JP2001072607A - New capillary endothelial function improvement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicinal composition that can ameliorate the morbid states of capillary endothelial function, hypertension and arteriosclerosis in an animal model onset the diseases accompanied by deterioration in capillary endothelial function by incorporating the recombinant vector incorporating the klotho gene. SOLUTION: The objective medicinal composition includes an effective amount of a recombinant vector incorporating the cDNA of the klotho gene. In a preferred embodiment, the cDNA of the klotho gene is a DNA having the base sequence selected from the base sequence of the formula or the like or another DNA that can hybridize with the DNA under the stringent conditions. The recombinant vector is preferably prepared by incorporating the cDNA of the klotho gene into a vector selected from-plasmid vectors or a virus vectors. The resultant medicinal composition is useful for amelioration or prophylaxis of the morbid states caused by the morbid states of capillary endothelial function (for example, hypertension and arteriosclerosis in an animal model onset the diseases accompanied by deterioration in capillary endothelial function.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for improving a disease state caused by deterioration of vascular endothelial function or preventing the state from falling into the state, a pharmaceutical composition, and a recombinant vector used for them. More specifically, a gene DN for improving vascular endothelial function represented by the closo gene
A method for ameliorating (treating) the disease state or preventing the disease state from being administered by administering an expression vector containing A to a mammal having the disease state or having the possibility of becoming the disease state, and a pharmaceutical composition used in the method. And recombinant vectors used for them.

[0002]

2. Description of the Related Art Closo gene is a transgenic mouse exhibiting remarkable and various early aging symptoms such as shortening of life span, calcification of various organs, arteriosclerosis, atrophy of reproductive organs and the like.
(Croso mouse) is a gene that is present in the foreign gene insertion site and has been identified as a causative gene that causes the aforementioned aging symptoms in mice when its expression decreases. Up to three weeks after birth, Kuroso mice grow like wild-type mice, but then stop growing and show various signs of aging. At the age of 6 weeks, the activity of the closo mouse decreased to about 50% of that of the wild type, and Parkinson's disease-like walking was observed. The gonads are atrophied and infertile. Osteoporosis is remarkable, and ectopic calcification occurs in the aortic valve, bronchi, and choroid plexus of the brain. In the arterial system, there is a Menkeberg-type arteriosclerotic finding characteristic of aging, such as intimal thickening of the blood vessel and calcification of the media. In addition, atrophy of the skin and emphysema are also observed [WO 98/29544, Nature, 390 , 45 (199
7)]. From the analysis of the cDNA of the Closo gene, two types of mRNA were transcribed due to differences in splicing of the Closo gene,
Two types of proteins are translated from the mRNA (hereinafter, the proteins encoded by the closo gene, including these proteins, are referred to as closoproteins).

[0003] Of the above two types of mRNA, one of the closoproteins is a type 1 membrane protein having a structure having an N-terminal signal sequence region, an extracellular domain region and a C-terminal transmembrane domain region ( The extracellular domain has two domains (KL which are homologous to bacterial or plant β-glucosidase).
1, KL2). On the other hand, the other protein is a secretory protein having an N-terminal signal sequence region and a KL1 domain region (hereinafter, referred to as secretory closoprotein).
(Biochem. Biophys. Res.
Commun., 242 , 626 (1998)].

[0004] The mechanism by which the mutation of the Closo gene causes various aging symptoms and the molecular function of the Closo protein have not yet been elucidated. Although the expression of the closo gene is high in the kidney, severe aging symptoms affect systemic organs including the lung, bone, stomach wall, skin and the like. From this, it has been presumed that some secretory factor exists in the molecular function and exerts an effect [Nature, 390 , 45 (1997)].

[0005] The aging of Closo mice has been demonstrated by crossing between Closo mice and Closo gene-expressing transgenic mice, and experiments in which Closo proteins are expressed in Closo mice using an adenovirus vector containing a cDNA of the membrane-bound Closo gene. Has been achieved, it has been suggested that treatment of a disease derived from dysfunction of closoprotein can be achieved by enhancing the closogene using some means [Nature, 39
0 , 45 (1997), WO 98/29544]. Vascular endothelial cells not only regulate vascular tone by releasing endothelium-dependent vasorelaxant and endothelium-dependent vasoconstrictor, but are also deeply involved in intimal permeability and platelet aggregation, It plays an important role in the development of thrombotic arteriovenous disease. Examples of the vascular endothelium-dependent vasorelaxant include nitric oxide (NO), prostaglandin I ₂ , C-type natriuretic peptide, adrenomedullin, and vascular endothelium-derived hyperpolarizing factor. On the other hand, thromboxane A ₂ , angiotensin II, endothelin, prostaglandin H _{2 and the} like are known as endothelium-dependent vasoconstrictor factors.

[0006] When acetylcholine is applied to an extirpated specimen of a rabbit artery, NO specifically causes a relaxation reaction only in a specimen retaining endothelial cells [Nature, 288 : 373].
(1980)] as one of the vascular endothelium-derived vasorelaxant (EDRF). NO is NO present in the vascular endothelium
Synthase (NOS) is released by oxidizing L-arginine. NO reaches adjacent vascular smooth muscle cells, activating its guanylate cyclase, increasing cGMP and leading to vasorelaxation. NOS includes neural NOS (nNOS),
Endothelial NOS (eNOS) and inducible NOS (iN
The existence of three isoforms of OS) has been demonstrated. Among them, the endothelial NOS-deficient mouse lacks an endothelial-dependent vasodilator response and has a 20 mmHg higher blood pressure than a healthy mouse, and therefore has a NO-dependent vasoconstriction regulation mechanism essential for regulation of blood flow and blood pressure. Has been shown to exist [Nature, 377 , 239 (1995)].

As described above, there is a close relationship between the vascular endothelial function and the amount of NO produced by endothelial cells, and it is a clinically important problem to measure the ability to produce NO. But N
O is present in a very small amount and is unstable.
Attempts to directly measure have not been applied to the clinical field. As a result, the kinetics of NO is generally estimated by measuring the function of vascular endothelium. The function of the vascular endothelium is generally performed by examining an endothelium-dependent vasodilatory reaction [Ayumi of Medicine 189 , 517 (1999)]. This involves administering an endothelium-dependent vasodilator such as acetylcholine to the arteries, and controlling the endothelial N
This is a method for evaluating O secretion ability. When the vascular endothelial function is measured using the vascular endothelium-dependent relaxation response to acetylcholine as an index in the Closo mouse, the maximal vasodilator response is lower in the artery of the Closo mouse than in the normal mouse with high expression of the Closo gene. In addition, the Closo heterozygous mouse, in which the expression of the Closo gene is reduced by about half compared to the normal mouse, has a decreased maximal vasodilator response, although not as much as the Closo mouse, indicating that the expression amount of the Closo gene and the function of vascular endothelium are reduced. Is suggested to be correlated. It has been shown that impaired vascular function in Closo mice is related to impaired NO production in vascular endothelial cells [BBRC, 248 , 324 (1998)]. Indeed, when normal and closohetero mice are surgically combined and fluid exchange between them is performed, the function of the vascular endothelium of the closohetero mouse is restored to the same level as the normal mouse (BBRC, 248 , 324 (1998))
Therefore, it is expected that vascular endothelial function can be improved by enhancing the closoprotein. However, there has been no report that in a disease in which vascular endothelial function has been reduced, for example, in a disease such as arteriosclerosis or hypertension, treatment or prevention of such a disease has been performed by externally administering a closogene or a closoprotein.

[0008]

The etiology of pathologies such as hypertension and arteriosclerosis is complicated, and therapeutic and prophylactic agents based on new mechanisms of action are selected so that treatments can be selected according to the patient's condition and complications. Development is desired.

[0009]

Means for Solving the Problems By administering a recombinant vector incorporating a closogene cDNA to an animal model which has developed diseases such as hypertension or arteriosclerosis accompanied by a decrease in vascular endothelial function, these diseases are obtained. The present inventors have found that the disease state of vascular endothelial function, hypertension and arteriosclerosis of a model animal can be improved, and have completed the present invention.

The present invention relates to the following (1) to (10). (1) A pharmaceutical composition for ameliorating or preventing a disease state caused by deterioration of vascular endothelial function in a mammal, which comprises an effective amount of a recombinant vector into which the closo gene cDNA has been incorporated. (2) The pharmaceutical composition according to (1), wherein the cDNA of the closo gene is the senescence-suppressing gene cDNA described in WO98 / 29544. (3) Closo gene cDNA has SEQ ID NO: 1, 2, 3, 4
2. The pharmaceutical composition according to 1, which is a DNA having a base sequence selected from the base sequences represented by and 5 or a DNA that hybridizes with the DNA under stringent conditions. (4) The pharmaceutical composition according to (1), wherein the disease state due to vascular endothelial function deterioration is a disease selected from the group consisting of hypertension, arteriosclerosis, hypercholesterolemia, diabetes, myocardial infarction and cerebral infarction. (5) The pharmaceutical composition according to (1), wherein the recombinant vector is obtained by incorporating the closo gene cDNA into a vector selected from a plasmid vector and a virus vector. (6) the virus vector is a retrovirus vector,
2. The pharmaceutical composition according to claim 1, which is selected from an adenovirus vector, an adeno-associated virus vector and a herpes virus vector. (7) A recombinant vector of a Closo gene obtained by incorporating a cDNA of the Closo gene into a viral vector. (8) the virus vector is a retrovirus vector,
8. The Closo gene recombinant vector according to 7, which is selected from an adenovirus vector, an adeno-associated virus vector and a herpes virus vector. (9) by administering an effective amount of the pharmaceutical composition according to any one of Items 1 to 6 to a mammal having a disease state due to vascular endothelial function deterioration or a mammal likely to fall into the disease state, A method for treating or preventing the above condition. (10) The method according to (9), wherein the pathological condition due to vascular endothelial function deterioration is a disease selected from the group consisting of hypertension, arteriosclerosis, hypercholesterolemia, diabetes, myocardial infarction and cerebral infarction.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention,
A pharmaceutical composition for improving or preventing vascular endothelial function, such as hypertension or arteriosclerosis, comprising a pathological condition due to vascular endothelial function deterioration, comprising a disease state due to vascular endothelial function deterioration in a mammal, comprising an effective amount of a recombinant vector incorporating DNA. By administering to a mammal or a mammal that may fall into the disease state, the disease state can be treated or prevented.

[0012] The recombinant vector is a cloned cDNA
Can be obtained by incorporating it into a vector. A plasmid vector or a virus vector (virus vector) can be used as a vector used for the production of the recombinant vector. However, a virus vector is preferably used in order to introduce the closogene cDNA into a mammal and efficiently express it. . Viral vectors include Adenoviridae, Retroviridae, Parvoviridae, Herpesviridae, Poxviridae,
Any of the group consisting of Papovaviridae, Hepadnaviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabdoviridae, Paramyxoviridae, Orthomyxoviridae, Banyaviridae, Arenaviridae, and Reoviridae And viruses derived from these viruses, and adenovirus dodecahedron vectors (Fender et al., Nat.
ure Biotech 15:. 52 (1997 ) derived vector from the viral proteins such as] a vector combining viral proteins to the liposome (e.g., Sendai virus and liposomes vectors, etc.), and the like, human adenoviruses preferably Used.

For example, a specific example of a method for improving the disease state of a patient having the disease state by using an adenovirus as a vector is described below. (i) Construction of Recombinant Cosmid Containing Closo Gene cDNA
A, and a poly-A addition signal, further comprising E1A,
Adenovirus type 5 genome D lacking E1B and E3
Preferably, it contains NA.

PAxCAwt [Nucl. Acids Res., 23 , 3816 (19)
95)]. As the Closo gene cDNA, any DNA described in WO98 / 29544 can be used, but it is not limited thereto. Specific examples include a DNA having a base sequence selected from the base sequences represented by SEQ ID NOs: 1, 2, 3, 4, and 5, or a DNA that hybridizes with the DNA under stringent conditions. .

Here, the DNA that can hybridize under stringent conditions refers to a DNA having the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 5 as a probe by colony hybridization, plaque hybridization,
D obtained by using a hybridization method or a Southern blot hybridization method
NA, specifically, 0.7% using a filter on which DNA derived from colonies or plaques was immobilized.
After hybridization at 65 ° C. in the presence of ~ 1.0 M sodium chloride, 0.1- to 2-fold concentration of SSC
DNA that can be identified by washing the filter with a solution (the composition of a 1-fold concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate) at 65 ° C. can be mentioned. Hybridization is performed using Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practica.
l Approach, Second Edition, Oxford University (199
It can be performed according to the method described in 5).
Specifically, as a hybridizable DNA, FAS
A DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NOs: 1, 2, 3, 4 and 5 when calculated using T, preferably a DNA having 80% or more homology; More preferably, a DNA having 95% or more homology can be mentioned.

Unless otherwise specified, phage, plasmids, DNA, various enzymes, Escherichia coli, cultured cells, and other operations are performed unless otherwise specified.
nual, T. Maniatis et al., 2nd edition (1989), Cold Spri
ng Harbor Laboratory (hereinafter abbreviated as Molecular Cloning 2nd Edition). By a conventional method, the cosmid appropriate restriction enzyme, and cleaved for example Swa I like to prepare a recombinant cosmid by connecting the closo gene cDNA. (ii) Preparation of Recombinant Adenovirus Containing Closo Gene cDNA Recombinant adenovirus can be prepared, for example, by the method of Miyake et al. [Proc. Natl. Acad. Sci. USA, 93, 1320 (1996)]. it can.

Specifically, the closo gene c prepared in (i)
Recombinant cosmid containing DNA and adenovirus type 5 deficient in E3, E1A, E1B deficient Ad5dIX DNA [e.g., Eco T22I] [J. Virology, 54 , 711 (19
85)], and use the calcium phosphate method or the lipofection method [Japanese Patent Application Laid-Open No. 2-227075, Experimental Medicine Separate Volume New Genetic Engineering Handbook, Yodosha (19996)], for example.
It is introduced into a cell line containing the 1A and E1B genes, for example, 293 cells derived from human fetus [J. Gen. Virol., 36 , 59 (1977)]. If recombination of cosmid and adenovirus DNA occurs in cells, recombinant adenovirus is produced and the cells are killed. Therefore, the production of the recombinant adenovirus containing the closogene cDNA using the death of the cells as an index. Confirm. The dead cells are collected, and the cells are disrupted, for example, by repeated freezing and thawing or by using a cell disrupter, to obtain a recombinant adenovirus solution containing the closogene cDNA.

The DNA of the recombinant adenovirus is extracted from the resulting solution by a conventional method, and a restriction enzyme such as XhoI is used.
The structure is confirmed by cutting with. (iii) Purification of Recombinant Adenovirus The obtained recombinant adenovirus DNA can be obtained, for example, by the method of Kanegae et al. [Jpn. J. Med. Sci. Biol., 47 , 157 (199
4)], purify by cesium chloride density gradient twice, suspend in a solution such as PBS containing 10% glycerol, HEPES-MgCl ₂ containing 10% glycerol, and HEPES-EDTA containing 10% glycerol, 80
It can be stored at ℃ and used as appropriate. (iv) administration to a disease state model of a recombinant adenovirus containing the closogene cDNA to remove glycerol by dialysis of the recombinant virus solution expressing the closogene cDNA obtained in (iii),
Recombinant virus is obtained by diluting to an appropriate amount with physiological saline. Otsuka Long showing obesity, hyperinsulinemia, hypertension and hyperlipidemia, and a condition similar to obese human NIDDM (non-insulin dependent diabetes mellitus)
-Evans Tokushima Fatty (OLE
TF) Rats [Diabetes, 41 , 1422 (1992)] (30 weeks old) were intramuscularly injected with 5 × 10 ⁸ pfu of the recombinant virus once a week for 3 weeks. Check for improvements. The above shows a specific example of the method of the present invention using an adenovirus, but any virus other than adenovirus can be used as long as the object of the present invention can be achieved.

The preparation of a recombinant virus vector using another virus vector is performed by using a general recombinant DNA preparation technique (see Molecular Cloning, 2nd Edition, etc.) for DNA encoding the proteins constituting the virus. This can be done by replacing the coding region of the relevant viral protein to encode the protein. Preparation of cosmids, extraction of DNA, and other gene recombination techniques can be performed, for example, by the methods described in the following documents.

Wolff ed., Gene therapeutics: Methods
and applications of direct genetransfer.Birkhaeus
er, Boston, 1994; Kaplitt and Loewy eds., Viral v
ectors: Gene therapy and neuroscience application
s. Academic Press, San Diego, 1995; Liu et al. ed
s., DNA vaccines: A new era in vaccinology. Annals
of the New York Academy of Sciences vol. 772. The
New York Academy of Sciences, New York, 1995; Glu
zman and Hughes eds., Viral vectors: Current commu
nications in molecular biology Cold Spring Harbor
Laboratory, New York, 1988; Roth ed., Methods in
cell biology. vol. 43. Protein expression in anim
al cells. Academic Press, San Diego 1994. A pharmaceutical composition containing a recombinant vector can be used alone with the vector, but is usually prepared by combining the vector with one or more pharmacologically acceptable carriers. Desirably, they are mixed together and provided as a pharmaceutical formulation prepared by any of the methods used in the pharmaceutical arts. Preferably, a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, glucose, human albumin or the like is used. Also, pharmacologically acceptable additives such as buffering agents and tonicity agents for bringing the formulation solution closer to physiological conditions, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid Sodium and the like can be added. Alternatively, it can be stored after being freeze-dried and dissolved in an appropriate solvent before use.

The administration route is preferably the one that is most effective in the treatment, and is preferably administered orally or intraorally.
Parenteral administration, such as in the respiratory tract, rectally, subcutaneously, intramuscularly and intravenously, can be mentioned. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as, for example, emulsions and syrups include water, sucrose, sorbitol, sugars such as fructose, polyethylene glycol,
It can be produced using glycols such as propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, starch,
Disintegrators such as sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin are used as additives. Can be manufactured.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, injections
It is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Also,
Sprays are prepared using the vector itself or a carrier which does not irritate the oral and respiratory mucosa of the recipient and which disperses the vector as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the vector and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added. The dose or frequency of administration depends on the intended therapeutic effect,
Depending on the age, body weight, type of virus vector, etc., it is usually 10 ³ -1
0 ¹⁵ are administered. In the present invention, the closo gene c
Diseases can also be treated by administering DNA to patients using gene therapy methods. Using the above method, the effect of treating a disease condition is a decrease in blood pressure,
Evaluation is based on improvement of vascular endothelial function. A general method for measuring the function of vascular endothelium includes a method for examining endothelium-dependent vasodilatory response. This is a method of administering an endothelium-dependent vasodilator such as acetylcholine to an artery and evaluating the endothelial NO secretion ability based on the degree of vasodilation at that time [Ayumi of Medicine 189 , 517 (199)
9)].

Hereinafter, using a human type 5 adenovirus (Ad5) as a virus vector and a mouse-derived closogene cDNA as a gene to be integrated, obesity, hyperinsulinemia, hypertension and hyperlipidemia occur, and obesity L exhibiting a pathology similar to that of human NIDDM
ong-Evans Tokushima Fatty
(OLETF) The present invention will be specifically described using examples in which the vascular endothelial function of rats has been improved, but the present invention is not limited to these examples.

[0024]

EXAMPLES Example 1: Improvement of vascular function by administration of recombinant adenovirus containing the closo gene cDNA. (Step 1) The production of a recombinant adenovirus containing the mouse-derived closogene cDNA was basically performed by the method of Miyake et al.
c. Natl Acad. Sci. USA., 93 , 1320 (1996)]. Specifically, plasmid pNKM101 (FERM BP-576) containing mouse-derived closo gene cDNA
5, WO98 / 29544) by Not I and Xba I
And the obtained 3.1 kb fragment containing the gene was DN
Both ends were blunted with A Blunting kit (Takara Shuzo).

Cosmid pAxCAwt containing 3 μg of the fragment and a type 5 adenovirus genome lacking the E3, E1A, and E1B regions, a cytomegalovirus enhancer, and a chimeric promoter of the chicken β-actin promoter (CAG promoter) [Kanegae et al., Nucl. Acids Res. .,
23, T4DN and Swa I fragment 1μg of 3816 (1995)]
A ligase buffer was dissolved in 20 μl, and T4DN was added to the solution.
One unit of A ligase was added, and the binding reaction was performed at 16 ° C. for 18 hours.

The ligase reaction solution and Gigapack II XL Pac
In vitro packaging was performed using a kaging Extract (manufactured by Stratagene), and the obtained phage was transformed into E. coli DH5α [J. Bacteriology, 170 , 611].
(1988)] to obtain a recombinant cosmid. In addition, mouse-derived closo gene cDN in recombinant cosmid
Check the direction of to the promoter of A is, the cosmid B
After digestion with am HI, detection was performed by detecting a 1.6 kb fragment.

With 8 μg of the cosmid thus obtained,
E3, E1A, E1B cleaved by Eco T22I
Type 5 adenovirus Ad5dIX DNA [J. Virol
ogy., 54 , 711 (1985)].
Using a Transfection Kit (Pharmacia Biotech), transfection was performed on human fetal-derived 293 cells by the calcium phosphate method. Thereafter, a recombinant virus was obtained according to the method of Kanegae et al. [Bio Manual Series, 4 , 43-58, Yodosha (1994)].

The recombinant virus was obtained by the method of Kanegae et al. [Jp.
n. J. Med. Sci. Biol., 47 , 157 (1994)], twice purified by cesium chloride density gradient, suspended in PBS containing 10% glycerol, stored at -80 ° C and used as appropriate. . The virus titer of the recombinant virus solution was calculated according to the method of Kanegae et al. [Jpn. J. Med. Sci. Biol., 47 , 157 (1994)] and found to contain 1 × 10 ⁹ pfu / ml of virus. I understood.

(Step 2) The recombinant virus solution obtained in step 1 is dialyzed to remove glycerol, and diluted with physiological saline to obtain a recombinant virus expressing mouse full-length closo. Was. Otsuka Long-Eva which causes hypertension and hyperlipidemia with obesity and hyperinsulinemia, and shows a condition similar to obese human NIDDM
ns Tokushima Fatty (OLETF) rat [Diabetes, 41 , 1422 (1992)] (30 weeks old) was intramuscularly injected with 5 × 10 ⁸ pfu of recombinant virus 3 times a week.
Administered for a week. As a result, the blood pressure of the rat was 156.3 ± 4 mmHg (n = 8) in the non-administration group, but was improved to 139.4 ± 7 mmHg (n = 5) in the administration group.

(Step 3) Sodium pentobarbital (50 mg / kg) was added to the OLETF rat obtained in step 2
Is administered intraperitoneally, and after anesthesia, the chest is incised to remove the thoracic aorta (3 mm). The isolated thoracic aorta (aortic ring) was placed between two steel wires and
0 ml of Krebs' bicarbonate solution [120 mM NaCl, 5.2 mM KCl, 2.4 m
MCaCl ₂ , 1.2 mM MgSO ₄ .7H ₂ O, 25
mM NaHCO ₃ , 0.03 mM Na ₂ -EDTA, 1
1 mM dextrose (pH 7.4)]. The solution was saturated with oxygen through bubbles of 95% oxygen, 5% carbon dioxide. One end of the wire is fixed and the other end is Biochem. Biophys. Res. Commun., 248 , 324
(1998) and connected to a mechanical transducer. A 2.0 g weight is added to the aortic ring, tension is applied, the mixture is left for 90 minutes, and then 0.1 μM norepinephrine is added to contract. Endothelial function was added to this solution at a final concentration of 10 μM.
When acetylcholine was added, the percentage of the contracted length relaxed and recovered. (OLETF) Aorta prepared by administering once a week to a 30-week-old rat an adenovirus expressing full-length mouse closo-encoded by DNA having the nucleotide sequence of SEQ ID NO: 3 for 3 weeks The relaxation rate of the ring was 83 ± 3%. In contrast, untreated OLETF rats showed a clear improvement at 67 ± 2%.

[0031]

According to the present invention, an expression vector containing a nucleic acid sequence encoding a therapeutic or prophylactic agent for a disease such as arteriosclerosis or hypertension, such as the closo gene, is administered to a host to encode a therapeutic agent. It has been shown that by carrying DNA having a nucleic acid sequence into a host, vascular endothelial function is improved, and it is possible to treat or prevent the onset of arteriosclerosis, hypertension, and other conditions involving the closo gene.

[0032]

[Sequence List] Sequence Listing <110> KYOWA HAKKO KOGYO CO., LTD. <120> NOVEL METHOD OF IMPROVING ENDOTHELIAL FUNCTION <130> H11-127 <140> <141> <160> 5 <170> PatentIn Ver.2.0

<210> 1 <211> 3163 <212> DNA <1> Homo sapiens <220> <221> CDS <1> (9) ... (3047) <400> 1 cgcgcagc atg ccc gcc agc gcc ccg ccg cgc cgc ccg cgg ccg ccg ccg 50 Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro 1 5 10 ccg tcg ctg tcg ctg ctg ctg gtg ctg ctg ggc ctg ggc ggc cgc Lecu Le Seru Ser Ser Val Leu Leu Gly Leu Gly Gly Arg Arg 15 20 25 30 ctg cgt gcg gag ccg ggc gac ggc gcg cag acc tgg gcc cgt gtc tcg 146 Leu Arg Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Val Ser 35 40 45 cgg cct cct gcc ccc gag gcc gcg ggc ctc ttc cag ggc acc ttc ccc 194 Arg Pro Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly Thr Phe Pro 50 55 60 gac ggc ttc ctc tgg gcc gtg ggc agc gcc gcc tac ggc 242 Asp Gly Phe Leu Trp Ala Val GLyser Ala Ala Tyr Gln Thr Glu Gly 65 70 75 ggc tgg cag cag cac ggc aag ggt gcg tcc atc tgg gac acg ttc acc 290 Gly Trp Gln Gln His Gly Lys Gly Ala Ser Ile Asp Thr Phe Thr 80 85 90 cac cac ccc ctg gca ccc ccg gga gac tcc cgg aac gcc agt c tg ccg 338 His His Pro Leu Ala Pro Pro Gly Asp Ser Arg Asn Ala Ser Leu Pro 95 100 105 110 ttg ggc gcc ccg tcg ccg ctg cag ccc gcc acc ggg gac gta gcc agc 386 Leu Gly Ala Pro Ser Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser 115 120 125 gac agc tac aac aac gtc ttc cgc gac acg gag gcg ctg cgc gag ctc 434 Asp Ser Tyr Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg Glu Leu 130 135 140 ggg gtc act cac tac cgc ttc tcc atc tcg tgg gcg cga gtg ctc ccc 482 Gly Val Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro 145 150 155 aat ggc agc gcg ggc gtc ccc aac cgc gag ggg ctg cgc tac tac tac Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg 160 165 170 cgc ctg ctg gag cgg ctg cgg gag ctg ggc gtg cag ccc gtg gtc acc 578 Arg Leu Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Pro Val Thr 175 180 185 190 ctg tac cac tgg gac ctg ccc cag cgc ctg cag gac gcc tac ggc ggc 626 Leu Tyr His Trp Asp Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly Gly 195 200 205 tgg gcc aac cgc gcc cg gcc gcc ag g gat tac gcg gag ctc 674 Trp Ala Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu 210 215 220 tgc ttc cgc cac ttc ggc ggt cag gtc aag tac tgg atc acc atc gac 722 Cys Phe Arg His Phe Gln Val Lys Tyr Trp Ile Thr Ile Asp 225 230 235 aac ccc tac gtg gtg gcc tgg cac ggc tac gcc acc ggg cgc ctg gcc 770 Asn Pro Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala 240 245 250 ccc ggc atc cgg ggc agc ccg cgg ctc ggg tac ctg gtg gcg cac aac 818 Pro Gly Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val Ala His Asn 255 260 265 270 270 ctc ctc ctg gct cat gcc aaa gtc tgg cat ctc tac ttc 866 Leu Leu Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe 275 280 285 cgt ccc act cag gga ggt cag gtg tcc att gcc cta agc tct cac tgg 914 Arg Pro Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His Trp 290 295 300 atc aat cct cga aga atg acc gac cac agc atc aaa gaa tgt caa aaa 962 Ile Asn Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu Cys Gln Lys 305 310 315 tct ctg gac ttt gta cta ggt tg g ttt gcc aaa ccc gta ttt att gat 1010 Ser Leu Asp Phe Val Leu Gly Trp Phe Ala Lys Pro Val Phe Ile Asp 320 325 330 ggt gac tat ccc gag agc atg aag aat aac ctt tca tct att ctg ctg cct 1058 Gly Asp Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro 335 340 345 350 350 gat ttt act gaa tct gag aaa aag ttc atc aaa gga act gct gac ttt 1106 Asp Phe Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp Phe 355 360 365 ttt gct ctt tgc ttt gga ccc acc ttg agt ttt caa ctt ttg gac cct 1154 Phe Ala Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp Pro 370 375 380 cac atg aag ttc cgc caa ttg gaa tct ccc agg caa ctg ctt 1202 His Met Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu 385 390 395 tcc tgg att gac ctt gaa ttt aac cat cct caa ata ttt att gtg gaa 1250 Ser Trp Ile Asp Leu Glu Phe Asn Pro Gln Ile Phe Ile Val Glu 400 405 410 aat ggc tgg ttt gtc tca ggg acc acc aag aga gat gat gcc aaa tat 1298 Asn Gly Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr 415 420 425 430 430 atg tat tac ctc aaa aag ttc atc atg gaa acc tta aaa gcc atc aag 1346 Met Tyr Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys 435 440 445 ctg gat ggg gtg gat gtc atc ggg tat acc gca tgg tcc 1394 Leu Asp Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp 450 455 460 ggt ttc gag tgg cac aga ggt tac agc atc agg cgt gga ctc ttc tat 1442 Gly Phe Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr 465 470 475 gtt gac ttt cta agc cag gac aag atg ttg ttg cca aag tct tca gcc 1490 Val Asp Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser Ser Ala 480 485 490 ttg ttc tac caa aag ag atg aaa aat ggc ttc cct cct tta cct 1538 Leu Phe Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe Pro Pro Leu Pro 495 500 505 510 gaa aat cag ccc cta gaa ggg aca ttt ccc tgt gac ttt gct tgg gga 1586 Glu Asn Gln Leu Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly 515 520 525 gtt gtt gac aac tac att caa gta gat acc act ctg tct cag ttt acc 1634 Val Val Asp Asn Tyr Ile Gln VAlasp Thr Thr Leu Ser Gln Phe Thr 5 30 535 540 gac ctg aat gtt tac ctg tgg gat gtc cac cac agt aaa agg ctt att 1682 Asp Leu Asn Val Tyr Leu Trp Asp Val His His Ser Lys Arg Leu Ile 545 550 555 aaa gtg gat ggg gtt gtg acc aag aag agg aaa tcc tac tgt gtt gac 1730 Lys Val Asp Gly Val Val Thr Lys Lys ArGLys Ser Tyr Cys Val Asp 560 565 570 ttt gct gcc atc cag ccc cag atc gct tta ctc cag gaa atg cac gtt 1778 Phe Ala Ala Ila Gln Pro Gln Ile Ala Leu Leu Gln Glu Met His Val 575 580 585 590 aca cat ttt cgc ttc tcc ctg gac tgg gcc ctg att ctc cct ctg ggt 1826 Thr His Phe Arg Phe Ser Leu Asp Trp Ala Leu Ile Leu Pro Leu Gly 595 600 605 aac cag tcc cag gtg aac cac acc atc ctg cag tac tat cgc tgc atg 1874 Asn Gln Ser Gln Val Asn His Thr Ile Leu Gln Tyr Tyr Arg Cys Met 610 615 620 gcc agc gag ctt gtc cgt gtc aac atc acc cca gtg ggg gcc 19 Ala Ser Glu Leu Val Arg Val Asn Ile Thr Pro Val Val Ala Leu Trp 625 630 635 cag cct atg gcc ccg aac caa gga ctg ccg cgc ctc ctg gcc agg cag 1970 Gln Pro Met Ala Pro Asn Gln Gly Leu Pro Arg Leu Leu Ala Arg Gln 640 645 650 ggc gcc tgg gag aac ccc tac act gcc ctg gcc ttt gca gag tat gcc 2018 Gly Ala Trp Glu Asn Pro Tyr Thr Ala Leu Ala Phe Ala Glu Tyr Ala 655 660 665 670 670 cga ctg tgag ttt ca ctc ggc cat cac gtc aag ctt tgg ata acg 2066 Arg Leu Cys Phe Gln Glu Leu Gly His His Val Lys Leu Trp Ile Thr 675 680 685 atg aat gag ccg tat aca agg aat atg aca tac agt gct ggcc cac aac Glun Aset Pro Tyr Thr Arg Asn Met Thr Tyr Ser Ala Gly His Asn 690 695 700 ctt ctg aag gcc cat gcc ctg gct tgg cat gtg tac aat gaa aag ttt 2162 Leu Leu Lys Ala His Ala Leu Ala Trp His Val Tyr Asn Glu Lys Phe 705 710 715 agg cat gct cag aat ggg aaa ata tcc ata gcc ttg cag gct gat tgg 2210 Arg His Ala Gln Asn Gly Lys Ile Ser Ile Ala Leu Gln Ala Asp Trp 720 725 730 ata gaa cct gcc tgc cct ttc tcc caa aag gac gag gtg gcc gag 2258 Ile Glu Pro Ala Cys Pro Phe Ser Gln Lys Asp Lys Glu Val Ala Glu 735 740 745 750 aga gtt ttg gaa ttt gac att ggc tgg ctg gct gag ccc att ttc ggc 2306 Arg Val Leu Glu Ashe Ile Gly Trp Leu Ala Glu Pro Ile Phe Gly 755 760 765 tct gga gat tat cca tgg gtg atg agg gac tgg ctg aac caa aga aac 2354 Ser Gly Asp Tyr Pro Trp Val Met Arg Asp Trp Leu Asn Gln Arg Asn 770 775 780a ttt ctt ctt cct tat ttc act gaa gat gaa aaa aag cta atc cag 2402 Asn Phe Leu Leu Pro Tyr Phe Thr Glu Asp Glu Lys Lys Leu Ile Gln 785 790 795 ggt acc ttt gac ttt ttg gct tta agc cat tat acc acccc gta 2450 Gly Thr Phe Asp Phe Leu Ala Leu Ser His Tyr Thr Thr Ile Leu Val 800 805 810 gac tca gaa aaa gaa gat cca ata aaa tac aat gat tac cta gaa gtg 2498 Asp Ser Glu Lys Glu Asp Pro Ile Lys Tyr Asn Asp Tyr Leu Glu Val 815 820 825 830 caa gaa atg acc gac atc acg tgg ctc aac tcc ccc agt cag gtg gcg 2546 Gln Glu Met Thr Asp Ile Thr Trp Leu Asn Ser Pro Ser Gln Val Ala 835 840 845 gta gtg ccc tgg gggtt cgc aaa gtg ctg aac tgg ctg aag ttc aag 2594 Val Val Pro Trp Gly Leu ArGLys Val Leu Asn Trp Leu Lys Phe Lys 850 855 860 tac gga gac ctc ccc atg tac ata ata tcc aac gga atc gat gac ggg 2642 y Asp Leu Pro Met Tyr Ile Ile Ser Asn Gly Ile Asp Asp Gly 865 870 875 ctg cat gct gag gac gac cag ctg agg gtg tat tat atg cag aat tac 2690 Leu His Ala Glu Asp Asp Gln Leu Arg Val Tyr Tyr Met Gln Asn Tyr 880 885 890 ata aac gaa gct ctc aaa gcc cac ata ctg gat ggt atc aat ctt tgc 2738 Ile Asn Glu Ala Leu Lys Ala His Ile Leu Asp Gly Ile Asn Leu Cys 895 900 905 910 gga tac ttt gct tat tcg cgc aca gct ccg agg ttt ggc 2786 Gly Tyr Phe Ala Tyr Ser Phe Asn Asp Arg Thr Ala Pro Arg Phe Gly 915 920 925 ctc tat cgt tat gct gca gat cag ttt gag ccc aag gca tcc atg aaa 2834 Leu Tyr Ala Tyr Ayr Asp Gln Phe Glu Pro Lys Ala Ser Met Lys 930 935 940 cat tac agg aaa att att gac agc aat ggt ttc ccg ggc cca gaa act 2882 His Tyr Arg Lys Ile Ile Asp Ser Asn Gly Phe Pro Gly Pro Glu Thr 945 950 955 ctg gaa aga ttt tgt cca gaa gaa ttc acc gtg tgt act gag tgc agt 2930 Leu Glu Arg Phe Cys Pro Glu Glu Phe Thr Val Cys Thr Glu Cys Ser 960 965 970 ttt ttt cac acc cga aag tct tta ctg gct ttc ata gct cta ttt 2978 Phe Phe His Thr Arg Lys Ser Leu Leu Ala Phe Ile Ala Phe Leu Phe 975 980 985 990 ttt gct tct att att tct ctc tcc ctt ata ttt tac tac tcg aag aaa 3026 Phe Ala Ser Ile Ile Ser Leu Ser Leu Ile Phe Tyr Tyr Ser Lys Lys 995 1000 1005 ggc aga aga agt tac aaa tagttctgaa catttttcta ttcattcatt 3074 Gly Arg Arg Ser Tyr Lys 1010 ttgaaataat tatgcagaca catcagctgt taaccatttg cacctctaag tgttgttt3 ct ct gt catag 3 ct ct gt gt gt ct gt gt gt ct gt gt gtt gtt gtt gtt gtt tt ct gt gtt gt gtt gtt gtt gtt tt gt gtt gtt gtt gtt tt gt gtt tt ct gt gtt gtt gtt gtt tt gt gtt gtt gtt tt gt gtt att gt gtat

<210> 2 <211> 3435 <212> DNA <1> Homo sapiens <220> <221> CDS <222> (9) ... (1655) <400> 2 cgcgcagc atg ccc gcc agc gcc ccg ccg cgc cgc ccg cgg ccg ccg ccg 50 Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro 1 5 10 ccg tcg ctg tcg ctg ctg ctg gtg ctg ctg ggc ctg ggc ggc cgc Lecu Le Seru Ser Ser Val Leu Leu Gly Leu Gly Gly Arg Arg 15 20 25 30 ctg cgt gcg gag ccg ggc gac ggc gcg cag acc tgg gcc cgt gtc tcg 146 Leu Arg Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Val Ser 35 40 45 cgg cct cct gcc ccc gag gcc gcg ggc ctc ttc cag ggc acc ttc ccc 194 Arg Pro Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly Thr Phe Pro 50 55 60 gac ggc ttc ctc tgg gcc gtg ggc agc gcc gcc tac ggc 242 Asp Gly Phe Leu Trp Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu Gly 65 70 75 ggc tgg cag cag cac ggc aag ggt gcg tcc atc tgg gac acg ttc acc 290 Gly Trp Gln Gln His Gly Lys Gly Ala Ser Ile Trp Asp Thr Phe Thr 80 85 90 cac cac ccc ctg gca ccc ccg gga gac tcc cgg aac gcc ag t ctg ccg 338 His His Pro Leu Ala Pro Pro Gly Asp Ser Arg Asn Ala Ser Leu Pro 95 100 105 110 ttg ggc gcc ccg tcg ccg ctg cag ccc gcc acc ggg gac gta gcc agc 386 Leu Gly Ala Pro Ser Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser 115 120 125 gac agc tac aac aac gtc ttc cgc gac acg gag gcg ctg cgc gag ctc 434 Asp Ser Tyr Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg Glu Leu 130 135 140 ggg gtc act cac tac cgc ttc tcc atc tcg tgg gcg cga gtg ctc ccc 482 Gly Val Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro 145 150 155 aat ggc agc gcg ggc gtc ccc aac cgc gag ggg ctg cgc Astc Gly Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg 160 165 170 cgc ctg ctg gag cgg ctg cgg gag ctg ggc gtg cag ccc gtg gtc acc 578 Arg Leu Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Val Thr 175 180 185 190 ctg tac cac tgg gac ctg ccc cag cgc ctg cag gac gcc tac ggc ggc 626 Leu Tyr His Trp Asp Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly Gly 195 200 205 tgg gcc aac cgc gcc ccg gcc ttc agg gat tac gcg gag ctc 674 Trp Ala Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu 210 215 220 tgc ttc cgc cac ttc ggc ggt cag gtc aag tac tgg atc acc atc gac 722 Cys Phe Gg His Ply Gln Val Lys Tyr Trp Ile Thr Ile Asp 225 230 235 aac ccc tac gtg gtg gcc tgg cac ggc tac gcc acc ggg cgc ctg gcc 770 Asn Pro Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala 240 245 250 ccc ggc atc cgg ggc agc ccg cgg ctc ggg tac ctg gtg gcg cac aac 818 Pro Gly Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val Ala His Asn 255 260 265 270 270 ctc ctc ctg gct cat gcc aaa gtc tgg cat ctc tac ttc 866 Leu Leu Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe 275 280 285 cgt ccc act cag gga ggt cag gtg tcc att gcc cta agc tct cac tgg 914 Arg Pro Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His Trp 290 295 300 atc aat cct cga aga atg acc gac cac agc atc aaa gaa tgt caa aaa 962 Ile Asn Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu Cys Gln Lys 305 310 315 tct ctg gac ttt gta cta ggt tgg ttt gcc aaa ccc gta ttt att gat 1010 Ser Leu Asp Phe Val Leu Gly Trp Phe Ala Lys Pro Val Phe Ile Asp 320 325 330 ggt gac tat ccc gag agc atg aag aat aac ctt tca tct att ctg cct 1058 Gly Asp Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro 335 340 345 350 350 gat ttt act gaa tct gag aaa aag ttc atc aaa gga act gct gac ttt 1106 Asp Phe Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp Phe 355 360 365 ttt gct ctt tgc ttt gga ccc acc ttg agt ttt caa ctt ttg gac cct 1154 Phe Ala Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp Pro 370 375 380 cac atg aag ttc cgc caa ttg gaa tct ccc agg caa ctg ctt 1202 His Met Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu 385 390 395 tcc tgg att gac ctt gaa ttt aac cat cct caa ata ttt att gtg gaa 1250 Ser Trp Ile Asp Leu Glu Phe Asn Pro Gln Ile Phe Ile Val Glu 400 405 410 aat ggc tgg ttt gtc tca ggg acc acc aag aga gat gat gcc aaa tat 1298 Asn Gly Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr 415 420 425 430 atg tat tac ctc aaa aag ttc atc atg gaa acc tta aaa gcc atc aag 1346 Met Tyr Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys 435 440 445 ctg gat ggg gtg gat gtc atc ggg tcc acc gca tgg gat 1394 Leu Asp Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp 450 455 460 ggt ttc gag tgg cac aga ggt tac agc atc agg cgt gga ctc ttc tat 1442 Gly Phe Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr 465 470 475 gtt gac ttt cta agc cag gac aag atg ttg ttg cca aag tct tca gcc 1490 Val Asp Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser Ser Ala 480 485 490 490 tttt ttc tac at ag aaag gag aaa aat ggc ttc cct cct tta cct 1538 Leu Phe Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe Pro Pro Leu Pro 495 500 505 510 gaa aat cag ccc cta gaa ggg aca ttt ccc tgt gac ttt gct tgg gga 15lu Glun Pro Leu Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly 515 520 525 gtt gtt gac aac tac att caa gta agt cag ctg aca aaa cca atc agc 1634 Val Val Asp Asn Tyr Ile Gln Val Ser Gln Leu Thr Lys Pro Ile S er 530 535 540 agt ctc acc aag ccc tat cac tagtagatac cactctgtct cagtttaccg 1685 Ser Leu Thr Lys Pro Tyr His 545 acctgaatgt ttacctgtgg gatgtccacc acagtaaaag gcttattaaa gtggatgggg 1745 ttgtgaccaa gaagaggaaa tcctactgtg ttgactttgc tgccatccag ccccagatcg 1805 ctttactcca ggaaatgcac gttacacatt ttcgcttctc cctggactgg gccctgattc 1865 tccctctggg taaccagtcc caggtgaacc acaccatcct gcagtactat cgctgcatgg 1925 ccagcgagct tgtccgtgtc aacatcaccc cagtggtggc cctgtggcag cctatggccc 1985 cgaaccaagg actgccgcgc ctcctggcca ggcagggcgc ctgggagaac ccctacactg 2045 ccctggcctt tgcagagtat gcccgactgt gctttcaaga gctcggccat cacgtcaagc 2105 tttggataac gatgaatgag ccgtatacaa ggaatatgac atacagtgct ggccacaacc 2165 ttctgaaggc ccatgccctg gcttggcatg tgtacaatga aaagtttagg catgctcaga 2225 atgggaaaat atccatagcc ttgcaggctg attggataga acctgcctgc cctttctccc 2285 aaaaggacaa agaggtggcc gagagagttt tggaatttga cattggctgg ctggctgagc 2345 ccattttcgg ctctggagat tatccatggg tgatgaggga ctggctgaac caaagaaaca 2405 attttcttct tccttatttc actgaagatg aa aaaaagct aatccagggt acctttgact 2465 ttttggcttt aagccattat accaccatcc ttgtagactc agaaaaagaa gatccaataa 2525 aatacaatga ttacctagaa gtgcaagaaa tgaccgacat cacgtggctc aactccccca 2585 gtcaggtggc ggtagtgccc tgggggttgc gcaaagtgct gaactggctg aagttcaagt 2645 acggagacct ccccatgtac ataatatcca acggaatcga tgacgggctg catgctgagg 2705 acgaccagct gagggtgtat tatatgcaga attacataaa cgaagctctc aaagcccaca 2765 tactggatgg tatcaatctt tgcggatact ttgcttattc gtttaacgac cgcacagctc 2825 cgaggtttgg cctctatcgt tatgctgcag atcagtttga gcccaaggca tccatgaaac 2885 attacaggaa aattattgac agcaatggtt tcccgggccc agaaactctg gaaagatttt 2945 gtccagaaga attcaccgtg tgtactgagt gcagtttttt tcacacccga aagtctttac 3005 tggctttcat agcttttcta ttttttgctt ctattatttc tctctccctt atattttact 3065 actcgaagaa aggcagaaga agttacaaat agttctgaac atttttctat tcattcattt 3125 tgaaataatt atgcagacac atcagctgtt aaccatttgc acctctaagt gttgtgaaac 3185 tgtaaatttc atacatttga cttctagaaa acatttttgt ggcttatgac agaggttttg 3245 aaatgggcat aggtgatcgt aaaatattga ataatgcg aa tagtgcctga atttgttctc 3305 tttttgggtg attaaaaaac tgacaggcac tataatttct gtaacacact aacaaaagca 3365 tgaaaaatag gaaccacacc aatgcaacat ttgtgcagaa atttgaatga caagattagg 3425 aatattttct 3435

<210> 3 <211> 5032 <212> DNA <1> Mouse <220> <221> CDS <1> (19) ... (3060) <400> 3 cctcccggct cccgcagc atg cta gcc cgc gcc cct cct cgc cgc ccg ccg 51 Met Leu Ala Arg Ala Pro Pro Arg Arg Pro Pro 1 5 10 cgg ctg gtg ctg ctc cgt ttg ctg ttg ctg cat ctg ctg ctg ctc gcc 99 Arg Leu Val Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Ala 15 20 25 ctg cgc gcc cgc tgc ctg agc gct gag ccg ggt cag ggc gcg cag acc 147 Leu Arg Ala Arg Cys Leu Ser Ala Glu Pro Gly Gln Gly Ala Gln Thr 30 35 40 tgg gct cgc ttc gcg ccc gcc cca gag gcc gct ggc ctc ctc 195 Trp Ala Arg Phe Ala Arg Ala Pro Ala Pro Glu Ala Ala Gly Leu Leu 45 50 55 cac gac acc ttc ccc gac ggt ttc ctc tgg gcg gta ggc agc gcc gcc Phe Hesp Thr Asp Gly Phe Leu Trp Ala Val Gly Ser Ala Ala 60 65 70 75 tat cag acc gag ggc ggc tgg cga cag cac ggc aaa ggc gcg tcc atc 291 Tyr Gln Thr Glu Gly Gly Trp Arg Gln His Gly Lys Gly Ala Ser Ile 80 85 90 tgg gac act ttc acc cat cac tct ggg gcg gcc ccg tcc gac tcc ccg 339 Trp Asp Thr Phe Thr His His Ser Gly Ala Ala Pro Ser Asp Ser Pro 95 100 105 atc gtc gtg gcg ccg tcg ggt gcc ccg tcg cct ccc ctg tcc tcc act 387 Ile Val Val Ala Pro Ser Gly Ala Pro Ser Pro Pro Leu Ser Ser Thr 110 115 120 gga gat gtg gcc agc gat agt tac aac aac gtc tac cgc gac aca gag 435 Gly Asp Val Ala Ser Asp Ser Tyr Asn Asn Val Tyr Arg Asp Thr Glu 125 130 135 ggg ctg cgc gaa ctg ggg gtc acc cac tcg ttc tcc ata tcg tgg 483 Gly Leu Arg Glu Leu Gly Val Thr His Tyr Arg Phe Ser Ile Ser Trp 140 145 150 155 gcg cgg gtg ctc ccc aat ggc acc gcg ggc act ccc aac cgc gag ggg 531 Ala Arg Val Leu Pro Asn Thr Ala Gly Thr Pro Asn Arg Glu Gly 160 165 170 ctg cgc tac tac cgg cgg ctg ctg gag cgg ctg cgg gag ctg ggc gtg 579 Leu Arg Tyr Tyr Arg Arg Leu Leu Glu Arg Leu Arg Glu Leu Gly Val cc 180 185 ca gtg gtt acc ctg tac cat tgg gac ctg cca cag cgc ctg cag 627 Gln Pro Val Val Thr Leu Tyr His Trp Asp Leu Pro Gln Arg Leu Gln 190 195 200 gac acc tat ggc gga tgg gcc aat cgc gcc ctg gcc gac cat ttc agg675 Asp Thr Tyr Gly Gly Trp Ala Asn Arg Ala Leu Ala Asp His Phe Arg 205 210 215 gat tat gcc gag ctc tgc ttc cgc cac ttc ggt ggt cag gtc aag tac 723 Asp Tyr Ala Glu Leu Cys Phe Arg His Phe Gly Val Lys Tyr 220 225 230 235 tgg atc acc att gac aac ccc tac gtg gtg gcc tgg cac ggg tat gcc 771 Trp Ile Thr Ile Asp Asn Pro Tyr Val Val Ala Trp His Gly Tyr Ala 240 245 250 acc ggg cgc ctg gcc ccg ggc gtg agg ggc agc tcc agg ctc ggg tac 819 Thr Gly Arg Leu Ala Pro Gly Val Arg Gly Ser Ser Arg Leu Gly Tyr 255 260 265 ctg gtt gcc cac aac cta ctt ttg gct cat gcc aaa gtc tgg cat ctc 867 Leu Val Ala His Asn Leu Leu Leu Ala His Ala Lys Val Trp His Leu 270 275 280 tac aac acc tct ttc cgc ccc aca cag gga ggc cgg gtg tct atc gcc 915 Tyr Asn Thr Ser Phe Arg Pro Thr Gln Gly Gly Arg Val Ser Ile Ala 285 290 295 tta agc tcc cat tgg atc aat cct cga aga atg act gac tat aat atc 963 Leu Ser Ser His Trp Ile Asn Pro Arg Arg Met Thr Asp Tyr Asn Ile 300 305 310 315 aga gaa tgc cag aag tct ctt gac ttt gtg cta ggctgg ttt gcc aaa 1011 Arg Glu Cys Gln Lys Ser Leu Asp Phe Val Leu Gly Trp Phe Ala Lys 320 325 330 ccc ata ttt att gat ggc gac tac cca gag agt atg aag aac aac ctc 1059 Pro Ile Phe Ile Asp Gly Asp Tyr Pro Glu Ser Met Lys Asn Asn Leu 335 340 345 tcg tct ctt ctg cct gat ttt act gaa tct gag aag agg ctc atc aga 1107 Ser Ser Leu Leu Pro Asp Phe Thr Glu Ser Glu Lys Arg Leu Ile Arg 350 355 360 gga act gct gac ttt ttt gct ctc tcc ttc gga cca acc ttg agc ttt 1155 Gly Thr Ala Asp Phe Phe Ala Leu Ser Phe Gly Pro Thr Leu Ser Phe 365 370 375 cag cta ttg gac cct aac atg aag ttc cgc caa ttg gag tct ccc acac Leu Leu Asp Pro Asn Met Lys Phe Arg Gln Leu Glu Ser Pro Asn 380 385 390 395 ctg agg cag ctt ctg tct tgg ata gat ctg gaa tat aac cac cct cca 1251 Leu Arg Gln Leu Leu Ser Trp Ile Asp Leu Glu Tyr Asn Pro Pro 400 405 410 ata ttt att gtg gaa aat ggc tgg ttt gtc tcg gga acc acc aaa agg 1299 Ile Phe Ile Val Glu Asn Gly Trp Phe Val Ser Gly Thr Thr Lys Arg 415 420 425 gat gat gcc aaa tat atg tat t at ctc aag aag ttc ata atg gaa acc 1347 Asp Asp Ala Lys Tyr Met Tyr Tyr Leu Lys Lys Phe Ile Met Glu Thr 430 435 440 tta aaa gca atc aga ctg gat ggg gtc gac gtc att ggg tac acc gcg A395 Leu Arg Leu Asp Gly Val Asp Val Ile Gly Tyr Thr Ala 445 450 455 tgg tcg ctc atg gac ggt ttc gag tgg cat agg ggc tac agc atc cgg 1443 Trp Ser Leu Met Asp Gly Phe Glu Trp His Arg Gly Tyr Ser Ile Arg 460 465 470 475 cga gga ctc ttc tac gtt gac ttt ctg agt cag gac aag gag ctg ttg 1491 Arg Gly Leu Phe Tyr Val Asp Phe Leu Ser Gln Asp Lys Glu Leu Leu 480 485 490 cca aag tct tcg gcc ttg actc tac ag gag gac aat ggc 1539 Pro Lys Ser Ser Ala Leu Phe Tyr Gln Lys Leu Ile Glu Asp Asn Gly 495 500 505 ttt cct cct tta cct gaa aac cag ccc ctt gaa ggg aca ttt ccc tgt 1587 Phe Pro Pro Leu Pro Glu Asn Gln Pro Leu Glu Gly Thr Phe Pro Cys 510 515 520 gac ttt gct tgg gga gtt gtt gac aac tac gtt caa gtg gac act act 1635 Asp Phe Ala Trp Gly Val Val Asp Asn Tyr Val Gln Val Asp Thr Thr 525 530 535 ctc tct ca g ttt act gac ccg aat gtc tat ctg tgg gat gtg cat cac 1683 Leu Ser Gln Phe Thr Asp Pro Asn Val Tyr Leu Trp Asp Val His His 540 545 550 555 555 agt aag agg ctt att aaa gta gac ggg gtt gta gcc aag aag aga aaa 1731 Ser Lys Arg Leu Ile Lys Val Asp Gly Val Val Ala Lys Lys Arg Lys 560 565 570 cct tac tgt gtt gat ttc tct gcc atc cgg cct cag ata acc tta ctt 1779 Pro Tyr Cys Val Asp Phe Ser Ala Ile Arg Pro Gln Ile Thr Leu Leu 575 580 585 cga gaa atg cgg gtc acc cac ttt cgc ttc tcc ctg gac tgg gcc ctg 1827 Arg Glu Met Arg Val Thr His Phe Arg Phe Ser Leu Asp Trp Ala Leu 590 595 600 atc ttg cct cg ggt acc caa gtg aac cac acg gtt ctg cac 1875 Ile Leu Pro Leu Gly Asn Gln Thr Gln Val Asn His Thr Val Leu His 605 610 615 ttc tac cgc tgc atg atc atg agc gag ctg gtg cac gcc aac atc act cca 1923 Phe Tyr Arg Met Ile Ser Glu Leu Val His Ala Asn Ile Thr Pro 620 625 630 635 gtg gtg gcc ctg tgg cag cca gca gcc ccg cac caa ggc ctg cca cat 1971 Val Val Ala Leu Trp Gln Pro Ala Ala Pro His Gln Gly Leu Pro His 640 645 650 gcc ctt gca aaa cat ggg gcc tgg gag aac ccg cac act gct ctg gcg 2019 Ala Leu Ala Lys His Gly Ala Trp Glu Asn Pro His Thr Ala Leu Ala 655 660 665 ttt gca gac tac gca aac ctg tgt ttt aaa gag ttg ggt cac tgg gtc 2067 Phe Ala Asp Tyr Ala Asn Leu Cys Phe Lys Glu Leu Gly His Trp Val 670 675 680 aat ctc tgg atc acc atg aac gag cca aac aca cgg aac atg acc tat 2115 Asn Leu Trp Ile Thr Met Asn Glu Pro Asn Thr Arg Asn Met Thr Tyr 685 690 695 cgt gcc ggg cac cac ctc ctg aga gcc cat gcc ttg gct tgg cat ctg 2163 Arg Ala Gly His His Leu Leu Arg Ala His Ala Leu Ala Trp His Leu 700 705 710 715 715 tac gat gac aag ttt agg gcg gct cag aaa ggc aaa ata tcc atc gcc 2211 Tyr Asp Asp Lys Phe Arg Ala Ala Gln Lys Gly Lys Ile Ser Ile Ala 720 725 730 ttg cag gct gac tgg ata gaa ccg gcc tgc cct ttc 2259 Leu Gln Ala Asp Trp Ile Glu Pro Ala Cys Pro Phe Ser Gln Asn Asp 735 740 745 aaa gaa gtg gcc gag aga gtt ttg gaa ttt gat ata ggc tgg ctg gca 2307 Lys Glu Val Ala Glu Arg Val Leu Glu Phe Asp Ile Gly Trp Leu Ala 750 755 760 gag cct att ttt ggt tcc gga gat tat cca cgt gtg atg agg gac tgg 2355 Glu Pro Ile Phe Gly Ser Gly Asp Tyr Pro Arg Val Met Arg Asp Trp 765 770 775 775 ctg aac aa aaat ttt ctt ttg ccc tat ttc acc gaa gat gaa 2403 Leu Asn Gln Lys Asn Asn Phe Leu Leu Pro Tyr Phe Thr Glu Asp Glu 780 785 790 795 aaa aag cta gtc cgg ggt tcc ttt gac ttc ctg gcg gtg agt cat Leu Val Arg Gly Ser Phe Asp Phe Leu Ala Val Ser His Tyr 800 805 810 acc acc att ctg gta gac tgg gaa aag gag gat ccg atg aaa tac aac 2499 Thr Thr Ile Leu Val Asp Trp Glu Lys Glu Asp Pro Met Lys Tyr Asn 815 820 825 gat tac ttg gag gta cag gag atg act gac atc aca tgg ctc aac tct 2547 Asp Tyr Leu Glu Val Gln Glu Met Thr Asp Ile Thr Trp Leu Asn Ser 830 835 840 ccc agt cag gtg gca gtg gtg cct tgg cgc aaa gtg ctc aac 2595 Pro Ser Gln Val Ala Val Val Pro Trp Gly Leu Arg Lys Val Leu Asn 845 850 855 tgg cta agg ttc aag tac gga gac ctc ccg atg tat gtg aca gcc aat 2643 Trp Leu Arg Phe Lys Tyr G ly Asp Leu Pro Met Tyr Val Thr Ala Asn 860 865 870 875 gga atc gat gat gac ccc cac gcc gag caa gac tca ctg agg atc tat 2691 Gly Ile Asp Asp Asp Pro His Ala Glu Gln Asp Ser Leu Arg Ile Tyr 880 885 890 tat att aag aat tat gtg aat gag gct ctg aaa gcc tac gtg ttg gac 2739 Tyr Ile Lys Asn Tyr Val Asn Glu Ala Leu Lys Ala Tyr Val Leu Asp 895 900 905 gac atc aac ctt tgt ggc tac tat gcg tat tca cgc tca 2787 Asp Ile Asn Leu Cys Gly Tyr Phe Ala Tyr Ser Leu Ser Asp Arg Ser 910 915 920 gct ccc aag tct ggc ttt tat cga tat gct gcg aat cag ttt gag ccc 2835 Ala Pro Lys Ser Gly Phe Tyr Arg Tyr Ala Ala Asn Gln Phe Glu Pro 925 930 935 aaa cca tct atg aaa cat tac agg aga att att gac agc aat ggc ttc 2883 Lys Pro Ser Met Lys His Tyr Arg Arg Ile Ile Asp Ser Asn Gly Phe 940 945 950 955 ctg ggt tct gga ctg gga agg ttt tgt cca gaa gaa tac act gtg 2931 Leu Gly Ser Gly Thr Leu Gly Arg Phe Cys Pro Glu Glu Tyr Thr Val 960 965 970 tgc acc gaa tgt gga ttt ttt caa acc cgg aag tct ttg ctg gtc ttc 29 s Thr Glu Cys Gly Phe Phe Gln Thr Arg Lys Ser Leu Leu Val Phe 975 980 985 atc tcg ttt ctt gtt ttt act ttt att att tct ctt gct ctc att ttt 3027 Ile Ser Phe Leu Val Phe Thr Phe Ile Ile Ser Leu Ala Leu Ile Phe 990 995 1000 cac tac tcc aag aaa ggc cag aga agt tat aag taatgtgaac gtctgcctgg 3080 His Tyr Ser Lys Lys Gly Gln Arg Ser Tyr Lys 1005 1010 ccattcgctt tgggatcaag atgtacacgc cgtcagccgt ttgcacctct ctgtgttgtg 3140 agccgcattc cacacatttc gattctagaa aacccttttt gtcatgggtg gtagaggttt 3200 taaacaggaa ttggtgagaa taaaatattg cagggtgaat ggtatctgaa tctgctctct 3260 ttggtggcaa ttacggaatt atactcacca cagtttctac agtgccccgg aatggaaggc 3320 atagaatacg gtagggataa cagtgccaag cagacagaag tttaaagaac aactttaggg 3380 acttgtttat ccatggccat ttttaaattc actcctgttg gggagtaaca ctctctcaat 3440 taccatctta acacctggac tttacctgat ccagttttac aaggtgaagt agaaaaatat 3500 ccagtaaagg tggccaagag ccctgagtcc agagcagccc attaagaagc actattccta 3560 ccaaatgctg ctaatgtcaa tttacaaata tacttagaaa gcacattatg gacatttgta 3620 ttcttgtgaa tgttt ttgag gtgtgcccta aaccccagat ccttgagggc tttctcttac 3680 caactttcct ttcagagcct gcttgttgga gattcttccc cagccccctt cccctttccc 3740 tcttgctctg ccccacctcg ctccacccag cttgctccag cccaaagatt ctttatttgt 3800 ttctcattac cgaaggttgt gagccaccat gtggtttctg ggatttgaac tcatgacctc 3860 cggaggagct gtcatgctct taaccagccc atgttgaaga ttcttttgat aaatattcac 3920 aaaaaataaa gatgagccat gagctgttgg cctcttcgga agcggaaact gagtgatttg 3980 attgaacatc cttttatctt tgaccagacc ttggaatgaa tgcaatgacc tttcccacag 4040 gaagaaggag gagctctcag tcaaactgta aagaatgcct cttcagaata tgctgtcagt 4100 gcttggatgc catgatgttc aactttctta gtcgatccgg cagcaatcac agtgtgagca 4160 cactgggaac ctgtccttgc ggccgccgag atctaccgtg tgcttctgtg aagaggcttt 4220 gacgtagccc ctctttgagc tcttacacca tgctactgac ttctagaaag gctaattagg 4280 tcttcttcta cacctaatac cctaagtctt actgactctc acgggagaag tctctgtgct 4340 acacctgagt ggtcttattg ataaccctga taccagatca ggcaagataa atccgtcata 4400 gcaggcatgg ctacccttgc tgccacaggg tcacagcaca tagctcatca ccctgttatt 4460 cttcatcttg caatgtggta tggttttcct ggtgaatgat cagcttttgc tgtggtattc 4520 tttatacatc tggacttatt attgaaatca aatgctatag aatcaatagt ttattttatg 4580 tctatttttc ttgatcgcag agtaatatat attaattgta aaaaatttaa gaaacaaaaa 4640 ctatatgtaa agaaaaaatt ataatataat acagagatgc tgctgacagt tcctatgtgt 4700 tgtgttttgt atactgagat catgtgatac gtaggcatac atcttcttgg gtttttttgt 4760 ttttgttttt tgttttgttt tgttttgttt tggttttttg agatagggtt tctctgtata 4820 gccctggctg tcctggaact cactttgcag accaggctag cctcaaactc ttattcattt 4880 ttactgaagt aatttttctg tcattagtct tcaagagcaa aactttaata gttatggaga 4940 atattgccag aacagctcaa aactgtttta tttgttggtc caatttccca ttaattagtt 5000 caataataaa tatcatttag aaataaaaaa aa 5032

<210> 4 <211> 1650 <212> DNA <1> Mouse <220> <221> CDS <1> (1) ... (1650) <400> 4 atg cta gcc cgc gcc cct cct cgc cgc ccg ccg cgg ctg gtg ctg ctc 48 Met Leu Ala Arg Ala Pro Pro Arg Arg Pro Pro Arg Leu Val Leu Leu 1 5 10 15 cgt ttg ctg ttg ctg cat ctg ctg ctg ctc gcc ctg cgc Lec 96g Leu His Leu Leu Leu Leu Ala Leu Arg Ala Arg Cys 20 25 30 ctg agc gct gag ccg ggt cag ggc gcg cag acc tgg gct cgc ttc gcg 144 Leu Ser Ala Glu Pro Gly Gln Gly Ala Gln Thr Trp Ala Arg Phe Ala 35 40 45 cgc gct cct gcc cca gag gcc gct ggc ctc ctc cac gac acc ttc ccc 192 Arg Ala Pro Ala Pro Glu Ala Ala Gly Leu Leu His Asp Thr Phe Pro 50 55 60 gac ggt ttc ctc tgg gcg gta ggc agc gcc gcc tat acc gag ggc 240 Asp Gly Phe Leu Trp Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu Gly 65 70 75 80 ggc tgg cga cag cac ggc aaa ggc gcg tcc atc tgg gac act ttc acc 288 Gly Trp Arg Gln His Gly Lys Gly Ala Ser Ile Trp Asp Thr Phe Thr 85 90 95 cat cac tct ggg gcg gcc ccg tcc gac tcc ccg atc gtc g tg gcg ccg 336 His His Ser Gly Ala Ala Pro Ser Asp Ser Pro Ile Val Val Ala Pro 100 105 110 tcg ggt gcc ccg tcg cct ccc ctg tcc tcc act gga gat gtg gcc agc 384 Ser Gly Ala Pro Ser Pro Pro Leu Ser Ser Thr Gly Asp Val Ala Ser 115 120 125 gat agt tac aac aac gtc tac cgc gac aca gag ggg ctg cgc gaa ctg 432 Asp Ser Tyr Asn Asn Val Tyr Arg Asp Thr Glu Gly Leu Arg Glu Leu 130 135 140 ggg gtc acc cac tac cgc ttc tcc ata tcg tgg gcg cgg gtg ctc ccc 480 Gly Val Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro 145 150 155 160 aat ggc acc gcg ggc act ccc aac cgc gag ggg ctg cgc tac tac cgg Gly Thr Ala Gly Thr Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg 165 170 175 cgg ctg ctg gag cgg ctg cgg gag ctg ggc gtg cag ccg gtg gtt acc 576 Arg Leu Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Val Thr 180 185 190 ctg tac cat tgg gac ctg cca cag cgc ctg cag gac acc tat ggc gga 624 Leu Tyr His Trp Asp Leu Pro Gln Arg Leu Gln Asp Thr Tyr Gly Gly 195 200 205 tgg gcc aat cgc gcc ctg gcc gac cat ttc a gg gat tat gcc gag ctc 672 Trp Ala Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu 210 215 220 tgc ttc cgc cac ttc ggt ggt cag gtc aag tac tgg atc acc att gac 720 Cys Phe Arg His Phe Gln Val Lys Tyr Trp Ile Thr Ile Asp 225 230 235 240 aac ccc tac gtg gtg gcc tgg cac ggg tat gcc acc ggg cgc ctg gcc 768 Asn Pro Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala 245 250 255 ccg ggc gtg agg ggc agc tcc agg ctc ggg tac ctg gtt gcc cac aac 816 Pro Gly Val Arg Gly Ser Ser Arg Leu Gly Tyr Leu Val Ala His Asn 260 265 270 cta ctt ttg gct cat gcc aaa gtc tgg cat ctc tac aac acc ttc 864 Leu Leu Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe 275 280 285 cgc ccc aca cag gga ggc cgg gtg tct atc gcc tta agc tcc cat tgg 912 Arg Pro Thr Gln Gly Gly Arg Val Ser Ile Ala Leu Ser Ser His Trp 290 295 300 atc aat cct cga aga atg act gac tat aat atc aga gaa tgc cag aag 960 Ile Asn Pro Arg Arg Met Thr Asp Tyr Asn Ile Arg Glu Cys Gln Lys 305 310 315 320 tct ctt gac ttt gtg cta g gc tgg ttt gcc aaa ccc ata ttt att gat 1008 Ser Leu Asp Phe Val Leu Gly Trp Phe Ala Lys Pro Ile Phe Ile Asp 325 330 335 ggc gac tac cca gag agt atg aag aac aac ctc tcg tct ctt ctg cct 1056 Gly Asp Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Leu Leu Pro 340 345 350 gat ttt act gaa tct gag aag agg ctc atc aga gga act gct gac ttt 1104 Asp Phe Thr Glu Ser Glu Lys Arg Leu Ile Arg Gly Thr Ala Asp Phe 355 360 365 ttt gct ctc tgc ttc gga cca acc ttg agc ttt cag cta ttg gac cct 1152 Phe Ala Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp Pro 370 375 380 aac atg aag ttc cgc caa ttg gag tcc agg cag ctt cts 1200 Asn Met Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu 385 390 395 400 tct tgg ata gat ctg gaa tat aac cac cct cca ata ttt att gtg gaa 1248 Ser Trp Ile Asp Len Glu His Pro Pro Ile Phe Ile Val Glu 405 410 415 aat ggc tgg ttt gtc tcg gga acc acc aaa agg gat gat gcc aaa tat 1296 Asn Gly Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr 420 425 430 atg ta t tat ctc aag aag ttc ata atg gaa acc tta aaa gca atc aga 1344 Met Tyr Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Arg 435 440 445 ctg gat ggg gtc gac gtc att ggg tac acc gcg tgg gac 1392 Leu Asp Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp 450 455 460 ggt ttc gag tgg cat agg ggc tac agc atc cgg cga gga ctc ttc tac 1440 Gly Phe Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr 465 470 475 480 gtt gac ttt ctg agt cag gac aag gag ctg ttg cca aag tct tcg gcc 1488 Val Asp Phe Leu Ser Gln Asp Lys Glu Leu Leu Pro Lys Ser Ser Ala 485 490 495 495 ttg ttc tac aca ata gag gac aat ggc ttt cct cct tta cct 1536 Leu Phe Tyr Gln Lys Leu Ile Glu Asp Asn Gly Phe Pro Pro Leu Pro 500 505 510 gaa aac cag ccc ctt gaa ggg aca ttt ccc tgt gac ttt gct tgg gga 1584 Glu Asn Pro Leu Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly 515 520 525 gtt gtt gac aac tac gta caa gta agt cct ttg aca aaa ccc agt gtc 1632 Val Val Asp Asn Tyr Val Gln Val Ser Pro Leu Thr Lys Pro Ser Val 530 535 540 ggc ctc ttg ctt cct cac 1650 Gly Leu Leu Leu Pro His 545 550

<210> 5 <211> 3460 <212> DNA <1> Homo sapiens <220> <221> CDS <1> (60) ... (3107) <400> 5 cagggaatga atggattttc ttcagcactg atgaaataac cacacgctat aggaataca 59 atg tcc aac ggg gga ttg caa aga tct gtc atc ctg tca gca ctt att 107 Met Ser Asn Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile 1 5 10 15 ctg cta cga gct gtt act gga ttc tct gga gat gga aga gct ata tgg 155 Leu Leu Arg Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp 20 25 30 tct aaa aat cct aat ttt act ccg gta aat gaa agt cag ctg ttt ctc 203 Ser Lys Asn Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu 35 40 45 tat ggc act ttc cct aaa aac ttt ttc tgg ggt att ggg act gga gca 251 Tyr Gly Thr Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thrhr Gly Ala 50 55 60 ttg caa gtg gaa ggg agt tgg aag aag gat gga aaa gga cct tct ata 299 Leu Gln Val Glu Gly Ser Trp Lys Lys Asp Gly Lys Gly Pro Ser Ile 65 70 75 80 tgg gat cat ttc atc cac aca cac ctt aaa aat gtc agc agc acg aat 347 Trp Asp His Phe Ile His Thr His Leu Lys Asn Val Ser Ser Thr Asn 85 90 95 ggt tcc agt gac agt tat att ttt ctg gaa aaa gac tta tca gcc ctg 395 Gly Ser Ser Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Ser Ala Leu 100 105 110 gat ttt ata gga gtt tct ttt tat caa ttt tca att tcc tgg cca agg 443 Asp Phe Ile Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg 115 120 125 ctt ttc ccc gat gga ata gta aca gtt gcc aac gca aaa ggt ctg cag 491 Leu Phe Pro Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln 130 135 140 tac tac agt act ctt ctg gac gct cta gtg ctt aga aac att gaa cct 539 Tyr Tyr Ser Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Pro 145 150 155 160 ata gtt act tta tac cac tgg gat ttg cct ttg gca cta caa gaa aaa 587 Ile Val Thr Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys 165 170 175 tat ggg ggg tgg aaa aat gat acc ata ata gat atc ttc aat gac tat 635 Tyr Gly Gly Trp Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn Asp Tyr 180 185 190 gcc aca tac tgt ttc cag atg ttt ggg gac cgt gtc aaa tat tgg att 683 Ala Thr Tyr Cys Phe Gln Met Phe Asp Arg Val Lys Tyr Trp Ile 195 200 205 aca att cac aac cca tat cta gtg gct tgg cat ggg tat ggg aca ggt 731 Thr Ile His Asn Pro Tyr Leu Val Ala Trp His Gly Tyr Gly Thr Gly 210 215 220 atg cat gcc cct gga gag aag gga aat tta gca gct gtc tac act gtg 779 Met His Ala Pro Gly Glu Lys Gly Asn Leu Ala Ala Val Tyr Thr Val 225 230 235 240 gga cac aac ttg atc aag gct cac tcg aaa gtt tgg cat aac tac aac 827 Gly His Asn Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asn 245 250 255 aca cat ttc cgc cca cat cag aag ggt tgg tta tcg atc acg ttg gga 875 Thr His Phe Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly 260 265 270 tct cat tgg atc gag cca aac cgg tcg gaa aac acg atg gat ata ttc 923 Ser His Trp Ile Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe 275 280 285 285 aaa tgt caa caa tcc atg gtt tct gtg ctt gga tgg ttt gcc aac cct 971 Lys Cys Gln Gln Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn Pro 290 295 300 atc cat ggg gat ggc gac tat cca gag ggg atg aga aag aag ttg ttc 1019 Ile His Gly Asp Gly Asp Ty r Pro Glu Gly Met Arg Lys Lys Leu Phe 305 310 315 320 tcc gtt cta ccc att ttc tct gaa gca gag aag cat gag atg aga ggc 1067 Ser Val Leu Pro Ile Phe Ser Glu Ala Glu Lys His Glu Met Arg Gly 325 330 335 aca gct gat ttc ttt gcc ttt tct ttt gga ccc aac aac ttc aag ccc 1115 Thr Ala Asp Phe Phe Ala Phe Ser Phe Gly Pro Asn Asn Phe Lys Pro 340 345 350 cta aac acc atg gct aaa atg gga caa aat gtt tca ctt aat tta aga 1163 Leu Asn Thr Met Ala Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg 355 360 365 gaa gcg ctg aac tgg att aaa ctg gaa tac aac aac cct cga atc ttg 1211 Glu Ala Leu Asn Trp Ile Lys Leu Glu Asn Pro Arg Ile Leu 370 375 380 att gct gag aat ggc tgg ttc aca gac agt cgt gtg aaa aca gaa gac 1259 Ile Ala Glu Asn Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp 385 390 395 395 400 acc acg gcc atc tac atg atg aag aat ttc ctc agc cag gtg ctt caa 1307 Thr Thr Ala Ile Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln 405 410 415 gca ata agg tta gat gaa ata cga gtg ttt ggt tat act gcc tgg tct 1355 A Ile Arg Leu Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Ser 420 425 430 ctc ctg gat ggc ttt gaa tgg cag gat gct tac acc atc cgc cga gga 1403 Leu Leu Asp Gly Phe Glu Trp Gln Asp Ala Tyr Thr Ig Arg Gly 435 440 445 tta ttt tat gtg gat ttt aac agt aaa cag aaa gag cgg aaa cct aag 1451 Leu Phe Tyr Val Asp Phe Asn Ser Lys Gln Lys Glu Arg Lys Pro Lys 450 455 460 tct tca gca cac tac tac aaa cag atc ata cga gaa aat ggt ttt tct 1499 Ser Ser Ala His Tyr Tyr Lys Gln Ile Ile Arg Glu Asn Gly Phe Ser 465 470 475 475 480 tta aaa gag tcc acg cca gat gtg cag ggc cag ttt ccc tgt gac ttc 1547 Leu Lys Glu Ser Thr Pro Asp Val Gln Gly Gln Phe Pro Cys Asp Phe 485 490 495 tcc tgg ggt gtc act gaa tct gtt ctt aag ccc gag tct gtg gct tcg 1595 Ser Trp Gly Val Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser 500 505 510 tcc cca cag ttc agc gat cct cat ctg tac gtg tgg aac gcc act ggc 1643 Ser Pro Gln Phe Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly 515 520 525 aac aga ctg ttg cac cga gtg gaa ggg gtg agg ctg aaa ca cga ccc 1691 Asn Arg Leu Leu His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro 530 535 540 gct caa tgc aca gat ttt gta aac atc aaa aaa caa ctt gag atg ttg 1739 Ala Gln Cys Thr Asp Phe Val Asn Ile Lys Lys Gln Leu Glu Met Leu 545 550 555 560 gca aga atg aaa gtc acc cac tac cgg ttt gct ctg gat tgg gcc tcg 1787 Ala Arg Met Lys Val Thr His Tyr Arg Phe Ala Leu Asp Trp Ala Ser 565 570 570 575 gtc ctt cact ggc aac ctg tcc gcg gtg aac cga cag gcc ctg agg 1835 Val Leu Pro Thr Gly Asn Leu Ser Ala Val Asn Arg Gln Ala Leu Arg 580 585 590 tac tac agg tgc gtg gtc agt gag ggg ctg aag Ttt gc atc883 Tyr Arg Cys Val Val Ser Glu Gly Leu Lys Leu Gly Ile Ser Ala 595 600 605 atg gtc acc ctg tat tat ccg acc cac gcc cac cta ggc ctc ccc gag 1931 Met Val Thr Leu Tyr Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu 610 615 620 cct ctg ttg cat gcc gac ggg tgg ctg aac cca tcg acg gcc gag gcc 1979 Pro Leu Leu His Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala 625 630 635 640 ttc cag gcc tac gct ggg ctg tg c ttc cag gag ctg ggg gac ctg gtg 2027 Phe Gln Ala Tyr Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val 645 650 655 aag ctc tgg atc acc atc aac gag cct aac cgg cta agt gac atc tac 2075 Lysu Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr 660 665 670 aac cgc tct ggc aac gac acc tac ggg gcg gcg cac aac ctg ctg gtg 2123 Asn Arg Ser Gly Asn Asp Thr Tyr Gly Ala Ala His Asn Leu Leu Val 675 685 gcc cac gcc ctg gcc tgg cgc ctc tac gac cag cag ttc agg ccg tca 2171 Ala His Ala Leu Ala Trp Arg Leu Tyr Asp Gln Gln Phe Arg Pro Ser 690 695 700 cag cgc ggg gcc gtg tcg ctg tgg gcg gaa ccc 2219 Gln Arg Gly Ala Val Ser Leu Ser Leu His Ala Asp Trp Ala Glu Pro 705 710 715 720 gcc aac ccc tat gct gac tcg cac tgg agg gcg gcc gag cgc ttc ctg 2267 Ala Asn Pro Tyr Ala Asp Ser His Trp Arg Ala Ala Glu Arg Phe Leu 725 730 735 cag ttc gag atc gcc tgg ttc gcc gag ccg ctc ttc aag acc ggg gac 2315 Gln Phe Glu Ile Ala Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp 740 745 g750 tac c gcc atg agg gaa tac att gcc tcc aag cac cga cgg ggg 2363 Tyr Pro Ala Ala Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly 755 760 765 ctt tcc agc tcg gcc ctg ccg cgc ctc acc gag agg gag agg Leu Ser Ser Ser Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu 770 775 780 ctc aag ggc acg gtc gac ttc tgc gcg ctc aac cac ttc acc act agg 2459 Leu Lys Gly Thr Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr Arg 785 790 795 800 ttc gtg atg cac gag cag ctg gcc ggc agc cgc tac gac tcg gac agg 2507 Phe Val Met His Glu Gln Leu Ala Gly Ser Arg Tyr Asp Ser Asp Arg 805 810 815 815 gac atc cag ttt gc cag acc cgc ctg agc tcc ccc acg cgc 2555 Asp Ile Gln Phe Leu Gln Asp Ile Thr Arg Leu Ser Ser Pro Thr Arg 820 825 830 ctg gct gtg att ccc tgg ggg gtg cgc aag ctg ctg cgg tgg gtc cgg I603 Pro A Trp Gly Val Arg Lys Leu Leu Arg Trp Val Arg 835 840 845 agg aac tac ggc gac atg gac att tac atc acc gcc agt ggc atc gac 2651 Arg Asn Tyr Gly Asp Met Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp 850 855 860 gac cag gct ctg gag gat gac cgg ctc cgg aag tac tac cta ggg aag 2699 Asp Gln Ala Leu Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys 865 870 875 880 tac ctt cag gag gtg tca tca gca tca gat aaa gtc aga atc 2747 Tyr Leu Gln Glu Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile 885 890 895 aaa ggc tat tat gca ttc aaa ctg gct gaa gag aaa tct aaa ccc aga 2795 Lys Gly Tyr Tyr Ayr Pyr Ala Glu Glu Lys Ser Lys Pro Arg 900 905 910 ttt gga ttc ttc aca tct gat ttt aaa gct aaa tcc tca ata caa ttt 2843 Phe Gly Phe Phe Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln Phe 915 920 925 tac aac aaa gtg atc agc agc agg ggc ttc cct ttt gag aac agt agt 2891 Tyr Asn Lys Val Ile Ser Ser Arg Gly Phe Pro Phe Glu Asn Ser Ser 930 935 940 tct aga tgc agt cag acc caa gaa aat aca gag tgc act gtc tg Ser Arg Cys Ser Gln Thr Gln Glu Asn Thr Glu Cys Thr Val Cys Leu 945 950 955 960 ttc ctt gtg cag aag aaa cca ctg ata ttc ctg ggt tgt tgc ttc ttc 2987 Phe Leu Val Gln Lys Lys Pro Leu Ile Phe Leu Gly Cys Cys Phe Phe 965 970 975 tcc acc ctg gtt cta ctc tta tca att gcc att ttt caa agg cag aag 3035 Ser Thr Leu Val Leu Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys 980 985 990 aga aga aaga tag ttt tgg aaa gca aaa aac tta caa cac ata cca tta aag 3083 Arg Arg Lys Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys 995 1000 1005 aaa ggc aag aga gtt gtt agc taaactgatc tgtctgcatg atagacagt10 3134 Lys Vally Lys taaaaattca tcccagttcc atatgctggt aacttacagg agatatacct gtattataga 3194 aagacaatct gagatacagc tgtaaccaag gtgatgacaa ttgtctctgc tgtgtggttc 3254 aaagaacatt cccttaggtg ttgacatcag tgaactcagt tcttggatgt aaacataaag 3314 gcttcatcct gacagtaagc tatgaggatt acatgctaca ttgcttctta aagtttcatc 3374 aactgtattc catcattctg ctttagcttt catctctacc aatagctact tgtggtacaa 3434 taaattattt ttaagaagaa aaaaaa 3460

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/10 101 A61P 9/10 101 9/12 9/12 25/00 25/00 C12N 15/09 ZNA C12N 15/00 ZNAA (72) Inventor Takuko Iida 3-6-6 Asahicho, Machida-shi, Tokyo Kyowa Hakko Kogyo Co., Ltd. Tokyo Research Laboratory F-term (reference) 4B024 AA01 BA80 CA04 DA02 EA02 EA04 GA11 HA17 4C084 AA13 ZA362 ZA422 ZA452 ZC332 ZC352 ZC522 4C087 AA01 AA02 BC83 CA12 ZA36 ZA42 ZA45 ZC33 ZC35 ZC52

Claims (10)

[Claims] 1. A pharmaceutical composition for ameliorating or preventing a disease state caused by deterioration of vascular endothelial function in a mammal, comprising a effective amount of a recombinant vector into which the cDNA of closo gene has been incorporated. 2. The method of claim 1 wherein the cDNA of the closo gene is WO98 / 295.
44. The aging suppression gene cDNA according to claim 44.
A pharmaceutical composition according to claim 1. 3. The cDNA of the Closo gene according to SEQ ID NOs: 1, 2,
The pharmaceutical composition according to claim 1, which is a DNA having a base sequence selected from the base sequences represented by 3, 4, and 5, or a DNA that hybridizes with the DNA under stringent conditions. 4. The disease state due to vascular endothelial function deterioration is hypertension,
2. A disease selected from the group consisting of arteriosclerosis, hypercholesterolemia, diabetes, myocardial infarction and cerebral infarction.
A pharmaceutical composition according to claim 1. 5. The recombinant vector according to claim 1, wherein said recombinant vector comprises
2. The pharmaceutical composition according to claim 1, wherein said is incorporated into a vector selected from a plasmid vector and a virus vector. 6. The pharmaceutical composition according to claim 1, wherein the virus vector is selected from a retrovirus vector, an adenovirus vector, an adeno-associated virus vector and a herpes virus vector. 7. A recombinant vector of a Closo gene in which the cDNA of the Closo gene has been incorporated into a viral vector. 8. The closo recombinant vector according to claim 7, wherein the virus vector is selected from a retrovirus vector, an adenovirus vector, an adeno-associated virus vector and a herpes virus vector. 9. An effective amount of the pharmaceutical composition according to any one of claims 1 to 6 is administered to a mammal having a pathological condition due to deterioration of vascular endothelial function or a mammal having the possibility of falling into the pathological condition. And a method for treating or preventing the above condition. 10. The method according to claim 9, wherein the pathological condition due to deterioration of vascular endothelial function is a disease selected from the group consisting of hypertension, arteriosclerosis, hypercholesterolemia, diabetes, myocardial infarction and cerebral infarction.