WO2011049207A1 - Moyamoya disease-related gene and utilization of same - Google Patents
Moyamoya disease-related gene and utilization of same Download PDFInfo
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- WO2011049207A1 WO2011049207A1 PCT/JP2010/068737 JP2010068737W WO2011049207A1 WO 2011049207 A1 WO2011049207 A1 WO 2011049207A1 JP 2010068737 W JP2010068737 W JP 2010068737W WO 2011049207 A1 WO2011049207 A1 WO 2011049207A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/136—Screening for pharmacological compounds
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to novel genes and SNPs that are useful for diagnosis before the onset of moyamoya disease. Furthermore, the present invention relates to a non-human animal having a functional defect of the gene, a cell having a functional defect of the gene, a method for determining the risk of developing familial moyamoya disease using the gene, the onset of ischemic heart disease The present invention relates to a method for determining risk, a method for determining the possibility of birth of identical twins, and a reagent useful for carrying out these determination methods.
- Moyamoya disease also called Willis artery occlusion, is a stenotic lesion at the terminal end of the internal carotid artery that develops in childhood and adulthood.
- Moyamoya disease is a disease that develops all over the world (Non-Patent Document 1), but the incidence of Moyamoya disease is particularly high in East Asia such as Japan, Korea, and China (Non-Patent Documents 2 and 3). In Japan, the latest morbidity and annual incidence are reported to be 10.5 and 0.94 per 100,000 (Non-patent Document 2). In Europe, the incidence is estimated to be about 1/10 of the incidence in Japan (Non-patent Documents 2 and 3). In the United States, the incidence is approximately 0.086 per 100,000, and is higher in Asian Americans and African Americans than in Caucasian Americans (Non-patent Documents 2 and 3).
- Non-patent Document 4 chromosomal region 17q25.3 is linked to the onset of moyamoya disease.
- the nucleotide sequence of the human chromosome has basically been determined by the comprehensive sequence of the human genome. From the sequence, a large number including CARD14, Raptor, AATK, KIAA1618, C17orf27, etc. in the chromosomal region 17q25.3. The presence of genes is expected.
- Raptor [regulatory associated protein of mammalian target of rapamycin (mTOR)] is a 150 kDa mTOR cofactor essential for TOR signaling (Non-patent Document 6). Raptor is associated with tissue hypertrophy (Non-patent document 7), is a regulator of hypoxia-inducible factor (Non-patent document 8), and is involved in vascular endothelial cell proliferation mediated by HLA class I antibodies (Non-patent document 7). Reference 9).
- Non-patent Document 10 Apoptosis is one of the phenomena observed in stenotic lesions in patients with MMD (Non-patent Document 11).
- amino acid sequences of KIAA1618 and C17orf27 are registered as accession numbers NP_066005.2 and NP_065965.3, respectively, and are published on the NCBI Internet homepage.
- these genes are “predicted genes” predicted to be structural genes based on nucleotide sequence analysis by a computer, and are disclosed as two independent genes, but functional analysis is not performed at all. Not.
- Patent Document 1 discloses a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4.
- the polypeptide is a membrane protein having a transmembrane domain, and is described to have a Zn finger domain and an ATP binding motif A (P loop).
- the purpose of the present invention is to identify SNPs that correlate with the onset of moyamoya disease, and to establish a pre-onset diagnosis method for moyamoya disease based on this. Furthermore, the present invention obtains a gene that may be related to the onset of moyamoya disease from the information of the SNPs, creates a non-human animal or animal cell containing a functional defect of the gene, and functions of the gene The purpose is to clarify. Moreover, this invention aims at providing the further use of this gene and SNPs.
- the present inventors conducted detailed linkage analysis on 17 Moyamoya disease families and succeeded in identifying a core region of about 2.1 Mb linked to the onset of Moyamoya disease from among the chromosome region 17q25.3. .
- the core region contained about 38 genes.
- SNP1 C17orf27
- SNP2 C17orf27
- SNP3 FLJ3520
- SNP4 NPTX1
- SNP5 KIAA1303
- zebrafish mysterin 1 antisense morpholino homologous to human mysterin is injected into a zebrafish fertilized egg to suppress the protein expression of mysterin 1.
- mysterin 1 is injected into a zebrafish fertilized egg to suppress the protein expression of mysterin 1.
- embryos injected with antisense morpholino of mysterin 1 early angiogenesis progressed normally, but subsequent angiogenesis was abnormal.
- characteristic abnormal angiogenesis was observed around the eyes.
- SNP2 has been shown to be associated with the frequency of ischemic heart disease and the frequency of delivery of identical twins.
- new SNPs that correlate with the onset of moyamoya disease were also found in Caucasian families. Based on the above findings, the present invention has been completed.
- a polynucleotide comprising a nucleotide sequence encoding the polypeptide according to [1].
- An expression vector comprising the polynucleotide according to [2].
- a transformant transformed with the expression vector according to [3].
- a non-human animal comprising a functional defect of a gene encoding the polypeptide according to [1].
- a screening method for a candidate substance for angiogenesis regulating agent comprising evaluating whether or not a test substance can regulate the expression or function of a gene encoding the polypeptide of [1].
- a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5) are included, and the allele of the SNP included in the partial sequence is a minor allele
- the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
- a method for determining the risk of developing ischemic heart disease comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
- An imaginary comprising a nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP A diagnostic agent for the risk of developing blood heart disease.
- a method for determining the possibility of delivery of identical twins comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
- a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution). [19] A polynucleotide comprising a nucleotide sequence encoding the polypeptide of [18].
- a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) A polynucleotide comprising at least one SNP selected from wherein the allele of the SNP contained in the partial sequence is a minor allele and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
- SNP6 The diagnostic agent according to [25], wherein the SNP is 55977 G> A (SNP6).
- the SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5)
- the nucleic acid probe or primer according to [27] which is at least one of [29]
- the nucleic acid probe or primer according to [27] wherein the SNP is 55977 G> A (SNP6).
- a polypeptide of a novel gene mysterin a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided.
- Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus. From the analysis of zebrafish in which expression of the mysterin gene was suppressed, it became clear that mysterin has a function of regulating angiogenesis.
- the present invention also provides new SNPs that exist at or near the mysterin locus.
- the SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
- FIG. 1 Schematic diagram of the structure of human mysterin. Expression of human mysterin protein in HEK293 cells. Intracellular distribution of human mysterin protein in Hela cells. Self-ubiquitination of human mysterin in HEK293 cells. The sequence of amino acid positions 2359 to 2613 of human mysterin. The underline indicated by ⁇ indicates a region predicted to constitute an alpha helix, and the underline indicated by ⁇ indicates a region predicted to constitute a beta sheet. ATPase activity of the Walker domain of human mysterin. Amplification of mysterin MO target region by RT-PCR. 1: Wild-type embryo, 2: Embryo injected with mysterin MO. Each arrow indicates the following information.
- Genome DNA, WT allele: wild-type PCR product, spMO allele: PCR product with exons skipped.
- Multiple alignment of sequences around mysterin AAA Sequences corresponding to the following vertebrate AAA domain peripheral sequences (amino acids 2412 to 2910 in human mysterin) were extracted and subjected to multiple alignment. “*” Indicates an invariant site.
- Mysterin polypeptide provides any polypeptide selected from the following (1) to (3): (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine); (2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity.
- the present invention also provides the following polypeptides (4) to (7): (4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with asparagine); (5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No.
- amino acid sequence represented by SEQ ID NO: 2 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid); (6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution).
- polypeptides of the above (1) to (3) in a further aspect, the above (1) to (7)
- the gene encoding the polypeptide are collectively referred to as “Mysterin”. To do.
- the polypeptide of (1) above is a typical human mysterin polypeptide. Because of a newly found SNP within the mysterin locus with amino acid substitution (SNP2 will be described in detail below), the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine. Good.
- amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is an SNP with amino acid substitution (detailed below as SNP6). Aspartic acid at position 4013 in the sequence may be substituted with asparagine.
- the amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is a SNP with amino acid substitution (detailed below as SNP7). Asparagine at position 3962 in the sequence may be substituted with aspartic acid.
- the amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is a SNP with amino acid substitution (detailed below as SNP8). Arginine of No. 4062 in the sequence may be substituted with glutamine.
- amino acid sequence represented by SEQ ID NO: 2 may contain 2, 3 or 4 substitutions selected from the group consisting of R4810K, D4013N, N3962D and R4062Q.
- Mysterin polypeptide has ubiquitin ligase activity and ATPase activity.
- the presence or absence of ubiquitin ligase activity is determined by combining an expression vector capable of expressing the target polypeptide tagged with HA or the like with an expression vector capable of expressing ubiquitin (for example, mammalian ubiquitin) and appropriate animal cells (for example, HEK293 cells) and immunoprecipitation using an antibody specifically recognizing the tag for the lysate of the cell, followed by Western blotting using an antibody specifically recognizing ubiquitin. It can be determined by evaluating whether or not the obtained polypeptide is self-ubiquitinated.
- ubiquitin for example, mammalian ubiquitin
- appropriate animal cells for example, HEK293 cells
- the presence or absence of ATPase activity can be determined by the following method, for example.
- the polypeptide of interest is added to a physiological buffer (eg, PBS) containing magnesium ions (eg, 1 mM) and ATP (eg, 5 mM) and incubated at 37 ° C. for 30 minutes to a final concentration of 1%
- the reaction is stopped by adding PCA.
- the coloration due to the binding of free phosphate and malachite green is measured by absorbance at 620 nm. If the tested polypeptide has ATPase activity, a significant color reaction due to the release of phosphate accompanying the hydrolysis of ATP is observed.
- the polypeptide of (2) above is 45% or more, preferably 70% or more, more preferably 95% or more, still more preferably 99.5% or more, and even more preferably 99% with the amino acid sequence represented by SEQ ID NO: 2. Amino acid sequences having 9% or more (for example, 99.96% or more) identity are included.
- identity refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue to the total overlapping amino acid residues (in which one or both of the gaps can be considered).
- amino acid sequence identity in this specification is determined by using the homology calculation algorithm Blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) published on the Internet website of NCBI as the default condition. Can be calculated.
- Blastp http://blast.ncbi.nlm.nih.gov/Blast.cgi
- the algorithm for determining amino acid sequence identity for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is NBLAST and XBLAST (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol.
- the amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, for example, (1) SEQ ID NO: 1 or more (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1 to a number in the amino acid sequence shown in FIG.
- (2-5) amino acid deleted amino acid sequence (2) one or more (preferably 1-500, more preferably 1-100, more preferably 1) amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence to which 1 to 30, even more preferably 1 to 10, most preferably 1 to several (2 to 5) amino acids are added; (3) one or more amino acid sequences shown in SEQ ID NO: 2 A plurality of amino acids (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1 to several (2 to 5)) amino acids Inserted amino acid sequence, (4) one or more (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, and even more preferably) in the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence in which 1 to 10 amino acids, most preferably 1 to several (2 to 5) amino acids are substituted with other amino acids, or (5) amino acids in which the mutations (1) to (4) above are combined
- the total number of amino acids added is preferably 1-500, more preferably 1-100, more
- the RING finger domain (corresponding to amino acids 3997 to 4035 of SEQ ID NO: 2) important for the activity is conserved in the polypeptides (2) and (3) above. Is preferred. The degree of conservation is usually 50% or more, preferably 54% or more, more preferably 69% or more, still more preferably 90% or more (eg 97% or more), and most preferably 100% as amino acid sequence identity. .
- the introduction is preferably performed so that the amino acid “*” shown in FIG. 10 is completely retained.
- amino acids with similar chemical properties include, for example, aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids ( Amino acids that fall into the same group such as Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met) Is mentioned. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Examples of conservative amino acid substitutions are well known in the art and are described in various references (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
- the AAA domain peripheral sequence (corresponding to human 2412 to 2910 amino acids) important for the activity is conserved. preferable.
- the degree of conservation is usually 60% or more, preferably 72% or more, more preferably 80% or more, still more preferably 90% or more (for example, 98% or more), and most preferably 100% as amino acid sequence identity.
- the introduction is preferably performed so that the amino acid “*” shown in FIG. 9 is completely retained.
- the amino acid “:” or “.” Shown in FIG. 9 is completely retained or the chemical properties are similar. It is preferable to introduce the amino acid so that the amino acid is substituted.
- the definition of “amino acids with similar chemical properties” is as described above.
- polypeptides of (2) and (3) above are natural mysterin polypeptides of animals. “Natural” means that the amino acid sequence constituting the polypeptide exists in nature.
- polypeptides of (2) and (3) above include polypeptides having a different amino acid sequence from the polypeptide of (1) generated by the polymorphism of the human mysterin gene.
- the polypeptides (2) and (3) above include orthologs of mysterin in animals other than humans.
- “non-human animals” include mammals other than humans, birds, reptiles, amphibians, fish, insects, and the like.
- mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits; domestic animals such as pigs, cows, goats, horses, sheep, minks; pets such as dogs and cats Primates such as, but not limited to, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees;
- the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons.
- Examples of fish include zebrafish, puffer fish, medaka, goldfish and loach.
- An ortholog of mysterin in a non-human animal refers to a nucleotide sequence (nucleotide sequence represented by SEQ ID NO: 1) encoding human mysterin disclosed herein, and a nucleic acid probe capable of specifically detecting mysterin Can be obtained by designing a primer or primer and using a well-known genetic engineering technique such as plaque hybridization or RACE PCR.
- Preferred examples of the polypeptides (2) and (3) above include mysterin polypeptides that are homologous to human mysterin of mammals, typical birds, or fish. If the evolution time is long, the preservation of the complete gene structure is not necessarily observed due to diversity among species due to mutation such as insertion or deletion into the protein. However, functionally important parts are highly conserved between species. Peripheral sequence of the myelin AAA domain (corresponding to human amino acids 2412 to 2910) and RING finger domain sequence (corresponding to human amino acids 3997 to 4035), human, chimpanzee, mouse, rat, chicken, puffer, and zebrafish ( 2 domains exist in the genome), and multi-full alignment was performed for each domain. The alignment results are shown in FIGS.
- the identity to the human mysterin amino acid sequence is as follows: chimpanzee 98%, mouse 82%, rat 80%, chicken 72%, puffer 67%, and zebrafish 68%, 63% (respectively mysterin 1 , 2).
- identity to the human mysterin amino acid sequence is 97% chimpanzee, 69% mouse, 74% rat, 54% chicken, 51% puffer fish, and 53% and 42% zebrafish (mysterin 1 respectively) , 2).
- mysterin 1 zRNF213
- mysterin 2 zRNF213.1
- a representative amino acid sequence of a zebrafish mysterin 1 polypeptide is shown in SEQ ID NO: 4.
- the length of the polypeptide of the present invention is not particularly limited as long as it has ubiquitin ligase activity and ATPase activity, and a polypeptide having a desired length can be selected according to the purpose of use.
- the polypeptide of the present invention has a length of 6000 amino acids or less, 5500 amino acids or less, 5400 amino acids or less, 5300 amino acids or less, 5250 amino acids or less, 5220 amino acids or less, 5210 amino acids The following can be selected as appropriate.
- the polypeptide of the present invention usually has a length of 4800 amino acids or more, preferably 5000 amino acids or more, more preferably 5200 amino acids or more, and most preferably 5207 amino acids or more.
- the polypeptide of the present invention may have one or more tag polypeptides or signal sequences.
- the tag polypeptide refers to a polypeptide that is added to facilitate the detection and purification of the polypeptide.
- tag polypeptides include, but are not limited to, epitope tags, fluorescent polypeptides, immunoglobulin Fc regions, and the like.
- An epitope tag refers to a peptide that is specifically recognized by an antibody or other binding partner, specifically, a Flag tag, a polyhistidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, an MBP tag. Etc. can be mentioned.
- the fluorescent polypeptide include GFP, YFP, RFP, CFP, BFP, EGFP and the like.
- tag polypeptides are well known to those skilled in the art, and various antibodies that specifically recognize the tag polypeptides are commercially available.
- the signal sequence refers to a polypeptide sequence that directs the transport and localization of the polypeptide from the synthesis site to a specific site inside the cell or outside the cell simultaneously with or after translation of the polypeptide.
- the signal sequence include a leader sequence that induces secretion of the polypeptide, a nuclear translocation signal sequence (for example, a nuclear translocation signal sequence of SV40 T antigen), a nuclear translocation signal sequence, a nucleolus localization signal, and the like. However, it is not limited to these.
- Such signal sequences are well known to those skilled in the art, and can be appropriately selected according to the purpose.
- the polypeptide of the present invention may be modified.
- modifications include addition of lipid chains (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.
- the polypeptide of the present invention contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase). , Peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin) Etc.).
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase.
- polypeptide is used to mean a salt thereof.
- Polypeptide salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. preferable.
- Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
- the polypeptide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed.
- the purity of the isolated or purified polypeptide of the present invention is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, substantially 100%).
- polypeptides of the invention are listed below: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine); (C) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine); (D) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine at No.
- SEQ ID NO: 2 comprises any one combination of substitutions selected from the following group; R4810K + D4013N, R4810K + N3962D, R4810K + R4062Q, D4013N + N3962D, D4013N + R4062Q, N3962D + R4062Q, R4810K + D4013N + N3962D, R4810K + D4013N + R4062Q, R4810K + N3962D + R4062Q, R4810K + N3962D + R4062Q, R4810K + N3962D + R4062Q, D4013N + N3962D + R4062Q, D4013N + N3962D + R4062Q, and R4810K + D40
- the method for producing the polypeptide of the present invention is not particularly limited, and may be produced according to a known peptide synthesis method or may be produced using a known genetic recombination technique.
- the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. If the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining portion, and the product has a protecting group, the protecting polypeptide can be eliminated to produce the desired polypeptide.
- polypeptide of the present invention When the polypeptide of the present invention is produced using genetic recombination technology, first, a polynucleotide encoding the polypeptide of the present invention as described later is obtained, and the host cell is expressed using an expression vector capable of expressing the polypeptide.
- the polypeptide can be produced by transforming and culturing the resulting transformant. A method for producing the polypeptide of the present invention using the polynucleotide and gene recombination techniques will be described later.
- the polypeptide of the present invention can be used to screen for candidate substances for angiogenesis regulating agents, to determine the risk of developing Moyamoya disease, to determine the risk of developing ischemic heart disease, to determine the likelihood of delivery of identical twins, This is useful for determining angiogenesis.
- polynucleotide Encoding Mysterin Polypeptide The present invention provides a polynucleotide comprising a nucleotide sequence encoding the above-described polypeptide of the present invention.
- the polynucleotide of the present invention may be DNA or RNA, or may be a DNA / RNA chimera.
- the polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.
- polynucleotide of the present invention examples include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or 3.
- the nucleotide sequence represented by SEQ ID NO: 1 encodes a human mysterin polypeptide (SEQ ID NO: 2)
- the nucleotide sequence represented by SEQ ID NO: 3 encodes a zebrafish mysterin 1 polypeptide (SEQ ID NO: 4).
- SNP2 zebrafish mysterin 1 polypeptide
- the 14429th guanine in the nucleotide sequence represented by SEQ ID NO: 1 was replaced with adenine. May be.
- SNP6 SNP6
- No. 12037 guanine in the nucleotide sequence represented by SEQ ID NO: 1 is converted to adenine. May be substituted.
- adenine at position 11884 in the nucleotide sequence represented by SEQ ID NO: 1 is converted to guanine. May be substituted.
- SNP8 because of a newly found SNP with an amino acid substitution in the mysterin locus (described in detail below as SNP8), 12185th guanine in the nucleotide sequence represented by SEQ ID NO: 1 is converted to adenine. May be substituted.
- the nucleotide sequence represented by SEQ ID NO: 1 is , G14429a, g12037a, a11884g and g12185a may comprise 2, 3 or 4 substitutions.
- the polynucleotide of the present invention can be easily produced by utilizing a known gene recombination technique based on the sequence information described in the sequence listing of the present specification. For example, by designing an appropriate primer based on the sequence information, RT-PCR using cDNA prepared from total RNA isolated from cells expressing mysterin (eg, renal cells such as 293 cells) as a template. Polynucleotides can be produced. Alternatively, the polynucleotide of the present invention may be synthesized by a polynucleotide synthesizer based on the sequence information described in the sequence listing of the present specification.
- the obtained polynucleotide encoding the polypeptide of the present invention can be used as it is depending on the purpose, or after digestion with a restriction enzyme or adding a linker as desired.
- the polynucleotide may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
- the polynucleotide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed.
- the purity of the isolated or purified polynucleotide of the present invention is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, substantially 100%).
- the polynucleotide of the present invention is useful for producing a mysterin polypeptide. Further, by analyzing SNP2 and SNP6 contained in the polynucleotide of the present invention, it is possible to determine the onset risk of moyamoya disease and ischemic heart disease and the possibility of delivery of identical twins (detailed below). ).
- Expression vector and transformant The present invention provides an expression vector containing the polynucleotide of the present invention and a transformant containing the expression vector.
- the expression vector can be produced by operably linking the polynucleotide of the present invention downstream of a promoter in an appropriate expression vector.
- the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host cell to be used.
- Host cells include prokaryotic cells and eukaryotic cells.
- prokaryotic cells include Escherichia bacteria (such as Escherichia coli), Bacillus bacteria (such as Bacillus subtilis), and the like.
- eukaryotic cells include yeast (Saccharomyces cerevisiae, etc.), insect cells (stolen larvae-derived cell lines (Spodoptera frugiperda cell; Sf cells), etc.), mammalian cells (human cells (293, etc.)) Monkey cells (such as COS-7), Chinese hamster cells (such as CHO cells)), and the like.
- mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
- Plasmid vectors derived from E. coli eg, pBR322, pBR325, pUC12, pUC13
- plasmid vectors derived from Bacillus subtilis eg, pUB110, pTP5, pC194
- yeast-derived plasmid vectors eg, pSH19, pSH15
- the type of virus vector can be appropriately selected according to the type of host cell used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.
- a promoter that can initiate transcription in the host cell can be selected according to the type of host cell to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SR ⁇ promoter and the like are preferable.
- the expression vector of the present invention may contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like in a functional manner, if desired.
- selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (sometimes abbreviated as Amp r ), neomycin resistance gene (Neo). G418 resistance) which may be abbreviated as r ).
- the expression vector of the present invention is preferably isolated or purified.
- the expression vector of the present invention is useful for producing the polypeptide of the present invention because it can express the polypeptide of the present invention in an appropriate host cell.
- the above-described expression vector of the present invention is prepared according to a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, protoplast fusion method, electroporation method, DEAE dextran method, gene transfer method using Gene Gun).
- a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, protoplast fusion method, electroporation method, DEAE dextran method, gene transfer method using Gene Gun).
- a transformant into which the expression vector has been introduced (the transformant of the present invention) can be produced.
- the transformant can express the polypeptide of the present invention.
- the transformant of the present invention is useful for producing the polypeptide of the present invention.
- the polypeptide of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the type of host and isolating the polypeptide of the present invention from the culture.
- the transformant whose host is Escherichia is cultured in an appropriate medium such as LB medium or M9 medium, usually at about 15 to 43 ° C. for about 3 to 24 hours.
- the transformant whose host is Bacillus is cultured in an appropriate medium, usually at about 30 to 40 ° C. for about 6 to 24 hours.
- Cultivation of the transformant whose host is yeast is usually carried out at about 20 ° C. to 35 ° C. for about 24 to 72 hours in a suitable medium such as a Burkholder medium.
- Cultivation of transformants whose host is an insect cell or an insect is carried out in a suitable medium such as Grace's Insect medium supplemented with about 10% bovine serum, usually at about 27 ° C for about 3 to 5 days.
- the transformant whose host is an animal cell is cultured in an appropriate medium such as a MEM medium supplemented with about 10% bovine serum, usually at about 30 ° C. to 40 ° C. for about 15 to 60 hours. In any culture, aeration and agitation may be performed as necessary.
- the cell lysate or the culture supernatant is subjected to a plurality of chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. Can be achieved.
- Non-human animal containing functional defect of mysterin The present invention provides a non-human animal comprising a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
- Examples of the species of the animal of the present invention include mammals other than humans, birds, reptiles, amphibians, fish, and insects, with mammals, birds and fish being preferred.
- mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits; domestic animals such as pigs, cows, goats, horses, sheep, minks; pets such as dogs and cats Primates such as, but not limited to, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees;
- the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons.
- fish include zebrafish, medaka, goldfish and loach.
- a preferred non-human mammal is a mouse.
- a preferred bird is a chicken.
- a functional defect of a mysterin gene refers to a state in which normal functions (ubiquitin ligase activity, ATPase activity, etc.) inherent to the mysterin gene cannot be sufficiently exerted, for example, a state in which the mysterin gene is not expressed at all, or a mysterin gene
- the mysterin gene is in a state where its expression level is reduced to the extent that it cannot exert its normal function, or the function of the mysterin gene product has been completely lost, or to the extent that the normal function inherent in the mysterin gene cannot be exerted
- a state in which the function of the product is reduced is mentioned.
- the animal of the present invention has various characteristics associated with a functional defect of the mysterin gene.
- the animals of the present invention have delayed embryogenesis and abnormalities in early angiogenesis and subsequent angiogenesis compared to wild type animals. Furthermore, it exhibits a particularly remarkable abnormality in angiogenesis of the head and eyes.
- the animal of the present invention may be an animal accompanied with a modification of genomic DNA, a so-called genetically modified animal.
- the genetically modified animal of the present invention may be a mysterin gene-deficient heterozygote or a mysterin gene-deficient homozygote.
- the genetically modified animal of the present invention can be produced by a method known per se. First, a method for producing a chimeric animal useful for producing the genetically modified animal of the present invention will be described.
- the animals of the present invention include chimeric animals useful for producing the genetically modified animals of the present invention.
- the chimeric animal of the present invention can be produced, for example, by a method including the following steps (a) to (c): (A) providing an embryonic stem cell containing a functional defect of the mysterin gene; (B) introducing the embryonic stem cells into an embryo to obtain a chimeric embryo; and (c) transplanting the chimeric embryo into an animal to obtain a chimeric animal.
- an embryonic stem cell (ES cell) containing a functional defect of the mysterin gene for example, the following 5.
- ES cell embryonic stem cell
- Those prepared by the method described in the section can be used.
- the animal species from which the embryo is derived may be the same as the genetically modified animal species of the present invention, and is preferably the same as the animal species from which the embryonic stem cells to be introduced are derived.
- embryos include blastocysts and 8-cell stage embryos.
- Embryos can be obtained by mating female animals that have undergone superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) and the like.
- hormone agents for example, using PMSG having FSH-like action and hCG having LH action
- methods for introducing embryonic stem cells into the embryo include micromanipulation methods and aggregation methods.
- the chimeric embryo can be transferred to the uterus or fallopian tube of the animal.
- the animal into which the chimeric embryo is transplanted is preferably a pseudopregnant animal.
- a pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like. The animal into which the chimeric embryo has been introduced becomes pregnant and gives birth to the chimeric animal.
- the animal born is a chimeric animal. Whether or not the animal born is a chimeric animal can be confirmed by a method known per se, for example, by body color or coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed.
- the genetically modified animal of the present invention can be produced, for example, by a method including the following steps (a) to (d): (A) providing an embryonic stem cell containing a functional defect of the mysterin gene; (B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo; (C) transplanting the chimeric embryo into an animal to obtain a chimeric animal; and (d) mating the chimeric animal to obtain a mysterin gene-deficient heterozygote.
- Steps (a) to (c) of the above method can be performed in the same manner as the method for producing a chimeric animal described above.
- step (d) of the above method mating is performed after the chimeric animal obtained in step (c) has matured.
- the mating can preferably take place between wild-type animals and chimeric animals or between chimeric animals.
- a mysterin gene deficiency has been introduced into germline cells of a chimeric animal and a mysterin gene deficient heterozygous offspring can be obtained can be confirmed using various traits as indicators, for example, the body of a progeny animal It can be distinguished by color and coat color.
- DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed.
- a mysterin gene-deficient homozygote can be produced by crossing the mysterin gene-deficient heterozygotes thus obtained.
- a progeny animal having a genotype in which a gene derived from an embryonic stem cell and a gene derived from an animal used for mating are obtained is obtained.
- the obtained mysterin gene deficient animal is backcrossed with a pure animal strain for about 5 to 8 generations. It is preferable to do.
- backcrossing is carried out only by natural mating, it may take a long time, so in vitro fertilization techniques can be used as appropriate when it is desired to speed up generational changes.
- the animal of the present invention may be an animal that is not accompanied by genomic DNA modification.
- Such an animal can be obtained by, for example, administering a substance that specifically suppresses the expression or function of the mysterin gene (eg, antisense nucleic acid, siRNA, expression vector capable of expressing these nucleic acids) to the animal or forced expression in the animal.
- a substance that specifically suppresses the expression or function of the mysterin gene eg, antisense nucleic acid, siRNA, expression vector capable of expressing these nucleic acids
- Administration can be carried out using a suitable delivery means such as, for example, microinjection, liposomes. It is also possible to achieve a cell-specific mysterin gene functional defect.
- morpholino antisense nucleic acids is advantageous from the viewpoint of ease of operation and time cost in the production of fish (eg, mysterin 1 in the case of zebrafish) in which expression of the mysterin gene is suppressed (Nucleic® Acid).
- the morpholino antisense nucleic acid is complementary to the partial sequence of mysterin mRNA (here, the partial sequence within 25 bases of the splicing acceptor and donor boundary sequence) (the number of mismatches is usually 3 bases or less, preferably It consists of a nucleotide sequence of about 25 bases in length (1 base or less, most preferably 0).
- the synthesized morpholino antisense nucleic acid is microinjected into the cytoplasm or yolk of a fertilized egg (embryo) at the 1-8 cell stage, the morpholino antisense nucleic acid inhibits splicing of mysterin mRNA in the nucleus, and exon skipping. The resulting frameshifted protein is produced.
- the animal of the present invention is useful as an animal model for diseases associated with abnormal neovascularization.
- Diseases related to abnormalities in angiogenesis include various factors such as occlusive diseases such as arteriosclerosis, arteriovenous malformations, neovascularization in tumors, inflammatory obstructive diseases, diabetic vascular disorders, and age-related macular degeneration Examples include vascular lesions and abnormalities in blood vessel construction due to a single gene such as Osler's disease.
- the animal of the present invention is also useful for analysis of the mysterin gene and angiogenesis, and screening for substances that can regulate angiogenesis and those that can treat angiogenesis abnormalities.
- gene expression profile is measured by means (for example, microarray) that enables comprehensive analysis of gene expression, and the gene of a control animal (same species or heterogeneous animal) such as a wild type animal is measured.
- the gene expression profile of the animal of the present invention can be traced over time to evaluate the linkage between expression and progression of phenotype (abnormal neovascularization) and changes in gene expression profile.
- Animal cell containing functional defect of mysterin The present invention provides an animal cell containing a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
- Examples of the animal species of the present invention include mammals including humans, birds, reptiles, amphibians, fish, and insects, with mammals, birds and fish being preferred.
- Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats;
- Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
- Examples of the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons.
- Examples of fish include zebrafish, medaka, goldfish and loach. Preferred mammals are humans or mice.
- a preferred bird is a chicken.
- a preferred fish is zebrafish.
- the animal cell of the present invention can also be a cell derived from any tissue, for example, a somatic cell in which a mysterin gene is expressed (for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta, Prostate, salivary gland, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels, vascular endothelial cells, vascular smooth muscle cells, pancreatic islets of Langerhans), spermatogonia, sperm, ovum, fertilization Examples include germline cells such as eggs, embryonic cells, and embryonic stem cells.
- a somatic cell in which a mysterin gene is expressed for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta, Prostate, salivary gland, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels,
- the animal cell of the present invention may be either a primary cultured cell or a cell line.
- An embryonic cell refers to a cell collected from an embryo and a cell generated by the division and not a embryo.
- the animal cell of the present invention may be a cell accompanied by a modification of genomic DNA, a so-called genetically modified cell.
- the genetically modified cell of the present invention can be, for example, a mysterin gene-deficient homozygote or a mysterin gene-deficient heterozygote.
- the genetically modified cell of the present invention can be produced by a method known per se.
- the animal cell of the present invention can be produced by a method comprising the following steps (a) to (c): (A) providing a targeting vector capable of inducing homologous recombination of the mysterin gene; (B) introducing the targeting vector into animal cells; and (c) selecting cells that have undergone homologous recombination from the cells into which the targeting vector has been introduced.
- the targeting vector capable of inducing homologous recombination of the mysterin gene used in step (a) of the above method is a mysterin cDNA sequence (SEQ ID NO: 1) disclosed in the sequence listing of the present specification, a genomic DNA sequence ( Based on the information of SEQ ID NO: 5), genomic DNA of the mysterin gene of the desired animal can be obtained, and can be easily produced by those skilled in the art according to a general genetic engineering technique in creating a knockout mouse.
- the targeting vector is introduced into the animal cell.
- the method for introducing the targeting vector into animal cells include the calcium phosphate method, the lipofection method / liposome method, and the electroporation method.
- the targeting vector is introduced into an animal cell, homologous recombination of genomic DNA containing the mysterin gene occurs in the animal cell.
- animal cell into which the targeting vector is introduced one produced by a method known per se, one commercially available, or one available from a predetermined organization can be used.
- the types of animal cells are as described above.
- an embryonic stem cell when used as an animal cell into which a targeting vector is introduced, the embryonic stem cell can be established by culturing an inner cell mass separated from a blastocyst of any animal on a feeder cell.
- existing embryonic stem cells can be obtained commercially or from a predetermined organization. Examples of existing mouse embryonic stem cells include ES-D3 cells, ES-E14TG2a cells, SCC-PSA1 cells, TT2 cells, AB-1 cells, J1 cells, R1 cells, E14.1 cells, RW-4 cells, etc. Is mentioned.
- embryonic stem cells derived from mammals such as humans, mink, hamsters, pigs, cattle, marmoset, rhesus monkeys, etc. have been established at present. You can also.
- somatic cells such as kidney cells are used as animal cells into which the targeting vector is introduced
- the somatic cells into which the targeting vector is introduced may be either primary cultured cells or cell lines.
- Primary cultured cells and cell lines can be prepared by a method known per se (for example, Current Protocols in Cell Biology, John John Wiley and Sons, Inc. (2001)).
- step (c) of the above method in order to select animal cells in which homologous recombination has occurred with genomic DNA containing the mysterin gene, the animal cells after introduction of the targeting vector are screened. For example, after selection by positive selection, negative selection, etc., screening based on genotype (for example, PCR method, Southern blot hybridization method) is performed.
- genotype for example, PCR method, Southern blot hybridization method
- a karyotype analysis of the recombinant embryonic stem cell is further performed.
- Karyotype analysis confirms that there is no chromosomal abnormality in the selected recombinant embryonic stem cells.
- Karyotype analysis can be performed by a method known per se.
- the karyotype of embryonic stem cells is preferably confirmed in advance before introducing the targeting vector.
- the genetically modified cells of the present invention can also be isolated from the genetically modified animals of the present invention. Since the genetically modified animal of the present invention contains a functional defect of the mysterin gene, the genetically modified cell isolated from the genetically modified animal of the present invention also contains a functional defect of the mysterin gene.
- cells isolated from the genetically modified animal of the present invention may be modified by a method such as genetic engineering techniques. Isolation and modification of cells can be performed by a method known per se (for example, Current Protocols in Cell Biology, John Wiley and Sons, Inc. (2001)).
- the animal cell of the present invention may be a cell that is not accompanied by genomic DNA modification.
- a cell can be prepared, for example, by introducing a substance that specifically suppresses the expression or function of the mysterin gene (for example, an antisense nucleic acid, siRNA, or an expression vector capable of expressing these nucleic acids) into the cell.
- a substance that suppresses the expression or function of the mysterin gene into cells can be performed by a method known per se, such as microinjection, calcium phosphate method, lipofection method / liposome method, electroporation method and the like.
- the animal cell of the present invention is useful for analysis of mysterin gene and angiogenesis, screening for a substance that can regulate angiogenesis and a substance that can treat angiogenesis abnormality, and production of the animal of the present invention.
- the present invention also includes a method for screening a candidate substance for an angiogenesis regulator, comprising evaluating whether a test substance can regulate the expression or function of a mysterin gene, and a blood vessel obtained by the screening method.
- a candidate substance for angiogenesis regulator is provided.
- test substance to be used for the screening method may be any known compound or novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, prepared using combinatorial chemistry technology Examples thereof include a compound library, a random peptide library prepared by solid phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
- the screening method of the present invention includes the following steps (a) to (c): (A) contacting the test substance with a cell capable of measuring the expression of the mysterin gene; (B) measuring the expression level of the mysterin gene in the cell contacted with the test substance, and comparing the expression level with the expression level of the mysterin gene in the control cell not contacted with the test substance; and (c) above ( A step of selecting a test substance that regulates the expression level of the mysterin gene based on the comparison result of b).
- step (a) of the above method the test substance is placed under contact with cells capable of measuring mysterin gene expression.
- Contact of the test substance with a cell capable of measuring the expression of the mysterin gene can be performed in a culture medium.
- a cell capable of measuring the expression of a mysterin gene refers to a cell capable of directly or indirectly evaluating the expression level of a mysterin gene product, for example, a transcription product or a translation product.
- a cell that can directly evaluate the expression level of a mysterin gene product can be a cell that can naturally express a mysterin gene (eg, a cell that naturally expresses a mysterin gene), while a product of a mysterin gene.
- a cell capable of indirectly assessing the expression level of can be a cell that allows a reporter assay for the mysterin gene transcriptional regulatory region.
- Cells that can measure the expression of the mysterin gene are animal cells, such as mammalian cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, humans, avian cells such as chickens, and fish cells such as zebrafish. obtain.
- mammalian cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, humans, avian cells such as chickens, and fish cells such as zebrafish. obtain.
- the cells that can naturally express the mysterin gene are not particularly limited as long as they potentially express the mysterin gene.
- Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. Examples of cell lines in which the mysterin gene is expressed include 293 cells.
- the mysterin gene is known to be expressed in the tissues or cells described above, such tissues or cells (for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta) , Prostate cells, salivary glands, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels, vascular endothelial cells, vascular smooth muscle cells, pancreatic Langerhans pancreatic B cells) May be.
- tissues or cells for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta
- Prostate cells salivary glands, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels, vascular endothelial cells, vascular smooth muscle cells, pancreatic Langerhans pancreatic B cells
- a cell that enables a reporter assay for a mysterin gene transcription regulatory region is a cell that contains a mysterin gene transcription regulatory region and a reporter gene operably linked to the region.
- the mysterin gene transcription regulatory region and reporter gene can be inserted into an expression vector.
- the mysterin gene transcription regulatory region is not particularly limited as long as it is a region capable of controlling mysterin gene expression. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the nucleotide sequence of this region is missing. Examples include a region consisting of a lost, substituted or added base sequence and having the ability to control transcription of the mysterin gene.
- the reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance.
- a GFP green fluorescent protein
- GUS ⁇ -glucuronidase
- LUC luciferase
- CAT Chloramphenicol acetyltransferase
- a cell into which a mysterin gene transcription regulatory region and a reporter gene operably linked to the region are introduced can quantitatively analyze the expression level of the reporter gene as long as the mysterin gene transcription regulatory function can be evaluated.
- the introduced cell is a cell that can naturally express the mysterin gene ( For example, cells that naturally express the mysterin gene) are preferred.
- the culture medium in which the test substance is contacted with the cells capable of measuring the expression of the mysterin gene is appropriately selected according to the type of cells used, and includes, for example, about 5 to 20% fetal bovine serum.
- the culture conditions are also appropriately determined according to the type of cells to be used. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.
- step (b) of the above method first, the expression level of the mysterin gene in the cell contacted with the test substance is measured.
- the expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing the mysterin gene is used as a cell capable of measuring the expression of the mysterin gene, the expression level is a method known per se for a mysterin gene product, for example, a transcription product or a translation product. Can be measured.
- the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like.
- the expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique.
- a radioisotope immunoassay RIA method
- an ELISA method Methods in Enzymol. 70: 419-439 (1980)
- a fluorescent antibody method or the like
- a cell capable of performing a reporter assay for the mysterin gene transcriptional regulatory region is used as a cell capable of measuring mysterin gene expression
- the expression level can be measured based on the signal intensity of the reporter.
- the expression level of the mysterin gene in the cells contacted with the test substance is compared with the expression level of the mysterin gene in the control cells not contacted with the test substance.
- the comparison of expression levels is preferably performed based on the presence or absence of a significant difference.
- the expression level of the mysterin gene in the control cells that are not contacted with the test substance is the expression level measured at the same time, even if the expression level is measured in advance compared to the measurement level of the mysterin gene expression level in the cell that is in contact with the test substance.
- it may be an amount, it is preferably an expression amount measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
- a test substance that regulates the expression level of the mysterin gene is selected.
- the regulation of the expression level of the mysterin gene can be an increase or decrease in the expression level.
- a test substance that increases (promotes expression) the expression level of a mysterin gene can be a candidate substance for a prophylactic or therapeutic agent for angiogenesis abnormality.
- a test substance that decreases (suppresses expression) the expression level of the mysterin gene can be an inducer of abnormal angiogenesis. Therefore, it becomes possible to select a candidate substance for a research reagent such as a drug such as a prophylactic or therapeutic agent for angiogenesis abnormality or an angiogenesis abnormality inducer, using the expression level of the mysterin gene as an index.
- the screening method of the present invention comprises the following steps (a) to (c): (A) contacting the test substance with a mysterin polypeptide; (B) measuring the function of the mysterin polypeptide in the presence of the test substance and comparing the function with the function of the mysterin polypeptide in the absence of the test substance; and (c) (b) above A step of selecting a test substance that modulates the function of the mysterin polypeptide based on the result of.
- step (a) of the above method the test substance is brought into contact with the mysterin polypeptide.
- Contact of the test substance with the polypeptide can be performed by mixing the test substance and the mysterin polypeptide in an appropriate buffer.
- Mysterin polypeptide can be prepared by a method known per se.
- a mysterin polypeptide can be isolated and purified from the above-described mysterin gene expression tissue.
- the recombinant polypeptide can It can be produced by the method described in the section.
- Examples of the function of the mysterin polypeptide in step (b) of the above method include ubiquitin ligase activity and ATPase activity.
- the ubiquitin ligase activity and ATPase activity are as described in 1. above. It can be measured by the method described in the section.
- the function of the mysterin polypeptide in the presence of the test substance is compared with the function of the mysterin polypeptide in the absence of the test substance.
- the comparison of functions is preferably performed based on the presence or absence of a significant difference.
- Measurement of mysterin polypeptide function in the absence of the test substance is performed simultaneously with measurement of mysterin polypeptide function in the presence of the test substance, even if it was performed in advance. However, it is preferable to measure simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
- a test substance that regulates the function of mysterin polypeptide is selected.
- Modulation of the function of a mysterin polypeptide can be promotion or suppression of function.
- a test substance that promotes the function of mysterin polypeptide can be a candidate substance for a prophylactic or therapeutic agent for angiogenesis abnormality.
- a test substance that suppresses the function of mysterin polypeptide can be an inducer of abnormal angiogenesis. Accordingly, it is possible to select a candidate substance for a research reagent such as a pharmaceutical agent such as a prophylactic or therapeutic agent for angiogenesis abnormality or an inducer of angiogenesis abnormality using the function of mysterin polypeptide as an index. .
- the screening method of the present invention includes the following steps (a) to (c): (A) contacting the test substance with a mysterin polypeptide; (B) measuring the binding ability of the test substance to the mysterin polypeptide; and (c) selecting a test substance having the binding ability to the mysterin polypeptide based on the result of (b) above.
- step (a) of the above method the test substance is brought into contact with the mysterin polypeptide.
- Contact of the test substance with the polypeptide can be performed by mixing the test substance and the mysterin polypeptide in an appropriate buffer.
- step (b) of the above method the binding ability of the test substance to the mysterin polypeptide is measured.
- the binding ability can be measured by a method known per se, for example, a binding assay, a method using surface plasmon resonance (for example, use of Biacore (registered trademark)).
- a test substance capable of binding to the mysterin polypeptide is selected.
- a test substance capable of binding to the mysterin polypeptide may be a substance capable of regulating (for example, promoting or suppressing) the function of the mysterin gene. Therefore, this methodology can be useful, for example, as a first screening for substances that can regulate the function of the mysterin gene.
- a test substance that efficiently regulates the function of the mysterin polypeptide can be reliably selected.
- the screening method of the present invention can also be performed by administering a test substance to an animal.
- the animal include mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, and the animals of the present invention.
- a test substance that regulates the expression level of the mysterin gene can be selected.
- the screening method of the present invention enables screening for candidate substances for regulators of angiogenesis. Therefore, the screening method of the present invention is useful for the development of a pharmaceutical agent such as a preventive or therapeutic agent for angiogenesis abnormality, or the development of a research reagent such as an angiogenesis abnormality inducer.
- a pharmaceutical agent such as a preventive or therapeutic agent for angiogenesis abnormality
- a research reagent such as an angiogenesis abnormality inducer.
- a polynucleotide comprising a SNP associated with moyamoya disease is a polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 4766 T> C ( SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917G> A (SNP4) and 232102 G> A (SNP5), and at least one SNP selected from the partial sequence
- SNP allele included in the present invention is a minor allele, and the partial sequence or its complementary sequence provides a polynucleotide having a length of at least 12 nucleotides.
- the present invention provides a polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G.
- SEQ ID NO: 5 is a partial nucleotide sequence of human chromosome 17 DNA containing a mysterin gene and genes in its peripheral region (FLJ3520, NPTX1, CARD14, and Raptor (KIAA1303)). Contig # NT010783 registered in NCBI. It corresponds to the 15th nucleotides 43560001 to 43795000.
- the nucleotide sequence represented by SEQ ID NO: 5 includes the following novel SNPs associated with moyamoya disease, which are present on human chromosome 17.
- SNP at position 4766 which is T or C (abbreviated herein as 4766 T> C, or SNP1)
- SNP at position 73097 which is G or A (in this specification, 73097 G> A, or SNP2)
- SNP at position 152917 which is G or A abbreviated as 152917 G> A or SNP4 herein
- SNP at position 232102 which is G or A (232102 G> A or SNP5 herein) Abbreviated).
- the nucleotide sequence represented by SEQ ID NO: 5 also includes the following novel SNPs associated with moyamoya disease that are present on human chromosome 17.
- SNP at position 55977 which is G or A (abbreviated as 55977 G> A or SNP6 in this specification), SNP at position 55712 which is A or G (abbreviated as 55712 A> G or SNP7 in this specification), and SNP at position 57483 which is G or A (57483G> A or SNP8 in this specification) Abbreviated).
- the position of the SNP is described based on the position of the nucleotide in the nucleotide sequence represented by SEQ ID NO: 5.
- SNP at position 4766 means the SNP at the nucleotide at position 4766 in the nucleotide sequence represented by SEQ ID NO: 5.
- nucleotide sequence is described as a DNA sequence unless otherwise specified.
- polynucleotide is RNA
- thymine (T) is appropriately replaced with uracil (U).
- the polynucleotide of the present invention includes at least one (1, 2, 3, 4 or 5, preferably 1) SNP selected from the group consisting of the above five SNPs. That is, in a preferred embodiment, the polynucleotide of the present invention includes SNP1, SNP2, SNP3, SNP4 or SNP5.
- the polynucleotide of the present invention has at least one selected from the group consisting of the above 8 SNPs (1, 2, 3, 4, 5, 6, 7 or 8, preferably 1,). SNPs are included. That is, in a preferred embodiment, the polynucleotide of the present invention includes SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 or SNP8.
- the SNP allele contained in the polynucleotide of the present invention is a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “C”; when SNP2 is included, the SNP is “A”; when SNP3 is included, the SNP is “A”. If SNP4 is included, the SNP is “A”; if SNP5 is included, the SNP is “A”.
- the SNP when the polynucleotide of the present invention includes SNP6, the SNP is “A”; when SNP7 is included, the SNP is “G”; when SNP8 is included, the SNP is “A”.
- the polynucleotide of the present invention may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA.
- the nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
- the polynucleotide of the present invention includes a complementary sequence of a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5, the complementarity of the complementary sequence is 100%.
- the length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide of the present invention is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, Preferably it is 20 nucleotides or more (for example, 25 nucleotides or more).
- the upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
- the length of the polynucleotide of the present invention is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more.
- the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
- the polynucleotide of the present invention may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
- the polynucleotide of the present invention contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), etc. It may be labeled with.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase
- the polynucleotide of the present invention is preferably isolated or purified.
- the polynucleotide of the present invention can be used as a nucleic acid probe or primer for detecting SNPs 1 to 5, it is useful as a diagnostic agent for the onset risk of moyamoya disease associated with these SNPs.
- SNP2 correlates with ischemic heart disease and the frequency of delivery of identical twins
- the polynucleotide of the present invention contains SNP2
- the polynucleotide contains the risk of developing ischemic heart disease, It is useful as a diagnostic agent for the possibility of delivery of identical twins (see sections 8 to 10 below).
- the polynucleotide of the present invention can be used as a nucleic acid probe or primer for detecting SNPs 1 to 8, and thus is useful as a diagnostic agent for the onset risk of moyamoya disease associated with these SNPs. .
- the present invention relates to 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A in the nucleotide sequence represented by SEQ ID NO: 5. (SNP4) and 232102 G> A (SNP5)
- SNP5 A method for determining the risk of developing Moyamoya disease comprising detecting at least one SNP selected from the group consisting of G> A (SNP5) is provided.
- the present invention provides 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 152917 G> A (SNP4) in the nucleotide sequence represented by SEQ ID NO: 5. Detecting at least one SNP selected from the group consisting of 232102 G> A (SNP5), 55777 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8), A method for determining the risk of developing moyamoya disease is provided.
- the determination method of the present invention includes the following steps (a) and (b): (A) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, and SNP5 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal; (B) A step of evaluating the risk of developing Moyamoya disease based on the detected SNP type.
- the determination method of the present invention includes the following steps (a ′) and (b ′): (A ′) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal The step of: (B ') The process of evaluating the onset risk of moyamoya disease based on the detected SNP type.
- At least one SNP selected from the group consisting of SNP1-5 (or SNP1-8) is detected in a biological sample collected from an animal (subject). Measure the type of the SNP.
- an animal the above-mentioned 5. Mammals listed in the section are preferred, and humans are particularly preferred.
- the human race is not particularly limited, but is preferably East Asian (East Asian / Mongoloid).
- the human race is not particularly limited, but is preferably Caucasian (white).
- the human race is preferably East Asian (East Asian / Mongoloid).
- the human race is preferably Caucasian (white).
- race is a group that can be distinguished as a specific subgroup in the Homo sapiens species. Race has a unique and distinguishable combination of genes and is identified by the characteristics (both mental and physical) created by the combination of genes. Members of the same race share a distinct genetic feature because they share a common genetic ancestry and, as a result, share similar gene combinations.
- East Asian means a person who originated in Japan, Korea, China, Taiwan or Mongolia.
- the East Asian is preferably a Japanese, a Korean, or a Chinese.
- a person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
- any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used.
- hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
- the SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
- RFLP restriction fragment length polymorphism
- PCR-SSCP single-stranded DNA higher-order structure polymorphism analysis
- ASO Allele Specific Oligonucleotide hybridization method
- sequencing method ARMS (Amplification Refracting Mutation System)
- denaturing gradient gel electrophoresis Den
- a minor allele (adenine) of SNP2 it can be determined that the risk of developing moyamoya disease is extremely high compared to the case where the minor allele is not detected.
- SNP7 or 8 Since mutation of SNP7 or 8 is accompanied by amino acid substitution of mysterin, the amino acid substitution of mysterin caused by this minor allele of SNP7 or 8 may affect the onset of moyamoya disease. Thus, in one preferred embodiment of the invention, SNP 7 or 8 is detected.
- DNA usually has a double helix structure consisting of two strands complementary to each other. Therefore, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, taking SNP1 as an example, even when the base on the complementary strand corresponding to the base at position 4766 of the nucleotide sequence shown in SEQ ID NO: 5 is G (guanine), the subject is at risk of developing Moyamoya disease. , Can be determined to be relatively high.
- nucleotide sequence disclosed in the present specification for example, the nucleotide sequence represented by SEQ ID NO: 5 and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5).
- Slight difference between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof
- nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject.
- nucleotide sequence disclosed in the present specification for example, the nucleotide sequence represented by SEQ ID NO: 5
- the information on the SNP to use the nucleotide sequence disclosed in the sequence listing (SEQ ID NO: 5
- a slight difference (deletion, substitution, addition) between the nucleotide sequence represented by (1) or a contiguous partial sequence thereof or a complementary sequence thereof and the nucleotide sequence of the corresponding site on the subject chromosome 17 DNA , Increase or decrease in the number of repetitive sequences, etc.) the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence derived from the subject are aligned to obtain the nucleotide derived from the subject.
- the SNP may be detected for at least one of the chromosome 17 of the subject.
- the method of the present invention is selected from the group consisting of cytosine in SNP1, adenine in SNP2, adenine in SNP3, adenine in SNP4, adenine in SNP5, adenine in SNP6, guanine in SNP7, and adenine in SNP8. If at least one minor allele is detected in at least one allele, it can be determined that the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected. That is, in the method of the present invention, the SNP may be detected for at least one of the chromosome 17 of the subject.
- the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was The risk of developing moyamoya disease can also be determined by identification.
- the mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography.
- the amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum.
- the major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identifying the amino acid No. 4810, when lysine is detected, the risk of developing Moyamoya disease is relatively higher than when lysine is not detected (that is, when only arginine is detected). Can be determined.
- the mutations in SNPs 6, 7 and 8 are amino acid substitution No. 4013 (aspartic acid ⁇ asparagine), amino acid substitution No. 3962 (aspartic acid ⁇ aspartic acid) and amino acid No. 4062 of human mysterin, respectively. Since the substitution (arginine ⁇ glutamine) is involved, the mysterin polypeptide is isolated from the subject, and the amino acids No. 4013, No. 3962 and No. 4062 are identified, respectively. It can be judged. The mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography. Identification of amino acids Nos.
- 4013, 3962, and 4062 can be performed by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum.
- the major allele of SNP6 gives aspartic acid as amino acid 4013 of human mysterin polypeptide, and the minor allele of SNP6 gives asparagine as amino acid 4013 of human mysterin polypeptide. Accordingly, as a result of identifying amino acid No. 4013, when asparagine is detected, the risk of developing moyamoya disease is relatively higher than when asparagine is not detected (that is, when only aspartic acid is detected). It can be determined that the price is high.
- the major allele of SNP7 gives asparagine as amino acid 3962 of human mysterin polypeptide, and the minor allele of SNP7 gives aspartic acid as amino acid 3962 of human mysterin polypeptide. Accordingly, as a result of identifying the amino acid No. 3962, when aspartic acid is detected, the risk of developing moyamoya disease is relative to when aspartic acid is not detected (that is, when only asparagine is detected). It can be determined to be high.
- the major allele of SNP8 gives arginine as amino acid 4062 of human mysterin polypeptide, and the minor allele of SNP8 gives glutamine as amino acid 4062 of human mysterin polypeptide. Therefore, as a result of identifying amino acid No. 4062, when glutamine is detected, the risk of developing Moyamoya disease is relatively higher than when glutamine is not detected (that is, when only arginine is detected). Can be determined.
- the present invention also provides a diagnostic agent for the onset risk of moyamoya disease, comprising a reagent for detecting at least one SNP selected from the group consisting of SNPs 1 to 5.
- a nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of SNP5) preferably SNP2
- a primer capable of specifically amplifying a region containing the SNP. I can do it.
- the present invention provides a diagnostic agent for the risk of developing Moyamoya disease, comprising a reagent for detecting at least one SNP selected from the group consisting of SNPs 1-8.
- SNPs 1 to 8 As a reagent for detecting at least one SNP selected from the group consisting of SNPs 1 to 8, (1 ′) 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A in the nucleotide sequence represented by SEQ ID NO: 5 (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) are specifically detected at least one SNP (preferably SNP2 or SNP6) selected from the group consisting of And (2 ′) a primer that can specifically amplify the region containing the SNP.
- hybridization conditions include low stringency conditions.
- the low stringent conditions are, for example, conditions of 42 ° C., 5 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS in washing after hybridization. is there. More preferable hybridization conditions include highly stringent conditions.
- High stringent conditions are, for example, 65 ° C., 0.1 ⁇ SSC, and 0.1% SDS.
- factors affecting the stringency of hybridization include multiple factors such as temperature and salt concentration, and those skilled in the art can realize the same stringency by selecting these factors as appropriate. is there.
- the nucleic acid probe of (1) or (1 ′) is preferably an affinity for the polynucleotide represented by SEQ ID NO: 5 wherein the SNP targeted by the nucleic acid probe is a major allele, and the SNP is a minor allele. There is a difference between the affinity for the polynucleotide represented by SEQ ID NO: 5 and it hybridizes to one of the major allele and minor allele under appropriate conditions (for example, highly stringent conditions). However, it does not hybridize to the other.
- the probe of (1) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5), wherein the SNP included in the partial sequence comprises at least one SNP selected from the group consisting of Examples of the polynucleotide include a major allele or a minor allele, and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
- the polynucleotide includes at least one (1, 2, 3, 4 or 5, preferably 1) SNP selected from the group consisting of the above five SNPs. That is, in a preferred embodiment, the polynucleotide includes SNP1, SNP2, SNP3, SNP4 or SNP5. When detecting SNP1, the polynucleotide includes SNP1, when detecting SNP2, the polynucleotide includes SNP2, and when detecting SNP3, the polynucleotide includes SNP3. When SNP4 is detected, the polynucleotide includes SNP4, and when SNP5 is detected, the polynucleotide includes SNP5.
- the SNP allele contained in the polynucleotide is a major allele or a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “T” or “C”; when SNP2 is included, the SNP is “G” or “A”; and SNP3 is included. The SNP is “G” or “A”; when SNP4 is included, the SNP is “G” or “A”; when SNP5 is included, the SNP is “G” or “A”. It is.
- the probe of (1 ′) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, and the partial sequence is 476647T> C ( SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 15292929G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) And at least one SNP selected from the group consisting of 57483 G> A (SNP8), the allele of the SNP included in the partial sequence is a major allele or a minor allele, and the partial sequence or its complementary sequence is Exemplified are polynucleotides having a length of at least 12 nucleotides.
- the polynucleotide includes at least one (1, 2, 3, 4, 5, 6, 7 or 8, preferably 1) SNP selected from the group consisting of the above 6 SNPs. That is, in a preferred embodiment, the polynucleotide includes SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 or SNP8. When detecting SNP1, the polynucleotide includes SNP1, when detecting SNP2, the polynucleotide includes SNP2, and when detecting SNP3, the polynucleotide includes SNP3. When SNP4 is detected, the polynucleotide includes SNP4.
- the polynucleotide when detecting SNP5, the polynucleotide includes SNP5, and when detecting SNP6, the polynucleotide includes SNP4.
- the nucleotide includes SNP6.
- SNP7 is detected, the polynucleotide includes SNP7.
- SNP8 is detected, the polynucleotide includes SNP8.
- the SNP allele contained in the polynucleotide is a major allele or a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “T” or “C”; when SNP2 is included, the SNP is “G” or “A”; and SNP3 is included.
- the SNP is “G” or “A”; when SNP4 is included, the SNP is “G” or “A”; when SNP5 is included, the SNP is “G” or “A”.
- SNP6 is included, the SNP is “G” or “A”; when SNP7 is included, the SNP is “A” or “G”; when SNP8 is included, the SNP is “G” or “A”.
- the polynucleotide that is the nucleic acid probe of (1) or (1 ') may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA.
- the nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
- the complementarity of the complementary sequence is preferably 100%.
- the length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, still more preferably 20 nucleotides or more (for example, 25 nucleotides or more).
- the upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
- the length of the polynucleotide is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more.
- the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
- the polynucleotide may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
- the polynucleotide also contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ - Labeled with glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) May be.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ - Labeled with glucosidase, alkaline phosphatase, peroxidase, mal
- telomere amplification of SNP by primer refers to PCR amplification of a region containing a specific SNP of a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 5 in a primer. This region means that PCR amplification is not performed.
- the primer of the above (2) starts complementary strand synthesis toward the selected SNP site using a polynucleotide (for example, genomic DNA) containing at least one SNP selected from the group consisting of SNPs 1 to 5 as a template.
- Primers that can be included are included.
- the primer can also be expressed as a primer for providing an origin of replication on the 3 'side of the SNP site in the polynucleotide containing the SNP site.
- the interval between the region where the primer hybridizes and the SNP site is arbitrary. A suitable length can be selected for the interval between the two according to the analysis method of the base at the SNP site.
- the primer is designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region including the SNP site. be able to.
- a person skilled in the art can design a primer suitable for each analysis method based on nucleotide sequence information about the surrounding DNA region including the SNP site.
- the nucleotide sequence constituting the primer of the above (2) can be appropriately modified as well as the nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
- the primer (2 ′) mentioned above is directed toward the selected SNP site using a polynucleotide (eg, genomic DNA) containing at least one SNP selected from the group consisting of SNPs 1 to 8 as a template. Primers capable of initiating complementary strand synthesis are included.
- the primer can also be expressed as a primer for providing an origin of replication on the 3 'side of the SNP site in the polynucleotide containing the SNP site.
- the interval between the region where the primer hybridizes and the SNP site is arbitrary. A suitable length can be selected for the interval between the two according to the analysis method of the base at the SNP site.
- the primer is designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region including the SNP site. be able to.
- a person skilled in the art can design a primer suitable for each analysis method based on nucleotide sequence information about the surrounding DNA region including the SNP site.
- the nucleotide sequence constituting the primer (2 ') can be appropriately modified as well as a nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
- the primers (2) and (2 ') may be DNA, RNA, or DNA / RNA chimera, but are preferably DNA.
- the nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
- the length of the region in which the primers (2) and (2 ′) can hybridize to the nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably Is 18 nucleotides or more, more preferably 20 nucleotides or more (for example, 25 nucleotides or more).
- the upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. It is.
- the lengths of the primers (2) and (2 ') are at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more.
- the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. .
- the primers (2) and (2 ') may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
- the primer may be a suitable labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase). , Alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ -glucosidase.
- Alkaline phosphatase eg, peroxidase, malate dehydrogenase
- various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method.
- the enzyme an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown.
- the buffer solution a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected.
- the enzyme substrate for example, a substrate for complementary strand synthesis is used.
- the diagnostic agent of the present invention When the diagnostic agent of the present invention is used, the risk of developing moyamoya disease can be easily determined by the determination method of the present invention.
- the present invention provides a method for determining the risk of developing ischemic heart disease, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. To do.
- the determination method of the present invention includes the following steps (a) and (b): (A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal (subject); (B) A step of evaluating the risk of developing ischemic heart disease based on the detected SNP type.
- Ischemic heart disease is a general term for diseases in which blood flow to the myocardium is blocked by coronary artery occlusion or stenosis, causing damage to the heart.
- Examples of the ischemic heart disease include myocardial infarction and angina.
- step (a) of the above method SNP2 is detected in a biological sample collected from an animal, and the type of the SNP is measured.
- the animal the above-mentioned mammals are preferable, and a human is particularly preferable.
- the human race is not particularly limited but is preferably East Asian (East Asian / Mongoloid).
- East Asian means a person who originated in Japan, Korea, China, Taiwan or Mongolia.
- the East Asian is preferably a Japanese, a Korean, or a Chinese.
- a person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
- Human sex is not particularly limited, but is preferably a woman who seems to have few general risk factors.
- the age of a human is not particularly limited, but is preferably 35 to 54 years old.
- any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used, but from the viewpoint of easy availability and minimal invasiveness, Hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
- the SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
- RFLP restriction fragment length polymorphism
- PCR-SSCP single-stranded DNA higher-order structure polymorphism analysis
- ASO Allele Specific Oligonucleotide hybridization method
- sequencing method ARMS (Amplification Refracting Mutation System)
- denaturing gradient gel electrophoresis Den
- DNA usually has a double helix structure consisting of two strands complementary to each other. Therefore, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, even when the base type of the site on the complementary strand corresponding to the site at position 73097 of the nucleotide sequence set forth in SEQ ID NO: 5 is T (thymine), the subject is at risk of developing ischemic heart disease, It can be determined that it is relatively high.
- nucleotide sequence disclosed in the present specification for example, the nucleotide sequence represented by SEQ ID NO: 5 and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5).
- Slight difference between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof
- the nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject. It is possible to accurately identify whether the base corresponds to SNP2 and detect SNP2. Such a case is also included in “detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5”.
- SNP2 may be detected for at least one of the chromosome 17 of the subject.
- the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was Identification can also determine the onset risk of ischemic heart disease.
- the mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography.
- the amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum.
- the major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identifying the amino acid at position 4810, when lysine is detected, the risk of developing ischemic heart disease is relative to when lysine is not detected (that is, when only arginine is detected). It can be determined to be high.
- the present invention also provides a diagnostic agent for the risk of developing ischemic heart disease, comprising a reagent for detecting SNP2.
- the probe of (1) is preferably represented by SEQ ID NO: 5 having an affinity for the polynucleotide represented by SEQ ID NO: 5 in which SNP2 is a major allele (guanine), and the SNP2 is a minor allele (adenine). And hybridizes to one of the major and minor alleles under appropriate conditions (eg, the above highly stringent conditions), but to the other Does not hybridize.
- the probe of (1) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 73097 G> A (SNP2),
- SNP2 73097 G> A
- Examples of the polynucleotide include that the allele of the SNP contained in the partial sequence is a major allele or a minor allele, and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
- the allele of SNP2 contained in the polynucleotide is a major allele (guanine) or a minor allele (adenine).
- the polynucleotide may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA.
- the nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
- the complementarity of the complementary sequence is preferably 100%.
- the length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, still more preferably 20 nucleotides or more (for example, 25 nucleotides or more).
- the upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
- the length of the polynucleotide is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more.
- the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
- the polynucleotide may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
- the polynucleotide also contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ - Labeled with glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) May be.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ - Labeled with glucosidase, alkaline phosphatase, peroxidase, mal
- telomere amplification of SNP by primer refers to PCR amplification of a region containing a specific SNP of a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 5 in a primer. This region means that PCR amplification is not performed.
- the primer (2) includes a primer that can initiate complementary strand synthesis toward SNP2 using a polynucleotide containing SNP2 (eg, genomic DNA) as a template.
- the primer can also be expressed as a primer for providing a replication origin on the 3 'side of SNP2 in a polynucleotide containing SNP2.
- region which a primer hybridizes and SNP2 is arbitrary. A suitable length can be selected for the interval between the two according to the SNP base analysis method.
- the primer in the case of a primer for analysis by DNA chip or direct sequencing, the primer should be designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region containing SNP2. Can do.
- a person skilled in the art can design a primer according to the analysis method based on the nucleotide sequence information about the peripheral DNA region containing SNP2.
- the nucleotide sequence constituting the primer of the above (3) can be appropriately modified as well as a nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
- the primer (2) may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA.
- the nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
- the length of the region where the primer (2) can hybridize to the nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, More preferably, it is 20 nucleotides or more (for example, 25 nucleotides or more).
- the upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. It is.
- the length of the primer (2) is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more.
- the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. .
- the primer (2) above may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence.
- the primer may be a suitable labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase). , Alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ -glucosidase.
- Alkaline phosphatase eg, peroxidase, malate dehydrogenase
- various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method.
- the enzyme an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown.
- the buffer solution a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected.
- the enzyme substrate for example, a substrate for complementary strand synthesis is used.
- the diagnostic agent of the present invention When the diagnostic agent of the present invention is used, the risk of developing ischemic heart disease can be easily determined by the determination method of the present invention.
- the present invention includes the possibility of delivery of identical twins, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. A determination method is provided.
- the determination method of the present invention includes the following steps (a) and (b): (A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal; (B) A step of evaluating the feasibility of identical twins based on the detected SNP type.
- step (a) of the above method SNP2 is detected in a biological sample collected from an animal, and the type of the SNP is measured.
- the animal the above-mentioned mammals are preferable, and a human is particularly preferable.
- the human race is not particularly limited but is preferably East Asian (East Asian / Mongoloid).
- East Asian means a person who originated in Japan, Korea, China, Taiwan or Mongolia.
- the East Asian is preferably a Japanese, a Korean, or a Chinese.
- a person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
- any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used, but from the viewpoint of easy availability and minimal invasiveness, Hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
- the SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
- RFLP restriction fragment length polymorphism
- PCR-SSCP single-stranded DNA higher-order structure polymorphism analysis
- ASO Allele Specific Oligonucleotide hybridization method
- sequencing method ARMS (Amplification Refracting Mutation System)
- denaturing gradient gel electrophoresis Den
- DNA usually has a double helix structure consisting of two strands complementary to each other. Accordingly, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, even when the base type is T (thymine) for the site on the complementary strand of the sequence corresponding to the site at position 73097 of the nucleotide sequence shown in SEQ ID NO: 5, It can be determined that the frequency of delivery is relatively high.
- T thymine
- nucleotide sequence disclosed in the present specification for example, the nucleotide sequence represented by SEQ ID NO: 5 and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5).
- Slight difference between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof
- the nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject. It is possible to accurately identify whether the base corresponds to SNP2 and detect SNP2. Such a case is also included in “detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5”.
- SNP2 may be detected for at least one of the chromosome 17 of the subject.
- the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was Identification can also determine the feasibility of identical twins.
- the mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography.
- the amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum.
- the major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identification of amino acid No. 4810, when lysine was detected, the relative feasibility of identical twins compared to the case where lysine was not detected (that is, only arginine was detected). Can be determined to be high.
- the present invention also provides a diagnostic agent for the feasibility of identical twins, including a reagent for detecting SNP2.
- various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method.
- the enzyme an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown.
- the buffer solution a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected.
- the enzyme substrate for example, a substrate for complementary strand synthesis is used.
- the diagnostic agent of the present invention When the diagnostic agent of the present invention is used, the possibility of delivery of identical twins can be easily determined by the determination method of the present invention.
- Example 1 Identification of new SNPs and analysis of allele frequency of each SNP in patients with moyamoya disease Study population This study was approved by the Ethics Committee of Kyoto University School of Medicine and was written by all subjects. Obtained informed consent by. Two types of moyamoya disease case participants were enrolled in this study. The first type is a family unit participant selected to participate in this study due to the clear presence of one or more moyamoya disease cases in relatives. Thirty-four moyamoya probands, including 31 Japanese and 3 Koreans, were enrolled in this study. To verify the diagnosis, a history and risk factors for vascular disease were collected from the proband and its family members. recruited founder family members who were admitted to Kyoto University Hospital or other partner hospitals.
- Genotyping, mapping and haplotype estimation were performed as previously described (Neurology vol70: 2357-2363, 2008). Briefly, genomic DNA was extracted from patient blood samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany).
- Genomic DNA extracted from peripheral blood was amplified by PCR with fluorescently labeled markers, and the PCR products were analyzed using ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, Calif.) And Genescan program. Detailed mapping markers were designed according to physical location information from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Linkage analysis was performed using a multipoint parametric linkage method assuming an autosomal dominant genetic model (J Neurol Neurosurg Psychiatry, vo. 77, pages 1025-1029, 2006 and Neurology vol70: 2357-2363, 2008). As previously reported, inevitable carriers were treated as those who developed (Neurology vol70: 2357-2363, 2008).
- the phenotype of non-onset individuals was “unknown” and the “unrelated” spouse was “non-onset”. Based on an observed morbidity of 6.03 per 100,000 (Stroke, vol. 39, pages 42-47, 2008), the disease allele frequency should be set to 0.00003015, but even if asymptomatic In view of the recent increase in the number of patients diagnosed with moyamoya disease by magnetic resonance imaging (MRI) and MRA, the allele frequency is calculated from a lower LOD score by linkage analysis. It was set to 0.0001, which is considered to be strict (J Clin Neurosci, vol. 13, pages 334-338, 2006;). The phenotype replication frequency was assumed to be 0.00001.
- the allele frequency of each microsatellite marker was estimated from all unrelated spouses using the Merlin software (Nat Genet, vol. 30, pages 97-101, 2002). Analysis was performed using GENEHUNTER version 2.0 (http://www.broad.mit.edu/ftp/distribution/software/genehunter/) (Am J Hum Genet, vol. 58, pages 1347-1363, 1996) .
- Raptor was sequenced for a control region approximately 2 kbp upstream of the first exon. After PCR amplification and purification, sequencing was performed using an ABI Prism 3100 Avant DNA Sequencer (Applied Biosystems). We checked the Single Nucleotide Polymorphism database (dbSNP) for reference (http://www.ncbi.nlm.nih.gov/SNP/index.html).
- SNPs Five mutations (SNPs) were identified in the 4 individuals who developed the pedigree in the sequence (Table 1).
- the frequency of minor alleles of SNP1 to SNP5 was about 52% or more in non-familial moyamoya disease patients and extremely high, about 79% or more in familial moyamoya disease patients.
- all familial moyamoya patients had minor alleles.
- the frequency of minor alleles SNP1 to SNP5 remained around 2%. Therefore, it was shown that the risk of moyamoya disease can be determined by determining SNP1 to SNP5.
- the minor allele of SNP2 may be a founder mutation of moyamoya disease in Asians.
- the odds ratios for each SNPs are summarized in Table 11.
- Example 2 Cloning and functional analysis of mysterin (cloning of mysterin) The predicted gene C17orf27 where SNP2 is located and the predicted gene KIAA1618 where SNP1 is located were cloned. Six cDNA fragments were obtained from mRNA recovered from the Human Embryonic Kidney 293 cell line by reverse transcription. They were combined using an endogenous restriction enzyme cleavage site to obtain a full-length cDNA.
- KIAA1618 and C17orf27 are disclosed on the Internet homepage of NCBI as two independent genes (accession numbers: NP_066005.2 and NP_065965.3), but surprisingly, C17orf27 and KIAA1618 are complete structural genes respectively. Rather, it became clear that C17orf27 and KIAA1618 were linked together to constitute one structural gene encoding a huge protein consisting of about 5000 amino acids (FIG. 1). This new gene was named mysterin.
- the cDNA sequence of human mysterin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2, respectively.
- ATPase activity of mysterin The amino acid sequence of human mysterin was analyzed for homology using the BLAST program. As a result, the regions of amino acids 2415-2436 and 2481-2494 showed strong homology with the known motifs Walker A and Walker B, respectively (FIG. 5). These motifs are conserved in dynein, proteasome, p97, FtsH, etc., and are NTP binding motif and divalent cation binding motif, respectively. These two motifs are known to have ATPase activity by conjugation. ing. From this, it was predicted that mysterin has ATPase activity.
- a protein in which a GST tag is fused to the N-terminus of the region containing these motifs contained in human mysterin is expressed in E. coli and purified using glutathione sepharose. ATPase activity was measured.
- the fusion protein is mixed with magnesium ions and ATP at a final concentration of 5 mM at the final concentration shown in FIG. 6, incubated for 30 minutes, mixed with PCA at a final concentration of 1% to stop the reaction, and then reacted with malachite green for 30 minutes. Incubated.
- Example 3 Preparation of mysterin-deficient zebrafish and analysis thereof (site setting and preparation of mysterin-complementary DNA fragment) Since the mysterin ortholog gene of zebrafish (Danio rerio) was unidentified, the mysterin gene of zebrafish was identified based on the cDNA sequence of human mysterin, and the cDNA was cloned. As a result, two types of mysterin 1 (zRNF213) and mysterin 2 (zRNF213.1) existed, and mysterin 1 (zRNF213) was found to be a human homologous gene.
- the cDNA sequence of zebrafish mysterin 1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4, respectively.
- the nucleotides at positions 2538 to 10462 of SEQ ID NO: 3 (corresponding to amino acids at positions 847 to 3487 in SEQ ID NO: 4) were actually cloned and the nucleotide sequence was determined experimentally. The remaining portion was subjected to amino acid prediction based on the nucleotide sequence that was gene-predicted by GENSCAN based on the zebrafish genomic sequence.
- mysterin antisense morpholino hereinafter abbreviated as mysterin MO
- mysterin MO a complementary DNA fragment for preparing a mysterin gene deficient.
- the nucleotide sequence of mysterin MO is shown in SEQ ID NOs: 6 and 7.
- zRNF213spMO1-A ACTCGTTGATGTCTGAAGTGATAAA (SEQ ID NO: 6)
- zRNF213spMO1-D AGCTAGGAGAAAGTCCTACCAATTT (SEQ ID NO: 7)
- zebrafish embryos 5 ng mysterin Mysterin MO for exon acceptor site and mysterin MO for donor site were implanted into zebrafish embryos at 1 to 8 cell stage.
- the genetic background of the zebrafish embryo used at this time is a line expressing GFP specifically in vascular endothelial cells, fli-EGFP (Tg (fl1: egfp)) (Lawson ND and Weinstein BM, Developmental Biology 243: 307 -318 (2002)), this line is useful as a blood vessel imaging analysis tool.
- Zebrafish were reared under artificial conditions of 14 h light period, 10 h dark period, and 28 ° C. Mating was performed by isolating zebrafish males and females before the incubator enters the dark period, and conversely with the males and females the next morning immediately after the light period. Eggs were collected within 30 minutes of fertilization and the above-described antisense morpholinos were injected into 60-180 eggs per test. The injected eggs were incubated in water containing methylene blue and 0.03% sea salt at 28 ° C.
- the injected egg was rinsed with water containing methylene blue and 0.03% seawater salt and cultured in an incubator with an internal temperature of 28.5 ° C. Fluorescence was observed and confirmed in developing embryos after 72 hours of culture.
- Example 4 Relationship between risk of developing ischemic heart disease and SNP2 Based on the frequency of the National Basic Life Survey conducted by the Ministry of Health, Labor and Welfare in 1998, the prevalence of ischemic heart disease was also observed in the age group. Compared with value. As a result, the prevalence of ischemic heart disease in relatively young women aged 35-54, who are generally assumed to be at low risk for ischemic heart disease, with minor alleles (founder mutations) of SNP2 was found to be significantly higher (Table 13).
- Example 5 Possibility of delivery of identical twins and association with SNP2
- Example 6 New SNPs related to moyamoya disease found in Caucasian families Genetic analysis was conducted on white spouses from the Czech Republic (CAU-Ped1, Fig. 11) and 12 sporadic cases in Germany.
- the proband of the family (CAU_Ped1_12) developed a mild ischemic stroke at the age of 30, and his mother died at the age of 35 due to ischemic stroke.
- the proband had four offspring with two unrelated spouses.
- the second child (CAU_Ped1_122) with the first spouse was diagnosed with moyamoya disease at the age of five.
- Genomic DNA was extracted from the blood of the proband father, and direct sequencing was performed on all coding regions of the seven genes SLC26A11, KIAA1618, LOC100287062, C17orf27, FLJ35220, LOC728991, and NPTX1, and 165 variant (registered: 133) Pieces, unregistered: 32 pieces).
- an unregistered variant present in the coding region is one of C17orf27 (SEQ ID NO: 5, 55777 position G> A; human mysterin No. 4013 amino acid substitution (aspartic acid ⁇ asparagine) (D4013N)) It was only. This variant was inherited by three children with moyamoya disease (FIG. 11).
- D4013N, N3962D, R4062Q allele was not found, and the allele frequency in the Caucasian population was 1% or less, It was considered a mutation. Based on the above, it was concluded that D4013N allele is a mutation causing causative Moyamoya disease. D4013N is located in the RING finger domain, but biochemical analysis did not affect ubiquitin ligase activity.
- a polypeptide of a novel gene mysterin a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided.
- Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus. Analysis of knockdown zebrafish of the mysterin gene revealed that mysterin has a function of regulating angiogenesis.
- the present invention also provides new SNPs that exist at or near the mysterin locus.
- the SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
- This application is based on Japanese Patent Application No. 2009-244938 filed in Japan (filing date: October 23, 2009), the contents of which are incorporated in full herein.
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Abstract
Provided are a novel gene "Mysterin" which participates in arterial obstruction at the terminal portion of the internal carotid artery and neogenesis that are the fundamental lesions of moyamoya disease, a Mysterin polypeptide, a polynucleotide encoding said polypeptide and so on. Also provided are multiple SNPs correlating with moyamoya disease which occur at the Mysterin locus and in the neighborhood thereof. By detecting these SNPs, the onset risk of moyamoya disease can be determined.
Description
本発明は、モヤモヤ病発症前診断に有用な新規遺伝子及びSNPsに関する。更に、本発明は、該遺伝子の機能的欠損を有する非ヒト動物、該遺伝子の機能的欠損を有する細胞、該遺伝子を利用した、家族性モヤモヤ病発症リスクの判定方法、虚血性心疾患の発症リスクの判定方法、一卵性双生児の出生可能性の判定方法、及びこれらの判定方法の実施に有用な試薬等に関する。
The present invention relates to novel genes and SNPs that are useful for diagnosis before the onset of moyamoya disease. Furthermore, the present invention relates to a non-human animal having a functional defect of the gene, a cell having a functional defect of the gene, a method for determining the risk of developing familial moyamoya disease using the gene, the onset of ischemic heart disease The present invention relates to a method for determining risk, a method for determining the possibility of birth of identical twins, and a reagent useful for carrying out these determination methods.
モヤモヤ病(Moyamoya disease: MMD)は、ウィリス動脈輪閉塞症とも呼ばれ、小児期及び成人期に発症する内頸動脈終末部の狭窄病変である。
Moyamoya disease (MMD), also called Willis artery occlusion, is a stenotic lesion at the terminal end of the internal carotid artery that develops in childhood and adulthood.
モヤモヤ病は世界中で発症する疾患であるが(非特許文献1)、モヤモヤ病の発症率は、日本、韓国及び中国などの東アジアにおいて特に高い(非特許文献2及び3)。日本においては、最新の罹患率及び年間発症率が、100,000人につき10.5人及び0.94人と報告されている(非特許文献2)。ヨーロッパにおいては、発症率は日本における発症率のおよそ1/10と見積もられている(非特許文献2及び3)。アメリカにおいては、発症率は100,000人につきおよそ0.086人であり、コーカサス系アメリカ人よりも、アジア系アメリカ人及びアフリカ系アメリカ人において高い(非特許文献2及び3)。
Moyamoya disease is a disease that develops all over the world (Non-Patent Document 1), but the incidence of Moyamoya disease is particularly high in East Asia such as Japan, Korea, and China (Non-Patent Documents 2 and 3). In Japan, the latest morbidity and annual incidence are reported to be 10.5 and 0.94 per 100,000 (Non-patent Document 2). In Europe, the incidence is estimated to be about 1/10 of the incidence in Japan (Non-patent Documents 2 and 3). In the United States, the incidence is approximately 0.086 per 100,000, and is higher in Asian Americans and African Americans than in Caucasian Americans (Non-patent Documents 2 and 3).
従来から遺伝的素因が疑われてきたが、感受性遺伝子の同定はなされていない。本発明者らは、これまでの解析において、染色体領域17q25.3がモヤモヤ病の発症と連鎖することを見出している(非特許文献4)。
Although genetic predisposition has been suspected in the past, no susceptibility gene has been identified. In the analysis so far, the present inventors have found that chromosomal region 17q25.3 is linked to the onset of moyamoya disease (Non-patent Document 4).
ヒトゲノムの網羅的シークエンスにより、基本的にはヒト染色体の全てのヌクレオチド配列が決定されており、その配列から、上記染色体領域17q25.3においては、CARD14、Raptor、AATK、KIAA1618、C17orf27等を含む数多くの遺伝子の存在が予想されている。
The nucleotide sequence of the human chromosome has basically been determined by the comprehensive sequence of the human genome. From the sequence, a large number including CARD14, Raptor, AATK, KIAA1618, C17orf27, etc. in the chromosomal region 17q25.3. The presence of genes is expected.
Bertinらは、ルシフェラーゼレポーター解析に基づき、CARD14がIKKG(IKBKG;300248)又はIKKBを通してNFKB活性を誘導すること、そしてこの誘導にはCARD14のN末端を要することを示している。CARD14のC末端ドメインの欠損により増強されたNFKB活性が生じることを報告している(非特許文献5)。従って、NFKB媒介経路のためのネガティブ制御機能が存在し、これが免疫反応において鍵となる役割を果たしていることが示唆される。
Bertin et al., Based on luciferase reporter analysis, indicate that CARD14 induces NFKB activity through IKKG (IKKBKG; 3000024) or IKKB, and that this induction requires the N-terminus of CARD14. It has been reported that enhanced NFKB activity occurs due to deletion of the C-terminal domain of CARD14 (Non-patent Document 5). Thus, there is a negative regulatory function for the NFKB-mediated pathway, suggesting that this plays a key role in the immune response.
Raptor[regulatory associated protein of mammalian target of rapamycin (mTOR)]は、TORシグナリングに必須の150kDaのmTOR補因子である(非特許文献6)。Raptorは、組織の肥大と関連し(非特許文献7)、hypoxia-inducible factorの制御因子であり(非特許文献8)、HLAクラスI抗体により媒介される血管内皮細胞増殖に関与する(非特許文献9)。
Raptor [regulatory associated protein of mammalian target of rapamycin (mTOR)] is a 150 kDa mTOR cofactor essential for TOR signaling (Non-patent Document 6). Raptor is associated with tissue hypertrophy (Non-patent document 7), is a regulator of hypoxia-inducible factor (Non-patent document 8), and is involved in vascular endothelial cell proliferation mediated by HLA class I antibodies (Non-patent document 7). Reference 9).
AATKは、アポトーシスに関与することが報告されている(非特許文献10)。アポトーシスは、MMDの患者の狭窄病変において観察される現象の1つである(非特許文献11)。
AATK has been reported to be involved in apoptosis (Non-patent Document 10). Apoptosis is one of the phenomena observed in stenotic lesions in patients with MMD (Non-patent Document 11).
KIAA1618及びC17orf27のアミノ酸配列は、それぞれアクセッション番号NP_066005.2及びNP_065965.3として登録され、NCBIのインターネットホームページ上に公開されている。しかしながら、これらの遺伝子は、コンピューターによるヌクレオチド配列解析に基づき構造遺伝子であろうと予測された「予測遺伝子(predicted gene)」であり、独立した2つの遺伝子として開示されているが、機能解析はまったくなされていない。
The amino acid sequences of KIAA1618 and C17orf27 are registered as accession numbers NP_066005.2 and NP_065965.3, respectively, and are published on the NCBI Internet homepage. However, these genes are “predicted genes” predicted to be structural genes based on nucleotide sequence analysis by a computer, and are disclosed as two independent genes, but functional analysis is not performed at all. Not.
特許文献1において、配列番号4で表されるアミノ酸配列からなるポリペプチドが開示されている。該ポリペプチドは膜貫通ドメインを有する膜タンパク質であり、Znフィンガードメイン及びATP結合モチーフA(Pループ)を有することが記載されている。
Patent Document 1 discloses a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4. The polypeptide is a membrane protein having a transmembrane domain, and is described to have a Zn finger domain and an ATP binding motif A (P loop).
本発明の目的は、モヤモヤ病の発症と相関するSNPsを同定し、それに基づきモヤモヤ病の発症前診断方法を確立することである。更に、本発明は、当該SNPsの情報から、モヤモヤ病の発症に関連する可能性のある遺伝子を取得し、該遺伝子の機能的欠損を含む非ヒト動物や動物細胞を作成し、該遺伝子の機能を明らかにすることを目的とする。また、本発明は、該遺伝子及びSNPsの更なる用途を提供することを目的とする。
The purpose of the present invention is to identify SNPs that correlate with the onset of moyamoya disease, and to establish a pre-onset diagnosis method for moyamoya disease based on this. Furthermore, the present invention obtains a gene that may be related to the onset of moyamoya disease from the information of the SNPs, creates a non-human animal or animal cell containing a functional defect of the gene, and functions of the gene The purpose is to clarify. Moreover, this invention aims at providing the further use of this gene and SNPs.
本発明者らは、17のモヤモヤ病家系について詳細な連鎖解析を行い、染色体領域17q25.3の中から、モヤモヤ病の発症と連鎖する、約2.1Mbのコア領域を特定することに成功した。該コア領域には約38個の遺伝子が含まれていた。
このコア領域のヌクレオチド配列を網羅的に解析したところ、5つの新たなSNPsが、KIAA1618(SNP1)、C17orf27(RNF213)(SNP2)、FLJ3520(SNP3)、NPTX1(SNP4)及びKIAA1303(SNP5)の各遺伝子内又はその近傍において同定された。これらのSNPsのマイナーアレルの頻度は、モヤモヤ病の発症とよく相関した。試験を行った全ての家族性のモヤモヤ病患者は、SNP2のマイナーアレルを保有していることから、SNP2のマイナーアレルが、アジア人のモヤモヤ病の創始者変異である可能性が示唆された。
このSNP2が位置する予測遺伝子C17orf27及び、SNP1が位置する予測遺伝子KIAA1618のクローニングを行ったところ、驚くべきことに、C17orf27及びKIAA1618はそれぞれ完全な構造遺伝子ではなく、C17orf27とKIAA1618とが1つにつながって、約5000アミノ酸からなる巨大なタンパク質をコードする1つの構造遺伝子を構成していることが明らかとなった。この新規遺伝子をミステリン(Mysterin)と命名した。
ミステリンの機能を解析したところ、E3リガーゼ活性及びATPase活性を有していた。
生体内でのミステリンの機能を解析するため、ヒトのミステリンに相同なゼブラフィッシュミステリン1のアンチセンスモルフォリノをゼブラフィッシュの受精卵に注入することにより、ミステリン1の蛋白発現が抑制されたゼブラフィッシュを作成した。ミステリン1のアンチセンスモルフォリノを注入した胚においては、初期血管発生は正常に進行したものの、それに続く血管新生に異常が見られた。また、ミステリン1の発現が抑制されたゼブラフィッシュにおいては目の周辺に特徴的な異常血管新生が見られた。これらの表現型から、ヒトのミステリンは血管構築の形成に重要な機能を有することが示唆された。
更に、SNP2が、虚血性心疾患の発症頻度や一卵性双生児の分娩頻度と関連することが示された。
更に、白人の家系においてもモヤモヤ病の発症と相関する新たなSNPsを見出した。
以上の知見に基づき、本発明が完成された。 The present inventors conducted detailed linkage analysis on 17 Moyamoya disease families and succeeded in identifying a core region of about 2.1 Mb linked to the onset of Moyamoya disease from among the chromosome region 17q25.3. . The core region contained about 38 genes.
When the nucleotide sequence of this core region was comprehensively analyzed, five new SNPs were found to be KIAA1618 (SNP1), C17orf27 (RNF213) (SNP2), FLJ3520 (SNP3), NPTX1 (SNP4) and KIAA1303 (SNP5). Identified within or near the gene. The frequency of minor alleles of these SNPs correlated well with the onset of moyamoya disease. All familial Moyamoya disease patients tested had a SNP2 minor allele, suggesting that the SNP2 minor allele may be the founder mutation of Asian Moyamoya disease.
When the predicted gene C17orf27 where SNP2 is located and the predicted gene KIAA1618 where SNP1 is located were cloned, surprisingly, C17orf27 and KIAA1618 are not complete structural genes, and C17orf27 and KIAA1618 are linked together. Thus, it was revealed that one structural gene encoding a huge protein consisting of about 5000 amino acids was constructed. This new gene was named mysterin.
When the function of mysterin was analyzed, it had E3 ligase activity and ATPase activity.
In order to analyze the function of mysterin in vivo,zebrafish mysterin 1 antisense morpholino homologous to human mysterin is injected into a zebrafish fertilized egg to suppress the protein expression of mysterin 1. Created a fish. In embryos injected with antisense morpholino of mysterin 1, early angiogenesis progressed normally, but subsequent angiogenesis was abnormal. In zebrafish in which the expression of mysterin 1 was suppressed, characteristic abnormal angiogenesis was observed around the eyes. These phenotypes suggested that human mysterin has an important function in the formation of vascular architecture.
Furthermore, SNP2 has been shown to be associated with the frequency of ischemic heart disease and the frequency of delivery of identical twins.
Furthermore, new SNPs that correlate with the onset of moyamoya disease were also found in Caucasian families.
Based on the above findings, the present invention has been completed.
このコア領域のヌクレオチド配列を網羅的に解析したところ、5つの新たなSNPsが、KIAA1618(SNP1)、C17orf27(RNF213)(SNP2)、FLJ3520(SNP3)、NPTX1(SNP4)及びKIAA1303(SNP5)の各遺伝子内又はその近傍において同定された。これらのSNPsのマイナーアレルの頻度は、モヤモヤ病の発症とよく相関した。試験を行った全ての家族性のモヤモヤ病患者は、SNP2のマイナーアレルを保有していることから、SNP2のマイナーアレルが、アジア人のモヤモヤ病の創始者変異である可能性が示唆された。
このSNP2が位置する予測遺伝子C17orf27及び、SNP1が位置する予測遺伝子KIAA1618のクローニングを行ったところ、驚くべきことに、C17orf27及びKIAA1618はそれぞれ完全な構造遺伝子ではなく、C17orf27とKIAA1618とが1つにつながって、約5000アミノ酸からなる巨大なタンパク質をコードする1つの構造遺伝子を構成していることが明らかとなった。この新規遺伝子をミステリン(Mysterin)と命名した。
ミステリンの機能を解析したところ、E3リガーゼ活性及びATPase活性を有していた。
生体内でのミステリンの機能を解析するため、ヒトのミステリンに相同なゼブラフィッシュミステリン1のアンチセンスモルフォリノをゼブラフィッシュの受精卵に注入することにより、ミステリン1の蛋白発現が抑制されたゼブラフィッシュを作成した。ミステリン1のアンチセンスモルフォリノを注入した胚においては、初期血管発生は正常に進行したものの、それに続く血管新生に異常が見られた。また、ミステリン1の発現が抑制されたゼブラフィッシュにおいては目の周辺に特徴的な異常血管新生が見られた。これらの表現型から、ヒトのミステリンは血管構築の形成に重要な機能を有することが示唆された。
更に、SNP2が、虚血性心疾患の発症頻度や一卵性双生児の分娩頻度と関連することが示された。
更に、白人の家系においてもモヤモヤ病の発症と相関する新たなSNPsを見出した。
以上の知見に基づき、本発明が完成された。 The present inventors conducted detailed linkage analysis on 17 Moyamoya disease families and succeeded in identifying a core region of about 2.1 Mb linked to the onset of Moyamoya disease from among the chromosome region 17q25.3. . The core region contained about 38 genes.
When the nucleotide sequence of this core region was comprehensively analyzed, five new SNPs were found to be KIAA1618 (SNP1), C17orf27 (RNF213) (SNP2), FLJ3520 (SNP3), NPTX1 (SNP4) and KIAA1303 (SNP5). Identified within or near the gene. The frequency of minor alleles of these SNPs correlated well with the onset of moyamoya disease. All familial Moyamoya disease patients tested had a SNP2 minor allele, suggesting that the SNP2 minor allele may be the founder mutation of Asian Moyamoya disease.
When the predicted gene C17orf27 where SNP2 is located and the predicted gene KIAA1618 where SNP1 is located were cloned, surprisingly, C17orf27 and KIAA1618 are not complete structural genes, and C17orf27 and KIAA1618 are linked together. Thus, it was revealed that one structural gene encoding a huge protein consisting of about 5000 amino acids was constructed. This new gene was named mysterin.
When the function of mysterin was analyzed, it had E3 ligase activity and ATPase activity.
In order to analyze the function of mysterin in vivo,
Furthermore, SNP2 has been shown to be associated with the frequency of ischemic heart disease and the frequency of delivery of identical twins.
Furthermore, new SNPs that correlate with the onset of moyamoya disease were also found in Caucasian families.
Based on the above findings, the present invention has been completed.
即ち、本発明は以下に関する。
[1]以下の(1)~(3)から選択されるいずれかのポリペプチド:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1~500個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。
[2][1]記載のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチド。
[3][2]記載のポリヌクレオチドを含む発現ベクター。
[4][3]記載の発現ベクターで形質転換された形質転換体。
[5][1]記載のポリペプチドをコードする遺伝子の機能的欠損を含む非ヒト動物。
[6]血管新生異常の動物モデルとしての、[5]記載の非ヒト動物の使用。
[7][1]記載のポリペプチドをコードする遺伝子の機能的欠損を含む動物細胞。
[8]被検物質が[1]記載のポリペプチドをコードする遺伝子の発現または機能を調節し得るか否かを評価することを含む、血管新生調節剤の候補物質のスクリーニング方法。
[9]配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチド。
[10]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法。
[11]該SNPが73097 G>A(SNP2)である、[10]記載の方法。
[12]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、モヤモヤ病の発症リスクの診断剤。
[13]該SNPが73097 G>A(SNP2)である、[12]記載の診断剤。
[14]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、虚血性心疾患の発症リスクの判定方法。
[15]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、虚血性心疾患の発症リスクの診断剤。
[16]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、一卵性双生児の分娩可能性の判定方法。
[17]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、一卵性双生児の分娩可能性の診断剤。
[18]以下の(4)~(7)から選択されるいずれかのポリペプチド:
(4)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(5)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい);
(6)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい);及び
(7)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい)。
[19][18]記載のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチド。
[20][19]記載のポリヌクレオチドを含む発現ベクター。
[21][20]記載の発現ベクターで形質転換された形質転換体。
[22]配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチド。
[23]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法。
[24]該SNPが55977 G>A(SNP6)である、[23]記載の方法。
[25]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、モヤモヤ病の発症リスクの診断剤。
[26]該SNPが55977 G>A(SNP6)である、[25]記載の診断剤。
[27]モヤモヤ病の発症リスクの診断において使用するための、配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。
[28]該SNPが4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つである、[27]記載の核酸プローブ又はプライマー。
[29]該SNPが73097 G>A(SNP2)である、[27]記載の核酸プローブ又はプライマー。
[30]該SNPが55977 G>A(SNP6)である、[27]記載の核酸プローブ又はプライマー。
[31]虚血性心疾患の発症リスクの診断において使用するための、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。
[32]一卵性双生児の分娩可能性の診断において使用するための、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。 That is, the present invention relates to the following.
[1] Any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which 1 to 500 amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity.
[2] A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to [1].
[3] An expression vector comprising the polynucleotide according to [2].
[4] A transformant transformed with the expression vector according to [3].
[5] A non-human animal comprising a functional defect of a gene encoding the polypeptide according to [1].
[6] Use of the non-human animal according to [5] as an animal model of abnormal angiogenesis.
[7] An animal cell containing a functional defect of a gene encoding the polypeptide according to [1].
[8] A screening method for a candidate substance for angiogenesis regulating agent, comprising evaluating whether or not a test substance can regulate the expression or function of a gene encoding the polypeptide of [1].
[9] A polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5) are included, and the allele of the SNP included in the partial sequence is a minor allele And the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
[10] 4766 T> C (SNP1), 73097 G> A (SNP2), 12076G G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) A method for determining the risk of developing Moyamoya disease, comprising detecting at least one SNP selected from the group consisting of SNP5).
[11] The method according to [10], wherein the SNP is 73097 G> A (SNP2).
[12] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A in the nucleotide sequence represented by SEQ ID NO: 5 ( A diagnostic agent for the risk of developing Moyamoya disease, comprising a nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of SNP5), or a primer capable of specifically amplifying a region containing the SNP.
[13] The diagnostic agent according to [12], wherein the SNP is 73097 G> A (SNP2).
[14] A method for determining the risk of developing ischemic heart disease, comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
[15] An imaginary comprising a nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP A diagnostic agent for the risk of developing blood heart disease.
[16] A method for determining the possibility of delivery of identical twins, comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
[17] A nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP, A diagnostic agent for the feasibility of delivery of egg-shaped twins.
[18] Any polypeptide selected from the following (4) to (7):
(4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid);
(6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution).
[19] A polynucleotide comprising a nucleotide sequence encoding the polypeptide of [18].
[20] An expression vector comprising the polynucleotide according to [19].
[21] A transformant transformed with the expression vector according to [20].
[22] A polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) A polynucleotide comprising at least one SNP selected from wherein the allele of the SNP contained in the partial sequence is a minor allele and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
[23] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7), and 57483 G> A (SNP8), which comprises detecting at least one SNP selected from the group consisting of: Judgment method.
[24] The method according to [23], wherein the SNP is 55977 G> A (SNP6).
[25] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) SNP5), 55777 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8), or a nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of A diagnostic agent for the risk of developing moyamoya disease, comprising a primer that can specifically amplify a region containing.
[26] The diagnostic agent according to [25], wherein the SNP is 55977 G> A (SNP6).
[27] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3) in the nucleotide sequence represented by SEQ ID NO: 5 for use in diagnosis of the onset risk of moyamoya disease, 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) A nucleic acid probe capable of specifically detecting the SNP, or a primer capable of specifically amplifying a region containing the SNP.
[28] The SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5) The nucleic acid probe or primer according to [27], which is at least one of
[29] The nucleic acid probe or primer according to [27], wherein the SNP is 73097 G> A (SNP2).
[30] The nucleic acid probe or primer according to [27], wherein the SNP is 55977 G> A (SNP6).
[31] A nucleic acid probe capable of specifically detecting a SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 for use in diagnosis of the risk of developing ischemic heart disease, or the SNP A primer capable of specifically amplifying a region containing.
[32] a nucleic acid probe capable of specifically detecting a SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 for use in the diagnosis of the feasibility of monozygotic twins, or A primer capable of specifically amplifying a region containing SNP.
[1]以下の(1)~(3)から選択されるいずれかのポリペプチド:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1~500個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。
[2][1]記載のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチド。
[3][2]記載のポリヌクレオチドを含む発現ベクター。
[4][3]記載の発現ベクターで形質転換された形質転換体。
[5][1]記載のポリペプチドをコードする遺伝子の機能的欠損を含む非ヒト動物。
[6]血管新生異常の動物モデルとしての、[5]記載の非ヒト動物の使用。
[7][1]記載のポリペプチドをコードする遺伝子の機能的欠損を含む動物細胞。
[8]被検物質が[1]記載のポリペプチドをコードする遺伝子の発現または機能を調節し得るか否かを評価することを含む、血管新生調節剤の候補物質のスクリーニング方法。
[9]配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチド。
[10]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法。
[11]該SNPが73097 G>A(SNP2)である、[10]記載の方法。
[12]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、モヤモヤ病の発症リスクの診断剤。
[13]該SNPが73097 G>A(SNP2)である、[12]記載の診断剤。
[14]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、虚血性心疾患の発症リスクの判定方法。
[15]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、虚血性心疾患の発症リスクの診断剤。
[16]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、一卵性双生児の分娩可能性の判定方法。
[17]配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、一卵性双生児の分娩可能性の診断剤。
[18]以下の(4)~(7)から選択されるいずれかのポリペプチド:
(4)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(5)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい);
(6)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい);及び
(7)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい)。
[19][18]記載のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチド。
[20][19]記載のポリヌクレオチドを含む発現ベクター。
[21][20]記載の発現ベクターで形質転換された形質転換体。
[22]配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチド。
[23]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法。
[24]該SNPが55977 G>A(SNP6)である、[23]記載の方法。
[25]配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、モヤモヤ病の発症リスクの診断剤。
[26]該SNPが55977 G>A(SNP6)である、[25]記載の診断剤。
[27]モヤモヤ病の発症リスクの診断において使用するための、配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。
[28]該SNPが4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つである、[27]記載の核酸プローブ又はプライマー。
[29]該SNPが73097 G>A(SNP2)である、[27]記載の核酸プローブ又はプライマー。
[30]該SNPが55977 G>A(SNP6)である、[27]記載の核酸プローブ又はプライマー。
[31]虚血性心疾患の発症リスクの診断において使用するための、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。
[32]一卵性双生児の分娩可能性の診断において使用するための、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマー。 That is, the present invention relates to the following.
[1] Any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which 1 to 500 amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity.
[2] A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to [1].
[3] An expression vector comprising the polynucleotide according to [2].
[4] A transformant transformed with the expression vector according to [3].
[5] A non-human animal comprising a functional defect of a gene encoding the polypeptide according to [1].
[6] Use of the non-human animal according to [5] as an animal model of abnormal angiogenesis.
[7] An animal cell containing a functional defect of a gene encoding the polypeptide according to [1].
[8] A screening method for a candidate substance for angiogenesis regulating agent, comprising evaluating whether or not a test substance can regulate the expression or function of a gene encoding the polypeptide of [1].
[9] A polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5) are included, and the allele of the SNP included in the partial sequence is a minor allele And the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
[10] 4766 T> C (SNP1), 73097 G> A (SNP2), 12076G G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) A method for determining the risk of developing Moyamoya disease, comprising detecting at least one SNP selected from the group consisting of SNP5).
[11] The method according to [10], wherein the SNP is 73097 G> A (SNP2).
[12] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A in the nucleotide sequence represented by SEQ ID NO: 5 ( A diagnostic agent for the risk of developing Moyamoya disease, comprising a nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of SNP5), or a primer capable of specifically amplifying a region containing the SNP.
[13] The diagnostic agent according to [12], wherein the SNP is 73097 G> A (SNP2).
[14] A method for determining the risk of developing ischemic heart disease, comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
[15] An imaginary comprising a nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP A diagnostic agent for the risk of developing blood heart disease.
[16] A method for determining the possibility of delivery of identical twins, comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
[17] A nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP, A diagnostic agent for the feasibility of delivery of egg-shaped twins.
[18] Any polypeptide selected from the following (4) to (7):
(4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid);
(6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution).
[19] A polynucleotide comprising a nucleotide sequence encoding the polypeptide of [18].
[20] An expression vector comprising the polynucleotide according to [19].
[21] A transformant transformed with the expression vector according to [20].
[22] A polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) A polynucleotide comprising at least one SNP selected from wherein the allele of the SNP contained in the partial sequence is a minor allele and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
[23] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7), and 57483 G> A (SNP8), which comprises detecting at least one SNP selected from the group consisting of: Judgment method.
[24] The method according to [23], wherein the SNP is 55977 G> A (SNP6).
[25] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) SNP5), 55777 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8), or a nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of A diagnostic agent for the risk of developing moyamoya disease, comprising a primer that can specifically amplify a region containing.
[26] The diagnostic agent according to [25], wherein the SNP is 55977 G> A (SNP6).
[27] 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3) in the nucleotide sequence represented by SEQ ID NO: 5 for use in diagnosis of the onset risk of moyamoya disease, 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) A nucleic acid probe capable of specifically detecting the SNP, or a primer capable of specifically amplifying a region containing the SNP.
[28] The SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5) The nucleic acid probe or primer according to [27], which is at least one of
[29] The nucleic acid probe or primer according to [27], wherein the SNP is 73097 G> A (SNP2).
[30] The nucleic acid probe or primer according to [27], wherein the SNP is 55977 G> A (SNP6).
[31] A nucleic acid probe capable of specifically detecting a SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 for use in diagnosis of the risk of developing ischemic heart disease, or the SNP A primer capable of specifically amplifying a region containing.
[32] a nucleic acid probe capable of specifically detecting a SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 for use in the diagnosis of the feasibility of monozygotic twins, or A primer capable of specifically amplifying a region containing SNP.
本発明により、新規遺伝子ミステリンのポリペプチドや該ポリペプチドをコードするポリヌクレオチド、発現ベクター、ミステリンの機能的欠損を含む動物等が提供される。ヒトゲノム遺伝子配列の網羅的な解析から、ミステリンの遺伝子座には、C17orf27及びKIAA1618の2つの独立した構造遺伝子の存在が予測されていたが、C17orf27及びKIAA1618はそれぞれ完全な構造遺伝子ではなく、C17orf27とKIAA1618とが1つにつながってミステリン遺伝子座を構成していることが明らかとなった。
ミステリン遺伝子を発現抑制したゼブラフィッシュの解析等から、ミステリンは血管新生を調節する機能を有することが明らかとなった。従って、ミステリンの発現や機能を調節する物質をスクリーニングすることにより、新規機序に立脚した血管新生調節剤の候補物質を得ることが出来る。
また、本発明により、ミステリン遺伝子座またはその近傍に存在する新たなSNPsが提供される。該SNPsはモヤモヤ病、虚血性心疾患、一卵性双生児の分娩頻度と相関することから、このSNPsを解析することにより、モヤモヤ病や虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性を判定することが可能である。 According to the present invention, a polypeptide of a novel gene mysterin, a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided. Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus.
From the analysis of zebrafish in which expression of the mysterin gene was suppressed, it became clear that mysterin has a function of regulating angiogenesis. Therefore, by screening for substances that regulate the expression and function of mysterin, candidate substances for angiogenesis regulators based on a novel mechanism can be obtained.
The present invention also provides new SNPs that exist at or near the mysterin locus. The SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
ミステリン遺伝子を発現抑制したゼブラフィッシュの解析等から、ミステリンは血管新生を調節する機能を有することが明らかとなった。従って、ミステリンの発現や機能を調節する物質をスクリーニングすることにより、新規機序に立脚した血管新生調節剤の候補物質を得ることが出来る。
また、本発明により、ミステリン遺伝子座またはその近傍に存在する新たなSNPsが提供される。該SNPsはモヤモヤ病、虚血性心疾患、一卵性双生児の分娩頻度と相関することから、このSNPsを解析することにより、モヤモヤ病や虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性を判定することが可能である。 According to the present invention, a polypeptide of a novel gene mysterin, a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided. Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus.
From the analysis of zebrafish in which expression of the mysterin gene was suppressed, it became clear that mysterin has a function of regulating angiogenesis. Therefore, by screening for substances that regulate the expression and function of mysterin, candidate substances for angiogenesis regulators based on a novel mechanism can be obtained.
The present invention also provides new SNPs that exist at or near the mysterin locus. The SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
1.ミステリンポリペプチド
本発明は、以下の(1)~(3)から選択されるいずれかのポリペプチドを提供するものである:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。 1. Mysterin polypeptide The present invention provides any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity.
本発明は、以下の(1)~(3)から選択されるいずれかのポリペプチドを提供するものである:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。 1. Mysterin polypeptide The present invention provides any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity.
更なる局面において、本発明は以下の(4)~(7)のポリペプチドをも提供する:
(4)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されていてもよい);
(5)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい);
(6)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい);及び
(7)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい)。 In a further aspect, the present invention also provides the following polypeptides (4) to (7):
(4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with asparagine);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid);
(6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution).
(4)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されていてもよい);
(5)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい);
(6)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい);及び
(7)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい)。 In a further aspect, the present invention also provides the following polypeptides (4) to (7):
(4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with asparagine);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid);
(6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution).
本明細書において、上記(1)~(3)(更なる局面においては、上記(1)~(7))のポリペプチド、及び該ポリペプチドをコードする遺伝子を「ミステリン(Mysterin)」と総称する。
In the present specification, the polypeptides of the above (1) to (3) (in a further aspect, the above (1) to (7)) and the gene encoding the polypeptide are collectively referred to as “Mysterin”. To do.
上記(1)のポリペプチドは、代表的なヒトミステリンポリペプチドである。ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP2として、下記に詳述)のため、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい。
The polypeptide of (1) above is a typical human mysterin polypeptide. Because of a newly found SNP within the mysterin locus with amino acid substitution (SNP2 will be described in detail below), the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine. Good.
更なる局面において、上記(4)のポリペプチドにおいては、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP6として、下記に詳述)のため、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されていてもよい。
In a further aspect, in the polypeptide of (4) above, the amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is an SNP with amino acid substitution (detailed below as SNP6). Aspartic acid at position 4013 in the sequence may be substituted with asparagine.
更なる局面において、上記(5)のポリペプチドにおいては、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP7として、下記に詳述)のため、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい。
In a further aspect, in the polypeptide of (5) above, the amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is a SNP with amino acid substitution (detailed below as SNP7). Asparagine at position 3962 in the sequence may be substituted with aspartic acid.
更なる局面において、上記(6)のポリペプチドにおいては、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP8として、下記に詳述)のため、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい。
In a further aspect, in the polypeptide of (6) above, the amino acid represented by SEQ ID NO: 2 is newly found in the mysterin locus and is a SNP with amino acid substitution (detailed below as SNP8). Arginine of No. 4062 in the sequence may be substituted with glutamine.
更なる局面において、上記(7)のポリペプチドにおいては、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴う4つのSNPs(SNP2、6、7及び8として、下記に詳述)のため、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい。
In a further aspect, in the polypeptide of (7) above, because of four SNPs with amino acid substitutions newly found in the mysterin locus (detailed below as SNP2, 6, 7, and 8), The amino acid sequence represented by SEQ ID NO: 2 may contain 2, 3 or 4 substitutions selected from the group consisting of R4810K, D4013N, N3962D and R4062Q.
ミステリンポリペプチドは、ユビキチンリガーゼ活性及びATPase活性を有する。ユビキチンリガーゼ活性の有無は、HA等でタグ標識した目的とするポリペプチドを発現し得る発現ベクターを、ユビキチン(例えば、哺乳動物のユビキチン)を発現し得る発現ベクターとともに、適切な動物細胞(例えば、HEK293細胞)に導入し、該細胞のライセートについて該タグを特異的に認識する抗体を用いて免疫沈降を行った後、ユビキチンを特異的に認識する抗体を用いてウエスタンブロットを行うことにより、導入したポリペプチドが自己ユビキチン化されたか否かを評価することにより決定することができる。
Mysterin polypeptide has ubiquitin ligase activity and ATPase activity. The presence or absence of ubiquitin ligase activity is determined by combining an expression vector capable of expressing the target polypeptide tagged with HA or the like with an expression vector capable of expressing ubiquitin (for example, mammalian ubiquitin) and appropriate animal cells (for example, HEK293 cells) and immunoprecipitation using an antibody specifically recognizing the tag for the lysate of the cell, followed by Western blotting using an antibody specifically recognizing ubiquitin. It can be determined by evaluating whether or not the obtained polypeptide is self-ubiquitinated.
ATPase活性の有無は、例えば、以下の方法により決定することができる。目的とするポリペプチドを、マグネシウムイオン(例えば1mMの濃度)及びATP(例えば5mMの濃度)を含む生理的な緩衝液(例えばPBS)に加え、37℃にて30分間インキュベートし、最終濃度1%のPCAを加えることにより、反応を止める。反応混合物にマラカイトグリーンを加え、更に30分間インキュベートする。遊離リン酸とマラカイトグリーンの結合による呈色を620nmの吸光で計測する。試験したポリペプチドがATPase活性を有していると、ATPの加水分解にともなうリン酸の遊離による有意な呈色反応が観察される。
The presence or absence of ATPase activity can be determined by the following method, for example. The polypeptide of interest is added to a physiological buffer (eg, PBS) containing magnesium ions (eg, 1 mM) and ATP (eg, 5 mM) and incubated at 37 ° C. for 30 minutes to a final concentration of 1% The reaction is stopped by adding PCA. Add malachite green to the reaction mixture and incubate for an additional 30 minutes. The coloration due to the binding of free phosphate and malachite green is measured by absorbance at 620 nm. If the tested polypeptide has ATPase activity, a significant color reaction due to the release of phosphate accompanying the hydrolysis of ATP is observed.
上記(2)のポリペプチドは、配列番号2で表されるアミノ酸配列と45%以上、好ましくは70%以上、より好ましくは95%以上、更に好ましくは99.5%以上、より更に好ましくは99.9%以上(例えば、99.96%以上)の同一性を有するアミノ酸配列を含む。
The polypeptide of (2) above is 45% or more, preferably 70% or more, more preferably 95% or more, still more preferably 99.5% or more, and even more preferably 99% with the amino acid sequence represented by SEQ ID NO: 2. Amino acid sequences having 9% or more (for example, 99.96% or more) identity are included.
ここで「同一性」とは、当該技術分野において公知の数学的アルゴリズムを用いて2つのアミノ酸配列をアラインさせた場合の、最適なアラインメント(好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方もしくは両方へのギャップの導入を考慮し得るものである)における、オーバーラップする全アミノ酸残基に対する、同一アミノ酸残基の割合(%)を意味する。
As used herein, “identity” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue to the total overlapping amino acid residues (in which one or both of the gaps can be considered).
本明細書におけるアミノ酸配列の同一性は、NCBIのインターネットホームページ上に公開されている相同性計算アルゴリズムBlastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi)を用い、デフォルト条件にて計算することができる。アミノ酸配列の同一性を決定するためのアルゴリズムとしては、例えば、Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) に記載のアルゴリズム[該アルゴリズムはNBLASTおよびXBLASTプログラム(version 2.0) に組み込まれている(Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))]、Needleman et al., J. Mol. Biol., 48:444-453 (1970) に記載のアルゴリズム[該アルゴリズムはGCGソフトウェアパッケージ中のGAPプログラムに組み込まれている]、Myers and Miller, CABIOS, 4: 11-17 (1988) に記載のアルゴリズム[該アルゴリズムはCGC配列アラインメントソフトウェアパッケージの一部であるALIGNプログラム (version 2.0) に組み込まれている]、Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) に記載のアルゴリズム[該アルゴリズムはGCGソフトウェアパッケージ中のFASTAプログラムに組み込まれている]等が挙げられるが、それらに限定されない。
The amino acid sequence identity in this specification is determined by using the homology calculation algorithm Blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) published on the Internet website of NCBI as the default condition. Can be calculated. As an algorithm for determining amino acid sequence identity, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is NBLAST and XBLAST (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 ( 1970) [The algorithm is incorporated in the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [The algorithm is a CGC sequence alignment software Embedded in the ALIGN program (version 2.0) that is part of the package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) GCG software E is incorporated in the FASTA program in the A package], etc. include, but are not limited to.
上記(3)のポリペプチドに含まれるアミノ酸配列は、配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列、例えば、(1)配列番号2に示されるアミノ酸配列中の1又は複数(好ましくは1~500個、より好ましくは1~100個、さらに好ましくは1~30個、更により好ましくは1~10個、最も好ましくは1~数(2~5)個)のアミノ酸が欠失したアミノ酸配列、(2)配列番号2に示されるアミノ酸配列に1又は複数(好ましくは1~500個、より好ましくは1~100個、さらに好ましくは1~30個、更により好ましくは1~10個、最も好ましくは1~数(2~5)個)のアミノ酸が付加されたアミノ酸配列、(3)配列番号2に示されるアミノ酸配列に1又は複数(好ましくは1~500個、より好ましくは1~100個、さらに好ましくは1~30個、更により好ましくは1~10個、最も好ましくは1~数(2~5)個)のアミノ酸が挿入されたアミノ酸配列、(4)配列番号2に示されるアミノ酸配列中の1又は複数(好ましくは1~500個、より好ましくは1~100個、さらに好ましくは1~30個、更により好ましくは1~10個、最も好ましくは1~数(2~5)個)のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、または(5)上記(1)~(4)の変異が組み合わせれたアミノ酸配列(欠失、置換、挿入又は付加されたアミノ酸の総数が、好ましくは1~500個、より好ましくは1~100個、さらに好ましくは1~30個、更により好ましくは1~10個、最も好ましくは1~数(2~5)個)である。
The amino acid sequence contained in the polypeptide of (3) above is an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2, for example, (1) SEQ ID NO: 1 or more (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1 to a number in the amino acid sequence shown in FIG. (2-5) amino acid deleted amino acid sequence, (2) one or more (preferably 1-500, more preferably 1-100, more preferably 1) amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence to which 1 to 30, even more preferably 1 to 10, most preferably 1 to several (2 to 5) amino acids are added; (3) one or more amino acid sequences shown in SEQ ID NO: 2 A plurality of amino acids (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably 1 to several (2 to 5)) amino acids Inserted amino acid sequence, (4) one or more (preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, and even more preferably) in the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence in which 1 to 10 amino acids, most preferably 1 to several (2 to 5) amino acids are substituted with other amino acids, or (5) amino acids in which the mutations (1) to (4) above are combined The total number of amino acids added (deleted, substituted, inserted or added) is preferably 1 to 500, more preferably 1 to 100, still more preferably 1 to 30, even more preferably 1 to 10, most preferably Preferably 1 to a number ( To 5)) is.
ユビキチンリガーゼ活性を保持するため、上記(2)及び(3)のポリペプチドにおいては、その活性に重要なRINGフィンガードメイン(配列番号2の第3997~4035アミノ酸に該当する)が保存されていることが好ましい。保存の程度は、アミノ酸配列の同一性として、通常50%以上、好ましくは54%以上、より好ましくは69%以上、更に好ましくは90%以上(例えば97%以上)、最も好ましくは100%である。ヒトのAAAドメイン周辺配列へ欠失、置換、挿入又は付加を導入する場合には、図10に示す「*」のアミノ酸が完全に保持されるように導入を行うことが好ましい。また、ヒトのAAAドメイン周辺配列へ欠失、置換、挿入又は付加を導入する場合には、図10に示す「:」又は「.」のアミノ酸が完全に保持されるか、化学的性質が類似したアミノ酸へ置換されるように導入を行うことが好ましい。「化学的性質が類似したアミノ酸」としては、例えば、芳香族アミノ酸(Phe、Trp、Tyr)、脂肪族アミノ酸(Ala、Leu、Ile、Val)、極性アミノ酸(Gln、Asn)、塩基性アミノ酸(Lys、Arg、His)、酸性アミノ酸(Glu、Asp)、水酸基を有するアミノ酸(Ser、Thr)、側鎖の小さいアミノ酸(Gly、Ala、Ser、Thr、Met)などの同じグループに分類されるアミノ酸が挙げられる。このような類似アミノ酸による置換はタンパク質の表現型に変化をもたらさない(即ち、保存的アミノ酸置換である)ことが予測される。保存的アミノ酸置換の具体例は当該技術分野で周知であり、種々の文献に記載されている(例えば、Bowieら,Science,247:1306-1310 (1990)を参照)。
In order to retain the ubiquitin ligase activity, the RING finger domain (corresponding to amino acids 3997 to 4035 of SEQ ID NO: 2) important for the activity is conserved in the polypeptides (2) and (3) above. Is preferred. The degree of conservation is usually 50% or more, preferably 54% or more, more preferably 69% or more, still more preferably 90% or more (eg 97% or more), and most preferably 100% as amino acid sequence identity. . When a deletion, substitution, insertion or addition is introduced into the human AAA domain peripheral sequence, the introduction is preferably performed so that the amino acid “*” shown in FIG. 10 is completely retained. In addition, when deletions, substitutions, insertions or additions are introduced into the human AAA domain peripheral sequence, the amino acid “:” or “.” Shown in FIG. 10 is completely retained or the chemical properties are similar. It is preferable to introduce the amino acid so that the amino acid is substituted. “Amino acids with similar chemical properties” include, for example, aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids ( Amino acids that fall into the same group such as Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met) Is mentioned. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Examples of conservative amino acid substitutions are well known in the art and are described in various references (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
また、ATPase活性を保持するため、上記(2)及び(3)のポリペプチドにおいては、その活性に重要なAAAドメイン周辺配列(ヒトの第2412~2910アミノ酸に相当)が保存されていることが好ましい。保存の程度は、アミノ酸配列の同一性として、通常60%以上、好ましくは72%以上、より好ましくは80%以上、更に好ましくは90%以上(例えば98%以上)、最も好ましくは100%である。ヒトのAAAドメイン周辺配列へ欠失、置換、挿入又は付加を導入する場合には、図9に示す「*」のアミノ酸が完全に保持されるように導入を行うことが好ましい。また、ヒトのAAAドメイン周辺配列へ欠失、置換、挿入又は付加を導入する場合には、図9に示す「:」又は「.」のアミノ酸が完全に保持されるか、化学的性質が類似したアミノ酸へ置換されるように導入を行うことが好ましい。「化学的性質が類似したアミノ酸」の定義は、上述の通りである。
In addition, in order to retain the ATPase activity, in the polypeptides of (2) and (3) above, the AAA domain peripheral sequence (corresponding to human 2412 to 2910 amino acids) important for the activity is conserved. preferable. The degree of conservation is usually 60% or more, preferably 72% or more, more preferably 80% or more, still more preferably 90% or more (for example, 98% or more), and most preferably 100% as amino acid sequence identity. . When a deletion, substitution, insertion or addition is introduced into the human AAA domain peripheral sequence, the introduction is preferably performed so that the amino acid “*” shown in FIG. 9 is completely retained. In addition, when a deletion, substitution, insertion or addition is introduced into the sequence around the human AAA domain, the amino acid “:” or “.” Shown in FIG. 9 is completely retained or the chemical properties are similar. It is preferable to introduce the amino acid so that the amino acid is substituted. The definition of “amino acids with similar chemical properties” is as described above.
1つの好ましい態様において、上記(2)及び(3)のポリペプチドは、動物の天然のミステリンポリペプチドである。「天然の」とは、ポリペプチドを構成するアミノ酸配列が天然に存在することをいう。
In one preferred embodiment, the polypeptides of (2) and (3) above are natural mysterin polypeptides of animals. “Natural” means that the amino acid sequence constituting the polypeptide exists in nature.
遺伝子を構成するヌクレオチド配列には通常多型(個体差)が存在することが知られている。上記(2)及び(3)のポリペプチドには、ヒトミステリン遺伝子の多型により生じた上記(1)のポリペプチドとはアミノ酸配列が異なるポリペプチドが含まれる。
It is known that there are usually polymorphisms (individual differences) in the nucleotide sequence constituting a gene. The polypeptides of (2) and (3) above include polypeptides having a different amino acid sequence from the polypeptide of (1) generated by the polymorphism of the human mysterin gene.
また、上記(2)及び(3)のポリペプチドには、ヒト以外の動物におけるミステリンのオルソログが含まれる。「ヒト以外の動物」としては、ヒト以外の哺乳動物、鳥類、爬虫類、両生類、魚類、昆虫などを挙げることができる。ヒト以外の哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物;ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜;イヌ、ネコ等のペット;サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。鳥類としては、ニワトリ、ウズラ、アヒル、ガチョウ、シチメンチョウ、エミュ、ダチョウ、ホロホロ鳥、ハト等を挙げることができる。魚類としては、ゼブラフィッシュ、フグ、メダカ、キンギョ、ドジョウ等が挙げられる。ヒト以外の動物におけるミステリンのオルソログは、本明細書に開示されたヒトミステリンをコードするヌクレオチド配列(配列番号1で表されるヌクレオチド配列)を参照し、ミステリンを特異的に検出し得る核酸プローブやプライマーを設計し、プラークハイブリダイゼーションや、RACE PCR等の周知の遺伝子工学的手法を用いることにより、獲得することが出来る。
The polypeptides (2) and (3) above include orthologs of mysterin in animals other than humans. Examples of “non-human animals” include mammals other than humans, birds, reptiles, amphibians, fish, insects, and the like. Examples of mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits; domestic animals such as pigs, cows, goats, horses, sheep, minks; pets such as dogs and cats Primates such as, but not limited to, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees; Examples of the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons. Examples of fish include zebrafish, puffer fish, medaka, goldfish and loach. An ortholog of mysterin in a non-human animal refers to a nucleotide sequence (nucleotide sequence represented by SEQ ID NO: 1) encoding human mysterin disclosed herein, and a nucleic acid probe capable of specifically detecting mysterin Can be obtained by designing a primer or primer and using a well-known genetic engineering technique such as plaque hybridization or RACE PCR.
上記(2)及び(3)のポリペプチドの好適な例としては、例えば、哺乳動物、代表的な鳥類又は魚類のヒトミステリンと相同なミステリンポリペプチドを挙げることができる。進化時間が長くなると、タンパク質への挿入あるいは欠損など変異による種間での多様性のため、必ずしも完全な遺伝子構造の保存は見られない。しかし機能的に重要な部分については種間で高度に保存される。ミステリンのAAAドメイン周辺配列(ヒトの第2412~2910アミノ酸に相当)およびRINGフィンガードメイン配列(ヒトの第3997~4035アミノ酸に相当)をヒト、チンパンジー、マウス、ラット、ニワトリ、フグ、およびゼブラフィッシュ(ゲノム中に2遺伝子存在する)より抽出し、それぞれのドメインについてマルチフルアライメントを行った。アライメントの結果を図9及び10に示す。AAAドメイン周辺配列については、ヒトミステリンアミノ酸配列に対する同一性は、チンパンジー98%、マウス82%、ラット80%、ニワトリ72%、フグ67%、およびゼブラフィッシュ68%、63%(それぞれ、ミステリン1,2に相当)である。RINGフィンガードメイン配列については、ヒトミステリンアミノ酸配列に対する同一性は、チンパンジー97%、マウス69%、ラット74%、ニワトリ54%、フグ51%、およびゼブラフィッシュ53%、42%(それぞれ、ミステリン1,2に相当)である。ゼブラフィッシュにおいては、ミステリン1(zRNF213)、ミステリン2(zRNF213.1)の2種類があり、前者がヒトのミステリンに相当する。ゼブラフィッシュのミステリン1ポリペプチドの代表的なアミノ酸配列を配列番号4に示す。
Preferred examples of the polypeptides (2) and (3) above include mysterin polypeptides that are homologous to human mysterin of mammals, typical birds, or fish. If the evolution time is long, the preservation of the complete gene structure is not necessarily observed due to diversity among species due to mutation such as insertion or deletion into the protein. However, functionally important parts are highly conserved between species. Peripheral sequence of the myelin AAA domain (corresponding to human amino acids 2412 to 2910) and RING finger domain sequence (corresponding to human amino acids 3997 to 4035), human, chimpanzee, mouse, rat, chicken, puffer, and zebrafish ( 2 domains exist in the genome), and multi-full alignment was performed for each domain. The alignment results are shown in FIGS. For the AAA domain peripheral sequence, the identity to the human mysterin amino acid sequence is as follows: chimpanzee 98%, mouse 82%, rat 80%, chicken 72%, puffer 67%, and zebrafish 68%, 63% (respectively mysterin 1 , 2). For the RING finger domain sequence, identity to the human mysterin amino acid sequence is 97% chimpanzee, 69% mouse, 74% rat, 54% chicken, 51% puffer fish, and 53% and 42% zebrafish (mysterin 1 respectively) , 2). There are two types of zebrafish: mysterin 1 (zRNF213) and mysterin 2 (zRNF213.1), and the former corresponds to human mysterin. A representative amino acid sequence of a zebrafish mysterin 1 polypeptide is shown in SEQ ID NO: 4.
本発明のポリペプチドは、ユビキチンリガーゼ活性及びATPase活性を有する限り、その長さは特に制限されず、使用目的に応じて所望の長さのポリペプチドを選択することが出来る。例えば、本発明のポリペプチドとして、長さが6000アミノ酸以下のもの、5500アミノ酸以下のもの、5400アミノ酸以下のもの、5300アミノ酸以下のもの、5250アミノ酸以下のもの、5220アミノ酸以下のもの、5210アミノ酸以下のもの等を適宜選択することが出来る。本発明のポリペプチドは、通常4800アミノ酸以上、好ましくは5000アミノ酸以上、より好ましくは5200アミノ酸以上、最も好ましくは5207アミノ酸以上の長さを有する。
The length of the polypeptide of the present invention is not particularly limited as long as it has ubiquitin ligase activity and ATPase activity, and a polypeptide having a desired length can be selected according to the purpose of use. For example, the polypeptide of the present invention has a length of 6000 amino acids or less, 5500 amino acids or less, 5400 amino acids or less, 5300 amino acids or less, 5250 amino acids or less, 5220 amino acids or less, 5210 amino acids The following can be selected as appropriate. The polypeptide of the present invention usually has a length of 4800 amino acids or more, preferably 5000 amino acids or more, more preferably 5200 amino acids or more, and most preferably 5207 amino acids or more.
本発明のポリペプチドは、1個以上のタグポリペプチド又はシグナル配列を有していてもよい。
The polypeptide of the present invention may have one or more tag polypeptides or signal sequences.
タグポリペプチドとは、ポリペプチドの検出や精製等を容易ならしめるために付加されるポリペプチドをいう。タグポリペプチドとしては、エピトープタグ、蛍光ポリペプチド、イムノグロブリンFc領域等を挙げることが出来るがこれらに限定されない。エピトープタグとは、抗体または他の結合パートナーによって特異的に認識されるペプチドをいい、具体的には、Flagタグ、ポリヒスチジンタグ、c-Mycタグ、HAタグ、AU1タグ、GSTタグ、MBPタグ等を挙げることが出来る。蛍光ポリペプチドとしては、GFP、YFP、RFP、CFP、BFP、EGFP等を挙げることが出来る。このようなタグポリペプチドは当業者に周知であり、当該タグポリペプチドを特異的に認識する多様な抗体が市販されている。
The tag polypeptide refers to a polypeptide that is added to facilitate the detection and purification of the polypeptide. Examples of tag polypeptides include, but are not limited to, epitope tags, fluorescent polypeptides, immunoglobulin Fc regions, and the like. An epitope tag refers to a peptide that is specifically recognized by an antibody or other binding partner, specifically, a Flag tag, a polyhistidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, an MBP tag. Etc. can be mentioned. Examples of the fluorescent polypeptide include GFP, YFP, RFP, CFP, BFP, EGFP and the like. Such tag polypeptides are well known to those skilled in the art, and various antibodies that specifically recognize the tag polypeptides are commercially available.
シグナル配列とは、ポリペプチドの翻訳と同時にまたは翻訳後に、合成部位から細胞内部の特定部位、又は細胞外部へのポリペプチドの運搬や局在を指示するポリペプチド配列をいう。シグナル配列には、ポリペプチドの分泌を誘導するリーダー配列、核移行シグナル配列(例えば、SV40 T抗原の核移行シグナル配列)、核外移行シグナル配列、核小体局在シグナル等を挙げることが出来るがこれらに限定されない。このようなシグナル配列は当業者に周知であり、目的に応じて適宜選択することが出来る。
The signal sequence refers to a polypeptide sequence that directs the transport and localization of the polypeptide from the synthesis site to a specific site inside the cell or outside the cell simultaneously with or after translation of the polypeptide. Examples of the signal sequence include a leader sequence that induces secretion of the polypeptide, a nuclear translocation signal sequence (for example, a nuclear translocation signal sequence of SV40 T antigen), a nuclear translocation signal sequence, a nucleolus localization signal, and the like. However, it is not limited to these. Such signal sequences are well known to those skilled in the art, and can be appropriately selected according to the purpose.
本発明のポリペプチドは修飾されていてもよい。該修飾としては、脂質鎖の付加(脂肪族アシル化(パルミトイル化、ミリストイル化等)、プレニル化(ファルネシル化、ゲラニルゲラニル化等)等)、リン酸化(セリン残基、スレオニン残基、チロシン残基等におけるリン酸化)、アセチル化、糖鎖の付加(Nグリコシル化、Oグリコシル化)等を挙げることが出来る。
The polypeptide of the present invention may be modified. Such modifications include addition of lipid chains (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.
また、本発明のポリペプチドは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)、アフィニティタグ(例:ビオチン等)などで標識されていてもよい。
In addition, the polypeptide of the present invention contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase). , Peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin) Etc.).
また、本明細書において用語「ポリペプチド」は、その塩をも含む意味として用いられる。ポリペプチドの塩としては生理学的に許容される酸(例:無機酸、有機酸)や塩基(例:アルカリ金属塩)などとの塩が用いられ、とりわけ生理学的に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸(例えば、塩酸、リン酸、臭化水素酸、硫酸)との塩、あるいは有機酸(例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸)との塩などが挙げられる。
In addition, in this specification, the term “polypeptide” is used to mean a salt thereof. Polypeptide salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
本発明のポリペプチドは単離又は精製されていることが好ましい。「単離又は精製」とは、目的とする成分以外の成分を除去する操作が施されていることを意味する。単離又は精製された本発明のポリペプチドの純度(全ポリペプチド重量に対する、本発明のポリペプチドの重量の割合)は、通常50%以上、好ましくは70%以上、より好ましくは90%以上、最も好ましくは95%以上(例えば実質的に100%)である。
The polypeptide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. The purity of the isolated or purified polypeptide of the present invention (ratio of the weight of the polypeptide of the present invention to the total polypeptide weight) is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, substantially 100%).
代表的な本発明のポリペプチドを以下に列挙する:
(a)配列番号2で表されるアミノ酸配列からなるポリペプチド;
(b)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されている);
(c)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(d)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されている);
(e)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されている);
(f)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、以下の群から選択されるいずれか1つの置換の組み合わせを含む;
R4810K+D4013N、
R4810K+N3962D、
R4810K+R4062Q、
D4013N+N3962D、
D4013N+R4062Q、
N3962D+R4062Q、
R4810K+D4013N+N3962D、
R4810K+D4013N+R4062Q、
R4810K+N3962D+R4062Q、
D4013N+N3962D+R4062Q、及び
R4810K+D4013N+N3962D+R4062Q);
(g)配列番号2で表されるアミノ酸配列を含むポリペプチド;
(h)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されている);
(i)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(j)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されている);
(k)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されている);及び
(l)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、以下の群から選択されるいずれか1つの置換の組み合わせを含む;
R4810K+D4013N、
R4810K+N3962D、
R4810K+R4062Q、
D4013N+N3962D、
D4013N+R4062Q、
N3962D+R4062Q、
R4810K+D4013N+N3962D、
R4810K+D4013N+R4062Q、
R4810K+N3962D+R4062Q、
D4013N+N3962D+R4062Q、及び
R4810K+D4013N+N3962D+R4062Q)。 Representative polypeptides of the invention are listed below:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine);
(C) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(D) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine at No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with aspartic acid);
(E) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 (here, the 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 is substituted with glutamine);
(F) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 includes any one substitution combination selected from the following group;
R4810K + D4013N,
R4810K + N3962D,
R4810K + R4062Q,
D4013N + N3962D,
D4013N + R4062Q,
N3962D + R4062Q,
R4810K + D4013N + N3962D,
R4810K + D4013N + R4062Q,
R4810K + N3962D + R4062Q,
D4013N + N3962D + R4062Q and R4810K + D4013N + N3962D + R4062Q);
(G) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(H) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine);
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(J) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine at No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with aspartic acid);
(K) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 is substituted with glutamine); and (l) SEQ ID NO: A polypeptide comprising the amino acid sequence represented by 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 comprises any one combination of substitutions selected from the following group;
R4810K + D4013N,
R4810K + N3962D,
R4810K + R4062Q,
D4013N + N3962D,
D4013N + R4062Q,
N3962D + R4062Q,
R4810K + D4013N + N3962D,
R4810K + D4013N + R4062Q,
R4810K + N3962D + R4062Q,
D4013N + N3962D + R4062Q, and R4810K + D4013N + N3962D + R4062Q).
(a)配列番号2で表されるアミノ酸配列からなるポリペプチド;
(b)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されている);
(c)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(d)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されている);
(e)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されている);
(f)配列番号2で表されるアミノ酸配列からなるポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、以下の群から選択されるいずれか1つの置換の組み合わせを含む;
R4810K+D4013N、
R4810K+N3962D、
R4810K+R4062Q、
D4013N+N3962D、
D4013N+R4062Q、
N3962D+R4062Q、
R4810K+D4013N+N3962D、
R4810K+D4013N+R4062Q、
R4810K+N3962D+R4062Q、
D4013N+N3962D+R4062Q、及び
R4810K+D4013N+N3962D+R4062Q);
(g)配列番号2で表されるアミノ酸配列を含むポリペプチド;
(h)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されている);
(i)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸はアスパラギンに置換されている);
(j)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されている);
(k)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されている);及び
(l)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、以下の群から選択されるいずれか1つの置換の組み合わせを含む;
R4810K+D4013N、
R4810K+N3962D、
R4810K+R4062Q、
D4013N+N3962D、
D4013N+R4062Q、
N3962D+R4062Q、
R4810K+D4013N+N3962D、
R4810K+D4013N+R4062Q、
R4810K+N3962D+R4062Q、
D4013N+N3962D+R4062Q、及び
R4810K+D4013N+N3962D+R4062Q)。 Representative polypeptides of the invention are listed below:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine);
(C) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(D) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine at No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with aspartic acid);
(E) a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 (here, the 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 is substituted with glutamine);
(F) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 includes any one substitution combination selected from the following group;
R4810K + D4013N,
R4810K + N3962D,
R4810K + R4062Q,
D4013N + N3962D,
D4013N + R4062Q,
N3962D + R4062Q,
R4810K + D4013N + N3962D,
R4810K + D4013N + R4062Q,
R4810K + N3962D + R4062Q,
D4013N + N3962D + R4062Q and R4810K + D4013N + N3962D + R4062Q);
(G) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(H) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine);
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(J) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine at No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with aspartic acid);
(K) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 is substituted with glutamine); and (l) SEQ ID NO: A polypeptide comprising the amino acid sequence represented by 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 comprises any one combination of substitutions selected from the following group;
R4810K + D4013N,
R4810K + N3962D,
R4810K + R4062Q,
D4013N + N3962D,
D4013N + R4062Q,
N3962D + R4062Q,
R4810K + D4013N + N3962D,
R4810K + D4013N + R4062Q,
R4810K + N3962D + R4062Q,
D4013N + N3962D + R4062Q, and R4810K + D4013N + N3962D + R4062Q).
本発明のポリペプチドの製造方法については特に制限はなく、公知のペプチド合成法に従って製造してもよく、また公知の遺伝子組み換え技術を用いて製造してもよい。ペプチド合成法は、例えば、固相合成法、液相合成法のいずれであってもよい。本発明のポリペプチドを構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合し、生成物が保護基を有する場合は保護基を脱離することにより目的とするポリペプチドを製造することができる。
The method for producing the polypeptide of the present invention is not particularly limited, and may be produced according to a known peptide synthesis method or may be produced using a known genetic recombination technique. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. If the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining portion, and the product has a protecting group, the protecting polypeptide can be eliminated to produce the desired polypeptide.
遺伝子組み換え技術を用いて本発明のポリペプチドを製造する場合には、先ず後述するような本発明のポリペプチドをコードするポリヌクレオチドを取得し、該ポリペプチドを発現し得る発現ベクターで宿主細胞を形質転換し、得られる形質転換体を培養することによって、該ポリペプチドを製造することができる。該ポリヌクレオチド、遺伝子組み換え技術を用いた本発明のポリペプチドの製造方法については後述する。
When the polypeptide of the present invention is produced using genetic recombination technology, first, a polynucleotide encoding the polypeptide of the present invention as described later is obtained, and the host cell is expressed using an expression vector capable of expressing the polypeptide. The polypeptide can be produced by transforming and culturing the resulting transformant. A method for producing the polypeptide of the present invention using the polynucleotide and gene recombination techniques will be described later.
本発明のポリペプチドは、血管新生調節剤の候補物質のスクリーニング、モヤモヤ病の発症リスクの判定、虚血性心疾患の発症リスクの判定、一卵性双生児の分娩可能性の判定、病理組織標本での血管新生の判定等に有用である。
The polypeptide of the present invention can be used to screen for candidate substances for angiogenesis regulating agents, to determine the risk of developing Moyamoya disease, to determine the risk of developing ischemic heart disease, to determine the likelihood of delivery of identical twins, This is useful for determining angiogenesis.
2.ミステリンポリペプチドをコードするポリヌクレオチド
本発明は上記本発明のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチドを提供するものである。 2. Polynucleotide Encoding Mysterin Polypeptide The present invention provides a polynucleotide comprising a nucleotide sequence encoding the above-described polypeptide of the present invention.
本発明は上記本発明のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチドを提供するものである。 2. Polynucleotide Encoding Mysterin Polypeptide The present invention provides a polynucleotide comprising a nucleotide sequence encoding the above-described polypeptide of the present invention.
本発明のポリヌクレオチドは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよい。また、該ポリヌクレオチドは二本鎖であっても、一本鎖であってもよい。二本鎖の場合は、二本鎖DNA、二本鎖RNAまたはDNA:RNAのハイブリッドでもよい。
The polynucleotide of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.
本発明のポリヌクレオチドとしては、配列番号1又は3で表されるヌクレオチド配列を含むポリヌクレオチドを挙げることが出来る。配列番号1で表されるヌクレオチド配列はヒトミステリンポリペプチド(配列番号2)を、配列番号3で表されるヌクレオチド配列は、ゼブラフィッシュミステリン1ポリペプチド(配列番号4)をそれぞれコードする。尚、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP2として、下記に詳述)のため、配列番号1で表されるヌクレオチド配列における第14429番のグアニンはアデニンに置換されていてもよい。
Examples of the polynucleotide of the present invention include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or 3. The nucleotide sequence represented by SEQ ID NO: 1 encodes a human mysterin polypeptide (SEQ ID NO: 2), and the nucleotide sequence represented by SEQ ID NO: 3 encodes a zebrafish mysterin 1 polypeptide (SEQ ID NO: 4). In addition, because of a newly found SNP in the mysterin locus with amino acid substitution (described in detail below as SNP2), the 14429th guanine in the nucleotide sequence represented by SEQ ID NO: 1 was replaced with adenine. May be.
更なる局面において、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP6として、下記に詳述)のため、配列番号1で表されるヌクレオチド配列における第12037番のグアニンはアデニンに置換されていてもよい。
In a further aspect, because of a newly found SNP with an amino acid substitution in the mysterin locus (described in detail below as SNP6), No. 12037 guanine in the nucleotide sequence represented by SEQ ID NO: 1 is converted to adenine. May be substituted.
更なる局面において、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP7として、下記に詳述)のため、配列番号1で表されるヌクレオチド配列における第11884番のアデニンはグアニンに置換されていてもよい。
In a further aspect, because of a newly found SNP with an amino acid substitution in the mysterin locus (described in detail below as SNP7), adenine at position 11884 in the nucleotide sequence represented by SEQ ID NO: 1 is converted to guanine. May be substituted.
更なる局面において、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴うSNP(SNP8として、下記に詳述)のため、配列番号1で表されるヌクレオチド配列における第12185番のグアニンはアデニンに置換されていてもよい。
In a further aspect, because of a newly found SNP with an amino acid substitution in the mysterin locus (described in detail below as SNP8), 12185th guanine in the nucleotide sequence represented by SEQ ID NO: 1 is converted to adenine. May be substituted.
更なる局面において、ミステリン遺伝子座内に新たに見出した、アミノ酸置換を伴う4つのSNPs(SNP2、6、7及び8として、下記に詳述)のため、配列番号1で表されるヌクレオチド配列は、g14429a、g12037a、a11884g及びg12185aからなる群から選択される、2,3又は4個の置換を含んでいてもよい。
In a further aspect, because of four newly discovered SNPs with amino acid substitutions in the mysterin locus (detailed below as SNPs 2, 6, 7, and 8), the nucleotide sequence represented by SEQ ID NO: 1 is , G14429a, g12037a, a11884g and g12185a may comprise 2, 3 or 4 substitutions.
本発明のポリヌクレオチドは、本明細書の配列表に記載された配列情報に基づき、公知の遺伝子組換え技術を利用することにより容易に製造することが出来る。例えば、配列情報に基づき適当なプライマーを設計し、ミステリンを発現する細胞(例えば、293細胞等の腎細胞)から単離した全RNAから調製したcDNAを鋳型とするRT-PCRにより、本発明のポリヌクレオチドを製造することが出来る。或いは、本明細書の配列表に記載された配列情報に基づいて、ポリヌクレオチド合成装置により本発明のポリヌクレオチドを合成してもよい。
The polynucleotide of the present invention can be easily produced by utilizing a known gene recombination technique based on the sequence information described in the sequence listing of the present specification. For example, by designing an appropriate primer based on the sequence information, RT-PCR using cDNA prepared from total RNA isolated from cells expressing mysterin (eg, renal cells such as 293 cells) as a template. Polynucleotides can be produced. Alternatively, the polynucleotide of the present invention may be synthesized by a polynucleotide synthesizer based on the sequence information described in the sequence listing of the present specification.
取得された本発明のポリペプチドをコードするポリヌクレオチドは、目的によりそのまま、または所望により制限酵素で消化するか、リンカーを付加した後に、使用することができる。該ポリヌクレオチドはその5’末端側に翻訳開始コドンとしてのATGを有し、また3’末端側には翻訳終止コドンとしてのTAA、TGAまたはTAGを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成DNAアダプターを用いて付加することができる。
The obtained polynucleotide encoding the polypeptide of the present invention can be used as it is depending on the purpose, or after digestion with a restriction enzyme or adding a linker as desired. The polynucleotide may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
本発明のポリヌクレオチドは、好ましくは単離又は精製されている。「単離又は精製」とは、目的とする成分以外の成分を除去する操作が施されていることを意味する。単離又は精製された本発明のポリヌクレオチドの純度(全ポリヌクレオチド重量に対する、本発明のポリヌクレオチドの重量の割合)は、通常50%以上、好ましくは70%以上、より好ましくは90%以上、最も好ましくは95%以上(例えば実質的に100%)である。
The polynucleotide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. The purity of the isolated or purified polynucleotide of the present invention (ratio of the weight of the polynucleotide of the present invention to the total weight of the polynucleotide) is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, substantially 100%).
本発明のポリヌクレオチドは、ミステリンポリペプチドの製造に有用である。また、本発明のポリヌクレオチドに含まれるSNP2やSNP6を解析することにより、モヤモヤ病や虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性を判定することが出来る(下記に詳述)。
The polynucleotide of the present invention is useful for producing a mysterin polypeptide. Further, by analyzing SNP2 and SNP6 contained in the polynucleotide of the present invention, it is possible to determine the onset risk of moyamoya disease and ischemic heart disease and the possibility of delivery of identical twins (detailed below). ).
3.発現ベクター及び形質転換体
本発明は、上記本発明のポリヌクレオチドを含む発現ベクター及び該発現ベクターを含む形質転換体を提供するものである。 3. Expression vector and transformant The present invention provides an expression vector containing the polynucleotide of the present invention and a transformant containing the expression vector.
本発明は、上記本発明のポリヌクレオチドを含む発現ベクター及び該発現ベクターを含む形質転換体を提供するものである。 3. Expression vector and transformant The present invention provides an expression vector containing the polynucleotide of the present invention and a transformant containing the expression vector.
該発現ベクターは、本発明のポリヌクレオチドを適当な発現ベクター中のプロモーターの下流に機能可能に連結することにより製造することができる。ベクターの種類としては、プラスミドベクター、ウイルスベクター等を挙げることができ、用いる宿主細胞に応じて適宜選択することが出来る。
The expression vector can be produced by operably linking the polynucleotide of the present invention downstream of a promoter in an appropriate expression vector. Examples of the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host cell to be used.
宿主細胞には、原核生物細胞及び真核生物細胞が含まれる。原核生物細胞としては、エシェリヒア属菌(エシェリヒア・コリ(Escherichia coli)等)、バチルス属菌(バチルス・サブチルス(Bacillus subtilis)等)等が用いられる。真核生物細胞としては、酵母(サッカロマイセス セレビシエ(Saccharomyces cerevisiae)等)、昆虫細胞(夜盗蛾の幼虫由来株化細胞(Spodoptera frugiperda cell;Sf細胞)等)、哺乳動物細胞(ヒト細胞(293等)、サル細胞(COS-7等)、チャイニーズハムスター細胞(CHO細胞等)等)などが用いられる。
Host cells include prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include Escherichia bacteria (such as Escherichia coli), Bacillus bacteria (such as Bacillus subtilis), and the like. Examples of eukaryotic cells include yeast (Saccharomyces cerevisiae, etc.), insect cells (stolen larvae-derived cell lines (Spodoptera frugiperda cell; Sf cells), etc.), mammalian cells (human cells (293, etc.)) Monkey cells (such as COS-7), Chinese hamster cells (such as CHO cells)), and the like.
哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物;ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜;イヌ、ネコ等のペット;ヒト、サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。
Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
プラスミドベクターとしては、大腸菌由来のプラスミドベクター(例、pBR322,pBR325,pUC12,pUC13)、枯草菌由来のプラスミドベクター(例、pUB110,pTP5,pC194)、酵母由来プラスミドベクター(例、pSH19,pSH15)等を挙げることができ、用いる宿主の種類や使用目的に応じて適宜選択することが出来る。
Plasmid vectors derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmid vectors derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmid vectors (eg, pSH19, pSH15), etc. And can be appropriately selected according to the type of host used and the purpose of use.
ウイルスベクターの種類は、用いる宿主細胞の種類や使用目的に応じて適宜選択することが出来る。例えば、宿主として昆虫細胞を用いる場合には、バキュロウイルスベクター等を用いることが出来る。また、宿主として哺乳動物細胞を用いる場合には、モロニーマウス白血病ウイルスベクター、レンチウイルスベクター、シンドビスウイルスベクター等のレトロウイルスベクター、アデノウイルスベクター、ヘルペスウイルスベクター、アデノ随伴ウイルスベクター、パルボウイルスベクター、ワクシニアウイルスベクター、センダイウイルスベクター等を用いることが出来る。
The type of virus vector can be appropriately selected according to the type of host cell used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.
また、プロモーターは、用いる宿主細胞の種類に対応して、該宿主細胞内で転写を開始可能なものを選択することが出来る。例えば、宿主がエシェリヒア属菌である場合、trpプロモーター、lacプロモーター、T7プロモーターなどが好ましい。宿主がバチルス属菌である場合、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。宿主が酵母である場合、PHO5プロモーター、PGKプロモーターなどが好ましい。宿主が昆虫細胞である場合、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。宿主が哺乳動物細胞である場合、サブゲノミック(26S)プロモーター、CMVプロモーター、SRαプロモーターなどが好ましい。
Also, a promoter that can initiate transcription in the host cell can be selected according to the type of host cell to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SRα promoter and the like are preferable.
本発明の発現ベクターは、所望によりエンハンサー、スプライシングシグナル、ポリA付加シグナル、選択マーカー、SV40複製オリジン(以下、SV40oriと略称する場合がある)などを、それぞれ機能可能な態様で含有していてもよい。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素(以下、dhfrと略称する場合がある)遺伝子〔メソトレキセート(MTX)耐性〕、アンピシリン耐性遺伝子(Amprと略称する場合がある)、ネオマイシン耐性遺伝子(Neorと略称する場合がある、G418耐性)等が挙げられる。
The expression vector of the present invention may contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like in a functional manner, if desired. Good. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (sometimes abbreviated as Amp r ), neomycin resistance gene (Neo). G418 resistance) which may be abbreviated as r ).
本発明の発現ベクターは好ましくは単離又は精製されている。
The expression vector of the present invention is preferably isolated or purified.
本発明の発現ベクターは、適切な宿主細胞内において、本発明のポリペプチドを発現し得るので、本発明のポリペプチドの製造に有用である。
The expression vector of the present invention is useful for producing the polypeptide of the present invention because it can express the polypeptide of the present invention in an appropriate host cell.
上記本発明の発現ベクターを、自体公知の遺伝子導入法(例えば、リポフェクション法、リン酸カルシウム法、マイクロインジェクション法、プロトプラスト融合法、エレクトロポレーション法、DEAEデキストラン法、Gene Gunによる遺伝子導入法等)に従って上記宿主細胞へ導入することにより、該発現ベクターが導入された形質転換体(本発明の形質転換体)を製造することができる。該形質転換体は本発明のポリペプチドを発現し得る。本発明の形質転換体は、本発明のポリペプチドの製造等に有用である。
The above-described expression vector of the present invention is prepared according to a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, protoplast fusion method, electroporation method, DEAE dextran method, gene transfer method using Gene Gun). By introducing it into a host cell, a transformant into which the expression vector has been introduced (the transformant of the present invention) can be produced. The transformant can express the polypeptide of the present invention. The transformant of the present invention is useful for producing the polypeptide of the present invention.
本発明の形質転換体を、宿主の種類に応じて、自体公知の方法で培養し、培養物から本発明のポリペプチドを単離することにより、本発明のポリペプチドを製造することが出来る。宿主がエシェリヒア属菌である形質転換体の培養は、LB培地やM9培地等の適切な培地中、通常約15~43℃で、約3~24時間行なわれる。宿主がバチルス属菌である形質転換体の培養は、適切な培地中、通常約30~40℃で、約6~24時間行なわれる。宿主が酵母である形質転換体の培養は、バークホールダー培地等の適切な培地中、通常約20℃~35℃で、約24~72時間行なわれる。宿主が昆虫細胞または昆虫である形質転換体の培養は、約10%のウシ血清が添加されたGrace’s Insect medium等の適切な培地中、通常約27℃で、約3~5日間行なわれる。宿主が動物細胞である形質転換体の培養は、約10%のウシ血清が添加されたMEM培地等の適切な培地中、通常約30℃~40℃で、約15~60時間行なわれる。いずれの培養においても、必要に応じて通気や撹拌を行ってもよい。培養物からの本発明のポリペプチドの単離又は精製は、例えば、菌体溶解液や培養上清を、逆相クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィーなどの複数のクロマトグラフィーに供することにより達成することができる。
The polypeptide of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the type of host and isolating the polypeptide of the present invention from the culture. The transformant whose host is Escherichia is cultured in an appropriate medium such as LB medium or M9 medium, usually at about 15 to 43 ° C. for about 3 to 24 hours. The transformant whose host is Bacillus is cultured in an appropriate medium, usually at about 30 to 40 ° C. for about 6 to 24 hours. Cultivation of the transformant whose host is yeast is usually carried out at about 20 ° C. to 35 ° C. for about 24 to 72 hours in a suitable medium such as a Burkholder medium. Cultivation of transformants whose host is an insect cell or an insect is carried out in a suitable medium such as Grace's Insect medium supplemented with about 10% bovine serum, usually at about 27 ° C for about 3 to 5 days. The transformant whose host is an animal cell is cultured in an appropriate medium such as a MEM medium supplemented with about 10% bovine serum, usually at about 30 ° C. to 40 ° C. for about 15 to 60 hours. In any culture, aeration and agitation may be performed as necessary. For isolation or purification of the polypeptide of the present invention from the culture, for example, the cell lysate or the culture supernatant is subjected to a plurality of chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. Can be achieved.
4.ミステリンの機能的欠損を含む非ヒト動物
本発明は、上記本発明のポリペプチドをコードする遺伝子(ミステリン遺伝子)の機能的欠損を含む非ヒト動物を提供する。 4). Non-human animal containing functional defect of mysterin The present invention provides a non-human animal comprising a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
本発明は、上記本発明のポリペプチドをコードする遺伝子(ミステリン遺伝子)の機能的欠損を含む非ヒト動物を提供する。 4). Non-human animal containing functional defect of mysterin The present invention provides a non-human animal comprising a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
本発明の動物の種としては、ヒト以外の哺乳動物、鳥類、爬虫類、両生類、魚類、昆虫などを挙げることができるが、哺乳動物、鳥類又は魚類が好ましい。ヒト以外の哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物;ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜;イヌ、ネコ等のペット;サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。鳥類としては、ニワトリ、ウズラ、アヒル、ガチョウ、シチメンチョウ、エミュ、ダチョウ、ホロホロ鳥、ハト等を挙げることができる。魚類としては、ゼブラフィッシュ、メダカ、キンギョ、ドジョウ等が挙げられる。好ましい非ヒト哺乳動物は、マウスである。好ましい鳥類は、ニワトリである。好ましい魚類は、ゼブラフィッシュである。
Examples of the species of the animal of the present invention include mammals other than humans, birds, reptiles, amphibians, fish, and insects, with mammals, birds and fish being preferred. Examples of mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits; domestic animals such as pigs, cows, goats, horses, sheep, minks; pets such as dogs and cats Primates such as, but not limited to, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees; Examples of the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons. Examples of fish include zebrafish, medaka, goldfish and loach. A preferred non-human mammal is a mouse. A preferred bird is a chicken. A preferred fish is zebrafish.
ミステリン遺伝子の機能的欠損とは、ミステリン遺伝子が本来有する正常な機能(ユビキチンリガーゼ活性、ATPase活性等)が十分に発揮できない状態をいい、例えば、ミステリン遺伝子が全く発現していない状態、またはミステリン遺伝子が本来有する正常な機能が発揮できない程度にその発現量が低下している状態、あるいはミステリン遺伝子産物の機能が完全に喪失した状態、またはミステリン遺伝子が本来有する正常な機能が発揮できない程度にミステリン遺伝子産物の機能が低下した状態が挙げられる。
A functional defect of a mysterin gene refers to a state in which normal functions (ubiquitin ligase activity, ATPase activity, etc.) inherent to the mysterin gene cannot be sufficiently exerted, for example, a state in which the mysterin gene is not expressed at all, or a mysterin gene The mysterin gene is in a state where its expression level is reduced to the extent that it cannot exert its normal function, or the function of the mysterin gene product has been completely lost, or to the extent that the normal function inherent in the mysterin gene cannot be exerted A state in which the function of the product is reduced is mentioned.
本発明の動物は、ミステリン遺伝子の機能的欠損に伴う種々の特徴を有する。例えば、本発明の動物は、野生型動物に比して、胚発生が遅延し、初期の血管発生及びそれに続く血管新生に異常を呈する。さらには、頭部、眼部の血管新生に特に顕著な異常を呈する。
The animal of the present invention has various characteristics associated with a functional defect of the mysterin gene. For example, the animals of the present invention have delayed embryogenesis and abnormalities in early angiogenesis and subsequent angiogenesis compared to wild type animals. Furthermore, it exhibits a particularly remarkable abnormality in angiogenesis of the head and eyes.
一実施形態では、本発明の動物は、ゲノムDNAの改変を伴う動物、いわゆる遺伝子改変動物であり得る。本発明の遺伝子改変動物は、ミステリン遺伝子欠損ヘテロ接合体、又はミステリン遺伝子欠損ホモ接合体であり得る。
In one embodiment, the animal of the present invention may be an animal accompanied with a modification of genomic DNA, a so-called genetically modified animal. The genetically modified animal of the present invention may be a mysterin gene-deficient heterozygote or a mysterin gene-deficient homozygote.
本発明の遺伝子改変動物は、自体公知の方法により製造できる。先ず、本発明の遺伝子改変動物の作製に有用なキメラ動物の作製法について説明する。なお、本発明の動物は、本発明の遺伝子改変動物の作製に有用なキメラ動物をも含む。
The genetically modified animal of the present invention can be produced by a method known per se. First, a method for producing a chimeric animal useful for producing the genetically modified animal of the present invention will be described. The animals of the present invention include chimeric animals useful for producing the genetically modified animals of the present invention.
本発明のキメラ動物は、例えば下記の工程(a)~(c)を含む方法により製造できる:
(a)ミステリン遺伝子の機能的欠損を含む胚性幹細胞を提供する工程;
(b)該胚性幹細胞を胚に導入し、キメラ胚を得る工程;及び
(c)該キメラ胚を動物に移植し、キメラ動物を得る工程。 The chimeric animal of the present invention can be produced, for example, by a method including the following steps (a) to (c):
(A) providing an embryonic stem cell containing a functional defect of the mysterin gene;
(B) introducing the embryonic stem cells into an embryo to obtain a chimeric embryo; and (c) transplanting the chimeric embryo into an animal to obtain a chimeric animal.
(a)ミステリン遺伝子の機能的欠損を含む胚性幹細胞を提供する工程;
(b)該胚性幹細胞を胚に導入し、キメラ胚を得る工程;及び
(c)該キメラ胚を動物に移植し、キメラ動物を得る工程。 The chimeric animal of the present invention can be produced, for example, by a method including the following steps (a) to (c):
(A) providing an embryonic stem cell containing a functional defect of the mysterin gene;
(B) introducing the embryonic stem cells into an embryo to obtain a chimeric embryo; and (c) transplanting the chimeric embryo into an animal to obtain a chimeric animal.
上記方法の工程(a)では、ミステリン遺伝子の機能的欠損を含む胚性幹細胞(ES細胞)としては、例えば、下記の5.の項に記載した方法にて作製されたものを使用できる。
In the step (a) of the above method, as an embryonic stem cell (ES cell) containing a functional defect of the mysterin gene, for example, the following 5. Those prepared by the method described in the section can be used.
上記方法の工程(b)では、胚が由来する動物種は、本発明の遺伝子改変動物種と同様であり得、また、導入される胚性幹細胞が由来する動物種と同一であることが好ましい。胚としては、例えば胚盤胞、8細胞期胚などが挙げられる。胚はホルモン剤(例えばFSH様作用を有するPMSGおよびLH作用を有するhCGを使用)等により過排卵処理を施した雌動物を、雄動物と交配させること等により得ることができる。胚性幹細胞を胚に導入する方法としては、マイクロマニピュレーション法、凝集法などが挙げられる。
In step (b) of the above method, the animal species from which the embryo is derived may be the same as the genetically modified animal species of the present invention, and is preferably the same as the animal species from which the embryonic stem cells to be introduced are derived. . Examples of embryos include blastocysts and 8-cell stage embryos. Embryos can be obtained by mating female animals that have undergone superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) and the like. Examples of methods for introducing embryonic stem cells into the embryo include micromanipulation methods and aggregation methods.
上記方法の工程(c)では、キメラ胚が動物の子宮又は卵管に移入され得る。キメラ胚が移植される動物は好ましくは偽妊娠動物である。偽妊娠動物は、正常性周期の雌動物を、精管結紮等により去勢した雄動物と交配することにより得ることができる。キメラ胚が導入された動物は、妊娠し、キメラ動物を出産する。
In step (c) of the above method, the chimeric embryo can be transferred to the uterus or fallopian tube of the animal. The animal into which the chimeric embryo is transplanted is preferably a pseudopregnant animal. A pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like. The animal into which the chimeric embryo has been introduced becomes pregnant and gives birth to the chimeric animal.
次いで、出生した動物がキメラ動物か否かが確認される。出生した動物がキメラ動物であるか否かは自体公知の方法により確認でき、例えば、体色や被毛色で判別できる。また、判別のために、体の一部からDNAを抽出し、サザンブロット解析やPCRアッセイを行ってもよい。
Next, it is confirmed whether the animal born is a chimeric animal. Whether or not the animal born is a chimeric animal can be confirmed by a method known per se, for example, by body color or coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed.
本発明の遺伝子改変動物は、例えば下記の工程(a)~(d)を含む方法により製造できる:
(a)ミステリン遺伝子の機能的欠損を含む胚性幹細胞を提供する工程;
(b)該胚性幹細胞を胚に導入し、キメラ胚を得る工程;
(c)該キメラ胚を動物に移植し、キメラ動物を得る工程;及び
(d)該キメラ動物を交配させ、ミステリン遺伝子欠損ヘテロ接合体を得る工程。 The genetically modified animal of the present invention can be produced, for example, by a method including the following steps (a) to (d):
(A) providing an embryonic stem cell containing a functional defect of the mysterin gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transplanting the chimeric embryo into an animal to obtain a chimeric animal; and (d) mating the chimeric animal to obtain a mysterin gene-deficient heterozygote.
(a)ミステリン遺伝子の機能的欠損を含む胚性幹細胞を提供する工程;
(b)該胚性幹細胞を胚に導入し、キメラ胚を得る工程;
(c)該キメラ胚を動物に移植し、キメラ動物を得る工程;及び
(d)該キメラ動物を交配させ、ミステリン遺伝子欠損ヘテロ接合体を得る工程。 The genetically modified animal of the present invention can be produced, for example, by a method including the following steps (a) to (d):
(A) providing an embryonic stem cell containing a functional defect of the mysterin gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transplanting the chimeric embryo into an animal to obtain a chimeric animal; and (d) mating the chimeric animal to obtain a mysterin gene-deficient heterozygote.
上記方法の工程(a)~(c)は、上述したキメラ動物の作製方法と同様にして行うことができる。
Steps (a) to (c) of the above method can be performed in the same manner as the method for producing a chimeric animal described above.
上記方法の工程(d)では、工程(c)で得られたキメラ動物が成熟した後に交配させる。交配は好ましくは、野生型動物とキメラ動物との間で、又はキメラ動物同士で行われ得る。ミステリン遺伝子欠損が、キメラ動物の生殖系列細胞へ導入され、ミステリン遺伝子欠損ヘテロ接合体子孫が得られたか否かは、自体公知の方法により種々の形質を指標として確認でき、例えば、子孫動物の体色や被毛色により判別できる。また、判別のために、体の一部からDNAを抽出し、サザンブロット解析やPCRアッセイを行ってもよい。さらに、このようにして得られたミステリン遺伝子欠損ヘテロ接合体同士を交配させることにより、ミステリン遺伝子欠損ホモ接合体を作製できる。
In step (d) of the above method, mating is performed after the chimeric animal obtained in step (c) has matured. The mating can preferably take place between wild-type animals and chimeric animals or between chimeric animals. Whether or not a mysterin gene deficiency has been introduced into germline cells of a chimeric animal and a mysterin gene deficient heterozygous offspring can be obtained can be confirmed using various traits as indicators, for example, the body of a progeny animal It can be distinguished by color and coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed. Furthermore, a mysterin gene-deficient homozygote can be produced by crossing the mysterin gene-deficient heterozygotes thus obtained.
一般的に、遺伝子改変動物の作製の過程では、胚性幹細胞に由来する遺伝子と、交配に用いた動物に由来する遺伝子とが交雑した遺伝子型を有する子孫動物が得られるため、結果としてミステリン遺伝子が欠損することのみによる特有の効果を調べることが困難となってしまう場合がある。そこで、ミステリン遺伝子欠損特有の効果のみをより適切に抽出するために、得られたミステリン遺伝子欠損動物(ヘテロ接合体またはホモ接合体)を純系の動物系統と、5世代~8世代程度にわたり戻し交配することが好ましい。また、自然交配のみにより戻し交配を行うと長い年月がかかる場合があるので、世代交代を早めたい場合には体外受精技術を適宜用いることもできる。
Generally, in the process of producing a genetically modified animal, a progeny animal having a genotype in which a gene derived from an embryonic stem cell and a gene derived from an animal used for mating are obtained is obtained. In some cases, it may be difficult to examine the peculiar effect only due to lack of. Therefore, in order to more appropriately extract only the effects peculiar to the mysterin gene deficiency, the obtained mysterin gene deficient animal (heterozygote or homozygote) is backcrossed with a pure animal strain for about 5 to 8 generations. It is preferable to do. In addition, when backcrossing is carried out only by natural mating, it may take a long time, so in vitro fertilization techniques can be used as appropriate when it is desired to speed up generational changes.
別の実施形態では、本発明の動物は、ゲノムDNAの改変を伴わない動物であり得る。かかる動物は、例えば、ミステリン遺伝子の発現または機能を特異的に抑制する物質(例えば、アンチセンス核酸、siRNA、これらの核酸を発現し得る発現ベクター)の動物への投与又は動物での強制発現により作製できる。投与は、例えば、マイクロインジェクション、リポソーム等の適切な送達手段を使用して行われ得る。また、細胞特異的なミステリン遺伝子の機能的欠損を達成することも可能である。
In another embodiment, the animal of the present invention may be an animal that is not accompanied by genomic DNA modification. Such an animal can be obtained by, for example, administering a substance that specifically suppresses the expression or function of the mysterin gene (eg, antisense nucleic acid, siRNA, expression vector capable of expressing these nucleic acids) to the animal or forced expression in the animal. Can be made. Administration can be carried out using a suitable delivery means such as, for example, microinjection, liposomes. It is also possible to achieve a cell-specific mysterin gene functional defect.
特に、ミステリン遺伝子を発現抑制した魚類(例、ゼブラフィッシュの場合はミステリン1)の作成においては、操作の簡便性や時間コストの観点から、モルフォリノアンチセンス核酸の使用が有利である(Nucleic Acid Drug Dev., vol. 7, pages 187-195, 1997;Nat Genet., vol. 26, pages 216-220, 2000)。モルフォリノアンチセンス核酸は、ミステリンmRNAの部分配列(ここでは、スプライシングアクセプターおよびドナーの境界配列25塩基の範囲内の部分配列)に相補的な(ミスマッチの数が、通常3塩基以下、好ましくは1塩基以下、最も好ましくは0である)約25塩基長のヌクレオチド配列からなる。合成したモルフォリノアンチセンス核酸を、1~8細胞期の受精卵(胚)の細胞質又は卵黄内にマイクロインジェクションすると、モルフォリノアンチセンス核酸が核内のミステリンmRNAのスプライシングを阻害し、エクソンスキップの結果によるフレームシフトされたタンパク質が産生される。この受精卵(胚)から、ミステリン遺伝子の発現が抑制された個体が生じる。モルフォリノアンチセンス核酸が適切にミステリン遺伝子の発現を抑制しているか否かは、RT-PCR等の方法により、得られた個体におけるミステリン遺伝子の発現パターンを測定することにより確認することが出来る。
In particular, the use of morpholino antisense nucleic acids is advantageous from the viewpoint of ease of operation and time cost in the production of fish (eg, mysterin 1 in the case of zebrafish) in which expression of the mysterin gene is suppressed (Nucleic® Acid). Drug Dev., Vol. 7, pages 187-195, 1997; Nat Genet., Vol. 26, pages 216-220, 2000). The morpholino antisense nucleic acid is complementary to the partial sequence of mysterin mRNA (here, the partial sequence within 25 bases of the splicing acceptor and donor boundary sequence) (the number of mismatches is usually 3 bases or less, preferably It consists of a nucleotide sequence of about 25 bases in length (1 base or less, most preferably 0). When the synthesized morpholino antisense nucleic acid is microinjected into the cytoplasm or yolk of a fertilized egg (embryo) at the 1-8 cell stage, the morpholino antisense nucleic acid inhibits splicing of mysterin mRNA in the nucleus, and exon skipping. The resulting frameshifted protein is produced. From this fertilized egg (embryo), an individual in which expression of the mysterin gene is suppressed is generated. Whether or not the morpholino antisense nucleic acid appropriately suppresses mysterin gene expression can be confirmed by measuring the expression pattern of the mysterin gene in the obtained individual by a method such as RT-PCR.
本発明の動物は、血管新生異常に関連する疾患の動物モデルとして有用である。血管新生の異常に関連する疾患としては、例えば、動脈硬化など閉塞性疾患、動静脈奇形、腫瘍における血管新生、炎症性閉塞疾患、糖尿病性血管障害、加齢性黄斑変性症など多様な要因による血管病変をはじめとし、オスラー病など単一遺伝子による血管構築異常等が挙げられる。
The animal of the present invention is useful as an animal model for diseases associated with abnormal neovascularization. Diseases related to abnormalities in angiogenesis include various factors such as occlusive diseases such as arteriosclerosis, arteriovenous malformations, neovascularization in tumors, inflammatory obstructive diseases, diabetic vascular disorders, and age-related macular degeneration Examples include vascular lesions and abnormalities in blood vessel construction due to a single gene such as Osler's disease.
本発明の動物はまた、ミステリン遺伝子および血管新生の解析、並びに血管新生を調節し得る物質や、血管新生異常を治療し得る物質のスクリーニングなどに有用である。例えば、本発明の動物における遺伝子発現を網羅的に解析することで、血管新生に関与する他の遺伝子(例えば、ミステリン遺伝子の発現様式と連動する発現様式を示す遺伝子)の同定が可能となる。この場合、例えば、本発明の動物において、遺伝子発現の網羅的解析を可能にする手段(例えば、マイクロアレイ)により遺伝子発現プロフィールが測定され、野生型動物等のコントロール動物(同種又は異種動物)の遺伝子発現プロフィールと比較される。また、本発明の動物の遺伝子発現プロフィールを経時的に追跡し、表現型(血管新生の異常)の発現、進行と遺伝子発現プロフィールの変化との連動性を評価することもできる。
The animal of the present invention is also useful for analysis of the mysterin gene and angiogenesis, and screening for substances that can regulate angiogenesis and those that can treat angiogenesis abnormalities. For example, by comprehensively analyzing gene expression in the animal of the present invention, it is possible to identify other genes involved in angiogenesis (for example, genes showing an expression pattern linked to the expression pattern of mysterin gene). In this case, for example, in the animal of the present invention, the gene expression profile is measured by means (for example, microarray) that enables comprehensive analysis of gene expression, and the gene of a control animal (same species or heterogeneous animal) such as a wild type animal is measured. Compared to expression profile. In addition, the gene expression profile of the animal of the present invention can be traced over time to evaluate the linkage between expression and progression of phenotype (abnormal neovascularization) and changes in gene expression profile.
5.ミステリンの機能的欠損を含む動物細胞
本発明は、上記本発明のポリペプチドをコードする遺伝子(ミステリン遺伝子)の機能的欠損を含む動物細胞を提供する。 5. Animal cell containing functional defect of mysterin The present invention provides an animal cell containing a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
本発明は、上記本発明のポリペプチドをコードする遺伝子(ミステリン遺伝子)の機能的欠損を含む動物細胞を提供する。 5. Animal cell containing functional defect of mysterin The present invention provides an animal cell containing a functional defect of a gene (mysterin gene) encoding the polypeptide of the present invention.
本発明の動物の種としては、ヒトを含む哺乳動物、鳥類、爬虫類、両生類、魚類、昆虫などを挙げることができるが、哺乳動物、鳥類又は魚類が好ましい。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物;ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜;イヌ、ネコ等のペット;ヒト、サル、カニクイザル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることが出来るが、これらに限定されるものではない。鳥類としては、ニワトリ、ウズラ、アヒル、ガチョウ、シチメンチョウ、エミュ、ダチョウ、ホロホロ鳥、ハト等を挙げることができる。魚類としては、ゼブラフィッシュ、メダカ、キンギョ、ドジョウ等が挙げられる。好ましい哺乳動物は、ヒト又はマウスである。好ましい鳥類は、ニワトリである。好ましい魚類は、ゼブラフィッシュである。
Examples of the animal species of the present invention include mammals including humans, birds, reptiles, amphibians, fish, and insects, with mammals, birds and fish being preferred. Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. Examples of the birds include chickens, quails, ducks, geese, turkeys, emu, ostriches, guinea fowls, and pigeons. Examples of fish include zebrafish, medaka, goldfish and loach. Preferred mammals are humans or mice. A preferred bird is a chicken. A preferred fish is zebrafish.
本発明の動物細胞はまた、任意の組織に由来する細胞であり得、例えば、ミステリン遺伝子が発現している体細胞(例えば心臓、脳、肝臓、膵臓、筋肉、肺、副腎、骨髄、胎盤、前立腺、唾液腺、胸腺、甲状腺、気管、子宮、脾臓、血管を構成している体細胞や、血管内皮細胞、血管平滑筋細胞、膵ランゲルハンス島膵B細胞)、精原細胞、精子、卵子、受精卵等の生殖系列細胞、胚性細胞、並びに胚性幹細胞などが挙げられる。好ましくは、血管を構成している体細胞、血管内皮細胞又は血管平滑筋細胞である。また、本発明の動物細胞は、初代培養細胞、細胞株のいずれであってもよい。なお、胚性細胞とは、胚から採取された細胞とその分裂によって生じた細胞で、胚でないものをいう。
The animal cell of the present invention can also be a cell derived from any tissue, for example, a somatic cell in which a mysterin gene is expressed (for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta, Prostate, salivary gland, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels, vascular endothelial cells, vascular smooth muscle cells, pancreatic islets of Langerhans), spermatogonia, sperm, ovum, fertilization Examples include germline cells such as eggs, embryonic cells, and embryonic stem cells. Preferably, it is a somatic cell, vascular endothelial cell or vascular smooth muscle cell constituting a blood vessel. The animal cell of the present invention may be either a primary cultured cell or a cell line. An embryonic cell refers to a cell collected from an embryo and a cell generated by the division and not a embryo.
一実施形態では、本発明の動物細胞は、ゲノムDNAの改変を伴う細胞、いわゆる遺伝子改変細胞であり得る。本発明の遺伝子改変細胞は、例えば、ミステリン遺伝子欠損ホモ接合体、又はミステリン遺伝子欠損ヘテロ接合体であり得る。
In one embodiment, the animal cell of the present invention may be a cell accompanied by a modification of genomic DNA, a so-called genetically modified cell. The genetically modified cell of the present invention can be, for example, a mysterin gene-deficient homozygote or a mysterin gene-deficient heterozygote.
本発明の遺伝子改変細胞は、自体公知の方法により製造できる。例えば、本発明の動物細胞は、下記の工程(a)~(c)を含む方法により製造できる:
(a)ミステリン遺伝子の相同組換えを誘導し得るターゲティングベクターを提供する工程;
(b)該ターゲティングベクターを動物細胞に導入する工程;及び
(c)該ターゲティングベクターを導入した細胞から、相同組換えを生じた細胞を選別する工程。 The genetically modified cell of the present invention can be produced by a method known per se. For example, the animal cell of the present invention can be produced by a method comprising the following steps (a) to (c):
(A) providing a targeting vector capable of inducing homologous recombination of the mysterin gene;
(B) introducing the targeting vector into animal cells; and (c) selecting cells that have undergone homologous recombination from the cells into which the targeting vector has been introduced.
(a)ミステリン遺伝子の相同組換えを誘導し得るターゲティングベクターを提供する工程;
(b)該ターゲティングベクターを動物細胞に導入する工程;及び
(c)該ターゲティングベクターを導入した細胞から、相同組換えを生じた細胞を選別する工程。 The genetically modified cell of the present invention can be produced by a method known per se. For example, the animal cell of the present invention can be produced by a method comprising the following steps (a) to (c):
(A) providing a targeting vector capable of inducing homologous recombination of the mysterin gene;
(B) introducing the targeting vector into animal cells; and (c) selecting cells that have undergone homologous recombination from the cells into which the targeting vector has been introduced.
上記方法の工程(a)において使用されるミステリン遺伝子の相同組換えを誘導し得るターゲティングベクターは、本明細書の配列表に開示されたミステリンのcDNA配列(配列番号1)や、ゲノムDNA配列(配列番号5)の情報に基づき、所望の動物のミステリン遺伝子のゲノムDNAを取得し、ノックアウトマウス作成において汎用の遺伝子工学的手法に従い、当業者であれば容易に製造することができる。
The targeting vector capable of inducing homologous recombination of the mysterin gene used in step (a) of the above method is a mysterin cDNA sequence (SEQ ID NO: 1) disclosed in the sequence listing of the present specification, a genomic DNA sequence ( Based on the information of SEQ ID NO: 5), genomic DNA of the mysterin gene of the desired animal can be obtained, and can be easily produced by those skilled in the art according to a general genetic engineering technique in creating a knockout mouse.
上記方法の工程(b)では、ターゲティングベクターが動物細胞中に導入される。ターゲティングベクターを動物細胞に導入する方法としては、例えば、リン酸カルシウム法、リポフェクション法/リポソーム法、エレクトロポレーション法などが挙げられる。ターゲティングベクターが動物細胞中に導入されると、当該動物細胞中でミステリン遺伝子を含むゲノムDNAの相同組換えが生じる。
In step (b) of the above method, the targeting vector is introduced into the animal cell. Examples of the method for introducing the targeting vector into animal cells include the calcium phosphate method, the lipofection method / liposome method, and the electroporation method. When the targeting vector is introduced into an animal cell, homologous recombination of genomic DNA containing the mysterin gene occurs in the animal cell.
ターゲティングベクターが導入される動物細胞としては、自体公知の方法で作製したもの、あるいは市販のもの、又は所定の機関より入手可能なものを使用できる。動物細胞の種類は、上述の通りである。
As the animal cell into which the targeting vector is introduced, one produced by a method known per se, one commercially available, or one available from a predetermined organization can be used. The types of animal cells are as described above.
例えば、ターゲティングベクターが導入される動物細胞として胚性幹細胞を使用する場合、胚性幹細胞は、任意の動物の胚盤胞から分離した内部細胞塊をフィーダー細胞上で培養することにより樹立してもよいが、市販または所定の機関より既存の胚性幹細胞を入手できる。既存のマウス胚性幹細胞としては、例えば、ES-D3細胞、ES-E14TG2a細胞、SCC-PSA1細胞、TT2細胞、AB-1細胞、J1細胞、R1細胞、E14.1細胞、RW-4細胞などが挙げられる。また、胚性幹細胞としては、現時点で、マウス胚性幹細胞以外に、ヒト、ミンク、ハムスター、ブタ、ウシ、マーモセット、アカゲザル等の哺乳動物由来のものなどが樹立されているので、これらを用いることもできる。
For example, when an embryonic stem cell is used as an animal cell into which a targeting vector is introduced, the embryonic stem cell can be established by culturing an inner cell mass separated from a blastocyst of any animal on a feeder cell. Although it is good, existing embryonic stem cells can be obtained commercially or from a predetermined organization. Examples of existing mouse embryonic stem cells include ES-D3 cells, ES-E14TG2a cells, SCC-PSA1 cells, TT2 cells, AB-1 cells, J1 cells, R1 cells, E14.1 cells, RW-4 cells, etc. Is mentioned. In addition to mouse embryonic stem cells, embryonic stem cells derived from mammals such as humans, mink, hamsters, pigs, cattle, marmoset, rhesus monkeys, etc. have been established at present. You can also.
また、ターゲティングベクターが導入される動物細胞として腎臓細胞等の体細胞を使用する場合、ターゲティングベクターが導入される体細胞は、初代培養細胞、細胞株のいずれでもよい。初代培養細胞および細胞株は、自体公知の方法により作製できる(例えば、Current Protocols in Cell Biology, John Wiley & Sons, Inc.(2001))。
In addition, when somatic cells such as kidney cells are used as animal cells into which the targeting vector is introduced, the somatic cells into which the targeting vector is introduced may be either primary cultured cells or cell lines. Primary cultured cells and cell lines can be prepared by a method known per se (for example, Current Protocols in Cell Biology, John John Wiley and Sons, Inc. (2001)).
上記方法の工程(c)では、ミステリン遺伝子を含むゲノムDNAで相同組換えが生じた動物細胞を選別するため、ターゲティングベクター導入後の動物細胞がスクリーニングされる。例えば、ポジティブ選別、ネガティブ選別等により選別を行った後に、遺伝子型に基づくスクリーニング(例えば、PCR法、サザンブロットハイブリダイゼーション法)を行う。
In step (c) of the above method, in order to select animal cells in which homologous recombination has occurred with genomic DNA containing the mysterin gene, the animal cells after introduction of the targeting vector are screened. For example, after selection by positive selection, negative selection, etc., screening based on genotype (for example, PCR method, Southern blot hybridization method) is performed.
動物細胞が胚性幹細胞である場合には、好ましくは、組換え胚性幹細胞の核型分析がさらに行なわれる。核型分析では、選別された組換え胚性幹細胞において染色体異常がないことが確認される。核型分析は、自体公知の方法により行うことができる。なお、胚性幹細胞の核型は、ターゲティングベクターの導入前に予め確認しておくことが好ましい。
When the animal cell is an embryonic stem cell, preferably, a karyotype analysis of the recombinant embryonic stem cell is further performed. Karyotype analysis confirms that there is no chromosomal abnormality in the selected recombinant embryonic stem cells. Karyotype analysis can be performed by a method known per se. The karyotype of embryonic stem cells is preferably confirmed in advance before introducing the targeting vector.
本発明の遺伝子改変細胞はまた、本発明の遺伝子改変動物から単離できる。本発明の遺伝子改変動物はミステリン遺伝子の機能的欠損を含むので、本発明の遺伝子改変動物から単離された遺伝子改変細胞もまた、ミステリン遺伝子の機能的欠損を含む。また、本発明の遺伝子改変動物から単離された細胞を、遺伝子工学的手法等の方法により改変してもよい。細胞の単離および改変は、自体公知の方法により行うことができる(例えば、Current Protocols in Cell Biology, John Wiley & Sons, Inc.(2001))。
The genetically modified cells of the present invention can also be isolated from the genetically modified animals of the present invention. Since the genetically modified animal of the present invention contains a functional defect of the mysterin gene, the genetically modified cell isolated from the genetically modified animal of the present invention also contains a functional defect of the mysterin gene. In addition, cells isolated from the genetically modified animal of the present invention may be modified by a method such as genetic engineering techniques. Isolation and modification of cells can be performed by a method known per se (for example, Current Protocols in Cell Biology, John Wiley and Sons, Inc. (2001)).
別の実施形態では、本発明の動物細胞は、ゲノムDNAの改変を伴わない細胞であり得る。かかる細胞は、例えば、ミステリン遺伝子の発現または機能を特異的に抑制する物質(例えば、アンチセンス核酸、siRNA、これらの核酸を発現し得る発現ベクター)の細胞への導入により作製できる。ミステリン遺伝子の発現または機能を抑制する物質の細胞への導入は、自体公知の方法により行うことができ、例えば、マイクロインジェクション、リン酸カルシウム法、リポフェクション法/リポソーム法、エレクトロポレーション法などが用いられる。
In another embodiment, the animal cell of the present invention may be a cell that is not accompanied by genomic DNA modification. Such a cell can be prepared, for example, by introducing a substance that specifically suppresses the expression or function of the mysterin gene (for example, an antisense nucleic acid, siRNA, or an expression vector capable of expressing these nucleic acids) into the cell. Introduction of a substance that suppresses the expression or function of the mysterin gene into cells can be performed by a method known per se, such as microinjection, calcium phosphate method, lipofection method / liposome method, electroporation method and the like.
特に、ミステリン遺伝子の機能的欠損を含む魚類(例、ゼブラフィッシュ)の作成においては、操作の簡便性や時間コストの観点から、モルフォリノアンチセンス核酸の使用が有利である(上述の4.の項参照)。
In particular, in the production of fish (eg, zebrafish) containing a functional defect of the mysterin gene, it is advantageous to use a morpholino antisense nucleic acid from the viewpoint of ease of operation and time cost (see 4. above). Section).
本発明の動物細胞は、ミステリン遺伝子および血管新生の解析、血管新生を調節し得る物質や血管新生異常を治療し得る物質のスクリーニング、並びに本発明の動物の製造などに有用である。
The animal cell of the present invention is useful for analysis of mysterin gene and angiogenesis, screening for a substance that can regulate angiogenesis and a substance that can treat angiogenesis abnormality, and production of the animal of the present invention.
6.血管新生調節剤の候補物質のスクリーニング方法
後述の実施例に示すように、ミステリンの発現を抑制した動物においては、血管新生の異常が認められる。従って、本発明はまた、被検物質がミステリン遺伝子の発現または機能を調節し得るか否かを評価することを含む、血管新生調節剤の候補物質のスクリーニング方法、ならびに当該スクリーニング方法により得られる血管新生調節剤の候補物質を提供する。 6). Method for Screening Candidate Substance for Angiogenesis Regulator As shown in the examples described later, abnormal angiogenesis is observed in animals in which mysterin expression is suppressed. Therefore, the present invention also includes a method for screening a candidate substance for an angiogenesis regulator, comprising evaluating whether a test substance can regulate the expression or function of a mysterin gene, and a blood vessel obtained by the screening method. A candidate substance for angiogenesis regulator is provided.
後述の実施例に示すように、ミステリンの発現を抑制した動物においては、血管新生の異常が認められる。従って、本発明はまた、被検物質がミステリン遺伝子の発現または機能を調節し得るか否かを評価することを含む、血管新生調節剤の候補物質のスクリーニング方法、ならびに当該スクリーニング方法により得られる血管新生調節剤の候補物質を提供する。 6). Method for Screening Candidate Substance for Angiogenesis Regulator As shown in the examples described later, abnormal angiogenesis is observed in animals in which mysterin expression is suppressed. Therefore, the present invention also includes a method for screening a candidate substance for an angiogenesis regulator, comprising evaluating whether a test substance can regulate the expression or function of a mysterin gene, and a blood vessel obtained by the screening method. A candidate substance for angiogenesis regulator is provided.
スクリーニング方法に供される被検物質は、いかなる公知化合物及び新規化合物であってもよく、例えば、核酸、糖質、脂質、蛋白質、ペプチド、有機低分子化合物、コンビナトリアルケミストリー技術を用いて作製された化合物ライブラリー、固相合成やファージディスプレイ法により作製されたランダムペプチドライブラリー、あるいは微生物、動植物、海洋生物等由来の天然成分等が挙げられる。
The test substance to be used for the screening method may be any known compound or novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, prepared using combinatorial chemistry technology Examples thereof include a compound library, a random peptide library prepared by solid phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
一実施形態(態様I)では、本発明のスクリーニング方法は、下記の工程(a)~(c)を含む:
(a)被検物質とミステリン遺伝子の発現を測定可能な細胞とを接触させる工程;
(b)被検物質を接触させた細胞におけるミステリン遺伝子の発現量を測定し、該発現量を被検物質を接触させない対照細胞におけるミステリン遺伝子の発現量と比較する工程;及び
(c)上記(b)の比較結果に基づいて、ミステリン遺伝子の発現量を調節する被検物質を選択する工程。 In one embodiment (Aspect I), the screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring the expression of the mysterin gene;
(B) measuring the expression level of the mysterin gene in the cell contacted with the test substance, and comparing the expression level with the expression level of the mysterin gene in the control cell not contacted with the test substance; and (c) above ( A step of selecting a test substance that regulates the expression level of the mysterin gene based on the comparison result of b).
(a)被検物質とミステリン遺伝子の発現を測定可能な細胞とを接触させる工程;
(b)被検物質を接触させた細胞におけるミステリン遺伝子の発現量を測定し、該発現量を被検物質を接触させない対照細胞におけるミステリン遺伝子の発現量と比較する工程;及び
(c)上記(b)の比較結果に基づいて、ミステリン遺伝子の発現量を調節する被検物質を選択する工程。 In one embodiment (Aspect I), the screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring the expression of the mysterin gene;
(B) measuring the expression level of the mysterin gene in the cell contacted with the test substance, and comparing the expression level with the expression level of the mysterin gene in the control cell not contacted with the test substance; and (c) above ( A step of selecting a test substance that regulates the expression level of the mysterin gene based on the comparison result of b).
上記方法の工程(a)では、被検物質がミステリン遺伝子の発現を測定可能な細胞と接触条件下におかれる。ミステリン遺伝子の発現を測定可能な細胞に対する被検物質の接触は、培養培地中で行われ得る。
In step (a) of the above method, the test substance is placed under contact with cells capable of measuring mysterin gene expression. Contact of the test substance with a cell capable of measuring the expression of the mysterin gene can be performed in a culture medium.
ミステリン遺伝子の発現を測定可能な細胞とは、ミステリン遺伝子の産物、例えば、転写産物、翻訳産物の発現レベルを直接的又は間接的に評価可能な細胞をいう。ミステリン遺伝子の産物の発現レベルを直接的に評価可能な細胞は、ミステリン遺伝子を天然で発現可能な細胞(例、ミステリン遺伝子を天然で発現している細胞)であり得、一方、ミステリン遺伝子の産物の発現レベルを間接的に評価可能な細胞は、ミステリン遺伝子転写調節領域についてレポーターアッセイを可能とする細胞であり得る。ミステリン遺伝子の発現を測定可能な細胞は、動物細胞、例えばマウス、ラット、ハムスター、モルモット、ウサギ、イヌ、サル、ヒト等の哺乳動物細胞、ニワトリ等の鳥類細胞、ゼブラフィッシュ等の魚類細胞であり得る。
A cell capable of measuring the expression of a mysterin gene refers to a cell capable of directly or indirectly evaluating the expression level of a mysterin gene product, for example, a transcription product or a translation product. A cell that can directly evaluate the expression level of a mysterin gene product can be a cell that can naturally express a mysterin gene (eg, a cell that naturally expresses a mysterin gene), while a product of a mysterin gene. A cell capable of indirectly assessing the expression level of can be a cell that allows a reporter assay for the mysterin gene transcriptional regulatory region. Cells that can measure the expression of the mysterin gene are animal cells, such as mammalian cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, humans, avian cells such as chickens, and fish cells such as zebrafish. obtain.
ミステリン遺伝子を天然で発現可能な細胞(例、ミステリン遺伝子を天然で発現している細胞)は、ミステリン遺伝子を潜在的に発現するものである限り特に限定されない。かかる細胞は、当業者であれば容易に同定でき、初代培養細胞、当該初代培養細胞から誘導された細胞株、市販の細胞株、セルバンクより入手可能な細胞株などを使用できる。ミステリン遺伝子が発現している細胞株としては、例えば、293細胞などが挙げられる。なお、ミステリン遺伝子は、上述の組織または細胞で発現していることが知られているので、このような組織または細胞(例えば、心臓、脳、肝臓、膵臓、筋肉、肺、副腎、骨髄、胎盤、前立腺、唾液腺、胸腺、甲状腺、気管、子宮、脾臓、血管を構成している体細胞や、血管内皮細胞、血管平滑筋細胞、膵ランゲルハンス島膵B細胞)由来の細胞または細胞株を使用してもよい。
The cells that can naturally express the mysterin gene (eg, cells that naturally express the mysterin gene) are not particularly limited as long as they potentially express the mysterin gene. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. Examples of cell lines in which the mysterin gene is expressed include 293 cells. Since the mysterin gene is known to be expressed in the tissues or cells described above, such tissues or cells (for example, heart, brain, liver, pancreas, muscle, lung, adrenal gland, bone marrow, placenta) , Prostate cells, salivary glands, thymus, thyroid, trachea, uterus, spleen, somatic cells that make up blood vessels, vascular endothelial cells, vascular smooth muscle cells, pancreatic Langerhans pancreatic B cells) May be.
ミステリン遺伝子転写調節領域についてレポーターアッセイを可能とする細胞は、ミステリン遺伝子転写調節領域、当該領域に機能可能に連結されたレポーター遺伝子を含む細胞である。ミステリン遺伝子転写調節領域、レポーター遺伝子は、発現ベクター中に挿入され得る。ミステリン遺伝子転写調節領域は、ミステリン遺伝子の発現を制御し得る領域である限り特に限定されないが、例えば、転写開始点から上流約2kbpまでの領域、あるいは該領域の塩基配列において1以上の塩基が欠失、置換若しくは付加された塩基配列からなり、且つミステリン遺伝子の転写を制御する能力を有する領域などが挙げられる。レポーター遺伝子は、検出可能な蛋白質又は検出可能な物質を生成する酵素をコードする遺伝子であればよく、例えばGFP(緑色蛍光蛋白質)遺伝子、GUS(β-グルクロニダーゼ)遺伝子、LUC(ルシフェラーゼ)遺伝子、CAT(クロラムフェニコルアセチルトランスフェラーゼ)遺伝子等が挙げられる。
A cell that enables a reporter assay for a mysterin gene transcription regulatory region is a cell that contains a mysterin gene transcription regulatory region and a reporter gene operably linked to the region. The mysterin gene transcription regulatory region and reporter gene can be inserted into an expression vector. The mysterin gene transcription regulatory region is not particularly limited as long as it is a region capable of controlling mysterin gene expression. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the nucleotide sequence of this region is missing. Examples include a region consisting of a lost, substituted or added base sequence and having the ability to control transcription of the mysterin gene. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, a GUS (β-glucuronidase) gene, a LUC (luciferase) gene, a CAT (Chloramphenicol acetyltransferase) gene and the like.
ミステリン遺伝子転写調節領域、当該領域に機能可能に連結されたレポーター遺伝子が導入される細胞は、ミステリン遺伝子転写調節機能を評価できる限り、即ち、該レポーター遺伝子の発現量が定量的に解析可能である限り特に限定されない。しかしながら、ミステリン遺伝子に対する生理的な転写調節因子を発現し、ミステリン遺伝子の発現調節の評価により適切であると考えられることから、該導入される細胞としては、ミステリン遺伝子を天然で発現可能な細胞(例、ミステリン遺伝子を天然で発現している細胞)が好ましい。
A cell into which a mysterin gene transcription regulatory region and a reporter gene operably linked to the region are introduced can quantitatively analyze the expression level of the reporter gene as long as the mysterin gene transcription regulatory function can be evaluated. There is no particular limitation. However, since it expresses a physiological transcriptional regulatory factor for the mysterin gene and is considered to be more suitable for evaluation of the expression regulation of the mysterin gene, the introduced cell is a cell that can naturally express the mysterin gene ( For example, cells that naturally express the mysterin gene) are preferred.
被検物質とミステリン遺伝子の発現を測定可能な細胞とが接触される培養培地は、用いられる細胞の種類などに応じて適宜選択されるが、例えば、約5~20%のウシ胎仔血清を含む最少必須培地(MEM)、ダルベッコ改変最少必須培地(DMEM)、RPMI1640培地、199培地などである。培養条件もまた、用いられる細胞の種類などに応じて適宜決定されるが、例えば、培地のpHは約6~約8であり、培養温度は通常約30~約40℃であり、培養時間は約12~約72時間である。
The culture medium in which the test substance is contacted with the cells capable of measuring the expression of the mysterin gene is appropriately selected according to the type of cells used, and includes, for example, about 5 to 20% fetal bovine serum. Minimum essential medium (MEM), Dulbecco's modified minimum essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells to be used. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.
上記方法の工程(b)では、先ず、被検物質を接触させた細胞におけるミステリン遺伝子の発現量が測定される。発現量の測定は、用いた細胞の種類などを考慮し、自体公知の方法により行われ得る。例えば、ミステリン遺伝子の発現を測定可能な細胞として、ミステリン遺伝子を天然で発現可能な細胞を用いた場合、発現量は、ミステリン遺伝子の産物、例えば、転写産物又は翻訳産物を対象として自体公知の方法により測定できる。例えば、転写産物の発現量は、細胞からtotal RNAを調製し、RT-PCR、ノザンブロッティング等により測定され得る。また、翻訳産物の発現量は、細胞から抽出液を調製し、免疫学的手法により測定され得る。免疫学的手法としては、放射性同位元素免疫測定法(RIA法)、ELISA法(Methods in Enzymol. 70: 419-439 (1980))、蛍光抗体法などが使用できる。一方、ミステリン遺伝子の発現を測定可能な細胞として、ミステリン遺伝子転写調節領域についてレポーターアッセイを可能とする細胞を用いた場合、発現量は、レポーターのシグナル強度に基づき測定され得る。
In step (b) of the above method, first, the expression level of the mysterin gene in the cell contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing the mysterin gene is used as a cell capable of measuring the expression of the mysterin gene, the expression level is a method known per se for a mysterin gene product, for example, a transcription product or a translation product. Can be measured. For example, the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the mysterin gene transcriptional regulatory region is used as a cell capable of measuring mysterin gene expression, the expression level can be measured based on the signal intensity of the reporter.
次いで、被検物質を接触させた細胞におけるミステリン遺伝子の発現量が、被検物質を接触させない対照細胞におけるミステリン遺伝子の発現量と比較される。発現量の比較は、好ましくは、有意差の有無に基づいて行なわれる。被検物質を接触させない対照細胞におけるミステリン遺伝子の発現量は、被検物質を接触させた細胞におけるミステリン遺伝子の発現量の測定に対し、事前に測定した発現量であっても、同時に測定した発現量であってもよいが、実験の精度、再現性の観点から同時に測定した発現量であることが好ましい。
Next, the expression level of the mysterin gene in the cells contacted with the test substance is compared with the expression level of the mysterin gene in the control cells not contacted with the test substance. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the mysterin gene in the control cells that are not contacted with the test substance is the expression level measured at the same time, even if the expression level is measured in advance compared to the measurement level of the mysterin gene expression level in the cell that is in contact with the test substance. Although it may be an amount, it is preferably an expression amount measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
上記方法の工程(c)では、ミステリン遺伝子の発現量を調節する被検物質が選択される。ミステリン遺伝子の発現量の調節は、発現量の増加または減少であり得る。例えば、ミステリン遺伝子の発現量を増加させる(発現を促進する)被検物質は、血管新生の異常の予防又は治療剤の候補物質となり得る。一方、ミステリン遺伝子の発現量を減少させる(発現を抑制する)被検物質は、血管新生異常の誘発剤となり得る。従って、ミステリン遺伝子の発現量を指標として、血管新生の異常の予防又は治療剤等の医薬、または血管新生異常の誘発剤等の研究用試薬のための候補物質を選択することが可能となる。
In step (c) of the above method, a test substance that regulates the expression level of the mysterin gene is selected. The regulation of the expression level of the mysterin gene can be an increase or decrease in the expression level. For example, a test substance that increases (promotes expression) the expression level of a mysterin gene can be a candidate substance for a prophylactic or therapeutic agent for angiogenesis abnormality. On the other hand, a test substance that decreases (suppresses expression) the expression level of the mysterin gene can be an inducer of abnormal angiogenesis. Therefore, it becomes possible to select a candidate substance for a research reagent such as a drug such as a prophylactic or therapeutic agent for angiogenesis abnormality or an angiogenesis abnormality inducer, using the expression level of the mysterin gene as an index.
別の実施形態(態様II)では、本発明のスクリーニング方法は、下記の工程(a)~(c)を含む:
(a)被検物質をミステリンポリペプチドに接触させる工程;
(b)被検物質の存在下でのミステリンポリペプチドの機能を測定し、該機能を被検物質の不在下におけるミステリンポリペプチドの機能と比較する工程;及び
(c)上記(b)の結果に基づいて、ミステリンポリペプチドの機能を調節する被検物質を選択する工程。 In another embodiment (Aspect II), the screening method of the present invention comprises the following steps (a) to (c):
(A) contacting the test substance with a mysterin polypeptide;
(B) measuring the function of the mysterin polypeptide in the presence of the test substance and comparing the function with the function of the mysterin polypeptide in the absence of the test substance; and (c) (b) above A step of selecting a test substance that modulates the function of the mysterin polypeptide based on the result of.
(a)被検物質をミステリンポリペプチドに接触させる工程;
(b)被検物質の存在下でのミステリンポリペプチドの機能を測定し、該機能を被検物質の不在下におけるミステリンポリペプチドの機能と比較する工程;及び
(c)上記(b)の結果に基づいて、ミステリンポリペプチドの機能を調節する被検物質を選択する工程。 In another embodiment (Aspect II), the screening method of the present invention comprises the following steps (a) to (c):
(A) contacting the test substance with a mysterin polypeptide;
(B) measuring the function of the mysterin polypeptide in the presence of the test substance and comparing the function with the function of the mysterin polypeptide in the absence of the test substance; and (c) (b) above A step of selecting a test substance that modulates the function of the mysterin polypeptide based on the result of.
上記方法の工程(a)では、被検物質がミステリンポリペプチドと接触条件下におかれる。被検物質の該ポリペプチドに対する接触は、適切な緩衝液中での被検物質とミステリンポリペプチドとの混合により行われ得る。
In step (a) of the above method, the test substance is brought into contact with the mysterin polypeptide. Contact of the test substance with the polypeptide can be performed by mixing the test substance and the mysterin polypeptide in an appropriate buffer.
ミステリンポリペプチドは自体公知の方法により調製できる。例えば、上述したミステリン遺伝子の発現組織からミステリンポリペプチドを単離・精製できる。しかしながら、迅速、容易かつ大量にミステリンポリペプチドを調製するためには、遺伝子組換え技術により組換えポリペプチドを調製するのが好ましい。組換えポリペプチドは、上記2.の項に記載した方法により製造することが出来る。
Mysterin polypeptide can be prepared by a method known per se. For example, a mysterin polypeptide can be isolated and purified from the above-described mysterin gene expression tissue. However, in order to prepare mysterin polypeptides quickly, easily and in large quantities, it is preferable to prepare recombinant polypeptides by genetic recombination techniques. The recombinant polypeptide can It can be produced by the method described in the section.
上記方法の工程(b)における、ミステリンポリペプチドの機能としては、ユビキチンリガーゼ活性及びATPase活性を挙げることが出来る。ユビキチンリガーゼ活性及びATPase活性は、上記1.の項で説明した方法により測定することが出来る。
Examples of the function of the mysterin polypeptide in step (b) of the above method include ubiquitin ligase activity and ATPase activity. The ubiquitin ligase activity and ATPase activity are as described in 1. above. It can be measured by the method described in the section.
次いで、被検物質の存在下でのミステリンポリペプチドの機能が、被検物質の不在下でのミステリンポリペプチドの機能と比較される。機能の比較は、好ましくは、有意差の有無に基づいて行なわれる。被検物質の不在下でのミステリンポリペプチドの機能の測定は、被検物質の存在下でのミステリンポリペプチドの機能の測定に対し、事前に行われたものであっても、同時に行われたものであってもよいが、実験の精度、再現性の観点から同時に測定するのが好ましい。
Next, the function of the mysterin polypeptide in the presence of the test substance is compared with the function of the mysterin polypeptide in the absence of the test substance. The comparison of functions is preferably performed based on the presence or absence of a significant difference. Measurement of mysterin polypeptide function in the absence of the test substance is performed simultaneously with measurement of mysterin polypeptide function in the presence of the test substance, even if it was performed in advance. However, it is preferable to measure simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
上記方法の工程(c)では、ミステリンポリペプチドの機能を調節する被検物質が選択される。ミステリンポリペプチドの機能の調節は、機能の促進又は抑制であり得る。例えば、ミステリンポリペプチドの機能を促進させる被検物質は、血管新生の異常の予防又は治療剤の候補物質となり得る。一方、ミステリンポリペプチドの機能を抑制する被検物質は、血管新生異常の誘発剤となり得る。従って、ミステリンポリペプチドの機能を指標として、血管新生の異常の予防又は治療剤等の医薬、または血管新生異常の誘発剤等の研究用試薬のための候補物質を選択することが可能となる。
In step (c) of the above method, a test substance that regulates the function of mysterin polypeptide is selected. Modulation of the function of a mysterin polypeptide can be promotion or suppression of function. For example, a test substance that promotes the function of mysterin polypeptide can be a candidate substance for a prophylactic or therapeutic agent for angiogenesis abnormality. On the other hand, a test substance that suppresses the function of mysterin polypeptide can be an inducer of abnormal angiogenesis. Accordingly, it is possible to select a candidate substance for a research reagent such as a pharmaceutical agent such as a prophylactic or therapeutic agent for angiogenesis abnormality or an inducer of angiogenesis abnormality using the function of mysterin polypeptide as an index. .
更に、別の実施形態(態様III)では、本発明のスクリーニング方法は、下記の工程(a)~(c)を含む:
(a)被検物質をミステリンポリペプチドに接触させる工程;
(b)被検物質のミステリンポリペプチドに対する結合能を測定する工程;及び
(c)上記(b)の結果に基づいて、ミステリンポリペプチドに結合能を有する被検物質を選択する工程。 Furthermore, in another embodiment (Aspect III), the screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a mysterin polypeptide;
(B) measuring the binding ability of the test substance to the mysterin polypeptide; and (c) selecting a test substance having the binding ability to the mysterin polypeptide based on the result of (b) above.
(a)被検物質をミステリンポリペプチドに接触させる工程;
(b)被検物質のミステリンポリペプチドに対する結合能を測定する工程;及び
(c)上記(b)の結果に基づいて、ミステリンポリペプチドに結合能を有する被検物質を選択する工程。 Furthermore, in another embodiment (Aspect III), the screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a mysterin polypeptide;
(B) measuring the binding ability of the test substance to the mysterin polypeptide; and (c) selecting a test substance having the binding ability to the mysterin polypeptide based on the result of (b) above.
上記方法の工程(a)では、被検物質がミステリンポリペプチドと接触条件下におかれる。被検物質の該ポリペプチドに対する接触は、適切な緩衝液中での被検物質とミステリンポリペプチドとの混合により行われ得る。
In step (a) of the above method, the test substance is brought into contact with the mysterin polypeptide. Contact of the test substance with the polypeptide can be performed by mixing the test substance and the mysterin polypeptide in an appropriate buffer.
上記方法の工程(b)では、ミステリンポリペプチドに対する被検物質の結合能が測定される。結合能の測定は、自体公知の方法、例えば、バインディングアッセイ、表面プラズモン共鳴を利用する方法(例えば、Biacore(登録商標)の使用)により行われ得る。
In step (b) of the above method, the binding ability of the test substance to the mysterin polypeptide is measured. The binding ability can be measured by a method known per se, for example, a binding assay, a method using surface plasmon resonance (for example, use of Biacore (registered trademark)).
上記方法の工程(c)では、ミステリンポリペプチドに結合能を有する被検物質が選択される。ミステリンポリペプチドに結合能を有する被検物質は、ミステリン遺伝子の機能を調節(例えば、促進または抑制)し得る物質となる可能性がある。従って、本方法論は、例えば、ミステリン遺伝子の機能を調節し得る物質の1stスクリーニングとして有用であり得る。本方法論により得られた物質を上記態様(II)における被検物質として更に評価することにより、効率的にミステリンポリペプチドの機能を調節する被検物質を確実に選択することができる。
In step (c) of the above method, a test substance capable of binding to the mysterin polypeptide is selected. A test substance capable of binding to the mysterin polypeptide may be a substance capable of regulating (for example, promoting or suppressing) the function of the mysterin gene. Therefore, this methodology can be useful, for example, as a first screening for substances that can regulate the function of the mysterin gene. By further evaluating the substance obtained by this methodology as the test substance in the above embodiment (II), a test substance that efficiently regulates the function of the mysterin polypeptide can be reliably selected.
本発明のスクリーニング方法はまた、被検物質の動物への投与により行われ得る。該動物としては、例えば、マウス、ラット、ハムスター、モルモット、ウサギ、イヌ、サル等の哺乳動物、又は本発明の動物が挙げられる。動物を用いて本発明のスクリーニング方法が行われる場合、例えば、ミステリン遺伝子の発現量を調節する被検物質が選択され得る。
The screening method of the present invention can also be performed by administering a test substance to an animal. Examples of the animal include mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, and the animals of the present invention. When the screening method of the present invention is performed using an animal, for example, a test substance that regulates the expression level of the mysterin gene can be selected.
本発明のスクリーニング方法は、血管新生の調節剤の候補物質のスクリーニングを可能とする。従って、本発明のスクリーニング方法は、血管新生の異常の予防又は治療剤等の医薬、または血管新生異常の誘発剤等の研究用試薬の開発などに有用である。
The screening method of the present invention enables screening for candidate substances for regulators of angiogenesis. Therefore, the screening method of the present invention is useful for the development of a pharmaceutical agent such as a preventive or therapeutic agent for angiogenesis abnormality, or the development of a research reagent such as an angiogenesis abnormality inducer.
7.モヤモヤ病に関連するSNPを含むポリヌクレオチド
本発明は、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドを提供する。 7). A polynucleotide comprising a SNP associated with moyamoya disease The present invention is a polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 4766 T> C ( SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917G> A (SNP4) and 232102 G> A (SNP5), and at least one SNP selected from the partial sequence The SNP allele included in the present invention is a minor allele, and the partial sequence or its complementary sequence provides a polynucleotide having a length of at least 12 nucleotides.
本発明は、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドを提供する。 7). A polynucleotide comprising a SNP associated with moyamoya disease The present invention is a polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 4766 T> C ( SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917G> A (SNP4) and 232102 G> A (SNP5), and at least one SNP selected from the partial sequence The SNP allele included in the present invention is a minor allele, and the partial sequence or its complementary sequence provides a polynucleotide having a length of at least 12 nucleotides.
更なる局面において、本発明は、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>Aからなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドを提供する。
In a further aspect, the present invention provides a polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 T> C (SNP1), 73097 G. > A (SNP2), 120764 G> A (SNP3), 152917G> A (SNP4), 232102 G> A (SNP5), 55977AG> A (SNP6), 57712 A> G (SNP7) and 57483 G> A A polynucleotide comprising at least one SNP selected from the group, wherein the allele of the SNP contained in the subsequence is a minor allele, and the subsequence or its complementary sequence has a length of at least 12 nucleotides. provide.
配列番号5は、ミステリン遺伝子及びその周辺領域の遺伝子(FLJ3520、NPTX1、CARD14及びRaptor (KIAA1303))を含むヒト第17番染色体DNAの部分ヌクレオチド配列であり、NCBIに登録されているContig #NT010783.15の第43560001~43795000番目のヌクレオチドに相当する。
SEQ ID NO: 5 is a partial nucleotide sequence of human chromosome 17 DNA containing a mysterin gene and genes in its peripheral region (FLJ3520, NPTX1, CARD14, and Raptor (KIAA1303)). Contig # NT010783 registered in NCBI. It corresponds to the 15th nucleotides 43560001 to 43795000.
配列番号5で表されるヌクレオチド配列には、ヒト第17番染色体上に存在する、モヤモヤ病に関連する以下の新規なSNPsが含まれる:
T又はCである第4766位のSNP (本明細書中4766 T>C、又はSNP1と略記する)、
G又はAである第73097位のSNP (本明細書中73097 G>A、又はSNP2と略記する)、
G又はAである第120764位のSNP (本明細書中120764 G>A、又はSNP3と略記する)、
G又はAである第152917位のSNP (本明細書中152917 G>A、又はSNP4と略記する)、及び
G又はAである第232102位のSNP (本明細書中232102 G>A、又はSNP5と略記する)。 The nucleotide sequence represented by SEQ ID NO: 5 includes the following novel SNPs associated with moyamoya disease, which are present on human chromosome 17.
SNP at position 4766 which is T or C (abbreviated herein as 4766 T> C, or SNP1),
SNP at position 73097 which is G or A (in this specification, 73097 G> A, or SNP2),
SNP at 120764 which is G or A (120764 G> A in this specification, or SNP3),
SNP at position 152917 which is G or A (abbreviated as 152917 G> A or SNP4 herein) and SNP at position 232102 which is G or A (232102 G> A or SNP5 herein) Abbreviated).
T又はCである第4766位のSNP (本明細書中4766 T>C、又はSNP1と略記する)、
G又はAである第73097位のSNP (本明細書中73097 G>A、又はSNP2と略記する)、
G又はAである第120764位のSNP (本明細書中120764 G>A、又はSNP3と略記する)、
G又はAである第152917位のSNP (本明細書中152917 G>A、又はSNP4と略記する)、及び
G又はAである第232102位のSNP (本明細書中232102 G>A、又はSNP5と略記する)。 The nucleotide sequence represented by SEQ ID NO: 5 includes the following novel SNPs associated with moyamoya disease, which are present on human chromosome 17.
SNP at position 4766 which is T or C (abbreviated herein as 4766 T> C, or SNP1),
SNP at position 73097 which is G or A (in this specification, 73097 G> A, or SNP2),
SNP at 120764 which is G or A (120764 G> A in this specification, or SNP3),
SNP at position 152917 which is G or A (abbreviated as 152917 G> A or SNP4 herein) and SNP at position 232102 which is G or A (232102 G> A or SNP5 herein) Abbreviated).
更なる局面において、配列番号5で表されるヌクレオチド配列には、ヒト第17番染色体上に存在する、モヤモヤ病に関連する以下の新規なSNPも含まれる:
G又はAである第55977位のSNP (本明細書中55977 G>A、又はSNP6と略記する)、
A又はGである第55712位のSNP (本明細書中55712 A>G、又はSNP7と略記する)、及び
G又はAである第57483位のSNP (本明細書中57483 G>A、又はSNP8と略記する)。 In a further aspect, the nucleotide sequence represented by SEQ ID NO: 5 also includes the following novel SNPs associated with moyamoya disease that are present on human chromosome 17.
SNP at position 55977, which is G or A (abbreviated as 55977 G> A or SNP6 in this specification),
SNP at position 55712 which is A or G (abbreviated as 55712 A> G or SNP7 in this specification), and SNP at position 57483 which is G or A (57483G> A or SNP8 in this specification) Abbreviated).
G又はAである第55977位のSNP (本明細書中55977 G>A、又はSNP6と略記する)、
A又はGである第55712位のSNP (本明細書中55712 A>G、又はSNP7と略記する)、及び
G又はAである第57483位のSNP (本明細書中57483 G>A、又はSNP8と略記する)。 In a further aspect, the nucleotide sequence represented by SEQ ID NO: 5 also includes the following novel SNPs associated with moyamoya disease that are present on human chromosome 17.
SNP at position 55977, which is G or A (abbreviated as 55977 G> A or SNP6 in this specification),
SNP at position 55712 which is A or G (abbreviated as 55712 A> G or SNP7 in this specification), and SNP at position 57483 which is G or A (57483G> A or SNP8 in this specification) Abbreviated).
なお、本明細書において、SNPの位置は、配列番号5で表されるヌクレオチド配列におけるヌクレオチドの位置を基準に記載する。例えば、「第4766位のSNP」は、配列番号5で表されるヌクレオチド配列における第4766位のヌクレオチドにおけるSNPを意味する。「4766 T>C」等と記載する場合、「>」の記号の前にメジャーアレルの塩基(この場合T)を、後にマイナーアレルの塩基(この場合C)を記載することとする。
In this specification, the position of the SNP is described based on the position of the nucleotide in the nucleotide sequence represented by SEQ ID NO: 5. For example, “SNP at position 4766” means the SNP at the nucleotide at position 4766 in the nucleotide sequence represented by SEQ ID NO: 5. When “4766 T> C” or the like is described, the base of the major allele (T in this case) is described before the symbol “>”, and the base of the minor allele (C in this case) is described later.
また、本明細書においてヌクレオチド配列は、特にことわりのない限りDNAの配列として記載するが、ポリヌクレオチドがRNAである場合は、チミン(T)をウラシル(U)に適宜読み替えるものとする。
In this specification, the nucleotide sequence is described as a DNA sequence unless otherwise specified. However, when the polynucleotide is RNA, thymine (T) is appropriately replaced with uracil (U).
本発明のポリヌクレオチドには、上記5つのSNPsからなる群から選択される少なくとも1つ(1、2、3、4又は5つ、好ましくは1つ)のSNPが含まれる。即ち、好ましい態様において、本発明のポリヌクレオチドには、SNP1、SNP2、SNP3、SNP4又はSNP5が含まれる。
The polynucleotide of the present invention includes at least one (1, 2, 3, 4 or 5, preferably 1) SNP selected from the group consisting of the above five SNPs. That is, in a preferred embodiment, the polynucleotide of the present invention includes SNP1, SNP2, SNP3, SNP4 or SNP5.
更なる局面において、本発明のポリヌクレオチドには、上記8つのSNPsからなる群から選択される少なくとも1つ(1、2、3、4、5、6、7又は8つ、好ましくは1つ)のSNPが含まれる。即ち、好ましい態様において、本発明のポリヌクレオチドには、SNP1、SNP2、SNP3、SNP4、SNP5、SNP6、SNP7又はSNP8が含まれる。
In a further aspect, the polynucleotide of the present invention has at least one selected from the group consisting of the above 8 SNPs (1, 2, 3, 4, 5, 6, 7 or 8, preferably 1,). SNPs are included. That is, in a preferred embodiment, the polynucleotide of the present invention includes SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 or SNP8.
本発明のポリヌクレオチドに含まれるSNPのアレルはマイナーアレルである。即ち、本発明のポリヌクレオチドにSNP1が含まれる場合、該SNPは「C」であり;SNP2が含まれる場合、該SNPは「A」であり;SNP3が含まれる場合、該SNPは「A」であり;SNP4が含まれる場合、該SNPは「A」であり;SNP5が含まれる場合、該SNPは「A」である。
The SNP allele contained in the polynucleotide of the present invention is a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “C”; when SNP2 is included, the SNP is “A”; when SNP3 is included, the SNP is “A”. If SNP4 is included, the SNP is “A”; if SNP5 is included, the SNP is “A”.
更なる局面において、本発明のポリヌクレオチドにSNP6が含まれる場合、該SNPは「A」であり;SNP7が含まれる場合、該SNPは「G」であり;SNP8が含まれる場合、該SNPは「A」である。
In a further aspect, when the polynucleotide of the present invention includes SNP6, the SNP is “A”; when SNP7 is included, the SNP is “G”; when SNP8 is included, the SNP is “A”.
本発明のポリヌクレオチドは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、該核酸は二本鎖であっても、一本鎖であってもよいが、好ましくは一本鎖である。
The polynucleotide of the present invention may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
本発明のポリヌクレオチドが、配列番号5で表されるヌクレオチド配列の連続した部分配列の相補配列を含む場合、該相補配列が有する相補性は100%である。
When the polynucleotide of the present invention includes a complementary sequence of a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5, the complementarity of the complementary sequence is 100%.
本発明のポリヌクレオチドに含まれる、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列の長さは、12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上(例えば、25ヌクレオチド以上)である。また、該部分配列またはその相補配列の長さの上限は特に限定されないが、合成の容易さや、SNPの検出感度を高くする観点から、該長さは、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide of the present invention is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, Preferably it is 20 nucleotides or more (for example, 25 nucleotides or more). The upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
本発明のポリヌクレオチドの長さは、少なくとも12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上である。また、本発明のポリヌクレオチドの長さの上限は特に限定されないが、合成の容易さの観点から、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the polynucleotide of the present invention is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more. Further, the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
本発明のポリヌクレオチドは、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列に加えて、任意の付加的配列を含んでいてもよい。
The polynucleotide of the present invention may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
また、本発明のポリヌクレオチドは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C、32P、33P等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などで標識されていてもよい。
In addition, the polynucleotide of the present invention contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), etc. It may be labeled with.
本発明のポリヌクレオチドは、好ましくは単離又は精製されている。
The polynucleotide of the present invention is preferably isolated or purified.
本発明のポリヌクレオチドは、SNPs1~5を検出するための核酸プローブやプライマーとして使用することができるので、これらのSNPsが関連するモヤモヤ病の発症リスクの診断剤として有用である。また、SNP2は、虚血性心疾患や、一卵性双生児の分娩頻度と相関するため、本発明のポリヌクレオチドがSNP2を含む場合には、該ポリヌクレオチドは、虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性の診断剤として有用である(下記8~10の項を参照)。
Since the polynucleotide of the present invention can be used as a nucleic acid probe or primer for detecting SNPs 1 to 5, it is useful as a diagnostic agent for the onset risk of moyamoya disease associated with these SNPs. In addition, since SNP2 correlates with ischemic heart disease and the frequency of delivery of identical twins, when the polynucleotide of the present invention contains SNP2, the polynucleotide contains the risk of developing ischemic heart disease, It is useful as a diagnostic agent for the possibility of delivery of identical twins (see sections 8 to 10 below).
更なる局面において、本発明のポリヌクレオチドは、SNPs1~8を検出するための核酸プローブやプライマーとして使用することができるので、これらのSNPsが関連するモヤモヤ病の発症リスクの診断剤として有用である。
In a further aspect, the polynucleotide of the present invention can be used as a nucleic acid probe or primer for detecting SNPs 1 to 8, and thus is useful as a diagnostic agent for the onset risk of moyamoya disease associated with these SNPs. .
8.モヤモヤ病の発症リスクの判定方法
本発明は、配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法を提供する。 8). The present invention relates to 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A in the nucleotide sequence represented by SEQ ID NO: 5. (SNP4) and 232102 G> A (SNP5) A method for determining the risk of developing Moyamoya disease comprising detecting at least one SNP selected from the group consisting of G> A (SNP5) is provided.
本発明は、配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法を提供する。 8). The present invention relates to 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A in the nucleotide sequence represented by SEQ ID NO: 5. (SNP4) and 232102 G> A (SNP5) A method for determining the risk of developing Moyamoya disease comprising detecting at least one SNP selected from the group consisting of G> A (SNP5) is provided.
更なる局面において、本発明は、配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法を提供する。
In a further aspect, the present invention provides 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 152917 G> A (SNP4) in the nucleotide sequence represented by SEQ ID NO: 5. Detecting at least one SNP selected from the group consisting of 232102 G> A (SNP5), 55777 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8), A method for determining the risk of developing moyamoya disease is provided.
一実施形態では、本発明の判定方法は、以下の工程(a)、(b)を含む:
(a)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP1、SNP2、SNP3、SNP4及びSNP5からなる群から選択される少なくとも1つのSNPを検出する工程;
(b)検出したSNPのタイプに基づきモヤモヤ病の発症リスクを評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, and SNP5 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal;
(B) A step of evaluating the risk of developing Moyamoya disease based on the detected SNP type.
(a)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP1、SNP2、SNP3、SNP4及びSNP5からなる群から選択される少なくとも1つのSNPを検出する工程;
(b)検出したSNPのタイプに基づきモヤモヤ病の発症リスクを評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, and SNP5 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal;
(B) A step of evaluating the risk of developing Moyamoya disease based on the detected SNP type.
更なる局面において、本発明の判定方法は、以下の工程(a’)、(b’)を含む:
(a’)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP1、SNP2、SNP3、SNP4、SNP5、SNP6、SNP7及びSNP8からなる群から選択される少なくとも1つのSNPを検出する工程;
(b’)検出したSNPのタイプに基づきモヤモヤ病の発症リスクを評価する工程。 In a further aspect, the determination method of the present invention includes the following steps (a ′) and (b ′):
(A ′) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal The step of:
(B ') The process of evaluating the onset risk of moyamoya disease based on the detected SNP type.
(a’)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP1、SNP2、SNP3、SNP4、SNP5、SNP6、SNP7及びSNP8からなる群から選択される少なくとも1つのSNPを検出する工程;
(b’)検出したSNPのタイプに基づきモヤモヤ病の発症リスクを評価する工程。 In a further aspect, the determination method of the present invention includes the following steps (a ′) and (b ′):
(A ′) detecting at least one SNP selected from the group consisting of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 in the nucleotide sequence represented by SEQ ID NO: 5 from a biological sample collected from an animal The step of:
(B ') The process of evaluating the onset risk of moyamoya disease based on the detected SNP type.
上記方法の工程(a)及び(a’)では、動物(被検者)から採取された生体試料においてSNP1~5(又はSNP1~8)からなる群から選択される少なくとも1つのSNPを検出し、そのSNPのタイプを測定する。動物としては、上述の5.の項で列挙した哺乳動物が好ましく、ヒトが特に好ましい。
In steps (a) and (a ′) of the above method, at least one SNP selected from the group consisting of SNP1-5 (or SNP1-8) is detected in a biological sample collected from an animal (subject). Measure the type of the SNP. As an animal, the above-mentioned 5. Mammals listed in the section are preferred, and humans are particularly preferred.
ヒトの人種は、特に限定されないが、好ましくは東アジア人(イーストアジアン/モンゴロイド)である。
The human race is not particularly limited, but is preferably East Asian (East Asian / Mongoloid).
更なる局面において、ヒトの人種は、特に限定されないが、好ましくはコーカソイド(白人)である。
In a further aspect, the human race is not particularly limited, but is preferably Caucasian (white).
SNP1~5からなる群から選択される少なくとも1つのSNPを検出する場合、ヒトの人種は好ましくは東アジア人(イーストアジアン/モンゴロイド)である。SNP6、7又は8を検出する場合、ヒトの人種は、好ましくはコーカソイド(白人)である。
When detecting at least one SNP selected from the group consisting of SNPs 1 to 5, the human race is preferably East Asian (East Asian / Mongoloid). When detecting SNP6, 7 or 8, the human race is preferably Caucasian (white).
ここで、人種(race)は、ホモ・サピエンス種の中の、特定のサブグループとして区別可能な集団である。人種は、特有で、区別可能な、遺伝子の組合せを有し、その遺伝子の組合せによって作られる特徴(精神的、肉体的とも)によって同定される。同じ人種のメンバーは、共通の遺伝的祖先を共有し、その結果、類似の遺伝子組合せを共有するため、はっきり区別できる遺伝的特徴を共有している。
Here, the race is a group that can be distinguished as a specific subgroup in the Homo sapiens species. Race has a unique and distinguishable combination of genes and is identified by the characteristics (both mental and physical) created by the combination of genes. Members of the same race share a distinct genetic feature because they share a common genetic ancestry and, as a result, share similar gene combinations.
例えば、世界の主要な人類集団について、23種類の遺伝子の情報に基づき、遺伝的近縁関係が調べられ、アフリカン(ネグロイド)、コーカソイド(白人)、オセアニアン(オーストラロイド)、イーストアジアン(モンゴロイド)及びネイティブアメリカンの5種に分類されている。
For example, genetic relationships among major human populations around the world are examined based on information on 23 genes, African (Negroid), Caucasian (Caucasian), Oceanian (Australoid), East Asian (Mongoloid) And 5 types of Native American.
東アジア人とは、日本、朝鮮、中国、台湾及びモンゴルの人々のいずれかを起源に持つ人という意味である。東アジア人は、好ましくは、日本人、朝鮮人、又は中国人である。
“East Asian” means a person who originated in Japan, Korea, China, Taiwan or Mongolia. The East Asian is preferably a Japanese, a Korean, or a Chinese.
当業者であれば、個人の身体的特徴、出身国、先祖の起源に関する情報等に基づいてその個人の人種を容易に特定することが可能である。
A person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
上記方法の工程(a)及び(a’)において用いられる生体試料としては、ゲノムDNAを採取可能な任意の組織、細胞、体液等を使用することができるが、入手の容易性及び低侵襲性等の観点から、毛髪、爪、皮膚、粘膜、血液、血漿、血清、唾液などが好ましく用いられる。
As the biological sample used in steps (a) and (a ′) of the above method, any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used. In view of the above, hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
SNPの検出方法は、当該技術分野において周知である。例えば、RFLP(制限酵素切断断片長多型)法、PCR-SSCP(一本鎖DNA高次構造多型解析)法、ASO(Allele Specific Oligonucleotide)ハイブリダイゼーション法、シークエンス法、ARMS(Amplification Refracting Mutation System)法、変性濃度勾配ゲル電気泳動(Denaturing Gradient Gel Electrophoresis)法、RNAseA切断法、DOL(Dye-labeled Oligonucleotide Ligation)法、TaqMan PCR法、primer extension法、インベーダー法などが使用できる。
The SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
後述の実施例に示すように、上述の新規SNP1~5について、モヤモヤ病との関連解析を行った結果、モヤモヤ病の患者においては、SNP1~5のマイナーアレルの頻度が、健常者と比較して有意に高かった。特に、実施例1及び2において試験を行った全ての家族性のモヤモヤ病患者は、SNP2のマイナーアレルを保有していたことから、SNP2のマイナーアレルが、モヤモヤ病の創始者変異である可能性がある。また、SNP2の変異は、ミステリンのアミノ酸置換を伴うので、このSNP2のマイナーアレルにより生じるミステリンのアミノ酸置換が、モヤモヤ病の発症に影響する可能性がある。従って、本発明の1つの好ましい態様において、SNP2が検出される。
As shown in the examples described later, as a result of the analysis of association with Moyamoya disease for the above novel SNPs 1 to 5, the frequency of minor alleles of SNPs 1 to 5 in patients with Moyamoya disease was compared with that of healthy subjects. Was significantly higher. In particular, since all familial moyamoya disease patients tested in Examples 1 and 2 possessed a minor allele of SNP2, there is a possibility that the minor allele of SNP2 is a founder mutation of moyamoya disease. There is. Moreover, since the mutation of SNP2 is accompanied by amino acid substitution of mysterin, the amino acid substitution of mysterin caused by this minor allele of SNP2 may affect the onset of moyamoya disease. Thus, in one preferred embodiment of the invention, SNP2 is detected.
これらの結果に基づけば、
SNP1におけるシトシン、
SNP2におけるアデニン、
SNP3におけるアデニン、
SNP4におけるアデニン、及び
SNP5におけるアデニン
からなる群から選択される少なくとも1つ(好ましくは2つ、更に好ましくは3つ、よりこのましくは4つ、最も好ましくは5つ)のマイナーアレルが、当該SNPにおける少なくとも一方のアレルにおいて検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。特に、SNP2のマイナーアレル(アデニン)が検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、極めて高いと判定することが出来る。 Based on these results,
Cytosine in SNP1,
Adenine in SNP2,
Adenine in SNP3,
At least one (preferably 2, more preferably 3, more preferably 4, and most preferably 5) minor alleles selected from the group consisting of adenine in SNP4 and adenine in SNP5, When it is detected in at least one allele in the SNP, it can be determined that the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected. In particular, when a minor allele (adenine) of SNP2 is detected, it can be determined that the risk of developing moyamoya disease is extremely high compared to the case where the minor allele is not detected.
SNP1におけるシトシン、
SNP2におけるアデニン、
SNP3におけるアデニン、
SNP4におけるアデニン、及び
SNP5におけるアデニン
からなる群から選択される少なくとも1つ(好ましくは2つ、更に好ましくは3つ、よりこのましくは4つ、最も好ましくは5つ)のマイナーアレルが、当該SNPにおける少なくとも一方のアレルにおいて検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。特に、SNP2のマイナーアレル(アデニン)が検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、極めて高いと判定することが出来る。 Based on these results,
Cytosine in SNP1,
Adenine in SNP2,
Adenine in SNP3,
At least one (preferably 2, more preferably 3, more preferably 4, and most preferably 5) minor alleles selected from the group consisting of adenine in SNP4 and adenine in SNP5, When it is detected in at least one allele in the SNP, it can be determined that the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected. In particular, when a minor allele (adenine) of SNP2 is detected, it can be determined that the risk of developing moyamoya disease is extremely high compared to the case where the minor allele is not detected.
更なる局面において、後述の実施例に示すように、あるモヤモヤ病の家系についてミステリン遺伝子の変異の有無を解析したところ、モヤモヤ病を発症した全ての患者がSNP6のマイナーアレルを保有していた。SNP6の変異は、ミステリンのアミノ酸置換を伴うので、このSNP6のマイナーアレルにより生じるミステリンのアミノ酸置換が、モヤモヤ病の発症に影響する可能性がある。従って、本発明の1つの好ましい態様において、SNP6が検出される。また、孤発例のモヤモヤ病患者についてミステリン遺伝子の変異の有無を解析したところ、SNP7又はSNP8のマイナーアレルを保有している患者を見出した。SNP7又は8の変異は、ミステリンのアミノ酸置換を伴うので、このSNP7又は8のマイナーアレルにより生じるミステリンのアミノ酸置換が、モヤモヤ病の発症に影響する可能性がある。従って、本発明の1つの好ましい態様において、SNP7又は8が検出される。
In a further aspect, as shown in the examples described later, when the presence or absence of mutations in the mysterin gene was analyzed for a family with a certain Moyamoya disease, all patients who developed Moyamoya disease had a minor allele of SNP6. Since the mutation of SNP6 involves an amino acid substitution of mysterin, the amino acid substitution of mysterin caused by this minor allele of SNP6 may affect the onset of moyamoya disease. Thus, in one preferred embodiment of the invention, SNP6 is detected. Moreover, when the presence or absence of the mutation of the mysterin gene was analyzed about the sporadic case of the moyamoya disease patient, the patient who has the minor allele of SNP7 or SNP8 was discovered. Since mutation of SNP7 or 8 is accompanied by amino acid substitution of mysterin, the amino acid substitution of mysterin caused by this minor allele of SNP7 or 8 may affect the onset of moyamoya disease. Thus, in one preferred embodiment of the invention, SNP 7 or 8 is detected.
これらの結果に基づけば、
SNP1におけるシトシン、
SNP2におけるアデニン、
SNP3におけるアデニン、
SNP4におけるアデニン、
SNP5におけるアデニン、
SNP6におけるアデニン、
SNP7におけるグアニン、及び
SNP8におけるアデニン
からなる群から選択される少なくとも1つ(好ましくは2つ、更に好ましくは3つ、よりこのましくは4つ、さらにより好ましくは5つ、6つ、7つ、最も好ましくは8つ)のマイナーアレルが、当該SNPにおける少なくとも一方のアレルにおいて検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。特に、SNP2又はSNP6のマイナーアレル(SNP2のアデニン/SNP6のアデニン)が検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、極めて高いと判定することが出来る。 Based on these results,
Cytosine in SNP1,
Adenine in SNP2,
Adenine in SNP3,
Adenine in SNP4,
Adenine in SNP5,
Adenine in SNP6,
At least one selected from the group consisting of guanine in SNP7 and adenine in SNP8 (preferably 2, more preferably 3, more preferably 4, even more preferably 5, 6, 7 , Most preferably 8) minor alleles are detected in at least one allele in the SNP, the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected Can be determined. In particular, when a minor allele of SNP2 or SNP6 (SNP2 adenine / SNP6 adenine) is detected, the risk of developing moyamoya disease is determined to be extremely high compared to the case where the minor allele is not detected. I can do it.
SNP1におけるシトシン、
SNP2におけるアデニン、
SNP3におけるアデニン、
SNP4におけるアデニン、
SNP5におけるアデニン、
SNP6におけるアデニン、
SNP7におけるグアニン、及び
SNP8におけるアデニン
からなる群から選択される少なくとも1つ(好ましくは2つ、更に好ましくは3つ、よりこのましくは4つ、さらにより好ましくは5つ、6つ、7つ、最も好ましくは8つ)のマイナーアレルが、当該SNPにおける少なくとも一方のアレルにおいて検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。特に、SNP2又はSNP6のマイナーアレル(SNP2のアデニン/SNP6のアデニン)が検出された場合には、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、極めて高いと判定することが出来る。 Based on these results,
Cytosine in SNP1,
Adenine in SNP2,
Adenine in SNP3,
Adenine in SNP4,
Adenine in SNP5,
Adenine in SNP6,
At least one selected from the group consisting of guanine in SNP7 and adenine in SNP8 (preferably 2, more preferably 3, more preferably 4, even more preferably 5, 6, 7 , Most preferably 8) minor alleles are detected in at least one allele in the SNP, the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected Can be determined. In particular, when a minor allele of SNP2 or SNP6 (SNP2 adenine / SNP6 adenine) is detected, the risk of developing moyamoya disease is determined to be extremely high compared to the case where the minor allele is not detected. I can do it.
DNAは通常、互いに相補的な二本の鎖からなる二重らせん構造を有している。従って、本明細書において、便宜的に一方の鎖におけるDNA配列を示した場合であっても、当然の如く、該配列(塩基)に相補的な配列(100%の相補性)も開示したものと解釈される。当業者にとって、一方のDNA配列(塩基)が判れば、該配列(塩基)に相補的な配列(塩基)は自明である。従って、SNP1を例とすると、配列番号:5に記載のヌクレオチド配列の4766位の塩基に対応する相補鎖上の塩基がG(グアニン)である場合も、被検者はモヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。
DNA usually has a double helix structure consisting of two strands complementary to each other. Therefore, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, taking SNP1 as an example, even when the base on the complementary strand corresponding to the base at position 4766 of the nucleotide sequence shown in SEQ ID NO: 5 is G (guanine), the subject is at risk of developing Moyamoya disease. , Can be determined to be relatively high.
尚、当業者は、本明細書に開示されたヌクレオチド配列(例えば配列番号5で表されるヌクレオチド配列)およびSNPに関する情報を用いれば、配列表に開示されたヌクレオチド配列(配列番号5で表されたヌクレオチド配列またはその連続した部分配列、あるいはそれらの相補配列)と被検者の第17番染色体DNA上の対応部位のヌクレオチド配列との間に若干の相違(欠失、置換、付加、繰り返し配列の個数の増減等)が認められた場合であっても、配列番号5で表されたヌクレオチド配列と被検者由来のヌクレオチド配列とのアライメントを行うことにより、被検者由来のヌクレオチド配列においてどの塩基がSNP1~5に相当するか正確に特定し、当該SNPを検出することが可能である。このような場合も「配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを検出すること」に包含される。
It should be noted that those skilled in the art can use the nucleotide sequence disclosed in the present specification (for example, the nucleotide sequence represented by SEQ ID NO: 5) and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5). Slight difference (deletion, substitution, addition, repeat sequence) between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof) In the nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject. It is possible to accurately identify whether the base corresponds to SNP 1 to 5 and detect the SNP. Also in such a case, “4766 T> C (SNP1), 73097 G> A (SNP2), 12064 G> A (SNP3), 152917 G> A (SNP4) and 232102 G in the nucleotide sequence represented by SEQ ID NO: 5 > Detecting at least one SNP selected from the group consisting of> A (SNP5).
更なる局面において、当業者は、本明細書に開示されたヌクレオチド配列(例えば配列番号5で表されるヌクレオチド配列)およびSNPに関する情報を用いれば、配列表に開示されたヌクレオチド配列(配列番号5で表されたヌクレオチド配列またはその連続した部分配列、あるいはそれらの相補配列)と被検者の第17番染色体DNA上の対応部位のヌクレオチド配列との間に若干の相違(欠失、置換、付加、繰り返し配列の個数の増減等)が認められた場合であっても、配列番号5で表されたヌクレオチド配列と被検者由来のヌクレオチド配列とのアライメントを行うことにより、被検者由来のヌクレオチド配列においてどの塩基がSNP1~8に相当するか正確に特定し、当該SNPを検出することが可能である。このような場合も「配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを検出すること」に包含される。
In a further aspect, those skilled in the art can use the nucleotide sequence disclosed in the present specification (for example, the nucleotide sequence represented by SEQ ID NO: 5) and the information on the SNP to use the nucleotide sequence disclosed in the sequence listing (SEQ ID NO: 5 A slight difference (deletion, substitution, addition) between the nucleotide sequence represented by (1) or a contiguous partial sequence thereof or a complementary sequence thereof and the nucleotide sequence of the corresponding site on the subject chromosome 17 DNA , Increase or decrease in the number of repetitive sequences, etc.), the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence derived from the subject are aligned to obtain the nucleotide derived from the subject. It is possible to accurately identify which base corresponds to SNP 1 to 8 in the sequence, and to detect the SNP. In such a case, “4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 152917 G> A (SNP4), 232102 G in the nucleotide sequence represented by SEQ ID NO: 5 > A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8).
本発明の方法においては、SNP1におけるシトシン、SNP2におけるアデニン、SNP3におけるアデニン、SNP4におけるアデニン、及びSNP5におけるアデニンからなる群から選択される少なくとも1つのマイナーアレルが、少なくとも一方のアレルにおいて検出されれば、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。即ち、本発明の方法においては、被検者の第17番染色体の少なくとも一方について上記SNPを検出すればよい。
In the method of the present invention, if at least one minor allele selected from the group consisting of cytosine in SNP1, adenine in SNP2, adenine in SNP3, adenine in SNP4, and adenine in SNP5 is detected in at least one allele. As compared with the case where the minor allele is not detected, it can be determined that the risk of developing moyamoya disease is relatively high. That is, in the method of the present invention, the SNP may be detected for at least one of the chromosome 17 of the subject.
更なる局面において、本発明の方法においては、SNP1におけるシトシン、SNP2におけるアデニン、SNP3におけるアデニン、SNP4におけるアデニン、SNP5におけるアデニン、SNP6におけるアデニン、SNP7におけるグアニン、及びSNP8におけるアデニンからなる群から選択される少なくとも1つのマイナーアレルが、少なくとも一方のアレルにおいて検出されれば、該マイナーアレルが検出されない場合と比較して、モヤモヤ病の発症リスクが、相対的に高いと判定することが出来る。即ち、本発明の方法においては、被検者の第17番染色体の少なくとも一方について上記SNPを検出すればよい。
In a further aspect, the method of the present invention is selected from the group consisting of cytosine in SNP1, adenine in SNP2, adenine in SNP3, adenine in SNP4, adenine in SNP5, adenine in SNP6, guanine in SNP7, and adenine in SNP8. If at least one minor allele is detected in at least one allele, it can be determined that the risk of developing Moyamoya disease is relatively high compared to the case where the minor allele is not detected. That is, in the method of the present invention, the SNP may be detected for at least one of the chromosome 17 of the subject.
尚、上述のように、SNP2の変異は、ヒトミステリンの第4810番のアミノ酸置換(アルギニン→リジン)を伴うので、対象者から、ミステリンポリペプチドを単離し、その第4810番のアミノ酸を同定することによっても、モヤモヤ病の発症リスクを判定することが出来る。ミステリンポリペプチドの単離は、抗体カラムクロマトグラフィー等の生化学分野において周知の方法を用いることにより実施することが出来る。また、第4810番のアミノ酸の同定は、ペプチドシークエンサーや、マススペクトル等の生化学分野において周知の方法を用いることにより実施することが出来る。SNP2のメジャーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてアルギニンを与え、SNP2のマイナーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてリジンを与える。従って、第4810番のアミノ酸を同定した結果、リジンが検出された場合は、リジンが検出されない場合(即ちアルギニンのみが検出された場合)と比較して、モヤモヤ病の発症リスクが相対的に高いと判定することが出来る。
As described above, since the mutation of SNP2 involves the amino acid substitution of human mysterin at position 4810 (arginine → lysine), the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was The risk of developing moyamoya disease can also be determined by identification. The mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography. The amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum. The major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identifying the amino acid No. 4810, when lysine is detected, the risk of developing Moyamoya disease is relatively higher than when lysine is not detected (that is, when only arginine is detected). Can be determined.
更なる局面において、SNP6、7及び8の変異は、それぞれヒトミステリンの第4013番のアミノ酸置換(アスパラギン酸→アスパラギン)、第3962番のアミノ酸置換(アスパラギン→アスパラギン酸)及び第4062番のアミノ酸置換(アルギニン→グルタミン)を伴うので、対象者から、ミステリンポリペプチドを単離し、それぞれ、その第4013番、第3962番及び4062番のアミノ酸を同定することによっても、モヤモヤ病の発症リスクを判定することが出来る。ミステリンポリペプチドの単離は、抗体カラムクロマトグラフィー等の生化学分野において周知の方法を用いることにより実施することが出来る。また、第4013番、第3962番及び4062番のアミノ酸の同定は、ペプチドシークエンサーや、マススペクトル等の生化学分野において周知の方法を用いることにより実施することが出来る。SNP6のメジャーアレルはヒトミステリンポリペプチドの第4013番アミノ酸としてアスパラギン酸を与え、SNP6のマイナーアレルはヒトミステリンポリペプチドの第4013番アミノ酸としてアスパラギンを与える。従って、第4013番のアミノ酸を同定した結果、アスパラギンが検出された場合は、アスパラギンが検出されない場合(即ちアスパラギン酸のみが検出された場合)と比較して、モヤモヤ病の発症リスクが相対的に高いと判定することが出来る。SNP7のメジャーアレルはヒトミステリンポリペプチドの第3962番アミノ酸としてアスパラギンを与え、SNP7のマイナーアレルはヒトミステリンポリペプチドの第3962番アミノ酸としてアスパラギン酸を与える。従って、第3962番のアミノ酸を同定した結果、アスパラギン酸が検出された場合は、アスパラギン酸が検出されない場合(即ちアスパラギンのみが検出された場合)と比較して、モヤモヤ病の発症リスクが相対的に高いと判定することが出来る。SNP8のメジャーアレルはヒトミステリンポリペプチドの第4062番アミノ酸としてアルギニンを与え、SNP8のマイナーアレルはヒトミステリンポリペプチドの第4062番アミノ酸としてグルタミンを与える。従って、第4062番のアミノ酸を同定した結果、グルタミンが検出された場合は、グルタミンが検出されない場合(即ちアルギニンのみが検出された場合)と比較して、モヤモヤ病の発症リスクが相対的に高いと判定することが出来る。
In a further aspect, the mutations in SNPs 6, 7 and 8 are amino acid substitution No. 4013 (aspartic acid → asparagine), amino acid substitution No. 3962 (aspartic acid → aspartic acid) and amino acid No. 4062 of human mysterin, respectively. Since the substitution (arginine → glutamine) is involved, the mysterin polypeptide is isolated from the subject, and the amino acids No. 4013, No. 3962 and No. 4062 are identified, respectively. It can be judged. The mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography. Identification of amino acids Nos. 4013, 3962, and 4062 can be performed by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum. The major allele of SNP6 gives aspartic acid as amino acid 4013 of human mysterin polypeptide, and the minor allele of SNP6 gives asparagine as amino acid 4013 of human mysterin polypeptide. Accordingly, as a result of identifying amino acid No. 4013, when asparagine is detected, the risk of developing moyamoya disease is relatively higher than when asparagine is not detected (that is, when only aspartic acid is detected). It can be determined that the price is high. The major allele of SNP7 gives asparagine as amino acid 3962 of human mysterin polypeptide, and the minor allele of SNP7 gives aspartic acid as amino acid 3962 of human mysterin polypeptide. Accordingly, as a result of identifying the amino acid No. 3962, when aspartic acid is detected, the risk of developing moyamoya disease is relative to when aspartic acid is not detected (that is, when only asparagine is detected). It can be determined to be high. The major allele of SNP8 gives arginine as amino acid 4062 of human mysterin polypeptide, and the minor allele of SNP8 gives glutamine as amino acid 4062 of human mysterin polypeptide. Therefore, as a result of identifying amino acid No. 4062, when glutamine is detected, the risk of developing Moyamoya disease is relatively higher than when glutamine is not detected (that is, when only arginine is detected). Can be determined.
本発明はまた、SNP1~5からなる群から選択される少なくとも1つのSNPを検出するための試薬を含む、モヤモヤ病の発症リスクの診断剤を提供する。
The present invention also provides a diagnostic agent for the onset risk of moyamoya disease, comprising a reagent for detecting at least one SNP selected from the group consisting of SNPs 1 to 5.
SNP1~5からなる群から選択される少なくとも1つのSNPを検出するための試薬としては、
(1)配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNP(好ましくはSNP2)を特異的に検出し得る核酸プローブ、又は
(2)該SNPを含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting at least one SNP selected from the group consisting ofSNPs 1 to 5,
(1) 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) A nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of SNP5) (preferably SNP2), or (2) a primer capable of specifically amplifying a region containing the SNP. I can do it.
(1)配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNP(好ましくはSNP2)を特異的に検出し得る核酸プローブ、又は
(2)該SNPを含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting at least one SNP selected from the group consisting of
(1) 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (in the nucleotide sequence represented by SEQ ID NO: 5) A nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of SNP5) (preferably SNP2), or (2) a primer capable of specifically amplifying a region containing the SNP. I can do it.
更なる局面において、本発明は、SNP1~8からなる群から選択される少なくとも1つのSNPを検出するための試薬を含む、モヤモヤ病の発症リスクの診断剤を提供する。
In a further aspect, the present invention provides a diagnostic agent for the risk of developing Moyamoya disease, comprising a reagent for detecting at least one SNP selected from the group consisting of SNPs 1-8.
SNP1~8からなる群から選択される少なくとも1つのSNPを検出するための試薬としては、
(1’)配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNP(好ましくはSNP2又はSNP6)を特異的に検出し得る核酸プローブ、又は
(2’)該SNPを含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting at least one SNP selected from the group consisting ofSNPs 1 to 8,
(1 ′) 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A in the nucleotide sequence represented by SEQ ID NO: 5 (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) are specifically detected at least one SNP (preferably SNP2 or SNP6) selected from the group consisting of And (2 ′) a primer that can specifically amplify the region containing the SNP.
(1’)配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNP(好ましくはSNP2又はSNP6)を特異的に検出し得る核酸プローブ、又は
(2’)該SNPを含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting at least one SNP selected from the group consisting of
(1 ′) 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A in the nucleotide sequence represented by SEQ ID NO: 5 (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8) are specifically detected at least one SNP (preferably SNP2 or SNP6) selected from the group consisting of And (2 ′) a primer that can specifically amplify the region containing the SNP.
本明細書において、「核酸プローブによるSNPの特異的な検出」とは、核酸プローブが配列番号5で表されるヌクレオチド配列からなるDNAの特定のSNPを含む領域にハイブリダイズし、ヒトゲノムDNA中の他の領域にハイブリダイズしないことを意味する。このようなハイブリダイゼーションの条件は、当業者であれば適宜選択することができる。ハイブリダイゼーションの条件として、例えば低ストリンジェントな条件が挙げられる。低ストリンジェントな条件とは、ハイブリダイゼーション後の洗浄において、例えば42℃、5×SSC、0.1%SDSの条件であり、好ましくは50℃、2×SSC、0.1%SDSの条件である。より好ましいハイブリダイゼーションの条件としては、高ストリンジェントな条件が挙げられる。高ストリンジェントな条件とは、例えば65℃、0.1×SSC、0.1%SDSである。但し、ハイブリダイゼーションのストリンジェンシーに影響する要素としては、温度や塩濃度等の複数の要素があり、当業者はこれらの要素を適宜選択することで、同様のストリンジェンシーを実現することが可能である。
In the present specification, “specific detection of SNP with a nucleic acid probe” means that a nucleic acid probe hybridizes to a region containing a specific SNP of DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5, This means that it does not hybridize to other regions. Such hybridization conditions can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. The low stringent conditions are, for example, conditions of 42 ° C., 5 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS in washing after hybridization. is there. More preferable hybridization conditions include highly stringent conditions. High stringent conditions are, for example, 65 ° C., 0.1 × SSC, and 0.1% SDS. However, factors affecting the stringency of hybridization include multiple factors such as temperature and salt concentration, and those skilled in the art can realize the same stringency by selecting these factors as appropriate. is there.
(1)又は(1’)の核酸プローブは、好ましくは、当該核酸プローブが標的とするSNPがメジャーアレルである配列番号5で表されるポリヌクレオチドへの親和性と、当該SNPがマイナーアレルである配列番号5で表されるポリヌクレオチドへの親和性との間に差を有しており、適切な条件下(例えば高ストリンジェントな条件)で、メジャーアレル及びマイナーアレルの一方へハイブリダイズするが、他方へはハイブリダイズしない。
The nucleic acid probe of (1) or (1 ′) is preferably an affinity for the polynucleotide represented by SEQ ID NO: 5 wherein the SNP targeted by the nucleic acid probe is a major allele, and the SNP is a minor allele. There is a difference between the affinity for the polynucleotide represented by SEQ ID NO: 5 and it hybridizes to one of the major allele and minor allele under appropriate conditions (for example, highly stringent conditions). However, it does not hybridize to the other.
(1)のプローブとしては、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはメジャーアレル又はマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドが例示される。
The probe of (1) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 4766 T> C (SNP1), 73097 G> A (SNP2), 120764 G> A (SNP3), 152917 G> A (SNP4) and 232102 G> A (SNP5), wherein the SNP included in the partial sequence comprises at least one SNP selected from the group consisting of Examples of the polynucleotide include a major allele or a minor allele, and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
該ポリヌクレオチドには、上記5つのSNPsからなる群から選択される少なくとも1つ(1、2、3、4又は5つ、好ましくは1つ)のSNPが含まれる。即ち、好ましい態様において、該ポリヌクレオチドには、SNP1、SNP2、SNP3、SNP4又はSNP5が含まれる。SNP1を検出する場合には、該ポリヌクレオチドにはSNP1が含まれ、SNP2を検出する場合には、該ポリヌクレオチドにはSNP2が含まれ、SNP3を検出する場合には、該ポリヌクレオチドにはSNP3が含まれ、SNP4を検出する場合には、該ポリヌクレオチドにはSNP4が含まれ、SNP5を検出する場合には、該ポリヌクレオチドにはSNP5が含まれる。
The polynucleotide includes at least one (1, 2, 3, 4 or 5, preferably 1) SNP selected from the group consisting of the above five SNPs. That is, in a preferred embodiment, the polynucleotide includes SNP1, SNP2, SNP3, SNP4 or SNP5. When detecting SNP1, the polynucleotide includes SNP1, when detecting SNP2, the polynucleotide includes SNP2, and when detecting SNP3, the polynucleotide includes SNP3. When SNP4 is detected, the polynucleotide includes SNP4, and when SNP5 is detected, the polynucleotide includes SNP5.
該ポリヌクレオチドに含まれるSNPのアレルはメジャーアレル又はマイナーアレルである。即ち、本発明のポリヌクレオチドにSNP1が含まれる場合、該SNPは「T」又は「C」であり;SNP2が含まれる場合、該SNPは「G」又は「A」であり;SNP3が含まれる場合、該SNPは「G」又は「A」であり;SNP4が含まれる場合、該SNPは「G」又は「A」であり;SNP5が含まれる場合、該SNPは「G」又は「A」である。
The SNP allele contained in the polynucleotide is a major allele or a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “T” or “C”; when SNP2 is included, the SNP is “G” or “A”; and SNP3 is included. The SNP is “G” or “A”; when SNP4 is included, the SNP is “G” or “A”; when SNP5 is included, the SNP is “G” or “A”. It is.
更なる局面において、(1’)のプローブとしては、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはメジャーアレル又はマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドが例示される。
In a further aspect, the probe of (1 ′) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, and the partial sequence is 476647T> C ( SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 15292929G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) And at least one SNP selected from the group consisting of 57483 G> A (SNP8), the allele of the SNP included in the partial sequence is a major allele or a minor allele, and the partial sequence or its complementary sequence is Exemplified are polynucleotides having a length of at least 12 nucleotides.
該ポリヌクレオチドには、上記6つのSNPsからなる群から選択される少なくとも1つ(1、2、3、4、5、6、7又は8つ、好ましくは1つ)のSNPが含まれる。即ち、好ましい態様において、該ポリヌクレオチドには、SNP1、SNP2、SNP3、SNP4、SNP5、SNP6、SNP7又はSNP8が含まれる。SNP1を検出する場合には、該ポリヌクレオチドにはSNP1が含まれ、SNP2を検出する場合には、該ポリヌクレオチドにはSNP2が含まれ、SNP3を検出する場合には、該ポリヌクレオチドにはSNP3が含まれ、SNP4を検出する場合には、該ポリヌクレオチドにはSNP4が含まれ、SNP5を検出する場合には、該ポリヌクレオチドにはSNP5が含まれ、SNP6を検出する場合には、該ポリヌクレオチドにはSNP6が含まれ、SNP7を検出する場合には、該ポリヌクレオチドにはSNP7が含まれ、SNP8を検出する場合には、該ポリヌクレオチドにはSNP8が含まれる。
The polynucleotide includes at least one (1, 2, 3, 4, 5, 6, 7 or 8, preferably 1) SNP selected from the group consisting of the above 6 SNPs. That is, in a preferred embodiment, the polynucleotide includes SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 or SNP8. When detecting SNP1, the polynucleotide includes SNP1, when detecting SNP2, the polynucleotide includes SNP2, and when detecting SNP3, the polynucleotide includes SNP3. When SNP4 is detected, the polynucleotide includes SNP4. When detecting SNP5, the polynucleotide includes SNP5, and when detecting SNP6, the polynucleotide includes SNP4. The nucleotide includes SNP6. When SNP7 is detected, the polynucleotide includes SNP7. When SNP8 is detected, the polynucleotide includes SNP8.
該ポリヌクレオチドに含まれるSNPのアレルはメジャーアレル又はマイナーアレルである。即ち、本発明のポリヌクレオチドにSNP1が含まれる場合、該SNPは「T」又は「C」であり;SNP2が含まれる場合、該SNPは「G」又は「A」であり;SNP3が含まれる場合、該SNPは「G」又は「A」であり;SNP4が含まれる場合、該SNPは「G」又は「A」であり;SNP5が含まれる場合、該SNPは「G」又は「A」であり;SNP6が含まれる場合、該SNPは「G」又は「A」であり;SNP7が含まれる場合、該SNPは「A」又は「G」であり;SNP8が含まれる場合、該SNPは「G」又は「A」である。
The SNP allele contained in the polynucleotide is a major allele or a minor allele. That is, when SNP1 is included in the polynucleotide of the present invention, the SNP is “T” or “C”; when SNP2 is included, the SNP is “G” or “A”; and SNP3 is included. The SNP is “G” or “A”; when SNP4 is included, the SNP is “G” or “A”; when SNP5 is included, the SNP is “G” or “A”. When SNP6 is included, the SNP is “G” or “A”; when SNP7 is included, the SNP is “A” or “G”; when SNP8 is included, the SNP is “G” or “A”.
上記(1)又は(1’)の核酸プローブであるポリヌクレオチドは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、該核酸は二本鎖であっても、一本鎖であってもよいが、好ましくは一本鎖である。
The polynucleotide that is the nucleic acid probe of (1) or (1 ') may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
該ポリヌクレオチドが、配列番号5で表されるヌクレオチド配列の連続した部分配列の相補配列を含む場合、該相補配列が有する相補性は、好ましくは、100%である。
When the polynucleotide includes a complementary sequence of a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5, the complementarity of the complementary sequence is preferably 100%.
該ポリヌクレオチドに含まれる、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列の長さは、12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上(例えば、25ヌクレオチド以上)である。また、該部分配列またはその相補配列の長さの上限は特に限定されないが、合成の容易さや、SNPの検出感度を高くする観点から、該長さは、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, still more preferably 20 nucleotides or more (for example, 25 nucleotides or more). The upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
該ポリヌクレオチドの長さは、少なくとも12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上である。また、本発明のポリヌクレオチドの長さの上限は特に限定されないが、合成の容易さの観点から、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the polynucleotide is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more. Further, the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
該ポリヌクレオチドは、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列に加えて、任意の付加的配列を含んでいてもよい。
The polynucleotide may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
また、該ポリヌクレオチドは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C、32P、33P等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などで標識されていてもよい。
The polynucleotide also contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, β-galactosidase, β- Labeled with glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) May be.
本明細書において、「プライマーによるSNPの特異的な増幅」とは、プライマーが配列番号5で表されるヌクレオチド配列からなるDNAの特定のSNPを含む領域をPCR増幅するが、ヒトゲノムDNA中の他の領域はPCR増幅しないことを意味する。
In the present specification, “specific amplification of SNP by primer” refers to PCR amplification of a region containing a specific SNP of a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 5 in a primer. This region means that PCR amplification is not performed.
上記(2)のプライマーには、SNP1~5からなる群から選択される少なくとも1つのSNPを含むポリヌクレオチド(例えばゲノムDNA)を鋳型として、当該選択したSNP部位に向かって相補鎖合成を開始することができるプライマーが含まれる。該プライマーは、SNP部位を含むポリヌクレオチドにおける、SNP部位の3’側に複製開始点を与えるためのプライマーと表現することもできる。プライマーがハイブリダイズする領域とSNP部位との間隔は任意である。両者の間隔は、SNP部位の塩基の解析手法に応じて、好適な長さを選択することができる。たとえば、DNAチップやダイレクトシークエンシングによる解析のためのプライマーであれば、SNP部位を含む領域として、通常25~500、例えば50~200ヌクレオチドの長さの増幅産物が得られるようにプライマーをデザインすることができる。当業者は、SNP部位を含む周辺DNA領域についてのヌクレオチド配列情報を基に、各解析手法に適したプライマーをデザインすることができる。上記(2)のプライマーを構成するヌクレオチド配列は、配列番号5に記載のヌクレオチド配列の部分配列又はその相補配列に対して完全に相補的なヌクレオチド配列のみならず、適宜改変することができる。
The primer of the above (2) starts complementary strand synthesis toward the selected SNP site using a polynucleotide (for example, genomic DNA) containing at least one SNP selected from the group consisting of SNPs 1 to 5 as a template. Primers that can be included are included. The primer can also be expressed as a primer for providing an origin of replication on the 3 'side of the SNP site in the polynucleotide containing the SNP site. The interval between the region where the primer hybridizes and the SNP site is arbitrary. A suitable length can be selected for the interval between the two according to the analysis method of the base at the SNP site. For example, in the case of a primer for analysis by a DNA chip or direct sequencing, the primer is designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region including the SNP site. be able to. A person skilled in the art can design a primer suitable for each analysis method based on nucleotide sequence information about the surrounding DNA region including the SNP site. The nucleotide sequence constituting the primer of the above (2) can be appropriately modified as well as the nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
更なる局面において、上記(2’)のプライマーには、SNP1~8からなる群から選択される少なくとも1つのSNPを含むポリヌクレオチド(例えばゲノムDNA)を鋳型として、当該選択したSNP部位に向かって相補鎖合成を開始することができるプライマーが含まれる。該プライマーは、SNP部位を含むポリヌクレオチドにおける、SNP部位の3’側に複製開始点を与えるためのプライマーと表現することもできる。プライマーがハイブリダイズする領域とSNP部位との間隔は任意である。両者の間隔は、SNP部位の塩基の解析手法に応じて、好適な長さを選択することができる。たとえば、DNAチップやダイレクトシークエンシングによる解析のためのプライマーであれば、SNP部位を含む領域として、通常25~500、例えば50~200ヌクレオチドの長さの増幅産物が得られるようにプライマーをデザインすることができる。当業者は、SNP部位を含む周辺DNA領域についてのヌクレオチド配列情報を基に、各解析手法に適したプライマーをデザインすることができる。上記(2’)のプライマーを構成するヌクレオチド配列は、配列番号5に記載のヌクレオチド配列の部分配列又はその相補配列に対して完全に相補的なヌクレオチド配列のみならず、適宜改変することができる。
In a further aspect, the primer (2 ′) mentioned above is directed toward the selected SNP site using a polynucleotide (eg, genomic DNA) containing at least one SNP selected from the group consisting of SNPs 1 to 8 as a template. Primers capable of initiating complementary strand synthesis are included. The primer can also be expressed as a primer for providing an origin of replication on the 3 'side of the SNP site in the polynucleotide containing the SNP site. The interval between the region where the primer hybridizes and the SNP site is arbitrary. A suitable length can be selected for the interval between the two according to the analysis method of the base at the SNP site. For example, in the case of a primer for analysis by a DNA chip or direct sequencing, the primer is designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region including the SNP site. be able to. A person skilled in the art can design a primer suitable for each analysis method based on nucleotide sequence information about the surrounding DNA region including the SNP site. The nucleotide sequence constituting the primer (2 ') can be appropriately modified as well as a nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
上記(2)及び(2’)のプライマーは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、該核酸は二本鎖であっても、一本鎖であってもよいが、好ましくは一本鎖である。
The primers (2) and (2 ') may be DNA, RNA, or DNA / RNA chimera, but are preferably DNA. The nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
上記(2)及び(2’)のプライマーが配列番号1に記載のヌクレオチド配列又はその相補配列からなる核酸にハイブリダイズし得る領域の長さは、12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上(例えば、25ヌクレオチド以上)である。また、該部分配列またはその相補配列の長さの上限は特に限定されないが、合成の容易さの観点から、該長さは、通常100ヌクレオチド以下、好ましくは50ヌクレオチド以下、より好ましくは30ヌクレオチド以下である。
The length of the region in which the primers (2) and (2 ′) can hybridize to the nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably Is 18 nucleotides or more, more preferably 20 nucleotides or more (for example, 25 nucleotides or more). The upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. It is.
上記(2)及び(2’)のプライマーの長さは、少なくとも12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上である。また、本発明のポリヌクレオチドの長さの上限は特に限定されないが、合成の容易さの観点から、該長さは、通常100ヌクレオチド以下、好ましくは50ヌクレオチド以下、より好ましくは30ヌクレオチド以下である。
The lengths of the primers (2) and (2 ') are at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more. The upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. .
上記(2)及び(2’)のプライマーは、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列に加えて、任意の付加的配列を含んでいてもよい。
The primers (2) and (2 ') may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
また、該プライマーは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C、32P、33P等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などで標識されていてもよい。
The primer may be a suitable labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, β-galactosidase, β-glucosidase). , Alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be.
本発明の診断剤には、SNPの検出方法に応じて、各種の酵素、酵素基質、および緩衝液などを組み合わせることができる。酵素としては、DNAポリメラーゼ、DNAリガーゼ、あるいは制限酵素などの、上記のSNPの検出方法として例示した各種の解析方法に必要な酵素を示すことができる。緩衝液は、これらの解析に用いる酵素の活性の維持に好適な緩衝液が、適宜選択される。更に、酵素基質としては、例えば、相補鎖合成用の基質等が用いられる。
In the diagnostic agent of the present invention, various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method. As the enzyme, an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.
本発明の診断剤を用いると、上記本発明の判定方法により、容易にモヤモヤ病の発症リスクを判定することが出来る。
When the diagnostic agent of the present invention is used, the risk of developing moyamoya disease can be easily determined by the determination method of the present invention.
9.虚血性心疾患の発症リスクの判定方法
本発明は、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出することを含む、虚血性心疾患の発症リスクの判定方法を提供する。 9. The present invention provides a method for determining the risk of developing ischemic heart disease, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. To do.
本発明は、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出することを含む、虚血性心疾患の発症リスクの判定方法を提供する。 9. The present invention provides a method for determining the risk of developing ischemic heart disease, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. To do.
一実施形態では、本発明の判定方法は、以下の工程(a)、(b)を含む:
(a)動物(被検者)から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP2を検出する工程;
(b)検出したSNPのタイプに基づき虚血性心疾患の発症リスクを評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal (subject);
(B) A step of evaluating the risk of developing ischemic heart disease based on the detected SNP type.
(a)動物(被検者)から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP2を検出する工程;
(b)検出したSNPのタイプに基づき虚血性心疾患の発症リスクを評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal (subject);
(B) A step of evaluating the risk of developing ischemic heart disease based on the detected SNP type.
虚血性心疾患とは、冠動脈の閉塞や狭窄などにより心筋への血流が阻害され、心臓に障害が起こる疾患の総称である。虚血性心疾患としては、心筋梗塞、狭心症等が挙げられる。
Ischemic heart disease is a general term for diseases in which blood flow to the myocardium is blocked by coronary artery occlusion or stenosis, causing damage to the heart. Examples of the ischemic heart disease include myocardial infarction and angina.
上記方法の工程(a)では、動物から採取された生体試料においてSNP2を検出し、そのSNPのタイプを測定する。動物としては、上述の哺乳動物が好ましく、ヒトが特に好ましい。ヒトの人種は、特に限定されないが、好ましくは東アジア人(イーストアジアン/モンゴロイド)である。
In step (a) of the above method, SNP2 is detected in a biological sample collected from an animal, and the type of the SNP is measured. As the animal, the above-mentioned mammals are preferable, and a human is particularly preferable. The human race is not particularly limited but is preferably East Asian (East Asian / Mongoloid).
東アジア人とは、日本、朝鮮、中国、台湾及びモンゴルの人々のいずれかを起源に持つ人という意味である。東アジア人は、好ましくは、日本人、朝鮮人、又は中国人である。
“East Asian” means a person who originated in Japan, Korea, China, Taiwan or Mongolia. The East Asian is preferably a Japanese, a Korean, or a Chinese.
当業者であれば、個人の身体的特徴、出身国、先祖の起源に関する情報等に基づいてその個人の人種を容易に特定することが可能である。
A person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
ヒトの性別は特に限定されないが、好ましくは一般的なリスク要因が少ないと思われる女性である。
Human sex is not particularly limited, but is preferably a woman who seems to have few general risk factors.
ヒトの年齢は特に限定されないが、好ましくは35~54歳である。
The age of a human is not particularly limited, but is preferably 35 to 54 years old.
上記方法の工程(a)において用いられる生体試料としては、ゲノムDNAを採取可能な任意の組織、細胞、体液等を使用することができるが、入手の容易性及び低侵襲性等の観点から、毛髪、爪、皮膚、粘膜、血液、血漿、血清、唾液などが好ましく用いられる。
As a biological sample used in step (a) of the above method, any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used, but from the viewpoint of easy availability and minimal invasiveness, Hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
SNPの検出方法は、当該技術分野において周知である。例えば、RFLP(制限酵素切断断片長多型)法、PCR-SSCP(一本鎖DNA高次構造多型解析)法、ASO(Allele Specific Oligonucleotide)ハイブリダイゼーション法、シークエンス法、ARMS(Amplification Refracting Mutation System)法、変性濃度勾配ゲル電気泳動(Denaturing Gradient Gel Electrophoresis)法、RNAseA切断法、DOL(Dye-labeled Oligonucleotide Ligation)法、TaqMan PCR法、primer extension法、インベーダー法などが使用できる。
The SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
後述の実施例に示すように、SNP2について、虚血性心疾患との関連解析を行った結果、虚血性心疾患の患者(特に、35~54歳の女性)においては、SNP2のマイナーアレルの頻度が、一般人口における頻度と比較して有意に高かった。
As shown in the Examples described later, as a result of the analysis of the relationship between SNP2 and ischemic heart disease, the frequency of SNP2 minor alleles in patients with ischemic heart disease (especially women aged 35 to 54 years) However, it was significantly higher than the frequency in the general population.
これらの結果に基づけば、SNP2において、マイナーアレル(アデニン)が少なくとも一方のアレルについて検出された場合には、該マイナーアレルが検出されない場合と比較して、虚血性心疾患の発症リスクが、相対的に高いと判定することが出来る。
Based on these results, in SNP2, when the minor allele (adenine) is detected for at least one allele, the risk of developing ischemic heart disease is relative to the case where the minor allele is not detected. Can be determined to be high.
DNAは通常、互いに相補的な二本の鎖からなる二重らせん構造を有している。従って、本明細書において、便宜的に一方の鎖におけるDNA配列を示した場合であっても、当然の如く、該配列(塩基)に相補的な配列(100%の相補性)も開示したものと解釈される。当業者にとって、一方のDNA配列(塩基)が判れば、該配列(塩基)に相補的な配列(塩基)は自明である。従って、配列番号:5に記載のヌクレオチド配列の73097位の部位に対応する相補鎖上の部位の塩基種がT(チミン)である場合も、被検者は虚血性心疾患の発症リスクが、相対的に高いと判定することが出来る。
DNA usually has a double helix structure consisting of two strands complementary to each other. Therefore, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, even when the base type of the site on the complementary strand corresponding to the site at position 73097 of the nucleotide sequence set forth in SEQ ID NO: 5 is T (thymine), the subject is at risk of developing ischemic heart disease, It can be determined that it is relatively high.
尚、当業者は、本明細書に開示されたヌクレオチド配列(例えば配列番号5で表されるヌクレオチド配列)およびSNPに関する情報を用いれば、配列表に開示されたヌクレオチド配列(配列番号5で表されたヌクレオチド配列またはその連続した部分配列、あるいはそれらの相補配列)と被検者の第17番染色体DNA上の対応部位のヌクレオチド配列との間に若干の相違(欠失、置換、付加、繰り返し配列の個数の増減等)が認められた場合であっても、配列番号5で表されたヌクレオチド配列と被検者由来のヌクレオチド配列とのアライメントを行うことにより、被検者由来のヌクレオチド配列においてどの塩基がSNP2に相当するか正確に特定し、SNP2を検出することが可能である。このような場合も「配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出すること」に包含される。
It should be noted that those skilled in the art can use the nucleotide sequence disclosed in the present specification (for example, the nucleotide sequence represented by SEQ ID NO: 5) and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5). Slight difference (deletion, substitution, addition, repeat sequence) between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof) In the nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject. It is possible to accurately identify whether the base corresponds to SNP2 and detect SNP2. Such a case is also included in “detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5”.
本発明の方法においては、SNP2におけるアデニンが、少なくとも一方のアレルにおいて検出されれば、該マイナーアレルが検出されない場合と比較して、虚血性心疾患の発症リスクが、相対的に高いと判定することが出来る。即ち、本発明の方法においては、被検者の第17番染色体の少なくとも一方についてSNP2を検出すればよい。
In the method of the present invention, if adenine in SNP2 is detected in at least one allele, it is determined that the risk of developing ischemic heart disease is relatively high compared to the case where the minor allele is not detected. I can do it. That is, in the method of the present invention, SNP2 may be detected for at least one of the chromosome 17 of the subject.
尚、上述のように、SNP2の変異は、ヒトミステリンの第4810番のアミノ酸置換(アルギニン→リジン)を伴うので、対象者から、ミステリンポリペプチドを単離し、その第4810番のアミノ酸を同定することによっても、虚血性心疾患の発症リスクを判定することが出来る。ミステリンポリペプチドの単離は、抗体カラムクロマトグラフィー等の生化学分野において周知の方法を用いることにより実施することが出来る。また、第4810番のアミノ酸の同定は、ペプチドシークエンサーや、マススペクトル等の生化学分野において周知の方法を用いることにより実施することが出来る。SNP2のメジャーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてアルギニンを与え、SNP2のマイナーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてリジンを与える。従って、第4810番のアミノ酸を同定した結果、リジンが検出された場合は、リジンが検出されない場合(即ちアルギニンのみが検出された場合)と比較して、虚血性心疾患の発症リスクが相対的に高いと判定することが出来る。
As described above, since the mutation of SNP2 involves the amino acid substitution of human mysterin at position 4810 (arginine → lysine), the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was Identification can also determine the onset risk of ischemic heart disease. The mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography. The amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum. The major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identifying the amino acid at position 4810, when lysine is detected, the risk of developing ischemic heart disease is relative to when lysine is not detected (that is, when only arginine is detected). It can be determined to be high.
本発明はまた、SNP2を検出するための試薬を含む、虚血性心疾患の発症リスクの診断剤を提供する。
The present invention also provides a diagnostic agent for the risk of developing ischemic heart disease, comprising a reagent for detecting SNP2.
SNP2を検出するための試薬としては、
(1)配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を特異的に検出し得る核酸プローブ、又は
(2)SNP2を含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting SNP2,
(1) A nucleic acid probe capable of specifically detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5, or (2) a primer capable of specifically amplifying a region containing SNP2. I can do it.
(1)配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を特異的に検出し得る核酸プローブ、又は
(2)SNP2を含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting SNP2,
(1) A nucleic acid probe capable of specifically detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5, or (2) a primer capable of specifically amplifying a region containing SNP2. I can do it.
(1)のプローブは、好ましくは、SNP2がメジャーアレル(グアニン)である配列番号5で表されるポリヌクレオチドへの親和性と、当該SNP2がマイナーアレル(アデニン)である配列番号5で表されるポリヌクレオチドへの親和性との間に差を有しており、適切な条件下(例えば上述の高ストリンジェントな条件)で、メジャーアレル及びマイナーアレルの一方へハイブリダイズするが、他方へはハイブリダイズしない。
The probe of (1) is preferably represented by SEQ ID NO: 5 having an affinity for the polynucleotide represented by SEQ ID NO: 5 in which SNP2 is a major allele (guanine), and the SNP2 is a minor allele (adenine). And hybridizes to one of the major and minor alleles under appropriate conditions (eg, the above highly stringent conditions), but to the other Does not hybridize.
(1)のプローブとしては、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、73097 G>A(SNP2)を含み、該部分配列に含まれる該SNPのアレルはメジャーアレル又はマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチドが例示される。
The probe of (1) is a polynucleotide comprising a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, the partial sequence comprising 73097 G> A (SNP2), Examples of the polynucleotide include that the allele of the SNP contained in the partial sequence is a major allele or a minor allele, and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
該ポリヌクレオチドに含まれるSNP2のアレルはメジャーアレル(グアニン)又はマイナーアレル(アデニン)である。
The allele of SNP2 contained in the polynucleotide is a major allele (guanine) or a minor allele (adenine).
該ポリヌクレオチドは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、該核酸は二本鎖であっても、一本鎖であってもよいが、好ましくは一本鎖である。
The polynucleotide may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
該ポリヌクレオチドが、配列番号5で表されるヌクレオチド配列の連続した部分配列の相補配列を含む場合、該相補配列が有する相補性は、好ましくは、100%である。
When the polynucleotide includes a complementary sequence of a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5, the complementarity of the complementary sequence is preferably 100%.
該ポリヌクレオチドに含まれる、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列の長さは、12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上(例えば、25ヌクレオチド以上)である。また、該部分配列またはその相補配列の長さの上限は特に限定されないが、合成の容易さや、SNPの検出感度を高くする観点から、該長さは、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence contained in the polynucleotide is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, still more preferably 20 nucleotides or more (for example, 25 nucleotides or more). The upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis and detection sensitivity of SNP, the length is usually 1000 nucleotides or less, preferably 100 nucleotides or less. More preferably, it is 50 nucleotides or less, More preferably, it is 30 nucleotides or less.
該ポリヌクレオチドの長さは、少なくとも12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上である。また、本発明のポリヌクレオチドの長さの上限は特に限定されないが、合成の容易さの観点から、通常1000ヌクレオチド以下、好ましくは100ヌクレオチド以下、より好ましくは50ヌクレオチド以下、更に好ましくは30ヌクレオチド以下である。
The length of the polynucleotide is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more. Further, the upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, it is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 30 nucleotides or less. It is.
該ポリヌクレオチドは、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列に加えて、任意の付加的配列を含んでいてもよい。
The polynucleotide may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof.
また、該ポリヌクレオチドは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C、32P、33P等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などで標識されていてもよい。
The polynucleotide also contains an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, β-galactosidase, β- Labeled with glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) May be.
本明細書において、「プライマーによるSNPの特異的な増幅」とは、プライマーが配列番号5で表されるヌクレオチド配列からなるDNAの特定のSNPを含む領域をPCR増幅するが、ヒトゲノムDNA中の他の領域はPCR増幅しないことを意味する。
In the present specification, “specific amplification of SNP by primer” refers to PCR amplification of a region containing a specific SNP of a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 5 in a primer. This region means that PCR amplification is not performed.
上記(2)のプライマーには、SNP2を含むポリヌクレオチド(例えばゲノムDNA)を鋳型として、SNP2に向かって相補鎖合成を開始することができるプライマーが含まれる。該プライマーは、SNP2を含むポリヌクレオチドにおける、SNP2の3’側に複製開始点を与えるためのプライマーと表現することもできる。プライマーがハイブリダイズする領域とSNP2との間隔は任意である。両者の間隔は、SNPの塩基の解析手法に応じて、好適な長さを選択することができる。たとえば、DNAチップやダイレクトシークエンシングによる解析のためのプライマーであれば、SNP2を含む領域として、通常25~500、例えば50~200ヌクレオチドの長さの増幅産物が得られるようにプライマーをデザインすることができる。当業者においては、SNP2を含む周辺DNA領域についてのヌクレオチド配列情報を基に、解析手法に応じたプライマーをデザインすることができる。上記(3)のプライマーを構成するヌクレオチド配列は、配列番号5に記載のヌクレオチド配列の部分配列又はその相補配列に対して完全に相補的なヌクレオチド配列のみならず、適宜改変することができる。
The primer (2) includes a primer that can initiate complementary strand synthesis toward SNP2 using a polynucleotide containing SNP2 (eg, genomic DNA) as a template. The primer can also be expressed as a primer for providing a replication origin on the 3 'side of SNP2 in a polynucleotide containing SNP2. The space | interval of the area | region which a primer hybridizes and SNP2 is arbitrary. A suitable length can be selected for the interval between the two according to the SNP base analysis method. For example, in the case of a primer for analysis by DNA chip or direct sequencing, the primer should be designed so that an amplification product usually having a length of 25 to 500, for example, 50 to 200 nucleotides can be obtained as a region containing SNP2. Can do. A person skilled in the art can design a primer according to the analysis method based on the nucleotide sequence information about the peripheral DNA region containing SNP2. The nucleotide sequence constituting the primer of the above (3) can be appropriately modified as well as a nucleotide sequence completely complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence.
上記(2)のプライマーは、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよいが、好ましくはDNAである。また、該核酸は二本鎖であっても、一本鎖であってもよいが、好ましくは一本鎖である。
The primer (2) may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded, but is preferably single-stranded.
上記(2)のプライマーが配列番号1に記載のヌクレオチド配列又はその相補配列からなる核酸にハイブリダイズし得る領域の長さは、12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上(例えば、25ヌクレオチド以上)である。また、該部分配列またはその相補配列の長さの上限は特に限定されないが、合成の容易さの観点から、該長さは、通常100ヌクレオチド以下、好ましくは50ヌクレオチド以下、より好ましくは30ヌクレオチド以下である。
The length of the region where the primer (2) can hybridize to the nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof is 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, More preferably, it is 20 nucleotides or more (for example, 25 nucleotides or more). The upper limit of the length of the partial sequence or its complementary sequence is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. It is.
上記(2)のプライマーの長さは、少なくとも12ヌクレオチド以上、好ましくは15ヌクレオチド以上、より好ましくは18ヌクレオチド以上、更に好ましくは20ヌクレオチド以上である。また、本発明のポリヌクレオチドの長さの上限は特に限定されないが、合成の容易さの観点から、該長さは、通常100ヌクレオチド以下、好ましくは50ヌクレオチド以下、より好ましくは30ヌクレオチド以下である。
The length of the primer (2) is at least 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 18 nucleotides or more, and further preferably 20 nucleotides or more. The upper limit of the length of the polynucleotide of the present invention is not particularly limited, but from the viewpoint of ease of synthesis, the length is usually 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less. .
上記(2)のプライマーは、配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列に加えて、任意の付加的配列を含んでいてもよい。
The primer (2) above may contain an arbitrary additional sequence in addition to a continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or its complementary sequence.
また、該プライマーは、適当な標識剤、例えば、放射性同位元素(例:125I、131I、3H、14C、32P、33P等)、酵素(例:β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等)、蛍光物質(例:フルオレスカミン、フルオレッセンイソチオシアネート等)、発光物質(例:ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等)などで標識されていてもよい。
The primer may be a suitable labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, etc.), an enzyme (eg, β-galactosidase, β-glucosidase). , Alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.) It may be.
本発明の診断剤には、SNPの検出方法に応じて、各種の酵素、酵素基質、および緩衝液などを組み合わせることができる。酵素としては、DNAポリメラーゼ、DNAリガーゼ、あるいは制限酵素などの、上記のSNPの検出方法として例示した各種の解析方法に必要な酵素を示すことができる。緩衝液は、これらの解析に用いる酵素の活性の維持に好適な緩衝液が、適宜選択される。更に、酵素基質としては、例えば、相補鎖合成用の基質等が用いられる。
In the diagnostic agent of the present invention, various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method. As the enzyme, an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.
本発明の診断剤を用いると、本発明の判定方法により、容易に虚血性心疾患の発症リスクを判定することが出来る。
When the diagnostic agent of the present invention is used, the risk of developing ischemic heart disease can be easily determined by the determination method of the present invention.
10.一卵性双生児の分娩可能性の発症リスクの判定方法
本発明は、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出することを含む、一卵性双生児の分娩可能性の判定方法を提供する。 10. Method for Determining the Risk of Onset of Fetal Delivery of Monozygotic Twins The present invention includes the possibility of delivery of identical twins, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. A determination method is provided.
本発明は、配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出することを含む、一卵性双生児の分娩可能性の判定方法を提供する。 10. Method for Determining the Risk of Onset of Fetal Delivery of Monozygotic Twins The present invention includes the possibility of delivery of identical twins, comprising detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5. A determination method is provided.
一実施形態では、本発明の判定方法は、以下の工程(a)、(b)を含む:
(a)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP2を検出する工程;
(b)検出したSNPのタイプに基づき一卵性双生児の分娩可能性を評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal;
(B) A step of evaluating the feasibility of identical twins based on the detected SNP type.
(a)動物から採取した生体試料について、配列番号5で表されるヌクレオチド配列におけるSNP2を検出する工程;
(b)検出したSNPのタイプに基づき一卵性双生児の分娩可能性を評価する工程。 In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of detecting SNP2 in the nucleotide sequence represented by SEQ ID NO: 5 for a biological sample collected from an animal;
(B) A step of evaluating the feasibility of identical twins based on the detected SNP type.
上記方法の工程(a)では、動物から採取された生体試料においてSNP2を検出し、そのSNPのタイプを測定する。動物としては、上述の哺乳動物が好ましく、ヒトが特に好ましい。ヒトの人種は、特に限定されないが、好ましくは東アジア人(イーストアジアン/モンゴロイド)である。
In step (a) of the above method, SNP2 is detected in a biological sample collected from an animal, and the type of the SNP is measured. As the animal, the above-mentioned mammals are preferable, and a human is particularly preferable. The human race is not particularly limited but is preferably East Asian (East Asian / Mongoloid).
東アジア人とは、日本、朝鮮、中国、台湾及びモンゴルの人々のいずれかを起源に持つ人という意味である。東アジア人は、好ましくは、日本人、朝鮮人、又は中国人である。
“East Asian” means a person who originated in Japan, Korea, China, Taiwan or Mongolia. The East Asian is preferably a Japanese, a Korean, or a Chinese.
当業者であれば、個人の身体的特徴、出身国、先祖の起源に関する情報等に基づいてその個人の人種を容易に特定することが可能である。
A person skilled in the art can easily identify the individual's race based on information on the individual's physical characteristics, country of origin, origin of ancestors, and the like.
上記方法の工程(a)において用いられる生体試料としては、ゲノムDNAを採取可能な任意の組織、細胞、体液等を使用することができるが、入手の容易性及び低侵襲性等の観点から、毛髪、爪、皮膚、粘膜、血液、血漿、血清、唾液などが好ましく用いられる。
As a biological sample used in step (a) of the above method, any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used, but from the viewpoint of easy availability and minimal invasiveness, Hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like are preferably used.
SNPの検出方法は、当該技術分野において周知である。例えば、RFLP(制限酵素切断断片長多型)法、PCR-SSCP(一本鎖DNA高次構造多型解析)法、ASO(Allele Specific Oligonucleotide)ハイブリダイゼーション法、シークエンス法、ARMS(Amplification Refracting Mutation System)法、変性濃度勾配ゲル電気泳動(Denaturing Gradient Gel Electrophoresis)法、RNAseA切断法、DOL(Dye-labeled Oligonucleotide Ligation)法、TaqMan PCR法、primer extension法、インベーダー法などが使用できる。
The SNP detection method is well known in the art. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like.
後述の実施例に示すように、上述の新規SNP2について、一卵性双生児の分娩頻度との関連解析を行った結果、SNP2のマイナーアレルを保持しているヒト(女性)が一卵性双生児を分娩する頻度は、一般的に報告されている頻度よりも有意に高かった。
As shown in the examples described later, as a result of the analysis of the relationship between the above-mentioned new SNP2 and the frequency of delivery of identical twins, a human (female) holding a minor allele of SNP2 is identified as an identical twin. The frequency of delivery was significantly higher than the commonly reported frequency.
これらの結果に基づけば、SNP2において、マイナーアレル(アデニン)が少なくとも一方のアレルについて検出された場合には、該マイナーアレルが検出されない場合と比較して、一卵性双生児を分娩する頻度が、相対的に高いと判定することが出来る。
Based on these results, in SNP2, when a minor allele (adenine) is detected for at least one allele, the frequency of delivery of identical twins compared to the case where the minor allele is not detected is It can be determined that it is relatively high.
DNAは通常、互いに相補的な二本の鎖からなる二重らせん構造を有している。従って、本明細書において、便宜的に一方の鎖におけるDNA配列を示した場合であっても、当然の如く、該配列(塩基)に相補的な配列(100%の相補性)も開示したものと解釈される。当業者にとって、一方のDNA配列(塩基)が判れば、該配列(塩基)に相補的な配列(塩基)は自明である。従って、配列番号:5に記載のヌクレオチド配列の73097位の部位に対応する該配列の相補鎖上の部位について、塩基種がT(チミン)である場合も、被検者は一卵性双生児を分娩する頻度が、相対的に高いと判定することが出来る。
DNA usually has a double helix structure consisting of two strands complementary to each other. Accordingly, in the present specification, even when the DNA sequence in one strand is shown for convenience, the sequence (100% complementarity) complementary to the sequence (base) is also disclosed as a matter of course. Is interpreted. For those skilled in the art, if one DNA sequence (base) is known, a sequence (base) complementary to the sequence (base) is obvious. Therefore, even when the base type is T (thymine) for the site on the complementary strand of the sequence corresponding to the site at position 73097 of the nucleotide sequence shown in SEQ ID NO: 5, It can be determined that the frequency of delivery is relatively high.
尚、当業者は、本明細書に開示されたヌクレオチド配列(例えば配列番号5で表されるヌクレオチド配列)およびSNPに関する情報を用いれば、配列表に開示されたヌクレオチド配列(配列番号5で表されたヌクレオチド配列またはその連続した部分配列、あるいはそれらの相補配列)と被検者の第17番染色体DNA上の対応部位のヌクレオチド配列との間に若干の相違(欠失、置換、付加、繰り返し配列の個数の増減等)が認められた場合であっても、配列番号5で表されたヌクレオチド配列と被検者由来のヌクレオチド配列とのアライメントを行うことにより、被検者由来のヌクレオチド配列においてどの塩基がSNP2に相当するか正確に特定し、SNP2を検出することが可能である。このような場合も「配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を検出すること」に包含される。
It should be noted that those skilled in the art can use the nucleotide sequence disclosed in the present specification (for example, the nucleotide sequence represented by SEQ ID NO: 5) and information on the SNP, and the nucleotide sequence disclosed in the sequence listing (represented by SEQ ID NO: 5). Slight difference (deletion, substitution, addition, repeat sequence) between the nucleotide sequence of the corresponding site on the subject's chromosome 17 DNA and the nucleotide sequence of the subject nucleotide sequence or a continuous partial sequence thereof or the complementary sequence thereof) In the nucleotide sequence derived from the subject by aligning the nucleotide sequence represented by SEQ ID NO: 5 with the nucleotide sequence derived from the subject. It is possible to accurately identify whether the base corresponds to SNP2 and detect SNP2. Such a case is also included in “detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5”.
本発明の方法においては、SNP2におけるアデニンが、少なくとも一方のアレルにおいて検出されれば、該マイナーアレルが検出されない場合と比較して、一卵性双生児の分娩可能性が、相対的に高いと判定することが出来る。即ち、本発明の方法においては、被検者の第17番染色体の少なくとも一方についてSNP2を検出すればよい。
In the method of the present invention, if adenine in SNP2 is detected in at least one allele, it is determined that the probability of delivery of identical twins is relatively high compared to the case where the minor allele is not detected. I can do it. That is, in the method of the present invention, SNP2 may be detected for at least one of the chromosome 17 of the subject.
尚、上述のように、SNP2の変異は、ヒトミステリンの第4810番のアミノ酸置換(アルギニン→リジン)を伴うので、対象者から、ミステリンポリペプチドを単離し、その第4810番のアミノ酸を同定することによっても、一卵性双生児の分娩可能性を判定することが出来る。ミステリンポリペプチドの単離は、抗体カラムクロマトグラフィー等の生化学分野において周知の方法を用いることにより実施することが出来る。また、第4810番のアミノ酸の同定は、ペプチドシークエンサーや、マススペクトル等の生化学分野において周知の方法を用いることにより実施することが出来る。SNP2のメジャーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてアルギニンを与え、SNP2のマイナーアレルはヒトミステリンポリペプチドの第4810番アミノ酸としてリジンを与える。従って、第4810番のアミノ酸を同定した結果、リジンが検出された場合は、リジンが検出されない場合(即ちアルギニンのみが検出された場合)と比較して、一卵性双生児の分娩可能性が相対的に高いと判定することが出来る。
As described above, since the mutation of SNP2 involves the amino acid substitution of human mysterin at position 4810 (arginine → lysine), the mysterin polypeptide was isolated from the subject and the amino acid at position 4810 was Identification can also determine the feasibility of identical twins. The mysterin polypeptide can be isolated by using a well-known method in the biochemical field such as antibody column chromatography. The amino acid at position 4810 can be identified by using a peptide sequencer or a well-known method in the biochemical field such as mass spectrum. The major allele of SNP2 gives arginine as amino acid 4810 of human mysterin polypeptide, and the minor allele of SNP2 gives lysine as amino acid 4810 of human mysterin polypeptide. Therefore, as a result of identification of amino acid No. 4810, when lysine was detected, the relative feasibility of identical twins compared to the case where lysine was not detected (that is, only arginine was detected). Can be determined to be high.
本発明はまた、SNP2を検出するための試薬を含む、一卵性双生児の分娩可能性の診断剤を提供する。
The present invention also provides a diagnostic agent for the feasibility of identical twins, including a reagent for detecting SNP2.
SNP2を検出するための試薬としては、
(1)配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を特異的に検出し得る核酸プローブ、又は
(2)SNP2を含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting SNP2,
(1) A nucleic acid probe capable of specifically detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5, or (2) a primer capable of specifically amplifying a region containing SNP2. I can do it.
(1)配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)を特異的に検出し得る核酸プローブ、又は
(2)SNP2を含む領域を特異的に増幅し得るプライマー
等を挙げることが出来る。 As a reagent for detecting SNP2,
(1) A nucleic acid probe capable of specifically detecting 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5, or (2) a primer capable of specifically amplifying a region containing SNP2. I can do it.
(1)のプローブおよび、(2)のプライマーの定義は、9.の項と同一である。
The definition of the probe in (1) and the primer in (2) is 9. Is the same as
本発明の診断剤には、SNPの検出方法に応じて、各種の酵素、酵素基質、および緩衝液などを組み合わせることができる。酵素としては、DNAポリメラーゼ、DNAリガーゼ、あるいは制限酵素などの、上記のSNPの検出方法として例示した各種の解析方法に必要な酵素を示すことができる。緩衝液は、これらの解析に用いる酵素の活性の維持に好適な緩衝液が、適宜選択される。更に、酵素基質としては、例えば、相補鎖合成用の基質等が用いられる。
In the diagnostic agent of the present invention, various enzymes, enzyme substrates, buffers, and the like can be combined depending on the SNP detection method. As the enzyme, an enzyme necessary for various analysis methods exemplified as the method for detecting SNP, such as DNA polymerase, DNA ligase, or restriction enzyme, can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.
本発明の診断剤を用いると、本発明の判定方法により、容易に一卵性双生児の分娩可能性を判定することが出来る。
When the diagnostic agent of the present invention is used, the possibility of delivery of identical twins can be easily determined by the determination method of the present invention.
本明細書中で挙げられた特許および特許出願明細書を含む全ての刊行物に記載された内容は、本明細書での引用により、その全てが明示されたと同程度に本明細書に組み込まれるものである。
The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.
以下、実施例を示して本発明をより具体的に説明するが、本発明は以下に示す実施例によって何ら限定されるものではない。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples shown below.
実施例1:新規SNPsの同定及びモヤモヤ病患者における各SNPsのアレル頻度の解析
調査集団
本試験は、京都大学医学部の倫理委員会により承認されたものであり、全ての対象から書面によるインフォームドコンセントを取得した。本試験においては2つのタイプのモヤモヤ病症例参加者が登録された。第1のタイプは、血縁者に1人以上のモヤモヤ病症例が明確に存在するために、本研究へ参加するために選択された家系単位の参加者である。31人の日本人及び3人の韓国人を含む34人のモヤモヤ病家系発端者が本試験に登録された。診断の検証のため、血管病に属する病歴及び危険因子を発端者とその家系メンバーから収集した。京都大学病院又は他の協力病院へ入院した創始者家系のメンバーをリクルートした。患者の同意のもと、試料及び臨床データを採取し、非識別化し、蓄積した。第2のタイプは、家族歴が不明のモヤモヤ病の患者であり、同疾患に罹患した血縁者の存在が不明の症例として個人で本試験に参加した者であり、個人症例として分類された。これらの症例を、京都大学(n = 86)、韓国のソウル国立大学(n = 38)、中国の首都医科大学(n = 10)からリクルートした。対照として、384人の日本人、294人の韓国人、150人の中国人をリクルートした。核磁気共鳴血管撮影(MRA)スクリーニングを、全ての日本人対照について行ったが、韓国人については一部、中国人の対照については行わなかった。家系の創始者又は単発性の参加者についてのモヤモヤ病の診断は、もっぱら日本の診断基準である、いわゆるRCMJ(厚生労働省のモヤモヤ病の検討委員会、日本)診断基準に基づき行った(Clin Neurol Neurosurg, vol. 99, Suppl 2, pages S238-240, 1997)。 Example 1: Identification of new SNPs and analysis of allele frequency of each SNP in patients with moyamoya disease Study population This study was approved by the Ethics Committee of Kyoto University School of Medicine and was written by all subjects. Obtained informed consent by. Two types of moyamoya disease case participants were enrolled in this study. The first type is a family unit participant selected to participate in this study due to the clear presence of one or more moyamoya disease cases in relatives. Thirty-four moyamoya probands, including 31 Japanese and 3 Koreans, were enrolled in this study. To verify the diagnosis, a history and risk factors for vascular disease were collected from the proband and its family members. Recruited founder family members who were admitted to Kyoto University Hospital or other partner hospitals. Samples and clinical data were collected, de-identified and accumulated with patient consent. The second type is a patient with moyamoya disease whose family history is unknown, and an individual who participated in this study as a case where the presence of relatives suffering from the disease is unknown, and was classified as an individual case. These cases were recruited from Kyoto University (n = 86), Seoul National University (n = 38), Korea, and Capital Medical University (n = 10) in China. In contrast, 384 Japanese, 294 Korean, and 150 Chinese were recruited. Nuclear magnetic resonance angiography (MRA) screening was performed for all Japanese controls, but some for Koreans and not for Chinese controls. Diagnosis of moyamoya disease in the founder or single participant of the family was made based on the so-called RCMJ (Moyamoya disease study committee of the Ministry of Health, Labor and Welfare, Japan) diagnostic criteria which is exclusively Japanese diagnostic criteria (Clin Neurol Neurosurg, vol. 99,Suppl 2, pages S238-240, 1997).
調査集団
本試験は、京都大学医学部の倫理委員会により承認されたものであり、全ての対象から書面によるインフォームドコンセントを取得した。本試験においては2つのタイプのモヤモヤ病症例参加者が登録された。第1のタイプは、血縁者に1人以上のモヤモヤ病症例が明確に存在するために、本研究へ参加するために選択された家系単位の参加者である。31人の日本人及び3人の韓国人を含む34人のモヤモヤ病家系発端者が本試験に登録された。診断の検証のため、血管病に属する病歴及び危険因子を発端者とその家系メンバーから収集した。京都大学病院又は他の協力病院へ入院した創始者家系のメンバーをリクルートした。患者の同意のもと、試料及び臨床データを採取し、非識別化し、蓄積した。第2のタイプは、家族歴が不明のモヤモヤ病の患者であり、同疾患に罹患した血縁者の存在が不明の症例として個人で本試験に参加した者であり、個人症例として分類された。これらの症例を、京都大学(n = 86)、韓国のソウル国立大学(n = 38)、中国の首都医科大学(n = 10)からリクルートした。対照として、384人の日本人、294人の韓国人、150人の中国人をリクルートした。核磁気共鳴血管撮影(MRA)スクリーニングを、全ての日本人対照について行ったが、韓国人については一部、中国人の対照については行わなかった。家系の創始者又は単発性の参加者についてのモヤモヤ病の診断は、もっぱら日本の診断基準である、いわゆるRCMJ(厚生労働省のモヤモヤ病の検討委員会、日本)診断基準に基づき行った(Clin Neurol Neurosurg, vol. 99, Suppl 2, pages S238-240, 1997)。 Example 1: Identification of new SNPs and analysis of allele frequency of each SNP in patients with moyamoya disease Study population This study was approved by the Ethics Committee of Kyoto University School of Medicine and was written by all subjects. Obtained informed consent by. Two types of moyamoya disease case participants were enrolled in this study. The first type is a family unit participant selected to participate in this study due to the clear presence of one or more moyamoya disease cases in relatives. Thirty-four moyamoya probands, including 31 Japanese and 3 Koreans, were enrolled in this study. To verify the diagnosis, a history and risk factors for vascular disease were collected from the proband and its family members. Recruited founder family members who were admitted to Kyoto University Hospital or other partner hospitals. Samples and clinical data were collected, de-identified and accumulated with patient consent. The second type is a patient with moyamoya disease whose family history is unknown, and an individual who participated in this study as a case where the presence of relatives suffering from the disease is unknown, and was classified as an individual case. These cases were recruited from Kyoto University (n = 86), Seoul National University (n = 38), Korea, and Capital Medical University (n = 10) in China. In contrast, 384 Japanese, 294 Korean, and 150 Chinese were recruited. Nuclear magnetic resonance angiography (MRA) screening was performed for all Japanese controls, but some for Koreans and not for Chinese controls. Diagnosis of moyamoya disease in the founder or single participant of the family was made based on the so-called RCMJ (Moyamoya disease study committee of the Ministry of Health, Labor and Welfare, Japan) diagnostic criteria which is exclusively Japanese diagnostic criteria (Clin Neurol Neurosurg, vol. 99,
連鎖解析及びハプロタイプの推定
全部で17のモヤモヤ病家系について、17q25.3のゲノタイプを解析した。ゲノタイピング、マッピング及びハプロタイプ推定を以前記載したように実施した(Neurology vol70: 2357-2363,2008)。端的にいうと、ゲノムDNAを、QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany)を用いて、患者の血液試料から抽出した。17q25-qter連鎖領域について、5.1-Mbの間隔で、全部で13個のマーカー(D17S2195, D17S1847, D17S1806, D17S784, rs2071148, 125 rs2280147, rs2293099, D17S704, D17S668, D17S928, rs2291395, rs2279395 及び 126 rs2292971)についてゲノタイプした(Neurology vol70: 2357-2363,2008)。マーカーの位置は、NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/)から取得した。末梢血から抽出したゲノムDNAを蛍光でラベルをしたマーカーと共にPCRにより増幅し、PCR産物をABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA)及びGenescanプログラムを用いて解析した。詳細なマッピングマーカーは、NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/)からの物理位置情報に従い、デザインした。連鎖解析は、常染色体優性遺伝モデルを仮定した多点パラメトリック連鎖法を用いて行った(J Neurol Neurosurg Psychiatry, vo. 77, pages 1025-1029, 2006及びNeurology vol70: 2357-2363,2008)。以前に報告したように、必然的キャリアーは、発症した者として処理した(Neurology vol70: 2357-2363,2008)。当該家系において、非発症の個人の表現型は、「不明」とし、「非血縁」の配偶者は、「非発症」とした。100,000人あたり6.03人という観察された罹患率に基づけば(Stroke, vol. 39, pages 42-47, 2008)、疾患アレル頻度は、0.00003015に設定されるべきであるが、無症状であっても、磁気共鳴画像(MRI)及びMRAによりモヤモヤ病であると診断された患者の数が近年増加していることに鑑み、そのアレル頻度を、連鎖解析でLOD score を低めに算出する点から、より厳格と考えられる0.0001に設定した(J Clin Neurosci, vol. 13, pages 334-338, 2006;)。表現型模写頻度を0.00001と仮定した。各マイクロサテライトマーカーのアレル頻度は、全ての非血縁配偶者から、Merlinソフトウェアを用いて見積もった(Nat Genet, vol. 30, pages 97-101, 2002)。解析は、GENEHUNTER version 2.0 (http://www.broad.mit.edu/ftp/distribution/software/genehunter/)を用いて実施した(Am J Hum Genet, vol. 58, pages 1347-1363, 1996)。
家系20の個人132は、rs2280147とrs2293099との間での組換えを有していたので、更なる追加的なマーカー、即ち、rs71166116 (CHMP6), rs9896314 (AZI1), rs62075318 (AZI1) 及び P1026P (BAHCC1)により、3’隣接領域を決定した。家系20のハプロタイプは、4つのマーカーのアレル頻度を0.50に設定し、GENEHUNTER(Am J Hum Genet, vol. 58, pages 1347-1363, 1996)を用いて構築した。 Linkage analysis and estimation of haplotypes A total of 17 moyamoya disease families were analyzed for 17q25.3 genotypes. Genotyping, mapping and haplotype estimation were performed as previously described (Neurology vol70: 2357-2363, 2008). Briefly, genomic DNA was extracted from patient blood samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). For the 17q25-qter linkage region, a total of 13 markers (D17S2195, D17S1847, D17S1806, D17S784, rs2071148, 125 rs2280147, rs2293099, D17S704, D17S668, D17S928, rs2291395, and rs22293951 at 5.1-Mb intervals) Genotyped (Neurology vol70: 2357-2363, 2008). The position of the marker was obtained from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Genomic DNA extracted from peripheral blood was amplified by PCR with fluorescently labeled markers, and the PCR products were analyzed using ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, Calif.) And Genescan program. Detailed mapping markers were designed according to physical location information from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Linkage analysis was performed using a multipoint parametric linkage method assuming an autosomal dominant genetic model (J Neurol Neurosurg Psychiatry, vo. 77, pages 1025-1029, 2006 and Neurology vol70: 2357-2363, 2008). As previously reported, inevitable carriers were treated as those who developed (Neurology vol70: 2357-2363, 2008). In the family, the phenotype of non-onset individuals was “unknown” and the “unrelated” spouse was “non-onset”. Based on an observed morbidity of 6.03 per 100,000 (Stroke, vol. 39, pages 42-47, 2008), the disease allele frequency should be set to 0.00003015, but even if asymptomatic In view of the recent increase in the number of patients diagnosed with moyamoya disease by magnetic resonance imaging (MRI) and MRA, the allele frequency is calculated from a lower LOD score by linkage analysis. It was set to 0.0001, which is considered to be strict (J Clin Neurosci, vol. 13, pages 334-338, 2006;). The phenotype replication frequency was assumed to be 0.00001. The allele frequency of each microsatellite marker was estimated from all unrelated spouses using the Merlin software (Nat Genet, vol. 30, pages 97-101, 2002). Analysis was performed using GENEHUNTER version 2.0 (http://www.broad.mit.edu/ftp/distribution/software/genehunter/) (Am J Hum Genet, vol. 58, pages 1347-1363, 1996) .
Individuals 132 infamily 20 had recombination between rs2280147 and rs2293099, so additional additional markers, namely rs71166116 (CHMP6), rs9896314 (AZI1), rs62075318 (AZI1) and P1026P ( The 3 ′ flanking region was determined by BAHCC1). The haplotype of family 20 was constructed using GENEHUNTER (Am J Hum Genet, vol. 58, pages 1347-1363, 1996) with the allele frequency of the four markers set to 0.50.
全部で17のモヤモヤ病家系について、17q25.3のゲノタイプを解析した。ゲノタイピング、マッピング及びハプロタイプ推定を以前記載したように実施した(Neurology vol70: 2357-2363,2008)。端的にいうと、ゲノムDNAを、QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany)を用いて、患者の血液試料から抽出した。17q25-qter連鎖領域について、5.1-Mbの間隔で、全部で13個のマーカー(D17S2195, D17S1847, D17S1806, D17S784, rs2071148, 125 rs2280147, rs2293099, D17S704, D17S668, D17S928, rs2291395, rs2279395 及び 126 rs2292971)についてゲノタイプした(Neurology vol70: 2357-2363,2008)。マーカーの位置は、NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/)から取得した。末梢血から抽出したゲノムDNAを蛍光でラベルをしたマーカーと共にPCRにより増幅し、PCR産物をABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA)及びGenescanプログラムを用いて解析した。詳細なマッピングマーカーは、NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/)からの物理位置情報に従い、デザインした。連鎖解析は、常染色体優性遺伝モデルを仮定した多点パラメトリック連鎖法を用いて行った(J Neurol Neurosurg Psychiatry, vo. 77, pages 1025-1029, 2006及びNeurology vol70: 2357-2363,2008)。以前に報告したように、必然的キャリアーは、発症した者として処理した(Neurology vol70: 2357-2363,2008)。当該家系において、非発症の個人の表現型は、「不明」とし、「非血縁」の配偶者は、「非発症」とした。100,000人あたり6.03人という観察された罹患率に基づけば(Stroke, vol. 39, pages 42-47, 2008)、疾患アレル頻度は、0.00003015に設定されるべきであるが、無症状であっても、磁気共鳴画像(MRI)及びMRAによりモヤモヤ病であると診断された患者の数が近年増加していることに鑑み、そのアレル頻度を、連鎖解析でLOD score を低めに算出する点から、より厳格と考えられる0.0001に設定した(J Clin Neurosci, vol. 13, pages 334-338, 2006;)。表現型模写頻度を0.00001と仮定した。各マイクロサテライトマーカーのアレル頻度は、全ての非血縁配偶者から、Merlinソフトウェアを用いて見積もった(Nat Genet, vol. 30, pages 97-101, 2002)。解析は、GENEHUNTER version 2.0 (http://www.broad.mit.edu/ftp/distribution/software/genehunter/)を用いて実施した(Am J Hum Genet, vol. 58, pages 1347-1363, 1996)。
家系20の個人132は、rs2280147とrs2293099との間での組換えを有していたので、更なる追加的なマーカー、即ち、rs71166116 (CHMP6), rs9896314 (AZI1), rs62075318 (AZI1) 及び P1026P (BAHCC1)により、3’隣接領域を決定した。家系20のハプロタイプは、4つのマーカーのアレル頻度を0.50に設定し、GENEHUNTER(Am J Hum Genet, vol. 58, pages 1347-1363, 1996)を用いて構築した。 Linkage analysis and estimation of haplotypes A total of 17 moyamoya disease families were analyzed for 17q25.3 genotypes. Genotyping, mapping and haplotype estimation were performed as previously described (Neurology vol70: 2357-2363, 2008). Briefly, genomic DNA was extracted from patient blood samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). For the 17q25-qter linkage region, a total of 13 markers (D17S2195, D17S1847, D17S1806, D17S784, rs2071148, 125 rs2280147, rs2293099, D17S704, D17S668, D17S928, rs2291395, and rs22293951 at 5.1-Mb intervals) Genotyped (Neurology vol70: 2357-2363, 2008). The position of the marker was obtained from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Genomic DNA extracted from peripheral blood was amplified by PCR with fluorescently labeled markers, and the PCR products were analyzed using ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, Calif.) And Genescan program. Detailed mapping markers were designed according to physical location information from NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Linkage analysis was performed using a multipoint parametric linkage method assuming an autosomal dominant genetic model (J Neurol Neurosurg Psychiatry, vo. 77, pages 1025-1029, 2006 and Neurology vol70: 2357-2363, 2008). As previously reported, inevitable carriers were treated as those who developed (Neurology vol70: 2357-2363, 2008). In the family, the phenotype of non-onset individuals was “unknown” and the “unrelated” spouse was “non-onset”. Based on an observed morbidity of 6.03 per 100,000 (Stroke, vol. 39, pages 42-47, 2008), the disease allele frequency should be set to 0.00003015, but even if asymptomatic In view of the recent increase in the number of patients diagnosed with moyamoya disease by magnetic resonance imaging (MRI) and MRA, the allele frequency is calculated from a lower LOD score by linkage analysis. It was set to 0.0001, which is considered to be strict (J Clin Neurosci, vol. 13, pages 334-338, 2006;). The phenotype replication frequency was assumed to be 0.00001. The allele frequency of each microsatellite marker was estimated from all unrelated spouses using the Merlin software (Nat Genet, vol. 30, pages 97-101, 2002). Analysis was performed using GENEHUNTER version 2.0 (http://www.broad.mit.edu/ftp/distribution/software/genehunter/) (Am J Hum Genet, vol. 58, pages 1347-1363, 1996) .
Individuals 132 in
ダイレクトシークエンシング
KIAA1618、C17orf27(RNF213)、FLJ3520、NPTX1、CARD14、Raptor (KIAA1303)、AATK及びBAHCC1の、全てのコーディングエクソン、イントロン-エクソン境界、仮想的なプロモーター配列及び3’-UTRsを、4人の発症した個人(系統1、2、14及び15における個人12、1411、112及び12)についてのダイレクトシークエンシングにより解析した。コーディングエクソンのためのプライマーは、イントロン-エクソン境界から100ベースペア(bp)より離れたイントロン配列からデザインし、PROLIGO Primers & Probes (Kyoto, Japan; http://www.proligo.com)により商業的に合成した(Neurology vol70: 2357-2363,2008)。Raptorについては、第1エクソンの約2kbp上流の制御領域についてシークエンスした。PCR増幅及び精製の後に、ABI Prism 3100 Avant DNA Sequencer (Applied Biosystems)を用いてシークエンシングを実施した。我々は、Single Nucleotide Polymorphism database (dbSNP)を参考としてチェックした(http://www.ncbi.nlm.nih.gov/SNP/index.html)。 Direct sequencing KIAA1618, C17orf27 (RNF213), FLJ3520, NPTX1, CARD14, Raptor (KIAA1303), AATK and BAHCC1, all coding exons, intron-exon boundaries, virtual promoter sequences and 3'-UTRs Were analyzed by direct sequencing of individuals who developed the disease ( individuals 12, 1411, 112 and 12 in strains 1, 2, 14 and 15). Primers for coding exons are designed from intron sequences separated by 100 base pairs (bp) from the intron-exon boundary and are commercially available by PROLIGO Primers & Probes (Kyoto, Japan; http://www.proligo.com) (Neurology vol70: 2357-2363, 2008). Raptor was sequenced for a control region approximately 2 kbp upstream of the first exon. After PCR amplification and purification, sequencing was performed using an ABI Prism 3100 Avant DNA Sequencer (Applied Biosystems). We checked the Single Nucleotide Polymorphism database (dbSNP) for reference (http://www.ncbi.nlm.nih.gov/SNP/index.html).
KIAA1618、C17orf27(RNF213)、FLJ3520、NPTX1、CARD14、Raptor (KIAA1303)、AATK及びBAHCC1の、全てのコーディングエクソン、イントロン-エクソン境界、仮想的なプロモーター配列及び3’-UTRsを、4人の発症した個人(系統1、2、14及び15における個人12、1411、112及び12)についてのダイレクトシークエンシングにより解析した。コーディングエクソンのためのプライマーは、イントロン-エクソン境界から100ベースペア(bp)より離れたイントロン配列からデザインし、PROLIGO Primers & Probes (Kyoto, Japan; http://www.proligo.com)により商業的に合成した(Neurology vol70: 2357-2363,2008)。Raptorについては、第1エクソンの約2kbp上流の制御領域についてシークエンスした。PCR増幅及び精製の後に、ABI Prism 3100 Avant DNA Sequencer (Applied Biosystems)を用いてシークエンシングを実施した。我々は、Single Nucleotide Polymorphism database (dbSNP)を参考としてチェックした(http://www.ncbi.nlm.nih.gov/SNP/index.html)。 Direct sequencing KIAA1618, C17orf27 (RNF213), FLJ3520, NPTX1, CARD14, Raptor (KIAA1303), AATK and BAHCC1, all coding exons, intron-exon boundaries, virtual promoter sequences and 3'-UTRs Were analyzed by direct sequencing of individuals who developed the disease (
シークエンスをした4人の家系性で発症した個人において5つの変異(SNPs)が同定された(表1)。
Five mutations (SNPs) were identified in the 4 individuals who developed the pedigree in the sequence (Table 1).
上記5つのSNPsのアレルを、全ての参加者について決定した。結果を表2~10に示す。
The above 5 SNPs alleles were determined for all participants. The results are shown in Tables 2-10.
上述の表に示すように、SNP1~SNP5のマイナーアレルの頻度は、非家族性のモヤモヤ病患者において約52%以上であり、家族性のモヤモヤ病患者において約79%以上と極めて高かった。特に、SNP2については、家族性のモヤモヤ病患者の全てがマイナーアレルを保有していた。これに対して、対照群においては、SNP1~SNP5のマイナーアレルの頻度は、2%前後に留まっていた。従って、SNP1~SNP5を決定することにより、モヤモヤ病の発症リスクを判定することが可能であることが示された。また、SNP2のマイナーアレルは、アジア人におけるモヤモヤ病の創始者変異である可能性が示唆された。各SNPsのオッズ比を表11にまとめる。
As shown in the table above, the frequency of minor alleles of SNP1 to SNP5 was about 52% or more in non-familial moyamoya disease patients and extremely high, about 79% or more in familial moyamoya disease patients. In particular, for SNP2, all familial moyamoya patients had minor alleles. In contrast, in the control group, the frequency of minor alleles SNP1 to SNP5 remained around 2%. Therefore, it was shown that the risk of moyamoya disease can be determined by determining SNP1 to SNP5. Moreover, it was suggested that the minor allele of SNP2 may be a founder mutation of moyamoya disease in Asians. The odds ratios for each SNPs are summarized in Table 11.
実施例2:ミステリンのクローニング及び機能解析
(ミステリンのクローニング)
SNP2が位置する予測遺伝子C17orf27及びSNP1が位置する予測遺伝子KIAA1618のクローニングを行った。
Human Embryonic Kidney 293 細胞株から回収したmRNAから、逆転写により6つのcDNAフラグメントを得た。それらを内在性の制限酵素切断サイトを利用して結合し、全長cDNAを得た。 Example 2: Cloning and functional analysis of mysterin (cloning of mysterin)
The predicted gene C17orf27 where SNP2 is located and the predicted gene KIAA1618 where SNP1 is located were cloned.
Six cDNA fragments were obtained from mRNA recovered from the Human Embryonic Kidney 293 cell line by reverse transcription. They were combined using an endogenous restriction enzyme cleavage site to obtain a full-length cDNA.
(ミステリンのクローニング)
SNP2が位置する予測遺伝子C17orf27及びSNP1が位置する予測遺伝子KIAA1618のクローニングを行った。
Human Embryonic Kidney 293 細胞株から回収したmRNAから、逆転写により6つのcDNAフラグメントを得た。それらを内在性の制限酵素切断サイトを利用して結合し、全長cDNAを得た。 Example 2: Cloning and functional analysis of mysterin (cloning of mysterin)
The predicted gene C17orf27 where SNP2 is located and the predicted gene KIAA1618 where SNP1 is located were cloned.
Six cDNA fragments were obtained from mRNA recovered from the Human Embryonic Kidney 293 cell line by reverse transcription. They were combined using an endogenous restriction enzyme cleavage site to obtain a full-length cDNA.
KIAA1618及びC17orf27は、独立した2つの遺伝子としてNCBIのインターネットホームページ上に開示されているが(アクセッション番号:NP_066005.2及びNP_065965.3)、驚くべきことに、C17orf27及びKIAA1618はそれぞれ完全な構造遺伝子ではなく、C17orf27とKIAA1618とが1つにつながって、約5000アミノ酸からなる巨大なタンパク質をコードする1つの構造遺伝子を構成していることが明らかとなった(図1)。この新規遺伝子をミステリン(Mysterin)と命名した。ヒトミステリンのcDNA配列を配列番号1に、アミノ酸配列を配列番号2にそれぞれ示す。
KIAA1618 and C17orf27 are disclosed on the Internet homepage of NCBI as two independent genes (accession numbers: NP_066005.2 and NP_065965.3), but surprisingly, C17orf27 and KIAA1618 are complete structural genes respectively. Rather, it became clear that C17orf27 and KIAA1618 were linked together to constitute one structural gene encoding a huge protein consisting of about 5000 amino acids (FIG. 1). This new gene was named mysterin. The cDNA sequence of human mysterin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2, respectively.
(ミステリンのエクソン構造)
NCBIが開示しているKIAA1618及びC17orf27のエクソン構造と、今回同定されたミステリンのエクソン構造を比較した(表12)。KIAA1618のエクソン4及びC17orf27のエクソン13及び27が、ミステリンにおいてはスキップされていることが判明した。また、ストップコドンが存在すると考えられていたKIAA1618のエクソン18と、C17orf27のエクソン1との間に、8つのエクソンが存在することが明らかとなった。 (Exon structure of mysterin)
The exon structures of KIAA1618 and C17orf27 disclosed by NCBI were compared with the exon structure of mysterin identified this time (Table 12). It was found that exon 4 of KIAA1618 and exons 13 and 27 of C17orf27 were skipped in mysterin. It was also revealed that there were 8 exons between exon 18 of KIAA1618, which was thought to have a stop codon, andexon 1 of C17orf27.
NCBIが開示しているKIAA1618及びC17orf27のエクソン構造と、今回同定されたミステリンのエクソン構造を比較した(表12)。KIAA1618のエクソン4及びC17orf27のエクソン13及び27が、ミステリンにおいてはスキップされていることが判明した。また、ストップコドンが存在すると考えられていたKIAA1618のエクソン18と、C17orf27のエクソン1との間に、8つのエクソンが存在することが明らかとなった。 (Exon structure of mysterin)
The exon structures of KIAA1618 and C17orf27 disclosed by NCBI were compared with the exon structure of mysterin identified this time (Table 12). It was found that exon 4 of KIAA1618 and exons 13 and 27 of C17orf27 were skipped in mysterin. It was also revealed that there were 8 exons between exon 18 of KIAA1618, which was thought to have a stop codon, and
(ミステリンの発現)
C末端にMycエピトープを融合した全長ヒトミステリンタンパク質を、発現ベクターを用いてHEK293細胞に発現させた。推定550kDaの位置に発現がみられ、また、それらは細胞融解後、可溶性画分に回収された(図2)。 (Expression of mysterin)
A full-length human mysterin protein fused with a Myc epitope at the C-terminus was expressed in HEK293 cells using an expression vector. Expression was seen at an estimated 550 kDa position and they were recovered in the soluble fraction after cell lysis (FIG. 2).
C末端にMycエピトープを融合した全長ヒトミステリンタンパク質を、発現ベクターを用いてHEK293細胞に発現させた。推定550kDaの位置に発現がみられ、また、それらは細胞融解後、可溶性画分に回収された(図2)。 (Expression of mysterin)
A full-length human mysterin protein fused with a Myc epitope at the C-terminus was expressed in HEK293 cells using an expression vector. Expression was seen at an estimated 550 kDa position and they were recovered in the soluble fraction after cell lysis (FIG. 2).
(ミステリンの細胞内分布)
HAエピトープを融合した全長ヒトミステリンを、発現ベクターを用いてHela細胞株に発現させ、抗HA抗体を用いて細胞染色を行った。対照に用いたGFPは核を含む細胞質に一様に局在しているのに対してミステリンは主として細胞質ゾルに局在していた(図3)。 (Intracellular distribution of mysterin)
Full-length human mysterin fused with the HA epitope was expressed in a Hela cell line using an expression vector, and cell staining was performed using an anti-HA antibody. GFP used as a control was uniformly localized in the cytoplasm including the nucleus, whereas mysterin was mainly localized in the cytosol (FIG. 3).
HAエピトープを融合した全長ヒトミステリンを、発現ベクターを用いてHela細胞株に発現させ、抗HA抗体を用いて細胞染色を行った。対照に用いたGFPは核を含む細胞質に一様に局在しているのに対してミステリンは主として細胞質ゾルに局在していた(図3)。 (Intracellular distribution of mysterin)
Full-length human mysterin fused with the HA epitope was expressed in a Hela cell line using an expression vector, and cell staining was performed using an anti-HA antibody. GFP used as a control was uniformly localized in the cytoplasm including the nucleus, whereas mysterin was mainly localized in the cytosol (FIG. 3).
(ミステリンのユビキチンリガーゼ活性)
HAエピトープを融合した全長ヒトミステリン、あるいはRINGフィンガードメイン欠損ヒトミステリンおよび、Mycエピトープを融合したユビキチンを発現ベクターを用いてHEK293細胞株に共発現させ、抗HA抗体を用いて免疫沈降を行ったのち、抗Myc抗体を用いてウエスタンブロットを行った。自己ユビキチン化されたミステリンのシグナルがRINGフィンガードメイン依存的に検出されたことから、ミステリンがユビキチンリガーゼ活性を持つことが分かった(図4)。 (Ubiquitin ligase activity of mysterin)
Coexpression of full-length human mysterin fused with HA epitope or human mysterin lacking RING finger domain and ubiquitin fused with Myc epitope in HEK293 cell line using expression vector and immunoprecipitation using anti-HA antibody Thereafter, Western blotting was performed using an anti-Myc antibody. Since the signal of self-ubiquitinated mysterin was detected in a RING finger domain-dependent manner, it was found that mysterin has ubiquitin ligase activity (FIG. 4).
HAエピトープを融合した全長ヒトミステリン、あるいはRINGフィンガードメイン欠損ヒトミステリンおよび、Mycエピトープを融合したユビキチンを発現ベクターを用いてHEK293細胞株に共発現させ、抗HA抗体を用いて免疫沈降を行ったのち、抗Myc抗体を用いてウエスタンブロットを行った。自己ユビキチン化されたミステリンのシグナルがRINGフィンガードメイン依存的に検出されたことから、ミステリンがユビキチンリガーゼ活性を持つことが分かった(図4)。 (Ubiquitin ligase activity of mysterin)
Coexpression of full-length human mysterin fused with HA epitope or human mysterin lacking RING finger domain and ubiquitin fused with Myc epitope in HEK293 cell line using expression vector and immunoprecipitation using anti-HA antibody Thereafter, Western blotting was performed using an anti-Myc antibody. Since the signal of self-ubiquitinated mysterin was detected in a RING finger domain-dependent manner, it was found that mysterin has ubiquitin ligase activity (FIG. 4).
(ミステリンのATPase活性)
ヒトミステリンのアミノ酸配列について、BLASTプログラムを用いたホモロジー解析を行った。その結果、第2415-2436アミノ酸および第2481-2494アミノ酸の領域がそれぞれ、既知のモチーフであるWalker AおよびWalker Bモチーフと強い相同性を示した(図5)。これらのモチーフは、ダイニン、プロテアソーム、p97、FtsHなどにおいて保存されたモチーフで、それぞれNTP結合モチーフ、二価陽イオン結合モチーフであり、この二つのモチーフが共役してATPase活性をもつことが知られている。このことから、ミステリンがATPase活性をもつことが予想された。 (ATPase activity of mysterin)
The amino acid sequence of human mysterin was analyzed for homology using the BLAST program. As a result, the regions of amino acids 2415-2436 and 2481-2494 showed strong homology with the known motifs Walker A and Walker B, respectively (FIG. 5). These motifs are conserved in dynein, proteasome, p97, FtsH, etc., and are NTP binding motif and divalent cation binding motif, respectively. These two motifs are known to have ATPase activity by conjugation. ing. From this, it was predicted that mysterin has ATPase activity.
ヒトミステリンのアミノ酸配列について、BLASTプログラムを用いたホモロジー解析を行った。その結果、第2415-2436アミノ酸および第2481-2494アミノ酸の領域がそれぞれ、既知のモチーフであるWalker AおよびWalker Bモチーフと強い相同性を示した(図5)。これらのモチーフは、ダイニン、プロテアソーム、p97、FtsHなどにおいて保存されたモチーフで、それぞれNTP結合モチーフ、二価陽イオン結合モチーフであり、この二つのモチーフが共役してATPase活性をもつことが知られている。このことから、ミステリンがATPase活性をもつことが予想された。 (ATPase activity of mysterin)
The amino acid sequence of human mysterin was analyzed for homology using the BLAST program. As a result, the regions of amino acids 2415-2436 and 2481-2494 showed strong homology with the known motifs Walker A and Walker B, respectively (FIG. 5). These motifs are conserved in dynein, proteasome, p97, FtsH, etc., and are NTP binding motif and divalent cation binding motif, respectively. These two motifs are known to have ATPase activity by conjugation. ing. From this, it was predicted that mysterin has ATPase activity.
そこで、ヒトミステリンに含まれるこれらのモチーフを含む領域(第2359-2613アミノ酸)のN末端にGSTタグを融合させたタンパク質を大腸菌で発現させ、グルタチオンセファロースを用いて精製し、この融合タンパク質のATPase活性を計測した。融合タンパク質を、図6に示す最終濃度で、マグネシウムイオンおよび最終濃度5mMのATPと混合し、30分間インキュベートし、最終濃度1%のPCAと混合して反応をとめたのち、マラカイトグリーンと30分間インキュベートした。遊離リン酸とマラカイトグリーンの結合による呈色を620nmの吸光で計測したところ、融合タンパク質濃度に依存的なATPの加水分解にともなうリン酸の遊離による呈色が観察された(図6)。従って、ミステリンは、細胞内でATPase活性をもち、何らかの細胞内機能を果たすことが強く示唆された。
Therefore, a protein in which a GST tag is fused to the N-terminus of the region containing these motifs contained in human mysterin (amino acids 2359-2613) is expressed in E. coli and purified using glutathione sepharose. ATPase activity was measured. The fusion protein is mixed with magnesium ions and ATP at a final concentration of 5 mM at the final concentration shown in FIG. 6, incubated for 30 minutes, mixed with PCA at a final concentration of 1% to stop the reaction, and then reacted with malachite green for 30 minutes. Incubated. When the coloration due to the binding of free phosphate and malachite green was measured by absorbance at 620 nm, the coloration due to the release of phosphate accompanying the hydrolysis of ATP depending on the fusion protein concentration was observed (FIG. 6). Therefore, it was strongly suggested that mysterin has ATPase activity in cells and performs some intracellular functions.
実施例3:ミステリン欠損ゼブラフィッシュの作成及びその解析
(ミステリン相補DNA断片の部位設定および作成)
ゼブラフィッシュ(Danio rerio)のミステリンオーソログ遺伝子は未同定であったので、ヒトミステリンのcDNA配列に基づき、ゼブラフィッシュのミステリン遺伝子を同定し、cDNAをクローニングした。その結果、2種類のミステリン1(zRNF213)およびミステリン2(zRNF213.1)が存在し、ミステリン1(zRNF213)が、ヒト相同の遺伝子と判明した。ゼブラフィッシュミステリン1のcDNA配列を配列番号3に、アミノ酸配列を配列番号4に、それぞれ示す。尚、配列番号3の第2538~10462位のヌクレオチド(配列番号4では第847~3487位のアミノ酸に相当)については、実際にクローニングを行い、実験的にヌクレオチド配列を決定した。残りの部分については、ゼブラフィッシュのゲノム配列を元にGENSCANにより遺伝子予測を行ったヌクレオチド配列を元に、アミノ酸予測を行った。同定された配列情報を元に、ミステリン遺伝子欠損体作成のための相補DNA断片(以下ミステリンアンチセンスモルフォリノ、以下ミステリンMOと略す)の配列を決定し、合成した。ミステリンMOのヌクレオチド配列を配列番号6及び7に示す。
zRNF213spMO1-A:ACTCGTTGATGTCTGAAGTGATAAA(配列番号6)
zRNF213spMO1-D:AGCTAGGAGAAAGTCCTACCAATTT(配列番号7) Example 3: Preparation of mysterin-deficient zebrafish and analysis thereof (site setting and preparation of mysterin-complementary DNA fragment)
Since the mysterin ortholog gene of zebrafish (Danio rerio) was unidentified, the mysterin gene of zebrafish was identified based on the cDNA sequence of human mysterin, and the cDNA was cloned. As a result, two types of mysterin 1 (zRNF213) and mysterin 2 (zRNF213.1) existed, and mysterin 1 (zRNF213) was found to be a human homologous gene. The cDNA sequence ofzebrafish mysterin 1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4, respectively. The nucleotides at positions 2538 to 10462 of SEQ ID NO: 3 (corresponding to amino acids at positions 847 to 3487 in SEQ ID NO: 4) were actually cloned and the nucleotide sequence was determined experimentally. The remaining portion was subjected to amino acid prediction based on the nucleotide sequence that was gene-predicted by GENSCAN based on the zebrafish genomic sequence. Based on the identified sequence information, the sequence of a complementary DNA fragment (hereinafter referred to as mysterin antisense morpholino, hereinafter abbreviated as mysterin MO) for preparing a mysterin gene deficient was determined and synthesized. The nucleotide sequence of mysterin MO is shown in SEQ ID NOs: 6 and 7.
zRNF213spMO1-A: ACTCGTTGATGTCTGAAGTGATAAA (SEQ ID NO: 6)
zRNF213spMO1-D: AGCTAGGAGAAAGTCCTACCAATTT (SEQ ID NO: 7)
(ミステリン相補DNA断片の部位設定および作成)
ゼブラフィッシュ(Danio rerio)のミステリンオーソログ遺伝子は未同定であったので、ヒトミステリンのcDNA配列に基づき、ゼブラフィッシュのミステリン遺伝子を同定し、cDNAをクローニングした。その結果、2種類のミステリン1(zRNF213)およびミステリン2(zRNF213.1)が存在し、ミステリン1(zRNF213)が、ヒト相同の遺伝子と判明した。ゼブラフィッシュミステリン1のcDNA配列を配列番号3に、アミノ酸配列を配列番号4に、それぞれ示す。尚、配列番号3の第2538~10462位のヌクレオチド(配列番号4では第847~3487位のアミノ酸に相当)については、実際にクローニングを行い、実験的にヌクレオチド配列を決定した。残りの部分については、ゼブラフィッシュのゲノム配列を元にGENSCANにより遺伝子予測を行ったヌクレオチド配列を元に、アミノ酸予測を行った。同定された配列情報を元に、ミステリン遺伝子欠損体作成のための相補DNA断片(以下ミステリンアンチセンスモルフォリノ、以下ミステリンMOと略す)の配列を決定し、合成した。ミステリンMOのヌクレオチド配列を配列番号6及び7に示す。
zRNF213spMO1-A:ACTCGTTGATGTCTGAAGTGATAAA(配列番号6)
zRNF213spMO1-D:AGCTAGGAGAAAGTCCTACCAATTT(配列番号7) Example 3: Preparation of mysterin-deficient zebrafish and analysis thereof (site setting and preparation of mysterin-complementary DNA fragment)
Since the mysterin ortholog gene of zebrafish (Danio rerio) was unidentified, the mysterin gene of zebrafish was identified based on the cDNA sequence of human mysterin, and the cDNA was cloned. As a result, two types of mysterin 1 (zRNF213) and mysterin 2 (zRNF213.1) existed, and mysterin 1 (zRNF213) was found to be a human homologous gene. The cDNA sequence of
zRNF213spMO1-A: ACTCGTTGATGTCTGAAGTGATAAA (SEQ ID NO: 6)
zRNF213spMO1-D: AGCTAGGAGAAAGTCCTACCAATTT (SEQ ID NO: 7)
(ミステリンMOによるミステリン遺伝子の生体内操作)
ミステリンMOを受精卵に打ち込み、ゼブラフィッシュの発生初期に不完全なミステリンタンパク質を産生させることにより、生体内でミステリンが機能しない個体を作製した。詳細には、以下のようにして、ミステリン欠損ゼブラフィッシュを作成した。 (In-vivo manipulation of mysterin gene by mysterin MO)
An individual in which mysterin does not function in vivo was produced by implanting mysterin MO into a fertilized egg and producing incomplete mysterin protein in the early stage of zebrafish development. Specifically, a mysterin-deficient zebrafish was prepared as follows.
ミステリンMOを受精卵に打ち込み、ゼブラフィッシュの発生初期に不完全なミステリンタンパク質を産生させることにより、生体内でミステリンが機能しない個体を作製した。詳細には、以下のようにして、ミステリン欠損ゼブラフィッシュを作成した。 (In-vivo manipulation of mysterin gene by mysterin MO)
An individual in which mysterin does not function in vivo was produced by implanting mysterin MO into a fertilized egg and producing incomplete mysterin protein in the early stage of zebrafish development. Specifically, a mysterin-deficient zebrafish was prepared as follows.
5ngのミステリン エクソンアクセプターサイトに対するミステリンMO及びドナーサイトに対するミステリンMOを、1から8細胞段階のゼブラフィッシュ胚に打ち込んだ。この時に使用したゼブラフィッシュ胚の遺伝的背景は、血管内皮細胞特異的にGFPを発現するライン、fli-EGFP(Tg(fli1:egfp))であり(Lawson ND and Weinstein BM, Developmental Biology 243:307-318 (2002))、このラインは血管イメージング分析用ツールとして有用である。
5 ng mysterin Mysterin MO for exon acceptor site and mysterin MO for donor site were implanted into zebrafish embryos at 1 to 8 cell stage. The genetic background of the zebrafish embryo used at this time is a line expressing GFP specifically in vascular endothelial cells, fli-EGFP (Tg (fl1: egfp)) (Lawson ND and Weinstein BM, Developmental Biology 243: 307 -318 (2002)), this line is useful as a blood vessel imaging analysis tool.
ゼブラフィッシュの飼育は、明期14h、暗期10h、28℃という人工条件下で行った。飼育器が暗期に入る前にゼブラフィッシュの雄と雌を隔離し、翌朝、明期になった直後に逆に雄と雌を会わせることにより、交配を行った。受精から30分以内に卵を採集し、上述のアンチセンスモルフォリノを、一試験あたり60-180個の卵にインジェクションを行った。インジェクションを施した卵を、28℃の条件で、メチレンブルー及び0.03% 海水塩を含有する水中でインキュベートした。
Zebrafish were reared under artificial conditions of 14 h light period, 10 h dark period, and 28 ° C. Mating was performed by isolating zebrafish males and females before the incubator enters the dark period, and conversely with the males and females the next morning immediately after the light period. Eggs were collected within 30 minutes of fertilization and the above-described antisense morpholinos were injected into 60-180 eggs per test. The injected eggs were incubated in water containing methylene blue and 0.03% sea salt at 28 ° C.
インジェクションの施された卵をメチレンブルー及び0.03% 海水塩を含有する水ですすぎ、内部温度28.5℃のインキュベーター内で培養した。培養72時間後の発生中の胚において蛍光を観察確認した。
The injected egg was rinsed with water containing methylene blue and 0.03% seawater salt and cultured in an incubator with an internal temperature of 28.5 ° C. Fluorescence was observed and confirmed in developing embryos after 72 hours of culture.
(ミステリンMOによるミステリン遺伝子のエクソンスキップの確認)
ミステリンMOをインジェクションした胚およびインジェクションしていない野性型胚より全RNAを抽出し、ABI社の逆転写酵素およびランダムプライマー(High capacity cDNA reverse transcription Kit)を用いて、cDNAを合成した。続いてPCR法により、該当エクソン周辺のcDNAを増幅した。具体的にはタカラ社のtaqポリメラーゼ(eX Tag)を用いて反応系を調整し、ニップンテクノクラスタ社のサーマルサイクラー(Palm-Cycler)を用いてPCR(35サイクル)を行うことにより、エクソンスキップの効果を検証した。その結果、野性型胚ではターゲット配列から予測された通りの長さのPCR産物が得られることが確認された(図7)。一方、ミステリンMOをインジェクションした胚では、エクソンがスキップしたと思われる短いPCR産物が得られる結果となった。それぞれのPCR産物を単離しシークエンスを決定したところ、ミステリンMOは実験デザイン通りに有効に機能していることが示された。 (Confirmation of exon skip of mysterin gene by mysterin MO)
Total RNA was extracted from embryos injected with mysterin MO and wild-type embryos not injected, and cDNA was synthesized using ABI reverse transcriptase and random primers (High capacity cDNA reverse transcription Kit). Subsequently, cDNA around the exon was amplified by PCR. Specifically, the reaction system is adjusted using taq polymerase (eX Tag) manufactured by Takara, and PCR (35 cycles) is performed using a thermal cycler (Palm-Cycler) manufactured by Nipple Technocluster Co. The effect was verified. As a result, it was confirmed that a PCR product having a length predicted from the target sequence was obtained in the wild type embryo (FIG. 7). On the other hand, in the embryo injected with mysterin MO, a short PCR product that exon seems to have skipped was obtained. When each PCR product was isolated and the sequence was determined, it was shown that mysterin MO functions effectively as designed in the experiment.
ミステリンMOをインジェクションした胚およびインジェクションしていない野性型胚より全RNAを抽出し、ABI社の逆転写酵素およびランダムプライマー(High capacity cDNA reverse transcription Kit)を用いて、cDNAを合成した。続いてPCR法により、該当エクソン周辺のcDNAを増幅した。具体的にはタカラ社のtaqポリメラーゼ(eX Tag)を用いて反応系を調整し、ニップンテクノクラスタ社のサーマルサイクラー(Palm-Cycler)を用いてPCR(35サイクル)を行うことにより、エクソンスキップの効果を検証した。その結果、野性型胚ではターゲット配列から予測された通りの長さのPCR産物が得られることが確認された(図7)。一方、ミステリンMOをインジェクションした胚では、エクソンがスキップしたと思われる短いPCR産物が得られる結果となった。それぞれのPCR産物を単離しシークエンスを決定したところ、ミステリンMOは実験デザイン通りに有効に機能していることが示された。 (Confirmation of exon skip of mysterin gene by mysterin MO)
Total RNA was extracted from embryos injected with mysterin MO and wild-type embryos not injected, and cDNA was synthesized using ABI reverse transcriptase and random primers (High capacity cDNA reverse transcription Kit). Subsequently, cDNA around the exon was amplified by PCR. Specifically, the reaction system is adjusted using taq polymerase (eX Tag) manufactured by Takara, and PCR (35 cycles) is performed using a thermal cycler (Palm-Cycler) manufactured by Nipple Technocluster Co. The effect was verified. As a result, it was confirmed that a PCR product having a length predicted from the target sequence was obtained in the wild type embryo (FIG. 7). On the other hand, in the embryo injected with mysterin MO, a short PCR product that exon seems to have skipped was obtained. When each PCR product was isolated and the sequence was determined, it was shown that mysterin MO functions effectively as designed in the experiment.
(血管新生の観察)
蛍光実体顕微鏡、Leica MZ-16FA(ライカ マイクロシステムズ社)により、経時的にGFPの蛍光を観察した。その結果、野性型胚、ミステリンMOをインジェクションした胚ともに血管新生は生じた。一方、36hpf(hours post fertilization)あたりから、ミステリンMOをインジェクションした胚に発生の遅滞が起きていた。初期血管発生はほぼ正常に進行したものの、それに続く血管新生に異常が見られた(図8A及びB)。また、ミステリン発現抑制ゼブラフィッシュにおいては目の周辺に特徴的な異常血管新生が見られた。これらの表現型から、ミステリンは血管構築の形成に重要な機能を有することが示唆された。尾部および眼内における血管新生の異常は、対照(野性型)100個体を観察した中では全く認められなかったが、これに対して、実験群(ミステリンMO)では100匹中92匹にこの表現型が認められた。以上より、ミステリンは、血管構築に重要な機能を持つことが示唆された。 (Observation of angiogenesis)
Fluorescence of GFP was observed over time with a fluorescent stereomicroscope, Leica MZ-16FA (Leica Microsystems). As a result, angiogenesis occurred in both wild type embryos and embryos injected with mysterin MO. On the other hand, from around 36 hpf (hours post fertilization), development delay occurred in embryos injected with mysterin MO. Although initial angiogenesis progressed almost normally, abnormalities were observed in subsequent angiogenesis (FIGS. 8A and B). In the mysterin expression-suppressed zebrafish, characteristic abnormal angiogenesis was observed around the eyes. These phenotypes suggested that mysterin has an important function in the formation of vascular architecture. No abnormalities in angiogenesis in the tail and in the eye were observed in 100 control (wild type) individuals, whereas in the experimental group (mysterin MO) this expression was present in 92 out of 100 animals. The mold was recognized. From the above, it was suggested that mysterin has an important function for blood vessel construction.
蛍光実体顕微鏡、Leica MZ-16FA(ライカ マイクロシステムズ社)により、経時的にGFPの蛍光を観察した。その結果、野性型胚、ミステリンMOをインジェクションした胚ともに血管新生は生じた。一方、36hpf(hours post fertilization)あたりから、ミステリンMOをインジェクションした胚に発生の遅滞が起きていた。初期血管発生はほぼ正常に進行したものの、それに続く血管新生に異常が見られた(図8A及びB)。また、ミステリン発現抑制ゼブラフィッシュにおいては目の周辺に特徴的な異常血管新生が見られた。これらの表現型から、ミステリンは血管構築の形成に重要な機能を有することが示唆された。尾部および眼内における血管新生の異常は、対照(野性型)100個体を観察した中では全く認められなかったが、これに対して、実験群(ミステリンMO)では100匹中92匹にこの表現型が認められた。以上より、ミステリンは、血管構築に重要な機能を持つことが示唆された。 (Observation of angiogenesis)
Fluorescence of GFP was observed over time with a fluorescent stereomicroscope, Leica MZ-16FA (Leica Microsystems). As a result, angiogenesis occurred in both wild type embryos and embryos injected with mysterin MO. On the other hand, from around 36 hpf (hours post fertilization), development delay occurred in embryos injected with mysterin MO. Although initial angiogenesis progressed almost normally, abnormalities were observed in subsequent angiogenesis (FIGS. 8A and B). In the mysterin expression-suppressed zebrafish, characteristic abnormal angiogenesis was observed around the eyes. These phenotypes suggested that mysterin has an important function in the formation of vascular architecture. No abnormalities in angiogenesis in the tail and in the eye were observed in 100 control (wild type) individuals, whereas in the experimental group (mysterin MO) this expression was present in 92 out of 100 animals. The mold was recognized. From the above, it was suggested that mysterin has an important function for blood vessel construction.
実施例4:虚血性心疾患の発症リスクとSNP2との関連
1998年に厚生労働省により行われた国民基礎生活調査の頻度をもとに虚血性心疾患の有病率を年齢階層でもとめ、観察値と比較した。その結果一般的に虚血性心疾患のリスクが低いと想定される35-54歳の比較的若年の女性で、SNP2のマイナーアレル(創始者変異)を有するものでは虚血性心疾患の有病率が有意に高いことが見いだされた(表13)。 Example 4: Relationship between risk of developing ischemic heart disease and SNP2 Based on the frequency of the National Basic Life Survey conducted by the Ministry of Health, Labor and Welfare in 1998, the prevalence of ischemic heart disease was also observed in the age group. Compared with value. As a result, the prevalence of ischemic heart disease in relatively young women aged 35-54, who are generally assumed to be at low risk for ischemic heart disease, with minor alleles (founder mutations) of SNP2 Was found to be significantly higher (Table 13).
1998年に厚生労働省により行われた国民基礎生活調査の頻度をもとに虚血性心疾患の有病率を年齢階層でもとめ、観察値と比較した。その結果一般的に虚血性心疾患のリスクが低いと想定される35-54歳の比較的若年の女性で、SNP2のマイナーアレル(創始者変異)を有するものでは虚血性心疾患の有病率が有意に高いことが見いだされた(表13)。 Example 4: Relationship between risk of developing ischemic heart disease and SNP2 Based on the frequency of the National Basic Life Survey conducted by the Ministry of Health, Labor and Welfare in 1998, the prevalence of ischemic heart disease was also observed in the age group. Compared with value. As a result, the prevalence of ischemic heart disease in relatively young women aged 35-54, who are generally assumed to be at low risk for ischemic heart disease, with minor alleles (founder mutations) of SNP2 Was found to be significantly higher (Table 13).
実施例5:一卵性双生児の分娩可能性とSNP2との関連
SNP2のマイナーアレル(創始者変異)を有する家系では、264分娩中5例で一卵生双生児を認めた。日本における一卵性双生児の出生頻度はおおよそ1000分娩に対して4であると報告されているから(J Prev Soc Med, vol.30, pages 175-179, 1976;Int J Biometeorol, vol. 43, pages 91-95, 1999)、SNP2のマイナーアレルを有していると一卵性双生児の分娩可能性が高くなることが示された(オッズ比=約5、p<0.001)。 Example 5: Possibility of delivery of identical twins and association with SNP2 In families with a minor allele of SNP2 (founder mutation), 5 twins were found in 264 delivery. It is reported that the birth frequency of monozygotic twins in Japan is approximately 4 for 1000 deliveries (J Prev Soc Med, vol. 30, pages 175-179, 1976; Int J Biometeorol, vol. 43, pages 91-95, 1999), it was shown that having a minor allele of SNP2 increases the chance of delivery of identical twins (odds ratio = approximately 5, p <0.001).
SNP2のマイナーアレル(創始者変異)を有する家系では、264分娩中5例で一卵生双生児を認めた。日本における一卵性双生児の出生頻度はおおよそ1000分娩に対して4であると報告されているから(J Prev Soc Med, vol.30, pages 175-179, 1976;Int J Biometeorol, vol. 43, pages 91-95, 1999)、SNP2のマイナーアレルを有していると一卵性双生児の分娩可能性が高くなることが示された(オッズ比=約5、p<0.001)。 Example 5: Possibility of delivery of identical twins and association with SNP2 In families with a minor allele of SNP2 (founder mutation), 5 twins were found in 264 delivery. It is reported that the birth frequency of monozygotic twins in Japan is approximately 4 for 1000 deliveries (J Prev Soc Med, vol. 30, pages 175-179, 1976; Int J Biometeorol, vol. 43, pages 91-95, 1999), it was shown that having a minor allele of SNP2 increases the chance of delivery of identical twins (odds ratio = approximately 5, p <0.001).
実施例6:白人家系に見出されたモヤモヤ病に関連する新たなSNP
チェコ共和国の白人家系(CAU-Ped1、図11)とドイツの12の孤発例を対象に遺伝解析を行った。本家系の発端者(CAU_Ped1_12)は30歳時に軽度の虚血性脳卒中を発症しており、また彼の母は虚血性脳卒中により35歳時に死亡している。発端者は、2名の非血縁配偶者との間に4人の子孫をもうけた。第一の配偶者との第2子(CAU_Ped1_122)は5歳時にモヤモヤ病と診断された。第2の配偶者との第1子(CAU_Ped1_123)は9歳時に不随意運動ともなったモヤモヤ病を発症し、第2子(CAU_Ped1_124)は3歳時に虚血性脳卒中で発症しモヤモヤ病と診断された。モヤモヤ病の診断は磁気共鳴画像(MRI)にて行われ、発症者は全て日本のモヤモヤ病の診断基準を満たすことを確認した。遺伝形式から、典型的な常染色体優性遺伝形式に従うものと判断された。また我々はチェコ共和国のパラツキー大学、オロモウツ大学病院、ドイツのテュービンゲン大学、及びCoriell instituteより購入したCoriell Caucasian Panelより314人を白人対照群として解析を行った。対照群の平均年齢は31.8±14.0歳、男女比は129:185であり、MRIスクリーニングは未施行である。 Example 6: New SNPs related to moyamoya disease found in Caucasian families
Genetic analysis was conducted on white spouses from the Czech Republic (CAU-Ped1, Fig. 11) and 12 sporadic cases in Germany. The proband of the family (CAU_Ped1_12) developed a mild ischemic stroke at the age of 30, and his mother died at the age of 35 due to ischemic stroke. The proband had four offspring with two unrelated spouses. The second child (CAU_Ped1_122) with the first spouse was diagnosed with moyamoya disease at the age of five. The first child (CAU_Ped1_123) with the second spouse developed moyamoya disease that became involuntary movement at the age of nine, and the second child (CAU_Ped1_124) developed with ischemic stroke at the age of three and was diagnosed with moyamoya disease . The diagnosis of moyamoya disease was performed by magnetic resonance imaging (MRI), and it was confirmed that all the onset patients met the diagnostic criteria for moyamoya disease in Japan. From the inheritance format, it was judged to follow a typical autosomal dominant inheritance format. We also analyzed 314 white control groups from the Coriell Caucasian Panel purchased from the University of Palatsky, Olomouc University Hospital, the University of Tubingen, Germany, and the Coriell institute. The average age of the control group is 31.8 ± 14.0 years old, the male / female ratio is 129: 185, and MRI screening has not been performed.
チェコ共和国の白人家系(CAU-Ped1、図11)とドイツの12の孤発例を対象に遺伝解析を行った。本家系の発端者(CAU_Ped1_12)は30歳時に軽度の虚血性脳卒中を発症しており、また彼の母は虚血性脳卒中により35歳時に死亡している。発端者は、2名の非血縁配偶者との間に4人の子孫をもうけた。第一の配偶者との第2子(CAU_Ped1_122)は5歳時にモヤモヤ病と診断された。第2の配偶者との第1子(CAU_Ped1_123)は9歳時に不随意運動ともなったモヤモヤ病を発症し、第2子(CAU_Ped1_124)は3歳時に虚血性脳卒中で発症しモヤモヤ病と診断された。モヤモヤ病の診断は磁気共鳴画像(MRI)にて行われ、発症者は全て日本のモヤモヤ病の診断基準を満たすことを確認した。遺伝形式から、典型的な常染色体優性遺伝形式に従うものと判断された。また我々はチェコ共和国のパラツキー大学、オロモウツ大学病院、ドイツのテュービンゲン大学、及びCoriell instituteより購入したCoriell Caucasian Panelより314人を白人対照群として解析を行った。対照群の平均年齢は31.8±14.0歳、男女比は129:185であり、MRIスクリーニングは未施行である。 Example 6: New SNPs related to moyamoya disease found in Caucasian families
Genetic analysis was conducted on white spouses from the Czech Republic (CAU-Ped1, Fig. 11) and 12 sporadic cases in Germany. The proband of the family (CAU_Ped1_12) developed a mild ischemic stroke at the age of 30, and his mother died at the age of 35 due to ischemic stroke. The proband had four offspring with two unrelated spouses. The second child (CAU_Ped1_122) with the first spouse was diagnosed with moyamoya disease at the age of five. The first child (CAU_Ped1_123) with the second spouse developed moyamoya disease that became involuntary movement at the age of nine, and the second child (CAU_Ped1_124) developed with ischemic stroke at the age of three and was diagnosed with moyamoya disease . The diagnosis of moyamoya disease was performed by magnetic resonance imaging (MRI), and it was confirmed that all the onset patients met the diagnostic criteria for moyamoya disease in Japan. From the inheritance format, it was judged to follow a typical autosomal dominant inheritance format. We also analyzed 314 white control groups from the Coriell Caucasian Panel purchased from the University of Palatsky, Olomouc University Hospital, the University of Tubingen, Germany, and the Coriell institute. The average age of the control group is 31.8 ± 14.0 years old, the male / female ratio is 129: 185, and MRI screening has not been performed.
発端者である父の血液からゲノムDNAを抽出し、SLC26A11、KIAA1618、LOC100287062、C17orf27、FLJ35220、LOC728991及びNPTX1の7つの遺伝子の全コード領域についてダイレクトシークエンスを行い、165個のvariant(登録済み:133個、未登録:32個)を認めた。それらのvariantのうちコード領域に存在する未登録variantはC17orf27上の1つ (配列番号5の55977位G>A;ヒトミステリンの第4013番のアミノ酸置換(アスパラギン酸→アスパラギン)(D4013N))のみであった。このvariantはモヤモヤ病に罹患している三人の子供に遺伝していた(図11)。
Genomic DNA was extracted from the blood of the proband father, and direct sequencing was performed on all coding regions of the seven genes SLC26A11, KIAA1618, LOC100287062, C17orf27, FLJ35220, LOC728991, and NPTX1, and 165 variant (registered: 133) Pieces, unregistered: 32 pieces). Among these variants, an unregistered variant present in the coding region is one of C17orf27 (SEQ ID NO: 5, 55777 position G> A; human mysterin No. 4013 amino acid substitution (aspartic acid → asparagine) (D4013N)) It was only. This variant was inherited by three children with moyamoya disease (FIG. 11).
ドイツの孤発例でも同様に解析したところ、C17orf27上に以下の2つのvariantを得た:
配列番号5の55712位A>G;ヒトミステリンの第3962番のアミノ酸置換(アスパラギン→アスパラギン酸)(N3962D);及び
配列番号5の57483位G>A;ヒトミステリンの第4062番のアミノ酸置換(アルギニン→グルタミン)(R4062Q)。 A similar analysis of a sporadic case in Germany yielded the following two variants on C17orf27:
SEQ ID NO: 5 at position 57712 A>G; amino acid substitution at position 3962 of human mysterin (asparagine → aspartic acid) (N3962D); and position 57483 at SEQ ID NO: 5 at position G>A; amino acid at position 4062 of human mysterin Substitution (arginine → glutamine) (R4062Q).
配列番号5の55712位A>G;ヒトミステリンの第3962番のアミノ酸置換(アスパラギン→アスパラギン酸)(N3962D);及び
配列番号5の57483位G>A;ヒトミステリンの第4062番のアミノ酸置換(アルギニン→グルタミン)(R4062Q)。 A similar analysis of a sporadic case in Germany yielded the following two variants on C17orf27:
SEQ ID NO: 5 at position 57712 A>G; amino acid substitution at position 3962 of human mysterin (asparagine → aspartic acid) (N3962D); and position 57483 at SEQ ID NO: 5 at position G>A; amino acid at position 4062 of human mysterin Substitution (arginine → glutamine) (R4062Q).
更に、314人の白人対照者について制限酵素HpyCH4V、HinfIとMspIを用いたRFLP法によるスクリーニングを行ったがD4013N、N3962D、R4062Qアリルは見出されず、白人集団でのアリル頻度は1%以下であり、突然変異と考えられた。以上より、D4013Nアリルが本家系モヤモヤ病の原因となる突然変異であると結論づけた。D4013Nは、RINGフィンガードメインに位置しているが、生化学的な分析では、ユビキチンリガーゼ活性に影響を与えなかった。
Furthermore, 314 Caucasian controls were screened by the RFLP method using restriction enzymes HpyCH4V, HinfI and MspI, but D4013N, N3962D, R4062Q allele was not found, and the allele frequency in the Caucasian population was 1% or less, It was considered a mutation. Based on the above, it was concluded that D4013N allele is a mutation causing causative Moyamoya disease. D4013N is located in the RING finger domain, but biochemical analysis did not affect ubiquitin ligase activity.
本発明により、新規遺伝子ミステリンのポリペプチドや該ポリペプチドをコードするポリヌクレオチド、発現ベクター、ミステリンの機能的欠損を含む動物等が提供される。ヒトゲノム遺伝子配列の網羅的な解析から、ミステリンの遺伝子座には、C17orf27及びKIAA1618の2つの独立した構造遺伝子の存在が予測されていたが、C17orf27及びKIAA1618はそれぞれ完全な構造遺伝子ではなく、C17orf27とKIAA1618とが1つにつながってミステリン遺伝子座を構成していることが明らかとなった。
ミステリン遺伝子のノックダウンゼブラフィッシュの解析等から、ミステリンは血管新生を調節する機能を有することが明らかとなった。従って、ミステリンの発現や機能を調節する物質をスクリーニングすることにより、新規機序に立脚した血管新生調節剤の候補物質を得ることが出来る。
また、本発明により、ミステリン遺伝子座またはその近傍に存在する新たなSNPsが提供される。該SNPsはモヤモヤ病、虚血性心疾患、一卵性双生児の分娩頻度と相関することから、このSNPsを解析することにより、モヤモヤ病や虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性を判定することが可能である。
本出願は日本で出願された特願2009-244938(出願日:2009年10月23日)を基礎としており、その内容は本明細書に全て包含されるものである。 According to the present invention, a polypeptide of a novel gene mysterin, a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided. Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus.
Analysis of knockdown zebrafish of the mysterin gene revealed that mysterin has a function of regulating angiogenesis. Therefore, by screening for substances that regulate the expression and function of mysterin, candidate substances for angiogenesis regulators based on a novel mechanism can be obtained.
The present invention also provides new SNPs that exist at or near the mysterin locus. The SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
This application is based on Japanese Patent Application No. 2009-244938 filed in Japan (filing date: October 23, 2009), the contents of which are incorporated in full herein.
ミステリン遺伝子のノックダウンゼブラフィッシュの解析等から、ミステリンは血管新生を調節する機能を有することが明らかとなった。従って、ミステリンの発現や機能を調節する物質をスクリーニングすることにより、新規機序に立脚した血管新生調節剤の候補物質を得ることが出来る。
また、本発明により、ミステリン遺伝子座またはその近傍に存在する新たなSNPsが提供される。該SNPsはモヤモヤ病、虚血性心疾患、一卵性双生児の分娩頻度と相関することから、このSNPsを解析することにより、モヤモヤ病や虚血性心疾患の発症リスクや、一卵性双生児の分娩可能性を判定することが可能である。
本出願は日本で出願された特願2009-244938(出願日:2009年10月23日)を基礎としており、その内容は本明細書に全て包含されるものである。 According to the present invention, a polypeptide of a novel gene mysterin, a polynucleotide encoding the polypeptide, an expression vector, an animal containing a mysterin functional defect, and the like are provided. Comprehensive analysis of the human genome gene sequence predicted the existence of two independent structural genes, C17orf27 and KIAA1618, at the mysterin locus, but C17orf27 and KIAA1618 are not complete structural genes, respectively. It became clear that KIAA1618 was connected to one to constitute the mysterin locus.
Analysis of knockdown zebrafish of the mysterin gene revealed that mysterin has a function of regulating angiogenesis. Therefore, by screening for substances that regulate the expression and function of mysterin, candidate substances for angiogenesis regulators based on a novel mechanism can be obtained.
The present invention also provides new SNPs that exist at or near the mysterin locus. The SNPs correlate with the frequency of moyamoya disease, ischemic heart disease, and the frequency of delivery of identical twins. By analyzing this SNPs, the risk of developing moyamoya disease and ischemic heart disease, the delivery of identical twins It is possible to determine the possibility.
This application is based on Japanese Patent Application No. 2009-244938 filed in Japan (filing date: October 23, 2009), the contents of which are incorporated in full herein.
Claims (25)
- 以下の(1)及び(4)~(7)から選択されるいずれかのポリペプチド:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンがリジンに置換されている);
(4)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4013番のアスパラギン酸がアスパラギンに置換されている);
(5)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第3962番のアスパラギンはアスパラギン酸に置換されていてもよい);
(6)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4062番のアルギニンはグルタミンに置換されていてもよい);及び
(7)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列が、R4810K、D4013N、N3962D及びR4062Qからなる群から選択される、2、3又は4個の置換を含んでいてもよい)。 Any polypeptide selected from the following (1) and (4) to (7):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine);
(4) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, aspartic acid at position 4013 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with asparagine);
(5) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the asparagine of No. 3962 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with aspartic acid);
(6) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein 4062 arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with glutamine); and (7) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of R4810K, D4013N, N3962D and R4062Q, May include substitution). - 配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンがリジンに置換されている)である、請求項1記載のポリペプチド。 The polypeptide according to claim 1, which is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (wherein arginine at position 4810 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine). .
- 請求項1又は2記載のポリペプチドをコードするヌクレオチド配列を含むポリヌクレオチド。 A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to claim 1 or 2.
- 請求項3記載のポリヌクレオチドを含む発現ベクター。 An expression vector comprising the polynucleotide according to claim 3.
- 請求項4記載の発現ベクターで形質転換された形質転換体。 A transformant transformed with the expression vector according to claim 4.
- 以下の(1)~(3)から選択されるいずれかのポリペプチドをコードする遺伝子の機能的欠損を含む非ヒト動物:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。 Non-human animals containing a functional defect of a gene encoding any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity. - 血管新生異常の動物モデルとしての、請求項6記載の動物の使用。 Use of the animal according to claim 6 as an animal model of abnormal angiogenesis.
- 以下の(1)~(3)から選択されるいずれかのポリペプチドをコードする遺伝子の機能的欠損を含む動物細胞:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。 Animal cells containing a functional defect of a gene encoding any polypeptide selected from the following (1) to (3):
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity. - 被検物質が以下の(1)~(3)から選択されるいずれかのポリペプチドをコードする遺伝子の発現または機能を調節し得るか否かを評価することを含む、血管新生調節剤の候補物質のスクリーニング方法:
(1)配列番号2で表されるアミノ酸配列を含むポリペプチド(ここで、配列番号2で表されるアミノ酸配列における第4810番のアルギニンはリジンに置換されていてもよい);
(2)配列番号2で表されるアミノ酸配列と45%以上の同一性を有するアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド;及び
(3)配列番号2で表されるアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含み、且つユビキチンリガーゼ活性及びATPase活性を有するポリペプチド。 A candidate for angiogenesis-regulating agent, comprising evaluating whether a test substance can regulate the expression or function of a gene encoding any polypeptide selected from the following (1) to (3): Substance screening method:
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (here, the 4810th arginine in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with lysine);
(2) a polypeptide comprising an amino acid sequence having 45% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having ubiquitin ligase activity and ATPase activity; and (3) the amino acid represented by SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the sequence, and having ubiquitin ligase activity and ATPase activity. - 配列番号5で表されるヌクレオチド配列の連続した部分配列又はその相補配列を含むポリヌクレオチドであって、該部分配列は、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを含み、該部分配列に含まれる該SNPのアレルはマイナーアレルであり、且つ該部分配列又はその相補配列は少なくとも12ヌクレオチドの長さを有する、ポリヌクレオチド。 A polynucleotide comprising a contiguous partial sequence of the nucleotide sequence represented by SEQ ID NO: 5 or a complementary sequence thereof, wherein the partial sequence is 4766 > T> C (SNP1), 73097 G> A (SNP2), 1207664 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5), 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 及 び G> A (SNP8) A polynucleotide comprising at least one SNP, wherein the allele of the SNP contained in the partial sequence is a minor allele, and the partial sequence or its complementary sequence has a length of at least 12 nucleotides.
- 該SNPが、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つである、請求項10記載のポリヌクレオチド。 The SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 15291715G> A (SNP4) and 232102 G> A (SNP5). The polynucleotide of claim 10, wherein the polynucleotide is at least one.
- 該SNPが73097 G>A(SNP2)である、請求項10記載のポリヌクレオチド。 The polynucleotide according to claim 10, wherein the SNP is 73097 G> A (SNP2).
- 該SNPが55977 G>A(SNP6)である、請求項10記載のポリヌクレオチド。 The polynucleotide according to claim 10, wherein the SNP is 55977 G> A (SNP6).
- 配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを検出することを含む、モヤモヤ病の発症リスクの判定方法。 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5) in the nucleotide sequence represented by SEQ ID NO: 5, A method for determining the risk of developing Moyamoya disease, comprising detecting at least one SNP selected from the group consisting of 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8).
- 該SNPが、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つである、請求項14記載の方法。 The SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 15291715G> A (SNP4) and 232102 G> A (SNP5). 15. The method of claim 14, wherein there is at least one.
- 該SNPが73097 G>A(SNP2)である、請求項14記載の方法。 The method according to claim 14, wherein the SNP is 7309730G> A (SNP2).
- 該SNPが55977 G>A(SNP6)である、請求項14記載の方法。 The method according to claim 14, wherein the SNP is 55977 G> A (SNP6).
- 配列番号5で表されるヌクレオチド配列における4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)、232102 G>A(SNP5)、55977 G>A(SNP6)、55712 A>G(SNP7)及び57483 G>A(SNP8)からなる群から選択される少なくとも1つのSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、モヤモヤ病の発症リスクの診断剤。 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 152917 G> A (SNP4), 232102 G> A (SNP5) in the nucleotide sequence represented by SEQ ID NO: 5, A nucleic acid probe capable of specifically detecting at least one SNP selected from the group consisting of 55977 G> A (SNP6), 55712 A> G (SNP7) and 57483 G> A (SNP8), or a region containing the SNP A diagnostic agent for the risk of developing Moyamoya disease, comprising a primer capable of specifically amplifying the lysate.
- 該SNPが、4766 T>C(SNP1)、73097 G>A(SNP2)、120764 G>A(SNP3)、152917 G>A(SNP4)及び232102 G>A(SNP5)からなる群から選択される少なくとも1つである、請求項18記載の診断剤。 The SNP is selected from the group consisting of 4766 T> C (SNP1), 73097 G> A (SNP2), 12076 G> A (SNP3), 15291715G> A (SNP4) and 232102 G> A (SNP5). 19. The diagnostic agent according to claim 18, wherein the diagnostic agent is at least one.
- 該SNPが73097 G>A(SNP2)である、請求項18記載の診断剤。 The diagnostic agent according to claim 18, wherein the SNP is 73097 G> A (SNP2).
- 該SNPが55977 G>A(SNP6)である、請求項18記載の診断剤。 The diagnostic agent according to claim 18, wherein the SNP is 55977 G> A (SNP6).
- 配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、虚血性心疾患の発症リスクの判定方法。 A method for determining the risk of developing ischemic heart disease, comprising detecting an SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
- 配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、虚血性心疾患の発症リスクの診断剤。 Ischemic heart disease comprising a nucleic acid probe capable of specifically detecting SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5 or a primer capable of specifically amplifying a region containing the SNP A diagnostic agent for the risk of developing the disease.
- 配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを検出することを含む、一卵性双生児の分娩可能性の判定方法。 A method for determining the possibility of delivery of identical twins, comprising detecting a SNP of 73097 G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5.
- 配列番号5で表されるヌクレオチド配列における73097 G>A(SNP2)のSNPを特異的に検出し得る核酸プローブ、又は該SNPを含む領域を特異的に増幅し得るプライマーを含む、一卵性双生児の分娩可能性の診断剤。 An identical twin comprising a nucleic acid probe capable of specifically detecting a SNP of 730977G> A (SNP2) in the nucleotide sequence represented by SEQ ID NO: 5, or a primer capable of specifically amplifying a region containing the SNP A diagnostic agent for the possibility of delivery.
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CN110396525A (en) * | 2019-07-03 | 2019-11-01 | 中山大学附属第一医院 | RNF213 gene completely knocks out the preparation method and purposes of homozygote zebra fish |
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Cited By (5)
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KR20160010269A (en) * | 2014-12-01 | 2016-01-27 | 이화여자대학교 산학협력단 | Composition for diagnosing Moyamoya disease using CpG methylation status in promoter region of SNAR-G2 gene and uses thereof |
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WO2019225803A1 (en) * | 2018-05-25 | 2019-11-28 | 의료법인 성광의료재단 | Association between rnf213 single nucleotide polymorphism and risk of developing moyamoya disease in koreans |
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