JP2006121953A - CALCIUM CHANNEL, VOLTAGE-DEPENDENT, P/Q TYPE, ALPHA 1a SUBUNIT GENE-MUTANT RAT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new model animal for Cacna1a-related diseases useful for screening preventive or therapeutic materials for the Cacna1a associated diseases. <P>SOLUTION: A method for producing a Cacna1a-related disease model nonhuman vertebrate including modification of a chromosome to express a specific mutant Cacna1a polypeptide is provided. The Cacna1a-related disease model nonhuman vertebrate produced by the method, especially the Groggy rat is useful for screening preventive or therapeutic materials for the Cacna1a-related diseases or methods for treating the diseases. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a mutant Cacna1a polypeptide, a polynucleotide encoding the polypeptide, an antibody that specifically recognizes the mutant Cacna1a polypeptide, a method for producing a Cacna1a-related disease model non-human vertebrate, and a preventive / therapeutic substance for Cacna1a-related disease Screening methods, methods for screening Cacna1a-related diseases, methods for examining Cacna1a-related diseases, methods for identifying Groggy rats, and the like.

“Groggy rat” is a rat characterized by ataxia, which was discovered and established from the Wistar rat colony maintained at the Institute for Developmental Disorders in the Aichi Prefectural Psychosomatic Disorder Colony. The Groggy rat is derived from a mutant rat in which methyl nitrosourea is administered to the Scl: Wistar rat on the 4th day of gestation, and the main symptom is ataxia occurring in the litter.
Groggy rats usually have gait abnormalities around 15 days after birth, and show ataxia that drags or entangles the hind limbs. It becomes inactive from about 20 days after birth, and falls and extension of the hind limbs are observed. These phenotypes are inherited in autosomal recessive manner, and the causative gene is named groggy ( gry ).
Pathologically, no obvious macroscopic findings are observed in the brains of Groggy rats until about 40 days after birth, but the cerebellum of Groggy rats is smaller than controls (healthy rats) after about 60 days of age. . In addition, from around 20 to 60 days after birth, numerous nerve fiber degenerations are observed in various parts of the brain such as the pyramidal tract, hair band, trigeminal spinal tract, vestibular system, spinocerebellar tract, and cranial nerve. A number of neuronal degenerations are observed in the striatum from around 60 days after birth. In addition, with an electron microscope, concentric lamellar bodies are observed in the axon terminal part of the cerebellar nucleus, vestibular nucleus, and thin bundle nucleus, and in synaptic all axons (particularly, cerebellar Purkinje cell axons). In aged Groggy rats 180 days or older after birth, a large number of Purkinje cell axons are found in the cerebellar nucleus (see Non-Patent Documents 1-7).
On the other hand, the calcium channel subunit α1a (hereinafter sometimes referred to as Cacna1a, CACNA1A, etc.) is a protein that forms a voltage-dependent P / Q type calcium channel pore and is encoded by Cacna1a . The calcium channel subunit α1a is composed of four repeating structures (domains I to IV) each having six transmembrane sites (S1 to S6).
P / Q type calcium channels are high threshold active (HVA) calcium channels, expressed in the nervous system, especially in the presynaptic terminals of cerebellar Purkinje cells and granule cells, in the dendritic membrane, and release of neurotransmitters, cells It is known to regulate various functions such as excitatory transmission, cytoskeleton, intracellular metabolism, and gene expression.
In addition, it has been suggested that gene mutations in P / Q type calcium channels are associated with various diseases. For example, familial hemiplegic migraine (FHM), spinocerebellar ataxia type 6 (Spinocerebellar Diseases such as Ataxia 6, SCA6), paroxysmal ataxia type 2 (Episodic Ataxia 2, EA2), and idioplathic generalized epilepsy are caused by mutations in the Cacna1a gene (Non-patent Document 8).
Of these, familial hemiplegic migraine (FHM) refers to a familial hemiplegic migraine that shows signs of paralysis and sensory abnormalities before and after the headache. In families with familial hemiplegic migraine, families with cerebellar atrophy and patients with progressive cerebellar ataxia have also been reported. A mutation in the Cacna1a gene has been observed in about half of patients with familial hemiplegic migraine or in families (Non-patent Document 9).
Spinocerebellar ataxia is a general term for degenerative diseases whose main symptoms are ataxia and have a lesion head in the cerebellum and spinal cord, and are recognized as specific diseases. Among them, type 6 (SCA6) is considered to be caused by an increase in CAG repeat of the Cacna1a gene (Non-patent Document 10). In SCA6, nystagmus and cerebellar symptoms are observed.
Paroxysmal ataxia type 2 (EA2) is a disease characterized by ataxia with paroxysmal nystagmus, and often occurs in children. More than half of patients have dizziness, nausea and vomiting. Seizures are triggered by stress and emotional uplift. In EA2 patients, CAG repeat abnormalities have been reported in addition to missense mutations and nonsense mutations due to single base substitution of the Cacna1a gene (Non-patent Document 11). Nystagmus, dizziness, and recurrent seizure symptoms may also be seen in FHM or SCA6 patients.
Generalized epilepsy is a general term for seizures that are considered to start simultaneously from the entire cerebral hemisphere on both sides, and the one that is associated with a predisposition to birth is called idiopathic generalized epilepsy. Idiopathic generalized epilepsy accounts for about 60% of all epilepsy. In general epileptic seizures, epilepsy in which consciousness is interrupted for several seconds is called absence seizure. In recent years, point mutations in the Cacna1a gene have been discovered in epileptic patients with absence seizures, and it has been suggested that P / Q-type calcium channel dysfunction is related to the onset of epilepsy (Non-patent Document 12).
Thus, mutations in the Cacna1a gene cause various diseases, and several mouse strains having mutations in the Cacna1a gene that are useful as model mice for various diseases have been established.
For example, the “Tottering mouse” has a missense mutation (P → L) in the domain II pore-linking region of the Cacna1a gene, and exhibits symptoms such as paroxysmal dysmotility, absence seizure, and cortical spike-wave discharge ( Non-patent document 13).
“Leaner mice” have point mutations in the domain IV carboxyl terminus of the Cacna1a gene, resulting in splicing abnormalities. Leaner mice exhibit symptoms such as ataxia, absence seizures, cortical spike-wave discharge, and cerebellar nerve cell loss (Non-patent Document 13).
“Rolling mouse” has a missense mutation (R → G) in domain IIIS4 of Cacna1a gene, and exhibits symptoms such as ataxia and cerebellar nerve cell loss (Non-patent Document 14).
The “Rocker mouse” has a missense mutation (T → K) in the domain IIIS5-S6 transmembrane region of the Cacna1a gene, and exhibits symptoms such as ataxia, absence seizure, and cortical spike-wave discharge (Non-patent Document 15). ).
However, since the mouse has a small body size, it may not be suitable for observation over time or surgical procedures, and may not be suitable for observation of behavior, learning ability, etc. There has been a demand for the development of a higher model Cacna1a-related disease model animal having a large body size.
Takeuchi, IK et al., Acta Neuropathologica, (Germany), Vol. 102, No. 2, p. 191-194, 2001 Takeuchi, IK et al., Brain Research, (The Netherlands), 831, p. 292-296, 1999 Takeuchi, IK et al., Acta Neuropathologica, (Germany), Vol. 95, No. 5, p. 483-492, 1998 Takeuchi, IK et al., Journal of Electorn Microscopy, (Japan), vol. 44, No. 3, p. 140-144, 1995 Takeuchi, IK et al., Acta Neuropathologica, (Germany), Vol. 90, No. 5, p. 486-492, 1998 Takeuchi, IK et al., Acta Neuropathologica, (Germany), Vol. 87, No. 6, p. 628-634, 1994 Takeuchi, IK et al., Experimentia, (Switzerland), vol. 47, p. 1215-1218, 1991 Hidehiro Mizusawa, “Spinocerebellar degeneration and Ca channel abnormality”, Brain and Neurology, School of Medicine, Vol. 53, No. 1, p. 14-24, 2001 Ducross, A, Lancet Neurology, (UK), Volume 1, p. 285-293, 2002 Zuchenko, O et al., Nature Genetics (UK), Vol. 15, p. 62-69, 1997 Ophoff, RA et al., Cell, (USA), Vol. 87, p. 543-552, 1996 Chioza, B et al., Neurology, (USA), Vol. 56, p. 1245-1246, 2001 Fletcher, CF, et al., Cell, (USA), Vol. 87, p. 607-617, 1996 Mori, Y et al., The Journal of Neuroscience, (USA), Vol. 20, p. 5654-5562, 2000 Zwingman TA, et al., The Journal of Neuroscience, (USA), Vol. 21, p. 1169-1178, 2001

  An object of the present invention is to provide a new Cacna1a-related disease model animal useful for screening for a preventive / therapeutic substance for a Cacna1a-related disease. Another object of the present invention is to provide a simple and highly sensitive test method for Cacna1a-related diseases.

As a result of diligent research to achieve the above object, it was found that the disease-causing gene of the Groggy rat is the Cacna1a gene, and the rat is extremely useful as a Cacna1a-related disease model animal, and the present invention has been completed. That is, the present invention relates to the following.
(1) An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 amino acids or more including the lysine of amino acid number 251 A polypeptide comprising a partial sequence consisting of a continuous amino acid sequence.
(2) An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 amino acids or more including the lysine of amino acid number 251 A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a partial sequence consisting of a continuous amino acid sequence or a complementary sequence thereof.
(3) In the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which thymine at base number 752 is substituted with adenine, or a partial sequence consisting of a continuous nucleotide sequence of 15 bases or more including adenine at base number 752; Or a polynucleotide comprising a complementary sequence thereof.
(4) A recombinant vector containing the polynucleotide according to (2) or (3) above.
(5) A transformant transformed with the recombinant vector according to (4) above.
(6) An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence represented by SEQ ID NO: 2, or five or more amino acids including the lysine of amino acid number 251 Specifically recognizing a polypeptide comprising a partial sequence consisting of a continuous amino acid sequence of
It does not recognize a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof consisting of a continuous amino acid sequence of 5 amino acids or more including the methionine of amino acid number 251 antibody.
(7) Modification of the chromosome so that a polypeptide containing an amino acid sequence in which the methionine of amino acid number 251 is replaced with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 A method for producing a Cacna1a-related disease model non-human vertebrate.
(8) The method according to (7) above, wherein the non-human vertebrate is a rat.
(9) The Cacna1a-related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, nerve fiber degeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axis The disease selected from the group consisting of swollen cord, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dyskinesia, cortical spike wave emission and cerebellar nerve cell loss, The method according to 7).
(10) The method according to (7) above, wherein the Cacna1a-related disease is recessive hereditary.
(11) a non-human vertebrate capable of expressing a polypeptide comprising an amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and wherein the methionine of amino acid number 251 is substituted with lysine, or A screening method for a prophylactic / therapeutic substance for a Cacna1a-related disease, which comprises applying a test substance to a part of the living body and examining the effect of the substance on the pathological condition of the Cacna1a-related disease.
(12) The method according to (11) above, wherein the non-human vertebrate is a rat.
(13) The method according to (11) above, wherein the rat is a Groggy rat or a progeny thereof.
(14) The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, nerve fiber degeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axis The disease selected from the group consisting of swollen cord, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dyskinesia, cortical spike wave emission and cerebellar nerve cell loss, The method according to 11).
(15) The method according to (11) above, wherein the Cacna1a-related disease is recessive inherited.
(16) a non-human vertebrate capable of expressing a polypeptide comprising an amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, wherein the methionine of amino acid number 251 is substituted with lysine, or A screening method of a method for treating a Cacna1a-related disease, comprising applying the treatment to a part of the living body and assaying the effect of the treatment on the pathology of the Cacna1a-related disease.
(17) The method according to (16) above, wherein the non-human vertebrate is a rat.
(18) The method according to (16) above, wherein the rat is a Groggy rat or a progeny thereof.
(19) The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, nerve fiber degeneration, nerve cell degeneration, concentric lamellar body formation, Purkinje cell axis The disease selected from the group consisting of swollen cord, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dyskinesia, cortical spike wave emission and cerebellar nerve cell loss, The method according to 16).
(20) The method according to (16) above, wherein the Cacna1a-related disease is recessive inherited.
(21) A method for testing a Cacna1a-related disease, comprising detecting a mutation of amino acid number 251 methionine to lysine in an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2.
(22) Cacna1a-related comprising detecting a mutation of a codon encoding methionine of amino acid number 251 to a codon encoding lysine in the same or substantially the same amino acid sequence represented by SEQ ID NO: 2 Disease testing method.
(23) The Cacna1a-related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, nerve fiber degeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axis The disease selected from the group consisting of swollen cord, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dyskinesia, cortical spike wave emission and cerebellar nerve cell loss, 21) or the method according to (22).
(24) The method according to (21) or (22) above, wherein the Cacna1a-related disease is recessive hereditary.
(25) A reagent for testing Cacna1a-related disease, comprising means for detecting a mutation of amino acid number 251 methionine to lysine in an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2.
(26) Cacna1a-related comprising means for detecting a mutation of a codon encoding methionine of amino acid number 251 to a codon encoding lysine in the same or substantially the same amino acid sequence represented by SEQ ID NO: 2 Reagent for disease testing.
(27) A method for identifying a Groggy rat or a progeny thereof, which comprises detecting a mutation of amino acid number 251 methionine to lysine in the amino acid sequence represented by SEQ ID NO: 2.
(28) A method for identifying a Groggy rat or a progeny thereof, which comprises detecting a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.
(29) A reagent for identifying a Groggy rat or a progeny thereof, comprising means for detecting a mutation of methionine of amino acid number 251 in the amino acid sequence represented by SEQ ID NO: 2 to lysine.
(30) A reagent for identifying a Groggy rat or its progeny, comprising a means for detecting a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.
(31) An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 amino acids or more including the lysine of amino acid number 251 A prophylactic / therapeutic agent for a Cacna1a-related disease comprising a polynucleotide comprising a complementary sequence of a nucleotide sequence encoding a polypeptide comprising a partial sequence consisting of a continuous amino acid sequence.

  Vertebrate animals that can express the mutant Cacna1a polypeptide of the present invention, in particular, Groggy rats, are useful as model animals for various Cacna1a-related diseases. By using the model animals, prophylactic / therapeutic substances for Cacna1a-related diseases and the like can be obtained. It is possible to screen. Moreover, a Cacna1a-related disease can be examined by detecting a mutation similar to the mutation of the Cacna1a gene found in Groggy rats.

  The polypeptide of the present invention is an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (may be abbreviated as “M251K” in some cases). A polypeptide containing a partial sequence of M251K consisting of a continuous amino acid sequence of 5 amino acids or more including the lysine of amino acid number 251 (sometimes abbreviated as "mutant Cacna1a polypeptide of the present invention") Peptide (may be abbreviated as “partial peptide of mutant Cacna1a of the present invention”).

  The mutant Cacna1a polypeptide of the present invention may be a polypeptide derived from, for example, a vertebrate cell or any tissue or organ in which the cell is present, and may be chemically or cell-free protein as described below. A polypeptide synthesized biochemically using a synthetic system may also be used. Alternatively, it may be a recombinant polypeptide produced from a transformant introduced with a polynucleotide having a nucleotide sequence encoding the amino acid sequence.

  Examples of vertebrates include mammals, birds, fish, amphibians and reptiles. Examples of mammals include, but are not limited to, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits, domestic animals such as pigs, cows, goats, horses, sheep, minks, dogs, cats, etc. Primates such as pets, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees and the like. Examples of birds include chickens, quails, ducks, geese, turkeys, ostrichs, emu, ostriches, guinea fowls, pigeons and the like. The vertebrate is preferably a mammal.

  Examples of cells include hepatocytes, spleen cells, neurons, glial cells, Purkinje cells, granule cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth cells Muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes) ), Megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells.

  Examples of tissues or organs include the brain, each part of the brain (eg, olfactory bulb, amygdaloid nucleus, basal cerebral sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, Kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary , Placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle, tail, claws, mucous membrane and the like.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is about 50% or more, preferably about 60% or more, more preferably about 70% or more, with the amino acid sequence represented by SEQ ID NO: 2. More preferably, it is an amino acid sequence having a homology of about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, and the 251st amino acid (ie, methionine) in SEQ ID NO: 2 is conserved. And a polypeptide having the amino acid sequence has substantially the same activity as the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. As used herein, “homology” 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 percentage of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in (one or both of which can be considered introduction of a gap). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (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), etc. Examples include amino acids classified into groups. It is expected that such substitutions with similar amino acids do not change the phenotype of the polypeptide (ie, are conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
As an algorithm for determining the homology of amino acid sequences, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm includes the NBLAST and XBLAST programs ( version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970). [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0)], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated into the FASTA program in the GCG software package. Built-in It is to have], and the like, but not limited to.

  The substantially homogeneous activity includes voltage-dependent P / Q type calcium channel activity similar to that of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (sometimes abbreviated as “rat Cacna1a polypeptide”). Is mentioned. “Substantially homogeneous” means that their activities are qualitatively (eg, physiologically or pharmacologically) homogeneous. Accordingly, the quantitative factors such as the above-mentioned degree of activity are preferably equivalent, but may be different (for example, about 0.01 to about 100 times, preferably about 0.1 to about 10 times, more Preferably about 0.5 to about 2 times).

As an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, for example, (1) 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 2, Preferably, about 1 to 10 amino acids, more preferably 1 to 5 amino acid sequences deleted. (2) 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 2 (3) 1 or 2 or more (preferably 1 or more) in the amino acid sequence represented by SEQ ID NO: 2 (about 3 or more, preferably about 1 to 10, more preferably 1 to 5). About 30 amino acids, preferably about 1-10 amino acids, more preferably 1-5 amino acids, and (4) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 ( Preferably, about 1-30, preferably Or an amino acid sequence in which about 1 to 10 amino acids, more preferably 1 to 5 amino acids are substituted with other amino acids, or (5) an amino acid sequence combining them, and the 251st position in SEQ ID NO: 2 And a sequence in which the polypeptide having the amino acid sequence has substantially the same activity as the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the polypeptide having the amino acid sequence is not impaired.
Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 include the amino acid sequence of the Cacna1a ortholog polypeptide in the above vertebrates.

  “In the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, the methionine of amino acid number 251 is conserved” means that the amino acid sequence is substantially the same as the amino acid sequence represented by SEQ ID NO: 2. When the sequence is aligned with the amino acid sequence represented by SEQ ID NO: 2 by the above algorithm or the like, it means that the amino acid corresponding to the 251st methionine in SEQ ID NO: 2 is methionine. The methionine at amino acid number 251 is very highly conserved among the Cacna1a orthologs in the vertebrates described above.

The “methionine of amino acid number 251” means the 251st methionine in SEQ ID NO: 2 and the 251st methionine in SEQ ID NO: 2 in the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2. Means methionine corresponding to
“Lysine having amino acid number 251” refers to lysine produced by replacing methionine having amino acid number 251 with lysine.

  Specific examples of the mutant Cacna1a polypeptide of the present invention include a rat mutant Cacna1a polypeptide having an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2, and the like. .

  The partial peptide of the mutant Cacna1a of the present invention includes at least 5 amino acids including lysine of amino acid number 251 in M251K, for example, 8 amino acids, preferably 10 amino acids, more preferably 50 amino acids, even more preferably 100 amino acids or more. A polypeptide containing a partial sequence consisting of a sequence is exemplified.

In the present specification, the polynucleotide specified by the amino acid sequence is the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. In the polypeptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR).
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl C _6-12 aryl groups such as α-naphthyl, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl _{A 14} aralkyl group, a pivaloyloxymethyl group and the like are used.
When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Further, the polypeptides of the present invention, amino acid residues at the N-terminus: the amino group protecting group (e.g. methionine residue) (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _1-6 A group protected by an acyl group, an N-terminal glutamine residue produced by cleavage in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH) , Amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex polypeptide such as a so-called glycoprotein to which a sugar chain is bound.

  Salts of the polypeptides of the present invention are also within the scope of the present invention. As the salt of the polypeptide of the present invention, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and particularly physiologically acceptable. Acid addition salts are preferred. 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 mutant Cacna1a polypeptide of the present invention can be prepared from a vertebrate cell or tissue expressing the mutant Cacna1a polypeptide by a known protein purification method. Specifically, after homogenizing the animal tissue or cells, extraction with an acid or the like is performed, and the extract is purified and isolated by combining chromatography such as reverse phase chromatography or ion exchange chromatography. it can.
A vertebrate expressing the mutant Cacna1a polypeptide of the present invention can be produced, for example, according to the following method for producing a Cacna1a-related disease model non-human vertebrate.

The polypeptide of the present invention can also be produced according to a known peptide synthesis method.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid that can constitute the polypeptide of the present invention is condensed with the remaining part and the product has a protecting group, the protecting polypeptide is eliminated to produce the desired polypeptide. Here, the condensation and the removal of the protecting group are performed according to a method known per se, for example, the methods described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Yajima Haruaki and Sugawara Shunpei, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(5) Supervised by Haruaki Yajima, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten

For the synthesis of the polypeptide of the present invention, commercially available resins for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid in which the α-amino group and the side chain functional group are appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target polypeptide. At the end of the reaction, the protein or partial peptide is cut out from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or their amides. To do.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is directly added to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reactions, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid using acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the elimination of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as -butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

As another method for obtaining an amide form of a polypeptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is protected by amidation, and then the peptide (protein) chain is extended to the desired chain length on the amino group side. Thereafter, a protein or partial peptide from which only the α-amino group protecting group at the N-terminal of the peptide chain is removed and a protein or partial peptide from which only the protecting group at the C-terminal carboxyl group has been removed are produced, and these polypeptides Are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude polypeptide. This crude polypeptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide form of the desired polypeptide.
To obtain an ester of a polypeptide, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein or peptide of the desired protein or peptide is obtained in the same manner as the amide of the polypeptide. An ester body can be obtained.

  The partial peptide of mutant Cacna1a of the present invention can also be produced by cleaving the mutant Cacna1a polypeptide of the present invention obtained by any of the methods described above or below with an appropriate peptidase.

The polypeptide of the present invention thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the polypeptide obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when the polypeptide is obtained as a salt. Alternatively, the salt can be converted to a free form or other salt by a known method or a method analogous thereto.

The polypeptide of the present invention is obtained by culturing a transformant introduced with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the polypeptide (or a complementary sequence thereof) to produce the polypeptide of the present invention. The polypeptide can also be produced by separating and purifying the polypeptide from the resulting culture.
The polynucleotide containing the nucleotide sequence encoding the polypeptide of the present invention (or its complementary sequence) includes the nucleotide sequence encoding the above-described mutant Cacna1a polypeptide of the present invention or a partial peptide thereof (or its complementary sequence). Anything can be used. 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. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid.
The DNA containing the nucleotide sequence encoding the polypeptide of the present invention (or its complementary sequence) is derived from chromosomal DNA, the vertebrate cell expressing the polypeptide of the present invention, or any tissue or organ in which the cell is present. CDNA, synthetic DNA and the like. The chromosomal DNA and cDNA encoding the polypeptide of the present invention are polymerized chain reaction (hereinafter referred to as “PCR method”) using the chromosomal DNA fraction and the total RNA or mRNA fraction prepared from the cells and tissues as templates, respectively. It can also be directly amplified by Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”). Alternatively, chromosomal DNA and cDNA encoding the polypeptide of the present invention can be prepared by inserting chromosomal DNA, cDNA and total RNA or mRNA fragments prepared from the cells / tissues described above into an appropriate vector. It can also be cloned from the library and cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

Examples of the polynucleotide containing the nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention include, for example, a nucleotide sequence obtained by substituting adenine for the thymine at base number 752 in the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter referred to as “T752A”). Or a base sequence that hybridizes with T752A under highly stringent conditions, the adenine at base number 752 is conserved, and the mutant Cacna1a poly of the present invention described above is contained. Examples thereof include a polynucleotide encoding a polypeptide having substantially the same quality of activity as a peptide.
Examples of the polynucleotide capable of hybridizing with a polynucleotide containing T752A under highly stringent conditions include, for example, about 60% or more, preferably about 70% or more, more preferably about 80% or more, and particularly preferably about T752A. A polynucleotide containing a nucleotide sequence having 90% or more homology is used.
The homology of nucleotide sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining nucleotide sequence homology, the above-described amino acid sequence homology calculation algorithm is also preferably exemplified.

Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
High stringent conditions include, for example, conditions in which the sodium salt concentration is about 19 to about 40 mM, preferably about 19 to about 20 mM, and the temperature is about 50 to about 70 ° C., preferably about 60 to about 65 ° C. Can be mentioned. In particular, it is preferable that the sodium salt concentration is about 19 mM and the temperature is about 65 ° C. Those skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.

  A polynucleotide comprising a nucleotide sequence encoding a mutant Cacna1a polypeptide of the present invention is preferably a rat mutant Cacna1a containing a nucleotide sequence in which thymine at base number 752 is substituted with adenine in the nucleotide sequence represented by SEQ ID NO: 1. A polynucleotide or an allelic variant thereof.

A polynucleotide comprising a nucleotide sequence encoding a partial peptide of mutant Cacna1a of the present invention (or a complementary sequence thereof) includes a nucleotide sequence encoding a continuous amino acid sequence of 5 amino acids or more including the lysine of amino acid number 251 in M251K. Any thing can be used. Further, any of chromosomal DNA, cDNA derived from the cells and tissues described above, synthetic DNA, total RNA, and mRNA may be used.
Specifically, examples of the polynucleotide containing the nucleotide sequence encoding the partial peptide include, for example, the nucleotide sequence of nucleotide number 752 of the nucleotide sequence in which thymine of SEQ ID NO: 752 is substituted with adenine in the nucleotide sequence represented by SEQ ID NO: 1. Examples thereof include a polynucleotide comprising a partial sequence consisting of a continuous nucleotide sequence of 15 bases, preferably 30 bases, more preferably 150 bases, and even more preferably 300 bases or more, including adenine.

  A polynucleotide comprising a nucleotide sequence encoding the polypeptide of the present invention is amplified by PCR using a synthetic primer having a part of the nucleotide sequence encoding the polypeptide, or is incorporated into an appropriate expression vector Can be cloned by hybridization with a labeled nucleotide fragment or synthetic nucleic acid encoding part or all of the mutant Cacna1a polypeptide of the present invention. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

Alternatively, the polynucleotide comprising a nucleotide sequence encoding the polypeptide of the present invention has an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, or 5 amino acids or more including methionine of amino acid number 251 From a polynucleotide comprising a nucleotide sequence encoding a partial peptide consisting of a continuous amino acid sequence, from a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc. By converting a codon encoding methionine of amino acid number 251 to a codon encoding lysine according to a method known per se such as ODA-LA PCR method, Gapped duplex method, Kunkel method or the like, using Can also be obtained.

  The cloned polynucleotide can be used as it is or after digestion with a restriction enzyme or addition of a linker, if 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 nucleic acid adapter.

Producing a polypeptide by transforming a host with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the polypeptide of the present invention (or its complementary sequence) and culturing the resulting transformant. Can do.
The expression vector is obtained, for example, by cutting out a nucleic acid fragment of interest from a polynucleotide having a nucleotide sequence encoding a mutant Cacna1a polypeptide of the present invention and ligating the nucleic acid fragment downstream of a promoter in an appropriate expression vector. Can be manufactured.
As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); Bacteriophages; animal viruses such as retroviruses, vaccinia viruses and baculoviruses; pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter, Cacna1a gene promoter, and the like are used. Of these, the CMV promoter, the SRα promoter, the promoter of the Cacna1a gene, and the like are preferable.
When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp 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, GAP promoter, ADH promoter, etc. are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

As an expression vector, a vector containing 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 is used in addition to the above promoter. be able to.
If necessary, a nucleotide sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5 ′ end side of the nucleotide sequence encoding the polypeptide of the present invention.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.
Examples of the genus Escherichia include Escherichia coli K12 / DH1, JM103, JA221, HB101, and C600.
Examples of Bacillus bacteria include Bacillus subtilis MI114, 207-21, and the like.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia Used.

Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

Transformation can be performed according to a known method depending on the type of host.
Escherichia bacteria, for example, Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene ( Gene), Vol. 17, 107 (1982) and the like.
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
Yeast is, for example, Methods in Enzymology, 1
94, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), Proceedings of the National Academy of Sciences of the USA. Can be transformed according to the method described in the above.
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells are, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).

  The culture of the transformant can be performed according to a known method according to the type of the host, and the polypeptide of the present invention can be produced inside or outside the transformant.

The polypeptide of the present invention can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when the polypeptide of the present invention is extracted from cultured cells or cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc. A method of obtaining a crude extract of soluble protein by centrifugation or filtration after destroying cells or cells by the method is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ .
Isolation and purification of the polypeptide of the present invention contained in the soluble fraction thus obtained can be performed according to a method known per se. As such a method, a method using solubility such as salting out or solvent precipitation method; mainly using a difference in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

When the polypeptide of the present invention thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the polypeptide is obtained as a salt. The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
The polypeptide of the present invention produced by the transformant can be arbitrarily modified or partially removed by applying an appropriate protein modifying enzyme before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the polypeptide of the present invention thus obtained can be confirmed by an enzyme immunoassay or Western blotting using a specific antibody.

  Furthermore, the polypeptide of the present invention can be translated in vitro using a polynucleotide encoding it (particularly RNA) as a template using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate and the like. Can also be synthesized. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding the polypeptide of the present invention as a template. Cell-free protein (transcription / translation) translation systems can be commercially available, and methods known per se, specifically, Escherichia coli extract is edited by Pratt JM et al., “Transcription and Tranlation”, Hames BD and Higgins SJ , IRL Press, Oxford 179-209 (1984).

The polynucleotide containing “the nucleotide sequence encoding the polypeptide of the present invention or a complementary sequence thereof” refers not only to the polynucleotide encoding the mutant Cacna1a polypeptide of the present invention described above or a partial peptide thereof in frame, It is used in the sense of including partial nucleotide sequences that do not fit in the reading frame (or their complementary sequences).
A polynucleotide comprising a nucleotide sequence complementary to the target region of the target polynucleotide (complementary sequence), that is, a polynucleotide capable of hybridizing with the target polynucleotide is “antisense” to the target polynucleotide. It can be said. On the other hand, a polynucleotide comprising a base sequence having homology with the target region of the target polynucleotide can be said to be “sense” with respect to the target polynucleotide. Here, “having homology” or “complementary” means about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more between nucleotide sequences. Having identity or complementarity.

  A polynucleotide comprising a complementary sequence of a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention or a partial peptide thereof (hereinafter also referred to as “antisense mutant Cacna1a polynucleotide”) is a nucleotide encoding the mutant Cacna1a polypeptide of the present invention. Design and synthesis based on sequence information. Such polynucleotides can inhibit the replication or expression of the gene encoding the mutant Cacna1a polypeptide of the invention. That is, the antisense mutant Cacna1a polynucleotide can hybridize with RNA transcribed from the gene encoding the mutant Cacna1a polypeptide of the present invention, and inhibits mRNA synthesis (processing) or function (translation into protein). can do.

The target region of the antisense mutant Cacna1a polynucleotide includes a region encoding lysine of amino acid number 251 and hybridization to the antisense polynucleotide results in inhibition of translation into the mutant Cacna1a polypeptide of the present invention. The length is not particularly limited, and may be the entire sequence or a partial sequence of mRNA encoding the polypeptide. The short or about 15 bases long and the mRNA or initial transcript. Are all sequences. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, the 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, translation initiation codon, protein coding region of the nucleic acid encoding the mutant Cacna1a polypeptide of the present invention, A translation stop codon, 3 ′ end untranslated region, 3 ′ end palindromic region, 3 ′ end hairpin loop and the like can be selected as a target region, but as long as it includes a region encoding amino acid 251 lysine, Any region within the gene encoding the mutant Cacna1a polypeptide can be selected as a target. For example, it is also preferable to use the intron portion of the gene as a target region.
Furthermore, the antisense mutant Cacna1a polynucleotide not only hybridizes with mRNA or initial transcription product encoding the mutant Cacna1a polypeptide of the present invention to inhibit translation into the polypeptide, but also is a double-stranded DNA of the present invention. It may be one that can bind to a gene encoding the mutant Cacna1a polypeptide to form a triplex and inhibit RNA transcription.

  The antisense mutant Cacna1a polynucleotide can be a double-stranded DNA, a single-stranded DNA, a double-stranded RNA, a single-stranded RNA, or a DNA: RNA hybrid, and an unmodified polynucleotide (or unmodified oligonucleotide). ), With known modifications, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs Those with intramolecular nucleotide modifications, such as those with uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, Having phosphorodithioate, for example, protein (nuclea) , Nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side-current groups, intercurrent compounds (eg, acridine, psoralen, etc.) Etc.), containing chelate compounds (eg metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, having modified bonds (eg α-anomeric nucleic acid and the like.

  Antisense mutant Cacna1a polynucleotides may be altered, contain modified sugars, bases, linkages, be donated in specialized forms such as liposomes, microspheres, or applied in gene therapy Can be provided in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  A ribozyme capable of specifically cleaving mRNA or an initial transcript encoding the mutant Cacna1a polypeptide of the present invention within the coding region (including an intron in the case of the initial transcript) is also an antisense mutant Cacna1a polynucleotide. Can be included. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the nucleotide sequence of the enzyme active site also has a nucleic acid cleaving activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking chromosomal DNA since only RNA is the substrate. When the mRNA encoding the mutant Cacna1a polypeptide of the present invention has a double-stranded structure by itself, a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase can be used as a target. The sequence can be single stranded [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  Double-stranded oligo RNA (siRNA) having a nucleotide sequence complementary to a partial sequence (including an intron portion in the case of the initial transcript) of the mRNA encoding the mutant Cacna1a polypeptide of the present invention or the initial transcript Can also be included in antisense mutant Cacna1a polynucleotides. A phenomenon called so-called RNA interference (RNAi), in which short double-stranded RNA is introduced into a cell and mRNA complementary to one strand of the RNA is degraded, has been known in nematodes, insects, plants, etc. However, since it was confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.

  The present invention also provides an antibody against the mutant Cacna1a polypeptide of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as “anti-mutant Cacna1a antibody”). The anti-mutant Cacna1a antibody may be a monoclonal antibody or a polyclonal antibody as long as it specifically recognizes the mutant Cacna1a polypeptide of the present invention or a partial peptide thereof. The antibody can be produced according to a known method for producing an antibody or antiserum using a mutant Cacna1a polypeptide or a partial peptide thereof as an antigen.

  The antibody of the present invention specifically recognizes the mutant Cacna1a polypeptide of the present invention, but is a normal Cacna1a polypeptide (ie, a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2). (Peptide) is not recognized. Therefore, the antigen used for the production of the antibody is a partial sequence of an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2. A polypeptide containing a partial sequence consisting of a continuous amino acid sequence of 5 amino acids or more containing the lysine of amino acid number 251 is preferred.

In the production of a monoclonal antibody, the polypeptide of the present invention is administered to a mammal at a site where the antibody can be produced by administration, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. As mammals to be used, mice and rats are preferably used.
For example, an individual having an antibody titer is selected from a mammal immunized with an antigen, such as a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are allogeneic or Monoclonal antibody-producing hybridomas can be prepared by fusing with myeloma cells from different animals. The antibody titer in the antiserum can be measured, for example, by a method known per se, for example, ELISA method. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein (Nature, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

Examples of myeloma cells include mammalian myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
A monoclonal antibody-producing hybridoma can be prepared, for example, by adding a hybridoma culture supernatant to a solid phase (eg, a microplate) on which an antigen is adsorbed directly or together with a carrier, and then labeling with an anti-immunoglobulin antibody (cells) labeled with a radioactive substance or enzyme. When mouse used for fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A is added to detect monoclonal antibody bound to the solid phase; solid phase adsorbed with anti-immunoglobulin antibody or protein A The hybridoma culture supernatant is added to the mixture, the polypeptide of the present invention labeled with a radioactive substance or an enzyme is added, and the monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. The selection of the monoclonal antibody can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. Any medium can be used for the selection and breeding of the monoclonal antibody as long as the hybridoma can grow.
The monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, : Adsorption / desorption method by DEAE), ultracentrifugation method, gel filtration method, specific purification method in which only antibody is collected by antigen-binding solid phase or active adsorbent such as protein A or protein G and the binding is dissociated to obtain antibody ] Can be separated and purified according to the above.

A polyclonal antibody against the mutant Cacna1a polypeptide of the present invention or a partial peptide thereof can be produced according to a method known per se. For example, an immunizing antigen (for example, the polypeptide of the present invention) itself or a complex thereof and a carrier protein is prepared, and a mammal is immunized in the same manner as the above-described monoclonal antibody production method. It can be produced by collecting the antibody-containing material and separating and purifying the antibody.
Regarding the complex of immunizing antigen and carrier protein used to immunize mammals, the kind of carrier protein and the mixing ratio of carrier and hapten should be such that antibodies can be efficiently produced against hapten immunized by cross-linking to carrier. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 to about 1 by weight with respect to hapten 1. A method of coupling at a rate of 5 is used.
The condensation product is administered to a mammal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as that of the monoclonal antibody.

The polypeptide of the present invention is useful for the production of an antibody that specifically recognizes the polypeptide.
The polynucleotide of the present invention comprises (1) production of the polypeptide of the present invention (2) production of Cacna1a-related disease model non-human vertebrate (3) reagent for testing Cacna1a-related disease (4) prevention of Cacna1a-related disease -Therapeutic agent (5) Useful for identification of Groggy rats.
In addition, the antibody of the present invention is useful for (1) a reagent for testing Cacna1a-related diseases, and (2) identification of Groggy rats.

  In addition, the present invention can express a polypeptide comprising an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2. A method for producing a Cacna1a-related disease model non-human vertebrate comprising modifying a chromosome is provided.

As a result, the method for modifying a chromosome includes an amino acid sequence in which a non-human vertebrate has an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and methionine of amino acid number 251 is substituted with lysine. There is no particular limitation as long as the polypeptide can be expressed. As this method, for example,
(1) A method (Method I) of finding an individual capable of modifying a chromosome by allowing a mutagenic factor to act on a germ cell and expressing the mutant Cacna1a polypeptide of the present invention in offspring derived from the germ cell;
(2) A polynucleotide comprising a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention is introduced into the germ cell chromosome to obtain an individual capable of expressing the mutant Cacna1a polypeptide of the present invention in offspring derived from the germ cell. A method to perform (Method II);
(3) A polynucleotide containing a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention is introduced into the chromosome of an embryonic stem cell (ES cell), and the mutant Cacna1a polypeptide of the present invention is expressed in progeny derived from the ES cell. A method for obtaining possible individuals (Method III);
Etc. Each method will be described below.

In the above method I, mutagenic factors include N-ethyl-N-nitrosourea, methylnitrosourea, procarbazine hydrochloride, triethylenemelamine, acrylamide monomer, chlorambucil, melphalan, cyclophosphamide, diethyl sulfate, ethyl Methane sulfonate, methyl methane sulfonate, 6-mercaptopurine, mitomycin C, procarbazine, N-methyl-N′-nitro-N-nitrosoguanidine, ³ H ₂₀ , urethane, ultraviolet light, X-ray, γ-ray, etc. Is preferably N-ethyl-N-nitrosourea or methylnitrosourea.

  In Method I, the germ cell is not particularly limited, but is preferably a male germ cell. Examples of male germ cells include sperm and progenitor cells thereof (spermatogonia, sperm cells, spermatogonia, spermatocytes, sperm cells, etc.).

The method of causing the mutagenic factor to act on the germ cell is not particularly limited, but preferably, the mutagenic factor is brought into contact with the germ cell by administering the mutagenic factor to a non-human vertebrate having a germ cell. Let it work. As the vertebrate, any of the above-mentioned vertebrates excluding humans may be used. Preferably, the vertebrate is a rodent, and more preferably a rat.
The vertebrate is preferably a male.

  The mutagenic factor can be administered to non-human vertebrates according to a method known per se, for example, the method described in (Nat. Biotechnol., Vol. 21, p. 645-651, 2003), etc. A dose of a mutagenic factor capable of modifying the chromosomes of germ cells in the non-human vertebrate at an appropriate frequency is administered to the non-human vertebrate. As a result, a non-human vertebrate having germ cells whose chromosomes are randomly altered can be obtained.

  Next, a non-human vertebrate having a germ cell having a modified chromosome is mated with a wild-type animal to obtain offspring derived from the germ cell. The modified chromosome is transmitted to the somatic and germ cells of the progeny animal. The mating may be natural mating or artificial insemination (microinsemination, IVF, etc.), but it is preferably performed by natural mating in consideration of the ease of operation.

  Furthermore, from the obtained progeny, an individual into which the mutation has been introduced into the endogenous Cacna1a gene and the mutant Cacna1a polypeptide of the present invention can be expressed is selected.

  In the selection, for example, the above-mentioned cells derived from the obtained progeny animals or the chromosomal DNA fraction and the total RNA or mRNA fraction prepared from any tissue or organ in which the cells exist are used as templates, respectively. The Cacna1a gene of the animal is amplified by the RT method or the RT-PCR method. Whether or not a mutation capable of expressing the mutant Cacna1a polypeptide of the present invention was introduced by isolating the amplified Cacna1a gene and analyzing the nucleotide sequence after direct cloning or subcloning into an appropriate vector. Individuals who are determined and capable of expressing the desired polypeptide are selected.

  When the obtained individual has a mutation capable of expressing the mutant Cacna1a polypeptide of the present invention only in one of the homologous chromosomes (heterozygote), the heterozygous siblings of the heterozygote are crossed. Thus, an animal (homozygote) into which a mutation capable of expressing the mutant Cacna1a polypeptide of the present invention is introduced into both homologous chromosomes can be obtained.

  In the above method II, fertilized eggs of the above-mentioned non-human vertebrates, unfertilized eggs, sperm and their progenitor cells (primitive germ cells, oocytes, oocytes, eggs, spermatogonia, spermatocytes, spermatozoa, spermatozoa Cells, etc.), preferably in the early stages of embryo development of fertilized eggs (more preferably before the 8-cell stage), calcium phosphate coprecipitation, electroporation, lipofection, aggregation, A polynucleotide containing a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention is introduced by a gene introduction method such as a microinjection method, a gene gun (particle gun) method, or a DEAE-dextran method.

In introducing a polynucleotide comprising a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention into a germ cell, the target polypeptide is downstream of a promoter that can be expressed in the cells of the target non-human vertebrate. It is generally advantageous to use it as a linked gene construct (eg, vector, etc.).
Specifically, a target is a vector obtained by linking a polynucleotide containing a nucleotide sequence encoding the polypeptide downstream of various promoters derived from the above-mentioned non-human vertebrates and capable of expressing the target polypeptide. A transgenic non-human mammal that highly expresses the target polypeptide can be produced by microinjection into a fertilized egg (eg, a rat fertilized egg) of the non-human mammal.
Examples of the vector include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, and animal viruses such as vaccinia virus or baculovirus. Of these, plasmids derived from E. coli, plasmids derived from Bacillus subtilis, or plasmids derived from yeast are preferably used, and plasmids derived from E. coli are particularly preferable.

Examples of promoters that regulate expression include promoters of genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, Genes derived from rats, mice, etc.) and birds (such as chickens) (eg, albumin, endothelin, osteocalcin, muscle creatine kinase, type I and type II collagen, cyclic AMP-dependent protein kinase βI subunit, atrial natriuretic factor) , Dopamine β-hydroxylase, neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, smooth muscle α-actin, polypeptide chain elongation factor 1α (EF1-α), β-actin, α and β-mi Osin heavy chain, myosin light chain 1 and 2, myelin basic protein, serum amyloid P component, renin promoter, etc.).
Preferably, since the Cacna1a gene is normally expressed specifically in the nervous system, particularly cerebellar Purkinje cells and granule cells, the mutant Cacna1a polypeptide of the present invention is specifically expressed in the nervous system, particularly cerebellar Purkinje cells and granule cells. A promoter that can be highly expressed can be appropriately selected. The promoter can be the promoter of the Cacna1a gene itself.

The vector preferably has a sequence (poly A, generally referred to as a terminator) that terminates the transcription of the target mRNA in a non-human vertebrate. For example, it is derived from viruses, various mammals, and birds. Gene expression can be manipulated using the sequence of each gene. Preferably, a simian virus SV40 terminator or the like is used. In addition, for the purpose of further expressing the polypeptide of interest, a splicing signal of each gene, an enhancer region, a part of an intron of a eukaryotic gene, 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region It is also possible to connect 3 'downstream.
In addition, the vector further includes a selection marker gene (eg, a neomycin resistance gene, a hygromycin resistance gene, a drug resistance gene such as ampicillin resistance) for selecting a clone in which the introduced polynucleotide is stably integrated. It is preferable.

In a preferred embodiment, an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a mutant Cacna1a polypeptide of the present invention is introduced into a fertilized egg of a target non-human vertebrate by a microinjection method.
The fertilized eggs of the target non-human vertebrate are collected from in-vivo fertilized eggs obtained by mating male and female of the same non-human vertebrate, or eggs and sperm collected from male and female of the same non-human vertebrate in vitro. It can be obtained by fertilization.

  Microinjection of a polynucleotide into a fertilized egg can be performed according to a conventional method using a known device such as a micromanipulator. Briefly, a fertilized egg placed in a microdrop of embryo culture medium is aspirated and fixed with a holding pipette, and the polynucleotide solution is placed into a male or female pronucleus, preferably into the male pronucleus, using an injection pipette. Inject directly. The polynucleotide to be introduced is preferably highly purified by CsCl density gradient ultracentrifugation or the like. Moreover, it is preferable that the polynucleotide to be introduced is linearized by cleaving the vector portion using a restriction enzyme.

The fertilized egg after introduction of the polynucleotide is cultured from the 1st cell stage to the blastocyst stage in 5% carbon dioxide gas / 95% air in the medium for embryo culture by microdrop culture etc. Transplanted into the fallopian tube or uterus of female non-human vertebrates. The female non-human vertebrate for embryos may be of the same kind as the animal from which the early embryo to be transplanted is derived. As a method for making a female non-human vertebrate for embryo transfer into a pseudopregnant state, for example, mating with the same kind of vasectomized (ligated) male non-human mammal and selecting a vaginal plug confirmed The method is known.
Embryonic females may be naturally ovulated, or administered luteinizing hormone-releasing hormone (generally abbreviated as LHRH) or its analogs prior to mating with vasectomized (ligated) males. Alternatively, fertility induced may be used. The dose of LHRH or an analog thereof, and the timing of mating with a male non-human vertebrate after the administration differ depending on the type of non-human vertebrate.

Usually, when the early embryo to be transplanted is an 8-cell stage embryo or later, the embryo is transplanted into the uterus of a female for embryo reception, and before that (for example, 1-cell to 4-cell stage embryo). As the female for embryo transfer, a female that has passed a certain number of days from pseudopregnancy according to the stage of development of the transplanted embryo is appropriately used. After anesthesia (preferably Avertin or the like is used) for the embryo recipient female, incision is made to draw out the ovary, and early embryos (about 5 to about 10) suspended in the medium for embryo culture are used with an embryo transfer pipette. Inject into the tubal cavity or the uterine horn near the tubal junction.
If the transplanted embryo is successfully implanted and the embryo recipient female becomes pregnant, a pup non-human mammal can be obtained by natural delivery or caesarean section. It is only necessary to continue suckling to the naturally delivered embryo recipient female. If the baby is delivered by caesarean section, the offspring can be fed to a separately prepared female for suckling.

The introduction of a polynucleotide comprising a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention at the fertilized egg cell stage is such that the introduced polynucleotide is present in all germ line cells and somatic cells of the subject non-human vertebrate. Secured. Whether or not the introduced polynucleotide is incorporated into the chromosomal DNA can be assayed, for example, by screening the chromosomal DNA separated and extracted from the tail of the pup by Southern hybridization or PCR. The presence of a polynucleotide comprising a nucleotide sequence encoding the mutant Cacna1a polypeptide of the present invention in the chromosome of a germline cell of a non-human vertebrate (F ₀ ) obtained as described above indicates that a progeny (F ₁ ) In all the germline cells and somatic cells, the polynucleotide containing the nucleotide sequence encoding the polypeptide of interest is transmitted.

Usually, F ₀ animals are obtained as heterozygotes having a polynucleotide introduced into only one of the homologous chromosomes. Individual F ₀ individuals are randomly inserted on different chromosomes unless homologous recombination is used. In order to obtain a homozygote having a polynucleotide introduced into both homologous chromosomes, F ₀ animals and non-transgenic animals were crossed to produce F ₁ animals and introduced into only one of the homologous chromosomes. What is necessary is just to cross the siblings of the heterozygote which has a polynucleotide.
As a result, a non-human vertebrate capable of expressing the mutant Cacna1a polypeptide of the present invention can be obtained.

  In Method III, an ES cell clone in which a polynucleotide encoding the mutant Cacna1a polypeptide of the present invention is incorporated into the endogenous Cacna1a locus of ES cells by homologous recombination is selected, and progeny derived from the clone is derived. To win.

Specifically, for example, first, chromosomal DNA encoding the endogenous Cacna1a gene is isolated from a target non-human vertebrate according to a conventional method. Then, the region containing the ORF is excised using an appropriate restriction enzyme, and a polynucleotide encoding the mutant Cacna1a polypeptide of the present invention is inserted instead to construct a targeting vector. In the targeting vector, a positive selection drug resistance marker gene such as a neomycin resistance gene or a hygromycin resistance gene is inserted into the 3 ′ untranslated region of the Cacna1a gene, and the HSV-tk is outside the region homologous to the target sequence. A negative selection marker gene such as a gene or diphtheria toxin gene may be inserted.
In addition, since the expression of the mutant Cacna1a polypeptide of the present invention into which the positive selection marker gene has been introduced may be hindered, a targeting vector in which loxP sequences or frt sequences are arranged at both ends of the positive selection marker gene may be used. . In this case, a positive selection marker gene can be cut out by allowing Cre or Flp recombinase or the recombinase expression vector (eg, adenovirus vector) to act at an appropriate time.

  The targeting vector thus obtained can be used for calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method, gene gun (particle gun) Introduced into ES cells by the method, DEAE-dextran method or the like. Preferably, the electroporation method is generally selected from the viewpoint that a large number of cells can be easily treated.

  When the drug resistance marker gene for positive selection is inserted into the targeting vector, the ES cell in which the targeting vector has been introduced into the chromosome can be efficiently obtained by culturing the ES cell in a medium containing the corresponding drug. It is possible to select. For example, when a targeting vector containing a neomycin resistance gene is used, the ES cell after gene introduction treatment is cultured in a medium containing a neomycin antibiotic such as G418.

  In addition, when a drug resistance marker gene for negative selection is inserted into the targeting vector, ES cells in which the targeting vector has been introduced at random by culturing ES cells in a medium containing the corresponding drug It is possible to efficiently select only ES cells in which the desired homologous recombination has occurred.

  The selected ES cells are subsequently cultured, chromosomal DNA is isolated from a portion thereof, and the presence of the desired homologous recombination is confirmed by PCR or Southern hybridization.

A chimeric embryo is obtained by introducing ES cells in which the presence of the desired homologous recombination is confirmed into a host embryo. The host animal species is preferably the same as the ES cell species to be introduced. The embryo is not particularly limited, and examples thereof include a blastocyst and an 8-cell stage embryo.
Embryos can be obtained by mating female animals subjected to superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) and the like with male animals. As a method for introducing ES cells into a host embryo, a microinjection method and an aggregation method are known, but any method can be used.

  A chimeric non-human vertebrate is obtained by transferring the chimeric embryo into the uterus or oviduct of the host animal. The host animal is preferably a pseudopregnant animal. The host animal into which the chimeric embryo has been transferred becomes pregnant and gives birth to a chimeric non-human vertebrate.

  Furthermore, the chimeric non-human vertebrate is bred with a normal animal, and an individual having the desired homologous recombination is selected from the next generation (F1) individuals, whereby the mutation of the present invention is applied to only one homologous chromosome. A heterozygote having a polynucleotide encoding a Cacna1a polypeptide can be obtained. For selection of an individual in which the desired homologous recombination exists, various traits can be used as an index. For example, body color or coat color is used as an index. It is also possible to select by extracting chromosomal DNA from a part of the body and performing Southern blot analysis or PCR assay.

  Further, by mating the above-mentioned heterozygous siblings, a homozygote having a polynucleotide encoding the mutant Cacna1a polypeptide of the present invention in both homologous chromosomes can be obtained.

  In the above-mentioned methods I to III, an individual from which expression of an endogenous normal Cacna1a polypeptide (polypeptide consisting of the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2) has been removed is obtained. Methods I and III are preferred in that they can be made. In addition, Method III is preferable in that a target individual can be obtained with high efficiency, but Method I is preferable in that it can be applied to animal species for which it is difficult to obtain available ES cells.

  As a preferred non-human vertebrate obtained by the above-described methods I to III, a polypeptide comprising an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2 can be expressed. Examples include rats with modified chromosomes. The nucleotide sequence encoding the polypeptide may preferably be a nucleotide sequence in which thymine at base number 752 is substituted with adenine in the nucleotide sequence represented by SEQ ID NO: 1.

  Most preferred non-human vertebrates obtained by the above-mentioned methods I to III include Groggy rats and their progeny. “Groggy rat” is a rat obtained by Method I, and has a chromosome so that a polypeptide consisting of an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2 can be expressed. As a nucleotide sequence that is a modified rat and encodes the polypeptide, in the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence (in cDNA) in which the thymine at base number 752 is substituted with adenine is represented by Cacna1a Has at both loci.

  The fertilized egg of the Groggy rat was deposited on October 20, 2004 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 1-1-3 Higashi, Tsukuba, Ibaraki, Japan as receipt number FERM AP-20261. ing.

  “Groggy rat progeny” is a progeny derived from Gloggy rat, in which at least one (preferably both) of the Cacna1a locus is derived from Gloggy rat, and in the nucleotide sequence represented by SEQ ID NO: 1, Refers to a rat having a nucleotide sequence (but in cDNA) with 752 thymine replaced by adenine.

  Non-human vertebrates whose chromosomes have been modified to express the mutant Cacna1a polypeptide of the present invention obtained by the above-described methods I to III are extremely useful as a Cacna1a-related disease model.

  Cacna1a-related disease refers to a disease that can occur due to abnormal function of Cacna1a. Examples of Cacna1a-related diseases include migraine (especially familial hemiplegic headache), spinocerebellar ataxia (especially type 6), paroxysmal ataxia (especially type 2), epilepsy (especially general epilepsy, idiopathic general) Epilepsy or absence seizures), ataxia (eg gait abnormalities, falls, hindlimb extension, etc.), cerebellar atrophy, nerve fiber degeneration (eg pyramidal tract, hair band, trigeminal spinal tract, vestibular system, spinocerebellar tract, (In cranial nerves), neuronal degeneration (eg in striatum), concentric lamellar body formation (eg in cerebellar nucleus, vestibular nucleus, thin bundle nucleus axon terminal, synaptic axon, cerebellar Purkinje cell axon) ), Purkinje cell axon enlargement, cerebellar ataxia (especially progressive cerebellar ataxia), nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dysmotility, cortical spike- wave discharge Include cerebellar cell loss or the like.

Also, a Cacna1a-related disease that develops in a non-human vertebrate whose chromosome has been modified to express a mutant Cacna1a polypeptide of the invention can be inferior hereditary.
Therefore, when a non-human vertebrate whose chromosome has been modified to express the mutant Cacna1a polypeptide of the present invention is used as a Cacna1a-related disease model, the animal has an endogenous normal Cacna1a polypeptide (shown in SEQ ID NO: 2). It is preferable that it is an individual from which the expression of a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence to be determined is removed. More preferably, the animal is a homozygote having a polynucleotide encoding a mutant Cacna1a polypeptide of the present invention on both homologous chromosomes of the Cacna1a locus.

  A non-human vertebrate whose chromosome has been modified so that it can express the mutant Cacna1a polypeptide of the present invention is extremely useful as a Cacna1a-related disease model. Therefore, a test substance is applied to the animal or a part of its living body, By examining the effect of the substance on the pathological condition of the Cacna1a-related disease, a prophylactic / therapeutic substance for the Cacna1a-related disease can be screened.

  Specifically, first, a test substance is applied to a non-human vertebrate whose chromosome has been modified so that the mutant Cacna1a polypeptide of the present invention can be expressed or a part of its living body. The “part of the living body” is not particularly limited, and examples thereof include the above-described cells, tissues or organs.

  The test substance is not particularly limited, and examples thereof include nucleic acid molecules, peptide compounds, carbohydrates, lipids, low molecular organic compounds, inorganic compounds, mixed solutions thereof, compounds synthesized by combinatorial chemistry, natural products, and synthetic products. And extracts from animals and plants, fungi, algae, and microorganisms.

  When the test substance is a nucleic acid molecule, it may be DNA or RNA, and its base may be other naturally occurring nucleotides or artificially synthesized nucleotides in addition to general A, G, C, T. May be included. In addition, degradation resistant nucleic acid mimics such as PNA (peptide nucleic acid) and thiolated DNA may be used.

  Peptide compounds used as test substances include peptides composed of natural amino acids, peptides composed of non-natural amino acids, peptides composed of natural amino acids / non-natural amino acids, and antibodies and fragments thereof. Non-natural amino acids include natural amino acid D-form, β-amino acids, γ-amino acids and the like. Moreover, these mixtures can also be used as a test substance. Therefore, as a test substance, for example, a member of a random peptide library (see, for example, Lam, KS et al., 1991, Nature 354: 82-84; Houghten, R. et al., 1991, Nature 354: 84-86) , And soluble peptides, phosphopeptides and the like including a chemically derived molecular library in a combination of D / L-configuration amino acids.

  The test substance is applied to the non-human vertebrate or a part of the living body by a method known per se, for example, the test substance is administered to the non-human vertebrate, for example, orally / parenterally.

Next, whether the applied test substance can treat the pathological condition of a Cacna1a-related disease in a non-human vertebrate or a part of the living body whose chromosome has been modified so that the mutant Cacna1a polypeptide of the present invention can be expressed. Whether or not is tested.
The pathological condition of the Cacna1a-related disease in the non-human vertebrate to which the test substance is applied or a part of the living body thereof is Cacna1a-related in the non-human vertebrate or the part of the living body (negative control) to which the test substance is not applied. It is compared with the disease state of the disease, and the therapeutic effect of the test substance is tested based on the absolute or relative significant difference.

  As a result of the comparison, by selecting a substance that improves the pathological condition of the Cacna1a-related disease, a preventive / therapeutic substance for the Cacna1a-related disease can be identified. Furthermore, a substance that more effectively improves the pathological condition of the Cacna1a-related disease may be selected as a substance that has a higher preventive / therapeutic effect on the Cacna1a-related disease.

  Similarly, the treatment is applied to a non-human vertebrate whose chromosome has been modified so that the mutant Cacna1a polypeptide of the present invention can be expressed, or a part of its living body, and the effect of the treatment on the pathology of the Cacna1a-related disease. Can be used to screen for methods for treating Cacna1a-related diseases.

Specifically, first, treatment is applied to a non-human vertebrate whose chromosome has been modified so that the mutant Cacna1a polypeptide of the present invention can be expressed or a part of its living body.
The treatment is not particularly limited, and may include medical treatment such as surgery, anesthesia, physical therapy, transplantation, etc. in addition to the administration of the above-described test substance.

Next, whether the applied treatment can treat the pathology of a Cacna1a-related disease in a non-human vertebrate or a part of its organism whose chromosome has been modified to express the mutant Cacna1a polypeptide of the invention Is tested.
The pathology of the Cacna1a-related disease in the non-human vertebrate or part of the living body to which treatment is applied is the pathology of the Cacna1a-related disease in the non-human vertebrate or part of the living body (negative control) to which treatment is not applied. The therapeutic effects of the treatment are tested based on their absolute or relative significance.

  As a result of the comparison, a treatment method for Cacna1a-related disease can be identified by selecting a treatment that improves the pathological condition of the Cacna1a-related disease. Furthermore, a treatment that more effectively improves the pathological condition of the Cacna1a-related disease may be selected as a treatment method having a higher therapeutic effect on the Cacna1a-related disease.

  A vertebrate capable of expressing an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 develops the above-mentioned Cacna1a-related disease Therefore, by detecting a mutation of amino acid number 251 methionine to lysine in the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, the risk of suffering from Cacna1a-related disease is determined. Is possible. The present invention provides a method for testing such a Cacna1a-related disease.

  The detection of the mutation can be performed by directly detecting a mutation in the Cacna1a polypeptide or by detecting a mutation in the polynucleotide encoding the Cacna1a polypeptide, and is not particularly limited.

When the mutation in the polypeptide is directly detected, for example, the mutant Cacna1a polypeptide of the present invention can be detected by an immunological technique using an antibody that specifically recognizes the mutant Cacna1a polypeptide of the present invention (for example, the antibody of the present invention). Is detected immunospecifically.
For example, a sample capable of measuring the mutant Cacna1a polypeptide of the present invention is first prepared from a biological sample collected from a test vertebrate. As the biological sample, the above-described cells, tissues, organs, and the like are used, but it is preferable to use a site (nervous system, cerebellar Purkinje cell, granule cell, etc.) that can express Cacna1a.
When the mutant Cacna1a of the present invention is detected by an immunological method such as ELISA, Western blot method, spot method, aggregation method, antibody array, surface plasmon resonance, etc., a liquid sample is usually prepared from a biological sample. For example, when the biological sample is a tissue or a cell, a solubilizate is prepared by mechanically crushing them or treating them with a solubilizer. When the mutant Cacna1a polypeptide of the present invention is detected by an immunological method such as flow cytometry or immunological tissue staining, a biological sample such as tissue or cell is appropriately subjected to treatment such as fixation and stained with an antibody. Is done.

As a result of the detection, when the mutant Cacna1a polypeptide of the present invention is detected, it can be determined that the vertebrate individual to be measured has a high risk of suffering from a Cacna1a-related disease. On the other hand, when the mutant Cacna1a polypeptide of the present invention is not detected, it can be determined that the vertebrate individual to be measured has a low risk of suffering from a Cacna1a-related disease.
The Cacna1a associated disease may be recessive.

  When detecting a mutation in the polynucleotide encoding the Cacna1a polypeptide, first, chromosomal DNA, total RNA or mRNA is prepared from a biological sample collected from the test vertebrate. As the biological sample, the above-described cells, tissues, organs, and the like are used. In consideration of availability, peripheral blood, hair, nails, skin, mucous membranes, and the like are preferably used. Further, from the viewpoint of analysis of mutation, it is preferable to use a site (nervous system, cerebellar Purkinje cell, granule cell, etc.) capable of expressing Cacna1a polypeptide. Preparation of chromosomal DNA, total RNA, or mRNA from a biological sample can be performed by a method well known in the art, or a commercially available kit may be used.

It is determined whether or not the polynucleotide encoding the Cacna1a polypeptide contained in the chromosomal DNA, total RNA or mRNA obtained as described above has a mutation. The mutation may be a mutation of a codon encoding methionine of amino acid number 251 in the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 to a codon encoding lysine. The codon encoding methionine is usually ATG. The codon encoding lysine is usually AAA or AAG, but preferably AAG.
When the vertebrate is a rat, the mutation may be a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.

  The mutation can be analyzed by a well-known method. Examples of the method include RFLP (restriction fragment length polymorphism) method and PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method (see, for example, Biotechniques, 16, 296-297 (1994)). ASO (Allele Specific Oligonucleotide) hybridization method (see, for example, Clin. Chim. Acta, 189, 153-157 (1990), direct sequencing method (see, for example, Biotechniques, 11, 246-249 (1991)), ARMS ( Amplification Refracting Mutation System (for example, see Nuc. Acids. Res., 19, 3561-3567 (1991)), Denaturing Gradient Gel Electrophoresis (for example, Biotechniqus, 27, 1016-1018) (See 1999)), RNase A cleavage method (see, for example, DNA Cell. Biol., 14, 87-94 (1995)), chemical cleavage method (see, for example, Biotechniques, 21, 216-218 (1996)), DOL ( Dye-labeled Oligonucleotide Ligation (eg Genome Res., 8, 549-556 (199 8)), TaqMan PCR method (for example, see Genet. Anal., 14, 143-149 (1999)), Invader method (for example, Science, 5109, 778-783 (1993); Nat. Biotechnol., 17, 292-296 (1999)), MALDI-TOF / MS (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry) method (Genome Res., 7, 378-388 (1997)), TDI (Template-directed Dye) -terminator Incorporation) method (for example, Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)), Southern blotting method and the like.

  For example, by using the above-described polynucleotide of the present invention as a probe, it is possible to detect the presence or absence of mutation by an ASO hybridization method or an RNase A cleavage method. Alternatively, in a polynucleotide encoding an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (amino acid of amino acid number 251 is methionine or lysine), a codon encoding amino acid of amino acid number 251 By using a primer that can amplify a region containing, it is possible to detect the presence or absence of mutation by a direct sequencing method or the like.

  When the mutation is detected as a result of the detection, it can be determined that the vertebrate individual to be measured has a high risk of suffering from a Cacna1a-related disease. On the other hand, when the mutation is not detected, it can be determined that the vertebrate individual to be measured has a low risk of suffering from a Cacna1a-related disease.

  In addition, since the Cacna1a-related disease may be recessive, it is more likely to have a Cacna1a-related disease when the mutation is present in both homologous chromosomes than when the mutation is present in only one of the homologous chromosomes. It can be determined that the risk is high. Conversely, when the mutation is present only on one of the homologous chromosomes, it can be determined that the risk of suffering from a Cacna1a-related disease is lower than when the mutation is present on both homologous chromosomes.

  The present invention also relates to a reagent for testing Cacna1a-related disease, comprising means for detecting a mutation of amino acid number 251 methionine to lysine in the same or substantially the same amino acid sequence as represented by SEQ ID NO: 2. I will provide a. The means may be a means for detecting a mutation of a codon encoding methionine of amino acid number 251 to a codon encoding lysine in an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2. By using the test reagent, the above-described test method for Cacna1a-related diseases can be easily performed.

The means is not particularly limited, but for example,
(1) an antibody that specifically recognizes the mutant Cacna1a of the present invention (for example, the antibody of the present invention);
(2) the polynucleotide of the present invention;
(3) In a polynucleotide encoding an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (amino acid of amino acid number 251 is methionine or lysine), it encodes the amino acid of amino acid number 251 A primer capable of amplifying the region containing the codon;
Etc. can be mentioned.

  In addition to the above-mentioned means, various reagents used in the above-described inspection methods (for example, protein extraction reagents, labeled antibodies, chromosomal DNA preparation reagents, total RNA preparation reagents, mRNA preparation reagents, enzymes, reagents and biological samples are diluted. For example, a buffer solution, a positive control, a negative control, a reaction container, and an instruction sheet describing a test protocol). These elements can be mixed in advance if necessary. Moreover, a preservative and preservative can also be added to each element as needed. The test kit is extremely useful because it allows the method for testing Cacna1a-related diseases to be carried out easily.

  In addition, since the amino acid sequence of the Clogna1a polypeptide of Gloggy rat is an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2, in the amino acid sequence represented by SEQ ID NO: 2 By detecting the mutation of amino acid number 251 methionine to lysine, it is possible to identify the Groggy rat or its progeny. The present invention provides a method for identifying such Gloggy rats or their progeny.

  The detection of the mutation can be performed by directly detecting a mutation in the Cacna1a polypeptide or by detecting a mutation in the polynucleotide encoding the Cacna1a polypeptide, and is not particularly limited.

  In the case of directly detecting a mutation in the polypeptide, for example, the amino acid sequence represented by SEQ ID NO: 2 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2, as in the above-described method for testing Cacna1a-related diseases. The presence of the mutant Cacna1a polypeptide is immunospecifically detected by an immunological technique using an antibody that specifically recognizes the polypeptide (for example, the antibody of the present invention).

  As a result of the detection, when the mutant Cacna1a polypeptide is detected, the individual can be determined as a Groggy rat or a progeny thereof. Conversely, if the mutant Cacna1a polypeptide is not detected, it can be determined that the individual is not a Groggy rat or its offspring.

  When detecting a mutation in the polynucleotide encoding the Cacna1a polypeptide, first, chromosomal DNA, total RNA, or mRNA is prepared from a biological sample collected from the test rat in the same manner as described above.

Next, it is determined whether or not the polynucleotide encoding the Cacna1a polypeptide contained in the obtained chromosomal DNA, total RNA or mRNA has a mutation. The mutation may be a mutation of a codon encoding methionine of amino acid number 251 in the amino acid sequence represented by SEQ ID NO: 2 to a codon encoding lysine. The codon encoding methionine is usually ATG. The codon encoding lysine is usually AAA or AAG, but preferably AAG.
More preferably, the mutation is a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.

  The mutation can be analyzed by a well-known method in the same manner as the above-described method for testing a Cacna1a-related disease.

For example, in the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which thymine at base number 752 is substituted with adenine, or a partial sequence consisting of a continuous nucleotide sequence of 15 bases or more including adenine at base number 752, or these It is possible to detect the presence or absence of mutation by an ASO hybridization method or an RNase A cleavage method by using a polynucleotide containing a complementary sequence of.
Alternatively, in the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 (the amino acid of amino acid number 251 is methionine or lysine), a primer capable of amplifying a region containing a codon encoding the amino acid of amino acid number 251 is used. Thus, it is possible to detect the presence or absence of mutation by a direct sequencing method or the like.
Furthermore, in the nucleotide sequence represented by SEQ ID NO: 1, by using a primer that can amplify a region containing a restriction site newly generated by mutation (for example, PsiI site), the corresponding restriction enzyme is amplified after the region is amplified. It is also possible to detect the presence or absence of mutation by determining whether or not the amplified product can be digested.

  If the mutation is detected as a result of the detection, the rat can be determined to be a Groggy rat or its offspring. Conversely, if the mutation is not detected, it can be determined that the rat is not a Groggy rat or its progeny.

  In particular, when this identification method is used, when the above mutation is present only in one of the homologous chromosomes of the Cacna1a gene, the rat is a progeny of the Groggy rat even if the rat does not exhibit the phenotype normally possessed by the Groggy rat such as ataxia. This is extremely useful in the maintenance, storage, backcrossing, etc. of Groggy rats.

  The present invention also provides a reagent for identifying a Groggy rat or its progeny, comprising a means for detecting a mutation of amino acid number 251 methionine to lysine in the amino acid sequence represented by SEQ ID NO: 2. The means is means for detecting a mutation of a codon encoding methionine of amino acid number 251 in the amino acid sequence represented by SEQ ID NO: 2 to a codon encoding lysine, or base number 752 in the nucleotide sequence represented by SEQ ID NO: 1. It can be a means for detecting a mutation of thymine to adenine. By using the test reagent, it is possible to easily carry out the above-described method for identifying a Groggy rat or its progeny.

The means is not particularly limited, but for example,
(1) an antibody that specifically recognizes a polypeptide comprising an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the amino acid sequence represented by SEQ ID NO: 2 (for example, the antibody of the present invention);
(2) In the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which thymine at base number 752 is substituted with adenine, or a partial sequence consisting of a continuous nucleotide sequence of 15 bases or more including adenine at base number 752; Or a polynucleotide comprising a complementary sequence thereof;
(3) a primer capable of amplifying a region containing a codon encoding the amino acid of amino acid 251 in a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 (the amino acid of amino acid 251 is methionine or lysine);
(4) a primer capable of amplifying a region containing a restriction site newly generated by mutation (eg, a PsiI site) in the nucleotide sequence represented by SEQ ID NO: 1 and a corresponding restriction enzyme;
Etc. can be mentioned.

  In addition to the above-mentioned means, various reagents used in the above identification method (for example, protein extraction reagent, labeled antibody, chromosomal DNA preparation reagent, total RNA preparation reagent, mRNA preparation reagent, enzyme, reagent and biological sample are diluted. Buffer, positive control, negative control, reaction container, instructions describing the test protocol, etc.) may be included to provide a kit for identification of Groggy rats or their progeny. These elements can be mixed in advance if necessary. Moreover, a preservative and preservative can also be added to each element as needed. The identification kit is extremely useful because it enables the above-described method for identifying a Groggy rat or its progeny to be easily carried out.

  The present invention also includes an amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 containing the lysine of amino acid number 251 Provided is a prophylactic / therapeutic agent for a Cacna1a-related disease comprising a polynucleotide (antisense mutant Cacna1a polynucleotide) comprising a complementary sequence of a nucleotide sequence encoding a polypeptide comprising a partial sequence consisting of a continuous amino acid sequence of at least amino acids. .

When the antisense mutant Cacna1a polynucleotide is used as the prophylactic / therapeutic agent, it can be formulated according to conventional means.
On the other hand, when the antisense mutant Cacna1a polynucleotide is used as the prophylactic / therapeutic agent, the antisense mutant Cacna1a polynucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. Thereafter, it can be administered according to conventional means. The antisense mutant Cacna1a polynucleotide can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter.
For example, the antisense mutant Cacna1a polynucleotide is orally administered as a sugar-coated tablet, capsule, elixir, microcapsule or the like, or with water or other pharmaceutically acceptable liquid as necessary. It can be used parenterally in the form of sterile solutions or injections such as suspensions. For example, unit dosage forms required for the practice of accepted formulations with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., for antisense mutant Cacna1a polynucleotides Can be produced by mixing with. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, for example, by administering an effective amount thereof to the above-mentioned vertebrates, Cacna1a-related diseases can be prevented and treated.

  Although the dosage of the antisense mutant Cacna1a polynucleotide varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, for example, generally in humans (as 60 kg) of Cacana1a related diseases, About 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, it is usually a human with Cacanala-related disease (as 60 kg). In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other vertebrates, an amount converted per 60 kg can be administered.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

[1] Experimental method (1) Gene mapping
(Groggy X BN) F1 female rats and Groggy male rats were mated to produce 766 backcross pups. Homo ( gry / gry ) individuals were identified by gait abnormalities observed from around 3-4 weeks of age. Chromosomal DNA was extracted from the spleen of the animal, and positional cloning was performed using 100 microsatellite markers covering all rat chromosomes. PCR is performed using Taq polymerase (Takara) under conditions of 35 cycles (denaturation 94 ° C, 30 seconds, annealing 55 ° C, 1 minute, extension reaction 72 ° C, 5 seconds), and PCR products use ethidium bromide staining. Then, it was observed by 4% agarose gel electrophoresis.

(2) RT-PCR and sequence analysis
Total RNA was extracted from 9-week-old Groggy rat brain using ISOGEN (NIPPON GENE, Tokyo), and 5 μg of this was extracted with Superscript II reverse transcriptase (Invitrogen, Leek, Netherland). cDNA was synthesized. Using 1 μl of cDNA dissolved in 50-μl as a template, PCR reaction was performed using primers prepared based on rat CACNA1A cDNA base sequence (NM_012918) retrieved from GenBank (Table 1).

The PCR product was subjected to cycle sequencing using Big Dye Terninator Ready Reaction Mix (Applied Biosystems, Foster City, CArlif) and purified with Dye Ex ^™ 2.0 Spin Kit (QIAGEN). The purified product was dissolved in 60% formaldehyde and sequenced by ABI 3100 genetic analyzer (Applied Biosystems).

(3) Genome sequence
PCR reaction using primer set (5'-CGTCCTGAAGTCAATCATGAAG-3 '(SEQ ID NO: 29), 5'-AGTCTCAGCTGCTCTGTGGTT-3' (SEQ ID NO: 30)) sandwiching the gry gene locus using Pyrobest (Takara) for 30 cycles (Denaturation 98 ° C., 2 seconds, annealing 60 ° C., 30 minutes, extension reaction 72 ° C., 1 minute). As templates, chromosomal DNA of Groggy parent rats, backcross homozygous and heterozygous individuals, ACI, BN, DON, DRH, F344, IS and WTC rats were used. The obtained PCR product was sequenced by the method described above.

(4) Restriction enzyme reaction Restriction enzyme PSi I (Sib Enzyme) was added to the PCR product replicated from the chromosomal DNA and allowed to stand at 37 ° C. for 14 hours. Cleavage with PSi I was observed by 2% agarose gel electrophoresis.

(5) Electroencephalogram recording Under pentobarbital anesthesia, an electrode for electroencephalogram recording was implanted in the cerebral cortex of Groggy rats, and a miniature plug was placed in the middle of the head to connect with Thermal Array Recorder (Nihon Kohden). EEG recordings were made one week after surgery. The animals were allowed to acclimatize for 30 minutes in a shielded box for electroencephalogram recording (40X40X40 cm), and then electroencephalogram recording and behavioral observation were performed for 30 minutes. The number of occurrences and duration for 30 minutes were measured with a 7-8 Hz spine-slow-wave complex over 1 second on the electroencephalogram appearing in coincidence with the animal's movement stop. In order to maintain the animal's wakefulness, the back of the Groggy rat was breathed and stimulated every 5 minutes.

(6) Pharmacological effect test of antiepileptic drugs Ethosuximide (SIGMA) 200 mg / kg and phenytoin sodium salt (SIGMA) 20 mg / kg were used. These antiepileptic drugs are in clinical use, and absence seizures are suppressed by ethosuximide but not by phenytoin. Ethosuximide was dissolved in polyethylene glycol and then diluted with sterilized water, and phenytoin dissolved in sterilized water was used. As a control, a 5% polyethylene glycol liquid (solvent) was prepared. A 15-minute control electroencephalogram was recorded prior to administration of the antiepileptic drug. Subsequently, a solvent, ethosuximide, or phenytoin was intraperitoneally administered, and the number and duration of absence-like seizures for 15 minutes after 15-30 and 45-60 minutes after the administration were measured (FIG. 1).

[2] Results (1) Identification of gry mutation
To determine the position of the gry gene on the linkage map, 766 backcross pups obtained from Groggy X (Groggy X BN) F1 are used to cover rat chromosomes 1 to 20 Positional cloning was performed using 80 microsatellite markers. The gry locus was linked to markers D19rat36 and D19Rat25 . Furthermore, the gry gene was mapped between D19Rat122 and D19Rat120 on rat chromosome 19 by linkage analysis using 20 markers covering chromosome 19 (FIG. 2). In this region, the Calcium channel α1A subunit ( Cacna1a ) gene encoding the Cav2.1 subunit of the voltage-dependent P / Q type calcium channel is mapped. Focusing on the Cacna1a gene as a promising candidate gene for gry mutation, RT-PCR was performed using primers covering the nucleotide sequence of rat CACNA1A mRNA and a brain cDNA derived from Groggy rat. A point sequence mutation from thymidine (T) to adenine (A) was found in the coding region of the Groggy rat Cacna1a gene by sequential sequence analysis. This mutation replaced the 251st methionine of CACNA1A protein with lysine (Met251Lys), and this site corresponded to the extracellular region of the P-loop forming the pore of the channel. According to genomic sequencing, this missense mutation was found only in Groggy parent rats and backcross individuals, and was unique to Groggy rats (FIG. 3A). Therefore, it was presumed that the mutation trait by the gry gene was caused by the Met251Lys mutation of the Cacna1a gene (FIG. 3C). As a result of searching restriction enzyme sites using the analysis software GENEX, it was found that a cleavage site of Psi I appeared in that region due to gry mutation. In order to confirm this, a restriction enzyme reaction with Psi I was performed using a PCR product amplified from the genome, and as a result, cleavage of the PCR product was confirmed only in individuals having the gry allele (FIG. 3B).

(2) Groggy rat seizure
In CACNA1A gene mutant mice represented by tottering, a spine-slow wave complex is observed consistent with behavioral absence-like seizures. In Groggy rats, similar traits were suspected, and behavioral observation and EEG recording were performed. The animal suddenly stopped moving during the EEG recording, when a 7-8 Hz spike-slow wave complex was observed on the EEG. The seizure frequency was 0.47 / min and the total duration of 30 minutes was 200.3 seconds (Table 2, Figure 3).

  Table 2 shows the frequency of absence-like seizures observed in Groggy rats (O) and the duration per seizure (D).

  Furthermore, in a pharmacological effect test using antiepileptic drugs, Groggy rat seizure-like seizures were clearly suppressed by ethosuximide, but not by phenytoin (Table 3, Figure 5).

  Table 3 shows the effect of antiepileptic drugs on absence-like seizures in Groggy rats.

  Vertebrate animals that can express the mutant Cacna1a polypeptide of the present invention, in particular, Groggy rats, are useful as model animals for various Cacna1a-related diseases. By using the model animals, prophylactic / therapeutic substances for Cacna1a-related diseases and the like can be obtained. It is possible to screen. Moreover, a Cacna1a-related disease can be examined by detecting a mutation similar to the mutation of the Cacna1a gene found in Groggy rats.

1 shows a protocol for testing the effect of antiepileptic drugs on absence-like seizures in Groggy rats. The mapping of the gry gene is shown. A) shows a distribution map of haplotype of rat chromosome 19 using 766 backcross pups. The black box represents a gry allele homozygote and the white box represents a heterozygote. B) shows a linkage map of the gry locus region. The identification of the gry mutation is shown. A) shows the genomic sequence of BN and WTC rats, backcross homozygous ( gry / gry ) and heterozygous ( gry / +) individuals. B) shows a restriction enzyme reaction with PsiI. C) shows substitution of the 251st amino acid from methionine (Mt) to lysine (Ly) by the gry mutation. The cerebral cortex electroencephalogram of a Groggy rat (6 weeks old) is shown. Consistent with behavioral absence-like seizures, a bilateral spinal-slow wave complex is observed on the electroencephalogram. The effect of ethosuximide (200 mg / kg, intraperitoneal (ip) administration) and phenytoin (20 mg / kg, ip administration) on absence-like seizures in Groggy rats is shown.

Claims (31)

  An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 or more consecutive amino acids including the lysine of amino acid number 251 A polypeptide comprising its partial sequence consisting of sequences.   An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 or more consecutive amino acids including the lysine of amino acid number 251 A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a partial sequence thereof, or a complementary sequence thereof.   In the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence in which thymine at base number 752 is substituted with adenine, a partial sequence consisting of a continuous nucleotide sequence of 15 bases or more including adenine at base number 752, or a sequence thereof A polynucleotide comprising a complementary sequence.   A recombinant vector containing the polynucleotide according to claim 2 or 3.   A transformant transformed with the recombinant vector according to claim 4. In the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which methionine of amino acid number 251 is substituted with lysine, or five or more consecutive amino acids including the lysine of amino acid number 251 Specifically recognizing a polypeptide comprising a partial sequence consisting of an amino acid sequence,
It does not recognize a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof consisting of a continuous amino acid sequence of 5 amino acids or more including the methionine of amino acid number 251 antibody.   Modifying the chromosome so that a polypeptide comprising an amino acid sequence in which the methionine of amino acid number 251 is replaced with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 can be expressed. A method for producing a Cacna1a-related disease model non-human vertebrate, comprising:   8. The method of claim 7, wherein the non-human vertebrate is a rat.   The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, neurodegeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axon tumor 8. The disease selected from the group consisting of large, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dyskinesia, cortical spike wave emission, and cerebellar nerve cell loss. the method of.   8. The method of claim 7, wherein the Cacna1a related disease is recessive hereditary.   A non-human vertebrate that can express a polypeptide comprising an amino acid sequence that is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2, wherein the methionine of amino acid number 251 is replaced by lysine, or a living organism thereof A screening method for a prophylactic / therapeutic substance for a Cacna1a-related disease, which comprises applying a test substance to a part and examining the effect of the substance on the pathological condition of the Cacna1a-related disease.   The method of claim 11, wherein the non-human vertebrate is a rat.   12. The method of claim 11, wherein the rat is a Groggy rat or its progeny.   The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, neurodegeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axon tumor The disease selected from the group consisting of large, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dysmotility, cortical spike wave emission, and cerebellar nerve cell loss. the method of.   12. The method of claim 11, wherein the Cacna1a associated disease is recessive hereditary.   A non-human vertebrate that can express a polypeptide comprising an amino acid sequence that is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2, wherein the methionine of amino acid number 251 is replaced by lysine, or a living organism thereof A screening method for a method for treating a Cacna1a-related disease, which comprises applying the treatment to a part and assaying the effect of the treatment on the pathology of the Cacna1a-related disease.   The method of claim 16, wherein the non-human vertebrate is a rat.   17. The method of claim 16, wherein the rat is a Groggy rat or a progeny thereof.   The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, neurodegeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axon tumor The disease selected from the group consisting of large, cerebellar ataxia, nystagmus, cerebellar symptoms, dizziness, nausea, vomiting, paroxysmal dysmotility, cortical spike wave emission, and cerebellar nerve cell loss. the method of.   17. The method of claim 16, wherein the Cacna1a associated disease is recessive hereditary.   A method for examining a Cacna1a-related disease, comprising detecting a mutation of amino acid number 251 methionine to lysine in an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2.   A test for a Cacna1a-related disease, comprising detecting a mutation of a codon encoding methionine of amino acid number 251 to a codon encoding lysine in the same or substantially the same amino acid sequence as represented by SEQ ID NO: 2. Method.   The Cacna1a related diseases include migraine, spinocerebellar ataxia, paroxysmal ataxia, epilepsy, ataxia, cerebellar atrophy, neurodegeneration, neuronal degeneration, concentric lamellar body formation, Purkinje cell axon tumor The disease selected from the group consisting of large, cerebellar ataxia, nystagmus, cerebellar symptom, dizziness, nausea, vomiting, paroxysmal dysmotility, cortical spike wave emission, and cerebellar nerve cell loss. The method described in 1.   23. The method of claim 21 or 22, wherein the Cacna1a related disease is recessive hereditary.   A reagent for testing Cacna1a-related disease, comprising a means for detecting a mutation of amino acid number 251 methionine to lysine in the same or substantially the same amino acid sequence represented by SEQ ID NO: 2.   Examination of Cacna1a-related disease comprising means for detecting a mutation of a codon encoding methionine of amino acid number 251 in the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 to a codon encoding lysine Reagent.   A method for identifying a Groggy rat or its progeny, comprising detecting a mutation of methionine of amino acid number 251 to lysine in the amino acid sequence represented by SEQ ID NO: 2.   A method for identifying a Groggy rat or its progeny, comprising detecting a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.   A reagent for identifying a Groggy rat or its progeny, comprising a means for detecting a mutation of methionine of amino acid number 251 to lysine in the amino acid sequence represented by SEQ ID NO: 2.   A reagent for identifying a Groggy rat or its progeny, comprising a means for detecting a mutation of thymine at base number 752 to adenine in the nucleotide sequence represented by SEQ ID NO: 1.   An amino acid sequence in which the methionine of amino acid number 251 is substituted with lysine in the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or 5 or more consecutive amino acids including the lysine of amino acid number 251 A prophylactic / therapeutic agent for a Cacna1a-related disease comprising a polynucleotide comprising a complementary sequence of a nucleotide sequence encoding a polypeptide comprising the partial sequence consisting of the sequence.