JP2001224385A - Method for testing therapeutic or prophylactic agent for hyperlipidemia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for testing the effect of a therapeutic or prophylactic agent for hyperlipidemia and obtain a polynucleotide and an antibody used in the method. SOLUTION: This method is carried out for detecting the expression amount of mRNA containing the nucleotide sequence (however, t in the sequence is reading-replaced by u) shown from the nucleotide number 47 of the sequence number 1 in the sequence table (refer disclosure) to 1411 or the nucleotide number 78 of the sequence number 3 in the sequence table to 1457 on the cell obtained by cultivating a culture cell in the presence or absence of a test substance and selecting the test substance by which the expression amount of the detected mRNA is reduced on the cell cultured in the presence of the test substance more than the cell cultured in the absence of the test substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel test method for a therapeutic or preventive agent for hyperlipidemia, a nucleic acid probe, a primer and an antibody used in the method.

[0002]

2. Description of the Related Art There has been a remarkable increase in the number of hyperlipidemic patients due to excessive intake of fat and cholesterol accompanying recent changes in dietary habits. Hyperlipidemia is associated with serum lipids, namely cholesterol, triglycerides (TG),
It is a disease in which serum concentrations of phospholipids, free fatty acids, etc. are high, and is an important risk factor for arteriosclerosis. Furthermore, there is a high possibility that complications such as coronary atherosclerosis such as hypertension, angina pectoris and myocardial infarction and cerebral infarction will be caused.

[0003] To date, a large number of compounds exhibiting an anti-hyperlipidemic effect have been reported, but many of them have been synthesized by chemical methods, and due to their nature, various compounds have been used. Currently, side effects cannot be avoided.

[0004] On the other hand, pravastatin sodium which is currently commercially available is one of the powerful therapeutic agents for hyperlipidemia derived from microbial metabolites, and its action is HMG-CoA reduction, which is a rate-limiting enzyme in the cholesterol biosynthesis system. It is an inhibitor of the enzyme. As described above, if a protein associated with hyperlipidemia or a gene encoding the protein present in the living body can be identified, its function is directly inhibited or its expression is controlled to thereby reduce side effects. Although it is thought that a less (or less), more effective therapeutic agent for hyperlipidemia can be developed, a gene closely related to the onset of such hyperlipidemia was not known.

[0005] In addition, the same sequence as the nucleic acid probe used in the method of the present invention can be found on the Genbank database in "Angiopoiet-related protein 3 (angiopoiet-related protein 3)".
in-related protein 3) "and disclosed in International Patent Application Publication WO9
No. 9/55869 discloses the same nucleotide sequence as “z
alfa5 ", but nothing is known about the relationship between genes having these nucleotide sequences and hyperlipidemia. Therefore, “angiopoietin-related protein 3” to “za
part of the nucleotide sequence encoding "lpha5"
It is also not known to be useful for testing therapeutic or prophylactic agents for hyperlipidemia.

[0006]

An object of the present invention is to provide a novel method for testing an agent for treating or preventing hyperlipidemia and a nucleic acid probe, primer or antibody used in the method. .

[0007]

Means for Solving the Problems The present inventors aimed at searching for a target gene for an agent for treating or preventing hyperlipidemia,
By comparing the gene of the congenital hypolipidemic mouse with the gene of the hyperlipidemic mouse, the chromosomal location of the gene causing hyperlipidemia was narrowed down, and the gene highly expressed in the hyperlipidemic mouse Successfully identified. The cDNA derived from this gene was identified by Genbank as a result of homology search.
"Angiopoietin-related protein 3" in the database
However, the present inventors have found that when a gene having the nucleotide sequence is forcibly expressed in hypolipidemic mice, the concentration of neutral fat in the blood increases. Have confirmed that the gene has a novel function that has not been reported before. Thus, a new test method for a therapeutic or preventive agent for hyperlipidemia using a gene expression detection system using the nucleotide sequence or a part thereof as a probe or primer was successfully constructed. In addition, a novel test for a therapeutic or preventive agent for hyperlipidemia using an experimental system for preparing an antibody specific to the polypeptide encoded by the nucleotide sequence and detecting the production amount of the polypeptide using the antibody The method was built. Then, a model animal in which the nucleotide sequence was introduced into an experimental animal was prepared, a new test method for a therapeutic or preventive agent for hyperlipidemia using the model animal was successfully constructed, and the present invention was completed. Was.

The present invention provides (1) a method for testing the effect of a substance as an agent for treating or preventing hyperlipidemia, which comprises the following steps: 1) culturing cells in the presence or absence of a test substance; 2) In the cultured cells obtained in the above 1), the following a)
MRNA having the nucleotide sequence according to any one of (a) to (e), wherein t in the sequence is replaced with u.
A) Nucleotide number 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. A nucleotide sequence encoding a polypeptide having an activity of increasing soybean and increasing the concentration of neutral fat in the blood; and 3) cells cultured in the absence of the test substance as a result of the above step 2); Comparing the expression level of the detected mRNA with cells cultured under the following conditions: (2) characterized in that the cultured cells are derived from the liver;
(3) the method according to (1) or (2), wherein the cultured cells are derived from a primate or a rodent; (4) the cultured cells are human or (5) The method according to any one of (1) to (4), wherein the method for detecting the expression level of mRNA is a Northern blot. (6) The method according to any one of (1) to (4), wherein the method for detecting the expression level of mRNA is RT-P.
(7) The method according to any one of (1) to (4), wherein the method for detecting the expression level of mRNA is a ribonuclease protection assay. (8) The method according to any one of (1) to (4), wherein the method for detecting the expression level of mRNA is a run-on assay. (9) The following a: A) a polynucleotide having a nucleotide sequence that hybridizes under stringent conditions to an mRNA containing the nucleotide sequence according to any one of the above (where t in the sequence is replaced by u) To d) except for those containing the nucleotide sequence described in: a) nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
(1) nucleotide sequence of the DNA inserted into the phagemid retained in (1), (10) nucleotide number 47 of SEQ ID NO: 1 in the sequence listing
Consisting of at least 15 nucleotides, wherein one or both ends of the nucleotide sequence shown in SEQ ID NOS: to 1411 are deleted by one or more nucleotides
A, (11) nucleotide No. 78 of SEQ ID No. 3 in the sequence listing
Consisting of at least 15 nucleotides, wherein one or both termini of the nucleotide sequence shown in SEQ ID NO: 1457 are deleted by one or more nucleotides
A, (12) A method for testing the effect of a substance as an agent for treating or preventing hyperlipidemia, comprising the following steps: 1) culturing cultured cells in the presence or absence of a test substance 2) an amount of a polypeptide comprising the amino acid sequence encoded by the nucleotide sequence according to any one of the following a) to e) or a part thereof in the cultured cell supernatant obtained in 1) above: Is detected using an antibody that specifically recognizes the polypeptide: a) nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing.
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. A nucleotide sequence encoding a polypeptide having an activity of increasing soybean and increasing the concentration of neutral fat in the blood; and 3) cells cultured in the absence of the test substance as a result of the above step 2); Comparing the amount of the detected polypeptide with the cells cultured under the following conditions: (13) The method according to any one of (a) to (e), wherein An antibody that specifically recognizes a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence or a part thereof is obtained by combining the amino acid sequence of SEQ ID NO: 2 with amino acid sequence 17- 455 or a part thereof, a polypeptide consisting of the amino acid sequence shown in 19-455, or a part thereof or a polypeptide consisting of the amino acid sequence shown in amino acid Nos. 17-460 of SEQ ID NO: 4. (14) The method according to (12) or (13), wherein the peptide or the part thereof is specifically recognized. An antibody that specifically recognizes a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence or a part thereof can be obtained from the amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or the amino acid sequence represented by amino acid number 1 of SEQ ID NO: 10 (15) A method characterized in that it specifically recognizes the amino acid sequence shown in (14) to (14). ) To (14), wherein the production amount of the amino acid sequence encoded by the nucleotide sequence described in any one of (a) to (e) or a polypeptide consisting of a part thereof is determined. (16) The method according to any one of (12) to (14), wherein the operation of detecting using an antibody that specifically recognizes the polypeptide is a Western blot, a dot blot, or a slot blot. The method described above uses an antibody that specifically recognizes the production amount of a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof. A method characterized in that the operation of detecting by means of an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA); 7) an antibody that specifically recognizes a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence described in any one of the following a) to e) or a part thereof: a) SEQ ID NO: 1 in the sequence listing Nucleotides 47 to 1
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. (18) a nucleotide sequence encoding a polypeptide having an activity of increasing soybean and increasing the concentration of triglyceride in blood, which is the antibody according to (17), wherein amino acids 1 to 13 of SEQ ID NO: 9 in the sequence listing; (19) comprising the antibody according to (17) or (18), which specifically recognizes the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in amino acid numbers 1 to 14 of SEQ ID NO: 10. A kit for testing a therapeutic or prophylactic agent for hyperlipidemia, comprising: (20) a substance for treating or preventing hyperlipidemia; The following steps: 1) a nucleotide sequence obtained by genetic manipulation and described in any one of the following a) to e): a) SEQ ID NO: 1 in the sequence listing Nucleotides 47 to 1 of
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of the above a) to d) under stringent conditions. Tested on a non-human animal into which a foreign gene containing a nucleotide sequence encoding a polypeptide having activity to increase soybean triglyceride concentration in blood has been introduced so that the gene can be highly expressed. Administering the substance; and 2) measuring the neutral fat concentration in the blood of the animal of 1); (21) the method of (20), wherein the non-human animal of 1) is a mouse (22) The method according to (20), wherein the introduction of the foreign gene into the non-human animal according to (1) comprises the step of introducing the foreign gene into the non-human animal. Wherein the is due to infection of the animal adenovirus comprising adenoviral vector, (23) nucleotide number 78 in SEQ ID NO: 3 in the Sequence Listing
(1) DNA or RNA comprising an antisense sequence of a nucleotide sequence consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence shown in (1) to (2), (24) Hyperlipidemia containing the DNA or RNA according to (23) as an active ingredient A therapeutic agent for diseases.

That is, the present invention relates to SEQ ID NO: 1 in the sequence listing.
For testing the effect of a test substance as a therapeutic or prophylactic agent for hyperlipidemia using the expression of a gene involved in the regulation of neutral fat concentration in mammalian blood shown in , A nucleic acid probe, a primer and an antibody used in the method.

In the present invention, “hybridize under stringent conditions” refers to hybridization at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech), or under conditions equivalent thereto. It means to hybridize.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention is specifically,
411, or a gene having the nucleotide sequence shown in SEQ ID NO: 3 from nucleotides 78 to 1457, or a gene encoding a polypeptide having the same activity as the polypeptide encoded by them. , A test substance that is measured by specific detection of the nucleic acid (mRNA) or the polypeptide and reduces the expression level of the gene is selected as a candidate substance for a therapeutic or preventive agent for hyperlipidemia. . Hereinafter, an embodiment for detecting a nucleic acid and an embodiment for detecting a polypeptide will be described separately.

(A) Detection of Nucleic Acid 1) Probe Among the modes for detecting nucleic acid, the probe used in the method utilizing nucleic acid hybridization is D
NA or RNA, the nucleotide sequence of which is:
The following a) to e): a) nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) the nucleotide sequence of the DNA fragment inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence contained in the DNA fragment inserted into the phagemid retained in the phagemid; e) a polynucleotide having an antisense sequence of the nucleotide sequence described in any one of the above a) to d) under stringent conditions. A nucleotide sequence encoding a polypeptide having an activity of hybridizing and increasing the concentration of triglyceride in blood, wherein any one of the above (where t in the sequence is replaced by u) and a stringent Any nucleotide sequence that hybridizes under the following conditions may be used. For example, a polynucleotide having an antisense sequence of the nucleotide sequence described in any one of the above a) to e), at least 15 contiguous nucleotides in the antisense sequence A polynucleotide having a partial sequence consisting of nucleotides or By specifically hybridizing with a polyribonucleotide having a nucleotide sequence substantially as described in any one of the above a) to e) under stringent conditions, such as a variant of the antisense sequence, Anything that can be detected can be used in the method of the present invention. Among them, a polynucleotide having an antisense sequence of the nucleotide sequence described in the above a) can be obtained by a known method, for example, based on the nucleotide sequence information shown in SEQ ID NO: 1 from a mouse liver-derived cDNA library, for example, Plaque hybridization method,
A cDNA clone cloned by a colony hybridization method, a PCR method, or the like, which is directly labeled by a well-known method, or labeled in a replication or transcription reaction by a polymerase reaction using the cDNA clone as a template to obtain a labeled probe. Can be. The polynucleotide having the antisense sequence of the nucleotide sequence described in b) can be obtained by performing the same operation on the basis of the nucleotide sequence information shown in SEQ ID NO: 3 in the sequence listing from a cDNA library derived from human liver, for example. A labeled probe can be obtained from the cloned cDNA clone. On the other hand, a polynucleotide having an antisense sequence of the nucleotide sequence described in c) or d) above was internationally deposited on November 19, 1999 with the National Institute of Bioscience and Biotechnology,
Accession number FERM BP-6940 or FERM B
The transformed E. coli strain E.P. co
lipBK / m55-1 SANK 72199 strain or E. coli. coli pTrip / h55-1 SANK
It can be obtained from the recombinant phagemid carried by strain 72299.

Further, the polynucleotide having the antisense sequence of the nucleotide sequence described in the above e) can be obtained by, for example, probing the polynucleotide having the antisense sequence of the nucleotide sequence described in the above a) to d) obtained as described above. Can be obtained from a cDNA clone isolated by cloning a cDNA library derived from any mammal (preferably liver) by plaque hybridization or colony hybridization.

Further, the polynucleotide having a partial sequence consisting of at least 15 consecutive nucleotides in the antisense sequence of the nucleotide sequence described in any one of the above a) to e) is about several tens of nucleotides. If available, it can be obtained by chemical synthesis.
Alternatively, an arbitrary partial sequence in a cDNA clone having the nucleotide sequence according to any one of the above a) to e) obtained as described above may be used, for example, by PCR.
After subcloning as described above, a probe having an antisense sequence can be prepared in the same manner as described above.

For example, in a partial sequence consisting of several tens of consecutive nucleotides in the antisense sequence of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, a nucleotide sequence in which several nucleotides are added, deleted and / or added The polynucleotide can be used in the method of the present invention as long as it hybridizes with the polyribonucleotide described in any one of the above a) to e) under stringent conditions. Such polynucleotides can be obtained by chemical synthesis or polymerase chain reaction (hereinafter "P
CR ") and the like, using a well-known mutagenesis method in the technical field to which the present invention belongs.

The probe used in the method of the present invention is not limited to a probe consisting of a single nucleotide sequence. That is, in the method of the present invention, for example, a mixture of a plurality of types of nucleotide sequences satisfying the above requirements may be used as a probe, or multiplex detection may be performed using each of the plurality of types of nucleotide sequences individually.

2) Primers for RT-PCR Another aspect of the present invention for detecting nucleic acids is as follows:
First, a reverse transcriptase reaction using mRNA as a template is performed, and then PCR is performed to specifically amplify the DNA fragment.
This is a method of performing so-called RT-PCR. In this method, in order to specifically amplify the nucleotide sequence of interest, an antisense primer complementary to a specific partial sequence of the mRNA of interest and a sequence of cDNA generated by the reverse transcriptase from the antisense primer are used. A sense primer complementary to a specific partial sequence therein is used.

The antisense primer used for both the reverse transcriptase reaction and the PCR is substantially at least 18 nucleotides, preferably at least 23 nucleotides in the antisense sequence of the nucleotide sequence described in a) to e) above. Consisting of the nucleotide sequence of

On the other hand, the sequence of the sense primer used in the PCR is 5 'further than the most 5' terminal position in the sequence corresponding to the complementary strand of the antisense primer in the nucleotide sequences described in a) to e) above. It consists of any partial sequence of at least 18 nucleotides, preferably at least 21 nucleotides, contiguous in the sequence present in the terminal region. However, if the sense primer and the antisense primer have mutually complementary sequences, annealing of the primers will amplify non-specific sequences, which may hinder specific gene detection. It is preferable to design primers that avoid various combinations.

In each of the antisense primer and the sense primer, a nucleotide sequence irrelevant to the nucleotide sequences described in a) to e) above is added as a linker to the 5 ′ end of the nucleotide sequence defined above. You may. However, it is preferable that the linker does not cause non-specific annealing with the nucleic acid in the reaction solution during the reaction so as not to hinder the specific gene detection.

3) Cell or animal for detecting gene expression Next, the cultured cell used in the method of the present invention is a mammalian cell expressing the gene having the nucleotide sequence described in any one of the above a) to e). Any animal cell may be used. Preferably, a cultured cell derived from a mammalian liver (preferably, a primary cultured hepatocyte), for example, a cell into which a gene having the nucleotide sequence described in any one of the above a) to e) is introduced together with its promoter region. For example, artificially transformed cells (eg, CHO cells) can also be used. As the mammalian species, humans, mice, rats or hamsters are preferable, humans or mice are more preferable, and primary cells of KK mouse (available from CLEA Japan) which is a hyperlipidemic mouse are preferable cells. Cells include, but are not limited to. When it is judged that it is more preferable than using cultured cells, a test substance is administered to a mammal individual, and then the above-mentioned a) to e) of the organ or tissue cell extracted from the animal individual is used. A method for detecting the expression of a gene having the nucleotide sequence described in any one of the above may also be employed. At this time, the organ or tissue to be detected for gene expression may be any one that expresses the gene having the nucleotide sequence described in any one of the above a) to e), but is preferably liver. Preferred mammalian species in this embodiment are preferably humans, mice, rats or hamsters, more preferably humans or mice. For example, as the mouse, the KK mouse is preferably used, but the present invention is not limited to this.

The cultured cells used in the method of the present invention may be any conditions under which a gene having the nucleotide sequence described in any one of the above a) to e) can be expressed (provided that no test substance is added). Culture may be performed under any conditions. For example, when culture conditions established for the cultured cells are known and the cells express a gene having the nucleotide sequence according to any one of the above a) to e) under the conditions, Culture may be performed under conditions.

4) Addition of test substance During the culture of the cells, the test substance is added to the culture medium and cultured for a certain period. Examples of the test substance include compounds, metabolites of microorganisms, extracts of plant and animal tissues, derivatives thereof, and mixtures thereof. The dose and concentration of the test substance may be set as appropriate, or a plurality of doses may be set, for example, by preparing a dilution series. The period for culturing in the presence of the test substance may be appropriately set, but is preferably 30 minutes to 24 hours. When a test substance is administered to a mammalian individual, a dosage form such as oral administration, intravenous injection, intraperitoneal injection, transdermal administration, or subcutaneous injection is used depending on the physical properties of the test substance.

5) Preparation of Sample When extracting RNA from the cells cultured as described above, immediately after completion of the culture, the cells are subjected to a solvent for RNA extraction (for example, a component having an action to inactivate ribonuclease such as phenol). Is preferable. Alternatively, the cells may be carefully scraped off with a scraper or gently separated from the culture substrate using a protease such as trypsin so as not to destroy the cells. Move to process.

[0025] RNA extraction methods include guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloric acid method, guanidine thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N. ., (1987) Anal. Bio
Chem., 162, 156-159) and the like, but the guanidine acid thiocyanate-phenol-chloroform method is preferred.

A method for further purifying mRNA from the obtained RNA is as described below. That is, since it is known that many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 ′ end, for example, biotinylated oligo (dT)
The mRNA is adsorbed to the probe, and the paramagnetic particles on which streptavidin is immobilized capture the mRNA using the binding between biotin and streptavidin, and after the washing operation, the mRNA is eluted to purify the mRNA. be able to. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. However, for the method of the present invention, these mRNA purification steps are not essential, as long as the expression of the gene having the nucleotide sequence described in any one of the above a) to e) can be detected. Alternatively, total RNA can be used in subsequent steps.

6) Immobilization of Sample When performing detection by nucleic acid hybridization, the RNA sample is subjected to agarose electrophoresis to specifically detect a gene in the RNA sample obtained as described above. The sample is transferred to a hybridization experiment membrane (hereinafter simply referred to as “membrane”) (Northern blot method), or immobilized on the membrane by so-called dot blot method or slot blot method in which a sample is directly infiltrated into the membrane. Examples of the membrane include a nitrocellulose membrane (for example, High Bond-C Pure (manufactured by Amersham Pharmacia)), a positive charge nylon membrane (for example, High Bond-N + Amersham Pharmacia), or a hydrophilic nylon membrane (for example). For example, High Bond-N
/ NX (manufactured by Amersham Pharmacia) and the like.

Agarose electrophoresis for Northern blotting includes agarose formamide gel electrophoresis, a method in which a sample is treated with glyoxal and dimethyl sulfoxide, denatured, and then electrophoresed on an agarose gel prepared with a phosphate buffer. Agarose gel methylmercury electrophoresis (Maniatis, T. et al. (1982) in "M
olecular Cloning A Laboratory Manual "Cold Spring
Harbor Laboratory, NY.).
It is not limited to these.

From the gel after electrophoresis to RNA
As a so-called blotting method for transferring DNA, a capillary transfer method (Maniatis, T. et al. (1982)
in "Molecular Cloning A Laboratory Manual" Cold Sp
ring Harbor Laboratory, NY.), vacuum method, electrophoresis method (Maniatis, T. et al. (1989) in "MolecularCl
oning A Laboratory Manual, 2nd ed "Cold Spring Ha
rbor Laboratory, NY.). Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (manufactured by Bio-Rad)).

After the blotting, an operation for fixing the RNA transferred to the membrane to the membrane is performed (this operation differs depending on the material of the membrane, and depending on the product, the fixing operation may not be necessary).

7) Labeling of Probe In the detection by nucleic acid hybridization, a labeling method and a detection method of a probe for detecting a specific mRNA in an RNA sample immobilized as described above are described below. : I) Radioactive isotope labeling A nick translation method (for example, using a nick translation kit (manufactured by Amersham Pharmacia) or the like) using a DNA fragment or a vector carrying the same as a material or template, a random prime method (for example, ,
Multiprime DNA labeling system (Amersham-Pharmacia) and the like, end labeling method (for example, Megalabel (Takara Shuzo Co., Ltd.), 3'-end labeling kit (Amersham-Pharmacia) and the like) To prepare a labeled DNA probe, or SP in a vector in which DNA as a template is subcloned.
A labeled RNA probe is prepared by an in vitro transcription method using 6 promoter or T7 promoter. Detection of these probes can be performed by X-ray film or autoradiography using an imaging plate. In the case of X-ray film, densitometry (for example, GS-700 imaging densitometer (manufactured by Bio-Rad)) In the case of an imaging plate, quantification using a BAS2000II (manufactured by Fuji Film) system is also possible.

Ii) Enzyme labeling DNA or RNA fragments are directly enzyme-labeled. Enzymes used for labeling include, for example, alkaline phosphatase (using AlkPhos Direct system for chemiluminescence (manufactured by Amersham Pharmacia)), horseradish peroxidase (ECL)
Direct Nucleic Acid Labeling and Detection System (Amersham Pharmacia) or the like). The detection of the probe is performed by immersing the membrane in an enzyme reaction buffer containing a substrate capable of detecting the catalytic reaction of the labeled enzyme, for example, a chromogenic substance by the catalytic reaction or a substrate that emits light. . When a chromogenic substrate is used, it can be detected visually, and when a luminescent substrate is used, autoradiography using an X-ray film or an imaging plate or a photograph using an instant camera as in the case of radioisotope labeling It can be detected by photographing. Furthermore, when a luminescent substrate is used, quantification using densitometry or a BAS2000II system is also possible.

Iii) Labeling with other molecules Fluorescein labeling: Nick translation, random prime, or 3 ′ end labeling (available from Amersham Pharmacia, Inc.)
CL 3'-oligolabeling system). Alternatively, RNA is labeled by in vitro transcription with SP6, T7 promoters; biotin labeling: D
The 5 ′ end of NA is labeled (using an oligonucleotide biotin labeling kit commercially available from Amersham Pharmacia) or a DNA fragment is labeled by a nick translation method, a random prime method, or the like; a digoxigenin-modified dUTP label The DNA fragment is labeled by a nick translation method, a random prime method, or the like.

The detection of these labeled molecules includes, in each case, the operation of binding a molecule that specifically binds to the labeled molecule, labeled with a radioisotope or an enzyme, to a probe. Specific binding molecules include, for example, an anti-fluorescein antibody or an anti-digoxigenin antibody in the case of fluorescein or digoxigenin, and avidin or streptavidin in the case of biotin. After these are bound to the probe, detection can be carried out using the labeled radioisotope or enzyme according to the same method as described in i) or ii) above.

8) Hybridization Hybridization can be performed by a method well known in the art to which the present invention belongs. Regarding the relationship between the composition of the hybridization solution or washing solution in the present invention and the hybridization temperature or washing temperature,
For example, in the literature (Bio Experimental Illustrated 4, p14
8, Shujunsha), but preferred conditions are as follows: Hybridization solution: ExpressHyb Hybridiza
Action Solution (manufactured by Clontech); Final concentration of probe (for radiolabeled probe): 1 to 2 × 10 ⁶ cpm / ml (preferably 2 × 10 ⁶ cp)
hybridization temperature and time: 68 ° C., 1
~ 24 hours.

Conditions for washing the membrane: i) 0.1 to 5 × SSC (most preferably 2 × SS)
C), 0.05 to 0.1% SDS (most preferably 0.05%) sodium dodecyl sulfate (hereinafter "SDS")
) At room temperature to 42 ° C. (most preferably at room temperature) for 20 to 60 minutes (most preferably 20 minutes) by changing the washing solution 2 to 6 times (most preferably 3 times).
Ii) After the above i), 0.1 × SSC, 0.1% S
The operation of shaking at 50 to 65 ° C. in DS for 20 to 60 minutes is performed 2 to 6 times by exchanging the washing solution. Or
62 in 0.2 to 0.5 × SSC, 0.1% SDS
The operation of shaking at 乃至 65 ° C. for 20 to 60 minutes is performed 2 to 6 times while changing the washing solution. Most preferably, 0.1 ×
The operation of shaking in SSC, 0.1% SDS at 50 ° C. for 20 minutes is performed three times by changing the washing solution.

After completion of the washing, as described in the above 7),
Perform detection and quantification according to the probe labeling method. Also,
Genes known to have stable expression levels per cell (eg, 23 kDa highly basic protein, α-
Probes for detecting gene expression of tubulin, glyceraldehyde-3-dehydrogenase, hypoxanthine / guanine / phosphoribosyltransferase, phospholipase A2, ribosomal protein S9, ubiquitin and the like are commercially available). The expression level is measured simultaneously for the purpose of correcting variations caused by differences in RNA levels between the samples, and the relative value of the expression level of the gene to be detected based on the expression level of the stable expressed gene is determined. By comparing the cell group to which the test substance has been administered and the cell group to which the test substance has not been administered, more precise evaluation can be performed.

As a result, the test substance which decreases the expression level of the gene having the nucleotide sequence according to any one of the above a) to e) can be a therapeutic or preventive agent for hyperlipidemia.

9) RT-PCR reaction In an embodiment in which a nucleic acid is detected by RT-PCR,
The conditions of each reaction are as described below. Note that RT
-The sample for detection by PCR is usually poly (A) ⁺
It need not be purified to RNA.

I) Reverse transcriptase reaction Example of reaction solution composition (total volume: 20 μl): Total RNA as appropriate; magnesium chloride 2.5 to 5 mM (preferably 5 mM)
M); 1 × RNA PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C., 50 mM potassium chloride)); dNTPs 0.5 to 1 mM (preferably 1 mM) Antisense primer 1 μM (2.5 μM of a commercially available random primer or oligo (dT) primer (12-20 nucleotides) can be added as a substitute for the antisense primer); reverse transcriptase 0.25 to 1 unit / μl (preferably 0.25 units / μl); adjust to 20 μl with sterile water.

Reaction temperature conditions: After incubating at 30 ° C. for 10 minutes (only when a random primer is used), 42 to 60 ° C.
(Preferably 42 ° C.) for 15 to 30 minutes (preferably 30 minutes), and further heat at 99 ° C. for 5 minutes to inactivate the enzyme.
Cool for minutes.

Ii) Example of composition of PCR reaction solution: Magnesium chloride 2 to 2.5 mM (preferably 2.
1 × PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 at 25 ° C., preferably 8.3));
50 mM potassium chloride; dNTPs 0.2 to 0.25 mM (preferably 0.1 to 0.25 mM).
25 mM); antisense and sense primers
2 to 0.5 μM (preferably 0.2 μM); Taq polymerase 1 to 2.5 units (preferably 2.5 units); adjust the total volume to 80 μl by adding sterile water,
The PCR is started after the reverse transcription reaction has been added to the entire reaction mixture.

Reaction temperature conditions: First, heating at 94 ° C. for 2 minutes, and then at 90 to 95 ° C. (preferably 94 ° C.) for 30 seconds, 40 to 60 ° C. (preferably, a dissociation temperature (Tm calculated from the characteristics of the primer) ) To a temperature 20 degrees below it for 30 seconds at 70-75 ° C. (preferably 72 ° C.) for 1.5 minutes.
Repeat for 0 cycles (preferably 28 cycles) and then cool to 4 ° C.

After completion of the PCR, the reaction solution is subjected to electrophoresis to detect whether or not a band of a desired size has been amplified.
In order to perform quantitative detection, cDNA that has been serially diluted in advance
PCR is performed under the same conditions using the clone as a standard template DNA, and the number of temperature cycles at which quantitative detection is possible is determined, or, for example, a part of the reaction solution is sampled every 5 cycles and subjected to electrophoresis . Also, for example, PCR
By using radiolabeled dCTP during the reaction, quantification can be performed using the amount of radioactivity incorporated in the band as an index.

The detection results were compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance. The test substance in which the expression level of the gene having the nucleotide sequence described in (1) is reduced can be a therapeutic or preventive agent for hyperlipidemia.

10) Other Methods Other methods for measuring the expression level of the gene having the nucleotide sequence according to any one of the above a) to e) include the following methods.

I) Ribonuclease protection assay (RNas
e protection assay): The labeled probe is hybridized only to the mRNA having the nucleotide sequence described in any one of the above a) to e) (where t in the sequence is replaced with u) in the RNA sample. When a ribonuclease is added to the sample and incubated after the formation of the single-stranded polynucleotide, the mRNA to which the probe has hybridized forms a double-stranded strand, and thus is exempt from digestion by the ribonuclease. As a result of the digestion, only the double-stranded polynucleotide remains (if the probe is shorter than the mRNA to be detected, the double-stranded polynucleotide corresponding to the length of the probe remains). By quantifying the double-stranded polynucleotide, the expression level of the target gene is measured. Specifically, for example, the method described below is followed.

It is preferable that the surplus labeled probe is digested with ribonuclease in order to surely separate the surplus labeled probe without forming a double strand from the double-stranded polynucleotide and facilitate quantification. If a ribonuclease capable of digesting single-stranded DNA is used, the labeled probe may be DNA or RNA. The method for preparing the labeled probe is as described in the above 1) and 7), but the length of the probe used in this method is 50 to 5
About 00 nucleotides are preferred. Also, double-stranded DNA
Probes in which complementary strands are mixed, such as a probe which is directly denatured and thermally denatured, are not suitable for this method.

The RNA probe is prepared, for example, according to the following method. First, a template DNA is used as a bacteriophage promoter (T7, SP6, T3 promoter, etc.)
(For example, pGEM-T (promega)). Next, this recombinant plasmid vector is digested with a restriction enzyme so that it is cleaved only one place immediately downstream of the inserted fragment. Obtained linear DN
Using A as a template, an in vitro transcription reaction is performed in the presence of radiolabeled ribonucleotides. In this reaction, an enzyme such as T7, SP6, or T3 polymerase is used in accordance with the promoter in the vector. The above operations can be performed using, for example, Riboprobe System-T7, -SP6 or -T3 (all manufactured by Promega).

The steps up to the preparation of the RNA sample are as described in the above 3) to 5). A ribonuclease protection assay is performed using 10 to 20 μg of the prepared total RNA sample and an excess amount of the labeled probe corresponding to 5 × 10 ⁵ cpm. This operation is performed using a commercially available kit (HybSpeed R
PA Kit, manufactured by Ambion).
The obtained ribonuclease digested sample is electrophoresed on a 4 to 12% polyacrylamide gel containing 8 M urea, the gel is dried, and autoradiography is performed with an X-ray film. By the above operation, the band of the double-stranded polynucleotide which escaped the ribonuclease digestion can be detected, and the quantification can be carried out according to the method described in 7) i) above). Further, as in the case of the Northern blot analysis, if the expression level of the β-actin gene is simultaneously measured for the purpose of correcting variations caused by differences in RNA amounts between the samples, more precise evaluation will be performed. be able to.

As described above, the detection results are compared between the sample derived from the cells cultured in the presence of the test substance and the sample derived from the cells cultured in the absence of the test substance. The test substance in which the expression level of the gene having the nucleotide sequence according to any one of the above is reduced can be a therapeutic or preventive agent for hyperlipidemia.

Ii) Run-on assa
y, Greenberg, ME and Ziff, EB (1984) Nature 3
11, 433-438, and Groudine, M. et al. (1981) Mol.
This method is a method for isolating the nucleus from a cell and measuring the transcriptional activity of the gene of interest. In this method, the method for detecting mRNA in a cell as described above is used. However, in the present invention, it is included in the “method for detecting the expression level of a gene”. When a transcription reaction is performed in vitro using the isolated cell nucleus, only the reaction in which transcription is already started before the nucleus is isolated and the one where the mRNA chain is being generated elongates proceeds. I do. At the time when the nucleus was isolated by adding a radiolabeled ribonucleotide during this reaction to label the elongating mRNA and detecting the mRNA contained therein that hybridized to the unlabeled probe Can be measured for the transcription activity of the target gene. In order to use the data of the time when the influence of the test substance appears most remarkably for the determination, the time from the addition of the test substance to the cultured cells to the isolation of the nucleus, for example, 30 minutes after addition, 1 hour after addition, Two hours later,
After 4 hours, 8 hours, and 24 hours, nuclei can be isolated from cells and assayed, for example. The specific operation method is the same as that described in the above-mentioned reference except that a probe that is not labeled is prepared according to the description in the above 1). In this way, the detection results are compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance. A test substance having reduced transcription activity of the gene having the nucleotide sequence according to any one of the above can be a therapeutic or preventive agent for hyperlipidemia.

Iii) DNA chip analysis, DNA microarray analysis [1] Preparation of sample for obtaining probe First, a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance Is extracted and purified. The steps up to preparing the RNA sample are as described in the above 3) to 5).

[2] Labeling of Probes As a starting material for preparing probes for DNA chip analysis or DNA microarray analysis, unpurified total RNA can be used, but the method described in the above 5) is used. More preferably, it is poly (A) ⁺ RNA purified by

In performing detection by nucleic acid hybridization, a method for labeling and detecting a probe will be described below.

Probe for analysis using DNA chip manufactured by Affymetrix: DN manufactured by Affymetrix
According to the protocol attached to the A chip, a biotin-labeled cRNA probe is used.

Probe for analysis using DNA microarray: Poly (A) ⁺ RNA by reverse transcriptase reaction
When a cDNA is prepared from a fluorescent dye (for example, Cy)
The cDNA is fluorescently labeled by adding d-UTP or the like labeled with (3, Cy5, etc.). At this time, if a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance are labeled with different dyes, both are mixed during subsequent hybridization. Can be used. Probe for analysis using a membrane filter: When preparing cDNA from poly (A) ⁺ RNA by reverse transcriptase reaction, d-C labeled with a radioactive isotope (e.g. ³² P, ^{3 3} P)
The probe is labeled by adding TP or the like.

[3] Solid-Phase Immobilized Sample The following solid-phased sample to be hybridized with the labeled probe obtained in the above step [2] can be mentioned.

EST (expressed sequ
gene chip (manufactured by Affymetrix) on which an antisense oligonucleotide synthesized based on an ence tag) sequence or an mRNA sequence is immobilized (Lipshutz, RJ et a
l. (1999) Nature genet. 21, suppliment, 20-24):
The EST sequence or mRNA sequence is most preferably derived from an animal of the same species as the cell used for preparing the probe, but is not limited thereto. For example, those derived from an animal of a closely related species can also be used. However, the above a) to e)
A gene disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database, or a sequence containing a partial sequence of any one of them. Use phased ones.

An mRN obtained from an organ tissue (preferably a liver) of an animal of the same species as or related to the cells used for the preparation of the probe, or an isolated or established cell from the organ tissue
DNA microarray or membrane filter on which the cDNA or RT-PCR product prepared from A is immobilized: These cDNA or RT-PCR products are based on, for example, sequence information of an animal EST database or the like used as the material of mRNA. It was cloned by performing a reverse transcriptase reaction or PCR with the prepared primers. As a material for preparing a sample, a gene having the nucleotide sequence according to any one of the above a) to e), a gene disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” on the Genbank database Is used (preferably liver-derived). This cDNA and RT-P
As a CR product, mRNA having a different expression level is used in advance by a subtraction method (Diatchenko, L. et al. (1996) Pro
c. Natl. Acad. Sci. USA 93, 6025-6030), differential display method (Kato, K. (1995) Nuclei
c Acids Res. 23, 3685-3690) and the like. In addition, DNA microarrays and filters are used to detect genes to be detected, that is, Ge.
A commercially available product containing a gene disclosed as a nucleotide sequence encoding “angiopoietin-related protein 3” on the nbank database may be used, or any one of the above a) to e) may be used using a spotter. Having immobilized gene having nucleotide sequence as described above
A microarray or a filter can also be prepared (for example, GMS417 Alayer, manufactured by Takara Shuzo Co., Ltd.).

Using the probe prepared in the above [2], the immobilized sample is hybridized separately under the same conditions or simultaneously by mixing (Brown, PO et a).
l. (1999) Nature genet. 21, suppliment, 33-37).

[4] Analysis In the case of analysis using a DNA chip manufactured by Affymetrix: Hybridization and analysis are performed according to the protocol attached to the DNA chip manufactured by Affymetrix.

In the case of analysis using a DNA microarray: For example, when a commercial DNA microarray manufactured by Takara Shuzo Co., Ltd. is used, hybridization and washing are performed according to the protocol of the company, and a fluorescent signal detector (eg, GMS418 Array Scanner (Takara Shuzo ( Analyzes are performed after detecting the fluorescent signal by the method of).

In the case of analysis using a filter: Hybridization can be performed by a method well known in the art to which the present invention belongs. For example, after performing hybridization and washing, analysis is performed using an analyzer (for example, Atlas Image (manufactured by Clontech)).

In each of the above cases, a probe derived from a cell derived from a cell cultured in the presence of the test substance and a probe derived from a cell cultured in the absence of the test substance on the solid-phased sample of the same lot. Are hybridized. At this time, the hybridization conditions other than the probe used are the same. As described in [2] above,
When each probe is labeled with a different fluorescent dye, a mixture of both probes can be hybridized to one immobilized sample (Brown, PO et al. (199).
9) Nature genet. 21, suppliment, 33-37).

As a result of the analysis, the solid-phased sample was probed between a probe derived from a sample derived from cells cultured in the presence of the test substance and a probe derived from a sample derived from cells cultured in the absence of the test substance. The amount of the probe hybridized to the target gene, that is, the gene having the nucleotide sequence according to any one of the above a) to e) or the gene having the nucleotide sequence encoding the angiopoietin-related protein 3 is measured. As a result, the amount of the probe hybridized to the target gene was compared, and the probe derived from the sample derived from the cell cultured in the presence of the test substance was better than the probe derived from the sample derived from the cell cultured in the absence of the test substance. If the amount is less, the test substance is a substance that suppresses the expression of the gene having the nucleotide sequence according to any one of the above a) to e), and thus the therapeutic or prophylactic agent for hyperlipidemia. Can be

Iv) Others In addition to the above, any one of the above a) to e) is a sample derived from cells cultured in the presence of the test substance, compared to a sample derived from cells cultured in the absence of the test substance. The following methods can be mentioned as a method for detecting the identification of a test substance that reduces the expression level of the gene having the nucleotide sequence described in (1).

That is, in a single reaction, a technique known as “Taqman” combining PCR with hybridization probing (hereinafter referred to as “probing”) (Holland, PM et al. (1991) Proc. Natl. A
cad. Sci. USA 88, 7276-7280) or a method combining PCR with probing in a single reaction (Higuchie
tal. Biotechnology, 10, 413-417 (1992)) can also be used. In the latter method, ethidium bromide, a nucleic acid detection reagent that emits fluorescence when excited by ultraviolet light, is added to a PCR reaction solution. Since the fluorescence of ethidium bromide increases in the presence of double-stranded DNA, the increase in fluorescence detected upon irradiation with excitation light may correlate with the accumulation of double-stranded PCR product.

Further, as another method combining amplification by PCR and probing, a method described in EP-A-0 601 889 can be used. Furthermore, a light cycler system (manufactured by Roche Diagnostics, Japan Patent Publication 2000-312)
It is also possible to quantify the amount of mRNA using the method described in Japanese Patent Application Publication No. 600 (1999).

(B) Detection of Polypeptide Next, as another embodiment of the method of the present invention, a method for detecting a polypeptide encoded by a gene to be detected in the above-described embodiment for detecting gene expression is described. There is. In this embodiment, the polypeptide in the sample is immobilized on the inner bottom surface of a well of a 96-well plate, a membrane, or the like, and then detection using an antibody that specifically recognizes the target polypeptide is performed. Of these, a 96-well plate is generally used for enzyme-linked immunosorbent assay (ELIS).
A) and radioisotope immunoassay (RIA). On the other hand, as a method of immobilizing the sample on a membrane, a method of transferring a polypeptide to the membrane via polyacrylamide electrophoresis of a sample (Western blotting) or a method of directly impregnating the sample with a sample or a dilution thereof into the membrane is called a dot method. Blot method and slot blot method are mentioned.

1) Preparation of Sample The conditions for the type of cultured cells used in the embodiment for detecting a polypeptide as described above are the same as those in the embodiment for detecting gene expression described above. Alternatively, a method may be employed in which a test substance is administered to a mammal individual, and thereafter, serum or the like collected from the animal individual is used as a sample. Preferred mammalian species in this case are humans, mice, rats or hamsters, more preferably humans or mice. For example, as a mouse, a KK mouse which is a hyperlipidemic mouse is preferably used, but the present invention is not limited to this. The culture conditions of the cultured cells and the method of administering the test substance are the same as those in the embodiment in which gene expression is detected. The test substance to be tested for its activity as a therapeutic or preventive agent for hyperlipidemia includes compounds, metabolites of microorganisms, extracts of plant and animal tissues, derivatives thereof, and mixtures thereof. be able to.

As a material for preparing a sample for this embodiment, a culture supernatant or a cytoplasmic fraction of a cell culture cultured in the presence or absence of a test substance may be used. Is preferred. The culture supernatant is collected after the completion of the culture and, if necessary, is subjected to filter filtration sterilization treatment.
It is used for a preparation process of a sample for ISA / RIA and a sample for Western blot.

As a sample for ELISA / RIA, for example, the collected culture supernatant is used as it is, or a sample appropriately diluted with a buffer is used.

A method for preparing a sample for Western blot (for electrophoresis) is as follows. First, for example, the culture supernatant is treated with trichloroacetic acid to precipitate the protein, and the precipitate is obtained by centrifugation. The precipitate is washed with ice-cooled acetone, air-dried, and then dissolved in a sample buffer (manufactured by Bio-Rad) containing 2-mercatorethanol for SDS-polyacrylamide electrophoresis.

In the case of dot / slot blotting, for example, the collected culture supernatant itself or one appropriately diluted with a buffer is blotted using a blotting apparatus.
Adsorb directly to the membrane.

2) Immobilization of Sample In order to specifically detect the polypeptide in the sample obtained as described above, the sample is immobilized. Examples of the membrane used for the western blotting, dot blotting or slot blotting include nitrocellulose membranes (for example, manufactured by Bio-Rad) and nylon membranes (for example, Hybond-ECL (Amersham
Pharmacia), a cotton membrane (for example, a blot absorbent filter (manufactured by Bio-Rad), etc.) or polyvinylidene difluoride (PV
DF) membrane (for example, manufactured by Bio-Rad) and the like.

The so-called blotting method for transferring a polypeptide from a gel after electrophoresis to a membrane includes:
Wet blotting method (CURRENT PROTOCOLS IN I
MMUNOLOGY volume2 ed by JE Coligan, AM Kruis
beek, DH Margulies, EM Shevach, W. Strobe
r), semi-dry blotting method (CURRENT PRO
TOCOLS IN IMMUNOLOGY volume2). Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (Biorad)).

On the other hand, in order to perform detection and quantification by the ELISA method / RIA method, a sample or a diluent (for example, 0.1 μm) is placed in a dedicated 96-well plate (for example, Immunoplate Maxisorp (manufactured by Nunc Corporation)). By adding a phosphate-buffered saline solution containing 05% sodium azide (diluted with “PBS”) and allowing it to stand at 4 ° C. to room temperature overnight or at 37 ° C. for 1 to 3 hours, The polypeptide is adsorbed on the bottom surface of the well and solidified.

3) Antibody The antibody used in this embodiment is a polypeptide, preferably a polypeptide, produced by expressing the gene containing the nucleotide sequence described in a) to e) in (A) above in animal cells. Amino acid numbers 17-455 of SEQ ID NO: 2 in the column list
(Preferably, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 19-455) or a part thereof, or a polypeptide consisting of the amino acid sequence shown in amino acid numbers 17-460 of SEQ ID NO: 4, or a part thereof. Recognize. Suitable antibodies are, for example, amino acids 17-4 of SEQ ID NO: 2 in the sequence listing.
55 (more preferably 19-455) and the polypeptide consisting of the amino acid sequence of amino acids 17-460 of SEQ ID NO: 4, but bind to any other Antibodies that do not bind to mouse or human proteins can also be mentioned.

Antibodies for this embodiment can be prepared by a conventional method (for example, Shinsei Kagaku Kenkyusho 1, Protein 1, p.389-).
397, 1992), a protein serving as an antigen, or any polypeptide selected from the amino acid sequence of the protein is immunized to an animal, and an antibody produced in the living body is collected and purified. In addition, known methods (for example, Ko
hler and Milstein, Nature 256, 495-497, 1975, Kenn
et, R. ed., Monoclonal Antibody p.365-367, 1980, P
According to renum Press, NY), a hybridoma can be established by fusing an antibody-producing cell that produces an antibody against the protein of the present invention with a myeloma cell to obtain a monoclonal antibody.

As an antigen for preparing the antibody used in this embodiment, a polypeptide consisting of the amino acid sequence represented by amino acid Nos. 17-455 (preferably 19-455) of SEQ ID NO: 2 in the sequence listing or A polypeptide comprising at least six consecutive partial amino acid sequences thereof, or amino acids 17 to 460 of SEQ ID NO: 4.
Or a polypeptide consisting of at least six consecutive partial amino acid sequences thereof, or a derivative in which an arbitrary amino acid sequence or a carrier is added to the polypeptide, Preferably, a keyhole limpet hemocyanin is provided at the C-terminus of the polypeptide consisting of amino acid Nos. 1-14 of SEQ ID NO: 9 or the N-terminus of the polypeptide consisting of amino acid No. 1-14 of SEQ ID NO: 10 in the sequence listing. Are bonded as a carrier.

Amino acid number 17- of SEQ ID NO: 2 in the sequence listing
455 (preferably 19 to 455) or a polypeptide consisting of the amino acid sequence of amino acids 17 to 460 of SEQ ID NO: 4, for example, the nucleotide of SEQ ID NO: 1 in the sequence listing No. 47-1411 or nucleotide No. 78-1 of SEQ ID No. 3 in the sequence listing
The polypeptide can be obtained by producing a polypeptide encoded by the nucleotide sequence represented by 457 in a host cell by genetic manipulation. Specifically, other prokaryotic or eukaryotic host cells can be transformed by incorporating the DNA having the above nucleotide sequence into an appropriate vector DNA. Furthermore, the gene can be expressed in each host by introducing an appropriate promoter and a sequence involved in expression into these vectors.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
And the like. To transform the gene of interest into these host cells, the host cell is transformed with a plasmid vector containing replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

For example, K12 strain and the like are often used as Escherichia coli, and pBR322 or pUC-type plasmids are generally used as vectors, but are not limited thereto, and any known various strains and vectors can be used. .

The promoters include, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (t
ac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, and any promoter can be used for production of the target polypeptide.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura,
K. et al. (1984) J. Biochem. 95, 87-93) and the like, but are not limited thereto. Bacillus subtilis α
DN encoding the signal peptide sequence of amylase
By linking the A sequence, secretory expression outside the cells is also possible.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like.
For example, COS cells (Gluzman, Y. (1.
981) Cell 23, 175-182, ATCC CRL-165
0) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) deficient in dihydrofolate reductase (Urlaub, G. and Chasin, LA (1980) Proc.
Natl. Acad. Sci. USA 77, 4126-4220) and the like are often used, but are not limited thereto.

As the vertebrate cell expression promoter, those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of the gene to be expressed can be used. May have a replication origin. An example of such an expression vector is pSV2dhfr having the early promoter of SV40 (Subramani, S. et al. (198
1) Mol. Cell. Biol. 1,854-864).
It is not limited to this.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, Those having an RNA splice site can be used. The expression vector is diethylaminoethyl (D
EAE)-Dextran method (Luthman, H. and Magnusso)
n, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and
van der Eb, AJ (1973) Virology 52, 456-457),
And electric pulse drilling (Neumann, E. et al. (1982)
EMBO J. 1, 841-845) can be incorporated into COS cells, and thus desired transformed cells can be obtained. When CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1)
989): "Molecular CloningA Laboratory Manual" Cold
Spring Harbor Laboratory, NY) and pSV2-neo
(Southern, PJ and Berg, P. (1982) J. Mol. App
l. Genet. 1, 327-341) and the like, and selecting a G418-resistant colony, a transformed cell stably producing the desired polypeptide can be obtained.

When insect cells are used as host cells, cell lines derived from ovarian cells of Spodoptera frugiperda (Sf-9 or Sf-21) of the family Lepidoptera, Noctuidae, and Trichopl
High Five cells derived from usia ni egg cells (Wickham, TJ e
tal, (1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and the baculovirus transfer vector is pVL1392 / 1393 is frequently used (Kidd, IM and VC Emery (1993) The use of b
aculoviruses as expression vectors.Applied Bioche
mistry and Biotechnology 42, 137-159). In addition to these, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, A
By connecting the secretion signal sequence of the envelope surface protein GP67 of cNPV to the N-terminal side of the target protein,
It is also possible to express the recombinant protein as a secreted protein (Zhe-mei Wang, et al. (1998) Biol. Chem., 3
79, 167-174).

As an expression system using eukaryotic microorganisms as host cells, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevi
siae and petroleum yeast Pichia pastoris are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen,
L. and Hall, BD (1982) J. Biol. Chem. 257, 301
8-3025) and the promoter of the acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. S
ci. USA 80, 1-5) can be preferably used. Also,
When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site for an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretion signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. Type tryptase is known to be secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Ap
pl. Biochem. 28,125-131).

The transformant obtained as described above is
It can be cultured according to a conventional method.
Alternatively, the desired polypeptide is produced extracellularly. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of the above COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used.

The recombinant protein produced by the above culture inside or outside the cells of the transformant can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. can do. As the method, specifically, for example, treatment with a normal protein precipitant,
Examples include various liquid chromatography such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof. . In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified by a nickel affinity column. By combining the above methods, the polypeptide of the present invention can be easily produced in a large amount with high yield and high purity.

The antibody obtained as described above is
Method, ELISA method, fluorescent antibody method, passive hemagglutination method, etc., and various immunological measurement methods and immunohistological staining.

4) Detection The antibody obtained by the method described in the above 3) may be directly labeled or used as a primary antibody to specifically recognize the antibody (an antibody derived from the animal in which the antibody was prepared). Is used for detection in cooperation with a labeled secondary antibody.

Preferred examples of the type of label include, but are not limited to, an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation of further binding enzyme-labeled streptavidin to biotin of the secondary antibody is added). A variety of pre-labeled antibodies (or streptavidin) are commercially available for methods using a labeled secondary antibody (or labeled streptavidin). I for RIA
An antibody labeled with a radioisotope such as ¹²⁵ is used, and the measurement is performed using a liquid scintillation counter or the like.

By detecting the activity of these labeled enzymes, the amount of the polypeptide which is the antigen is measured.
In the case of alkaline phosphatase or horseradish peroxidase, substrates that emit color or emit light by the catalyst of those enzymes are commercially available.

When a substrate that develops color is used, it can be visually detected by Western blotting or dot / slot blotting. In the ELISA method, quantification is preferably performed by measuring the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. Also, preferably, a standard curve prepared by preparing a dilution series of the antigen used for producing the antibody in the above 3), performing a detection operation simultaneously with other samples using the dilution series as a standard antigen sample, and plotting the standard antigen concentration and the measured value It is possible to quantify the antigen concentration in other samples by preparing

On the other hand, when a substrate that emits light is used, in Western blotting or dot / slot blotting, detection is performed by autoradiography using an X-ray film or an imaging plate or photographing using an instant camera. Quantification using densitometry or BAS2000II system is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

5) Measurement procedure i) In the case of Western blot, dot blot or slot blot First, in order to prevent nonspecific adsorption of an antibody, a membrane was previously added to a substance that inhibits such nonspecific adsorption (skim milk, bovine). An operation (blocking) of immersing in a buffer containing serum albumin, gelatin, polyvinylpyrrolidone, etc. for a certain period of time is performed. The composition of the blocking solution is
For example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% skim milk, 0.05 to 0.1% Tween 20 is used. Instead of skim milk, 1 to 10% bovine serum albumin, 0.5 to 3% gelatin or 1% polyvinylpyrrolidone may be used. The blocking time is
16 to 24 hours at 4 ° C. or 1 to 3 hours at room temperature.

Next, 0.05 to 0.1% of the membrane
After washing with PBS or TBS containing Tween 20 (hereinafter referred to as “washing solution”) to remove excess blocking solution, the antibody prepared by the method described in 3) above is immersed in a solution appropriately diluted with the washing solution for a certain period of time. To bind the antibody to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary Western blotting experiment using a sample obtained by serially diluting the recombinant antigen described in 3) above. This antibody reaction operation is preferably performed at room temperature for 1 hour. After the completion of the antibody reaction operation, the membrane is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a labeled secondary antibody is commercially available, it is used after diluting it 2000 to 20,000 times with a washing solution (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, the membrane is immersed in a secondary antibody solution at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, exchanging the washing solution for a new one, shaking for 5 minutes, exchanging the washing solution again, and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

Ii) ELISA / RIA First, in order to prevent non-specific adsorption of the antibody to the bottom surface in the well of the plate on which the sample was immobilized by the method 2),
As in the case of Western blot, blocking is performed in advance. The blocking conditions are as described in the section of Western blot.

Next, 0.05 to 0.1%
After washing with PBS or TBS containing Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, a solution obtained by appropriately diluting the antibody prepared by the method described in 3) above with a washing solution is dispensed. Incubate for a certain time to allow the antibody to bind to the antigen. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary ELISA experiment using a sample obtained by serially diluting the recombinant antigen described in 3) above. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After completion of the antibody reaction operation, the inside of the well is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a labeled secondary antibody is commercially available, it is used after diluting it 2000 to 20,000 times with a washing solution (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, the secondary antibody solution is dispensed into the wells, incubated at room temperature for 1 to 3 hours, washed with a washing solution, and then washed.
Perform detection operation according to the labeling method. The washing operation is performed, for example, by first dispensing a washing solution into a well, shaking for 5 minutes, exchanging the washing solution for a new one and shaking for 5 minutes,
This is performed by replacing the washing solution again and shaking for 5 minutes.
If necessary, the washing solution may be further exchanged for washing.

In the present invention, the so-called sandwich ELISA can be carried out, for example, by the method described below. First, amino acid numbers 17 to 455 of SEQ ID NO: 2 in the sequence listing (preferably 19 to 455)
In any one of the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in amino acid Nos. 17-460 of SEQ ID NO: 4, two regions having high hydrophilicity are selected, and each region comprises at least six amino acid residues. A partial peptide is synthesized to obtain two kinds of antibodies using the partial peptide as an antigen. One of the antibodies is labeled as described in 4) above. The unlabeled antibody is immobilized on the bottom surface in the well of a 96-well ELISA plate according to the method described in 2) above. After blocking, the sample solution is placed in a well and incubated at room temperature for 1 hour. After washing the wells, the labeled antibody diluent is dispensed into each well and incubated. After the inside of the well is washed again, a detection operation according to the labeling method is performed.

6) Evaluation By the method described above, the detection results were compared between a sample derived from cells cultured in the presence of the test substance and a sample derived from cells cultured in the absence of the test substance. As a result, the test substance produced by the method described in the above 3) and having reduced production of the polypeptide to which the antibody specifically binds can be a therapeutic or preventive agent for hyperlipidemia. In addition, a kit for testing a therapeutic or prophylactic agent for hyperlipidemia is provided by combining the antibody prepared by the method described in the above 3) and other reagents used in the above series of methods.

The polypeptide to be detected in the method of the present invention can be obtained in accordance with the description of the above-mentioned method for obtaining an antigen for preparing an antibody, or can be a nucleotide represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing. Nucleotides 78-14 of SEQ ID NO: 3 of the sequence or sequence listing
Animal cells can also be produced using an adenovirus vector into which DNA having the nucleotide sequence shown in SEQ ID NO: 57 has been incorporated. As a method for constructing such a recombinant adenovirus vector, a commercially available kit (for example, adenovirus expression vector,
Kit, manufactured by Takara Shuzo Co., Ltd.). The gene to be detected in the method of the present invention is closely correlated with the blood neutral fat concentration of the mammal,
For example, when a mammal, such as a mouse, is infected with a recombinant adenovirus having the recombinant adenovirus vector obtained as described above, and the gene retained by the recombinant adenovirus vector is expressed, neutral neutral blood An increase in concentration was observed, and in congenital hypolipidemic mice, nucleotide number 47 of SEQ ID NO: 1 in the sequence listing
This is evidenced by the fact that the expression level of the gene having the nucleotide sequence shown at -1411 is lower than that in the hyperlipidemic mouse.

Further, a non-human gene, which is obtained by genetic manipulation and has a foreign gene containing the nucleotide sequence according to any one of the above a) to e), which is introduced so as to enable high expression of the gene, is introduced. A method for testing an agent for treating or preventing hyperlipidemia using an animal is also included in the present invention. Animals used in this method include, for example, KK / San mice infected with the above-mentioned recombinant adenovirus, the nucleotide sequence shown in nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing or SEQ ID NO: 3 in the sequence listing. Examples include, but are not limited to, transgenic mice in which a DNA having the nucleotide sequence of nucleotide numbers 78-1457 has been introduced into mice.

A transgenic animal can be obtained by obtaining a fertilized egg from the animal, transferring the gene, and transplanting the embryo into a pseudopregnant animal to generate the animal. [Emergency Engineering Experiment Manual (edited by Tatsuji Nomura, edited by Motoya Katsuki, 19
1987), mouse embryo operation manual ("Manipulating"
the Mouse Embryo, A Laboratory Manual "B. Hogan,
F. Costantini and E. Lacy, translated by Kazuya Yamauchi, Yutaka Toyoda, Yotsumori Mori and Yoichiro Iwakura, 1989), Tokuho 5-4809
No. 3 publication etc.]. Specifically, for example, in the case of a mouse,
First, after administration of an ovulation-inducing agent to a female mouse (preferably a mouse having a blood triglyceride concentration lower than usual, for example, KK / San mouse is preferable, but not limited thereto),
After crossing with males of the same strain, fertilized pronuclear eggs are collected from the oviduct of female mice the next day. Next, the DNA fragment solution to be introduced is injected into the pronucleus of a fertilized egg using a micro glass tube. In addition, as a regulatory gene such as a promoter or an enhancer for expressing a gene to be introduced in animal cells, any gene that functions in the cell of the introduced animal can be used. The fertilized egg into which the DNA was injected was implanted into the oviduct of a pseudopregnant foster female mouse (Slc: ICR, etc.),
Born 0 days later by spontaneous delivery or cesarean section. As a method for confirming that the thus obtained animal has the introduced gene, DN can be obtained from the tail of the animal or the like.
A and PCR using the DNA as a template and sense and antisense primers specific to the transgene
The DNA is digested with several types of restriction enzymes, gel electrophoresed, and the DNA in the gel is blotted on a nitrocellulose membrane or a nylon membrane. And a method of performing Southern blot analysis.
Whether or not the introduced gene is actually expressed in the animal body can be confirmed by measuring the concentration of neutral fat in peripheral blood. When the introduced gene is actually expressed in the animal, the blood triglyceride concentration will be higher than in the animal without the gene.

A test substance is administered to the transgenic animal thus obtained, and a substance that reduces the concentration of neutral fat in blood is selected (preferably, the test substance is administered to an animal that does not have the gene transfected). Then, the results are compared, and a substance that significantly lowers the blood neutral fat concentration in the transgenic animal is selected). In this method, not only a substance that suppresses or inhibits the expression of the introduced gene, but also the function of the polypeptide itself encoded by the gene or interacts with the polypeptide to contribute to the functional expression of the polypeptide Substances such as factors (for example, receptors) that inhibit any of the biological reactions related to an increase in blood triglyceride concentration by the action of the polypeptide can be found. Such a substance can also be a therapeutic or preventive agent for hyperlipidemia.

The present invention also relates to a polypeptide other than an antibody that specifically binds to a polypeptide to be detected in the method of the present invention (hereinafter, simply referred to as “polypeptide to be detected”), ie, a polypeptide to be detected. For the receptor. When the receptor is a protein present on the cell membrane, the receptor can be cloned, for example, according to the method described below. First, a recombinant consisting of a nucleotide sequence represented by nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the Sequence Listing or a nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the Sequence Listing can be expressed in a mammalian cell. A vector is constructed, introduced into COS-1, the culture supernatant is collected, and the recombinant polypeptide of the polypeptide to be detected is purified. The obtained recombinant polypeptide is fluorescein isothiocyanate (hereinafter referred to as "FITC").
Mark with. Next, the labeled recombinant polypeptide is added to cultured cells derived from various mammalian tissues, and cells that specifically bind the labeled recombinant polypeptide on the cell membrane, that is, cells that express the receptor, are identified. A cDNA library derived from the identified cells is incorporated into a vector that can be expressed in mammalian cells, and expressed in cells that do not express the receptor (preferably COS-1 or CHO cells, more preferably CHO cells). The above-mentioned FITC-labeled recombinant polypeptide is added to the cells in which the cDNA library has been expressed, and after culturing for a certain period of time, the cells are collected, and the cells to which the FITC-labeled recombinant polypeptides are bound by a cell sorter are selected. I do. The cDNA introduced from the obtained cells is cloned by PCR or the like. The above operation is repeated as necessary, and finally F
A cDNA encoding a receptor that specifically binds to the ITC-labeled recombinant polypeptide is cloned. In addition, the receptor itself can be recovered from the cells thus cloned.

In this way, if cDNA encoding the receptor of the polypeptide to be detected is obtained,
Utilizing the cells used as the material of the DNA library, a substance that inhibits the interaction between the polypeptide to be detected and the receptor, and exhibits the same function as the polypeptide to be detected by binding to the receptor material,
Alternatively, it can be used for screening for a substance that inhibits signal transduction initiated by the interaction between the polypeptide to be detected and the receptor. In particular,
For example, mouse genomic DNA is appropriately fragmented by restriction enzyme digestion or the like, and a green fluorescent protein (gree
n-fluorescent protein: pEGFP-1 (manufactured by Clontech) is commercially available as a vector for a reporter assay), and a vector linked thereto is introduced into the cells used as the material of the cDNA library. During the culture of the cells, a recombinant polypeptide (not labeled) of the polypeptide to be detected is added, and the cells producing the green fluorescent protein are selected with a cell sorter. The sorted cells produce green fluorescent protein in the presence of the recombinant polypeptide. The cells are cultured in a 96-well culture plate so that the number of cells per well is substantially the same, and only the test substance is added, or the recombinant polypeptide and the test substance are added simultaneously for a certain period of time. After culturing, the amount of green fluorescent protein produced is measured using a fluorescent plate reader or the like. If the test substance alone causes green fluorescent protein production,
The substance is considered to be an agonist of the polypeptide to be detected. On the other hand, when the amount of green fluorescent protein produced in the well to which the recombinant polypeptide and the test substance are simultaneously added is smaller than that in the well to which the recombinant polypeptide alone is added, the substance is an antagonist of the polypeptide to be detected or the same. It is a signal transduction inhibitor of polypeptide, and is considered to be useful as an agent for treating or preventing hyperlipidemia.

When the antagonist thus obtained is a protein or peptide, the polynucleotide having a nucleotide sequence encoding the protein or peptide can be used for gene therapy of hyperlipidemia. Such polynucleotides may be obtained, for example, by analyzing the amino acid sequence of the identified antagonist protein or polypeptide, synthesizing an oligonucleotide probe consisting of a nucleotide sequence encoding the amino acid sequence, and preparing various cDNA and genomic libraries. It can be obtained by performing screening. When the peptide having antagonist activity is derived from a randomly synthesized artificial peptide library, a DNA consisting of a nucleotide sequence encoding the amino acid sequence of the peptide is chemically synthesized. In gene therapy, a polynucleotide encoding the antagonist thus obtained is incorporated into, for example, a viral vector, and the virus (detoxified) having the recombinant viral vector is transmitted to a patient. Since an antagonist is produced in the patient and inhibits the function of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, the concentration of neutral fat in blood can be reduced.

Methods for introducing a gene therapy agent into cells include a gene transfer method using a viral vector and a non-viral gene transfer method (Nikkei Science, April 1994, pp. 20-45, Experimental Medicine). Extra number, 12 (15) (199
4), Experimental Medicine Supplement, “Basic Technologies for Gene Therapy,” Yodosha (1
996)) can be applied.

As a method for gene transfer using a viral vector, for example, DNA viruses or RNA viruses such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, and simbis virus, TR
4 or a mutant TR4. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is particularly preferable. Non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method,
Examples thereof include a calcium phosphate method and an electroporation method, and a DNA vaccine method and a liposome method are particularly preferable.

In order for the gene therapy agent to actually act as a medicine, the DNA is directly introduced into the body.
In vivo) method and ex vivo method in which certain cells are removed from a human, DNA is introduced outside the body into the cells, and the cells are returned to the body (Nikkei Science, April 1994).
Monthly, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Special Edition, 12 (15) (1994)).

For example, when the gene therapy agent is administered by an in vivo method, it is administered by an appropriate administration route such as intravenous, arterial, subcutaneous, intradermal, intramuscular, etc., depending on the disease or condition. When administered by the in vivo method, the gene therapy agent is generally used as an injection or the like, but if necessary, a conventional carrier may be added. In addition, liposomes or membrane-fused liposomes (such as Sendai virus-liposome)
In the case of the form (1), a liposome preparation such as a suspension, a cryoprotectant, a centrifugal concentrated cryopreservative and the like can be prepared.

Nucleotide No. 7 of SEQ ID No. 3 in Sequence Listing
A nucleotide sequence complementary to a partial sequence of the nucleotide sequence shown in 4-1457 can be used for so-called antisense therapy. The antisense molecule is a stable DNA such as a DNA consisting of usually 15 to 30 mer, or a phosphorothioate, methylphosphonate or morpholino derivative thereof, which is complementary to a part of the nucleotide sequence of nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing. DNA derivative, 2'-O-alkyl RNA
Etc. can be used as stable RNA derivatives. Such antisense molecules can be introduced into cells by methods well known in the art of the present invention, such as by microinjection, liposome encapsulation, or expression using a vector having an antisense sequence. Such antisense therapy is useful for the treatment of diseases in which it is useful to reduce the activity of the protein encoded by the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing, particularly for the treatment of hyperlipidemia. It is.

A composition useful as a medicine containing the above-mentioned antisense oligonucleotide can be produced by a known method such as mixing a pharmaceutically acceptable carrier. Examples of such carriers and manufacturing methods are described in Remington's Pharmaceut
ical Sciences. And SEQ ID NO: 3
Is administered to each individual in an amount sufficient for the treatment of hyperlipidemia in which the expression of the gene containing the nucleotide sequence represented by nucleotide numbers 78-1457 and the activity of the gene product thereof are abnormal. The effective amount can vary depending on various factors such as the condition, weight, sex, and age of each individual, and the administration method such as subcutaneous, topical, oral, and intramuscular. For example, for intravenous injection, 0.02-0.2 mg /
It can vary from 2 hours at kg / hr and from 1 to 200 mg / m ² / day for subcutaneous administration.

[0119]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In the following examples, unless otherwise specified, each operation relating to gene manipulation is referred to as “molecular cloning (Molecular Cloning)” [Sambrook, J., Fritsch, E., et al.
F. and Maniatis, T., Cold Spring Harbor Labora
tory Press, published in 1989], or in the case of using a commercially available reagent or kit, in accordance with the instructions of a commercially available product.

Reference Example 1 Cloning of cDNA Using mouse liver as a material, cDN having the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing according to the following method
A was obtained.

A) Extraction of mRNA from mouse liver Two 9-week-old KK mice (male, obtained from Hamamatsu Medical University attached animal experiment facility) were dissected and their livers were excised and immediately placed in liquid nitrogen. Quick frozen. The weight was measured, and 3.1 g was ground in a mortar in the presence of liquid nitrogen.
5.5 M guanidine thiocyanate (hereinafter GT) buffer (5.5 M guanidine thiocyanate, 25 mM
Sodium citrate (pH 7.0), 0.5% sarkosyl, 0.2 M β-mercaptoethanol (30 m
l), crush with a pestle, and add a fresh GT buffer (10 m
l) was added, and the mixture was crushed with a pestle, and the lysate was recovered. Also,
The mortar was washed with GT buffer (20 ml), and this solution was also recovered. 36 ml of the recovered solution is 3,000 rpm at 10 ° C. for 1 hour.
After centrifugation for 0 minutes, the supernatant was transferred to a new tube, and each was added for 18 minutes.
The suction and discharge were repeated 20 times with a gauge needle. Next, total RNA was separated by density gradient centrifugation using cesium trifluoroacetic acid (CsTFA). CsTFA stock solution (19 ml) was mixed with ribonuclease-free double distilled water (1
8.924 ml), and the diluted solution (6.18 m
l) was placed in six 13PA (manufactured by Beckman) tubes, and the previously collected sample (5.2 m per tube)
l) was overlaid. Using a swing rotor (P40ST manufactured by Hitachi Koki Co., Ltd.), this product was centrifuged (Hitachi SCP).
30000 rpm (about 125000 g) at 70H type),
After centrifugation at 20 ° C. for 20.5 hours, the supernatant was removed, and the resulting pellet was suspended in 3.3 ml of extraction buffer attached to an mRNA purification kit (Quick-prep mRNA purification kit manufactured by Amersham Pharmacia). The mRNA was purified by using the purification kit according to the attached protocol. 5 μg of mR thus obtained
A phage λ cDNA library was prepared using NA as a template and a cDNA library preparation kit (ZAP Express cDNA Gigapack III Gold Cloning Kit, manufactured by Stratagene) according to the protocol attached thereto.

B) Primary screening of cDNA library Escherichia coli infected with the λ phage cDNA library obtained by the method described in a) above was plated on an agar medium plate (NZY medium: 0) prepared in a 9 cm-diameter culture dish. .5
% Sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.5% yeast extract, 1% casein hydrolyzate, 1.5% agar) and 1.8 × 10 ⁵ per plate The cells were dispersed to form plaques and cultured at 37 ° C. for 8 hours. The agar medium was removed from the 14 plaque-formed agar medium together with the plaque using the bottom of a 250 μl wide diameter pipette tip (manufactured by RAININ), and the agar medium pieces were each 100 μl SM buffer solution (0. Place in a plastic centrifuge tube containing 1M sodium chloride, 8mM magnesium sulfate, 50mM Tris-HCl (pH 7.5), 0.01% gelatin) and vortex vigorously using a vortex mixer, then 1-2 hours at 4 ° C. After leaving
The supernatant was recovered by centrifugation at 12000 × g for 5 minutes to obtain a phage suspension.

On the other hand, as primers used for PCR,
The following nucleotide sequence: Primer 1: 5'-gactgatcaa atatgttgag ctt-3 '
(SEQ ID NO: 5 in the sequence listing); and primer 2: 5'-tgcatccaga gtggatccag a-
The oligonucleotide having 3 ′ (SEQ ID NO: 6 in the sequence listing) was subjected to a phosphoramidite method (Matteucci, MD, and) using an automatic DNA synthesizer (Model 394: manufactured by PerkinElmer Japan Applied Biosystems Division). Caruthers, MH (19
81) J. Am. Chem. Soc. 103, 3185-3191).

5 μl of the phage suspension obtained as described above was mixed with 2.5 μl of 10 × PCR buffer (attached to Taq polymerase manufactured by Takara Shuzo Co., Ltd.) and 4 μl of d
NTP mixture (2.5 mM each, Taq manufactured by Takara Shuzo Co., Ltd.)
Primer 1 and 2, adjusted to 1 μl each 7.5 μM each, 0.25 μl Taq
Polymerase (manufactured by Takara Shuzo Co., Ltd.), mixed with 11.25 μl of sterilized water, heated at 94 ° C. for 5 minutes, then heated at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The cycle was repeated 30 times and finally kept at 72 ° C for 7 minutes before storing at 4 ° C. The reaction was performed on a 4% agarose gel (NuSieve 3: 1 agarose (FMC
Prepared by Bio-Products, Inc.)
The amplification of the specific fragment was analyzed. As a result of screening 14 phage suspensions in this way, the desired cDN
Two positive samples were obtained in which the fragment of A was specifically amplified.

C) Secondary screening 100 ng of mouse genomic DNA (Clontech)
PCR was performed under the conditions described in b) above using
The amplified DNA fragment was recovered by agarose electrophoresis. Using this DNA fragment as a template, a reaction for generating a ³² P-labeled DNA fragment was performed using a multi-prime DNA labeling system (Amersham Pharmacia), and the reaction solution was converted to a nick column (Amersham Pharmacia). Was injected. 400 μl in this column
TE (10 mM Tris-HCl (pH 7.5), 1 m
M EDTA), wash once, and add another 400 μl
And the eluate was collected. The whole amount of the eluted fraction was used as a labeled probe in the following secondary screening.

On the other hand, the phage suspension determined to be positive in b) was diluted 100-fold with SM buffer, and 2 μm
E. coli infected with 1 minute of phage were dispersed on an agar plate prepared in a culture dish having a diameter of 9 cm, and
The cells were cultured at 7 ° C. for 8 hours. On this plaque-formed agar medium, a circular nylon membrane (Amersham Pharmacia, Hybond N
+), And the plaque was removed by leaving at 4 ° C. for 5 minutes. Using an 18G injection needle, penetrate three places of the membrane to the agar medium to mark the alignment, then peel off the membrane and place in an alkaline solution (1.5M sodium chloride, 0.5M sodium hydroxide). 5 minutes in a neutralization solution (1.5 M sodium chloride, 0.5 M Tris-HCl (pH 8.0)) for 2 minutes.
Min, 2xSSC, 0.2M Tris-HCl (p
After immersing in a solution containing H7.5) for 30 seconds, it was completely air-dried at room temperature.

This membrane was added to a 20 ml hybridization solution (ExpressHyb Hybridization Solutio
n, Clontech) at 68 ° C for 1 hour (prehybridization), and then replaced with an 8 ml hybridization solution containing a labeled probe and incubated at 68 ° C for 6 hours. Then, the membrane was washed with 2 × SSC, 0.05% SD.
The operation of washing with a solution containing S at room temperature for 15 minutes while gently shaking was repeated three times, and then 0.1 × S
The operation of washing with a solution containing SC and 0.1% SDS at 50 ° C. for 30 minutes was repeated three times.

The washed membrane was subjected to autoradiography, the original plaque at the position where the membrane was positive was recovered from the agar medium, and the phage suspension was subjected to PCR under the conditions described in b) above. As a result, specific amplification of the DNA fragment was observed in 6 out of 10 positive plaque samples.

Among the phage suspensions of the samples in which amplification was most strongly observed, ZAP Express
Using a helper phage and a host bacterium attached to the DNA Gigapack III Gold Cloning Kit (Stratagene), in vivo excision (In Vivo Excision) was performed according to the protocol attached to the kit, and the phagemid was placed on an agar medium. E. coli colonies were formed. These colonies were isolated, phagemids were respectively extracted, and PCR was performed by the method described in b) above. As a result, colonies showing specific amplification of the DNA fragment were selected and cultured, and a 1.6 kbp cDNA insert was selected. Transformed E. coli carrying phagemid # 55-1 having E. coli. coli pBK / m55-1 SANK 7
2199 was isolated.

As a result of analyzing the entire nucleotide sequence of the cDNA inserted into the obtained phagemid # 55-1, using an ABI prism 377 DNA sequencer or 3700 DNA sequencer manufactured by PerkinElmer Japan Applied Biosystems Co., Ltd. It turned out that it is the sequence shown by sequence number 1 of a sequence table (however, nucleotide number 1-8 of sequence number 1 of a sequence table is an adapter sequence derived from a vector). This sequence was identical to the sequence registered as mouse angiopoietin-related protein 3 in the GenBank database (accession number: AF162224). In addition, transformed E. coli E. coli harboring this phagemid # 55-1. c
oli pBK / m55-1 SANK 72199
Was internationally deposited with the National Institute of Bioscience and Human Technology on November 19, 1999, and the accession number F
ERM BP-6940 was attached.

Embodiment 1 Northern blot analysis a) Extraction of total RNA from mouse organs Northern blot analysis was performed for the purpose of examining the tissue expressing the cDNA obtained in Reference Example 1. First, KK
Mice (hyperlipidemic mice) and KK / San mice (hypolipidemic mutant mice; Shiraki et al., 7th Diabetes Animal Research Society (1993)) testis, spleen
n), total RNA was extracted from kidney (kidney), small intestine (small intestine), liver (liver) and brain (brain). Eighteen-week-old KK mice and KK / San mice were dissected, and each organ was excised, immediately placed in liquid nitrogen, rapidly frozen, and stored at -80 ° C. About 15 ml of TRIzol reagent (manufactured by Gibco BRL) was added to 0.5 g of the organ, and homogenized on ice using an ultra-high-speed homogenizer Polytron (manufactured by Kinematica) (scale 6: 2 minutes). After allowing this to stand at room temperature for 5 minutes, 3 ml of chloroform was added, and the mixture was mixed by inverting vigorously by hand for 15 seconds. Let stand at room temperature again for 3 minutes, then
Centrifugation was performed at 00 × g and 4 ° C. for 15 minutes. After centrifugation, the upper layer was recovered and mixed with 0.8 volume of ribonuclease-free isopropyl alcohol. This was allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 × g at 4 ° C. for 10 minutes. Then, the supernatant was removed, and ribonuclease-free 70% ethanol was added. This was centrifuged at 12000 × g at 4 ° C. for 10 minutes, the supernatant was removed, and the precipitate was dried and stored at −80 ° C.

B) Electrophoresis and blotting of total RNA The total RNA of each collected organ was adjusted to 4 µg / µl with ribonuclease-free double distilled water, and then 5 µl of this RNA solution was used.
And RNA sample buffer (1.15 × MOPS buffer (1 ×
The MOPS buffer is 20 mM MOPS, 5 mM sodium acetate, 1 mM ethylenediaminetetraacetic acid (hereinafter referred to as “E
DTA "), 2.4 M formaldehyde, 57% formamide, 7% glycerol, 18
μg / ml bromophenol blue, 18 μg / ml
Xylene cyanol, 0.18 mM EDTA) 16
After mixing at 65 ° C for 10 minutes, mix on ice for 5 minutes.
Let stand for minutes. The total amount of this sample solution was used as an agarose gel for electrophoresis containing 1.17% formalin (1 × MOPS buffer, 1.17% agarose (high strength, for analysis, manufactured by Bio-Rad), 0.66M formaldehyde). Injected into one well and electrophoresed. Electrophoresis is 500n
In a submarine electrophoresis tank containing a 1 × MOPS buffer containing g / ml ethidium bromide, electricity was supplied at 50 V for about 1 hour, and then electricity was supplied at 100 V for about 1.5 hours. After completion of the electrophoresis, the R in the agarose gel was
NA is transferred by capillary transfer method (Maniatis, T. e.)
t al. (1982) in "Molecular Cloning A Laboratory Man
ual "Cold Spring Harbor Laboratory, NY) and transferred overnight to a nylon membrane (Hybond N +, manufactured by Amersham Pharmacia).
0xSSC was used). This membrane is 2 × SSC
For 5 minutes, air-dried, and irradiated with ultraviolet rays (1200 J / cm ² ) using an ultraviolet irradiation device for crosslinking (Spectrolinker XL-1000, manufactured by Tommy Seiko Co., Ltd.) to fix the RNA.

C) Preparation of Probe Using the primer synthesized in b) of Reference Example 1, a thermal cycler (GeneAmp PCR System 9600,
(PerkinElmer Japan Applied Biosystems Division) under the following conditions. First, sterile water was added to primers (0.5 μM each in final concentration) and Tween 20 (manufactured by Sigma, 0.1% final concentration) to make 7.5 μl, and then 2 × PCR Solution Premix Taq (Takara Shuzo Co., Ltd.) ) Company: 0.05 units / μ
l Taq polymerase, 0.4 mM dNTPs, 2
6. 0 mM Tris-HCl (pH 8.3), 100 mM potassium chloride, 3 mM magnesium chloride).
5 μl was added. Further, 1 μl (corresponding to 100 ng) of mouse genomic DNA (manufactured by Clonetech) was added to prepare a reaction solution. The reaction solution was first heated at 94 ° C. for 3 minutes, and then subjected to a temperature cycle of 94 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C. for 45 seconds 35 times, and then kept at 4 ° C.

Take 1 μl of the reaction solution after the PCR and add
The amplified DNA fragment was cloned into a plasmid vector using a cloning kit (dual promoter version A, manufactured by Invitrogen) according to the attached protocol. Competent Escherichia coli was transformed with this recombinant plasmid vector and cultured on LB agar medium containing 50 μg / ml ampicillin. As a result, Escherichia coli colonies that showed resistance to ampicillin were selected and cultured overnight at 37 ° C. in 4 ml of a liquid LB medium containing 50 μg / ml of ampicillin. An automatic plasmid extractor (PI) was used from 3.5 ml of the culture solution.
-50, manufactured by Kurabo Industries Co., Ltd.). Nucleotide sequence analysis was performed on the obtained plasmid DNA, and the plasmid into which the desired PCR product had been inserted was used for the following operation.

8 μg of the selected plasmid DNA was
After digestion with the restriction enzyme EcoRI, phenol / chloroform extraction and ethanol precipitation were performed, and the obtained precipitate was dissolved in 10 μl of sterilized water. The dye solution (0.25
% Bromophenol blue, 0.25% xylene cyanol, 15% ficoll (type 400))
Then, the whole amount was subjected to polyacrylamide gel electrophoresis (gel concentration: 8%, 100 V, room temperature, 3 hours). After staining the gel after electrophoresis with ethidium bromide, a band corresponding to the target DNA (about 200 bp,
The gel piece of SEQ ID No. 11) in the sequence listing was cut off with a razor blade, transferred to a microcentrifuge tube and pulverized. 3 for this one
00 μl elution buffer (0.5 M ammonium acetate,
10 mM EDTA (pH 8.0), 0.1% SDS
After the mixture was kept at 37 ° C. overnight, phenol / chloroform extraction was performed twice and ethanol precipitation was performed once, and the precipitate was dissolved in 20 μl of sterilized water.

Using 5 μl of the obtained DNA solution, a probe (400 μl) labeled with ³² P was prepared by the method described in c) of Reference Example 1.

D) Hybridization The membrane prepared in b) was mixed with 20 ml of a hybridization solution (ExpressHyb Hybridization Solutio
n, manufactured by Clontech) at 68 ° C for 1 hour (prehybridization), and then incubated at 68 ° C overnight in a 20 ml hybridization solution containing a ³² P-labeled probe. Thereafter, the membrane is subjected to 2 × SSC, 0.05% SD.
Washed three times in a solution containing S at room temperature for 20 minutes, and further washed with a solution containing 0.1 × SSC and 0.1% SDS.
After washing three times at 20 ° C. for 20 minutes, autoradiography was performed.

As a result, the expression of the detected gene was found only in the liver, and the expression level was significantly lower in KK / San mice (hypolipidemic mice) than in KK mice (hyperlipidemic mice). (Fig. 1).

In the above experimental system, for example, an RNA sample was prepared from primary cultured hepatocytes of a KK mouse cultured in the presence or absence of a test substance, and the same operation as described above was carried out to obtain hyperlipidemia of the test substance. The effect as a therapeutic or prophylactic agent for the disease can be examined. A test substance that decreases the expression level of the gene detected in this experiment can be a therapeutic or preventive agent for hyperlipidemia. When performing multi-sample processing, dot blot or slot blot can be performed without electrophoresis.

Reference Example 2 Cloning of human cDNA 1) Preparation of probe In order to obtain a human cDNA corresponding to the mouse cDNA shown in SEQ ID NO: 1 in the sequence listing, the following nucleotide sequence: 5'-tcctctagtt atttcctccag-3 '(sequence number in the sequence listing) 7); and an oligonucleotide primer having 5′-tggtttgcca gcgatagatc-3 ′ (SEQ ID NO: 8 in the sequence listing) was synthesized.

Next, 1 μl of human genomic DNA (manufactured by Boehringer Mannheim, 200 mg / ml) and Taq polymerase (rTaq, manufactured by Takara Shuzo Co., Ltd., 5 units / μm)
l) 1 μl, 10 × PCR buffer (Takara Shuzo) 10 μl, dNTP mixed solution (2.5 mM each) 16 μl
l, 2 μl each of 20 μM primer, and 68 sterile water
μl was mixed. This reaction solution was heated at 94 ° C. for 5 minutes, and then heated at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C.
Temperature cycle of 30 seconds at ℃ 30 times, finally 7
After keeping the temperature at 2 ° C for 10 minutes, it was stored at 4 ° C. After electrophoresis of the reaction solution on a 2% agarose gel, the gel of the amplified DNA band was cut out and purified.
The DNA thus obtained was used as a DNA labeling kit (Bc
aBest Labeling Kit, manufactured by Takara Shuzo Co., Ltd.)
Those labeled with ³² P were used as probes in 2) below.

2) Primary screening of cDNA library A commercially available human liver-derived cDNA library (Human Live
r 5'-STRETCH cDNA Library, manufactured by Clontech), 1 × 10 ⁶ plaque DNA was immobilized on a nylon membrane. That is, Escherichia coli infected with the cDNA library was dispersed on 20 agar plates prepared in a culture dish having a diameter of 9 cm so that 5 × 10 ⁴ plaques were formed per plate and incubated at 37 ° C. for 8 hours. Cultured. A circular nylon membrane (Hybond N +, manufactured by Amersham-Pharmacia) was placed on the plaque-formed agar medium and adjusted to the inside diameter of the petri dish, and placed on the plate.
Plaques were removed by leaving at 5 ° C. for 5 minutes. Using an 18G syringe needle, three marks on the membrane were penetrated to the agar medium to mark the alignment, then the membrane was peeled off and the alkaline solution (1.5 M
2 minutes in sodium chloride, 0.5 M sodium hydroxide, then neutralization solution (1.5 M sodium chloride, 0.1 M).
The sample was immersed in 5M Tris-hydrochloric acid (pH 8.0) for 5 minutes and further in a solution containing 2 × SSC and 0.2M Tris-hydrochloric acid (pH 7.5) for 30 seconds, and then completely air-dried at room temperature. Then, the DNA was fixed by irradiating ultraviolet rays (1200 J / cm ² ) with an ultraviolet irradiation device for crosslink (Spectrolinker XL-1000, manufactured by Tommy Seiko Co., Ltd.).

[0142] The membrane thus prepared was
The mixture was incubated at 65 ° C. overnight in a hybridization solution (ExpressHyb solution, manufactured by Clontech) containing the labeled probe obtained in 1) above. Thereafter, the membrane is washed with a solution containing 2 × SSC and 0.05% SDS three times for 15 minutes each at room temperature, and then further washed with a solution containing 0.1 × SSC and 0.1% SDS for 30 minutes. After washing at 50 ° C. twice, autoradiography was performed.

The plaque at the position of the positive signal found as a result was collected together with the medium from the agar medium plate, and 100 μl of SM buffer (0.1 M
Sodium chloride, 8 mM magnesium sulfate, 50 mM
The suspension was put in Tris-hydrochloric acid (pH 7.5, 0.01% gelatin), suspended well, allowed to stand at 4 ° C. for 2 hours, and then centrifuged at 12,000 × g for 5 minutes to collect the supernatant.

Escherichia coli was again infected with the thus obtained primary positive phage solution, and 500 plaques were formed per a petri dish on an agar medium prepared in a culture dish of 9 cm in diameter. And secondary screening was performed by repeating the above procedure. The obtained secondary positive clone phage was transformed into E. coli strain BM.
The cells infected with 25.8 (manufactured by Clontech) were cultured at 37 ° C. on an agar medium to form E. coli colonies containing phagemid. The colony was isolated, cultured in a small amount in a liquid medium, phagemid was extracted, and the inserted fragment was analyzed by restriction enzyme digestion. As a result, E. coli E. coli having clone # h5-1 having an inserted fragment of 1.6 kbp was analyzed.
coli pTrip / h55-1 SANK 722
99 were isolated. As a result of analyzing the nucleotide sequence of the inserted fragment of this clone, it was confirmed that c was registered as a human angiopoietin-related protein 3 in GenBank.
This was consistent with the DNA sequence (accession number: AF152562) (SEQ ID NO: 3 in the sequence listing; however, nucleotide numbers 1 to 14 in SEQ ID NO: 3 in the sequence listing are vector-derived adapter sequences). The transformed E. coli E. coli harboring this phagemid # h5-1 was used. coli pTrip / h55-1
SANK 72299 was deposited on November 19, 1999 with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, and assigned the accession number FERM BP-6941.

Embodiment 2 FIG . Preparation of Polyclonal Antibody An amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence listing, that is, a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and SEQ ID NO: 4 in the sequence listing For the purpose of producing an antibody that recognizes both, two amino acid sequences selected from the regions conserved between the mouse and human polypeptides as antigens: Glu-Pro-Lys-Ser-Arg-Phe -Ala-Met-Leu-Asp-Asp-Val-Ly
s-Cys (55-1-N1, SEQ ID NO: 9 in the sequence listing); and Cys-Gly-Glu-Asn-Asn-Leu-Asn-Gly-Lys-Tyr-Asn-Lys-Pr
A peptide having o-Arg (55-1-C1, SEQ ID NO: 10 in the sequence listing) was synthesized by a chemical synthesis method (device used:
Model 433 manufactured by PerkinElmer Japan). However, the amino acid sequence of 55-1-N1 has a cysteine residue added to the C-terminal of the original amino acid sequence in order to bind keyhole limpet hemocyanin (hereinafter referred to as “KLH”) as a carrier. . On the other hand, 55
The N-terminal cysteine to which KLH is bound at -1-C1 is derived from the original amino acid sequence.

Next, the synthetic peptide 55-1-N11
1.1 mg and KLH 21.5 mg, or 55-
10.2 mg of 1-C1 and 21.2 mg of KLH were condensed using N- (6-maleimidocaproyloxy) succinimide (EMCS, manufactured by Dojindo Laboratories, Inc.) reagents (as a condensation reaction solvent). (Using 0.02 M phosphate buffer (pH 7.5) containing 8 M urea and 0.9% sodium chloride, the mixture was kept at room temperature for 15 hours). This reaction solution was put into an 8M urea solution, dialyzed against running water, further dialyzed against pure water, and then lyophilized to obtain a KLH-bound peptide antigen. To about 10 mg of these peptide antigens, 1 ml of physiological saline was added to form a fine suspension using an ultrasonic oscillator (sonicator), a vortex mixer, a glass rod, or the like. Thereafter, the total volume was made up to 7.5 ml each with physiological saline, and
Aliquots of each ml were stored in vials and stored frozen.

At the time of immunization, the antigen solution for one vial was melted, mixed with the same amount of adjuvant, and injected subcutaneously or intradermally into the back of two rabbits. The adjuvant is performed using Freund's complete adjuvant,
For the second and subsequent immunizations, incomplete Freund's adjuvant was used. Immunization was performed four times every two weeks, and after the second immunization, test blood was collected and the antibody titer in the serum was examined by enzyme immunoassay (ELISA) using a solid phase method. 96-hole ELI
An SA peptide (96-well H type, manufactured by Sumitomo Bakelite Co., Ltd.) was coated with each antigen peptide, and horseradish peroxidase-labeled anti-rabbit IgG antibody was used as a secondary antibody. Thirteen days after the fourth immunization, whole blood was collected.

After blood collection, the antibody was purified using an affinity column. That is, a carrier EMC-activated by reacting N- (6-maleimidocaproyloxy) succinimide (EMCS, manufactured by Dojindo Laboratories, Inc.) with aminoalkyl agarose (Bio-Rad, Affigel 102). Peptide (55) was added to agarose (about 5 ml).
-1-N1: 7.82 mg, 55-1-C1: 8.07
mg) was coupled. Unreacted EMC groups are 0.1 M mercaptoethylamine hydrochloride (including 5 mM EDTA)
For inactivation. 85 ml of antiserum was added to PBS (0.02 M phosphate buffer (pH 7.0),
The solution was diluted two-fold with 0.9% sodium chloride, and a precipitate was obtained by ammonium sulfate precipitation (final concentration: 40%). The precipitate was dissolved in PBS, desalted and dialyzed with PBS. Was used as a crude IgG fraction. The chromatographic operation was performed three times on the affinity column. That is, the operation of charging a 1/3 amount of the crude IgG fraction to the affinity column and re-injecting the passed fraction into the column was repeated three times. A total of 40 ml of the flow-through portion and the washing solution were collected as a non-adsorbed fraction. After thoroughly washing with 1 M sodium chloride-containing PBS to remove non-specifically bound to the column, 4 M magnesium chloride solution, 3.5 M
Potassium thiocyanate solution and 0.1 M glycine-
Hydrochloric acid buffer (pH 2.3) was sequentially passed through the column, and the antibody specifically bound to the peptide bound on the column carrier was eluted as an affinity purified antibody. 4M
Since the target antibody was contained in the eluates of the magnesium chloride solution and the 3.5 M potassium thiocyanate solution, each eluate dialyzed against PBS was used as an antibody in the following procedure.

Embodiment 3 FIG . Expression and expression in COS-1 cells
# H5-1 phagemid DNA obtained in Reference Example 2 and Western blot analysis was digested with restriction enzymes EcoRI and XbaI, subjected to 8% polyacrylamide gel electrophoresis, and subjected to the method described in Example 1 c). An approximately 1.6 kb fragment containing the cDNA was isolated and purified. On the other hand, the high expression vector pME18S (Ha
ra, T. et al. (1992) EMBO.J. 11, 1875-, edited by Takashi Yokota and Kenichi Arai, Bio Manual Series 3, Gene Cloning Experiments, Yodosha, p18-20)
Digested with XbaI and dephosphorylated at the ends.
The DNA fragment was ligated with a DNA ligation kit (version 2, Takara Shuzo Co., Ltd.). This DNA
, And the resulting transformant is analyzed by restriction enzyme analysis of the plasmid DNA contained therein, and a strain having a 1.6 kb DNA fragment is selected and p.
It was named MEh55-1.

Next, transformed Escherichia coli harboring pMEh55-1 was transformed with 100 μg / ml containing 50 μg / ml ampicillin.
The cells were cultured overnight at 37 ° C. in ml of liquid LB medium. From this culture solution, pMEh55-1 DNA was recovered using a plasmid purification kit (Wizard Purefection Plasmid DNA Purification System, manufactured by Promega) and purified by the cesium chloride method.

The thus obtained plasmid pME
COS-1 cells were transfected with h55-1. Transfection into COS-1 cells
This was performed by an electric perforation method using a gene transfer device GTE-1 manufactured by Shimadzu Corporation. That is, first, cells were recovered by trypsin-EDTA treatment from a flask in which COS-1 cells were grown until they reached semi-confluence.
The plate was washed with a BS (-) buffer (manufactured by Takara Shuzo Co., Ltd.). Next, the cells were washed with PBS (−) buffer at 6 × 10 ⁷ cells / m 2.
l. Plasmid DNA recovered by the above method
(PMEh55-1) was added to PBS (-)
g / ml. 20μ of cell suspension and DNA solution
The mixture was placed in a chamber with an electrode spacing of 2 mm, and a pulse of 600 V-30 μsec was applied twice at 1 second intervals. After cooling the chamber at 4 ° C. for 5 minutes, the cell-DNA mixture therein was diluted with D containing 10% fetal bovine serum.
Add 10 ml of MEM, transfer to a Petri dish, 37 ° C, 5
Cultured overnight under% CO ₂ . Thereafter, the culture supernatant was removed, and the cells were washed with a serum-free medium (DMEM).
10 ml was added and the cells were cultured for 3 days.

The culture supernatant was recovered from the cell culture thus obtained. Negative control plasmid pME18S or pMEh55- containing no cDNA
A 0.3 ml serum-free culture supernatant of COS-1 cells obtained by transfecting the cells was treated with trichloroacetic acid (hereinafter referred to as "TCA") to precipitate proteins, and the precipitate was obtained by centrifugation. The precipitate was washed with ice-cooled acetone, air-dried, and dissolved in a sample buffer (manufactured by Bio-Rad) containing 2-mercaptoethanol for SDS-polyacrylamide electrophoresis (SDS-PAGE).
SDS-PAGE was performed under reducing conditions using a 4-20% polyacrylamide density gradient gel (Multigel 4/20, manufactured by Daiichi Pure Chemicals Co., Ltd.).

After the electrophoresis, the band was separated from the polyacrylamide gel in a transfer buffer (192 mM glycine, 20% methanol, 25 mM Tris) using a gel membrane transfer device (manufactured by Marisol, NP7513) at 4 ° C. for 9 hours.
It was transferred to a nitrocellulose membrane (manufactured by Bio-Rad) at 200 mA for 0 minutes.

For the nitrocellulose membrane after transfer, the antibody obtained in Example 2 (hereinafter referred to as “55-1-N1
Antibody "or" 55-1-C1 antibody "). That is, first, a nitrocellulose membrane containing 0.05% Tween 20 in PBS (hereinafter referred to as “0.05% Tween 20-PB”) was used.
S ") (at room temperature, once for 15 minutes, then twice for 5 minutes), and put into a plastic bag (trade name: Hybrid Vac, manufactured by Cosmo Bio Co., Ltd.), and 5%
0.05% including skim milk (Snow Brand Milk Products Co., Ltd.)
Twenty 20-PBS (20 ml) was added and shaken at room temperature for 1 hour. After 1 hour, remove the membrane and remove
1x for 15 minutes in 05% Tween 20-PBS
Washing twice and then for 5 minutes × 2 times. After washing, the membrane was transferred to a new plastic bag, and the 55-1-N1 antibody or 55-1-C1 antibody (100-fold dilution), 1%
Twenty 0.05% Tween 20-PBS containing bovine serum albumin (hereinafter referred to as “BSA”, manufactured by Sigma) was added to 20
The mixture was shaken at room temperature for 1 hour. One hour later, the membrane was taken out and 0.05% Tween 20-PBS was removed.
The solution was washed once for 15 minutes × 2 times for 5 minutes × 2. Thereafter, the membrane was transferred to a new plastic bag, and 20 ml of a horseradish peroxidase-labeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia) diluted 2000 times with 0.05% Tween 20-PBS containing 1% BSA was added. Shake at room temperature for 1 hour. After 1 hour, remove the membrane and add 0.05% T
The wells were washed once with 20-PBS for 15 minutes and then 4 times for 5 minutes. After washing, the membrane was placed on a wrap film, and 55-1 was detected using an ECL western blotting detection solution (manufactured by Amersham Pharmacia).
The band to which the -N1 antibody or 55-1-C1 antibody was bound was detected (the membrane was placed on a wrap film, immersed in an ECL western blotting detection solution for 1 minute, and allowed to stand at room temperature for 1 hour to reduce the background. After attenuation, the X-ray film was exposed (3 seconds). As a result, pMEh55-
A specific band was detected in the COS-1 culture supernatant obtained by introducing 1 plasmid DNA (FIG. 2).

A similar experiment can be performed on the COS-1 cell culture supernatant expressing the mouse cDNA obtained in Reference Example 1. That is, the phagemid # 55-1 DNA obtained in Reference Example 1 was digested with the restriction enzymes EcoRI and Xba.
I, a clone obtained by incorporating about 1.6 kb of the inserted DNA fragment into pME18S (pME
55-1) is constructed, introduced into COS-1 cells in the same manner as described above, and the culture supernatant is collected. For the sample prepared from this culture supernatant, 55-1-N1 antibody and 55-N1 antibody
When Western blot analysis using the 1-C1 antibody is performed, a specific band is detected in the COS-1 culture supernatant obtained by introducing the pME55-1 plasmid DNA, regardless of the detection of any of the antibodies. .

In the above-described experimental system, for example, a sample is prepared from the culture supernatant of the primary culture of KK mouse hepatocytes cultured in the presence or absence of the test substance, and the same procedure is performed thereafter to obtain the test substance. The effect as a therapeutic or preventive agent for hyperlipidemia can be examined. A test substance that reduces the amount of antigen detected in this experiment can be a therapeutic or preventive agent for hyperlipidemia. When performing multi-sample processing, dot blot or slot blot can be performed without electrophoresis.

Embodiment 4 FIG . Production of recombinant adenovirus
Recombinant adenovirus for forced expression of mouse-derived cDNA obtained in Reference Example 1 and expression in cultured cells was obtained using a commercially available kit (Adenovirus Expression Vector Kit,
(Takara Shuzo Co., Ltd.). That is, the phagemid clone # 55-1 obtained in Reference Example 1 was digested with restriction enzymes EcoRI and NotI, and the obtained DNA fragment of about 1.7 kb was blunt-ended at the end to obtain an inserted DNA fragment. Used for the operation.

A cosmid vector pAxCAwt designed to be expressed by the cytomegalovirus enhancer and the chicken β-actin promoter.
A cosmid pAxCA / mAP5 was prepared in which an insert DNA fragment was inserted into the restriction enzyme SwaI recognition site of the adenovirus expression vector kit (attached to the adenovirus expression vector kit). pAxCA / mAP5 DNA and terminal protein-linked virus DNA (DNA-TPC, attached to the adenovirus expression vector kit) were transferred to 293 cells (ATCC) using a calcium phosphate transfection system (manufactured by Lifetech).
CRL1573), the recombinant adenovirus Ad / mAP-5 was isolated and further amplified in 293 cells. In addition, an adenovirus Ad / LacZ having a recombinant adenovirus vector into which the LacZ gene excised from the control cosmid pAxCAiLacZ was incorporated was similarly prepared and amplified in 293 cells. To recover the amplified virus from 293 cells, the virus-infected 293 cells were first subjected to sonication for 30 seconds × 4 times (using B-1200 manufactured by Branson), and then purified by cesium chloride density gradient centrifugation.
This was done by repeating the procedure several times. The obtained virus solution was added to PBS supplemented with 10% glycerol for 4 times.
After dialysis at ℃, it was frozen and stored at -70 ° C or lower until use.

The recombinant adenovirus thus obtained was introduced into HeLa cells (ATCC CCL2) in an amount of about 5 m.
o. i. (Multiplicity of infection)
And cultured in a serum-free medium (Dulbecco's modified Eagle's medium (DMEM)) for 3 to 4 days, and the culture supernatant was collected. 1 ml of the supernatant was placed in a 1.5 ml Eppendorf tube, 100 μl of trichloroacetic acid was added, and the mixture was allowed to stand at room temperature for 3 minutes.
and centrifuged for 5 minutes. The supernatant was removed, 0.5 ml of ice-cold acetone was added, and the mixture was stirred well.
Centrifuged at 5000 rpm for 2 minutes. The supernatant was removed, 0.5 ml of ice-cooled acetone was added again, and the mixture was stirred well. The mixture was centrifuged at 15000 rpm for 2 minutes using a tabletop centrifuge to remove the supernatant, and the precipitate was dried under vacuum. This precipitate is
SDS-PAGE sample buffer (manufactured by Bio-Rad) containing 2-mercaptoethanol dissolved in μl of distilled water
After heating at 99 ° C. for 5 minutes, a sample for electrophoresis was prepared. The sample was electrophoresed on a polyacrylamide density gradient gel having a gel concentration of 4 to 20% (electrophoresis buffer: 25 mM Tris, 192 mM glycine,
0.1% SDS) and then at 4 ° C. for 1 hour in transfer buffer.
It was transferred to a nitrocellulose membrane under the condition of 200 mA. The transferred membrane was blocked overnight at 4 ° C. with a PBS solution containing 0.5% skim milk, and washed three times with a washing solution (0.05% Tween 20-PBS). Next, the membrane was prepared by mixing the antiserum before purification of the 55-1-N1 antibody and the antiserum before purification of the 55-1-C1 antibody with 0.05% T fetal serum containing 5% fetal bovine serum.
After incubating at room temperature for 1 hour in a reaction solution diluted 10000-fold with ween 20-PBS, it was washed three times with a washing solution. Further, the membrane was prepared by adding horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (manufactured by Bio-Rad) to 0.05% containing 5% fetal bovine serum.
After incubation at room temperature for 1 hour in a reaction solution diluted 10000 times with 20% Tween 20-PBS, the plate was washed 5 times with a washing solution. Place the membrane on a wrap film and add 1 ml of ECL western blotting detection solution.
After soaking for 1 minute, the background was attenuated by leaving the plate at room temperature for 1 hour to expose the X-ray film (3 seconds). As a result, the recombinant adenovirus Ad / mAP-
A specific band was detected in the lane of the sample derived from the culture supernatant of the HeLa cell infected with No. 5 (FIG. 3).

On the other hand, a recombinant adenovirus (Ad / hAP) having the cDNA retained by the phagemid clone # h5-1 incorporated into an adenovirus vector
5) was prepared and the same experiment was performed. As a result of Western blot analysis of the culture supernatant expressed in HeLa cells, a specific band was similarly detected (FIG. 3).

Embodiment 5 FIG . Using recombinant adenovirus
Expression in vivo Recombinant adenovirus Ad purified as described above
/ MAP-5 or Ad / LacZ in PBS containing 10% glycerol at 2 × 10 ¹⁰ pfu (plaqueforming)
unit) / ml, and each 3 (Ad / mAP-
5) 100 μl (2 × 10 ⁹ pf) of two or 13 (Ad / LacZ) 13-14 week old male KK / San mice
u) Each was inoculated by tail vein injection. One day after inoculation, blood was collected from the fundus of each mouse using a hematocrit tube, centrifuged at 5200 rpm for 15 minutes using a tabletop centrifuge to separate plasma, and a neutral fat measurement kit (Triglyceride E-Test Wako, Wako Pharmaceutical Co., Ltd.) Was used to measure the neutral fat concentration. In the group of mice inoculated with Ad / LacZ, no significant difference was observed in the triglyceride concentration in blood, whereas the difference in Ad / m
There was a significant difference in blood neutral fat concentration between the AP-5 inoculated mouse group and the other two groups (FIG. 4).

On the other hand, a recombinant adenovirus (Ad / hAP) having a cDNA retained by the phagemid clone # h5-1 incorporated in an adenovirus vector
A similar experiment was performed in 5), and as a result of expression in male KK / San mice, a significant increase in blood triglyceride concentration was observed, indicating that the human-type molecule also functions in the mouse body (Fig. 4).

In addition, total RNA was recovered from the livers of a group of mice infected with adenovirus in which a significant difference was detected in the concentration of neutral fat in the blood, and Northern blot analysis was performed by the method described in Example 1. It was confirmed that the expressed gene was actually highly expressed (FIG. 5). The band detected in the group of KK / San mice infected with adenovirus is larger than the band detected in KK mice that have not been genetically engineered, but this is due to the effective transcription start site or poly ( A) It is considered that the additional signal is different from that on the original gene. In any case, immediately after the first translation start codon in the cDNA incorporated in the recombinant adenovirus vector, a termination codon is present on the same reading frame as the translation initiation codon, so that this mRNA The difference in size does not affect the translated amino acid sequence.

Embodiment 6 FIG . Purification and purification of recombinant proteins
And determination of the N-terminal amino acid sequence , prepared in Example 3, according to the method described below.
Expression vector pMEh55 into which human cDNA having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing has been inserted.
-1 is used to transform animal cells, and the recombinant protein secreted into the culture supernatant of the transformed cells is purified.
The terminal amino acid sequence was determined.

(1) Acquisition of Transformant and Preparation of Culture Supernatant 10% FCS (manufactured by Gibco BRL), 10 units / ml penicillin and 10 μg / ml streptomycin (manufactured by Gibco BRL) were used. ΑME including
M (manufactured by Gibco BRL) medium and dihydrofolate reductase-deficient CHO cells (ATCC CRL-909).
After growing 6), transfection reagent (Fu
GENE6: Roche Diagnostics) was used to transfect the pMEh55-1 plasmid at a rate of 1 μg / 10 ⁶ cells. Specifically, the cells were cultured in 200 cell culture dishes (150 mmφ, manufactured by Corning) at 1.5 × 10 ⁷ cells / dish,
15 μg per dish for transfection
PMEh55-1 plasmid was used. The cells after the transfection operation were cultured for 24 hours in the above-mentioned FCS-containing medium, and the medium was replaced with a serum-free αMEM medium (30 ml).
/ Dish), and further cultured for 3 days, and 6 liters of serum-free culture supernatant was collected.

(2) Purification of recombinant protein 1) 1.6 liters of Sephadex G25 (manufactured by Amersham Pharmacia Biotech) packed in a column (Streamline C-100: manufactured by Amersham Pharmacia Biotech) 600 ml of the culture supernatant obtained in the above (1) was injected into the gel (flow rate: 20 ml /
Minutes). The elution buffer (20 mM Tris-HCl (p
H7.5), 0.01% sodium azide (Sigma), 0.05% protease inhibitor mixture (Sigma), 0.05% Tween 20 (Sigma). Hereinafter, referred to as “solution A”) was passed through the column at a flow rate of 50 ml / min, and the first 1500 ml of eluate was collected. This operation was performed 10 times to desalt and remove low molecules of the 6 liter culture supernatant obtained in the above (1).

2) Next, the eluate obtained in the above 1) was subjected to ion exchange chromatography using an FPLC apparatus (BioPilot System, manufactured by Amersham-Pharmacia Biotech) under the conditions described below. Painted. Column: Q Sepharose Fast Flow (Amersham Pharmacia Biotech) 100
ml was packed in an XK50 / 100 column (manufactured by Amersham Pharmacia Biotech). Elution buffer composition: [Solution A] See above [Solution B] 20 mM Tris-HCl, 1 M sodium chloride (pH 7.5), 0.01% sodium azide,
0.05% protease inhibitor mixture, 0.0
5% Tween 20 Flow rate: 10 ml / min Fractionation: 10 ml / tube each Temperature: 4 ° C. Elution conditions: 100% solution A to 100% solution B: linear concentration gradient (60 minutes) Sodium chloride concentration 0.3-
The fraction eluted at 0.4M was collected.

3) Further, the fraction collected by the ion exchange chromatography in the above 2) was subjected to group-specific affinity chromatography under the following conditions using an FPLC apparatus. Column: Affigel Blue (Bio-Rad) 1
0 ml was packed in an XK16 / 40 column (manufactured by Amersham Pharmacia Biotech). Elution buffer composition: [C solution] 20 mM Tris-HCl, 0.5 M sodium chloride (pH 7.5), 0.01% sodium azide, 0.05% protease inhibitor mixture,
0.05% Tween20 [D solution] 20 mM Tris-HCl, 1 M sodium chloride (pH 7.5), 0.01% sodium azide,
0.05% protease inhibitor mixture, 0.0
5% Tween 20 Flow rate: 1.5 ml / min Temperature: 4 ° C. After passing the fraction collected in 2) above through the column, wash with 60 ml of solution C, then flow 100 ml of solution D, The eluate from was collected.

4) To the eluate obtained in the above 3), an equal amount (100 ml) of the solution A was added. About this, F
Using a PLC device, group-specific affinity chromatography under the conditions described below was performed. Column: 10 ml of Lentil Lectin Sepharose 4B (manufactured by Amersham Pharmacia Biotech) was packed in an XK16 / 40 column. Elution buffer composition: [Solution C] See above [Solution F] 20 mM Tris-HCl, 0.5 M sodium chloride, 0.3 M methyl mannopyranoside (pH 7.
5), 0.01% sodium azide, 0.05% protease inhibitor mixed solution, 0.05% Tween
n20 Flow rate: 1 ml / min Temperature: 4 ° C. After the sample was passed through the column, 50 ml of solution C was washed, followed by 50 ml of solution F, and the eluate from solution F was collected.

The eluate was transferred to a dialysis tube (exclusion limit molecular weight: 10 KDa: manufactured by Gibco BRL),
Dulbecco PBS containing 0.01% sodium azide and 0.1% protease inhibitor mixture
(−) (Manufactured by Nissui Pharmaceutical Co., Ltd.)
Dialysis at 0 ° C. overnight. Thereafter, the solution in the dialysis tube was collected, and 4 ml of the solution contained 1/10 volume (0.4 mg / ml TCA containing 4 mg / ml sodium deoxycholate).
ml), and the resulting precipitate was collected. Subsequently, acetone was added to collect the obtained precipitate, which was dissolved in 50 μl of sterilized ultrapure water.

(3) Determination of N-terminal amino acid sequence PNGaseF (manufactured by New England Biolab) was added to the sample purified in the above (2) to cleave N-linked sugar chains. Specifically, first, 6 μl was added to a 50 μl sample.
10x denaturing buffer (attached to PNGaseF reagent)
Was added and stirred and heated in a boiling water bath for 10 minutes. After returning this to room temperature, 6 μl of 10% Nonidet P-4 was added.
0 and 6 μl of 10 × G7 buffer (PNGa
(attached to the seF reagent) in that order and stirred. To this was added 3 μl of PNGaseF, and the mixture was stirred and kept in a water bath at 37 ° C. for 2 hours or more.

After the N-linked sugar chain cleavage reaction, the reaction solution was subjected to a 4 to 20% concentration gradient acrylamide gel (Multigel 4/20, manufactured by Daiichi Pure Chemicals Co., Ltd.) and a mini-slab electrophoresis apparatus (Nihon Eido) (Manufactured by K.K.) under reducing conditions. After the electrophoresis, using a gel membrane transfer device (Marisol),
The protein separated in the gel was transferred to a polyvinylidene difluoride (PVDF) membrane (manufactured by Bio-Rad) having a pore size of 0.2 μm ( ² mA / cm ² , 2 hours at 4 ° C.). After transfer, the membrane was wetted with 100% methanol (manufactured by Wako Pure Chemical Industries, Ltd.) for 10 seconds, washed with ultrapure water for 2 minutes, and then stained with Coomassie staining solution (Biorad) for 5 minutes. Further, as a result of immersing the membrane in methanol and decoloring for 2 minutes, a band corresponding to about 50 KDa was observed. The membrane of this band was cut out, and the N-terminal sequence was analyzed using a protein sequencer (PPSQ-10: manufactured by Shimadzu Corporation).

As a result, the N of the band of about 50 KDa
The terminal amino acid sequence was Ser-Arg-Ile-Asp-Gln-Asp-Asn-Ser-Ser-Phe-Asp (amino acids 17 to 27 of SEQ ID NO: 4 in the sequence listing). From this, the protein encoded by the nucleotide sequence represented by nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing,
The terminal 16 peptides (amino acid Nos. 1 to 16 of SEQ ID NO: 4 in the Sequence Listing) are cut off, and a serine residue immediately following them (amino acid No. 17 of SEQ ID NO: 4 in the Sequence Listing)
Was found to be secreted as a mature form having N at the N-terminus.

[0175]

As described above, according to the present invention, nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing can be obtained.
SEQ ID NO: 3 in the nucleotide sequence shown in
Is one of the genetic predispositions to increase the triglyceride concentration in blood, and therefore a substance that suppresses the expression level of the gene is hyperlipidemia. It was confirmed that it could be a therapeutic or prophylactic agent for. That is, the method of the present invention, the polynucleotide used as a probe or a primer in the method for detecting a nucleic acid in the method and the antibody used in the method for detecting a polypeptide in the method for treating hyperlipidemia or Useful for searching for prophylactic agents.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of Northern blot analysis on a sample derived from a KK mouse organ.

FIG. 2 is a diagram showing the results of Western blot analysis using a transgenic COS-1 cell culture supernatant as a sample.

FIG. 3 is a diagram showing the results of Western blot analysis using a transgenic HeLa cell culture supernatant as a sample.

FIG. 4. KK / S infected with recombinant adenovirus
The figure showing the measurement result of the neutral fat concentration in the peripheral blood of an mouse.

FIG. 5 shows the results of Northern blot analysis on samples derived from livers of KK mice and KK / San mice infected with recombinant adenovirus.

[Sequence List Free Text] SEQ ID NO: 9: Synthetic oligopeptide used as antigen for obtaining polyclonal antibody

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> Method of Testing Anti-hyperlipidemia Substances <130> 2000178SS <140> <141> <150> JP H11-349976 <151> 1999-12-09 <160 > 11 <170> PatentIn Ver. 2.0 <210> 1 <211> 1604 <212> DNA <213> Mus musculus <220> <221> CDS <222> (47) .. (1411) <400> 1 ggcacgaggt tccaaattgc ttaaaattga ataattgaga caaaaa atg cac aca 55 Met His Thr 1 att aaa tta ttc ctt ttt gtt gtt cct tta gta att gca tcc aga gtg 103 Ile Lys Leu Phe Leu Phe Val Val Pro Leu Val Ile Ala Ser Arg Val 5 10 15 gat cca gac ctt tca tca ttt gat tct gca cct tca gag cca aaa tca 151 Asp Pro Asp Leu Ser Ser Phe Asp Ser Ala Pro Ser Glu Pro Lys Ser 20 25 30 35 aga ttt gct atg ttg gat gat gtc aaa att tta gcg aat ggc ctc ctg 199 Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn Gly Leu Leu 40 45 50 cag ctg ggt cat gga ctt aaa gat ttt gtc cat aag act aag gga caa 247 Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr Lys Gly Gln 55 60 65 att aac gac ata ttt cag aag ctc aac ata ttt gat cag tct ttt tat 295 Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln Ser Phe Tyr 70 75 80 gac cta tca ctt cga acc aat gaa atc aaa gaa gag gaa aag gag cta 343 Asp Leu Ser Leu Arg Thr Asn Glu Ile Lys Glu Glu Glu Lys Glu Leu 85 90 95 aga aga act aca tct aca cta caa gtt aaa aac gag gag gtg aag aac 391 Arg Arg Thr Thr Ser Thr Leu Gln Val Lys Asn Glu Glu Val Lys Asn 100 105 110 115 atg tca gta gaa ctg aac tca aag ctt gag agt ctg ctg gaa gag aag 439 Met Ser Val Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys 120 125 130 aca gcc ctt caa cac aag gtc agg gct ttg gag gag cag cta acc aac Thr Ala Leu Gln His Lys Val Arg Ala Leu Glu Glu Gln Leu Thr Asn 135 140 145 tta att cta agc cca gct ggg gct cag gag cac cca gaa gta aca tca 535 Leu Ile Leu Ser Pro Ala Gly Ala Gln Glu His Pro Glu Val Thr Ser 150 155 160 ctc aaa agt ttt gta gaa cag caa gac aac agc ata aga gaa ctc ctc 583 Leu Lys Ser Phe Val Glu Gln Gln Asp Asn Ser Ile Arg Glu Leu Leu 165 170 175 cag agt gtg gaa gaa cagtat tta a gt caa cag cac atg cag 631 Gln Ser Val Glu Glu Gln Tyr Lys Gln Leu Ser Gln Gln His Met Gln 180 185 190 195 ata aaa gaa ata gaa aag cag ctc aga aag act ggt att caa gaa ccc 679 Ile Lys Glu Ile Glu Lys Gln Leu Arg Lys Thr Gly Ile Gln Glu Pro 200 205 210 tca gaa aat tct ctt tct tct aaa tca aga gca cca aga act act ccc 727 Ser Glu Asn Ser Leu Ser Ser Lys Ser Arg Ala Pro Arg Thr Thr Pro 215 220 225 cct ctt caa ctg aac gaa aca gaa aat aca gaa caa gat gac ctt cct 775 Pro Leu Gln Leu Asn Glu Thr Glu Asn Thr Glu Gln Asp Asp Leu Pro 230 235 240 gcc gac tgc tct gcc gtt tat aac aga ggc gagg cat aca agt gtg 823 Ala Asp Cys Ser Ala Val Tyr Asn Arg Gly Glu His Thr Ser Gly Val 245 250 255 tac act att aaa cca aga aac tcc caa ggg ttt aat gtc tac tgt gat 871 Tyr Thr Ile Lys Pro Arg Asn Ser Gln Gly Phe Asn Val Tyr Cys Asp 260 265 270 275 acc caa tca ggc agt cca tgg aca tta att caa cac cgg aaa gat ggc 919 Thr Gln Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Lys Asp Gly 280 285 290 tca cag gac ttc aac gaaaca tgg gaa aac tac gaa aag ggc ttt ggg 967 Ser Gln Asp Phe Asn Glu Thr Trp Glu Asn Tyr Glu Lys Gly Phe Gly 295 300 305 agg ctc gat gga gaa ttt tgg ttg ggc cta gag aag atc tat gg ata10 Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ala Ile 310 315 320 gtc caa cag tct aac tac att tta cga ctc gag cta caa gac tgg aaa 1063 Val Gln Gln Ser Asn Tyr Ile Leu Arg Leu Glu Leu Gln Asp Trp Lys 325 330 335 gac agc aag cac tac gtt gaa tac tcc ttt cac ctg ggc agt cac gaa 1111 Asp Ser Lys His Tyr Val Glu Tyr Ser Phe His Leu Gly Ser His Glu 340 345 350 355 acc aac tac acg cta cat gtg gct gag att gct ggc aat atc cct ggg 1159 Thr Asn Tyr Thr Leu His Val Ala Glu Ile Ala Gly Asn Ile Pro Gly 360 365 370 gcc ctc cca gag cac aca gac ctg atg ttt tct aca tgg aat cac aga 1207 Ala Leu Pro Glu His Thr Asp Leu Met Phe Ser Thr Trp Asn His Arg 375 380 385 gca aag gga cag ctc tac tgt cca gaa agt tac tca ggt ggc tgg tgg 1255 Ala Lys Gly Gln Leu Tyr Cys Pro Glu Ser Tyr Ser Gly Gly Trp Trp 390 395 400 tgg aa t gac ata tgt gga gaa aac aac cta aat gga aaa tac aac aaa 1303 Trp Asn Asp Ile Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 405 410 415 ccc aga acc aaa tcc aga cca gag aga aga aga ggg atc tac tgg aga 1351 Pro Arg Thr Lys Ser Arg Pro Glu Arg Arg Arg Gly Ile Tyr Trp Arg 420 425 430 435 cct cag agc aga aag ctc tat gct atc aaa tca tcc aaa atg atg ctc 1399 Pro Gln Ser Arg Lys Leu Tyr Ala Ile Lys Ser Ser Lys Met Met Leu 440 445 450 cag ccc acc acc taagaagctt caactgaact gagacaaaat aaaagatcaa 1451 Gln Pro Thr Thr 455 taaattaaat attaaagtcc tcccgatcac tgtagtaatc tggtattaaa attttaatgg 1511 aaagcttgag aattgaattt caattaggtt taaactcatt gttaagatca gatatcaccg 1571 aatcaacgta aacaaaattt atctttttca atc 1604 <210> 2 <211> 455 < 212> PRT <213> Mus musculus <400> 2 Met His Thr Ile Lys Leu Phe Leu Phe Val Val Pro Leu Val Ile Ala 1 5 10 15 Ser Arg Val Asp Pro Asp Leu Ser Ser Phe Asp Ser Ala Pro Ser Glu 20 25 30 Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45 Gly Leu Leu Gln Leu Gl y His Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 70 75 80 Ser Phe Tyr Asp Leu Ser Leu Arg Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95 Lys Glu Leu Arg Arg Thr Thr Ser Thr Leu Gln Val Lys Asn Glu Glu 100 105 110 Val Lys Asn Met Ser Val Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125 Glu Glu Lys Thr Ala Leu Gln His Lys Val Arg Ala Leu Glu Glu Gln 130 135 140 Leu Thr Asn Leu Ile Leu Ser Pro Ala Gly Ala Gln Glu His Pro Glu 145 150 155 160 Val Thr Ser Leu Lys Ser Phe Val Glu Gln Gln Asp Asn Ser Ile Arg 165 170 175 Glu Leu Leu Gln Ser Val Glu Glu Gln Tyr Lys Gln Leu Ser Gln Gln 180 185 190 His Met Gln Ile Lys Glu Ile Glu Lys Gln Leu Arg Lys Thr Gly Ile 195 200 205 Gln Glu Pro Ser Glu Asn Ser Leu Ser Ser Lys Ser Arg Ala Pro Arg 210 215 220 Thr Thr Pro Pro Leu Gln Leu Asn Glu Thr Glu Asn Thr Glu Gln Asp 225 230 235 240 Asp Leu Pro Ala Asp Cys Ser Ala Val Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Val Tyr Thr Ile Lys Pro Arg Asn Ser Gln Gly Phe Asn Val 260 265 270 270 Tyr Cys Asp Thr Gln Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 Lys Asp Gly Ser Gln Asp Phe Asn Glu Thr Trp Glu Asn Tyr Glu Lys 290 295 300 Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 305 310 315 320 Tyr Ala Ile Val Gln Gln Ser Asn Tyr Ile Leu Arg Leu Glu Leu Gln 325 330 335 Asp Trp Lys Asp Ser Lys His Tyr Val Glu Tyr Ser Phe His Leu Gly 340 345 350 Ser His Glu Thr Asn Tyr Thr Leu His Val Ala Glu Ile Ala Gly Asn 355 360 365 Ile Pro Gly Ala Leu Pro Glu His Thr Asp Leu Met Phe Ser Thr Trp 370 375 380 Asn His Arg Ala Lys Gly Gln Leu Tyr Cys Pro Glu Ser Tyr Ser Gly 385 390 395 400 400 Gly Trp Trp Trp Asn Asp Ile Cys Gly Glu Asn Asn Leu Asn Gly Lys 405 410 415 Tyr Asn Lys Pro Arg Thr Lys Ser Arg Pro Glu Arg Arg Arg Gly Ile 420 425 430 Tyr Trp Arg Pro Gln Ser Arg Lys Leu Tyr Ala Ile Lys Ser Ser Lys 435 440 445 Met Met Leu Gln Pro Thr Thr 450 455 <210> 3 <211> 1716 <212> DNA <213> Homo sapiens <220 > <221> CDS <222> (78). . (1457) <400> 3 gcggccgcgt cgacgtctag gtctgcttcc agaagaaaac agttccacgt tgcttgaaat 60 tgaaaatcaa gataaaa atg ttc aca att aag ctc ctt ctt ttt att gtt 110 Met Phe Thr Ile Lys Leu Let Lec Lec Lec Lec Lecc Lec att gat caa gac aat tca tca ttt gat 158 Pro Leu Val Ile Ser Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp 15 20 25 tct cta tct cca gag cca aaa tca aga ttt gct atg tta gac gat gta 206 Ser Leu Ser Pro Glu Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val 30 35 40 aaa att tta gcc aat ggc ctc ctt cag ttg gga cat ggt ctt aaa gac 254 Lys Ile Leu Ala Asn Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp 45 50 55 ttt gtc cat aag acg aag ggc caa att aat gac ata ttt caa aaa ctc 302 Phe Val His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu 60 65 70 75 aac ata ttt gat cag tct ttt tat gat cta tcg ctg caa acc agt gaa 350 Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu 80 85 90 atc aaa gaa gaa gaa aag gaa ctg aga aga act aca tat aaa cta caa 398 Ile Lys Glu Glu Glu Glu L ys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln 95 100 105 gtc aaa aat gaa gag gta aag aat atg tca ctt gaa ctc aac tca aaa 446 Val Lys Asn Glu Glu Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys 110 115 120 ctt gaa agc ctc cta gaa gaa aaa att cta ctt caa caa aaa gtg aaa 494 Leu Glu Ser Leu Leu Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys 125 130 135 tat tta gaa gag caa cta act aac tta att caa atat gaa act 542 Tyr Leu Glu Glu Gln Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr 140 145 150 155 cca gaa cac cca gaa gta act tca ctt aaa act ttt gta gaa aaa caa 590 Pro Glu His Pro Glu Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln 160 165 170 gat aat agc atc aaa gac ctt ctc cag acc gtg gaa gac caa tat aaa 638 Asp Asn Ser Ile Lys Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys 175 180 185 caa tta aac caa cag cat agt caa ata aaa gaa ata gaa aat cag ctc 686 Gln Leu Asn Gln Gln His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu 190 195 200 aga agg act agt att caa gaa ccc aca gaa att tct cta tct tcc aag 734 Arg T hr Ser Ile Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys 205 210 215 cca aga gca cca aga act act ccc ttt ctt cag ttg aat gaa ata aga 782 Pro Arg Ala Pro Arg Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg 220 225 230 235 aat gta aaa cat gat ggc att cct gct gaa tgt acc acc att tat aac 830 Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn 240 245 250 aga ggt gaa cat aca agt ggc atg tat gcc atc aga ccc agc aac tct 878 Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser 255 260 265 caa gtt ttt cat gtc tac tgt gat gtt ata tca ggt agt cca tgg aca 926 Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr 270 275 280 tta att caa cat cga ata gat gga tca caa aac ttc aat gaa acg tgg 974 Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp 285 290 295 295 gag aac tac aaa tat ggt ttt ggg agg ctt gat gga gaa ttt tgg ttg 1022 Glu Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu 300 305 310 315 ggc cta gag aag ata tac tcc ata gtg aag caa tct agtta 1070 Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu 320 325 330 cga att gag ttg gaa gac tgg aaa gac aac aaa cat tat att gaa tat 1118 Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr 335 340 345 tct ttt tac ttg gga aat cac gaa acc aac tat acg cta cat cta gtt 1166 Ser Phe Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu His Leu Val 350 355 360 gcg att act ggc aat gtc ccc aat gca atc ccg gaa aac aaa gat ttg 1214 Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu 365 370 375 gtg ttt tct act tgg gat cac aaa gca aaa gga cac ttc aac tgt cca 1262 Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His Phe Asn Cys Pro 380 385 390 395 gag ggt tat tca gga ggc tgg tgg tgg cat gat gag tgt gga gaa aac 1310 Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn 400 405 410 aac cta aat ggt aaa tat aac aaa cca aga gca aaa tct aag cca gag 1358 Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu 415 420 425 agg aga aga gaga tta tct tgg aag tct caa a at gga agg tta tac tct 1406 Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser 430 435 440 ata aaa tca acc aaa atg ttg atc cat cca aca gat tca gaa agc ttt 1454 Ile Lys Ser Thr Lys Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe 445 450 455 gaa tgaactgagg caaatttaaa aggcaataat ttaaacatta acctcattcc 1507 Glu 460 aagttaatgt ggtctaataa tctggtatta aatccttaag agaaagcttg agaaatagat 1567 tttttttatc ttaaagtcac tgtctattta agattaaaca tacaatcaca taaccttaaa 1627 gaataccgtt tacatttctc aatcaaaatt cttataatac tatttgtttt aaattttgtg 1687 atgtgggaat caattttaga tggtcacaa 1716 <210> 4 <211> 460 <212> PRT <213> Homo sapiens <400> 4 Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 10 15 Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30 Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45 Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 70 75 80 Ser P he Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu 85 90 95 Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu 100 105 110 Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125 Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140 Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu 145 150 155 160 Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys 165 170 175 Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln 180 185 190 His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile 195 200 205 Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg 210 215 220 Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp 225 230 235 240 Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 260 265 270 Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300 Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 305 310 315 320 Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 325 330 335 Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 340 345 350 Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn 355 360 365 Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp 370 375 380 Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 385 390 395 400 Gly Trp Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 405 410 415 Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu 420 425 430 Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 435 440 445 Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 450 455 460 <210> 5 <211> 23 <212> DNA <213> Mus musculus <400> 5 gactgatcaa atatgttgag ctt 23 <210> 6 <211> 21 <212> DNA <213> Mus musculus <400> 6 tgcatccaga gtggatccag a 21 <210> 7 <211> 21 <2 12> DNA <213> Homo sapiens <400> 7 tcctctagtt atttcctcca g 21 <210> 8 <211> 20 <212> DNA <213> Homo sapiens <400> 8 tggtttgcca gcgatagatc 20 <210> 9 <211> 14 <212 > PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligopeptide which is used as an antigen to obtain polyclonal antibody <400> 9 Glu Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Cys 15 10 <210> 10 <211> 14 <212> PRT <213> Mus musculus <400> 10 Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg 1 5 10 <210> 11 <211> 199 <212> DNA <213> Mus musculus <400> 11 ttgcatccag agtggatcca gacctttcat catttgattc tgcaccttca gagccaaaat 60 caagatttgc tatgttggat gatgtcaaaa ttttagcgaa tggcctcctg cagctgggtc catag tagca tca tca tca tca c gag catag tagca tca tca tca c gag

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/06 G01N 33/53 D C12Q 1/68 C12N 15/00 ZNAA G01N 33/53 5/00 E ( 72) Inventor Hidehiko Furukawa 1-58, Hiromachi, Shinagawa-ku, Tokyo, Japan Inside Sankyo Co., Ltd. (72) Inventor Toshihiko Fujiwara 1-2-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. 72) Inventor Kogetsu Daino Sankyo Co., Ltd. 1-58 Hiromachi, Shinagawa-ku, Tokyo

Claims (24)

[Claims] 1. A method for testing the effect of a substance as an agent for treating or preventing hyperlipidemia, comprising the following steps: 1) culturing cultured cells in the presence or absence of a test substance 2) the following a) in the cultured cells obtained in 1) above:
MRNA having the nucleotide sequence according to any one of (a) to (e), wherein t in the sequence is replaced with u.
A) Nucleotide number 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. A nucleotide sequence encoding a polypeptide having an activity of increasing soybean and increasing the concentration of neutral fat in the blood; and 3) cells cultured in the absence of the test substance as a result of the above step 2); Comparing the expression level of the detected mRNA with cells cultured below. 2. The method according to claim 1, wherein the cultured cells are derived from the liver. 3. The method according to claim 1, wherein the cultured cells are derived from a primate or a rodent. 4. The method according to claim 3, wherein the cultured cells are derived from human or mouse. 5. The method according to claim 1, wherein the method for detecting the expression level of mRNA is Northern blot, dot blot or slot blot. 6. The method according to claim 1, wherein the method for detecting the expression level of mRNA is RT.
-A method characterized by being PCR. 7. The method according to claim 1, wherein the method for detecting the expression level of mRNA is a ribonuclease protection assay. 8. The method according to claim 1, wherein the method for detecting the expression level of mRNA is a run-on assay. 9. It has a nucleotide sequence that hybridizes under stringent conditions with an mRNA containing a nucleotide sequence according to any one of the following a) to d) (where t in the sequence is replaced by u): Polynucleotides (excluding those containing the nucleotide sequences described in a) to d) below: a) Nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
The nucleotide sequence of the DNA inserted into the phagemid retained in 1). 10. A DNA comprising at least 15 nucleotides in which one or both ends of the nucleotide sequence shown in nucleotide numbers 47 to 1411 of SEQ ID NO: 1 in the sequence listing have been deleted by one or more nucleotides. 11. A DNA consisting of at least 15 nucleotides in which one or both ends of one or both nucleotides of nucleotides shown in nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing have been deleted. 12. A method for testing the effect of a substance as a therapeutic or preventive agent for hyperlipidemia, comprising the following steps: 1) culturing cultured cells in the presence or absence of a test substance. 2) an amount of a polypeptide comprising the amino acid sequence encoded by the nucleotide sequence according to any one of the following a) to e) or a part thereof in the cultured cell supernatant obtained in 1) above: Is detected using an antibody that specifically recognizes the polypeptide: a) nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing.
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. A nucleotide sequence encoding a polypeptide having an activity of increasing soybean and increasing the concentration of neutral fat in the blood; and 3) cells cultured in the absence of the test substance as a result of the above step 2); Comparing the amount of polypeptide detected with cells cultured underneath. 13. The method according to claim 12, wherein
An antibody that specifically recognizes an amino acid sequence encoded by the nucleotide sequence described in any one of (a) to (e) or a polypeptide consisting of a part thereof is identified by amino acids 17 to 455 of SEQ ID NO: 2 in the sequence listing. A polypeptide consisting of the amino acid sequence shown in, or a part thereof,
5. A polypeptide consisting of the amino acid sequence shown in 5, or a part thereof, or amino acids 17-46 of SEQ ID NO.
0. A method comprising the step of specifically recognizing a polypeptide consisting of the amino acid sequence shown in No. 0 or a part thereof. 14. The method according to claim 12 or 13, wherein the polypeptide comprising the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof is specifically recognized. The amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing
A method for specifically recognizing the amino acid sequence shown in (1). 15. The method according to any one of claims 12 to 14, wherein the polypeptide comprises the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof. An operation of detecting the amount of production using an antibody that specifically recognizes the polypeptide,
A method characterized by being a western blot, a dot blot or a slot blot. 16. The method according to claim 12, wherein the polypeptide comprises the amino acid sequence encoded by the nucleotide sequence according to any one of a) to e) or a part thereof. An operation of detecting the amount of production using an antibody that specifically recognizes the polypeptide,
A method characterized by being an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA). 17. An antibody that specifically recognizes a polypeptide consisting of an amino acid sequence encoded by the nucleotide sequence according to any one of the following a) to e) or a part thereof: a) a sequence in the sequence listing Nucleotide number 47 to 1 of number 1
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. A nucleotide sequence that encodes a polypeptide that has activity to soy and increase triglyceride levels in the blood. 18. The antibody according to claim 17, which is specific for an amino acid sequence represented by amino acid numbers 1 to 13 of SEQ ID NO: 9 or an amino acid sequence represented by amino acid numbers 1 to 14 of SEQ ID NO: 10 in the sequence listing. An antibody characterized by the fact that it is specifically recognized. 19. A kit for testing a therapeutic or prophylactic agent for hyperlipidemia, comprising the antibody according to claim 17 or 18. 20. A method for testing the effect of a substance as a therapeutic or preventive agent for hyperlipidemia, comprising the following steps: 1) Obtained by genetic manipulation, and any one of the following a) to e) Nucleotide sequence according to one of the following: a) nucleotide numbers 47 to 1 of SEQ ID NO: 1 in the sequence listing
411; a nucleotide sequence represented by SEQ ID NO: 3;
457; c) a transformed E. coli E. coli. coli pBK / m55-1
SANK 72199 (FERM BP-6940)
A) a nucleotide sequence contained in the DNA inserted into the phagemid carried by d. coli pTrip / h55
-1 SANK 72299 (FERM BP-694
1) a nucleotide sequence of the DNA inserted into the phagemid retained in 1); e) hybridizing with a polynucleotide comprising an antisense sequence of the nucleotide sequence described in any one of a) to d) above under stringent conditions. Tested on a non-human animal into which a foreign gene containing a nucleotide sequence encoding a polypeptide having activity to increase soybean triglyceride concentration in blood has been introduced so that the gene can be highly expressed. Administering the substance; and 2) measuring the neutral fat concentration in the blood of the animal of 1). 21. The method according to claim 20, wherein
The method according to 1), wherein the non-human animal is a mouse. 22. The method according to claim 20, wherein
(1) A method according to (1), wherein the introduction of the foreign gene into the non-human animal according to (1) is due to infection of the animal with an adenovirus containing an adenovirus vector into which the gene has been incorporated. 23. A DNA or RNA comprising an antisense sequence of a nucleotide sequence consisting of 15 to 30 contiguous nucleotides in the nucleotide sequence of nucleotide numbers 78 to 1457 of SEQ ID NO: 3 in the sequence listing. 24. The DNA or RN according to claim 23.
A therapeutic agent for hyperlipidemia containing A as an active ingredient.