JP2016098215A - Pain gene and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means for screening a pain inhibitor (an analgesic) based on a novel mechanism by identifying a novel responsible gene associated with pain using the gene or a product thereof; and to provide a novel and useful drug discovery target for pain by revealing the relation between SCN11A and pain.SOLUTION: We found out that, by performing a pedigree linkage analysis of a patient with juvenile periodic limb pain and a genome-wide exome sequencing, the responsible gene of the familial pain in the pedigree concerned is SCN11A, and that a missense mutation induces pain into a coding portion of a particular amino acid residue in the gene, and constructed a pain inhibitor screening system using such gene mutations.SELECTED DRAWING: None

Description

  The present invention relates to a gene involved in pain, a mutation of the gene that causes juvenile periodic limb pain, and a method for screening a pain inhibitor using the same.

  Pain (hereinafter sometimes referred to as “pain”) is broadly divided into acute pain and chronic pain. Acute pain acts as a defense system that protects the body from further injury and causes temporary pain due to injury, burns, etc., but disappears with the disappearance or healing of painful stimuli. On the other hand, chronic pain is sustained for a long period of time, even if the injury recovers, pain continues, diabetes, cancer, musculoskeletal systems such as spondylosis, low back pain, etc. It may accompany various diseases such as connective tissue diseases, neurological diseases, rheumatic diseases, diseases of unknown cause such as fibromyalgia.

Pain affects as many as 10% of the world population. Chronic pain, in which pain continues for a long period of time, has a great social loss, such as significantly lowering the quality of life of patients and causing difficulty in working. Therefore, appropriate measures against pain are required. However, the fundamental analgesic substance related to chronic pain has not been identified. Non-inflammatory limb joint pain is one of the chronic pains often seen in the elderly, but the mechanism is still unclear.
On the other hand, there are families with limbs and / or limb joint pain that periodically occur in childhood and the mechanism of which is unknown. Patients in this family have periodic limb / limb arthralgia beginning in infancy, remission in adulthood, and occasionally without migraine, other than cardiac conduction disturbances or epilepsy. Moreover, the cause of the pain (hereinafter sometimes abbreviated as “juvenile periodic limb pain” in the present specification) has not been known for a long time, for example, no inflammation findings were observed in clinical examination.

Single-gene hereditary pain disorders have improved molecular understanding of pain and have provided new analgesic drug targets.
Voltage-gated sodium channels (Nav) are essential for generating action potentials in excitable cells. Ten genes encoding the α-subunit of the sodium channel have been identified in mammals. Sodium channel voltage gated type XI alpha (SCN11A) is a voltage-gated sodium channel (Nav1.9) that has resistance to tetrodotoxin, and is a small-diameter sensation in the dorsal root and trigeminal ganglia. It encodes a nociceptive receptor localized in small-diameter sensory neurons. Since knockout mice of this gene show an analgesic phenotype, and various inflammatory mediators alleviate hyperalgesia (Non-Patent Document 1), Nav1.9 is a major cause of peripheral inflammatory hyperalgesia. It is considered to be an effector.
SCN9A and SCN10A are known as SCN11A paralog genes. SCN9A encodes Nav1.7, which has tetrodotoxin-sensitive gate characteristics, and inherited Erythromelalgia, paroxysmal extreme pain, congenital insensitivity to Pain, etc. Is the causative gene. On the other hand, SCN10A, like SCN11A, has a gate characteristic that is resistant to tetrodotoxin and encodes Nav1.8, which is presumed to be functionally similar. SCN10A is known to be a causative gene of small fiber neuropathy and Brugada syndrome. However, as for SCN11A, the human symptoms caused by the mutation were not known, and the elucidation of the function was delayed.

As described above, SCN9A and SCN10A have been elucidated for the pain and organ localization involved in the respective genes, and the development of inhibitors specific to the respective gene products has been performed. However, since SCN9A and SCN10A exist in addition to nerves, the effects on the autonomic nervous system and the cardiac conduction system are recognized as complications. Therefore, SCN11A inhibitors confined to dorsal root ganglia and trigeminal ganglia are considered promising as headache drugs, and a wide range of sodium channel inhibitors including the gene product (Nav1.9) have been reported so far. (For example, refer to Patent Document 1).
However, since there were no human cases of the nature of the SCN11A mutation that caused pain, the effects of treatment could not be predicted, and no specific inhibitors of SCN11A have been developed.

JP 2013-100374 A

Proc Natl Acad Sci USA Vol.102 No.26 Page.9382-9387 (2005)

An object of the present invention is to provide a means for screening a pain-inhibiting substance (analgesic agent) based on a novel mechanism by identifying a novel causal gene related to pain and using the gene or its product. .
Another object of the present invention is to clarify the relationship between SCN11A and pain, and thus to provide a novel and useful drug discovery target for pain.

As a hereditary pain disorder of unknown cause, the present inventors focused on juvenile periodic limb pain, and for three generations of patient families that complain of limb joint pain and limb pain periodically in childhood over 4 generations, In order to investigate the causative gene, linkage analysis and whole genome exome analysis were performed. As a result, only a person with onset of juvenile periodic limb pain in the family had a missense mutation (C A T (c.665G> A) or A GT (c) at the 222nd codon (CGT) of the SCN11A gene. .664C> A)) existed, and it was found that the 222th arginine of the SCN11A translation product was replaced with histidine (p.R222H) or serine (p.R222S) by the mutation. As a result of the mutation search in the general population in the area where these patients live, the above mutation was not found, so SCN11A is the causative gene of pain, and the cause of juvenile periodic limb pain in 3 families is the gene It was identified as a p.R222H or p.R222S mutation.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows:
[1] A protein of any one of the following (a) to (d).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids are deleted and / or substituted and / or inserted and / or added A protein having an amino acid sequence (excluding substitution to arginine at position 222) and having a pain-inducing action (c) a nucleic acid comprising a complementary strand sequence of the base sequence shown in SEQ ID NO: 1, and a stringent A protein encoded by a nucleic acid that hybridizes under conditions and having a pain-inducing action (d) an ortholog in another mammal of a protein comprising the amino acid sequence shown in SEQ ID NO: 2, A protein [2] [1] in which the amino acid residue corresponding to the position is an amino acid other than arginine and has a pain-inducing action Nucleic acid encoding the protein of the placement.
[3] A vector comprising the nucleic acid according to [2].
[4] A host cell transformed with the vector according to [3].
[5] The cell according to [4], wherein the host cell is a mammalian cell, and the nucleic acid encoding endogenous SCN11A is substituted with the nucleic acid according to [2].
[6] A method for producing the protein, comprising culturing the cell according to [4] and recovering the protein according to [1] from the culture.
[7] A transgenic non-human mammal that exogenously expresses the nucleic acid according to [2] and exhibits a pain phenotype or a part of the living body thereof.
[8] The animal according to [7] or a part of the living body thereof, wherein the nucleic acid encoding endogenous SCN11A of the non-human mammal is substituted with the nucleic acid according to [2].
[9] The animal or part of its living body according to [7] or [8], wherein the non-human mammal is a mouse or a rat.
[10] A test substance in which the test substance is brought into contact with the cell according to [5] or the animal according to any one of [7] to [9] or a part of the living body to improve the pain phenotype A method for screening a substance that suppresses pain, which comprises selecting
[11] A method for screening a substance that suppresses pain,
(A) contacting the test substance with the cell according to [5] or the non-human mammal according to any of [7]-[9] or a part of the living body thereof;
(B) a step of measuring the expression level of the protein according to [1] or the nucleic acid according to [2] in each of the case where the test substance is contacted and the case where the test substance is not contacted; and (c) (b) A method comprising: selecting a test substance having a reduced expression level as a substance that suppresses pain.
[12] An antibody or fragment thereof that specifically binds to the protein according to [1].
[13] A test for diagnosis of a pain-related disease in a subject characterized by detecting the presence of the protein according to [1] or the nucleic acid according to [2] in a biological sample collected from the subject Method.
[14] An agent for treating abnormal pain in a mammal having a pain-inducing mutation or an analgesia-inducing mutation in the SCN11A gene, the agent comprising a regulator of an ion channel composed of the gene product.
[15] The SCN11A gene having the pain-induced mutation is
(A) An ortholog in another mammal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, corresponding to position 222 of the amino acid sequence The agent according to [14], wherein the amino acid residue is an amino acid other than arginine and encodes a protein having a pain-inducing action.

  According to the present invention, SCN11A is a gene associated with pain, and its p.R222H or p.R222S mutation has been shown to cause juvenile periodic limb pain. Since SCN11A is restricted to dorsal root ganglia and trigeminal ganglia compared to paralogous genes SCN9A and SCN10A, the expression of SCN11A or the function of its gene product, Nav1.9, is selective. By inhibiting, it becomes possible to suppress pain.

FIG. 1 shows a family tree (upper) and an exome analysis result (lower) of a family (family 1) analyzed according to the present invention. ■ and ● indicate pain, □ and ○ indicate non-onset, ■ and □ indicate men, and ● and ○ indicate women. The arrow indicates the proband. FIG. 2 shows a family tree of the family line (family line 2) analyzed according to the present invention. Square symbols indicate men and circles indicate women. For each mark, the left half fill indicates a person with limb pain, and the right half fill indicates a headache. The arrow indicates the proband. Samples were collected from the circled individuals and analyzed. FIG. 3 shows a family tree of a family line (family line 3) analyzed according to the present invention. ■ and ● indicate pain, □ and ○ indicate non-onset, ■ and □ indicate men, and ● and ○ indicate women. The diagonal lines indicate that the symptoms are unknown because they have already died. P indicates a proband. The number + y indicates age. Samples were collected from the circled individuals and analyzed. FIG. 4 shows the sequencing results of the base sequence of the 222nd codon of the SCN11A gene in the proband of family 3 and their parents.

Details of the present invention will be described below.
In the present invention, “pain” or “pain” is defined by the International Pain Society (IASP, 1981) using a term that is related to or represents a substantial or potential injury to the tissue. It is used in the meaning of “expressed unpleasant sensory or emotional experience” and includes any of nociceptive pain (somatic pain, visceral pain), neuropathic pain, and psychogenic pain. Moreover, although it can include both acute pain and chronic pain, it mainly refers to the case of chronic pain.
In the present invention, “having pain-inducing action” means having an action of causing pain in mammals such as humans regardless of the degree of pain such as dull pain and severe pain and the kind of pain. For example, the degree of pain expressed by VAS (visual analogue pain scale) is statistically significant on the side of 1 mm or more, preferably 10 mm or more, more preferably 20 mm or more, and 100 mm (that is, the maximum pain that can be imagined). More preferably, it means that the degree of pain expressed by VAS is increased to 70 mm or more, more preferably 80 mm or less.
On the other hand, “suppressing pain” refers to reducing or eliminating pain regardless of the degree and type of the pain. For example, it means that the degree of pain expressed in VAS is statistically significantly changed to 1 mm or more, preferably 10 mm or more, more preferably 20 mm or more, and 0 mm (that is, no pain), More preferably, it means that the degree of pain expressed by VAS is reduced to 30 mm or less, more preferably 20 mm or less.
In the present invention, the “pain-inducing mutation” means a genetic mutation in which a mutant protein produced by the mutation has “pain-inducing action” described above. On the other hand, the “painless sensation-induced mutation” means a genetic mutation that causes a mutant protein caused by the mutation to blunt or lose pain sensation (analgesia).

  The voltage-gated sodium channel (Nav) is an ion channel that is essential for excitable cells to generate action potentials. The gate opens and closes due to changes in membrane potential, allowing Na ions to pass through according to the concentration difference inside and outside the cell. , Thereby a protein that changes the charge state of the cell membrane. Nav is composed of an α subunit and several β subunits that are responsible for most of its functions. To date, 10 subtypes (Nav1.1 to 1.9 and Nax) have been cloned as α subunits. Among them, Nav1.8 and Nav1.9 are specifically expressed in small nociceptive neurons that are present in peripheral sensory ganglia such as dorsal root ganglia (DRG) and trigeminal ganglia and are thought to transmit noxious stimulus information. Therefore, it may be involved in the development of pain. In fact, there have been many reports suggesting an association between Nav1.8 and inflammatory hyperalgesia and neuropathic pain. On the other hand, there are very few reports on the relationship between Nav1.9 and the development of pathological pain.

  The sodium channel voltage gated type XI alpha (SCN11A) gene encodes Nav1.9, and the human SCN11A gene encodes a protein consisting of 1791 amino acids. The nucleotide sequence of human SCN11A cDNA and the amino acid sequence of the translation product are registered in the National Center of Biotechnology Information (NCBI) database as NCBI RefSeq numbers NM_001287223 and NP_001274152, respectively.

The present invention provides a novel SCN11A gene having a pain-inducing mutation (hereinafter also referred to as “mutant SCN11A gene of the present invention”) and a mutant Nav1.9 protein having a pain-inducing action, which is a translation product thereof. The mutant Nav1.9 protein of the present invention is one of the following (a) to (d).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids are deleted and / or substituted and / or inserted and / or added A protein having an amino acid sequence (excluding substitution to arginine at position 222) and having a pain-inducing action (c) a nucleic acid comprising a complementary strand sequence of the base sequence shown in SEQ ID NO: 1, and a stringent A protein encoded by a nucleic acid that hybridizes under conditions and having a pain-inducing action (d) an ortholog in another mammal of a protein comprising the amino acid sequence shown in SEQ ID NO: 2, A protein in which the amino acid residue corresponding to the position is an amino acid other than arginine and has a pain-inducing action. "And / or" is used in a meaning including either one or both.
Here, the “amino acid sequence shown in SEQ ID NO: 2” is an amino acid sequence obtained by replacing arginine at position 222 with histidine (p.R222H) or serine (p.R222S) in the amino acid sequence of a known human Nav1.9 protein. is there. The “base sequence shown in SEQ ID NO: 1” is a known human SCN11A cDNA sequence in which the 665th G of the coding sequence (CDS) is A (corresponding to the p.R222H mutation) or the 664th C is A (Corresponding to p.R222S). That is, in the present invention, the causative gene of familial juvenile periodic limb pain having the following characteristics (i) to (vi) is the SCN11A gene, and the mutation that induces the pain is p.R222H (c.665G > A) or p.R222S (c.664C> A).
(i) Autosomal dominant.
(ii) Pain begins in infancy, remits in adulthood, and sometimes does not cause cardiac conduction injury or epilepsy other than leaving migraine.
(iii) Pain is strong spontaneous limb and limb joint pain that can be touched by bone.
(iv) No inflammatory findings.
(v) Pain is periodic and improves with warming induced by weather and cold exposure.
(vi) Non-steroidal anti-inflammatory analgesics mildly relieve pain.

Regarding (b) above, more specifically, (i) 1 to 50, preferably 1 to 20, more preferably 1 to number (5, 4, 3 or 2) an amino acid sequence in which one amino acid is deleted, (ii) 1 to 50, preferably 1 to 20, more preferably 1 to a number (5, 4, 3 or 2) in the amino acid sequence shown in SEQ ID NO: 2. ) Amino acid sequence added with amino acids, (iii) 1 to 50, preferably 1 to 20, more preferably 1 to several (5, 4, 3 or 2) amino acid sequence shown in SEQ ID NO: 2. (Iv) 1 to 50, preferably 1 to 20, more preferably 1 to several (5, 4, 3 or 2) amino acid sequences shown in SEQ ID NO: 2 An amino acid sequence in which the amino acid is replaced with another amino acid, or (v) a protein comprising an amino acid sequence combining them And the like. However, the case where position 222 is substituted with arginine in SEQ ID NO: 2 is excluded.
For example, human polymorphisms of human SCN11A that cause amino acid substitutions found in nature include glycine at position 481 substituted with glutamic acid (p.G481E; registered as rs12059805 in the NCBI dbSNP database), position 777 Methionine substituted with arginine (p.M777R; registered as rs4302324 in the NCBI dbSNP database), valine at position 909 replaced with isoleucine (p.V909I; registered as rs33985936 in the NCBI dbSNP database) ), Tyrosine at position 1198 is substituted with histidine (p.Y1198H; registered as rs12638601 in the NCBI dbSNP database), and the like.

  Furthermore, substitution of amino acids with similar properties (for example, glycine and alanine, valine and leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) If so, there may be a greater number of substitutions. When the amino acid is deleted, substituted or inserted as described above, the position of the deletion, substitution or insertion is not particularly limited as long as the pain-inducing action is maintained. Mutation can be introduced by a method known per se, such as site-directed mutagenesis, alanine scanning, or mutagenesis by PCR.

In the above (c), “stringent conditions” means nucleotide sequences having high identity, for example, 95% or more, preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, particularly preferably. Conditions under which nucleotide sequences having 99.5% or more identity are hybridized and nucleotide sequences with lower identity are not hybridized, specifically, Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1 -6.3.6, 1999, for example, hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., then at least once at 0.2 × SSC / 0.1% SDS / 50-65 ° C. However, those skilled in the art can appropriately select the hybridization conditions that give the same stringency.
Here, the “nucleotide sequence identity” 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; gap cost = Linear; filtering = ON; It can be calculated by match score = 1; mismatch score = -2).
Moreover, the Nav1.9 isoform encoded by the splicing variant of the SCN11A gene consisting of the base sequence shown in SEQ ID NO: 1 is also included as the protein of (c) above. For example, as an isoform of human Nav1.9, an isoform in which threonine at position 1444 is substituted with lysine and amino acids at positions 1445 to 1791 are deleted in the amino acid sequence shown in SEQ ID NO: 2 (UniprotKB identifier: Q9UI33-2) And an isoform (see UniprotKB identifier: Q9UI33-3) in which amino acids at positions 946 to 983 are deleted in the amino acid sequence shown in SEQ ID NO: 2.

  In the above (d), as the SCN11A gene ortholog derived from a mammal species other than human, in the amino acid sequence of mouse Nav1.9 protein registered as RefSeq number NP_036017 in the NCBI database, arginine at position 222 is another amino acid, preferably Has an amino acid sequence substituted with histidine or serine, in the amino acid sequence of rat Nav1.9 protein registered as RefSeq number NP_062138, amino acid sequence in which arginine at position 219 is substituted with another amino acid, preferably histidine or serine In the amino acid sequence of the bovine Nav1.9 protein registered as RefSeq number XP_002696965, having the amino acid sequence in which arginine at position 225 is replaced with another amino acid, preferably histidine or serine, accession number in UniprotKB Registered as H2QMB6 In the amino acid sequence of the chimpanzee Nav1.9 protein, those having an amino acid sequence in which arginine at position 222 is substituted with another amino acid, preferably histidine or serine, and the like are mentioned.

  In addition to the above sequences, the SCN11A gene or its gene product in other mammals can be obtained by querying the base sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 and / or the genome of non-human mammals and / or Alternatively, the nucleotide sequence and amino acid sequence of SCN11A derived from a desired species can be obtained by searching the cDNA database or protein database using BLAST or FASTA.

Mutant Nav1.9 protein having a pain-inducing action can be isolated from a small nociceptive neuron of a peripheral sensory ganglion of a mammal having the mutation, using a known membrane protein separation and purification method. Alternatively, mRNA is extracted from the cells, for example, a primer is designed based on the cDNA sequence information of human SCN11A shown in SEQ ID NO: 1, and the mutant SCN11A cDNA is cloned by performing RT-PCR using the mRNA as a template. It can also be obtained by inserting the cDNA into an appropriate expression vector, introducing and expressing it in a host cell, and recovering the mutant Nav1.9 protein from the culture of the host cell.
However, more simply, a normal SCN11A cDNA is cloned from a mammal having a normal SCN11A gene having no pain-inducing mutation by the same method as described above, and SEQ ID NO: 2 by a site-directed mutagenesis method known per se. Can be obtained by introducing a mutation that replaces the codon encoding the arginine residue corresponding to position 222 of the amino acid sequence shown in the above with another amino acid, preferably a histidine or serine residue.

  The mutant SCN11A gene of the present invention is not particularly limited as long as it is a nucleic acid containing a base sequence encoding the mutant Nav1.9 protein having the pain-inducing action described above. The nucleic acid may be any of double-stranded DNA, single-stranded DNA, single-stranded RNA, double-stranded RNA, and DNA / RNA hybrid, but is preferably double-stranded DNA or single-stranded RNA. More preferably, it is a double-stranded DNA. The mutant SCN11A gene of the present invention can be used by linking to a vector (cloning vector or expression vector). Furthermore, according to the purpose (for example, protein expression), a suitable one for the host cell into which the vector is introduced can be appropriately selected, and the vector can be transformed into the host cell. The vector can further contain an appropriate promoter, terminator region, a selection marker gene for selecting a transformant (drug resistance gene, gene complementing an auxotrophic mutation, etc.) and the like.

  In addition, the vector may contain a sequence encoding a tag sequence (for example, His tag, GST tag, FLAG (trademark) tag, etc.) useful for protein separation / purification. It may be incorporated into the genome.

  The type of vector is not particularly limited, but pBTrp2, pBTac1, pBTac2, pBluescript II SK (+), pBluescript II SK (-), pSTV28, pUC118, pUC19, pKK233-2, pSE280, pSupex, pUB110, pTP5, pC194 , PET vector, pAGE vector, pcDNA vector, pGEX vector, pMC1neo, λgt11, pkk223-3, pBlue BacIII, pVL1392, pVL1393 and the like.

Examples of the host cell include any cell capable of expressing the vector, for example, bacterial cell, fungal cell, insect cell, animal cell and the like.
Examples of bacterial cells include, but are not limited to, Escherichia coli, Bacillus, Brevibacillus, Corynebacterium, Streptomyces, and the like. Although it does not specifically limit as a fungal cell, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Aspergillus (Aspergillus) genus fungi, etc. are mentioned. Insect cells include, but are not limited to, silkworms (Bombyx mori), nettle squirrels (Trichoplusia ni), and the like. Examples of animal cells include, but are not limited to, CHO cells, HeLa cells, COS-1 cells, COS-7 cells, HEK293 cells, BALL-1 cells, HCT-15 cells, PC12 cells and other cell lines, dorsal root ganglia (DRG) Neuron primary culture, ES cells, iPS cells and the like.

  The introduction of the vector into the host cell may be carried out by a known transformation method such as competent cell method, protoplast method, calcium phosphate coprecipitation method, electroporation method, microinjection method, lipofection method, etc. according to each host cell. Can be used.

  When using transformed cells for the purpose of purification of mutant Nav1.9 protein, it is preferable to use cells that do not express the endogenous SCN11A gene as host cells in order to facilitate purification, but even when animal cells are used. Since there are some differences in the amino acid sequence of the SCN11A protein between humans, mice, rats, etc., for example, when producing recombinant human-derived mutant Nav1.9 protein, primates such as mice or rats, etc. Other mammalian cells can also be used.

  On the other hand, when the transformed cell is intended to be used, for example, as a screening system for a pain suppressor described later, it is preferable to use a mammalian cell, preferably a nerve cell, particularly a DRG neuron, etc. as a host cell. It is desirable to replace (knock in) the coding region of the sex SCN11A gene with a nucleic acid encoding a mutant Nav1.9 protein mounted on the vector. In order to easily obtain mutant SCN11A knock-in cells, ES cells or iPS cells can be used as host cells, knock-in ES or iPS cells can be produced by homologous recombination, and then differentiated into nerve cells by a known differentiation induction method. it can.

  The above transformed cells can be cultured by a method known per se. The medium used for the culture can be appropriately selected for each desired cell. For example, LB medium or the like can be used for E. coli culture, SD medium or the like can be used for yeast culture, and DMEM medium or EMEM medium can be used for mammalian culture. In addition, various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, etc.) and various types of medium (semi-solid medium, liquid medium, powder medium) are used as necessary. Amino acids (including natural amino acids or synthetic amino acids), nucleobases (purines, pyrimidines), inorganic salts (MgSO4, MnSO4, FeSO4, NaCl, etc.) antibiotics (ampicillin, kanamycin, hygromycin) and the like can be added.

  A person skilled in the art can appropriately determine the culture time, the culture temperature, and the like according to the type of cell. For example, the culture temperature can be set to 10 to 45 ° C., preferably 20 to 40 ° C., and the culture time can be set to 1 hour to 14 days, preferably 10 hours to 7 days. The pH can also be adjusted as appropriate.

  Mutant Nav1.9 protein obtained by the above method can be purified by a method known per se. For example, after the bacterial cells collected by centrifugation or the like are ground with ultrasonic waves or glass beads, solid substances such as cell debris are removed by centrifugation or the like, and a crude enzyme solution is prepared. A salting-out method using sodium or the like, ion exchange chromatography, gel filtration chromatography, chromatographic methods such as affinity chromatography, gel electrophoresis, or the like can be used.

  Since the mutated Nav1.9 protein encoded by the mutated SCN11A gene of the present invention has a pain-inducing action in mammals, a nucleic acid encoding the mutated Nav1.9 protein is introduced into a non-human mammalian cell to produce a transgenic animal. Can be used to provide a pain model animal. Therefore, the present invention also provides a transgenic (hereinafter also referred to as Tg) non-human mammal that exogenously expresses the mutant SCN11A gene of the present invention and exhibits a pain phenotype.

The Tg animal of the present invention is a fertilized egg of a non-human mammal, an unfertilized egg, a sperm and a progenitor cell thereof (primitive germ cell, oocyte cell, oocyte, egg cell, spermatogonia cell, spermatocyte, sperm cell) Etc.), preferably in the early stage of embryo development of fertilized eggs (more preferably before the 8-cell stage), calcium phosphate coprecipitation, electroporation, lipofection, aggregation, microinjection (micro It is produced by introducing the mutant SCN11A gene of the present invention by gene introduction methods such as injection), gene gun (particle gun), DEAE-dextran.
One embodiment of using a virus as an expression vector includes a method of infecting an early embryo or ES cell of a non-human mammal with a virus containing DNA encoding a mutant Nav1.9 protein (eg, Proc. Natl). Acad. Sci. USA, 99 (4): 2140-2145, 2002). For example, when using a retrovirus or lentivirus, seed the cells in a suitable incubator such as a dish, add the viral vector to the culture, and incubate for 1-2 days. For embryonic female non-human mammals transplanted into the oviduct or uterus, if ES cells, a selective agent such as G418 or hygromycin is added and culture is continued to select cells into which the vector has been incorporated.
Furthermore, as described in Proc. Natl. Acad. Sci. USA, 98: 13090-13095 (2001), a viral vector is used during co-culture of spermatogonia collected from male non-human mammals with STO feeder cells. After infecting, the exogenous mutant SCN11A gene hetero Tg (+/-) offspring can be obtained efficiently by injecting into the seminiferous tubule of male infertile non-human mammal and mating with female non-human mammal Can do.

  Further, by this gene introduction method, DNA containing the mutant SCN11A gene of the present invention can be introduced into somatic cells, tissues, organs, etc. of non-human mammals, and can be used for cell culture, tissue culture, etc. Tg animals can also be produced by fusing cells with the aforementioned embryonic (or reproductive) cells using known cell fusion methods. Alternatively, as in the case of producing a knockout animal, the embryonic stem cell (ES cell) or the induced pluripotent stem cell (iPS cell) of the non-human mammal contains the nucleic acid of the present invention using the above gene transfer method. After introducing a DNA and selecting a clone in which the DNA is stably integrated in advance, the ES cell is injected into a blastocyst or an ES cell mass and an 8-cell embryo are aggregated to produce a chimeric mouse. It is also possible to obtain Tg animals by selecting those in which the introduced DNA is transmitted to the germ line.

  Furthermore, the Tg animal is more preferably a knock-in (KI) animal in which the endogenous SCN11A gene is replaced with a sequence encoding a mutant human Nav1.9 protein. Therefore, the Tg animal of the present invention introduces the target DNA into ES or iPS cells using an appropriate targeting vector, and converts the endogenous SCN11A gene by homologous recombination into a sequence encoding a mutant human Nav1.9 protein. It can also be made by substitution.

The present invention relates to a part of a living body of a Tg animal (preferably KI animal) produced as described above (for example, (1) a cell (for example, a small nociceptive neuron) stably holding a chimeric SCN11A gene, tissue (E.g., dorsal root ganglia, trigeminal ganglia) etc. (2) cells or tissues derived from these, and subcultured as necessary, etc.) can also be used for the mutant human Nav1.9 protein of the present invention. The chimeric SCN11A gene capable of producing the human mutant Nav1.9 protein of the present invention can be expressed as a part of the living body of a transgenic non-human mammal that retains the chimeric SCN11A gene that can be produced. It can be used for the same purpose as the non-human mammal to be retained.
The Tg non-human mammal obtained by the above-described method or a part of the living body thereof can be used as a pain-inducing model animal or model cell.

The non-human mammal (recipient animal) that can be the subject of the present invention is not particularly limited as long as it is a mammal other than a human in which a transgenic system (knockout system in the case of KI animals) has been established. Rats, cows, monkeys, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters and the like. Preferably, rodents with relatively short ontogeny and biological cycle and easy breeding are more preferable in terms of production of disease model animals, especially mice (for example, C57BL / 6 strain, BALB / c strain, C3H strain as a pure strain) , FVB strain, DBA2 strain, etc., and B6C3F1 strain, BDF1 strain, B6D2F1 strain, ICR strain, etc.) and rats (eg, Wistar, SD, etc.) are preferred as hybrids.
In addition to mammals, birds such as chickens can be used for the same purpose as the non-human mammals targeted by the present invention.

  Furthermore, the present invention includes a method of screening for a substance that acts on the mutant Nav1.9 protein of the present invention. Substances that act include substances that bind to the mutant Nav1.9 protein, substances that are associated with the pain-inducing action of the mutant Nav1.9 protein, etc. The screening can be performed for the purpose of searching for a substance to be promoted.

  The method is not particularly limited, but the cells in the presence and absence of the test substance or the like for cells, tissues or transgenic non-human mammals expressing the above-mentioned mutant Nav1.9 protein or a fragment thereof, etc. By measuring the sodium ion current, action potential, etc. in the cells, etc. by a method known per se such as patch clamp method, and the like, and the sodium current in the presence and absence of the test substance A method for examining fluctuations in action potential and the like can be mentioned.

  Furthermore, the present invention includes a method of screening for a substance that suppresses pain. The method is not particularly limited. For example, the test substance is contacted with a cell, tissue or transgenic non-human mammal expressing the mutant Nav1.9 protein or a fragment thereof, and the test substance is not contacted. Examples include a method of measuring and comparing changes in pain phenotype in the cells, tissues, or animals, and selecting a test substance with improved pain phenotype as a candidate for a pain suppressing substance.

  The contact of the test substance is carried out by adding the test substance to a medium or buffer in the case of cells or tissues, and in the case of transgenic non-human mammals, the test substance is administered orally or parenterally. It can be performed by administering. The amount added to the medium and the like and the dose to the animal can be appropriately selected according to the test substance.

  The pain-inhibiting action can be evaluated using as an index the pain phenotype, for example, the change of sodium ion current in cells / tissues, and the reduction of pain symptoms in animals.

Alternatively, as another method of screening for a substance that suppresses pain, the following steps (a) to (c):
(A) contacting a test substance with a cell, tissue or transgenic non-human mammal expressing the above-described mutant Nav1.9 protein,
(B) a step of measuring the expression level of the mutant Nav1.9 protein or a nucleic acid encoding the same in the case where the test substance is brought into contact with the test substance, and (c) the expression level in (b). And a step of selecting the reduced test substance as a candidate for pain suppressing substance.

  The contact of the test substance in the step (a) can be performed in the same manner as described above. The measurement of the expression level in the step (b) can be carried out using a known protein or RNA detection method. The cells, tissues, and Tg animals used here are preferably those that do not express the endogenous SCN11A gene, and the expression of the mutant SCN11A gene is preferably controlled by the expression regulatory region of the endogenous SCN11A gene. Accordingly, in a preferred embodiment, a knock-in cell / tissue or animal in which the coding region of the endogenous SCN11A gene is replaced with a nucleic acid encoding a mutant Nav1.9 protein is used. However, even when using transformed cells or Tg animals that express the endogenous SCN11A gene, an anti-mutated Nav1.9 protein-specific antibody (that is, it can be used for clinical tests for the diagnosis of pain-related diseases described below (ie, An antibody that does not cross-react with the endogenous Nav1.9 protein) or a primer and / or probe that specifically detects only the mRNA encoding the mutant Nav1.9 protein, and encodes the mutant Nav1.9 protein It is possible to measure only mRNA.

  Test substances that can be tested in any of the above screening methods are not particularly limited, and examples thereof include low molecular compounds, high molecular compounds, proteins (cytokines, chemokines, antibodies, etc.), nucleic acids (DNA, RNA, etc.), viruses, Examples include compound libraries prepared using combinatorial chemistry techniques, random peptide libraries prepared by solid phase synthesis or phage display methods, natural components derived from microorganisms, animals, plants, marine organisms, and the like. Such a substance can be appropriately determined by those skilled in the art.

  The substance that suppresses pain obtained by the above-mentioned method is formulated with the substance as an active ingredient, added with excipients, binders, disintegrants, lubricants, etc., and used as a pain treatment or prevention agent You can also.

  In one aspect, the present invention provides a p.R222H (c.665G> A) or p.R222S (c.664C> A) mutation of the subject's SCN11A gene (if the subject is a mammal other than a human, Detecting a pain-related disease in the subject, comprising detecting a mutation in which an arginine residue corresponding to arginine at position 222 of the Nav1.9 protein is substituted with another amino acid, preferably histidine or serine) An inspection method is also provided.

In one embodiment, the mutation can be detected using any known SNP detection method. For example, using genomic DNA extracted from a subject's cells or the like as a sample, using a nucleic acid containing a continuous base sequence of about 15 to about 500 bases including any of the polymorphic site bases described above as a probe, for example, According to the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)), hybridization is carried out while accurately controlling stringency, and only sequences that are completely complementary to the probe are detected. The reaction temperature is gradually increased from the denaturation temperature using a method or a mixed probe in which one of the nucleic acid and the nucleic acid in which the polymorphic site in the nucleic acid is replaced with another base is labeled and the other is unlabeled. Hybridization while reducing the amount, and a method that prevents a cross-reaction with a probe having a mismatch by first hybridizing a completely complementary sequence with one probe. And the like. Examples of the labeling agent include radioisotopes (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenation) Enzymes, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, Cy3, Cy5, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) are used.

  Preferably, the detection of the polymorphism is performed by various methods described in, for example, WO 03/023063, such as RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturing agent concentration gradient gel electrophoresis. Electrophoresis, RNase A cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method , UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method and the like (see WO 03/023063, page 17, line 5 to page 28, line 20).

  As a kit suitable for carrying out the above-described inspection method, for example, p.R222H (c.665G> A) or p.R222S (mutant SCN11A gene) of the nucleic acid sequence encoding the mutant Nav1.9 protein described above is used. a kit containing a primer capable of amplifying a region containing c.664C> A). The primer can be prepared by a method known per se. For example, in the case of human, it has 15 or more consecutive nucleic acids of the base sequence shown in SEQ ID NO: 1, and p.R222H (c.665G> A) or p.R222S (c.664C> A) of the mutant SCN11A gene As a primer capable of amplifying a region containing.

  As another embodiment of detecting the p.R222H (c.665G> A) or p.R222S (c.664C> A) mutation of the SCN11A gene, there is a method for detecting the expression of the mutant Nav1.9 protein in the subject. It is done. For example, mutant Nav1.9 protein in a DRG biopsy sample is detected using immunological techniques such as Western blotting, radioisotope immunoassay (RIA), fluorescent antibody method, ELISA method, etc. be able to.

For the detection of the mutant Nav1.9 protein, an antibody or fragment thereof that specifically binds only to the mutant Nav1.9 protein and does not bind to the Nav1.9 protein that does not have the p.R222H or p.R222S mutation is used. The The method for producing the antibody is well known and can be produced by a method known per se. As the immunogen, the entire mutant Nav1.9 protein may be used, or a histidine at position 222 or a fragment thereof containing serine may be used. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. Examples of antibody fragments include, but are not limited to, scFv, Fab, Fab ′, (Fab ′) ₂ , Fv, Fd, scFv, sdFv, and the like.

Pain-related diseases include, but are not limited to, limb joint pain, neuropathy due to trauma or pain due to metabolic neuroinflammation, diabetic peripheral nerve injury, cancer pain, and the like. Preferred is familial juvenile periodic limb pain, and particularly preferred is familial juvenile periodic limb pain having the following characteristics (i) to (vi).
(i) Autosomal dominant.
(ii) Pain begins in infancy, remits in adulthood, and sometimes does not cause cardiac conduction injury or epilepsy other than leaving migraine.
(iii) Pain is strong spontaneous limb and limb joint pain that can be touched by bone.
(iv) No inflammatory findings.
(v) Pain is periodic and improves with warming induced by weather and cold exposure.
(vi) Non-steroidal anti-inflammatory analgesics mildly relieve pain.
Among these pain characteristics, (ii) it begins in infancy, remits in adulthood, without heart and autonomic abnormalities, and (v) is induced by cold exposure and can be improved by warming. 2 points are different from the pain of hereditary pain caused by mutations in SCN9A and SCN10A, which are paralogous genes of SCN11A, so it is unique due to SCN11A gene regardless of familial or sporadic (due to de novo mutation) The presence of pain based on this mechanism was shown.

The present invention is based on the first discovery that SCN11A is a causative gene of pain, and that specific gene mutation induces familial juvenile periodic limb pain. That is, it was demonstrated for the first time by the present invention that an abnormal function of the Nav1.9 sodium channel, which is the SCN11A gene product, can cause the development of pain or hypoalgesia. This is because, in mammals that have developed pain dysfunction with a pain-induced mutation or a blunting-induced mutation in the SCN11A gene, by regulating the function of the Nav1.9 sodium channel, which is the SCN11A gene product, the abnormal dysfunction is detected. It shows that it can be treated.
Therefore, the present invention is also a therapeutic agent for abnormal pain in a mammal having a pain-inducing mutation or a blunting-inducing mutation in the SCN11A gene, comprising a regulator of an ion channel composed of the gene product. An agent is provided.

  Mammals (eg, humans, monkeys, cows, pigs, dogs, cats, mice, rats, etc.) having pain-inducing mutations or blunting-inducing mutations in the SCN11A gene to be treated have symptoms of pain or hypoalgesia. For the mammals shown, it can be estimated by, for example, isolating genomic DNA or DRG neuron-derived mRNA and examining whether or not it has a mutation that causes an amino acid change in the coding region of the SCN11A gene. Pain-inducing mutation is p.R222H or p.R222S (in mammals other than humans, substitution of arginine residues corresponding to arginine at position 222 of human Nav1.9 protein with other amino acids, preferably histidine or serine) In such a case, the mutation can be detected using a method similar to the above-described test method for diagnosis of pain-related diseases.

  In order to characterize the sodium channel constituted by the mutant Nav1.9 protein encoded by the SCN11A gene with a pain-inducing mutation or an analgesia-inducing mutation, the identified mutant SCN11A gene can be used as a DRG neuron from mammals. In a cell expressing a mutant Nav1.9 protein obtained by introduction into a cell, etc., a per se known electrophysiological assay such as a patch clamp method can be performed to compare the characteristics of sodium channels with wild-type Nav1.9 .

  The action of the mutant Nav1.9 protein encoded by the identified mutant SCN11A gene to bring the channel characteristics (eg, sodium current magnitude, persistence, etc.) different from wild-type Nav1.9 closer to those of wild-type Nav1.9 By administering a modulator (modulator) of Nav1.9 sodium channel having the above to a mammal having the mutant SCN11A gene, it is possible to treat abnormal pain such as pain and hypoalgesia in the mammal. For example, when pain dysfunction is due to the generation of sodium current due to excessive mutation Nav1.9 compared to wild type Nav1.9, the symptoms can be alleviated by administering a blocker of Nav1.9 sodium channel . Examples of such channel blockers include compounds described in JP2013-100374, JP2012-149084, JP2012-126751, JP2012-126745, JP2012-126744, and the like. However, the present invention is not limited thereto, and other known channel blockers and novel drugs selected by the above-described screening method of the present invention can also be preferably used. Alternatively, siRNA, shRNA, miRNA, antisense oligonucleic acid, and ribozyme targeting SCN11A mRNA can be used for the purpose of selective inhibition of Nav1.9 sodium channel as an inhibitor of mutant Nav1.9 protein expression.

  The present invention will be described more specifically with reference to the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

In the examples below the sample, 3 patient families (family 1 to 3) were selected that complained of limb pain periodically in childhood over 4 generations. Informed consent was obtained from all subjects. This study was approved by the ethics committees of Kyoto University and Akita University.

Example 1 Linkage analysis and whole genome exome analysis of family 1 In this family, sudden limb pain occurs from the infancy. For limb pain until puberty, when the low pressure comes, the pain lasts for 30 to 40 minutes, then there is a painless time of about 1 hour and a half, and then it repeats the seizure of the 2 hour cycle that it becomes painful again Although it has changed, it has gradually improved since then, and it has a frequency of about 1 to 2 times / year in adulthood. Although pain is limited to the limbs, the pain site is indefinite and varies from a single limb only to all limbs, and may be only the upper limbs. There is no swelling / redness at the pain site, no pulsation pain, dull pain with a cold sensation, and slightly improves with warming. Bone and muscle changes were not observed, and nerve findings were also abnormal. No abnormalities in physique after adulthood. Fig. 1 (upper panel) shows the family tree (II-IV generation) of this family.

  Linkage analysis was performed in this family, and a linked region with a LOD score of 1.20 was found in the third chromosome region. The exome results of all 8 participants in the family are shown in Figure 1 (lower panel). New genetic polymorphisms that all patients have and no non-symptomatic individuals have are 5 genes ANKRD23, SCN11A, STAB1, RPAP1, and ITGA11. From the functions, SCN11A is predicted to be the causative gene. The mutation of the SCN11A gene was p.R222H. Details of Quality Control (QC) and Quality Assurance (QA) for exome analysis are shown below.

  Whole genome exome analysis was performed using Illumina HiSeq 2000, a next-generation sequencer. Genomic DNA was captured using Agilent's SureSelect Human All Exon V4 + UTRs Kit, and a library was prepared according to Illumina's protocol. The genomic DNA prepared from the subject was fragmented, and the genomic fragment of 150-200 bp (excluding the adapter) was linked to the capture to make a library. Multiplex sequencing was performed using a flow cell designated by Illumina. Read length was 100 bps. UCSC Genome Browser hg19 was used as a RefSeq database and analyzed using Burrows-Wheeler Aligner 0.6.2 (BWA). The Genome Analysis Toolkit 1.6-13-g91f02df (GATK) was used for analysis of SNP and In / del. The number of effective reads and bases averaged about 84.86 million read pairs and about 5.3 Gbp on a total of 11 samples including 3 samples from Family 2 described below. The analysis results are summarized in Table 1.

  Mutations in each sample including MS / NS / Indel / SS / FS / RT (MS, Missense; NS, Nonsense; Indel, Insertion or deletion; SS, Splice Site; FS, Fragile Sites; RT, Read through) Approximately 10,000 were found, and there were 61 heterozygous mutations that were common to the affected individuals and not present in the non-affected individuals. Among them, in the 1k genome, there were 23 mutations with a minor allele frequency (MAF) of 1% or less, and there were 11 mutations with a MAF of 1% or less in Japanese. Among them, 5 new mutations were not registered in dbSNP135.

Example 2 Linkage analysis of family 2 and whole genome exome analysis Family 2 (family diagram shown in Fig. 2) has already been reported as a paper (Brain Res., 2006, 28 (10): 660-662) Has almost the same symptoms as Family 1. Genomic DNA was collected from the brother (IV-2), brother (IV-3), mother (III-1) and father (III-1 spouse) of the proband (IV-1), of which 3 samples (IV -3, III-1 and III-1 spouses) were subjected to exome analysis in the same manner as in Example 1, and the results are summarized in Table 2.

About 10,000 mutations were observed in each specimen, including MS / NS / Indel / SS / FS / RT, and there were 1332 heterozygous mutations that were common to the affected individuals and not present in the non-affected individuals. . Among them, in the 1k genome, there were 238 mutations of MAF 1% or less, and 165 mutations of MAF 1% or less in Japanese. Among them, 59 new mutations were not registered in dbSNP135. Among these, the ion channel gene was only SCN11A, which is common to family 1, and the mutation was p.R222S. In this case, an increase in substance P (9.3-12.6 pg / ml; control 4.8 + 2.4 pg / ml), which is known to be released from the nerve endings present in the trigeminal ganglion at the time of onset, an increase in CGRP (20.9) -30.7 pg / ml; control 13.4 + 4.4 pg / ml).

Example 3 Analysis of Family 3 The fourth generation proband (IV-1) of the main family is a 2-year-old boy, 41 weeks, 2954 g, without asphyxia. There is a history of 5 febrile convulsions, but no electroencephalogram is observed. From about 1 year and 11 months ago, an episode of sudden crying appeared. I noticed that I started to complain that I couldn't put my leg on the floor. Pain is at the joints of the extremities, such as elbows, knees, ankles, wrists There are many knees. A single pain is about 5 minutes, but it occurs many times a day. There are no other accompanying symptoms during a pain attack (fever, redness, swelling, etc.). More in the evening when the weather is bad, when the air pressure drops, and during the day. He takes bufferin during pain. Father (III-5) also had the same symptoms since childhood, but belonged to the soccer club when he was a student, and the frequency of pain decreased dramatically in adulthood. In this family, at least II-4, III-5 and IV-1 have no headache, and III-3 and III-4 also have no headache according to III-5 (III-1, III- Details about 2 are unknown). III-5 and III-6 are not close relatives. I-2 has died and no symptoms have been confirmed. II-2 has no symptoms as long as III-5 knows.
In this family, the gene sequences of all exons of SCN11A were examined for the genomes of proband IV-1 and its parents (III-5 and III-6). R222H mutation was observed (FIG. 4). This mutation was not observed in the non-onset mother (III-6).

Example 4 Allele frequency of SCN11A gene mutation in general population of patient residence area In 150 general population in the patient residence area, the frequency of mutation alleles p.R222H and p.R222S was analyzed using restriction enzymes. In the general population of 150, two mutations were not observed and 95% CI was 0-0.0122.

  Although the present invention has been described with emphasis on preferred embodiments, it is obvious to those skilled in the art that the preferred embodiments can be modified.

  The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

To date, the pain associated with SCN11A has not been known. According to the present invention, it is clear that SCN11A is the causative gene and that the amino acid site involved in pain development is substitution of arginine at position 222 in three families that develop very characteristic juvenile periodic limb pain did. This demonstrated the possibility of drug discovery for pain relief with few side effects by a new mechanism effective for limb joint pain, chronic inflammation, and bone pain in patients with cancer. .
Since the expression of the SCN11A gene is confined to small nociceptive neurons in nerve cells, particularly peripheral sensory ganglia, it becomes possible to develop highly selective pain therapeutic agents that have very little effect on other organs. Therefore, the present invention is very useful industrially.

Claims (15)

Protein of any of the following (a)-(d).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids are deleted and / or substituted and / or inserted and / or added A protein having an amino acid sequence (excluding substitution to arginine at position 222) and having a pain-inducing action (c) a nucleic acid comprising a complementary strand sequence of the base sequence shown in SEQ ID NO: 1, and a stringent A protein encoded by a nucleic acid that hybridizes under conditions and having a pain-inducing action (d) an ortholog in another mammal of a protein comprising the amino acid sequence shown in SEQ ID NO: 2, A protein whose amino acid residue corresponding to the position is an amino acid other than arginine and has a pain-inducing action   A nucleic acid encoding the protein of claim 1.   A vector comprising the nucleic acid according to claim 2.   A host cell transformed with the vector according to claim 3.   The cell according to claim 4, wherein the host cell is a mammalian cell, and the nucleic acid encoding endogenous SCN11A is replaced with the nucleic acid according to claim 2.   A method for producing the protein comprising culturing the cell according to claim 4 and recovering the protein according to claim 1 from the culture.   A transgenic non-human mammal or a part of its living body that exogenously expresses the nucleic acid according to claim 2 and exhibits a pain phenotype.   The animal according to claim 7 or a part of its living body, wherein the nucleic acid encoding endogenous SCN11A of a non-human mammal is substituted with the nucleic acid according to claim 2.   The animal or part of its living body according to claim 7 or 8, wherein the non-human mammal is a mouse or a rat.   A test substance is contacted with the cell according to claim 5 or the animal according to any one of claims 7-9 or a part of the living body thereof, and the test substance with improved pain phenotype is selected. A method for screening a substance that suppresses pain. A method of screening for a substance that suppresses pain,
(A) contacting the test substance with the cell according to claim 5 or the non-human mammal according to any one of claims 7-9 or a part of the living body thereof;
(B) a step of measuring the expression level of the protein according to claim 1 or the nucleic acid according to claim 2 in each of the case where the test substance is brought into contact with the case where the test substance is not brought into contact; and (c) (b) A method comprising: selecting a test substance having a reduced expression level as a substance that suppresses pain.   An antibody or a fragment thereof that specifically binds to the protein of claim 1.   A test method for diagnosing a pain-related disease in a subject, which comprises detecting the presence of the protein according to claim 1 or the nucleic acid according to claim 2 in a biological sample collected from the subject.   An agent for treating abnormal pain in a mammal having a pain-inducing mutation or a blunting-inducing mutation in the SCN11A gene, the agent comprising an ion channel modulator comprising the gene product. The SCN11A gene having the pain-induced mutation is
(A) An ortholog in another mammal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, corresponding to position 222 of the amino acid sequence The agent according to claim 14, wherein the amino acid residue is an amino acid other than arginine and encodes a protein having a pain-inducing action.