WO2006085648A1

WO2006085648A1 - Novel narcolepsy-related gene

Info

Publication number: WO2006085648A1
Application number: PCT/JP2006/302453
Authority: WO
Inventors: Katsushi Tokunaga; Minae Kawashima; Hidetoshi Inoko; Yutaka Honda
Original assignee: The University Of Tokyo; Tokai University Educational System
Priority date: 2005-02-14
Filing date: 2006-02-13
Publication date: 2006-08-17
Also published as: JP2006217888A

Abstract

It is intended to provide a narcolepsy-related gene; a polypeptide encoded by this gene; and a medicinal composition to be used in diagnosing, preventing or treating narcolepsy which contains the above-described gene and the above-described polypeptide. By using genomewise designed microsatellite markers, a relativity analysis is conducted to thereby narrow the related region. Further, SNP markers showing particularly strong relativity in this area are determined. Then, linkage disequilibrium is analyzed on the above-described microsatellite markers showing particularly strong relativity with the SNP markers. By analyzing the linkage disequilibrium in these polymorphisms, a narcolepsy-related gene is identified.

Description

A novel narcolepsy-related gene

Technical field

[0001] The present invention relates to a narcolepsy-related gene. More specifically, a nalcholepsia-related gene identified by a gene mapping method using microsatellite and SNP (single nucleotide polymorphism), a polypeptide encoded by the gene, and the gene and the gene encoded by the gene Or a pharmaceutical composition comprising an agonist or an antagonist to the polypeptide.

Furthermore, the present invention relates to a method for determining the susceptibility to narcolepsy.

Background art

[0002] Narcolepsis, one of the typical hypersomnias, is repetitive intolerance that occurs during the day !, sleepiness (sleep seizures), emotional weakness attacks that suddenly lose muscle power triggered by strong emotional movements, It is characterized by abnormal REM sleep in which REM sleep is observed immediately after falling asleep. There are not many gender differences that occur in the teens, and the prevalence in Japanese is 0.16-0.18%. In addition, the agreement rate of identical twins was reported to be 25-31%, and the incidence of first-degree relatives was 1-2%, and human narcolepsy was caused by the involvement of multiple genetic and environmental factors. It is considered to be a multifactorial disease. The genetic factor that has been clarified so far is the HLA—DRB1 * 15 01 -DQBl * 0602 haplotype, which is present in the human leukocyte antigen (HLA) region, and almost all cases of Japanese patients with narcolepsy have this haplotype. (For example, non-patent documents 1 to 3). However, not all humans with this haplotype are affected by narcolepsy (about 10% in the healthy population), and many patients do not have this haplotype, especially in the African population. Therefore, although the HLA-DR-DQ haplotype shows an extremely strong association with human narcolepsy, it is considered not to be a sufficient condition.

Non-patent literature l: Matsui, K. et al., J. Clin. Invest., 76: 2078— 2083, 19 85

Non-Patent Document 2: Kuwata, S. et al., N. Engl. J. Med., 324: 271-272, 1991

Non-Patent Document 3: Honda, Y. et al., Genetic aspects of narcolepsy. In: Sleep and Sleep Disorders: From Molecule to Behavior. (Eds. Hay aishi, O., and Inoue, E.) Academic Press p. 341— 358, 199 8

Disclosure of the invention

Problems to be solved by the invention

[0003] In view of the above circumstances, the present inventors have conducted extensive research to search for susceptibility inheritance of narcolepsy diseases other than HLA-DR-DQ haplotypes, and as a result, identified a novel narcolepsy-related gene (polynucleotide). Succeeded in doing.

Therefore, an object of the present invention is to provide a novel narcolepsy-related gene. Another object of the present invention is to provide a polypeptide encoded by a novel narcolepsy-related gene.

Furthermore, an object of the present invention is to provide antibodies, agonists and Z or antagonists against the polypeptide.

A further object of the present invention is to provide a pharmaceutical composition comprising the gene, the polypeptide, the antibody, the agonist, and Z or the antagonist.

Another object of the present invention is to provide a screening method for the antigen and the antagonist.

Still another object of the present invention is to provide a method for determining the susceptibility to narcolepsy.

Means for solving the problem

[0004] So far, λ s (risk rate) in human narcolepsy has been reported to be 12. However, from the HLA typing results of the individual samples analyzed this time, s was estimated when it was assumed that only the HLA-DR-DQ haplotype was involved in the disease, and since 5.15, HLA-DR-DQ In addition to haplotypes, it was suggested that disease susceptibility genes may exist.

[0005] As a method for searching disease susceptibility genes for multifactorial diseases, Nonparametric linkage analysis for small families (affected sibling pair analysis), transmission disequilibrium test (TDT) for a large number of silkworms with one patient, and individual populations without family data Three main methods are used: targeted patient-control association analysis. Human narcolepsy is not suitable for the analysis of affected sib pairs with few frequent families. In addition, the ability of patient-control-related analysis is higher than that of TDT. It is difficult to list candidate genes for diseases whose onset / pathological mechanism is not clear, such as Narcolepsy. There is a problem that only known genes can be analyzed. Therefore, the present inventors have adopted a new strategy of “genome-wide association analysis method using poo led DNA using microsatellite markers”. This method enables comprehensive screening while maintaining high detection power by conducting a related analysis using a large number of microsatellite markers set at approximately equal intervals in the genome.

One of the microsatellite markers that showed a significant difference by the above-mentioned method. As a result of conducting a higher-density association analysis using a microsatellite polymorphism and SNP set at a higher density around one marker, We succeeded in identifying a novel gene with the relationship

That is, the present invention relates to the following (1) to (8).

(1) A first aspect of the present invention is an isolated polynucleotide comprising the following DNA (a) or (b) and a complementary strand thereof:

(a) DNA having a base sequence ability represented by SEQ ID NO: 8,

(b) A DNA that encodes a polypeptide that has the activity to bring about narcolepsy resistance and that is hybridized under stringent conditions with a DNA having a base sequence complementary to the DNA having the base sequence ability of (a).

(2) A second aspect of the present invention is an isolated polynucleotide comprising the following DNA (a) or (b) and a complementary strand thereof:

(a) DNA having a base sequence ability represented by SEQ ID NO: 9,

(b) A DNA having a base sequence ability of (a) and a DNA having a nucleotide sequence complementary to a DNA having a complementary base sequence and having an activity of bringing about resistance to narcolepsy under stringent conditions. DNA encoding the repeptide.

(3) A third aspect of the present invention is a polynucleotide containing the base sequence of the polynucleotide described in (1) or (2) above.

(4) A fourth aspect of the present invention is the polynucleotide according to (3) above, which comprises the base sequence represented by SEQ ID NO: 10 or 11.

(5) A fifth embodiment of the present invention is an isolated polynucleotide comprising the following DNA (a) or (b) and a complementary strand thereof:

(a) DNA having a nucleotide sequence represented by SEQ ID NO: 12,

(6) A sixth aspect of the present invention is a polynucleotide containing the base sequence of the polynucleotide described in (5) above.

(7) A seventh aspect of the present invention is the polynucleotide described in (6) above, which also has the base sequence ability represented by SEQ ID NO: 13.

(8) An eighth aspect of the present invention is a polypeptide encoded by the polynucleotide according to any one of (1) and (7) above.

(9) A ninth aspect of the present invention is a recombinant vector containing the polynucleotide according to any one of (1) and (7) above.

(10) The present invention

The tenth aspect of the present invention is an antigen against the polypeptide described in (8) above.

(11) An eleventh aspect of the present invention is an antagonist to the polypeptide described in (8) above.

(12) A twelfth aspect of the present invention is an antibody against the polypeptide described in (8) above.

(13) A thirteenth aspect of the present invention is a pharmaceutical composition comprising the vector according to (9) or the antigen according to (10) as an active ingredient, and used for the treatment of narcolepsy. It is a thing.

(14) A fifteenth aspect of the present invention uses the polypeptide or a salt thereof according to (8) above. A method for screening an agonist for the polypeptide or a salt thereof.

(15) A sixteenth aspect of the present invention uses the polypeptide described in (8) above or a salt thereof, and a method for screening an antagonist against the polypeptide or a salt thereof It is.

(16) A seventeenth aspect of the present invention is a method for determining a subject's susceptibility to narcolepsy, comprising the following steps:

(a) collecting a biological sample containing chromosomal DNA from the subject;

(b) determining the type of base at position 22.242 of human chromosome 21 at position 45242528 or the type of base at position 22.2345 of position 2 of human chromosome 21;

(c) step of determining the subject's susceptibility to narcolepsy based on the result obtained in step (b),

It is the method characterized by including.

(17) An eighteenth aspect of the present invention is a method for determining a subject's susceptibility to narcolepsy, which comprises the following steps:

(a) collecting a biological sample containing chromosomal DNA from the subject;

(b) determining a DNA sequence located at positions 877 to 879 in the sequence represented by SEQ ID NO: 21 in the chromosomal DNA;

It is the method characterized by including.

The invention's effect

[0007] By elucidating the function of the novel gene according to the present invention in more detail, it is possible to elucidate the onset mechanism of narcolepsy, and further, a novel therapeutic method, therapeutic agent or sleep inducer for narcolepsy Development can be promoted.

Brief Description of Drawings

[0008] Fig. 1 is a graph showing the distribution of p-values obtained by genome-wide association analysis using microsatellite markers. FIG. 2 is a graph showing P values obtained by association analysis with SNP markers in regions narrowed down by mapping with microsatellite, and odds values in SNPs showing significant differences.

[Figure 3] Shows the region narrowed by association analysis using microsatellite markers and SNPs.

FIG. 4 shows the result (P value) of the association analysis performed using the polymorphism detected by polymorphism analysis of the NLC1 region.

FIG. 5 shows the expression of NLC1-A and NLC1-C in the brain.

FIG. 6 shows the effect on promoter activity of C-7SNP polymorphism and NA3. L microsatellite polymorphism. The vertical axis shows the name of each polymorphic allele, and the horizontal axis shows the activity ratio with the null vector.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] 1. Polynucleotide of the present invention

The polynucleotide of the present invention comprises not only DNA having the nucleotide sequence represented by SEQ ID NO: 8 (NLC1-A) or 11 (NLC1-C), but also the nucleotide sequence represented by SEQ ID NO: 8 or 11. It also includes DNA consisting of DNA that has a complementary nucleotide sequence to DNA and DNA that encodes a polypeptide that hybridizes under stringent conditions and has the activity of bringing about resistance to narcolepsy. The polynucleotide of the present invention also includes the “gene” of the present invention. The gene of the present invention includes cDNA and genomic DNA, and includes not only exons and introns of the gene but also transcriptional regulatory regions such as promoters and enhancers. The polynucleotide of the present invention includes DNA and RNA, which may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA, double-stranded RNA or DNA and Hybrid with RNA! / RNA.

[0010] As a DNA that can be hybridized with a polynucleotide containing the base sequence represented by SEQ ID NO: 8 or 12 under stringent conditions, preferably about 70% or more of the base sequence represented by SEQ ID NO: 8 or 12 , More preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% polynucleotide sequence homology Examples thereof include DNA containing a base sequence.

Here, stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical calculations that depend on probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to re-anneal when the complementary strand is present in an environment near its melting point but below it.

Specifically, for example, as a low stringency condition, in a filter washing step after hybridization, under a temperature condition of 37 ° C. to 42 ° C., 0.1 X SSC, 0.1% For example, washing in an SDS solution can be raised. Further, as a high stringent condition, for example, in a washing step, washing in 65 ° C., 5 × SSC and 0.1% SDS can be mentioned. By making the stringent conditions higher, a highly homologous polynucleotide tide can be obtained.

[0012] The narcolepsy-related gene according to the present invention was also identified by gene mapping using a microsatellite marker and an SNP s marker. In other words, they were divided into two groups, nanorecoleptic patients and healthy individuals, and identified based on the presence or absence of significant differences in the frequency of alleles tested. If there is no difference in the frequency of a particular allele, it is considered that a narcolepsy-related gene is present near that allele.

If there is a difference in the frequency of a particular allele, a narcolepsy-related gene is considered to be present near that allele.

[0013] In general, as a gene polymorphism marker in gene mapping, SNPs (signature nucleotide polymoruphisms) marker and microsateful marker power S are known. Since microsatellite markers have a large number of alleles, narcolepsy-related gene strength is a feature that shows a correlation even if the marker is located at a certain distance, so narrow down the search region using genome-wide analysis. It is extremely effective in performing On the other hand, the SNP marker is an effective marker for specifying related genes in detail within a narrowed region. The narcolepsy-related gene according to the present invention uses both a microsatellite marker and an SNPs marker. I was able to identify.

[0014] The gene mapping method using microsatellite will be briefly described below, for example, the force described in detail in the pamphlet of WO01Z79482.

Genetic polymorphism means that there are two or more types of alleles at the target locus, and the frequency of major alleles is 99% or less. The gene locus is not limited to the region where the gene is expressed as long as it is a region on the genome. Microsatellite means a sequence that repeats 6 bases of 2 bases, and the number of repeats may vary among individuals, and the variation in repeats forms a polymorphism called STR (Short T andem Repeat) is doing. This number of repeats determines the microsatellite polymorphism. A typical microsatellite is a CA repeat.

[0015] First, microsatellite markers that are relatively rich in polymorphisms at a genome-wide suitable density, for example, 50 kb to 150 kb, preferably 80 kb to 120 kb, more preferably 90 to: one in L 10 kb Can be set at a rate of. As the number of alleles (araryl number) of microsatellite markers increases, the amount of information in analysis increases. By performing statistical analysis and related analysis on the distribution of the aryl frequency at each microsatellite locus and the deviation of the Nordic Weinberg equilibrium force, it is possible to search for microphone satellite markers showing a strong association. Strong association of microsatellite markers By setting markers at a high density in the vicinity of one and performing further association analysis, mapping of narcolepsy-related genes can be performed more accurately.

[0016] For detecting the polymorphism of the microsatellite, for example, a primer set for PCR that can amplify the microsatellite marker region can be used. Polymorphism detection methods using PCR include the RFLP (restriction fragment length polymorphism) method, the SS and P (single strana conformation polymorphism) method, the SSuP (sequence specific oligonucleotide probe) method, the RNase protection method. RDA (repre sentational difference analysis) method, RAPD (random amplified polymorphic DNA) method, AFLP (amplified fragment length polymorphism) method, etc. can be used.

[0017] Narcolepsy-related remains by gene mapping with microsatellite markers After narrowing the candidate area where there is likely to be a gene to some extent, set SNP strength at appropriate intervals and proceed with the related analysis. SNP exists in the genome at a ratio of 1 to 300-500 base pairs, and is 100 to 100 times higher than the appearance frequency of microsatellite markers V. Therefore, it is a candidate for a narcolepsy-related gene in a region narrowed to some extent It is a very effective means for specifying the area.

[0018] After the analysis with the microsatellite marker, in order to further narrow the candidate region where the narcolepsy-related gene is expected to exist, for example, using the SNP database, SNP markers are set at appropriate intervals. It is possible to identify narcolepsy-related gene candidates by conducting association analysis and analyzing linkage disequilibrium in the vicinity of SNPs where a significant association was observed.

[0019] Once a narcolepsy-related gene is identified using a gene mapping method, the gene can be used for examination of the disease, prevention and treatment of the disease. Cloning of the gene can be performed based on ordinary knowledge in the art. The gene can be obtained by screening a cDNA library prepared from cells that have developed and using the narcolepsy-related gene of the present invention or a part thereof as a probe. Alternatively, by preparing RNA from cells expressing the gene, synthesizing cDNA with reverse transcriptase, preparing PCR primers based on the gene sequence and amplifying the cDNA The cDNA may be obtained.

[0020] The cell that can be used for obtaining the polynucleotide of the present invention can be any cell as long as mRNA of the polypeptide is expressed. For example, in the case of NLC1-A, spleen, lung , Kidney, skeletal muscle, brain (^ I), hypothalamus, etc. In the case of NLC1-C, cells derived from tissues such as spleen, spleen, lung, brain (^ I), hypothalamus are used. preferable. The animal from which the cells are derived may be any animal (eg, human, chimpanzee, etc.) as long as it has the gene of the present invention.

Once the DNA sequence of the narcolepsy-related gene disclosed herein is confirmed, based on the common general technical knowledge in the field, the gene or the ortholog of the gene is, for example, usable animal tissue, but not limited thereto. Tissues such as humans and chimpanzees can be easily obtained from prepared cDNAs or cDNA libraries by the method described above. (For example, see Sambrook, J. 1989. Molecular cloning 2nd eds: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor.)

[0021] For preparation of mRNA and cDNA for obtaining the polynucleotide of the present invention, mRNA Purification Kit (Pharmasia), AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Use a commercially available kit.

[0022] 2. Polypeptide of the present invention and partial peptide thereof

The polypeptide of the present invention is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 18 (NLC1-A, long), SEQ ID NO: 19 (NLC1-A, short), or SEQ ID NO: 20 (NLC1-C). A polypeptide comprising the amino acid sequence of Here, the “polypeptide comprising substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. , More preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, It is a polypeptide comprising an amino acid sequence having 95%, 96%, 97%, 98%, most preferably about 99% amino acid identity, and having an activity of causing narcolepsy resistance. Here, the “activity that brings about nanorecoleptic resistance” refers to various symptoms of nanorecoleptic, but is not limited to, for example, living organisms for suppressing “unbearable sleepiness that repeats during the day” and cataplexy (weakness). Activity (eg, biochemical activity, physiological activity, etc.).

[0023] Alternatively, the polypeptide of the present invention includes a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. Examples of the polypeptide containing substantially the same amino acid sequence include one or several (preferably about 1 to 30, preferably about 1 to 30) of the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. A polypeptide having an amino acid sequence in which about 1 to 10, more preferably 1 to 5 amino acids are deleted, substituted or added, and has an activity of causing narcolepsy resistance is included.

The amino acid deletions, additions and substitutions may be present in the isolated natural polypeptide, and the gene encoding the polypeptide of the present invention may be obtained by a method known in the art. It may be newly introduced by modification by law. For example, substitution of a specific amino acid residue can be performed by using a commercially available kit (for example, Mutan ™ -G (TAKARA), Mutan ™ -K (TAKARA)) or the like, such as Guppedduplex method or Kunkel method. There is a method of! /, Which can be achieved by substituting the base by a method according to them.

[0024] In addition, the C-terminus of the polypeptide of the present invention is usually a carboxyl group (one COOH) or a force carboxylate (COO_), and the carboxyl group is an amide (CONH) or

2 It may be chemically modified to an ester (one COOR) or the like. Here, R in the ester is a C alkyl group (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl).

1-6

C) cycloalkyl group (for example, cyclopentyl, cyclohexyl), c aryl

3-8 1-6 group (for example, phenyl, naphthyl), ferro-C alkyl group (for example, ben

1-2

Diyl, phenethyl), a-naphthyl-C alkyl group (eg, a-naphthylmethyl)

1-2

Etc. In addition, it is possible to use bivalerooxymethyl ester which is widely used as an oral ester. When the polypeptide of the present invention has a carboxyl group in the polypeptide chain other than the c-terminus, the polypeptide of the present invention includes those in which the carboxyl group is amidized or esterified. Examples of esters in this case include the above-mentioned esters. Similarly, the N-terminus of the polypeptide of the present invention is usually an amino group (one NH 3), which is a formyl group, a acetyl group or the like.

2

It may be modified with C acyl group. In addition, the N-end side is cut in vivo

1 -6

Appropriate ones with pyroglutamic acid produced from the dartamyl group or substituents on the side chain of amino acids in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, gua-dino group, etc.) Polypeptides of the present invention include those that are chemically modified with various functional groups (eg, formyl group, acetyl), and those having a sugar chain attached thereto.

[0025] Peptides (also referred to as partial peptides) containing partial amino acid sequences in any of the above polypeptides according to the present invention are also included in the scope of the present invention. That is, as long as the partial peptide of the present invention includes a partial amino acid sequence of the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, It can be anything.

The number of amino acids constituting the partial peptide of the present invention is at least 10 or more, preferably 3 0 or more, more preferably 80 or more. Usually, the C-terminus of the partial peptide of the present invention is a carboxyl group (—COOH), and the N-terminus is an amino group (—NH 2).

2

May be.

[0026] The polypeptide of the present invention or a partial peptide thereof can be provided in the form of a salt, preferably in the form of a physiologically acceptable acid addition salt, if necessary. Such salts include inorganic acids, but are not limited to, for example, salts such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, organic acids, but are not limited to, for example, acetic acid, formic acid, propionic acid, Examples thereof include fumaric acid, maleic acid, succinic acid, tartaric acid, citrate, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like. Further, inorganic bases, but not limited to, for example, salts with alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, organic bases, but not limited to, for example, trimethylamine, And salts with triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine and the like.

[0027] The polypeptide of the present invention or a salt thereof is extracted from a cultured cell or tissue derived from a human animal (for example, chimpanzee etc.) expressing the polypeptide of the present invention by a conventional technique in the art. By 'cultivating a transformant that can be isolated, or in a state capable of expressing the DNA encoding the polypeptide of the present invention, as described later, it is extracted from the culture'. Can also be prepared. When preparing from human animal tissue or cells, homogenize human animal tissue or cells, extract with acid, etc., and extract the resulting extract using hydrophobic chromatography, reverse phase chromatography, ion chromatography. It can be isolated and purified by combining various types of chromatography such as exchange chromatography.

[0028] The partial peptide of the present invention or a salt thereof is produced by a known peptide synthesis method or by cleaving the polypeptide of the present invention with an appropriate peptidase (for example, trypsin, chymotrypsin, anoreginal endopeptidase). be able to. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining portion, and when the product has a protective group, the target peptide is removed by removing the protective group. (See, for example, Bondanszky et al., 1996; Schroeder et al., 1965). After the synthesis reaction, the partial peptide of the present invention can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, recrystallization and the like.

When the polypeptide of the present invention obtained by the above method or a partial peptide thereof is a free form, it can be converted into an appropriate salt by a known method. When it is obtained as a salt, it can be converted by a known method. Can be converted to free form.

[0029] 3. Production of recombinant vector and transformant

3- 1. Production of recombinant vectors

In the recombinant vector of the present invention, DNA encoding the polypeptide of the present invention (including polypeptides substantially the same as the polypeptide of the present invention and partial peptides thereof) is linked to an appropriate vector. Can be obtained. The vector for inserting the DNA sequence encoding the polypeptide of the present invention is not particularly limited as long as it can be replicated in a host when used for cloning. In addition, as a vector for expressing the polypeptide of the present invention, a vector that is replicable in a host and has a promoter that can express a DNA fragment encoding the polypeptide is used. Is possible.

[0030] Examples of vectors that can be used include plasmid DNA and phage DNA. Plasmid DNA includes plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194, etc.), and yeast-derived plasmids (eg, ΥΕρ13, ΥΕρ24, YCp50, YIp30, etc.), and phage DNA includes λ phage. Furthermore, animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus and toga virus can also be used.

[0031] The promoter used in the present invention is not particularly limited as long as it is an appropriate promoter corresponding to the host used for gene expression.

For example, when animal cells are used as the host, SRo; promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter, EF-1α Examples include promoters.

When the host is Escherichia coli, tac promoter, trp promoter, lac promoter, recA promoter, λ PL promoter, lpp promoter, etc. When the host is Bacillus subtilis, SPOl promoter, SP02 promoter, penP promoter, etc. Can be mentioned.

When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned.

When the host is an insect cell, polyhedrin promoter, P10 promoter, etc. are preferable.

[0032] In addition to the polypeptide coding sequence and promoter sequence of the present invention, the recombinant vector of the present invention includes a selection marker, a terminator, an enhancer, a splicing signal, a poly A addition signal, and a ribosome binding sequence (SD sequence). SV40 replication origin (SV40ori) can be linked.

Selectable markers include, but are not limited to, idaromomycin resistance marker (Hy _gr ), dihydrofolate reductase gene (dhfr), ampicillin resistance gene (Amp ¹ ), kanamycin resistance gene (Kan ¹ ), neomycin resistance gene (Neo ¹ , G418) etc. are available.

In addition, for the purpose of facilitating the isolation and purification of the recombinant protein, an appropriate signal sequence may be added to the N-terminal side of the polypeptide of the present invention! /.

When the host is Escherichia coli, alkaline phosphatase signal, OmpA signal, etc. can be used, and when the host is Bacillus subtilis, a amylase signal sequence, Suptilis signal sequence, etc. can be used. In the case of yeast, an α-factor signal sequence, an invertase signal sequence, etc. can be used. When the host is an animal cell, for example, an insulin signal sequence, an α interferon signal sequence, an antibody molecule signal sequence, etc. Is available.

[0033] The insertion of the polynucleotide encoding the polypeptide of the present invention into the vector described above means that the cloned DNA encoding the polypeptide of the present invention is digested as it is or with a restriction enzyme if desired, Add a linker, restriction vector DNA It can be performed by inserting into a site or multicloning site. The DNA to be ligated may have ATG as a translation initiation codon on the 5 ′ end side and TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codons and translation termination codons can also be added using an appropriate synthetic DNA adapter. The DNA to be ligated needs to be incorporated into a vector so that the polypeptide of the present invention encoded in the DNA is expressed in the host cell.

By the above method, a vector containing a DNA sequence encoding the polypeptide of the present invention can be constructed.

[0034] 3-2. Production of transformants

The transformant of the present invention can be obtained by introducing the recombinant expression vector of the present invention into a host so that the narcolepsy-related gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. Examples include Escherichia coli such as Escherichia coli, Bacillus genus such as Bacillus subtilis, Syudomonas genus such as Pseudomonas putida, and Rhizobium meliloti genus Rhizobium meliloti. Saccharomyces cerevisiae, Schizosaccharomyces pomDe, Pichia pastoris, and other yeasts, monkey cells COS-7, Vero, Chinese nymph ovary cells (CHO) Cell) or insect cells such as Sf9 and Sf21.

[0035] As a method for introducing a recombinant vector into Escherichia coli, a method using calcium ions (Cohen et al., 1972), an electoral position method (Shigekawa and Dower, 1988) and the like can be used. As a method for introducing a recombinant vector into yeast, the Elect Mouth Position method (B ecker et al., 1990), the Sugaguchi plast method (Hinnen et al., 1978), the lithium acetate method (Itoh et al., 1983), etc. can be used. . Methods for introducing thread-replaceable vectors into animal cells or animal cells include the DEAE dextran method (Lopata et al., 1984), the electopore position method, the phosphate phosphate method (Chen and Okayama, 1988), and cationic lipids. And the like (Elroy-Stein and Moss, 1990).

As described above, an expression vector into which a DNA encoding the polypeptide of the present invention has been inserted. Can be obtained.

[0036] 4. Production of polypeptide of the present invention and partial peptide thereof

The polypeptide of the present invention is produced by culturing a transformant introduced with an expression vector for a narcolepsy-related gene, expressing the polypeptide of the present invention from the gene, and isolating the polypeptide from the culture. can do. “Culture” means a culture supernatant, or cultured cells or cultured cells, or crushed cells or cells. The method of culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.

[0037] As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts and the like that can be assimilated by the microorganisms. Either a natural medium or a synthetic medium may be used as long as it can be efficiently cultured. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates, and other nitrogen-containing compounds. Noho force, peptone, meat extract, corn steep liquor, etc. are used. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like.

[0038] The culture is performed under conditions suitable for the host cell. For example, as a medium for culturing Escherichia coli, LB medium, M9 medium and the like are preferable. If desired, an agent such as isopropyl-1 thio β D-galactoside, 3 j8-indolylacrylic acid can be added to make the promoter work efficiently. In the case of Escherichia coli, the culture is usually carried out at about 15 to 37 ° C for about 3 to 24 hours, and if necessary, aeration or agitation can be added. When the host is Bacillus subtilis, cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, with aeration and agitation as necessary.

[0039] Examples of the medium for culturing yeast include SD medium and YPD medium. The pH of the medium is preferably adjusted to about 5-8. Incubate at about 20-35 ° C for about 24-72 hours Add aeration and agitation as necessary. When cultivating a transformant whose host is an insect cell or an insect, examples of the medium include a grace insect medium containing sushi serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Incubate at about 27 ° C for about 3 to 5 days, and add aeration or agitation as necessary.

[0040] When a transformant whose host is an animal cell is cultured, for example, a MEM medium, DMEM medium, RPMI1640 medium, or the like containing about 5 to 20% fetal bovine serum is used. The pH is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C for about 15-60 hours, with aeration and agitation as necessary. As described above, the polypeptide of the present invention can be produced in the transformant. Separation and purification of the polypeptide of the present invention from the culture can be performed, for example, by the following method.

[0041] When the cultured cells or cell force of the polypeptide of the present invention is also extracted, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and A method of obtaining a crude extract of the polypeptide of the present invention by centrifuging or filtering after destroying cells or cells by freezing and thawing or the like is appropriately used. The buffer may contain protein denaturing agents such as urea and guanidine hydrochloride, and surfactants such as Triton X-100. When the polypeptide of the present invention is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after completion of the culture, and the supernatant is prepared. Purification of the polypeptide of the present invention contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation and purification methods. These known separation and purification methods mainly use differences in molecular weight such as salting-out methods such as solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and SDS-PAGE. Methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, and methods that use hydrophobicity differences such as reversed-phase high-performance liquid chromatography. A method using the difference in isoelectric point, such as isoelectric focusing method, is used.

[0042] 5. Nucleic acid that inhibits the function of the gene encoding the polypeptide of the present invention

A nucleic acid that inhibits the function of the gene encoding the polypeptide of the present invention causes the sleep-introducing effect by losing the function of the gene encoding the polypeptide of the present invention. Seems to be able to. Examples of nucleic acids that inhibit gene functions include single-stranded nucleic acids such as antisense RNA or DNA and derivatives thereof, and short double-stranded RNA having a sequence complementary to a part of the gene region. it can.

Antisense acts as an effective inhibitor for the expression of the gene encoding the polypeptide of the present invention, which may be RNA or DNA or derivatives thereof. Antisense RNA is designed, for example, to hybridize with mRNA in vivo and inhibit translation from mRNA to GZF1 protein (Okano et al., 1991). The DNA oligonucleotide is designed to be complementary to the transcription initiation region of the narcolepsy-related gene of the present invention, and as a result, inhibits the expression of the gene (Cohen, 1989).

These antisense RNAs or DNAs can be introduced into cells such that they can function in vivo to inhibit the expression of the gene or polypeptide of the invention. When antisense DNA is used, it is preferably an oligonucleotide that binds to a position between about 10 and +10 of the target gene sequence, for example.

In addition, double-stranded RNA having a sequence complementary to the gene of the present invention can also be used for RNAi (RNA interference). RNAi is a phenomenon in which a target mRNA (eg, mRNA of a narcolepsy-related gene) is degraded by a double-stranded RNA having a sequence complementary to the target mRNA. By artificially introducing double-stranded RNA using this phenomenon, the expression of the target gene can be suppressed.

6. Antibodies against the polypeptides of the present invention

The antibody against the polypeptide of the present invention can bring about a sleep-introducing effect by inhibiting the function of the polypeptide of the present invention, or can promote the function of the polypeptide of the present invention to make it susceptible to narcolepsy. Can be suppressed.

The present invention includes an antibody that specifically binds to the polypeptide of the present invention, and an antibody fragment thereof such as Fab or F (a b ′).

2

The “antibody” (including both an antibody that promotes the activity of the polypeptide of the present invention and an antibody that inhibits the activity of the polypeptide of the present invention) includes a monoclonal specific antibody against the polypeptide of the present invention. Polyepitope specific antibodies, single chain antibodies, and fragments thereof. Be turned. These antibodies include, for example, monoclonal antibodies, polyclonal antibodies, human rabbit antibodies and the like.

6-1. Polyclonal antibodies

Polyclonal antibodies can be prepared, for example, by injecting a mixture of immunogen and adjuvant into a mammalian host animal. Usually, the immunogen and Z or adjuvant are ingested multiple times subcutaneously or intraperitoneally into the host animal. Immunogens include polypeptides of the invention and fusions with heterologous polypeptides or fragments thereof. Examples of adjuvants include complete Freud and monophosphoryl lipid A synthesized monotrehalose dicorynomycolate (MPL-TDM). In particular, in the case where the partial peptide of the polypeptide of the present invention is used as an immunogen, in order to enhance the immune response, keyhole limpet hemocyanin (KLH), serum albumin, cythyroglobulin, soybean trypsin inhibitor, etc. After binding the immunogenic protein to the immunogen, it may be injected.

Alternatively, it may be prepared using IgA molecules that produce IgY molecules (Schade et al., 1996). See, for example, Ausubel et al., 1987 or Harlow and Lane, 1988 for details on antibody production methods.

6-2 Monoclonal antibodies

Monoclonal antibodies can be prepared using the nobridoma method (Milstein and Cuello, 1983).

This method includes the following four steps: (i) immunizing the host animal or lymphocytes derived from the host animal, (ii) a monoclonal antibody secreting (or potentially secreting) linker. Collect spheres, (iii) fuse lymphocytes to immortalized cells, (iv) select cells that secrete the desired monoclonal antibody.

A mouse, rat, guinea pig, hamster, or other suitable host animal is selected as the immunized animal and immunogenic S-injected. Alternatively, lymphocytes obtained from immunized animals may be immunized in vitro. Peripheral blood lymphocytes (PBLs) are commonly used when human cells are desired. However, spleen cells or lymphocytes from other mammals are more common and preferred. Immunogens include polypeptides of the invention and their heterologous polypeptides and Fusions of these or fragments thereof are also included.

[0045] After immunization, the lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hyperpridoma cells (Goding, 1

996). As fusion cells, rodent, mouse, or human myeloma cells that are immortalized by transformation are used. Rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. Conventional techniques use parental cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hypridoma.

[0046] For the preparation of monoclonal antibodies, a preferred immortal cell line is the mouse myeloma line, which is available from the American Type Culture Collection (Manassas, VA). For production of human monoclonal antibodies by human myeloma and mouse-human heteromyeloma cell lines, see Kozbor et al., 1984; Schook, 1987.

Since the human and hybridoma cells secrete antibodies outside the cells, the presence or absence of production of the desired monoclonal antibody can be confirmed using the culture medium. The binding specificity of the monoclonal antibodies produced can be assessed by immunoprecipitation such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELIS A) or in vitro binding assays (Harlow and Lane, 1988; Harlow and Lane, 1999).

Monoclonal antibody-secreting hybridoma cells can be isolated as single clones by limiting dilution and subculture (Goding, 1996). Suitable media include Dulbecco's Modified Eagle Medium, RPMI-1640, and in some cases, protein-free or serum-free medium. Hypridoma cells may also be grown in the ascites of suitable host animals.

[0047] Monoclonal antibodies can be obtained from medium or ascites from protein A sepharose, hydoxyxpatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation, or affinity chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999). For those skilled in the art It is isolated and purified by a well-known method.

Monoclonal antibodies can also be produced by gene recombination technology (US Pat. No. 4166452, 1979) o Identification of the gene encoding the desired monoclonal antibody polypeptide from the hybridoma cell line secreting the antibody of interest For example, oligonucleotide probes that specifically bind to mouse heavy and light chain antibody genes may be used. As a result, when antibody heavy chain and light chain genes are obtained, the target antibody gene can be identified by determining the sequence of the genes. In order to express monoclonal antibodies, the isolated DNA fragments can be used to introduce an antibody gene into an appropriate expression vector and produce no other immunoglobulin protein.

COS—Transfects into host cells, such as 7 cells, Chinese nomstar ovary (CHO) cells, or myeloma cells. Isolated DNA fragments can be obtained, for example, by replacing coding sequences for human heavy and light chain constant domains with homologous mouse sequences (US Pat. No. 4,816,567; 1989; Morrison et al., 1987) or non-immune Modifications can be made by fusing the immunoglobulin coding sequence with all or part of the sequence encoding the globulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domain of the antibody to prepare a chimeric bivalent antibody or can be substituted for the constant domain of the antigen binding site.

6-3. Humanized and human antibodies

Polypeptide antibodies of the present invention include humanized or human antibodies. The human 匕 form of a non-human antibody is an antigen binding of a chimeric immunoglobulin, an immunoglobulin chain or a fragment thereof (Fv, Fab, Fab ', F (ab')) or other antibody containing a minimal sequence derived from a non-human immunoglobulin. Area)

2

It is.

In general, human rabbit antibodies have one or more amino acid residues into which non-human-derived immunoglobulin has been introduced. These non-human amino acid residues are often chosen from variable domain models. The human rabbit antibody can be prepared, for example, by substituting mouse CDRs or CDR (complementarity determining region) sequences with corresponding human antibody sequences (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al. , 1988). In other words, a humanized antibody is a residue in a specific human-derived CDR and a residue in a CDR of a non-human species such as mouse, rat, or rabbit that corresponds to the residue. A human antibody substituted with a group. In addition, nonhuman-derived residues may replace Fv framework residues of human immunoglobulin (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0049] 7. Agents and antagonists to the polypeptides of the present invention

The “agonist” in the present invention specifically binds to the polypeptide of the present invention, its receptor, or an interaction factor (binding partner) to promote the biological activity of the polypeptide of the present invention. And a part of the above-mentioned antibody is also included in the “agonist”. Examples of antibodies other than antibodies include, but are not limited to, polypeptides or fragments thereof, nucleic acids, and other low molecular compounds. In addition, the “antagonist” in the present invention means a substance that specifically binds to the polypeptide of the present invention, its receptor, or an interaction factor (binding partner) and suppresses the biological activity of the polypeptide of the present invention. This means that some of the above-mentioned antibodies are also included in “antagost”.

[0050] 8. Pharmaceutical composition

The polynucleotide, polypeptide or antibody of the present invention can be expected to exert an effect in the prevention or treatment of narcolepsy.

The polynucleotide, polypeptide or antibody of the present invention can be used as a therapeutic agent in the form of a pharmaceutical composition that does not adversely affect the living body. Such compositions typically include the nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like and are suitable for pharmaceutical administration. Yes (Gennaro, 2000). Examples of the carrier and preferred for diluting the carrier include, but are not limited to, water, saline, finger solution, dextrose solution, human serum albumin, and the like. Non-water soluble media such as ribosomes and non-volatile oils are also used. In addition, certain compounds that protect or promote the activity of the polynucleotides, polypeptides, or antibodies of the invention may be included in the composition.

[0051] The pharmaceutical composition according to the present invention includes intravenous, intradermal, subcutaneous, oral (for example, including inhalation), transdermal and transmucosal administration, and is suitable for a therapeutically appropriate route of administration. Formulation It becomes. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application include, but are not limited to, sterile diluents such as water for injection, saline solution, non-volatile oil, polyethylene. Glycol, glycerin, propylene glycol, or other synthetic solvents, benzyl alcohol or other preservatives such as methylparaben, antioxidants such as ascorbic acid or sodium sulfite, salt benzalcohol, Contains a soothing agent such as ethylenediaminetetraacetic acid (EDTA), a buffer such as acetate, citrate, or phosphate, and a drug for osmotic pressure adjustment such as sodium chloride or dextrose. But you can.

The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are stored in ampoules, glass or plastic use, disposable syringes or multi-dose vials.

8- 1. Injectable preparation

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (dispersible) or dispersion media and sterile powders to be prepared at the time of use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOP HOR EL ™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). When used as an injectable, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes and corrosion during preparation and storage and must prevent contamination from microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and a suitable mixture. For example, using a coating agent such as lectin, maintaining the required particle size in the dispersion medium, and maintaining a proper fluidity by using a surfactant. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can be used to prevent microbial contamination. In addition, sugars, polyalcohols such as mannitol and sorbitol, and isotonic agents such as sodium chloride may be included in the composition. Adsorption Compositions that can be delayed include agents such as aluminum monostearate and gelatin.

[0053] A sterile injectable solution may contain a necessary amount of the active compound (eg, a polynucleotide of the present invention, a polynucleotide of the present invention) in a suitable solvent, either alone or in combination with other components. It is prepared by adding sterilization (repeptides, antibodies, etc.). Generally, a dispersion medium is prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the other necessary ingredients discussed above. Sterile powder preparation methods for the preparation of sterile injectable solutions include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired components derived from the sterile solution. included.

[0054] 8— 2. Oral composition

Oral compositions usually include an inert diluent or a carrier that does not cause harm when taken into the body. Oral compositions are, for example, contained in gelatin capsules or compressed into tablets. For oral treatment, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. The oral composition can also be prepared using a fluid carrier, and the composition in the fluid carrier is applied orally. In addition, pharmaceutically compatible binding agents, and Z or adjuvant substances may be included.

Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds with similar properties: excipients such as microcrystalline cellulose, bindings such as gum arabic, tragacanth or gelatin Agents; alginic acid, such as starch or ratatose

, PRIMOGEL, or corn starch, etc .; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methylsalicylic acid or orange flavor Additive.

[0055] 8- 3. Carrier

The polynucleotide, polypeptide and antibody of the present invention should be prepared as a sustained-release preparation such as a delivery system encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body. Can do. Ethylene butyl acetate, polyanhydride Biodegradable, biocompatible polymers can be used such as products, polydalicholic acid, collagen, polyorthoesters, and polylactic acid. Such materials are readily available, such as ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), and can also be readily prepared by those skilled in the art. Ribosome suspensions can also be used as pharmaceutically acceptable carriers. Useful ribosomes are prepared as a lipid composition containing, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE), through a filter of appropriate pore size so that it is suitable for use and reversed. Purified by phase evaporation. For example, antibody Fab ′ fragments and the like may be conjugated to liposomes via a disulfide exchange reaction (Martin and Papahadjopoulos, 1982).

[0056] 8 -4. Dosage

In the treatment or prevention of a specific disease by the polypeptide of the present invention or a gene encoding the polypeptide, etc., an appropriate dosage level depends on the condition of the patient to be administered, the administration method, etc. If so, it can be easily optimized.

In the case of injection administration, for example, about 0.1 μg Zkg force per patient body weight per day, and preferably about 500 mg Zkg will generally be administered in one or more divided doses. Preferably, the dosage level is about 0.1 μg Zkg force per day as well as about 250 mg Zkg, more preferably about 0.5 to about lOO mg Zkg per day.

For oral administration, the composition is preferably provided in the form of a tablet containing from 1.0 to 10 mg of active ingredient, preferably the active ingredient is 1.0, 5.0, 10.0, 15.0. , 20. 0, 25. 0, 50. 0, 75. 0, 100. 0, 150. 0, 200. 0, 250. 0, 300. 0, 400. 0, 500. 0, 600. 0, 750 0, 800. 0, 900. 0 and 1000. Omg. The compound is administered on a regimen of 1 to 4 times, preferably once or twice a day.

[0057] 8-5. Unit dosage

A pharmaceutical composition or formulation should consist of uniform unit doses that ensure a certain dose. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. When determining the unit dosage of the present invention, the physical and chemical characteristics of the compound to be formulated, expected Influenced by therapeutic effects and precautions for formulation specific to the compound.

[0058] 8-6. Gene therapy composition

A method for introducing a nucleic acid molecule disclosed in the present invention (for example, a polynucleotide of the present invention, a vector into which the polynucleotide is inserted, and an antisense nucleic acid for a narcolepsy-related gene of the present invention) into a patient's cell is used. There are two main methods: in vivo or ex vivo. For in vivo delivery, it is injected directly into the part of the patient in need of treatment. In ex vivo treatment, cells at the site intended for treatment of the patient are isolated, the formulated nucleic acid molecule is introduced into the isolated cells, and the introduced cells are introduced directly into the patient or, for example, porous that is implanted in the patient. Can be administered in a capsule form (see US Pat. Nos. 4,892,538 and 5,283,187). The technique available for introducing the nucleic acid molecule into the living cell is selected depending on whether it is introduced into cultured cells or the like in vitro or introduced into a patient in vivo. Techniques suitable for introducing nucleic acid molecules into mammalian cells in vitro include ribosomes, electoporation, microinjection, transfection, cell fusion, the DEAE-dextran method, and the calcium phosphate precipitation method. . Transfection involves the binding of a recombinant virus (preferably retrovirus) particle to a cellular receptor, followed by the introduction of a nucleic acid molecule contained in the particle into the cell. A commonly used vector for ex vivo delivery of genes is retrovirus.

[0059] Currently preferred in vivo nucleic acid transfer technology! / Is a viral or non-viral vector (adenovirus, lentivirus, simple herpes I virus, or adeno-associated virus (AAV)), and cation Lipid-based systems (lipids useful for lipid-mediated transfer of genes are, for example, DOTMA DOPE ゝ and DC—Choi; for example, Tonkinson et al., Cancer In vestigation, 14 (1): 54-65 (1996) System using reference) is included. Most preferred for use in gene therapy, the vector is a virus, and of these, most preferably an adenovirus, AAV, lentivirus or retrovirus. Viral vectors, such as retroviral vectors, include at least one transcription promoter Z enhancer or position determining factor. Furthermore, virus vectors such as retrovirus vectors are examples. For example, when it is transcribed in a state containing a narcolepsy-related gene, it contains a cis element that enables translation of the encoded gene, that is, a nucleic acid sequence that functions as a translation initiation sequence. Such vector constructs contain a packaging signal, a terminal repeat (LTR) or part thereof suitable for the virus used. Further, these vectors usually contain a signal sequence for secreting a host cell force expressing polypeptide containing the vector. Preferably, the signal sequence for this purpose is a mammalian signal sequence. In some cases, the vector construct also includes polyadenylation as well as translation termination sequences. For example, it includes a 5 ′ LTR, a tRNA binding site, a knocking signal, an initiation point for DNA synthesis, and a 3 ′ LTR or a portion thereof. Other non-viral vectors can use, for example, cationic lipids, polylysine, and dendrimers.

In some cases, it is desirable to provide a nucleic acid used for treatment with a reagent that targets a target cell, for example, an antibody specific for a cell surface membrane protein, or a receptor ligand on a target cell. For a review of currently known! / Recognized gene labeling and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992).

9. Kit for pharmaceutical yarn

The pharmaceutical composition can be included in the kit, container, or container along with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different components of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately is to enable long-term storage without losing the function of the active component.

8- 1. Container

Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration. For example, a sealed glass ampoule contains a buffer packaged under a neutral, non-reactive gas such as nitrogen gas. The ampoule is composed of an organic polymer such as glass, polycarbonate, polystyrene, ceramic, metal, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include ampoules This includes simple bottles that are made of similar materials and packaging materials that are internally lined with foil such as aluminum or alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle with a stopper that can be penetrated by a hypodermic needle.

9- 2. Instructions for use

The kit also includes instructions for use. Instructions for use of the kit comprising the pharmaceutical composition are printed on paper or other material, and Z or floppy disk

It may be supplied as an electrically or electromagnetically readable medium such as a CD-ROM, DVD-ROM, Zip disk, video tape or audio tape. Detailed instructions for use may actually be included in the kit, or may be posted on a website designated by the kit manufacturer or distributor or notified by e-mail or the like.

10. Other medical and pharmaceutical applications such as screening of antagonists and antagonists against the polypeptide of the present invention

It is suggested that the polypeptide of the present invention may be a membrane protein by homology search. Therefore, the determination of an agonist or an antagonist for the polypeptide of the present invention and a partial peptide thereof or a salt thereof is expected to greatly contribute to the development of a therapeutic agent for narcolepsy and a sleep inducer.

The polypeptide of the present invention and the partial peptide thereof or a salt thereof, and the polynucleotide encoding them are: (i) determination of agoto or antagost for NLCl-A or NLCl-C polypeptide, (Iii) Narcolepsy prevention and Z or treatment agent, (iii) Sleep induction agent, (iv) Genetic diagnostic agent, (v) Quantification of antagost or antagost against NLCl-A or NLC1-C polypeptide, (Vi) screening for compounds that alter the binding properties of NLCl-A or NLC1-C polypeptide to agost or antagost; 《^ 1-eight or ^ [^ 1- preventive and Z or therapeutic agents for diseases containing compounds that alter the binding properties of polypeptides and antagonists or antagonists (viii ) Quantification of NLC1—A or NLC1—C polypeptide or its partial peptide; (ix) Neutralization with antibodies to NLCl—A or NLC1—C polypeptide and antagonist or its partial peptide; (x) NLCl— A or NLC1—C polybepetit It can be used to create a non-human animal that has a gene having a gene encoding or a deletion.

[0062] Specifically, the polypeptide of the present invention or a partial peptide thereof, or a salt thereof is a reagent for screening or identifying an agonist or an antagonist against the polypeptide of the present invention. Useful as. These reagents can be used as components of screening kits for substances that alter the binding properties of antigens or antagonists to the polypeptides of the present invention.

As described above, the present invention relates to an antagonist or antagonist of NLC1-A or NLC1-C polypeptide characterized by contacting a test substance with a polypeptide of the present invention or a partial peptide thereof, or a salt thereof. Provide a decision method. Examples of the test substance include a human or mammal (eg, chimpanzee, etc.) tissue extract, a cell culture supernatant, an artificially synthesized compound, and the like.

[0063] In order to carry out the method of determining the agonist or antagonist for the NLC1-A or NLC1-C polypeptide of the present invention, an appropriate NLC1-A or NLC1-C polypeptide fraction, label and A test substance (radiolabeled, fluorescent dye labeled, etc.) is used. The NLC1-A or NLC1-C polypeptide fraction may be a natural NLC1-A or N LCI-C polypeptide fraction or a thread-replaceable NLC1-A or NLC1-C polypeptide having equivalent activity. For example, in order to determine an antigen or antagonist against an NLC1-A or NLC1-C polypeptide or a salt thereof, cells containing NLC1-A or NLC1-C polypeptide Alternatively, prepare an NLC1-A or NLC1-C polypeptide preparation by suspending the membrane fraction of cells in a buffer appropriate for the determination method. The buffer may be any buffer as long as it does not inhibit the binding between the polypeptide such as a phosphate buffer and Tris monohydrochloride buffer and the argast or antagost. In order to reduce non-specific binding, surfactants such as CHAPS, Tween-80, and deoxycholate, and proteins such as ushi serum albumin and gelatin can be added to the buffer. A test substance with a certain amount of label (radiolabel, fluorescent dye label, etc.) coexists in a solution containing NLC 1-A or NLC 1-C polypeptide. To determine the amount of non-specific binding, use a large excess of unlabeled test compound. Prepare additional reaction tubes. The reaction is carried out at about 4-50 ° C, preferably about 4 ° C-37 ° C, for about 10 minutes to 24 hours, desirably about 30 minutes to 3 hours. After the reaction, it is filtered, washed with an appropriate amount of the same buffer, and the labeled test substance remaining on the filter paper is measured by a method suitable for detection of the labeled substance, such as a liquid scintillation counter. A test substance in which the count obtained by subtracting the non-specific binding amount from the total binding amount exceeds 0 can be selected as the antigen or antagonist of the present invention.

[0064] 10. Diagnosis

When an abnormality is observed in the expression of the gene of the present invention and the activity of the polypeptide encoded by the gene, it can be determined that narcolepsy has been developed or has a high risk of developing. In this case, a mutation (for example, point mutation, deletion, etc.) of the narcolepsy-related gene of the present invention (including not only the exon region but also an intron region and a promoter region) or a polypeptide encoded by the gene. ), The occurrence of narcolepsy and the risk of its occurrence can be predicted by examining the presence or absence of mutations in the expression control region of the gene. In particular, the NA3. L microsatellite polymorphism and C-7SNP disclosed in the present invention affect the regulation of NLCl-A gene expression and are expected to have an important effect on the development of narcolepsy. Is done.

[0065] To examine the expression level of the polypeptide of the present invention, an antibody specific for the polypeptide can be used. For example, a test sample is obtained from a narcolepsy patient or a patient suspected of developing narcolepsy, the antibody is brought into contact with the test sample, and the presence or absence of binding between the test sample and the antibody is detected. Therefore, it is possible to examine the onset or possibility of onset of narcolepsy. The presence or absence of binding between the test sample and the antibody can be confirmed by immunoprecipitation using antibodies, Western blotting, immunohistochemistry, ELISA, and the like. As a result of the confirmation, if it is determined that the polypeptide of the present invention is not detected at all or is present in an extremely small amount in the living body, the occurrence of narcolepsy is suspected.

[0066] In order to confirm the abnormality of the gene of the present invention, a primer or probe that can anneal to the cDNA sequence, genomic DNA sequence (including transcription control region, intron region, etc.) of the gene or a complementary strand thereof. Etc. can be used. One available primer is generally 15 bp to: LOO bp, preferably 17 bp to 30 bp in length, Any gene can be used as long as it can amplify at least a partial region of the coding region and non-coding region (including transcription control region, intron region, etc.) of the gene of the present invention.

[0067] The available probe generally has a length of 15 bp or more, and hybridizes with at least a partial region of the coding region and non-coding region (including transcription control region, intron region, etc.) of the gene of the present invention. Anything that can be used can be used. Further, in order to confirm that the probe hybridizes to the target DNA region, the probe can be used in a state where it can be detected by a fluorescent dye, a radiolabel, or the like.

[0068] Other methods for confirming the gene abnormality of the present invention include, for example, a narcolepsy patient or a patient who is suspected of developing narcolepsy, also obtaining a test sample and preparing narcolepsy prepared from the test sample. At least a portion of the coding region or non-coding region (including transcriptional control region, intron region, etc.) of the gene of the present invention by contacting the probe, primer, etc. The step of detecting the binding to the region or the step of amplifying the partial region is included. As a result of detection, when an abnormality is observed in the expression level of the gene of the present invention compared to a sample derived from a healthy subject, it is possible to predict the occurrence of narcolepsy or the risk of developing narcolepsy.

[0069] The narcolepsy test can also be performed by detecting a mutation or polymorphism in the narcolepsy-related gene of the present invention. Methods for detecting mutations or polymorphisms in the coding region and non-coding region (including transcription control region, intron region, etc.) of the gene include RFLP (restriction fr agment length polymorphism) method; 5SCP, smgle strand conformation polymorphism; method, S¾OP (.sequence specific oligonucleotide pr obe) method, RNase protection method, RDA (representational difference analysis) method, RAPD ( random amplified polymorphic DNA) method, AFLP (amplified fragment length polymorphism) method and the like. In these methods, the above primers can be used. The test by the above method is performed by, for example, extracting chromosomal DNA from a sample collected from a subject according to a standard method, and setting a primer set having the sequences represented by SEQ ID NO: 2 and SEQ ID NO: 3 to the chromosomal DNA. Is used to examine the sequence of microsatellite NA3. Shown in SEQ ID NO: 1, and using the primer set having the sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, 22 of human chromosome 21 45238073 at position 22.3 of human chromosome 21 by detecting SNP at position 45242528 or using a set of primers having the sequences represented by SEQ ID NO: 6 and SEQ ID NO: 7 This can be done by detecting the SNP of the location (the location of the SNP is the same in this specification by the UCSC Genomic QueryTool May 2004 version of NetAffx ™ Analysis Center).

[0070] Examples are shown below, but the present invention is not limited thereto.

Example

[0071] 1. Preliminary study on novel narcolepsy-related gene search:

Obtain 220 informed consent! /, Extract genomic DNA from peripheral blood obtained from a Japanese patient with narcolepsy, and concentration of double-stranded DNA using Cyber Green (PicoGreen, Molecular probes) Was measured accurately. Two sets of pooled DNA were prepared by mixing equal amounts of 110 people each, making 1st set and 2nd set respectively. Similarly, two sets of pooled DNA were prepared from the genomic DNA obtained from 420 control healthy subjects, each with 210 equivalents of pooled DNA. The 1st set pooled DNA was used for primary screening, and the 2nd set was used for secondary screening to remove false positives. Analysis of amplification products using pooled DNA in a cage was performed by an automatic sequencer using GeneScan software (Applied Biosystems).

[0072] 2. Association analysis with microsatellite markers on chromosome 6:

Association analysis was performed using 1,265 microsatellite markers (average gap 125.7 kb) covering the entire region of chromosome 6 where the HLA gene cluster is located. As a result of the primary screening of the 1st set of patients and the 1st set of controls, a significant difference was observed by a statistical analysis method using 2 x 2 Fisher's test method with 202 markers. When the second screening was performed on the 2nd set targeting these 202 markers, 42 types Still a significant difference was observed in the marker, also primary Te expected HLA-DR- DQ genes near marker Nitsuヽstrong association with secondary screening was found to ^{_{(p <10 "34) o}} HLA region In order to analyze in more detail, analysis was performed using microsatellite markers set at a high density, and 22 of the 30 markers covering 5.5 megabases showed significant differences. Significant differences were also found in the HLA—A, B, and C4A gene nearby markers located (p 10 ^_6 , p <10 " ⁹ , p <10" ²⁴ ) Based on the results above _o , it was set approximately every lOOkb It was considered possible to detect disease-susceptibility candidate regions by association analysis using a single microsatellite marker, while markers showing extremely strong associations other than the HLA region were not detected on chromosome 6. Ivy.

[0073] 3. Association analysis with genome-wide microsatellite markers:

A related analysis was conducted on 23,381 microsatellite markers (average gap 130.3 kb) (Table 1) distributed throughout the genome, and a high-density marker nearby was used for one microsatellite marker that was confirmed to be strongly related. The candidate area was narrowed by performing the related analysis.

[0074] [Table 1]

Micro-Sacho-Film used in analysis related to

Marker number and average gap for each chromosome

Number of chromosome markers (marker gap)

Chr. 1 1,949 (118.7kb)

Chr. 2 2,100

Chr. 3 1,745 (110.4kW m-car) Total number of markers:

Chr. 4 1,466 (118.8kb)

Chr. 5 1,467 (116.8kb / car) 23,381 car

Chr. 6 1,402 (125.7kb / power)

Chr. 7 1,430 (110.5kb)

Chr. 8 1,132 (118.1kb / power)

Chr average gap:

. 9 971 (120.4kb / machine)

Chr 10 1,145 (117.5 kb / power)

Chr 11 1,124 130.3

(124.3kb / ma-car) kbf marker

Chr 12 1,002 (127.9kb / power)

Chr 13 775 (125.9kb / m — Power one)

Chr 14 663 (132.7 kb / ma — Power one) Average heterozygosity

Chr 15 565 (142.8kb / car)

Chr 16 614 (124.4kb / power) 0.66

Chr 17 621 (131.0kb / ma-car)

Chr 18 634 (126.1kb / car)

Chr 19 451 (123.3kb / ma one power) Average number of Aril:

Chr 20 490 (122.8kb / ma-car)

Chr 21 284 (122.9kb / Ma one force one) 6 § ¹

Chr 22 255 (133.3W) / ma-car)

Chr. X 992 (137.4kW Ma-Power One)

Chr • Y 104 (287.0kb / car)

[0075] As shown in FIG. 1, a significant difference was observed by at least one statistical analysis for about 3,000 species by primary screening using the 1st set. As a result of secondary screening using these first and second sets, a significant difference was recognized again for over 300 markers!

In order to confirm the significance detected in the primary and secondary genome-wide association analysis using pooled DNA, patients with 91 markers with high reproducibility in the primary and secondary microsatellite peak patterns were identified. Individual samples of 95 people and 95 controls were typed. This analysis confirmed significant differences again for the 14 markers. When all individual samples were typed for these, three types of microsatellite markers were observed to have particularly low p values (p <0.0001).

[0076] Furthermore, in order to perform high-density mapping of these three regions, the area around each marker 500— When a new microsatellite marker polymorphism was screened at 700 kb, microsatellite markers could be set at an average interval of about 40 kb. Using these, <relationship analysis was performed, and a significant difference was also observed for markers located in the immediate vicinity of the three markers that were found to be related in the screening. In particular, the marker (NA3. L) (SEQ ID NO: 1), which was 70 bk away from NA3.4, showed a stronger association than the NA3.4 marker (P = 0. 00045). Table 2 shows the frequency of NA3.L for patients and healthy subjects. As a result, it was found that by examining the NA3. L microsatellite marker, the susceptibility to narcolepsy could be determined.

[¾2] Microsatellite NA3 丄 disease ^ "Control Aryl (%) (%)

(AC) ₈ 74 16.2 117 13.9

(AC) ₉ 277 60.5 515 61.3

(AC) _{1 ()} 4 0.9 39 4.6

(AC) _n 21 4.6 33 3.9

136 16.2

(AC) ₁₃ 1 0.2 0 0.0

458 100 840 100 p = 0.00045 Based on the above analysis, 70 kb around NA3.4 could be a disease-related candidate region. In order to further narrow these candidate regions for the NA3.4 region, a related analysis was performed by setting SNP markers at an average interval of 10 kb based on the SNP database. For 8 patients each A new SNP was screened. A related analysis of these SNPs using individual samples of 190 patients and 190 controls revealed 8 types of SNPs that showed significant differences (Figure 2). As a result of analysis using all the samples for the two SNPs in the vicinity of the NA3.L marker, which showed a particularly strong association, as shown in Fig. 3, it showed an equivalent or stronger association with the microsatellite marker ( p = 0. 0002,; p <0. 0001). Further analysis of linkage disequilibrium for these polymorphisms revealed that NA3.L and the two SNPs enter a single linkage disequilibrium block (Figure 4). These two SNPs are the CZT polymorphism (C-4,) and the AZG polymorphism (C-7), each of which has a nucleotide at position 22.242 of human chromosome 21 at position 45242528 or human chromosome 21. 22. Located at 45238073, third place. Table 3 and Table 4 show the phenotypic frequency and aryl frequency of each SNP for patients and healthy subjects. From this result, it was found that the susceptibility to narcolepsy could be determined by examining the SNPs at the two strengths.

[Table 3]

SNP C 4

Genotype frequency

330 (97.3%) 384 (91.4%)

9 (2.7%) 34 (8.1%)

0 (0%) 2 (0.5%) aryl frequency

c 669 (98.7%) 802 (95.5%)

t 9 (1.3%) 38 (4.5%) p = 0.00028

OR = 0.28 (95% C1: 0.1-0.6) [0080] [Table 4]

SNP C-7

/ Kushidon Λ ¾ Control Genotype frequency

aa 329 (97.1%) 384 (91.4%) ag 10 (2.9%) 34 (8.1%)

gg 0 (0%) 2 (0.5%)

a 668 (98.5%) 802 (95.5%)

10 (1.5%) 38 (4.5%) p = 0.00060

OR = 0.32 (95% C1: 0.2-0.6)

[0081] Therefore, it was suggested that some narcolepsy susceptibility gene (polymorphism) may exist in this block. There were no known genes in this block, and multiple ESTs and mRNAs were reported to the database at the same position, suggesting the existence of three new genes. The new genes were named NLC1-A, NLCl-B and NLCl-C. Among these genes, NLC1-A mRNA long form and short form are shown as SEQ ID NO: 10 and SEQ ID NO: 11, respectively. Furthermore, NLC1-C mRNA was shown as SEQ ID NO: 13. The predicted ORF sequence of NLC1-A is shown in SEQ ID NOs: 8 (long form) and 9 (short form), and the predicted ORF sequence of NLCl-C is shown in SEQ ID NO: 12. Furthermore, we confirmed that NLC1-A and NLC1-C were expressed in the brain and hypothalamus (Fig. 5).

[0082] A human organ poly A RNA sample purchased from Clontech Co., Ltd. When RT-PCR was performed using a primer set consisting of the sequence of SEQ ID NO: 15, an amplified product band corresponding to the ORF of the expected length of NLC1-A (Fig. 5A) could be confirmed. (Figure 5B). Similarly, when RT-PCR was performed using a primer set having the sequence power of SEQ ID NO: 16 and SEQ ID NO: 17 (amplification product band corresponding to the ORF of the expected length of NLC1-C (Fig. 5A) Was confirmed (Fig. 5B).

[0083] Furthermore, SEQ ID NO: 18 and SEQ ID NO: 19 show the predicted amino acid sequences of NLC1-A long form and short form, and SEQ ID NO: 20 shows the predicted amino acid sequence of NLC1-C.

When the characteristics of the NLC1-A long form shown in SEQ ID NO: 18 and the presence or absence of a specific motif were examined, most of the NLC1-A long form was found. Exists outside the membrane

Was expected to. Table 5 shows the expected structural characteristics of NLC1-A long form. In addition, the long form of NLC1-A has a binding protein-dependent transport systems inner member of the binding protein-dependent transport system that is a member of the superfamily of transporters found in humans. A region similar to the motif structure characteristic of the membrane component domain) was found.

[0084] [Table 5]

10 20, ... 30, ... 40, ... 50 60

ΑΑ MLSTAPTHHRICRPRAQLLLAGRRPAPPSDVQTSMGTLGLSTLLCPEPATIRVTQEPGAA

structure LLLLLLL L し LLLLLL L LLLL. ΕΕΕ LLLL. 70,... 80 90. ■, .. 100 110,.

GH QRGHVPCPRLPAPENGPGTPWSSGPRTAPDPPGTDPPGREGTPWSSGPRAAPDPPGTDPP

structure.. LLLLLLLLLLL. LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL

130 140 ... 150 ... 160 ... 170 ... 180.

ΑΑ GREGMNHIPDGGFRSESCQVTQVPCELLPGDGFRPWVYHSGCFPSAKPSLAFISPRTTVC

LLLLLLLLL ΕΕΕΕ LLLL EE. LLLLLLLLL LL.. 200. 190 200

AA FLSLWAYSGFLFYCLKL V

structure EEEE. LL

Helix

E = sheet

L = Loop Above NA3. L microsatellite polymorphism and C7SNP polymorphism are present in the promoter region and intron region, respectively. This may affect the transcription control of the image. Therefore, we investigated the effect of these two polymorphisms on promoter activity.

The DUAL—Luciferase Reporter Assay System (Promega) was used as an accessory thread, and experiments were performed according to the protocol attached to the kit.

First, the intron region (297 bp) containing the C-7SNP polymorphism and the promoter region (907 bp) containing the NA3. L microsatellite polymorphism were excised with Kpnl-Sacl, and the multicloning site of the pGL3-control vector (Kpnl — A test vector was prepared on the Sacl site using T4 DNA ligase. Escherichia coli (TOP10 strain) is transformed with the prepared vector, and E. coli clones containing the test vector by colony PCR method And whether or not the target clone could be selected was confirmed by agarose electrophoresis and direct sequencing. A test vector was isolated from the obtained clones according to a conventional method. Next, the isolated test vector and the control vector (pRL—SV40 vector) used as an internal standard were transduced into NB-1 cells or HeLa cells using Effectene (QIAGEN). Firefly luciferase activity and renilla luciferase activity of the obtained transduced strains were measured with a luminometer, and the influence of each polymorphism on promoter activity was examined based on the measured values (FIG. 6).

[0086] FIGS. 6A and B show the effect of each polymorphism on the promoter activity using NB-1 cells and HeLa cells, respectively. In the figure, the value of the test vector (null vect or) with nothing inserted is shown as 1.

In the case of g-aryl in C-7SNP, it was found that the promoter activity was lower than that of a-aryl. In the NA3. L microsatellite polymorphism, (AC) (SEQ ID NO: 22) aryl was found to be strongly associated with disease, and other aryl ((AC) (SEQ ID NO: 22

10 12

26), (AC) (SEQ ID NO: 24), (AC) (SEQ ID NO: 23))

9 8

As a result, it was found that the sex decreased to about 0.5 -0.6. The experiment was performed three times independently and similar results were obtained.

[0087] (References)

Ausubel et al., Urrent protocols in molecular biology. John Wiley & Sons, New Yor. 1 987.

Anderson et al., Science, 256: 808-813, 1992.

Becker et al., Methods. EnzymoL, 194: 182, 1990.

Bondanszky et al., Peptide Synthesis, Interscience Publishers, New York 199b.

Chen and Okayama, BioTechniques. 6: 632—638, 1988.

Cohen et al., Proc. Natl. Acad. Sci. USA 69: 2110, 1972.

Cohen et al., Oligodeoxynucleotides: Antisense inhibitors of gene expression. CRC Pres s, Boca Raton, FL. 255 pp. 1989.

Elroy- Stein and Moss, Proc. Natl. Acad. Sci. USA 87: 6743-6747, 1990.

Gennaro et al: The science and practice of pharmacy. Lippincott, Williams & Wilkins, P hiladelphia, PA. 2000.

Goding et al., Academic Press, San Diego. 492 pp. 1996.

Harlow and Lane, Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 726 pp, 1988.

Harlow and Lane, Using antibodies: A laboratory manual. Cold Spring Harbor Laboratory PRess, Cold Spring Harbor, New York. 1999.

Hinnen et al., Proc. Natl. Acad. Sci. USA. 75: 1929, 1978.

Honda et al., Genetic aspects of narcolepsy.In: Sleep and Sleep Disorders: From Mole cule to Behavior. (Eds.Hayaishi, O., and Inoue, E.)

Jones et al., Nature. 321: 522—5, 1986.

Kozbor et al., J Immunol. 133: 3001-5, 1984.

Kuwata et al., N. Engl. J. Med., 324: 271-272, 1991.

Lopata et al., Nucleic Acids Research. 12: 5707. 1984.

Martin and Papahadjopoulos, J Biol Chem. 257: 286-288, 1982.

Matsui et al., J. Clin. Invest., 76: 2078-2083, 1985.

Milstein and Cuello, Nature 305: 537-40, 1983.

Morrison et al., Genetically engineered antibody molecules and their application.Ann N

Y Acad Sci. 507: 187-98, 1987.

Okano et al., J Neurochem. 56: 560-7, 1991.

Presta et al., Curr Opin Biotechnol. 3: 394-8, 1992.

Riechmann et al., Nature 332: 323-7, 1988.

Sambrook, J. Molecular cloning: a laboratory manual. Cold Spring Harbor Laborator y, Cold Spring Harbor. 1989.

Schroeder et al., The peptide, Academic Press, New York 1965.

Shigekawa and Dower, BioTechniques. 6: 742—751, 1988.

Tonkinson et al., Cancer Investigation, 14 (1): 54-65, 1996.

Verhoeyen et al., Science. 239: 1534-6, 1988.

Claims

The scope of the claims

[I] An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:

(a) DNA having a base sequence ability represented by SEQ ID NO: 8,

[2] An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:

(a) DNA having a base sequence ability represented by SEQ ID NO: 9,

[3] A polynucleotide comprising the base sequence of the polynucleotide according to claim 1 or 2.

[4] The polynucleotide according to claim 2, which also has the nucleotide sequence represented by SEQ ID NO: 10 or 11.

[5] An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:

(a) DNA having a nucleotide sequence represented by SEQ ID NO: 12,

[6] A polynucleotide comprising the base sequence of the polynucleotide according to claim 5.

[7] The polynucleotide according to [6], comprising the base sequence represented by SEQ ID NO: 13.

[8] A polypeptide encoded by the polynucleotide according to any one of claims 1 to 7.

[9] A recombinant vector containing the polynucleotide according to any one of claims 1 and 7.

[10] An agonist for the polypeptide according to claim 8.

[II] An antagonist against the polypeptide according to claim 8.

[12] An antibody against the polypeptide according to claim 8.

[13] The vector according to claim 9 or the antigen according to claim 10 is contained as an active ingredient. A pharmaceutical composition for the treatment of narcolepsy.

[14] A method for screening an agonist for the polypeptide or a salt thereof, wherein the polypeptide or a salt thereof according to claim 8 is used.

[15] A method for screening for an antagonist to the polypeptide or a salt thereof, which comprises using the polypeptide or a salt thereof according to claim 8.

[16] A method for determining a subject's susceptibility to narcolepsy, comprising the following steps:

(a) collecting a biological sample containing chromosomal DNA from the subject;

A method comprising the steps of:

[17] A method for determining a subject's susceptibility to narcolepsy, comprising the following steps:

(a) collecting a biological sample containing chromosomal DNA from the subject;

(b) determining a DNA sequence located at positions 565 to 584 in the sequence represented by SEQ ID NO: 21 in the chromosomal DNA;

A method comprising the steps of: