra-rapid method.

  On the other hand, in the case of in vitro fertilization, ovulation was induced by administering follicle stimulating hormone and luteinizing hormone to female non-human mammals for egg collection (similar to those used in in vivo fertilization are preferably used) as described above. Thereafter, the ovum is collected and cultured in a fertilization medium (eg, TYH medium) in the atmosphere of 5% carbon dioxide / 95% by microdrop culture or the like until in vitro fertilization. On the other hand, the epididymis tail is taken out from the same kind of male non-human mammal (the same as in the case of in-vivo fertilization), and the sperm mass is collected and pre-cultured in a fertilization medium. Sperm after completion of preculture is added to a fertilization medium containing an egg, and cultured in a 5% carbon dioxide / 95% atmosphere by a microdrop culture method or the like, and then a fertilized egg having two pronuclei is observed under a microscope. Select. When DNA microinjection is not performed immediately, the obtained fertilized egg can be stored frozen by a slow method or an ultra-rapid method.

  Microinjection of DNA into a fertilized egg can be performed according to a conventional method using a known apparatus such as a micromanipulator. Briefly, a fertilized egg placed in a microdrop of embryo culture medium is aspirated and fixed with a holding pipette, and the DNA solution is directly injected into the male or female pronucleus, preferably into the male pronucleus, using an injection pipette. inject. The introduced DNA is preferably one that has been highly purified by CsCl density gradient ultracentrifugation or the like. The introduced DNA is preferably linearized by cleaving the vector portion using a restriction enzyme.

The fertilized egg after DNA introduction is cultured from the 1 cell stage to the blastocyst stage in 5% carbon dioxide gas / 95% air in the embryo culture medium by the microdrop culture method or the like, and then pseudopregnant female. Transplanted into the fallopian tube or uterus of a non-human mammal. The female non-human mammal for embryo transfer may be of the same species as the animal from which the early embryo to be transplanted is derived. For example, when transplanting a mouse early embryo, an ICR female mouse (preferably about 8 to about 10 weeks old) is preferably used. Examples of a method for bringing a female non-human mammal for embryo transfer into a pseudopregnant state include, for example, the same type of vasectomized (ligated) male non-human mammal (for example, in the case of a mouse, an ICR male mouse (preferably about 2). There is known a method of selecting those in which the presence of vaginal plug has been confirmed by mating with those of a month old or older)).
Embryonic females may be naturally ovulated, or administered luteinizing hormone-releasing hormone (generally abbreviated as LHRH) or its analogs prior to mating with vasectomized (ligated) males. Alternatively, fertility induced may be used. Examples of LHRH analogs include [3,5-DiI-Tyr ⁵ ] -LH-RH, [Gln ⁸ ] -LH-RH, [D-Ala ⁶ ] -LH-RH, [des-Gly ¹⁰ ]- Examples thereof include LH-RH, [D-His (Bzl) ⁶ ] -LH-RH, and their ethylamide. The dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after the administration vary depending on the type of non-human mammal. For example, when the non-human mammal is a mouse (preferably an ICR mouse or the like), it is usually preferable to cross with a male mouse about 4 days after administration of LHRH or an analog thereof. The dose of the analog is usually about 10-60 μg / individual, preferably about 40 μg / individual.

  Usually, when the early embryo to be transplanted is after the morula stage, the embryo is transplanted into the uterus of the female for embryos, and before that (for example, the 1-cell to 8-cell embryos). As a female for embryo transfer, a female whose a certain number of days has passed since pseudopregnancy is appropriately used depending on the stage of development of the transplanted embryo. For example, in the case of mice, female mice about 0.5 days after pseudopregnancy are preferable for transplanting 2-cell stage embryos, and female mice about 2.5 days after pseudopregnancy are preferable for transplanting blastocyst stage embryos. . After anesthesia (preferably Avertin or the like is used) for the embryo recipient female, incision is made to draw out the ovary, and early embryos (about 5 to about 10) suspended in the embryo culture medium are used with an embryo transfer pipette. Inject into the abdominal cavity or near the fallopian tube junction.

  If the transplanted embryo is successfully implanted and the recipient female becomes pregnant, a non-human mammal can be obtained by natural delivery or caesarean section. It is only necessary to continue suckling to the naturally delivered embryos, and if the baby is delivered by caesarean section, the offspring is a separately provided female female (for example, in the case of a mouse, a female mouse normally mated and delivered) Can be fed to ICR female mice))).

Gene transfer at the fertilized egg cell stage is ensured so that the transferred DNA is present in all germ line cells and somatic cells of the subject non-human mammal. Whether or not the introduced DNA is incorporated into the chromosomal DNA can be assayed, for example, by screening the chromosomal DNA separated and extracted from the tail of the litter by Southern hybridization or the PCR method. The presence of the introduced DNA in the germ line cells of the offspring non-human mammal (F ₀ ) obtained as described above means that all of the progeny (F ₁ ) animals have their germ line cells and somatic cells in all. It means that there is a gene under the control of a promoter containing the FRE of the present invention.
Usually, F ₀ animals are obtained as heterozygotes having the introduced DNA only on one of the homologous chromosomes. Individual F ₀ individuals are randomly inserted on different chromosomes unless homologous recombination is used. In order to obtain a homozygote having introduced DNA on both homologous chromosomes, F ₀ animals and non-transgenic animals are crossed to produce F ₁ animals, and heterozygotes having introduced DNA only on one of the homologous chromosomes. You only have to cross your brothers and sisters. If the introduced DNA is integrated only at one gene locus, 1/4 of the resulting F ₂ animals are homozygous.

  In another preferred embodiment, an expression vector containing a gene under the control of a promoter containing the FRE of the present invention is an embryo of a non-human mammal that is a subject by a known gene transfer method such as electroporation. Introduced into stem cells (ES cells).

ES cells are cells derived from the inner cell mass (ICM) of fertilized eggs at the blastocyst stage, and can be cultured and maintained in an undifferentiated state in vitro. ICM cells are cells that will form the embryo body in the future, and are stem cells that form the basis of all tissues including germ cells. As ES cells, those already established may be used, or newly established according to the method of Evans and Kaufman (Nature 292, 154, 1981). For example, in the case of mouse ES cells, currently, ES cells derived from 129 mice are generally used. However, since the immunological background is unclear, an alternative purely immunologically genetic gene is used. For the purpose of obtaining ES cells with a clear background, for example, C57BL / 6 mice and BDF ₁ mice (C57BL / 6 and DBA / ES cells established from F ₁ ) and 2 can also be used favorably. The BDF ₁ mouse has C57BL / 6 mice in the background in addition to the advantage that the number of eggs collected is large and the eggs are strong. Therefore, when the ES cells derived therefrom are used to produce disease model mice, the C57BL / 6 mice Can be advantageously used in that it can be backcrossed to replace its genetic background with C57BL / 6 mice.

ES cells can be prepared, for example, as follows. When using a female non-human mammal after mating [for example, a mouse (preferably an inbred mouse such as C57BL / 6J (B6), F _{1 of} B6 and other inbred line, etc.)] A blastocyst stage embryo is collected from the uterus of a female mouse (about 3.5 days of pregnancy is preferably used) of about 8 to about 10 weeks of age mated with a male mouse (or before the morula stage) After the initial embryo is collected from the fallopian tube, it may be cultured to the blastocyst stage in the same manner as described above in an embryo culture medium, and an appropriate feeder cell (for example, in the case of a mouse, a primary embryo prepared from a mouse embryo. When cultured on a layer such as a fibroblast or a known STO fibroblast cell line, some cells of the blastocyst gather to form an ICM that differentiates into an embryo in the future. This inner cell mass is trypsinized to dissociate single cells, and an ES cell is obtained by repeating dissociation and passage while maintaining a suitable cell density and exchanging the medium.

Both male and female ES cells may be used, but male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work. One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, the number of ES cells of about 1 colony (about 50) is required, while the number of cells required for karyotype analysis is about 10 ⁶ cells. It is possible to perform primary selection of ES cells in the early stage by discriminating between males and females, and by allowing male cells to be selected at an early stage, labor at the initial stage of culture can be greatly reduced.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical manipulation in establishing the cell line, normal cells (for example, mice) are introduced after gene introduction into the ES cell. Then, it is desirable to clone again into a cell having a chromosome number of 2n = 40.

The ES cell line thus obtained needs to be subcultured carefully in order to maintain the properties of undifferentiated stem cells. For example, on an appropriate feeder cell such as STO fibroblast, in a carbon dioxide incubator (preferably 5% carbon dioxide / 95) in the presence of LIF (1 to 10,000 U / ml) known as a differentiation inhibitor. Cultivated at about 37 ° C. with 5% oxygen or 5% oxygen / 5% carbon dioxide gas / 90% air), and at the time of passage, for example, trypsin / EDTA solution (usually 0.001 to 0.5% A single cell is obtained by treatment with trypsin / 0.1-5 mM EDTA (preferably about 0.1% trypsin / 1 mM EDTA) and seeded on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, the cells are observed, and if morphologically abnormal cells are observed, the cultured cells are desirably discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrology and Experimental Morphology, 87, 27, 1985], including the FRE of the present invention. The gene-expressing non-human mammalian cell obtained by differentiating an ES cell into which a gene under the control of a promoter is differentiated is an in vitro diet of the FRE of the present invention (eg, a high fructose diet). Useful in consideration of responsiveness to.

For gene transfer into ES cells, calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method (microinjection) method, gene gun (particle gun) method, Any of the DEAE-dextran methods and the like can be used, but the electroporation method is generally selected from the viewpoint that a large number of cells can be easily treated. For electroporation, the conditions used for gene transfer into normal animal cells may be used as they are. For example, after ES cells in the logarithmic growth phase are treated with trypsin and dispersed into single cells, 10 ⁶ 10 ⁸ transferred to a cuvette and suspended in medium to a cell / ml, the vector containing the introduced DNA was added 10-100 [mu] g, it can be carried out by applying an electric pulse of 200~600V / cm.

  ES cells into which the introduced DNA has been incorporated can be assayed by screening chromosomal DNA isolated from colonies obtained by culturing single cells on feeder cells by Southern hybridization or PCR, The greatest advantage of the transgenic system using ES cells is that a transformant can be selected at the cell stage using the expression of a drug resistance gene or a reporter gene as an index. Therefore, the introduction vector used here includes a drug resistance gene (eg, neomycin phosphotransferase II (nptII) gene, hygromycin phosphotransferase) in addition to the expression cassette of the gene under the control of the promoter containing the FRE of the present invention. (Hpt) gene) and a reporter gene (eg, β-galactosidase (lacZ) gene, chloramphenicol acetyltransferase (cat) gene, etc.) etc. For example, when a vector containing the nptII gene is used as a selectable marker gene, the ES cells after the gene transfer treatment are cultured in a medium containing a neomycin antibiotic such as G418, and the resulting resistant colonies are transferred to a culture plate. After repeated trypsinization and medium exchange, a part is left for culture, and the rest is subjected to PCR or Southern hybridization to confirm the presence of the introduced DNA.

  When the ES cell in which the introduced DNA has been confirmed to be integrated is returned into an embryo derived from the same kind of non-human mammal, it is incorporated into the ICM of the host embryo to form a chimeric embryo. A chimeric transgenic animal can be obtained by transplanting this to a foster parent (female for embryo transfer) and continuing the development. If ES cells contributed to the formation of primordial germ cells that will differentiate into eggs or sperm in the future in a chimeric animal, germline chimeras will be obtained, and the introduced DNA will be genetically fixed by mating. Transgenic non-human mammals can be produced.

The chimera embryos are prepared by adhering and collecting early embryos up to the morula stage (aggregate chimera method) and by microinjecting cells into the blastocyst dividing space (injection chimera method) However, in the production of chimeric embryos using ES cells, the latter has been widely used in the past. Recently, a method for producing an aggregate chimera by injecting ES cells into the zona pellucida of the 8-cell embryo, As a method that does not require a micromanipulator and is easy to operate, a method of producing an aggregate chimera by co-culturing and aggregating an ES cell mass and an 8-cell embryo from which the zona pellucida has been removed has also been performed.
In either case, the host embryo can be similarly collected from a non-human mammal that can be used as an egg-collecting female in gene transfer into a fertilized egg. It is preferable to collect a host embryo from a mouse having a different color from the strain from which the ES cells are derived so that the contribution rate can be determined by the coat color. For example, if ES cells are derived from 129 mice (hair color: Agouti), C57BL / 6 mice (hair color: black) and ICR mice (hair color: albino) are used as the female for egg collection, and ES cells are C57B / 6 or DBF. _{If it is} derived from ₁ mouse (hair color: black) or TT2 cells (derived from C57B / 6 and CBA F ₁ (hair color: Agouti)), ICR mouse or BALB / c mouse (hair color: albino) is used as a female for egg collection. be able to.
In addition, since the germline chimera-forming ability largely depends on the combination of ES cells and the host embryo, it is more preferable to select a combination having a high germline chimera-forming ability. For example, in the case of a mouse, it is preferable to use a host embryo derived from the C57B / 6 line for ES cells derived from the 129 line, and a host embryo derived from the BALB / c line for ES cells derived from the C57B / 6 line. Etc. are preferred.
The female mouse for egg collection is preferably about 4 to 6 weeks of age, and the male mouse for mating is preferably of the same strain of about 2 to about 8 months of age. The mating may be natural mating, but is preferably performed after inducing superovulation by administering a gonadotropin (follicle stimulating hormone and then luteinizing hormone).

In the case of blastocyst injection, a blastocyst stage embryo (for example, in the case of a mouse, about 3.5 days after mating) is collected from the uterus of a female for egg collection (or an early embryo before the morula stage is removed from the oviduct. After harvesting, it may be cultured to the blastocyst stage in the above-described embryo culture medium), and the gene under the control of the promoter containing the FRE of the present invention in the split space of the blastocyst using a micromanipulator After injecting ES cells (about 10 to about 15 cells) into which embryos have been introduced, they are transplanted into the uterus of a female non-human mammal for embryo transfer that has been pseudopregnant. As the female non-human mammal for embryo transfer, a non-human mammal that can be used as a female for embryo transfer in gene transfer into a fertilized egg can be used as well.
In the case of the co-culture method, 8-cell stage embryos and morulae (for example, about 2.5 days after mating in the case of mice) are collected from the oviduct and uterus of the female egg collection (or early stage before the 8-cell stage). The embryo may be collected from the oviduct and then cultured in the above-described embryo culture medium until the 8-cell stage or morula stage) After dissolving the zona pellucida in acidic tyrode solution, it was overlaid with mineral oil An ES cell mass (about 10 to about 15 cells) into which a gene under the control of a promoter containing the FRE of the present invention has been introduced into microdrops of an embryo culture medium, and further the above 8-cell embryo or mulberry Seed embryos (preferably 2) and co-culture overnight. The obtained morula or blastocyst is transplanted into the uterus of a female non-human mammal for embryo reception in the same manner as described above.

If the transplanted embryo is successfully implanted and the recipient female becomes pregnant, a chimeric non-human mammal can be obtained by natural delivery or caesarean section. It is only necessary to continue suckling for naturally delivered embryos, and if a baby is delivered by caesarean section, the offspring are fed to a separately prepared nursing female (a female non-human mammal that is normally mated and delivered). be able to.
In the selection of germline chimeras, first, if the sexes of ES cells have been discriminated in advance, a chimeric animal of the same sex as the ES cells is selected (usually male ES cells are used, so male chimeric animals are selected. Then, a chimeric animal (for example, 50% or more) having a high ES cell contribution rate is selected from the phenotype such as hair color. For example, in the case of a chimeric mouse obtained from a chimeric embryo of D3 cells, which are male ES cells derived from a 129 mouse, and a host embryo derived from a C57B / 6 mouse, the male mouse having a high proportion of agouti's coat color is selected. preferable. Check selected chimeric non-human mammal of whether germline chimeras can be performed based on the phenotype of the F ₁ animal obtained by crossing with the appropriate strains of the same animal species. For example, in the case of the above chimeric mouse, Agouti is dominant over black, so when crossed with female C57B / 6 mice, the hair color of F ₁ obtained is Agouti if the selected male mouse is a germline chimera. .

A germline chimeric non-human mammal (founder) into which a gene under the control of a promoter containing the FRE of the present invention obtained as described above is usually heterozygous having the introduced DNA only in one of the homologous chromosomes. Obtained as a body. Individual founders are randomly inserted on different chromosomes unless homologous recombination is used. In order to obtain a homozygote having a gene under the control of a promoter containing the FRE of the present invention in both homologous chromosomes, the introduced DNA is introduced into only one of the homologous chromosomes of the F ₁ animal obtained as described above. What is necessary is just to cross the heterozygous brothers and sisters which have. The selection of the heterozygote can be assayed, for example, by screening chromosomal DNA isolated and extracted from the tail of the F ₁ animal by Southern hybridization or PCR. If the introduced DNA is integrated only at one gene locus, 1/4 of the resulting F ₂ animals are homozygous.

  The FRE-Tg animal of the present invention is useful for examining the expression response of a gene (for example, SREBP-1c gene) under the control of a promoter containing the FRE of the present invention to a sugar (particularly fructose) load. There is no problem if the structural gene used for the transgene is a reporter gene that is not endogenous to the host animal. For example, when the SREBP-1c gene is used as the structural gene (particularly, SREBP- containing the FRE of the present invention in the promoter region). When the 1c gene is used as “a gene under the control of a promoter containing the FRE of the present invention”, it is desirable to inactivate the endogenous SREBP-1c gene. The FRE-Tg animal of the present invention in which the endogenous SREBP-1c gene is inactivated (SREBP-1c gene knockout animal) can be obtained by known methods (for example, Lee SS et al., Molecular and Cellular Biology (Mol. Cell. Biol.), Vol. 15, p. 3012, 1995) ES cells in which the SREBP-1c gene selected is selected, or SREBP-1c knockouts produced from the ES cells according to the method described above It can be obtained by introducing a gene into an early embryo or ES cell derived from an animal according to the method described above. As a specific means for knocking out the SREBP-1c gene, the SREBP-1c gene derived from the subject non-human mammal is isolated according to a conventional method, and, for example, other DNA fragments (for example, neomycin resistance gene, hygroglobulin) Insertion of drug resistance gene such as mycin resistance gene, reporter gene such as lacZ (β-galactosidase gene), cat (chloramphenicol acetyltransferase gene), etc. Incorporation of the introduced DNA can be selected by using drug resistance or reporter gene expression as an index), and using the Cre-loxP system or Flp-frt system, all or part of the SREBP-1c gene is excised. Or complete by inserting a stop codon into the protein coding region. Result by either disabling the translation of the correct protein or by inserting a DNA sequence (eg polyA addition signal) that terminates the transcription of the gene into the transcribed region and disabling complete messenger RNA synthesis. A method in which a DNA strand having a DNA sequence constructed so as to inactivate a gene (hereinafter abbreviated as a targeting vector) is incorporated into the SREBP-1c locus of the target non-human mammal by homologous recombination is preferred. Can be mentioned.

  Usually, gene recombination in mammals is mostly non-homologous, and the introduced DNA is randomly inserted at any position on the chromosome. Therefore, depending on the selection such as detection of drug resistance or reporter gene expression, it is not possible to efficiently select only clones targeted to the endogenous SREBP-1c gene targeted by homologous recombination. For these clones, it is necessary to confirm the integration site by Southern method or PCR method. Therefore, if a herpes simplex virus-derived thymidine kinase (HSV-tk) gene that confers sensitivity to ganciclovir, for example, is linked outside the region homologous to the target sequence of the targeting vector, cells into which the vector is randomly inserted Has a HSV-tk gene and cannot grow in a ganciclovir-containing medium, but cells targeted to the endogenous SREBP-1c locus by homologous recombination do not have the HSV-tk gene and are selected to be resistant to ganciclovir. Alternatively, if, for example, a diphtheria toxin gene is linked instead of the HSV-tk gene, cells into which the vector has been randomly inserted will be killed by the toxin produced by the vector, so that homologous recombinants can be removed in the absence of a drug. You can also choose.

Alternatively, the transgenic animal of the present invention in which the expression of the endogenous SREBP-1c gene has been inactivated is endogenous to the SREBP-1c gene containing the FRE of the present invention in the promoter region by gene targeting using homologous recombination. It may be a knock-in animal in which the SREBP-1c gene is replaced. That is, the present invention also provides that the endogenous SREBP-1c gene that does not have the FRE of the present invention in the promoter region (for example, contains the mutant FRE of the present invention in the promoter region) is under the control of the promoter containing the FRE of the present invention. FRE-Tg animals substituted with certain SREBP-1c genes are provided.
Examples of host non-human animals having an endogenous SREBP-1c gene that does not have the FRE of the present invention in the promoter region include non-human animals that include the mutant FRE of the present invention in the promoter region. Non-human animal strains (for example, C57BL, DBA mice in the case of mice) that do not increase the expression of the SREBP-1c gene in response to a load (particularly fructose load) or do not show a tendency of metabolic disorders such as serum lipid elevation Can be mentioned.

Knock-in animals can be prepared according to basically the same procedure as knock-out animals. A targeting vector containing a SREBP-1c gene under the control of a promoter containing the FRE of the present invention is introduced into ES cells derived from the subject non-human mammal according to the method described above, and endogenous SREBP- of the animal is homologously recombined. An ES cell clone in which the SREBP-1c gene under the control of a promoter containing the FRE of the present invention is selected at the 1c locus may be selected. Clone selection can be performed using PCR or Southern method. For example, a marker gene for positive selection such as neomycin resistance gene is inserted into the 3 ′ untranslated region of the SREBP-1c gene of the targeting vector, If a marker gene for negative selection such as HSV-tk gene or diphtheria toxin gene is inserted outside the region homologous to the sequence, homologous recombinants can be selected using drug resistance as an index.
In addition, since it may interfere with the expression of SREBP-1c into which a positive selection marker gene has been introduced, a targeting vector in which loxP sequences or frt sequences are arranged at both ends of the positive selection marker gene is used. It is preferable to excise a marker gene for positive selection by allowing Cre or Flp recombinase or the recombinase expression vector (eg, adenovirus vector) to act at an appropriate time. Alternatively, instead of using the Cre-loxP system or Flp-frt system, sequences that are homologous to the target sequence are repeatedly arranged in the same direction at both ends of the positive selection marker gene, and intragenic recombination between the sequences is used. Then, the marker gene for positive selection may be excised.

  The transgenic animal of the present invention having the SREBP-1c gene substituted with the promoter thus obtained (hereinafter referred to as “the FRE-KI animal of the present invention”) has a fructose response of the present invention in the SREBP-1c promoter region. It has an element. As shown in Examples below, in the hepatocytes of DBA / 2 mice, the expression of the transcriptional regulatory factor of the present invention that can bind to the FRE of the present invention is suppressed regardless of fasting and after eating. Therefore, by examining the expression of the transcriptional regulatory factor of the present invention in the FRE-KI animal of the present invention, the difference between the animal strains in the expression of the transcriptional regulatory factor can be elucidated, and thus the transcriptional regulatory factor and SREBP- It may be possible to clarify the whole expression control mechanism of sugar / lipid metabolism-related genes including 1c gene.

For the same purpose as described above, the present invention provides a non-human transgenic animal into which a gene under the control of a promoter containing the mutant FRE of the present invention has been introduced, or an endogenous SREBP containing the FRE of the present invention in the promoter region. A transgenic animal in which the -1c gene is replaced with a SREBP-1c gene that does not have the FRE of the present invention in the promoter region (for example, a SREBP-1c gene under the control of a promoter containing the mutant FRE of the present invention) is provided .
A non-human transgenic animal into which a gene under the control of a promoter containing the mutant FRE of the present invention is introduced, except that the mutant FRE of the present invention is used instead of the FRE of the present invention. It can be produced in the same manner as animals.
Examples of host non-human animals having an endogenous SREBP-1c gene containing the FRE of the present invention in the promoter region include, for example, increased expression of the SREBP-1c gene in response to sugar load (particularly fructose load), or serum lipids Non-human animal strains (eg, CBA, C3H mice in the case of mice) exhibiting a tendency of metabolic disorders such as elevation can be mentioned.
The knock-in animal can be produced in the same manner as the FRE-KI animal of the present invention described above.

The present invention also provides a non-human transgenic animal into which DNA encoding the transcriptional regulatory factor of the present invention has been introduced, and a non-human animal (knockout animal) in which the DNA encoding the transcriptional regulatory factor is inactivated. .
Such transgenic animals and knockout animals can be prepared by the same methods as those for the FRE-Tg animals and SREBP-1c gene knockout animals of the present invention described above.

A non-human transgenic animal into which a DNA encoding the transcriptional regulatory factor of the present invention (normal DNA) has been introduced (hereinafter referred to as “TF-Tg animal of the present invention”) is highly expressed in the transcriptional regulatory factor of the present invention. In some cases, the function of the endogenous normal DNA is promoted to eventually cause hyperfunction of the transcriptional regulator, which can be used as a disease model animal. For example, by using the normal DNA-transferred animal of the present invention, elucidation of the hyperfunction of the transcriptional regulatory factor of the present invention and the pathological mechanism of the disease associated with the transcriptional regulatory factor, and the examination of a treatment method for these diseases It is possible.
In addition, since the TF-Tg animal of the present invention has an increased symptom of the transcriptional regulatory factor of the present invention, diseases associated with hyperfunction of the transcriptional regulatory factor, for example, metabolic disorders, particularly sugar / lipid metabolic disorders (eg, : Prevention and treatment for diseases such as hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity, impaired glucose tolerance, fasting blood glucose disorder, hyperinsulinemia, hypertension, albuminuria) It can also be used for screening tests of substances.

On the other hand, a non-human mammal having DNA (abnormal DNA) encoding an abnormal transcription regulatory factor of the present invention (that is, a mutant protein of the transcription regulatory factor of the present invention that does not exhibit a transcriptional regulatory function) (hereinafter referred to as “the present invention”). "Abnormal TF-Tg animal") is highly expressed in the abnormal DNA, and finally inhibits the function of endogenous normal DNA (for example, transcription promoting activity such as SREBP-1c gene), The transcriptional regulatory factor of the present invention may become a functionally inactive refractory disease and can be used as a disease state model animal. For example, by using the abnormal TF-Tg animal of the present invention, it is possible to elucidate the pathological mechanism of the functional inactive refractory of the transcriptional regulatory factor and to examine a method for treating this refractory.
Further, as a specific applicability, the abnormal TF-Tg animal of the present invention is capable of inhibiting the function of a normal transcription regulatory factor (dominant negative) by an abnormal transcription regulatory factor in the functional inactive refractory of the transcriptional regulatory factor of the present invention. Model).
Moreover, since the abnormal TF-Tg animal of the present invention has the function-inhibiting symptom of the transcriptional regulatory factor of the present invention, it can be used in a therapeutic agent screening test for the functional inactive refractory of the transcriptional regulatory factor.
Furthermore, in order to develop a prophylactic / therapeutic agent for diseases related to the transcriptional regulatory factor, including the functional inactive refractory of the transcriptional regulatory factor of the present invention, using the TF-Tg animal of the present invention. Thus, it is possible to provide an effective and rapid screening method for a prophylactic / therapeutic agent for diseases using the above-described test methods and quantitative methods. Moreover, it is possible to examine and develop a gene therapy method for a disease associated with the transcriptional regulator using the TF-Tg animal of the invention or the DNA expression vector encoding the transcriptional regulator of the invention.

A non-human animal in which the DNA encoding the transcriptional regulatory factor of the present invention is inactivated (hereinafter referred to as “TF-KO animal of the present invention”) is a deficient or damaged DNA encoding the transcriptional regulatory factor of the present invention. It can be used for screening for compounds having therapeutic / preventive effects against diseases caused by the disease.
That is, the present invention is characterized by administering a test compound to the TF-KO animal of the present invention and observing and measuring changes in the animal, such as a deficiency or damage of DNA encoding the transcriptional regulatory factor of the present invention. A screening method for a compound having a therapeutic / prophylactic effect on a disease caused by the disease or a salt thereof is provided.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
Specifically, by administering a test compound to a TF-KO animal of the present invention and comparing changes in each organ, tissue, disease symptom, etc. of the animal with a control animal not administered with the test compound, The therapeutic / preventive effect can be tested.
As a method for administering a test compound to a TF-KO animal, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptom of the TF-KO animal, the nature of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

In the screening method, when a test compound is administered to a TF-KO animal, for example, when the symptom of the TF-KO animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, The test compound can be selected as a compound having a therapeutic / prophylactic effect on the above-mentioned diseases.
The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds, such as a safe and low-toxic therapeutic / preventive agent for diseases caused by deficiency or damage of the transcriptional regulatory factor of the present invention. It can be used as a medicine. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt, and the salt of the compound is physiological with an acid (eg, inorganic acid, organic acid, etc.) or a base (eg, alkali metal, etc.). Acceptable salts are used, and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
The pharmaceutical containing the compound obtained by the screening method or a salt thereof can be formulated in the same manner as the above-mentioned inhibitor of the transcriptional regulatory factor of the present invention and administered to a mammal.

According to the present invention, there is provided a compound or a salt thereof that promotes or inhibits the activity of the transcriptional regulator gene promoter of the present invention, comprising administering a test compound to the TF-KO animal of the present invention and detecting the expression of a reporter gene. A screening method is provided.
In the above screening method, the TF-KO animal of the present invention is inactivated by introducing a reporter gene into the transcriptional regulatory factor gene of the present invention, and the reporter gene is under the control of the transcriptional regulatory factor gene promoter of the present invention. Those that can be expressed in are used.
Examples of the test compound are the same as described above.
As the reporter gene, for example, β-galactosidase gene (lacZ), soluble alkaline phosphatase gene or luciferase gene is suitable.
In the TF-KO animal of the present invention in which the transcriptional regulatory factor gene of the present invention is replaced with a reporter gene, the reporter gene is present under the control of the transcriptional regulatory factor gene promoter of the present invention, so that the expression of the substance encoded by the reporter gene is expressed. By tracing the above, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the transcriptional regulatory factor of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the present invention is inherently a tissue expressing the transcriptional regulatory factor of the present invention. Β-galactosidase is expressed in place of the transcriptional regulatory factor. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), the present invention can be easily performed. It is possible to observe the expression state of the transcriptional regulatory factor in the animal body. Specifically, the transcriptional regulatory factor-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or 37. After reacting at about 30 ° C. for about 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the transcriptional regulator gene promoter activity of the present invention.
The compound obtained by the screening method may form a salt, and the salt of the compound is physiologically acceptable with an acid (eg, inorganic acid) or a base (eg, organic acid). In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

The compound or its salt that promotes the transcriptional regulator gene promoter activity of the present invention can promote the expression of the transcriptional regulatory factor of the present invention and promote the function of the transcriptional regulatory factor of the present invention. It can be used as a medicine such as a prophylactic / therapeutic agent for diseases associated with dysfunction of transcriptional regulators.
On the other hand, the compound or its salt that inhibits the transcriptional regulator gene promoter activity of the present invention can inhibit the expression of the transcriptional regulatory factor of the present invention and inhibit the function of the transcriptional regulatory factor of the present invention. The present invention is useful as a pharmaceutical agent such as a prophylactic / therapeutic agent for diseases associated with excessive expression of the transcriptional regulatory factor of the present invention. Specifically, metabolic disorders, particularly sugar / lipid metabolic disorders (eg, hyper-TG, hyper-LDL-C, hypo-HDL-C, obesity, impaired glucose tolerance, fasting glycemic disorder, hyperinsulinemia It can be used as a low-toxic and safe medicine such as a preventive / therapeutic agent for diseases such as symptom, hypertension, and albuminuria.
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The pharmaceutical containing the compound obtained by the screening method or a salt thereof can be formulated in the same manner as the above-mentioned inhibitor of the transcriptional regulatory factor of the present invention and administered to a mammal.

Thus, the TF-KO animal of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the transcriptional regulator gene promoter of the present invention, and encodes the transcriptional regulatory factor of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by deficient expression of DNA or the development of preventive / therapeutic agents.
In addition, by using DNA containing the promoter region of the transcriptional regulatory factor gene of the present invention, genes encoding various proteins are ligated downstream thereof, and this is injected into an egg cell of an animal so-called transgenic animal (gene transfer). If (animal) is produced, the protein can be synthesized in a tissue and / or time-specific manner, and the action in the living body can be examined. Furthermore, if an appropriate reporter gene is bound to the above promoter portion and a cell line in which it is expressed is established, the ability to specifically promote or suppress the production ability of the transcriptional regulatory factor of the present invention itself in the body is reduced. It can be used as a search system for molecular compounds.

In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid *: Corresponding to stop codon Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenol Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The base sequence of the SREBP-1c promoter region (from immediately before the presumed transcription start point to 574 bases upstream) derived from a CBA mouse is shown.
[SEQ ID NO: 2]
The base sequence encoding mouse-derived NBP is shown.
[SEQ ID NO: 3]
The amino acid sequence of mouse-derived NBP is shown.
[SEQ ID NO: 4]
The base sequence which codes mouse-derived RBMX-like protein is shown.
[SEQ ID NO: 5]
1 shows the amino acid sequence of a mouse-derived RBMX-like protein.
[SEQ ID NO: 6]
The base sequence of the SREBP-1c promoter region (from TATA-like sequence to about 1.2 kb upstream) derived from a CBA mouse is shown.
[SEQ ID NO: 7]
The base sequence of the oligonucleotide designed as a primer for amplifying the SREBP-1c promoter region (from TATA-like sequence to about 1.2 kb upstream) is shown.
[SEQ ID NO: 8]
The base sequence of the oligonucleotide designed as a primer for amplifying the SREBP-1c promoter region (from TATA-like sequence to about 1.2 kb upstream) is shown.
[SEQ ID NO: 9]
The base sequence of the oligonucleotide corresponding to the fructose response element in the SREBP-1c promoter of CBA / JN Crj mouse is shown.
[SEQ ID NO: 10]
The base sequence of the oligonucleotide corresponding to the mutant fructose response element in the SREBP-1c promoter of DBA / 2 JN Crj mouse is shown.
[SEQ ID NO: 11]
The base sequence of the oligonucleotide corresponding to the sense strand of the fructose response element in the SREBP-1c promoter of CBA / JN Crj mouse is shown.
[SEQ ID NO: 12]
The base sequence of the oligonucleotide corresponding to the antisense strand of the fructose response element in the SREBP-1c promoter of CBA / JN Crj mouse is shown.
[SEQ ID NO: 13]
The base of the human-derived SREBP-1c promoter region [corresponding to the complementary strand sequence of the base sequence represented by base numbers 1666061-16566560 in the base sequence of human chromosome 17 (registration number: NT_010718) registered in GenBank] Indicates the sequence.

The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by these examples.
All materials were reagent grade and purchased from Nacalai Tesque or Sigma Chemical unless otherwise specified. Unless otherwise specified, numerical data is expressed as an average value ± standard deviation. Significant differences between groups were determined using Tukey-Welsh's tapering multiple comparison. P <0.05 was considered significant.

Comparison of metabolic responses of various mouse strains to diet
5 lines [BALB / c Cr Slc (purchased from Japan SLC); C3H / HeJ (purchased from Charles River Japan); C57BL / 6J Jcl, DBA / 2N Crj and CBA / JN Crj (purchased from CLEA Japan)] Inbred mice (5 weeks old, male) with different age were placed in a breeding room controlled by a 12-hour light / dark cycle and allowed free access to laboratory food and water.
The animals were divided into 2 groups (normal diet group and high fructose diet group) and fed in parallel for 8 weeks. A normal diet (oriental yeast) consists of 58% carbohydrates (without fructose), 12% lipids and 30% protein, and a high fructose diet (oriental yeast) consists of 67% carbohydrates (98% fructose), 13% lipids and Contains 20% protein (% values indicate calories%).
The day before the experiment, food was removed from all animals at 8 pm. The animals were then divided into 4 groups (4-8 animals per group) as follows: 1) normal diet without refeeding (control-fasting; CF), 2) normal diet with refeeding (control-) Refeeding; CR), 3) High fructose diet without refeeding (fructose-fasting; FF), High fructose diet with refeeding (fructose-refeeding; FR). The mice in the CR and FR groups were re-fed in the dark between 6 am and 8 am. The CF and FF groups continued to fast. After measuring the body weight (BW) of each mouse, it was anesthetized at 10:00 am, and the incision, liver and epididymal fat were excised, and the weight (FW) of epididymal fat was measured. In addition, various blood tests for blood glucose (BS), triglyceride (TG), total cholesterol (CHO), and insulin (INS) were performed according to conventional methods. Table 1 shows the results of the CR group, and Table 2 shows the results of the FR group.

[Table 1]

Mouse BW FW BS TG CHO INS
Strain (g) (g) (mg / dl) (mg / dl) (mg / dl) (ng / ml)
C3H / HeJ 23 ± 2 0.37 ± 0.11 288 ± 31 154 ± 12 90 ± 7 0.3 ± 0.01
C57BL / 6J 23 ± 0 0.3 ± 0 302 ± 1 103 ± 4 63 ± 1 0.1 ± 0
BALB / c Cr SLC 21 ± 2 0.53 ± 0.01 269 ± 7 106 ± 15 240 ± 7 1.3 ± 0.3
CBA / JN Crj 28 ± 2 0.75 ± 0.07 252 ± 8 262 ± 35 77 ± 2 1.2 ± 0.4
DBA / 2JN Crj 23 ± 1 0.31 ± 0.06 223 ± 22 118 ± 9 91 ± 3 0.1 ± 0

[Table 2]

Mouse BW FW BS TG CHO INS
Strain (g) (g) (mg / dl) (mg / dl) (mg / dl) (ng / ml)
C3H / HeJ 33 ± 1 ^a 1.06 ± 0.12 ^b 328 ± 30 133 ± 16 153 ± 16 2.3 ± 0 ^b
C57BL / 6J 23 ± 0 0.5 ± 0 ^b 301 ± 30 101 ± 1 113 ± 3 ^a 0.1 ± 0
BALB / c Cr SLC 27 ± 1 0.77 ± 0.3 317 ± 7 ^a 153 ± 28 316 ± 7 ^a 2.4 ± 0 ^b
CBA / JN Crj 31 ± 1 1.41 ± 0.12 ^a 331 ± 19 ^a 392 ± 43 ^a 113 ± 7 ^b 6.5 ± 0.2 ^b
DBA / 2JN Crj 25 ± 1 0.5 ± 0.13 242 ± 15 160 ± 27 110 ± 4 0.2 ± 0
a: p <0.05 vs CR; b: p <0.01 vs CR

  In CBA / JN Crj mice, the high fructose diet significantly increased epididymal fat weight, blood glucose level, TG, CHO, and INS compared to the normal diet, whereas DBA / 2JN Crj mice There was no significant difference from the high fructose diet.

Correlation between metabolic response of various mouse strains to diet and SREBP-1c promoter sequence
5 strains [C3H / He Slc (purchased from Japan SLC); C57BL / 6N Jcl, DBA / 1JN Crj, DBA / 2N Crj and CBA / JN Crj (purchased from Japan Claire)] different inbred mice (5 weeks) (Age, male) were fed using the same feeding method as in Example 1 (4-6 animals per group), then anesthetized at 10 am, the liver was excised, immediately frozen in liquid nitrogen and stored at -80 ° C did. In addition, serum triglyceride (TG) concentration was measured according to a conventional method.
On the other hand, genomic DNA was isolated from frozen liver using DNeasy kit (QIEGEN). Based on the known mouse SREBP-1c promoter sequence, primers (sense: 5'-GCTGGACAGAACGGTGTCAT-3 '(SEQ ID NO: 7); antisense: 5'-TAAGAGCTCGGTACCTCCCCTAGGGC-3' (SEQ ID NO: 8)) were synthesized, and each genome PCR was performed using DNA as a template to amplify an SREBP-1c promoter fragment of about 1.2 kb. The amplified fragment was subcloned into TA-Cloning vector (Invitogen). The base sequence of each insert was determined using a DNA automatic sequencer DSQ 1000 (Shimadzu Corporation). As a result, the SREBP-1c promoter of C3H / He Slc and CBA / JN Crj mice has the nucleotide sequence represented by SEQ ID NO: 6, and in DBA / 2N Crj, DBA / 1JN Crj and C57BL / 6N Jcl mice It was revealed that guanine (G ₁₁₂ ) represented by base number 749 (base number 112 in the base sequence represented by SEQ ID NO: 1) in the base sequence represented by SEQ ID NO: 6 was substituted with adenine. Examination of the relationship between the metabolism response and the base substitution of mice against diet in C3H / the He Slc and CBA / JN Crj mice having SREBP-1c promoter comprising G _112, as compared to fasting (FF group) postprandial whereas serum TG concentration (FR group) is remarkably increased, DBA / 2N Crj with SREBP-1c promoter G ₁₁₂ is replaced with adenine, the DBA / 1JN Crj and C57BL / 6N Jcl mice, fasting There was no significant difference in blood TG concentration between (FF group) and after meal (FR group) (Table 3)

[Table 3]

Mouse strain 112th base serum TG concentration (mg / dl)
(SEQ ID NO: 1) Fasting (FF) After meal (FR)
C3H / He Slc G 80.3 ± 22.5 181 ± 19.9 ^*
CBA / JN Crj G 142 ± 22.7 419 ± 17.8 ^*
DBA / 2N Crj A 108 ± 63.2 126 ± 56.2
DBA / 1JN Crj A 67.5 ± 20.6 92.3 ± 26.3
C57BL / 6N Jcl A 54 ± 13.9 56.5 ± 10.9
*: P <0.001 vs FF

Differences between mouse strains in the response of lipid metabolism-related genes to diet
DBA / 2N Crj and CBA / JN Crj mice (5 weeks old, male; purchased from CLEA Japan) were raised with the same feeding method as in Example 1 (4-6 mice per group), then anesthetized and liver at 10 AM Were excised, immediately frozen in liquid nitrogen and stored at -80 ° C. In addition, serum triglyceride (TG) concentration was measured according to a conventional method.
Total RNA was isolated from frozen liver using TRIzol reagent (GIBCO-BRL Life Technologies, Inc.). RNA was run on a 1% agarose gel containing formamide and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech). Probes for SREBP-1, PPAR-α and fatty acid synthase (FAS) mRNA were prepared according to the method described in Am J Physiol Endocrinol Metab 282: E1180-E1190,2002. The probe was labeled with [α- ³² P] dCTP (New England Nuclear Research Products) using a labeling kit (Takara). The membrane was hybridized with a radiolabeled probe in Perfecthyb Buffer (Toyobo) and washed in 1 × SSC, 0.1% SDS at 68 ° C. for 1 hour. The blot was exposed to Kodak Biomax MR (Eastman Kodak) film at -80 ° C. The signal was quantified with a densitometer and the loading difference was normalized to the signal obtained using a probe for 18S ribosomal RNA. The results are shown in FIG.
In the DBA / 2 Crj mice, there was no significant difference in serum TG concentration among any of the 4 groups (CF, CR, FF, FR), whereas in the CBA / JN Crj mice, both normal and high fructose diets Serum TG concentration significantly increased after meals (CR, FR) compared to fasting (CF, FF), and high fructose diet (FR) increased TG concentration compared to normal diet (CR) I let you. No significant difference was found between the normal diet (CF) and the high fructose diet (FF) in the starved state (FIG. 1A). The expression level of SREBP-1c mRNA correlated well with the serum TG concentration (FIG. 1B). In addition, FAS, which is known to be regulated by SREBP-1c, showed the same expression behavior as SREBP-1c (FIG. 1D). On the other hand, the expression of PPARα mRNA, which regulates the expression of lipolytic enzymes, was not different between DBA / 2 Crj and CBA / JN Crj, both of which decreased after meal compared to fasting (Fig. 1C).

Difference between mouse strains in response to expression of lipid metabolism-related genes in response to sugar stimulation in primary hepatocytes CF and FR mice reared in the same manner as in Example 1 (DBA / 2 Crj and CBA / JN Crj (purchased from CLEA Japan)) Thus, hepatocytes were isolated by partially modifying the collagenase method. The animals were anesthetized and each liver was perfused with Krebs-Ringer buffer (KRB) in situ through the portal vein. The liver was then perfused with 100 ml of KRB containing collagenase (Sigma-Aldrich). Dissociated cells were shaken and dispersed, then in ice-cold DMEM (GIBCO-BRL Life Technology) containing 10% (v / v) fetal calf serum (FCS), 100 μg / ml streptomycin and 100 U / ml penicillin after shaking And filtered through a gauge at 4 ° C.
Cells were precipitated and washed twice at 4 ° C. using the same medium. Aliquots of 8 × 10 ⁶ cells were added to William's E medium (Shigma-Aldrich) supplemented with 10% (v / v) FCS, 1 nM insulin, 100 nM triiodothyronine, 100 nM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin. ) Medium was plated on a 6-well plate coated with rat collagen. After 3 hours incubation at 37 ° C. in 9% CO ₂ , the cells were washed twice with PBS and William E supplemented with 1 nM insulin, 100 nM triiodothyronine, 100 nM dexamethasone, 100 U / ml penicillin and 100 μg / ml streptomycin. Incubated in medium. After 16 hours of incubation, cells were transferred to William's E medium supplemented with 5 mM glucose, 5 mM fructose or 5 mM glucose + 100 nM insulin. After incubation for a certain period of time, the cells were collected, and extraction of total RNA and Northern hybridization using SREBP-1c or FAS cDNA probe were performed in the same manner as in Example 3. The results are shown in FIG.
Even in in vitro experiments using primary hepatocytes, CAB / JN Crj mice showed enhanced expression of SREBP-1c (Fig. 2A) and FAS (Fig. 2B) mRNA to the same level as that of insulin stimulation by single stimulation of fructose. However, in DBA / 2 JN Crj mice, there was no significant difference in SREBP-1c mRNA expression between each stimulus, and there was no difference in FAS mRNA expression between glucose stimulation and fructose stimulation. The expression of FAS mRNA is also increased by insulin stimulation in DBA / 2 JN Crj mice, suggesting that regulatory factors other than SREBP-1c are involved in the control of FAS expression response to insulin.

Identification of a transcriptional regulatory factor that binds to the fructose response element in the SREBP-1c promoter From CF and FR group mice (DBA / 2 Crj and CBA / JN Crj (purchased from Clea Japan)) reared in the same manner as in Example 1. The liver was excised and hepatocyte nuclear protein extract was isolated according to the method of Gorski et al. (Cell 47: 767-776, 1986). Suspend nuclear extract in 20 mM HEPES (pH 7.9), 330 mM NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol and 0.2 mM PMSF and freeze aliquots in liquid nitrogen And stored at -80 ° C. Meanwhile, CBA / JN Crj mice SREBP-1c of G ₁₁₂ and the vicinity thereof in the promoter nucleotide sequence (5'-CTAAAGGCAGCTATTGGCCT-3 '; SEQ ID NO: 9) and and DBA / 2 JN Crj mouse SREBP-1c promoter in Two types of radiolabeled double-stranded oligonucleotides (referred to as CBA probe and DBA probe, respectively) consisting of the base sequence of the corresponding region (5′-CTAAAGGCAACTATTGGCCT-3 ′; SEQ ID NO: 10) were synthesized. These were used for electrophoretic mobility shift assay. Nucleic extract 10 μg, poly (dI-dC) 1 μg, 10 mM HEPES (pH 7.9), 60 mM KCL, 1 mM EDTA, 7% glycerol and 100,000 cpm labeled probe are mixed and incubated at room temperature for 20 minutes, protein-DNA binding The reaction was performed. After incubation, the sample was loaded onto a 6% polyacrylamide gel in 0.25 × Tris-Borate-EDTA (TBE) buffer and run at a voltage of 150V. After electrophoresis, the gel was dried and exposed to film. A competition assay using unlabeled oligonucleotide (cold probe) was also performed, and the band disappeared in the presence of the cold probe was identified as the band of the transcriptional regulator bound specifically to the probe. The results are shown in FIG.
A band (FIG. 3A; arrow) observed when a nuclear extract from a CBA / JN Crj mouse of FR group was reacted with a CBA probe was not observed when reacted with a DBA probe. Thus, the presence of transcriptional regulators that specifically binds to CBA / JN Crj mouse SREBP-1c G ₁₁₂ and its vicinity of the base sequence in the promoter of was confirmed.
In addition, when a nuclear extract derived from DBA / 2 JN Crj mice was reacted with a CBA probe, a band observed when a nuclear extract derived from CBA / JN Crj mice was reacted with a CBA probe (FIG. 3B; arrow) )), The SREBP-1c expression does not increase in response to high fructose diet load in DBA / 2 JN Crj mice due to not only the promoter mutation but also the binding protein expression deficiency. It is suggested that
Furthermore, when a nuclear extract from a CBA / JN Crj mouse in the CF group is reacted with a CBA probe, a band observed when a nuclear extract from a CBA / JN Crj mouse in the FR group is reacted with a CBA probe. The signal of (FIG. 3C; arrow) was very weak and correlated well with the expression of SREBP-1c mRNA in each group. Therefore, it was suggested that the SREBP-1c expression response to the diet in CBA / JN Crj mice is regulated by the expression of a transcriptional regulator that binds to the fructose response element in the SREBP-1c promoter.

Amino acid sequence analysis of a transcriptional regulator that binds to the fructose response element in the SREBP-1c promoter
The liver was extracted from CBA / JN Crj mice at the time of fasting and 2 hours after ingestion of the fructose diet, and the nucleoprotein was extracted in the same manner as in Example 5. Two synthetic DNA (5'-AATTCTAAAGGCAGCTATTGGCCT-3 ' : SEQ ID NO: 11; 5'-AATTGGCCAATAGCTGCCTTTAG-3' : SEQ ID NO: 12) were hybridized with the G ₁₁₂ in SREBP-1c promoter CBA / JN Crj mice that A double-stranded DNA containing a nearby base sequence was prepared and bound to EasyAnchor EcoRI-N (Nippon Gene, Tokyo) using a Takara ligation kit. EasyAnchor 50 μl and nucleoprotein extract 200 μg were allowed to stand at room temperature for 30 minutes in the binding buffer used in the gel shift analysis. After centrifugation (15,000 rpm for 1 min at 4 ° C), the precipitate is washed at 4 ° C with washing buffer (100 mM KCl, 15 mM HEPES-KOH (pH 7.9), 25 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% (w / v) glycerol) After washing 5 times with 100 ml of elution buffer (1.5 M KCl, 15 mM HEPES-KOH (pH 7.9), 25 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% (w / v) glycerol) at room temperature The protein was eluted. After centrifugation (15,000 rpm for 1 min at 4 ° C.), the salt in the supernatant was removed by a conventional method, and then developed by SDS-PAGE. After electrophoresis, the protein band was visualized by silver staining, and two bands around 41-45 kDa, which showed a large difference between the fasting sample and the sample after ingesting the fructose diet, were excised from the gel. TOF-MS analysis (consigned to Shimadzu Corporation and Tsukuba) was performed to analyze the primary structure of the protein. As a result, these proteins are known Nonamer Binding Protein (GenBank accession number: AAA81558 (SEQ ID NO: 3); cDNA is M88489 (SEQ ID NO: 2), and RNA binding motif protein, X chromosome retrogene-like protein (GenBank accession number: AAH11441 (SEQ ID NO: 5); cDNA was found to be identical to BC011441 (SEQ ID NO: 4).

Search for human SREBP-1c promoter sequence homologous to fructose response element in mouse SREBP-1c promoter
The SREBP-1c promoter sequence of CBA / JN Crj mouse and the base numbers 16566061-16566560 in the base sequence of human chromosome 17 (registration number: NT_010718) registered in GenBank corresponding to the promoter region of human SREBP-1c gene Was compared with the complementary strand sequence (SEQ ID NO: 13) of the base sequence represented by the DNASIS-homology search program. The results are shown in FIG. A sequence homologous to the mouse fructose response element (“homology site” in FIG. 4B) was also found in the human promoter.

  The fructose responsive element of the present invention and the transcriptional regulatory factors that interact with them, and the non-human animals into which they are introduced or inactivated, are not only extremely useful for elucidating the mechanism of induction of metabolic disorders, but also metabolizing. It is also useful for diagnosis of genetic susceptibility to disorders and screening for preventive and therapeutic agents for metabolic disorders.

Serum TG concentration (A), SREBP-1c mRNA level (B), PPARα mRNA level (C) and FAS mRNA level in each feeding group of DBA / 2 JN crj mouse (left) and CBA / JN Crj mouse (right) ( It is a figure which shows D). In A, B, C, and D, the vertical axis represents serum TG concentration (mg / dl), SREBP-1c mRNA level (relative value with CF group as 1 unit), and PPARα mRNA level (CF group as 1 unit), respectively. Relative values) and FAS mRNA levels (relative values with the CF group as one unit), and each bar graph shows the CF group, CR group, FF group and FR group from the left. In the figure, * indicates p <0.01, and ** indicates p <0.05. It is a figure which shows the SREBP-1c mRNA level (A) and FAS mRNA level (B) in each stimulation group of the primary hepatocytes derived from DBA / 2 JN Crj mouse (left) and CBA / JN Crj mouse (right). In A and B, the vertical axis indicates the SREBP-1c mRNA level (relative value with 1 unit of glucose stimulation group) and FAS mRNA level (relative value with 1 unit of glucose stimulation group), and each bar graph is from the left. A glucose stimulation group, a fructose stimulation group, and a glucose + insulin stimulation group are shown. In the figure, * indicates p <0.01, and ** indicates p <0.05. It is a photograph which shows the result of the electrophoretic mobility shift assay (EMSA) which shows the presence of the transcriptional regulatory factor couple | bonded with the fructose response element of this invention, and its expression control. A shows EMSA of a CBA / JN Crj mouse-derived nuclear extract from the FR group and a CBA probe or DBA probe. Lanes 1 and 2: CBA probe, Lanes 3 and 4: DBA probe; Lanes 1 and 3: cold probe coexistence, Lanes 2 and 4: cold probe non-coexistence. B shows EMSA of a CBA probe with a CBA / JN Crj mouse or DBA / 2 JN Crj mouse-derived nuclear extract from the FR group. Lane 1: CBA / JN Crj mouse-derived nuclear extract; Lane 2: DBA / 2 JN Crj mouse-derived nuclear extract. C shows EMSA of CBA / JN Crj mouse-derived nuclear extract and CBA probe of CF group or FR group. Lanes 1 and 2: CF group, Lanes 3 and 4: FR group; Lanes 1 and 4: cold probe non-coexistence, lanes 2 and 3: cold probe coexistence It is a figure which shows the comparison of the base sequence of the SREBP-1c promoter of a mouse | mouth (A) and a human (B). candidate binding site: candidate binding site of the transcriptional regulatory factor of the present invention (fructose response element); SNP (G-> A): G → A polymorphism (SNP) is shown in bold “G”. LXRE: Liver X Receptor (LXR) response element; NF-Y: NF-Y binding site; E-box: E-box; SRE: sterol response element; SP1: SP1 binding site; homology site: the above candidate binding site Homologous sites

Claims (8)

Whether the guanine represented by base number 112 in the base sequence represented by SEQ ID NO: 1 or the guanine represented by base number 39 in the base sequence represented by SEQ ID NO: 13 in the SREBP-1c promoter is substituted with adenine or and detecting a test method for genetic susceptibility to glucose and lipid metabolic disorder in a subject animal. A method for screening a prophylactic / therapeutic substance for a sugar / lipid metabolism disorder , comprising detecting binding inhibition between the following (a) and the following (b) and / or (c) in the presence of a test substance : .
(a) DNA having a nucleotide sequence represented by SEQ ID NO: 1
(b) protein Shitsumata its salt containing the amino acid sequence represented by SEQ ID NO: 3
(c) protein Shitsumata its salt containing the amino acid sequence shown in SEQ ID NO: 5 An animal cell containing a gene under the control of a promoter containing a DNA having the base sequence represented by SEQ ID NO: 1 is loaded with sugar, and the expression of the gene in the presence and absence of the test substance is compared. A screening method for a prophylactic / therapeutic substance for a sugar / lipid metabolism disorder . The animal cell has the ability to produce a protein containing the amino acid sequence represented by SEQ ID NO: 3 or a salt thereof and / or a protein containing the amino acid sequence represented by SEQ ID NO: 5 or a salt thereof. The method of claim 3 . The method according to claim 3 , wherein the animal cell is a hepatocyte. The method according to claim 3 , wherein the sugar is fructose. Sugar was loaded in mice containing gene under the control of a promoter comprising a DNA having a nucleotide sequence represented by SEQ ID NO: 1, expression of the gene in the liver in administration with and without administration of a test substance 4. The method of claim 3 , wherein: The method according to claim 7 , wherein the sugar is fructose.

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