JP2006217888A - Novel narcolepsy related genes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a narcolepsy-related gene and a polypeptide encoded by the gene, and a pharmaceutical composition for diagnosis and prevention or treatment of narcolepsy comprising the gene and the polypeptide peptide.
SOLUTION: The present invention narrows down a region where association is recognized by using a microsatellite marker set genome-wide, further sets an SNP marker showing a particularly strong association in the region, Narcolepsy-related genes are identified by analyzing linkage disequilibrium for these microsatellite markers that showed particularly strong association and analyzing linkage disequilibrium of these polymorphisms.
[Selection figure] None

Description

The present invention relates to narcolepsy-related genes. More specifically, a narcolepsy-related gene identified by a gene mapping method using microsatellite and SNP (single nucleotide polymorphism), a polypeptide encoded by the gene, and a gene encoded by the gene and the gene The present invention relates to a pharmaceutical composition comprising a peptide or an agonist or antagonist for the polypeptide.
Furthermore, the present invention relates to a method for determining the susceptibility to narcolepsy.

Narcolepsy, a typical hypersomnia, has repetitive unbearable sleepiness (sleeping seizures) that occur during the day, emotional weakness that suddenly loses muscle strength triggered by strong emotional movements, and REM sleep immediately after falling asleep. Characterized by abnormal REM sleep. It often develops in the 10s, there is no gender difference, and the prevalence in Japanese is 0.16-0.18%. In addition, it is reported that the coincidence rate of identical twins is 25-31%, and the incidence rate of first-degree relatives is 1-2%. In human narcolepsy, multiple genetic and environmental factors are involved in each other. It is considered a multifactorial disease. As a genetic factor that has been clarified so far, there is an HLA-DRB1 * 1501-DQB1 * 0602 haplotype that exists in the human leukocyte antigen (HLA) region, and almost all patients with Japanese 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% frequency in the healthy population), and there are many patients who do not have this haplotype, especially in the African population. Therefore, although the HLA-DR-DQ haplotype shows a very strong association with human narcolepsy, it is considered not a sufficient condition.
Matsui, K .; J. et al. Clin. Invest. 76: 2078-2083, 1985 Kuwata, S .; Et al. Engl. J. et al. Med. , 324: 271-272, 1991 Honda, Y .; Et al., Genetic Aspects of Narcolepsy. In: Sleep and Sleep Disorders: From Molecule to Behavior. (Eds. Hayashi, O., and Inoue, E.) Academic Press p. 341-358, 1998

In light of the above circumstances, the present inventors have conducted extensive research to search for susceptibility inheritance of narcolepsy disease other than HLA-DR-DQ haplotype, and as a result, succeeded in identifying a novel narcolepsy-related gene (polynucleotide). .
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, this invention aims at providing the antibody, agonist, and / or antagonist with respect to this polypeptide.
Still another object of the present invention is to provide a pharmaceutical composition comprising the gene, the polypeptide, the antibody, the agonist and / or the antagonist.
Another object of the present invention is to provide a screening method for the agonist and the antagonist.
Furthermore, an object of the present invention is to provide a method for determining the susceptibility to narcolepsy.

  So far, λs (risk rate) in human narcolepsy has been reported to be 12. However, from the HLA typing results of the individual samples analyzed this time, λs when only the HLA-DR-DQ haplotype was assumed to be involved in the disease was estimated to be 5.15, so other than the HLA-DR-DQ haplotype It was also suggested that there may be a disease susceptibility gene.

  Non-parametric linkage analysis (affected sibling pair analysis) for a large number of small families with multiple patients, and transmission for a large number of families with a single patient Three main methods are used: a transmission disequilibrium test (TDT) and a patient-control association analysis for an individual population without pedigree data. Human narcolepsy has few frequent families and is not suitable for analysis of affected sibling pairs. In addition, patient-control-related analysis is more powerful than TDT, but it is difficult to list candidate genes for diseases such as narcolepsy whose onset / pathologic mechanism is not clear. There is a problem that only genes can be analyzed. Therefore, the present inventors have adopted a new strategy called “genome-wide association analysis method using pooled DNA using microsatellite markers”. This method can perform comprehensive screening while maintaining high detection power by performing association analysis using a large number of microsatellite markers set at approximately equal intervals in the genome.

As a result of conducting a higher-density association analysis using a microsatellite polymorphism set at a higher density and a SNP around one of the microsatellite markers that showed a significant difference by the above method, the relationship with narcolepsy Succeeded in identifying a novel gene having
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 comprising the base sequence represented by SEQ ID NO: 8,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance.
(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 comprising the base sequence represented by SEQ ID NO: 9,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance.
(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 aspect of the present invention is an isolated polynucleotide comprising the following DNA (a) or (b) and a complementary strand thereof:
(A) DNA comprising the base sequence represented by SEQ ID NO: 12,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance.
(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 according to (6) above, comprising the base sequence 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) to (7) above.
(9) A ninth aspect of the present invention is a recombinant vector containing the polynucleotide according to any one of (1) to (7) above.
(10) A tenth aspect of the present invention is an agonist for the polypeptide according to (8) above.
(11) An eleventh aspect of the present invention is an antagonist for the polypeptide according to (8) above.
(12) A twelfth aspect of the present invention is an antibody against the polypeptide according to (8) above.
(13) A thirteenth aspect of the present invention is a pharmaceutical composition comprising the vector according to (9) or the agonist according to (10) as an active ingredient and used for the treatment of narcolepsy.
(14) A fifteenth aspect of the present invention is a method for screening for an agonist for the polypeptide or a salt thereof, characterized by using the polypeptide or a salt thereof according to (8) above.
(15) A sixteenth aspect of the present invention is a method for screening for an antagonist to the polypeptide or a salt thereof, characterized by using the polypeptide or a salt thereof described in (8) above.
(16) A seventeenth 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 the type of base at position 2524528 of position 22.3 of human chromosome 21 or the position of position 45238073 of position 22.3 of human chromosome 21;
(C) based on the result obtained in step (b), determining the susceptibility of the subject to narcolepsy,
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;
(C) based on the result obtained in step (b), determining the susceptibility of the subject to narcolepsy,
It is the method characterized by including.

  By elucidating the function of the novel gene according to the present invention in more detail, it can be expected to elucidate the pathogenesis of narcolepsy, and further promote the development of a new treatment method, therapeutic agent or sleep-inducing agent for narcolepsy Can do.

1. Polynucleotide of the Present Invention The polynucleotide of the present invention is represented not only by the DNA consisting of the base sequence represented by SEQ ID NO: 8 (NLC1-A) or 11 (NLC1-C) but also by SEQ ID NO: 8 or 11. Also included are those consisting of DNA encoding a polypeptide that hybridizes with a DNA consisting of a base sequence and a DNA consisting of a complementary base sequence under stringent conditions and has an activity of causing narcolepsy resistance. 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 transcription regulatory regions such as promoters and enhancers. The polynucleotide of the present invention includes DNA and RNA, and may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA, double-stranded RNA or DNA and It may be a hybrid with RNA.

  The DNA capable of hybridizing under stringent conditions with the polynucleotide containing the base sequence represented by SEQ ID NO: 8 or 12, preferably about 70% or more, more preferably the base sequence represented by SEQ ID NO: 8 or 12. About 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, most preferably about 99% of the DNA containing a nucleotide sequence having a polynucleotide sequence homology.

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 reanneal when the complementary strand is present in an environment close to but below its melting point.
Specifically, for example, as a low stringency condition, in a filter washing step after hybridization, washing is performed in a 0.1 × SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. Can be raised. Examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step. By making the stringent conditions higher, a highly homologous polynucleotide can be obtained.

  The narcolepsy related genes according to the present invention were also identified by gene mapping using microsatellite markers and SNPs markers. That is, it divided into two groups, a narcolepsy patient and a healthy person, and identified based on the presence or absence of the significant difference in the frequency of the allele which was examined. If there is no difference in a particular allelic frequency, it is considered that the narcolepsy-related gene is not present near that allele, and if there is a difference in a particular allelic frequency, the narcolepsy-related gene is It is thought that it exists near.

  In general, SNPs (single nucleotide polymorphisms) markers and microsatellite markers are known as genetic polymorphism markers for gene mapping. Since microsatellite markers have a large number of alleles, even markers that are set at a certain distance from narcolepsy-related genes have characteristics that show correlation, so it is extremely difficult to narrow down the search region in genome-wide analysis. It is valid. On the other hand, the SNPs marker is an effective marker for specifying related genes in detail within a region narrowed down to some extent. The narcolepsy-related gene according to the present invention could be identified by using both microsatellite markers and SNPs markers.

The gene mapping method using microsatellite is described in detail in, for example, the pamphlet of WO01 / 79482, but will be briefly described below.
Gene polymorphism means that there are two or more types of alleles crowned at the target locus, and the frequency of major alleles is 99% or less. The gene locus may be any region as long as it is a region on the genome, and is not limited to the region where the gene is expressed. Microsatellite means a sequence in which 2 to 6 bases are repeated, and the number of repetitions may vary among individuals, and the variation in repetitions forms a polymorphism called STR (Short Tandem Repeat). ing. This number of repeats determines the microsatellite polymorphism. A typical microsatellite is a CA repeat.

  First, microsatellite markers that are relatively rich in polymorphisms should be set at a genome-wide appropriate density, for example, 50 kb to 150 kb, preferably 80 kb to 120 kb, more preferably 90 to 110 kb. Can do. As the number of alleles (the number of alleles) of the microsatellite marker increases, the degree of heterozygosity increases and the amount of information in the analysis increases. A microsatellite marker showing a strong association can be searched by performing a statistical analysis and a relational analysis on the distribution of allele frequencies at each microsatellite locus and the deviation from the Hardy Weinberg equilibrium. Narcolepsy-related genes can be mapped more accurately by setting markers at high density in the vicinity of highly related microsatellite markers and further performing association analysis.

  For detecting the polymorphism of the microsatellite, for example, a primer set for PCR that can amplify the microsatellite marker region can be used. As polymorphism detection methods using PCR, in addition to the RFLP (restriction fragment length polymorphism) method, the SSCP (single strand formation polymorphism) method, the SSOP (sequence specific nucleic acid prodrugation method, and the SSOP (sequence specific RNA production method) A differential analysis (RAPD) method, a random amplified polymorphic DNA (RAPD) method, an amplified fragment length polymorphism (AFLP) method, or the like can be used.

After narrowing a candidate region where a narcolepsy-related gene is likely to exist to some extent by gene mapping using a microsatellite marker, SNP markers are set at appropriate intervals, and further association analysis is performed. SNP exists at a rate of 1 to 300-500 base pairs on the genome, and it is -100 times higher than the appearance frequency of microsatellite markers, so it is very useful for identifying candidate regions for narcolepsy-related genes in a region narrowed to some extent. It is an effective means.

  After further analysis with a microsatellite marker, in order to further narrow the candidate region where narcolepsy-related genes are expected to exist, for example, using a SNP database, a SNP marker is set at an appropriate interval, and a related analysis is performed. By analyzing linkage disequilibrium in the vicinity of the SNP in which a significant association is recognized, it becomes possible to identify a narcolepsy-related gene candidate.

  When a narcolepsy-related gene is identified using a gene mapping method, it can be used for examination of the genetic 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 by preparing a cDNA library from the expressing cells and using the narcolepsy-related gene of the present invention or a part thereof as a probe. Alternatively, RNA is prepared from cells in which the gene is expressed, cDNA is synthesized by reverse transcriptase, PCR primers are prepared based on the gene sequence, and the cDNA is amplified to obtain the cDNA. May be.

Any cell can be used for obtaining the polynucleotide of the present invention as long as the mRNA of the polypeptide is expressed. For example, in the case of NLC1-A, the spleen, lung, kidney In the case of NLC1-C, cells derived from tissues such as spleen, pancreas, lung, brain (whole brain), hypothalamus are preferably used. Further, 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.
When the DNA sequence of the narcolepsy-related gene disclosed herein is determined, based on the common general technical knowledge in the art, the gene or the ortholog of the gene is, for example, usable animal tissue, but not limited to, human, It can be easily obtained from a cDNA or cDNA library prepared from a tissue such as chimpanzee by the above-described method (for example, Sambrook, J. 1989. Molecular cloning 2nd eds: a laboratory manual. Cold Spring Harbor Laboratory, (See Cold Spring Harbor.)

  For the preparation of mRNA and cDNA for obtaining the polynucleotide of the present invention, commercially available kits such as mRNA Purification Kit (Pharmasia), AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) are used. It may be used.

2. Polypeptide of the present invention and partial peptide thereof The polypeptide of the present invention is 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 an amino acid sequence identical or substantially identical to an amino acid sequence. Here, the “polypeptide comprising substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. Is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, A polypeptide comprising an amino acid sequence having 96%, 97%, 98%, most preferably about 99% amino acid identity, and having an activity of causing narcolepsy resistance. Here, the activity that brings about resistance to narcolepsy is not limited to various symptoms of narcolepsy, but for example, biological activity to bring about the suppression of “during unbearable sleepiness that repeats during the day” and the suppression of cataplexy (weakness) ( For example, biochemical activity, physiological activity, etc.).

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, more preferably 1) in 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 10 to 10 (more preferably 1 to 5) amino acids have been 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, or by modifying the gene encoding the polypeptide of the present invention by a technique known in the art. It may be newly introduced. For example, the substitution of a specific amino acid residue is performed using a commercially available kit (for example, Mutan ^™ -G (TAKARA), Mutan ^™ -K (TAKARA)) or the like, and a known method such as the Gupdedduplex method or the Kunkel method. Alternatively, it can be achieved by performing base substitution by a method according to them.

Further, C-terminal, of the polypeptide of the present invention is usually a carboxyl group (-COOH) or a carboxylate (-COO ^-) is a, the carboxyl group, an amide (-CONH ₂₎ or ester (-COOR) etc. It may be chemically modified. Here, as R in the ester, a C _1-6 alkyl group (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl), a C _3-8 cycloalkyl group (for example, cyclopentyl, cyclohexyl), C _1-6 aryl group (for example, phenyl, α-naphthyl), phenyl-C _1-2 alkyl group (for example, benzyl, phenethyl), α-naphthyl-C _1-2 alkyl group (for example, α-naphthylmethyl) and the like Is mentioned. In addition, pivaloyloxymethyl ester, which is widely used as an oral ester, can be used. 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 amidated or esterified. Examples of the ester in this case include the above-mentioned esters. Similarly, the N-terminus of the polypeptide of the present invention is usually an amino group (—NH ₂ ), which is chemically modified with a C _1-6 acyl group such as a formyl group or an acetyl group. Also good. In addition, the glutamyl group produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized, or a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group) , A guanidino group, etc.) that are chemically modified with an appropriate functional group (for example, formyl group, acetyl, etc.) and those that are linked to a sugar chain are also included in the polypeptide of the present invention.

Peptides (also referred to as partial peptides) containing a partial amino acid sequence in any of the above polypeptides according to the present invention are also included in the scope of the present invention. That is, the partial peptide of the present invention is not limited as long as it includes a part 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. There may be.
The number of amino acids constituting the partial peptide of the present invention is at least 10 or more, preferably 30 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 ), but may be chemically modified.

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

  The polypeptide of the present invention or a salt thereof can be extracted / separated from a cultured cell or tissue derived from a human or animal (for example, chimpanzee etc.) expressing the polypeptide of the present invention by a conventional technique in the art. Alternatively, as described later, it can also be prepared by culturing a transformant containing a DNA encoding the polypeptide of the present invention in a state capable of being expressed, and extracting and separating from the culture. When preparing from human or animal tissues or cells, homogenize human or animal tissues or cells, extract with acid, etc., and use the resulting extract for hydrophobic chromatography, reverse phase chromatography, ion exchange chromatography, etc. It can be isolated and purified by combining various types of chromatography.

The partial peptide of the present invention or a salt thereof can be produced by cleaving a known peptide synthesis method or the polypeptide of the present invention with an appropriate peptidase (for example, trypsin, chymotrypsin, arginyl endopeptidase). 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 can be produced 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 to an appropriate salt by a known method. When obtained by a salt, it is released by a known method. Can be converted into a body.

3. 3. Production of recombinant vector and transformant 3-1. Production of Recombinant Vector The recombinant vector of the present invention encodes the polypeptide of the present invention (including polypeptides substantially the same as the polypeptide of the present invention and partial peptides thereof, and so forth) on an appropriate vector. It can be obtained by ligating DNA. 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 can be replicated in a host and has a promoter capable of expressing a DNA fragment encoding the polypeptide can be used. is there.

  Examples of vectors that can be used include plasmid DNA and phage DNA. Plasmid DNA includes plasmids derived from E. coli (for example, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, etc.), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5, pC194, etc.), yeast-derived plasmids (for example, YEp13, YEp24, YCp50, YIp30, etc.) and the like, and examples of the phage DNA include λ phage. Furthermore, animal viruses such as retroviruses and vaccinia viruses, and insect virus vectors such as baculoviruses and togaviruses can also be used.

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, SRα promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter, EF-1α promoter and the like can be mentioned.
When the host is Escherichia coli, tac promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc. are mentioned. When the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter, etc. are mentioned. It is done.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

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, terminator, enhancer, splicing signal, poly A addition signal, ribosome binding sequence (SD sequence), SV40 replication origin ( SV40ori) or the like can be connected.
The selection markers include, but are not limited to, hygromycin resistance marker ^(Hyg r), dihydrofolate reductase gene (dhfr), the ampicillin resistance gene ^(Amp r), the kanamycin resistance gene ^(Kan r), neomycin resistant gene (Neo ^r , G418) can be used.
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. When the host is Bacillus subtilis, α-amylase signal sequence, subtilis signal sequence, etc. can be used. In some cases, α-factor signal sequence, invertase signal sequence, etc. can be used. When the host is an animal cell, for example, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. It is.

Inserting a polynucleotide encoding the polypeptide of the present invention into the above-described vector means adding a linker by digesting the cloned DNA encoding the polypeptide of the present invention as it is or, if desired, with a restriction enzyme. However, it can be carried out by inserting the vector DNA into a restriction enzyme site or a multicloning site. The DNA to be ligated may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter. The DNA 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.

3-2. Production of Transformant The transformant of the present invention can be obtained by introducing the recombinant expression vector of the present invention into a host so that a 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. For example, Escherichia genus such as Escherichia coli, Bacillus genus such as Bacillus subtilis, Pseudomonas putida genus Pseudomonas genus, Rhizobium merilloti genus Rhizobium genus Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), yeasts such as Pichia pastoris (Pichia pastoris), monkey cells COS-7, O hams S Insect cells such as

As a method for introducing a recombinant vector into Escherichia coli, a method using calcium ions (Cohen et al., 1972), an electroporation method (Shigekawa and Dower, 1988) and the like can be used. As a method for introducing a recombinant vector into yeast, an electroporation method (Becker et al., 1990), a spheroplast method (Hinnen et al., 1978), a lithium acetate method (Itoh et al., 1983) and the like can be used. As a method for introducing a recombinant vector into animal cells or animal cells, DEAE dextran method (Lopata et al., 1984), electroporation method, calcium phosphate method (Chen and Okayama, 1988), cationic lipid method (Elroy-Stein) And Moss, 1990).
As described above, a transformant containing an expression vector into which a DNA encoding the polypeptide of the present invention has been inserted can be obtained.

4). Production of polypeptide of the present invention and partial peptide thereof The polypeptide of the present invention is obtained by culturing a transformant introduced with an expression vector of a narcolepsy-related gene, expressing the polypeptide of the present invention from the gene, It can be produced by isolating the polypeptide. “Culture” means any of culture supernatant, cultured cells or cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.

  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, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. 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. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  Culturing is performed under conditions suitable for the host cell. For example, as the medium for culturing Escherichia coli, LB medium, M9 medium and the like are preferable. Agents such as isopropyl-1-thio-β-D-galactoside, 3β-indolylacrylic acid can be added to make the promoter work efficiently if desired. In the case of Escherichia coli, the culture is usually performed 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, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added as necessary.

  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. The culture is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary. When culturing a transformant whose host is an insect cell or an insect, examples of the medium include a grace insect medium containing bovine serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

  When cultivating a transformant whose host is an animal cell, for example, a MEM medium, DMEM medium, RPMI 1640 medium, or the like containing about 5 to 20% fetal bovine serum is used as the medium. The pH is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added 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.

  When the polypeptide of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw. For example, a method of obtaining a crude extract of the polypeptide of the present invention by centrifugation or filtration after destroying cells or cells by, for example, is suitably used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant 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 collected. Purification of the polypeptide of the present invention contained in the culture supernatant or extract thus obtained can be performed by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation, dialysis methods, ultrafiltration methods, gel filtration methods, and mainly using differences in molecular weight such as SDS-PAGE. Method, method utilizing the difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric point A method using the difference in isoelectric point such as electrophoresis is used.

5. Nucleic acid that inhibits the function of the gene encoding the polypeptide of the present invention The nucleic acid that inhibits the function of the gene encoding the polypeptide of the present invention causes sleep by losing the function of the gene encoding the polypeptide of the present invention. It seems that the introduction effect can be brought about. Examples of nucleic acids that inhibit gene function include single-stranded nucleic acids such as antisense RNA and DNA and derivatives thereof, and short double-stranded RNAs having a sequence complementary to a part of the gene region. .
Antisense may be RNA or DNA or derivatives thereof and acts as an effective inhibitor for the expression of the gene encoding the polypeptide of the present invention. Antisense RNA is designed, for example, to hybridize with mRNA in vivo and inhibit translation of mRNA to GZF1 protein (Okano et al., 1991). In addition, 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, for example, an oligonucleotide that binds to a position between about -10 and +10 of the target gene sequence is desirable.
Moreover, 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 target mRNA (for example, mRNA of a narcolepsy-related gene) is degraded by double-stranded RNA having a sequence complementary to the target mRNA. By using this phenomenon to artificially introduce double-stranded RNA, expression of the target gene can be suppressed.

6). Antibody against the polypeptide of the present invention The antibody against the polypeptide of the present invention inhibits the function of the polypeptide of the present invention to bring about a sleep-inducing effect or promote the function of the polypeptide of the present invention. Can suppress the susceptibility to narcolepsy.
The present invention includes antibodies that specifically bind to a polypeptide of the present invention, and antibody fragments thereof, such as Fab or F (ab ′) 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 monoepitope specific antibody against the polypeptide of the present invention, poly Epitope specific antibodies, single chain antibodies, and fragments thereof are included. These antibodies include, for example, monoclonal antibodies, polyclonal antibodies, humanized 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 / or adjuvant is injected 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 synthesis-trehalose dicorynomycolate (MPL-TDM). In particular, when a partial peptide of the polypeptide of the present invention is used as an immunogen, immunogenicity such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor is used to enhance the immune response. The protein may be injected after the protein is bound to the immunogen.
Alternatively, it may be prepared using chickens 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 hybridoma method (Milstein and Cuello, 1983).
This method includes the following four steps: (i) immunizing a host animal or lymphocytes from the host animal, (ii) monoclonal antibody secreting (or potentially secreting) lymphocytes Harvest, (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 the immunogen is injected. Alternatively, lymphocytes obtained from immunized animals may be immunized in vitro. If human cells are desired, peripheral blood lymphocytes (PBLs) are generally used. However, spleen cells or lymphocytes from other mammals are more common and preferred. Immunogens also include fusions with the polypeptides of the invention and heterologous polypeptides or fragments thereof.

  After immunization, lymphocytes obtained from the host animal are fused with an immortal cell line using a fusing agent such as polyethylene glycol to establish hybridoma cells (Goding, 1996). As fusion cells, rodent, bovine, or human myeloma cells immortalized by transformation are used, or 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 parent 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 hybridomas.

For the preparation of monoclonal antibodies, the preferred immortal cell line is the mouse myeloma line 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 hybridoma cells secrete antibodies outside the cells, the presence or absence of production of the desired monoclonal antibody can be confirmed using a culture solution. The binding specificity of the monoclonal antibodies produced can be assessed by immunoprecipitation such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) 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's Medium, RPMI-1640, and in some cases, protein-free or serum-free media. Hybridoma cells may also be grown in the ascites of a suitable host animal.

Monoclonal antibodies are well known to those skilled in the art, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999) from medium or ascites. Isolated and purified by
Monoclonal antibodies can also be produced by gene recombination techniques (US Pat. No. 4,166,452, 1979). To identify the gene encoding the monoclonal antibody polypeptide of interest from a hybridoma cell line secreting the antibody of interest, for example, using oligonucleotide probes that specifically bind to mouse heavy and light chain antibody genes Good. 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 a monoclonal antibody, the isolated DNA fragment introduces an antibody gene into an appropriate expression vector, and the vector does not produce any other immunoglobulin protein, such as simin COS-7 cells, Chinese hamster ovary (CHO). Transfect into cells or host cells such as 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 replace the constant domain of the antibody or can be replaced with the constant domain of one antigen binding site to prepare a chimeric bivalent antibody.

6-3. Humanized and human antibodies Polypeptide antibodies of the present invention include humanized or human antibodies. Humanized forms of non-human antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (Fv, Fab, Fab ′, F (ab ′) ₂ ) or other antibody antigen binding that contain minimal sequences derived from non-human immunoglobulin. Area).
In general, humanized antibodies have one or more amino acid residues introduced from non-human-derived immunoglobulin. These non-human amino acid residues are often selected from variable domains. Humanized antibodies can be produced, 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). ). That is, a humanized antibody is a human antibody in which a certain residue in a specific CDR derived from human and a residue in a CDR of a non-human species such as mouse, rat or rabbit corresponding to the residue are substituted. That is. In addition, nonhuman-derived residues may replace Fv framework residues of human immunoglobulin (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

7). Agonists and antagonists for the polypeptide of the present invention The “agonist” in the present invention specifically binds to the polypeptide of the present invention or its receptor, interacting factor (binding partner), and the biology of the polypeptide of the present invention. Is intended to promote pharmacological activity, and some of the above-mentioned antibodies are also included in the “agonist”. Examples of the agonist other than the antibody include, but are not limited to, a polypeptide or a fragment thereof, a nucleic acid, and other low molecular weight compounds. In addition, the “antagonist” in the present invention is 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. Meaning and some of the above-mentioned antibodies are also included in “antagonists”.

8). Pharmaceutical Composition The polynucleotide, polypeptide or antibody of the present invention can be expected to exhibit 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” refers to those suitable for pharmaceutical administration, including solvents, dispersion media, coating agents, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like. (Gennaro, 2000). Examples of such carriers and those preferred for diluting the carriers include, but are not limited to, water, saline, finger solutions, dextrose solutions, and human serum albumin. Non-aqueous media such as liposomes and non-volatile oils are also used. Furthermore, certain compounds that protect or promote the activity of the polynucleotides, polypeptides or antibodies of the present invention may be included in the composition.

The pharmaceutical composition according to the present invention includes intravenous, intradermal, subcutaneous, oral (including inhalation, etc.), transdermal and transmucosal administration, and is formulated to be suitable for a therapeutically appropriate route of administration. 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 solutions, non-volatile oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents, benzyl alcohol or other preservatives such as methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, soothing agents such as benzalkonium chloride, procaine hydrochloride, ethylenediaminetetraacetic acid Chelating agents such as (EDTA), buffering agents such as acetate, citrate, or phosphate, and agents for osmotic pressure adjustment such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are contained in ampoules, glass or plastic disposable syringes or multiple dose vials.

8-1. Injectable Formulations Pharmaceutical compositions suitable for injection include sterile injectable solutions or dispersion media in sterile aqueous (water soluble) or dispersion media and sterile powders for preparation at the time of use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL ^™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). When used as an injection, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes, corrosion, and the like during formulation and storage, and must prevent contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures. For example, using a coating agent such as lectin, maintaining a 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, polyalcohols such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition. Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.

  A sterile injectable solution may contain the required amount of active compound (eg, a polynucleotide, polypeptide, antibody, etc. of the invention) in a suitable solvent after the required component alone or in combination with other components. In addition, it is prepared by sterilization. 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 described 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 ingredients derived from the sterile solution. It is.

8-2. Oral Compositions Oral compositions usually include an inert diluent or a carrier that does not harm when incorporated 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. Oral compositions can also be prepared using a flowable carrier, and the composition in the flowable carrier is applied orally. In addition, pharmaceutically compatible binding agents, and / or adjuvant materials may be included.
Tablets, pills, capsules, troches and the like may contain any of the following components or compounds with similar properties: excipients such as microcrystalline cellulose, binding such as gum arabic, tragacanth or gelatin Agents; bulking agents such as alginic acid, PRIMOGEL or corn starch such as starch or lactose; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl A fragrance additive such as salicylic acid or orange flavor.

8-3. Carriers The polynucleotides, polypeptides, and antibodies of the present invention are prepared using sustained-release preparations such as delivery systems encapsulated in implantable tablets and microcapsules using carriers that can prevent immediate removal from the body. be able to. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be obtained from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), etc., and can also be easily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanol (PEG-PE) through a filter of appropriate pore size to obtain a size suitable for use, and reverse phase. Purified by evaporation. For example, Fab ′ fragments of antibodies and the like may be bound to liposomes via a disulfide exchange reaction (Martin and Papahadjopoulos, 1982).

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., the 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, it is preferable to administer about 0.1 μg / kg to about 500 mg / kg of the patient's body weight per day, and it will generally be possible to administer a single dose or divided into multiple doses. Preferably, the dosage level is about 0.1 μg / kg to about 250 mg / kg per day, more preferably about 0.5 to about 100 mg / kg per day.
For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 to 1000 mg of active ingredient, preferably 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 1000.0 mg. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily.

8-5. Unit Dosage A pharmaceutical composition or formulation must be composed of uniform unit dosages to ensure a certain dosage. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. The unit dosage of the present invention is determined by the physical and chemical characteristics of the compound to be formulated, the expected therapeutic effect, and the formulation considerations specific to the compound. .

8-6. Gene therapy composition The 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 the narcolepsy-related gene of the present invention) is introduced into a patient's cell. There are two main methods, in vivo or ex vivo. For in vivo delivery, it is injected directly into the site of the patient in need of treatment. In ex vivo treatment, a cell at a site intended for treatment of a patient is isolated, a formulated nucleic acid molecule is introduced into the isolated cell, and the introduced cell is directly inserted into the patient or, for example, porous that is implanted in the patient. Can be administered in an encapsulated form (see US Pat. Nos. 4,892,538 and 5,283,187). The technique available for introducing a nucleic acid molecule into a living cell is selected depending on whether it is introduced into a cultured cell or the like in vitro or introduced into a patient in vivo. Suitable techniques for introducing nucleic acid molecules into mammalian cells in vitro include liposomes, electroporation, microinjection, transfection, cell fusion, the DEAE-dextran method, and calcium phosphate precipitation. Transfection involves the binding of a recombinant viral (preferably retroviral) particle to a cellular receptor, followed by introduction of the nucleic acid molecule contained in the particle into the cell. A commonly used vector for ex vivo delivery of genes is a retrovirus.

Currently preferred in vivo nucleic acid transfer techniques are viral or non-viral vectors (adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV)), and cationic lipid-based systems (lipid mediated transfer of genes). Useful lipids include, for example, DOTMA, DOPE, and DC-Cho1; see, for example, Tonkinson et al., Cancer Investigation, 14 (1): 54-65 (1996)). The most preferred vector for use in gene therapy is a virus, of which adenovirus, AAV, lentivirus or retrovirus is most preferred. Viral vectors, such as retroviral vectors, include at least one transcription promoter / enhancer or locator. Furthermore, a viral vector such as a retroviral vector contains a cis element that enables translation of the encoded gene, that is, a nucleic acid sequence that functions as a translation initiation sequence when transcribed in a state including a narcolepsy-related gene. . Such vector constructs contain a packaging signal, a terminal repeat (LTR) or part thereof suitable for the virus used. In addition, these vectors usually contain a signal sequence that causes the expressed polypeptide to be secreted from the host cell 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 packaging 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 may be desirable to provide a nucleic acid for treatment with a reagent that targets the cell of interest, for example, an antibody specific for a cell surface membrane protein, or a ligand for a receptor on a target cell. For review of currently known gene labeling and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992).

9. Kits for pharmaceutical compositions The pharmaceutical compositions can be included in the kit, container or pack with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different constituents of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately in this way is to enable long-term storage without losing the function of the active component.
8-1. The reagent contained in the container kit is supplied in any kind of container in which the constituents maintain the activity effectively for a long period of time, are not adsorbed by the material of the container, and are not altered. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.
9-2. Instructions for use The instructions for use are also attached to the kit. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

10. Other medical and pharmaceutical applications such as screening of agonists and antagonists for the polypeptide of the present invention The homology search suggests that the polypeptide of the present invention may be a membrane protein. Therefore, the determination of agonists or antagonists for the polypeptide of the present invention and its partial peptides or salts thereof is expected to be very useful for the development of narcolepsy therapeutic agents and sleep induction agents.
The polypeptides of the present invention and partial peptides thereof or salts thereof, and the polynucleotides encoding them are (i) determination of agonists or antagonists to NLC1-A or NLC1-C polypeptides, (ii) prevention of narcolepsy and / or Or a therapeutic agent, (iii) sleep-inducing agent, (iv) genetic diagnostic agent, (v) quantification of antagonist or antagonist to NLC1-A or NLC1-C polypeptide, (vi) NLC1-A or NLC1-C polypeptide Screening for compounds that alter the binding properties of agonists or antagonists, (vii) prevention and / or prevention of diseases containing compounds that alter the binding properties of NLC1-A or NLC1-C polypeptides to agonists or antagonists A therapeutic agent, (viii) quantification of NLC1-A or NLC1-C polypeptide or a partial peptide thereof, (ix) neutralization by antibody against NLC1-A or NLC1-C polypeptide and antagonist or partial peptide thereof, (x) NLC1- It can be used to create a non-human animal having or deleting a gene encoding A or NLC1-C polypeptide.

Specifically, the polypeptide of the present invention or a partial peptide thereof, or a salt thereof is useful as a reagent for screening or identifying an agonist or antagonist for the polypeptide of the present invention. These reagents can be used as components of a screening kit for a substance that changes the binding property of an agonist or antagonist to the polypeptide of the present invention.
As described above, the present invention relates to an agonist or antagonist for NLC1-A or NLC1-C polypeptide, which comprises 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 tissue extracts of humans or mammals (for example, chimpanzees, etc.), cell culture supernatants, artificially synthesized compounds, and the like.

  In order to carry out the method for determining an agonist or antagonist for the NLC1-A or NLC1-C polypeptide of the present invention, an appropriate NLC1-A or NLC1-C polypeptide fraction and a label (radiolabel, fluorescent dye label, etc.) ) Test substances are used. Examples of the NLC1-A or NLC1-C polypeptide fraction include a natural NLC1-A or NLC1-C polypeptide fraction, or a recombinant NLC1-A or NLC1-C polypeptide fraction having an activity equivalent thereto. For example, to make an agonist or antagonist determination for an NLC1-A or NLC1-C polypeptide or salt thereof, first a cell or a membrane fraction of a cell containing the NLC1-A or NLC1-C polypeptide, NLC1-A or NLC1-C polypeptide preparations are prepared by suspending in a buffer suitable for the determination method. The buffer may be any buffer as long as it does not inhibit the binding between a polypeptide and an agonist or antagonist, such as a phosphate buffer or Tris-HCl buffer. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80, deoxycholate, or a protein such as bovine serum albumin or gelatin can be added to the buffer. A test substance labeled with a certain amount (such as radioactive label or fluorescent dye label) is allowed to coexist in a solution containing NLC1-A or NLC1-C polypeptide. In order to know the amount of non-specific binding, a reaction tube to which a large excess of unlabeled test compound is added is also prepared. The reaction is carried out at about 4-50 ° C., preferably about 4 ° C.-37 ° C., for about 10 minutes-24 hours, desirably about 30 minutes-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 agonist or antagonist of the present invention.

10. Diagnosis When abnormalities are found 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 developed or is at high risk of developing. In this case, the narcolepsy-related gene of the present invention (including not only the exon region but also the intron region and the promoter region) or a mutation of the polypeptide encoded by the gene (eg, point mutation, deletion, etc.), By examining the presence or absence of mutations in the expression control region, it is possible to predict the occurrence of narcolepsy and the risk of the onset. In particular, the NA3. The L microsatellite polymorphism and C-7 SNP affect the regulation of NLC1-A gene expression and are expected to have an important effect on the development of narcolepsy.

  In order to examine the expression level of the polypeptide of the present invention, an antibody specific to 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 contacted with the test sample, and the presence or absence of binding between the test sample and the antibody is detected. The onset or likelihood of onset can be examined. The presence or absence of binding between the test sample and the antibody can be confirmed by immunoprecipitation using an antibody, Western blotting, immunohistochemistry, ELISA, or the like. As a result of 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.

  In order to confirm the abnormality of the gene of the present invention, a primer, probe, etc. that can anneal with the cDNA sequence, genomic DNA sequence (including transcription control region, intron region, etc.) of these genes or their complementary strands are used. be able to. The primer that can be used is generally 15 bp to 100 bp, and preferably has a length of 17 bp to 30 bp. The coding region and non-coding region of the gene of the present invention (including transcription control region, intron region, etc.) Any material can be used as long as it can amplify at least a partial region.

  The available probe generally has a length of 15 bp or more and can hybridize 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.

Other methods for confirming the gene abnormality of the present invention include, for example, obtaining a test sample from a narcolepsy patient or a patient suspected of developing narcolepsy, and mRNA of a narcolepsy-related gene prepared from the test sample, or a step of contacting the probe, primer and the like with cDNA prepared from mRNA and detecting binding to 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; Alternatively, a 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, the occurrence of narcolepsy or the risk of developing narcolepsy can be predicted.

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 fragment length polymorphism) in addition to the method of directly determining the base sequence. In addition to the method, the SSCP (single strand conformation polymorphism) method, the SSOP (sequence specific amplification probe) method, the RNase protection method, the RDA (representation differential PD) method, and the RDA (representative differentiation method). Or the like can be mentioned length polymorphism) method. In these methods, the above primers can be used.
The test by the above method is, for example, extracting chromosomal DNA from a sample collected from a subject according to a standard method, and using the primer set having sequences represented by SEQ ID NO: 2 and SEQ ID NO: 3 for the chromosomal DNA, The microsatellite NA3. By examining the sequence of L, and detecting SNP at 45242528 position 22.3 of human chromosome 21 using a primer set having the sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, Alternatively, it can be carried out by detecting a SNP at position 45238073 at position 22.3 of human chromosome 21 using a primer set having the sequences represented by SEQ ID NO: 6 and SEQ ID NO: 7 (position of SNP) (According to UCSC Genomic QueryTool May 2004 version of NetAffx ^™ Analysis Center, as used herein).

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

1. Preliminary study on searching for new narcolepsy-related genes:
Genomic DNA was extracted from peripheral blood obtained from Japanese narcolepsy patients who obtained 220 informed consent, and the concentration of double-stranded DNA was accurately measured using Cyber Green (PicoGreen, Molecular probes). Two sets of pooled DNA were prepared by mixing equal amounts of 110 people each, and 1st set and 2nd set were made respectively. Similarly, two sets of pooled DNA were prepared from 210 genomic DNAs obtained from healthy controls, each of which was pooled in an equal amount of 210 people. 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 as a template was performed by an automatic sequencer using GeneScan software (Applied Biosystems).

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 on chromosome 6 where the HLA gene group is located. As a result of the primary screening for the 1st set of patients and the 1st set of controls, a significant difference was observed by a statistical analysis method using 2 × 2 Fisher test method with 202 markers. When secondary screening was performed on those 2nd sets targeting 202 markers, significant differences were still observed in 42 types of markers, and primary and secondary screening for HLA-DR-DQ gene neighboring markers as expected. A strong association was found (p <10 ⁻³⁴ ). In order to analyze the HLA region in more detail, analysis was performed using a microsatellite marker set at high density, and 22 markers out of 30 markers covering 5.5 megabases showed significant differences, and HLA class I or class III Significant differences were also observed in markers near the HLA-A, B, and C4A genes located in the region (p <10 ⁻⁶ , p <10 ⁻⁹ , p <10 ⁻²⁴ ). From the above results, it was considered possible to detect disease susceptibility candidate regions by association analysis using microsatellite markers set approximately every 100 kb. On the other hand, no marker showing a very strong association other than the HLA region was detected on chromosome 6.

3. Association analysis with genome-wide microsatellite markers:
A related analysis was performed on 23,381 microsatellite markers (average gap 130.3 kb) (Table 1) distributed genome-wide. For microsatellite markers that were confirmed to be strongly related, a high density marker nearby was used. The candidate area was narrowed by performing association analysis.

As shown in FIG. 1, significant differences were observed by at least one statistical analysis for about 3,000 markers by primary screening using the 1st set. As a result of secondary screening using these markers and the 2nd set, a significant difference was recognized again for more than 300 kinds of markers.
In order to confirm the significance detected in the primary and secondary genome-wide association analysis using pooled DNA, 91 markers with high reproducibility in the primary and secondary microsatellite peak patterns were identified. Individual samples of 95 people were typed. This analysis confirmed significant differences again for the 14 markers. When all individual samples were typed for these, particularly low p-values were observed for the three microsatellite markers (p <0.001).

Furthermore, in order to perform high-density mapping of these three regions, when a new microsatellite marker polymorphism was screened around 500-700 kb around each marker, microsatellite markers could be set at an average interval of about 40 kb. . When association analysis was performed using these, a significant difference was also observed for markers located in the immediate vicinity of the 3 markers that were found to be associated in the screening. In particular, the marker (NA3.L) (SEQ ID NO: 1) 70 bk away from the NA3.4 marker showed a stronger association than the NA3.4 marker (p = 0.00045). NA3. The allele frequency of L is shown in Table 2 for patients and healthy subjects. From this result, NA3. It was found that the susceptibility to narcolepsy can be determined by examining the L microsatellite marker.

Based on the above analysis, the area around NA3.4 of 70 kb could be a disease-related candidate region. For the area around NA3.4, in order to further narrow these candidate areas, association analysis was performed by setting SNP markers at an average interval of 10 kb based on the SNP database, and for the vicinity of SNPs where significant association was observed, New SNP screening was performed on samples of 8 patients and healthy individuals. When these SNPs were subjected to association analysis using individual samples of 190 patients and 190 controls, 8 types of SNPs showing significant differences were found (FIG. 2). NA3. Which showed particularly strong association. As a result of analysis using two samples for two SNPs in the vicinity of the L marker, as shown in FIG. 3, the same or stronger association with the microsatellite marker was shown (p = 0.0002, p <0. 0001). Furthermore, when linkage disequilibrium was analyzed about these polymorphisms, NA3. L and two SNPs were found to enter one linkage disequilibrium block (FIG. 4). These two SNPs are the C / T polymorphism (C-4,) and the A / G polymorphism (C-7), each of which has a base at position 45242528 at position 22.3 of human chromosome 21, or It exists at 45238073 of 22.3 position of chromosome 21. The phenotypic frequencies and allele frequencies of each SNP are shown in Tables 3 and 4 for patients and healthy subjects. From this result, it was found that the susceptibility to narcolepsy can be determined by examining the two SNPs.

Therefore, it was suggested that some narcolepsy susceptibility gene (polymorphism) may exist in this block. There are no known genes in this block, and multiple ESTs and mRNAs are reported in the database at the same position, suggesting the presence of three novel genes. The new genes were named NLC1-A, NLC1-B and NLC1-C. Among these genes, the long form and short form of NLC1-A mRNA are shown as SEQ ID NO: 10 and SEQ ID NO: 11, respectively. Further, NLC1-C mRNA is 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 NLC1-C is shown in SEQ ID NO: 12. Furthermore, it was confirmed that NLC1-A and NLC1-C were expressed in the brain and hypothalamus (FIG. 5).

  When RT-PCR was performed on a human organ poly A RNA sample purchased from Clontech using a primer set consisting of the sequences of SEQ ID NO: 14 and SEQ ID NO: 15, an ORF of the expected length of NLC1-A A band of an amplification product corresponding to (FIG. 5A) could be confirmed (FIG. 5B). Similarly, when RT-PCR was performed using a primer set comprising the sequences of SEQ ID NO: 16 and SEQ ID NO: 17 (a band of an amplification product corresponding to the ORF of the expected length of NLC1-C (FIG. 5A) Was confirmed (FIG. 5B).

Furthermore, the predicted amino acid sequences of NLC1-A long form and short form are shown in SEQ ID NO: 18 and SEQ ID NO: 19, and the predicted amino acid sequence of NLC1-C is shown in SEQ ID NO: 20.
When the characteristics on the amino acid sequence of the long form of NLC1-A shown in SEQ ID NO: 18 and the presence or absence of a specific motif were examined, most of the regions having at least one transmembrane region and constituting the long form of NLC1-A Was expected to be present outside the membrane. Table 5 shows the predicted structural features of the NLC1-A long form. In addition, the long form of NLC1-A has a binding protein-dependent transport systems inner membrane component domain that is a member of the superfamily of transporters found in humans. A region similar to the characteristic motif structure was found.

NA3. Since the L microsatellite polymorphism and the C-7 SNP polymorphism are present in the promoter region and the intron region, respectively, they affect the transcriptional control of the NLC1 gene rather than the polypeptide itself encoded by the NLC1 gene. May affect. Thus, the effect of these two polymorphisms on promoter activity was examined.
As an assay system, DUAL-Luciferase Reporter Assay System (Promega) was used, and experiments were performed according to the protocol attached to the kit.
First, an intron region (297 bp) containing a C-7 SNP polymorphism and NA3. The promoter region (907 bp) containing the L microsatellite polymorphism was excised with KpnI-SacI, and a test vector was prepared using T4 DNA ligase at the multicloning site of the pGL3-control vector (KpnI-SacI site). E. coli (TOP10 strain) is transformed with the prepared vector, E. coli clones containing the test vector are selected by colony PCR, and whether the target clone has been selected is confirmed by agarose electrophoresis and direct sequencing. went. From the obtained clone, a test vector was isolated 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). The firefly luciferase activity and Renilla luciferase activity of the obtained transduced strain were measured with a luminometer, and the influence of each polymorphism on the promoter activity was examined based on the measured values (FIG. 6).

6A and 6B show the influence of each polymorphism on the promoter activity using NB-1 cells and HeLa cells, respectively. In the values shown in the figure, the value of a test vector (null vector) into which nothing is inserted is shown as 1.
In the case of g-allyl in C-7 SNP, it was found that the promoter activity was lower than that of a-allyl. NA3. In the L microsatellite polymorphism, (AC) ₁₀ (SEQ ID NO: 22) allele was found to be strongly associated with the disease, and other alleles ((AC) ₁₂ (SEQ ID NO: 26), (AC) ₉ (SEQ ID NO: 24). ) And (AC) ₈ (SEQ ID NO: 23)), the promoter activity was found to be reduced to about 0.5-0.6. The experiment was performed three times independently and similar results were obtained.

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It is a graph which shows distribution of p value obtained by the genome-wide association analysis using a microsatellite marker. It is a graph which shows the p value obtained by the related analysis by the SNP marker in the area | region narrowed down by the mapping by a microsatellite, and the odds value in the SNPs by which the significant difference was seen. Regions narrowed by association analysis using microsatellite markers and SNPs are shown. The result (p value) of the related analysis performed using the polymorphism detected by performing the polymorphism analysis of NLC1 area | region is shown. It is a figure which shows the expression of NLC1-A and NLC1-C in a brain. C-7 SNP polymorphism and NA3. The influence with respect to the promoter activity of L microsatellite polymorphism is shown. The vertical axis indicates the allyl name of each polymorphism, and the horizontal axis indicates the activity ratio with the null vector.

Claims (17)

An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:
(A) DNA comprising the base sequence represented by SEQ ID NO: 8,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance. An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:
(A) DNA comprising the base sequence represented by SEQ ID NO: 9,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance.   A polynucleotide containing the base sequence of the polynucleotide according to claim 1 or 2.   The polynucleotide according to claim 2, comprising the base sequence represented by SEQ ID NO: 10 or 11. An isolated polynucleotide comprising the following DNA (a) or (b) and its complementary strand:
(A) DNA comprising the base sequence represented by SEQ ID NO: 12,
(B) DNA encoding a polypeptide that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and has an activity of bringing about narcolepsy resistance.   A polynucleotide comprising the base sequence of the polynucleotide according to claim 5.   The polynucleotide of Claim 6 consisting of the base sequence represented by SEQ ID NO: 13.   A polypeptide encoded by the polynucleotide according to any one of claims 1 to 7.   A recombinant vector containing the polynucleotide according to any one of claims 1 to 7.   An agonist for the polypeptide according to claim 8.   An antagonist to the polypeptide of claim 8.   Antibody against the polypeptide of claim 8   A pharmaceutical composition comprising the vector according to claim 9 or the agonist according to claim 10 as an active ingredient and used for the treatment of narcolepsy.   A method for screening for an agonist for the polypeptide or a salt thereof, which comprises using the polypeptide according to claim 8 or a salt thereof.   A method for screening for an antagonist to the polypeptide or a salt thereof, wherein the polypeptide or a salt thereof according to claim 8 is used. A method for determining a subject's susceptibility to narcolepsy, the following step (a) collecting a biological sample containing chromosomal DNA from the subject,
(B) determining the type of base at position 2524528 of position 22.3 of human chromosome 21 or the position of position 45238073 of position 22.3 of human chromosome 21;
(C) based on the result obtained in step (b), determining the susceptibility of the subject to narcolepsy,
A method comprising the steps of: A method for determining a subject's susceptibility to narcolepsy, the following step (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;
(C) based on the result obtained in step (b), determining the susceptibility of the subject to narcolepsy,
A method comprising the steps of:

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