JP4288345B2 - New epilepsy model animal - Google Patents

Description

【図面の簡単な説明】
【図１】ＳＰＡＲＣ融合タンパク質の全アミノ酸配列を示す図である。
【図２】ｐＥＴ３２（＋）−ＳＰＡＲＣベクターの構築を示す図である。
【図３】参考例のSPARC融合タンパク質の生産工程において得られた精製試料のＳＤＳ−ＰＡＧＥの結果を示す写真である。
【図４】ＳＰＡＲＣ融合タンパク質、生理的食塩水およびチオレドキシン（Ｔｒｘ）をそれぞれ脳内投与後、カイニン酸-一水和物を投与した場合における、痙攣回数の経時変化を測定した結果を示すグラフである。
【図５】ＳＰＡＲＣ融合タンパク質、生理的食塩水およびチオレドキシン（Ｔｒｘ）をそれぞれ脳内投与後、カイニン酸一水和物を投与した場合における、痙攣スコアの経時変化を測定した結果グラフである。
【図６】ＳＰＡＲＣ融合タンパク質、熱処理ＳＰＡＲＣ融合タンパク質、チオレドキシン（Ｔｒｘ−Ｔａｇ）およびベヒクルのみそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物を投与後９０分間の各合計痙攣回数を示すグラフである。
【図７】ＳＰＡＲＣ融合タンパク質、熱処理ＳＰＡＲＣ融合タンパク質、チオレドキシン（Ｔｒｘ−Ｔａｇ）およびベヒクルのみそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、痙攣回数の経時変化を測定した結果を示すグラフである。
【図８】ＳＰＡＲＣ融合タンパク質、熱処理ＳＰＡＲＣ融合タンパク質、チオレドキシン（Ｔｒｘ−Ｔａｇ）およびベヒクルのみそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物を投与後９０分間の各合計痙攣スコアを示すグラフである。
【図９】ＳＰＡＲＣ融合タンパク質、熱処理ＳＰＡＲＣ融合タンパク質、オレドキシン（Ｔｒｘ−Ｔａｇ）およびベヒクルのみそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物を投与後９０分間に呈した各痙攣程度の割合を示すグラフである。
【図１０】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物を投与した場合における、ＳＰＡＲＣ融合タンパク質の投与量とカイニン酸一水和物を投与後９０分間に呈した痙攣回数との関係を示すグラフである。
【図１１】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物を投与した場合において、ＳＰＡＲＣ融合タンパク質の各投与量毎に痙攣回数の経時変化を測定した結果を示すグラフである。
【図１２】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物を投与した場合における、ＳＰＡＲＣ融合タンパク質の投与量とカイニン酸一水和物投与後９０分間に呈した痙攣スコアの関係を示すグラフである。
【図１３】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物を投与後９０分間に呈した各痙攣程度の割合をＳＰＡＲＣ融合タンパク質の投与量毎に測定したグラフである。
【図１４】ＳＰＡＲＣ融合タンパク質、チオレドキシン（Ｔｒｘ−Ｔａｇ）およびベヒクルをそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物の投与量と痙攣回数の関係を示すグラフである。
【図１５】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物を投与した場合において、カイニン酸一水和物の各投与量毎に痙攣回数の経時変化を測定した結果を示すグラフである。
【図１６】ＳＰＡＲＣ融合タンパク質をそれぞれ脳内投与後、カイニン酸一水和物をそれぞれ投与した場合における、カイニン酸一水和物の投与量と痙攣スコアの関係を示すグラフである。
【図１７】ＳＰＡＲＣ融合タンパク質を脳内投与後、カイニン酸一水和物を投与した場合における、カイニン酸一水和物を投与後９０分間に呈した各痙攣程度の割合をカイニン酸一水和物の投与量毎に測定したグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an epilepsy model animal effective for searching for an antiepileptic drug or for elucidating the mechanism of epileptogenesis, a method for producing the epilepsy model animal, and a material for producing the animal.
[0002]
[Prior art]
A brain that has acquired chronic brain damage (epileptic disorder) that causes repeated epileptic seizures has acquired the property of easily transitioning to a hyperexcited state (convulsability) even during non-seizures. In addition, it is considered that an important factor in the onset of epileptic seizures is an imbalance between the inhibitory (GABA) and excitatory (glutamic acid) neurotransmission systems. In order to elucidate the mechanism of epilepsy onset, it is necessary to analyze a model animal that has acquired such “convulsant readiness”, and the following animal models are conventionally known.
[0003]
(1) Electric shock convulsion (kindling) model
-An animal model in which seizures are gradually enhanced by repeated electrical stimulation of a specific brain region and repeated post-launches, and secondary generalized seizures appear. It is considered as a secondary generalized seizure model of refractory human temporal epilepsy and has the characteristic that maintenance of seizure preparation is semi-permanent.
(2) Seizure-inducing animals (El mice, etc.)
The El mouse is an epilepsy model mouse found from a mutant of the ddY mouse, and is the model closest to human epilepsy involving genetic predisposition and acquired factors (seizure history). Abnormalities in the GABA inhibitory system and mutations in multiple genes have been shown to be involved in seizures.
[0004]
(3) Causative gene modified mouse
The 'glial glutamate transporter GLT-1 knockout mice present in astrocytes cause fatal seizures that closely resemble those observed when NMDA is injected subcutaneously. This is known to be caused by inhibition of glutamate uptake in the synaptic cleft.
[0005]
(4) Kainic acid model
Kainic acid, one of the representative ion channel glutamate receptor agonists, induces severe 1 imbic motor seizures by systemic administration such as subcutaneous injection or intravenous injection, and is selected for cerebral cortex, hippocampal CA1, amygdala, etc. Causes neuronal cell death. Convulsions caused by kainic acid are characteristic and intense, and are said to be a model of temporal epilepsy. From the anatomical analysis of the brain, 1) “hippocampal sclerosis” resulting from the proliferation of glial cells resulting from the loss of neurons due to epileptic seizures 2) changes in the pattern of hippocampal neural circuits due to abnormal germination of mossy fibers, 3 ) Changes in gene expression have been reported.
[0006]
The main cause of epilepsy onset is presumed to be an unbalanced state of the GABA inhibitory nervous system and the excitatory amino acid nervous system. Currently, drugs having an action point on either or both of these nervous systems are used as clinically used antiepileptic drugs. When antiepileptic drugs are categorized from the viewpoint of the epileptic seizure mechanism, 1) inhibitors of the inhibitory GABA nervous system, 2) inhibitors of excitatory amino acid nervous system, 3) Zen transmembrane excitability acting on ion channel system Classified as sex stabilizer. Group 1 antiepileptic drugs enhance the GABAergic system and are typically barbital drugs such as phentobarbital and benzodiazepine drugs such as diazepam, and GABA receptors and synaptic gap GABA uptake systems. Alternatively, the decomposition system is the point of action.
[0007]
These drugs are thought to enhance the inhibitory GABA nervous system by increasing GABA in the synaptic cleft and exert an antiepileptic effect. Antiepileptic drugs of the second group are drugs that exert antiepileptic effects by suppressing or blocking excitatory amino acid transmission in the nervous system, and inhibiting glutamate release by inhibiting voltage-gated Na + channels at presynaptic terminals. Representative drugs are phenytoin and lamotrigine. Group 3 antiepileptic drugs are drugs that act on ion channels existing on the cell membrane to induce stabilization of cell membrane excitability, and are voltage-dependent Na + channel antagonist zonisamide and T-type Ca2 + channel antagonists. The drug lamotrigine is a typical drug.
[0008]
[Problems to be solved by the invention]
However, for the intractable epilepsy, the drugs in the above three groups are not effective, and there are still many unclear points regarding the onset mechanism. Therefore, there is a need for the development of new drugs that have different mechanisms of action from neurotransmitter receptors, neurotransmitter uptake / degradation, and various ion channel agonists. Drug discovery is essential. Moreover, the epileptogenesis mechanism by GABA inhibitory nervous system abnormality is an important research subject also in the development of a new drug. The present invention is intended to present a new epileptic onset mechanism and onset factors related to GABA-inhibiting nervous system function, and at the same time opens up the possibility of new drug discovery based on the new mechanism.
[0009]
[Means for Solving the Problems]
As a result of diligent research, the present inventors have found that a secreted anti-adhesive factor, Secreted Protein Acidic and Rich in Cysteine (SPARC), newly identified as an epilepsy-causing factor in a kainic acid model, is a GABA-inhibitory neuron in the hippocampal stratum corneum region. Was found to cause epilepsy symptoms by up-regulation. Based on this knowledge, the present invention is based on 1) creation of a new animal model for elucidating the mechanism of abnormal GABA suppression, 2) development of new drugs and prevention methods for especially intractable epilepsy, and 3) foundation of new drug development. This paper presents basic technologies for identifying SPARC target factors and elucidating molecular physiological functions. That is, the present invention involves the following configurations.
[0010]
(1) Epilepsy model animal, characterized in that SPARC protein is administered into the brain (Excluding humans) .
(2) The epilepsy model animal according to (1) above, wherein the SPARC protein is a fusion protein with thioredoxin (Excluding humans) .
(3) SPARC protein including, Epilepsy model animal (Excluding humans) Production drug.
(4) The epilepsy model animal according to (3) above, wherein the SPARC protein is a fusion protein with thioredoxin (Excluding humans) Production drug.
(5) Epilepsy model animal characterized by administering SPARC protein in brain (Excluding humans) How to make.
(6) The epilepsy model animal according to (5) above, wherein the SPARC protein is a fusion protein with thioredoxin (Excluding humans) How to create.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The epilepsy model animal of the present invention is produced using a SPARC protein, a fusion protein of a SPARC protein and another protein, or a gene thereof.
This SPARC protein (secreted protein acidic and rich in cysteine) was first reported as Osteonectin, a major non-collagenous protein present in bone (Termine, JD et al., Cell, 26: 99-105, 1981), which has strong binding to collagen and hydroxyapatite, and has Ca2 + binding ability, has attracted attention as a functional bone-specific protein. The gene sequence of this SPARC has been determined from what has been reported as a 43 kD protein with a molecular weight of 43 kD that is secreted to the outside of cells in the presence of adherent cultured cells (Sage.H. et al., J. Biol Chem., 259, 3993-4007, 1984).
The amino acid sequence of this SPARC protein and the base sequence of the gene are respectively shown in the sequence listing.
SEQ ID NOs: 1 and 2 (Accession ID: X04017).
[0012]
SPARC was later observed to be secreted from various cultured cells, especially from normal fibroblasts and vascular endothelial cells (Lane. TF & Sage, EH, FASEB J., 8: 163-173, 1994). ). In addition, as a function of SPARC, it is known that addition of SPARC to a cell culture system inhibits cell adhesion to an extracellular matrix molecule in a concentration-dependent manner.
[0013]
On the other hand, the present inventors found a SPARC gene in a gene group that is specifically elevated in the amygdala during chronic administration of morphine, incorporated the SPARC gene downstream of the thioredoxin gene-containing vector, and expressed it in E. coli. In addition to obtaining a thioredoxin-SPARC fusion protein (hereinafter referred to as SPARC fusion protein), it has been found that it exhibits anti-cell adhesion activity and spheronization activity on nerve cells. Furthermore, the present inventor found that in mice in which a small amount of brain was administered to the lateral amygdala of this SPARC fusion protein, a single administration of morphine causes an increase in morphine locomotor activity, that is, changes in neuronal synapses. In addition, it has been found that the SPARC protein functions as a neuroplastic factor in the brain (Patent Nos. 3012930 and 3012931).
The amino acid sequence of the SPARC fusion protein and the base sequence of the gene are shown in SEQ ID NOs: 3 and 4, respectively.
[0014]
In the present invention, as a material for producing an epilepsy model animal, the SPARC protein having the amino acid sequence of SEQ ID NO: 1, the thioredoxin (Trx) -SPARC fusion protein having the amino acid sequence of SEQ ID NO: 3, or SEQ ID NO: 1 Alternatively, the amino acid sequence shown in 3 has an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added, and has the physiological activity of the SPARC protein.
[0015]
The SPARC protein is a secreted anti-adhesive protein rich in cysteine, and in order to express its physiological activity, it is necessary to accurately reconstitute intramolecular SS bonds. The technique of obtaining a fusion protein with thioredoxin (Trx) using genetic engineering techniques is accurate when the bioactive protein of an animal is produced using bacteria such as Escherichia coli as a host. It is used for maintaining the three-dimensional structure by reconstituting and obtaining the original physiological activity. However, when animal cells are used as a host, they can be used even if they are not fusion proteins.
[0016]
These proteins are administered into the mammalian brain, for example, the hippocampus, dentate gyrus, amygdala, cerebral cortex and the like, thereby obtaining an epilepsy model animal. The dose of these proteins in the brain is about several to several tens of pmoles, depending on the dose of drugs such as kainic acid and individual differences among animals. In the brain administration example of thioredoxin (Trx) -SPARC fusion protein to the hippocampus (radiation layer), significant enhancement of kainic acid sensitivity was induced by 5 to 10 pmoles of Trx-SPARC fusion protein in the brain. It has been found that
[0017]
Further, in the present invention, the gene encoding the SPARC protein having the amino acid sequence of SEQ ID NO: 1 or the SPARC fusion protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 1 or 3, Alternatively, a gene encoding a physiologically active protein of the SPARC protein having an amino acid sequence in which several amino acid sequences have been deleted, substituted, or added can be transferred to brain tissues such as hippocampus, amygdala, cerebral cortex, striatum. The protein may be introduced into the body and expressed in the brain. More specifically, these genes include those having the base sequence of SEQ ID NO: 2 or 4.
[0018]
These SPARC protein-encoding genes can be incorporated into various viruses and introduced into the brain to express the SPARC protein in the brain and produce epilepsy model animals. Examples of the virus to be used include Sindbis virus, herpes virus, adenovirus, and lentivirus. Further, as typical viral vectors for incorporating the gene, there are pSinrep5 (Invitorogen), HSV-PrpUC, pAxCAwt, (Takara Shuzo), pLenti6 / V5 (Invitorogen), and the like.
[0019]
A cDNA encoding the SPARC protein is inserted in vitro into a multicloning site present in the viral vector to produce a recombinant viral DNA. This recombinant DNA (or recombinant viral RNA prepared by reverse transcription from recombinant viral DNA) is transfected into host cells (BHK cells, HEK293 cells, etc.), and the recombinant virus that has grown in the host cells and released into the medium A high-titer SPARC gene-inserted recombinant virus is prepared by collection and purification.
In order to introduce the gene encoding the SPARC protein retained in these viruses or liposomes into the brain tissue, a known method such as electroporation, direct injection, or intracerebral administration may be applied.
[0020]
As another method for expressing the SPARC protein in the brain, cells and tissues that secrete the SPARC protein may be transplanted into the brain. The types of cells to be transplanted include primary neuronal cultured cells such as hippocampus, dentate gyrus, amygdala, and cerebral cortex, and various neuronal cultured cells derived from neurons and glial cells (NG108-15, N18TG- 2, C6Bu-1, PC12, etc.), cell groups such as neural stem cells. Examples of the tissue to be transplanted include a part of brain tissue (hippocampus, dentate gyrus, amygdala, cerebral cortex, etc.).
[0021]
Alternatively, the cells / tissues artificially constructed to overexpress the SPARC protein may be transplanted into the brain using a virus expression system or a genetically modified animal.
Increased expression of the protein by intracerebral administration of the SPARC protein, introduction of the gene of the protein into the brain, or transplantation of the protein secreting cell or tissue into the brain caused abnormal increase in GABA-inhibiting nerves, and was produced. Animals easily cause seizures such as convulsions due to administration of convulsants such as kainic acid, electrical stimulation, stress (throwing stimulation), etc., and exhibit epilepsy symptoms. That is, the epilepsy model animal in the present invention is in a state of “preparation of seizure” and is in a state corresponding to so-called “holding epilepsy”.
[0022]
As described above, refractory epilepsy is ineffective in the above three groups of drugs, and there is a strong possibility that abnormalities different from those in which these drugs act are generated in the brain. Abnormal increase in GABA-inhibitory nerves based on the increase in the level of suspicion is considered to be the cause of intractable epilepsy.
Therefore, according to the present invention, for example, administration of a test drug to an epilepsy model animal or antagonism against SPARC protein, etc., as described below, is used to prevent or treat epilepsy, in particular, prevent or treat refractory epilepsy. It becomes possible to screen or design drugs.
[0023]
(1) Screening for new antiepileptic drugs based on behavioral pharmacological evaluation
Administer the test drug (including natural extracts, proteins, peptides, nucleic acids, other inorganic and organic compounds) to the above epilepsy model animals (ascites, static artery administration, oral administration, intracerebral administration, etc.) Behavioral pharmacological observation of the number of convulsions caused by kainic acid administration, the degree of convulsions, etc. Alternatively, drugs that suppress or reduce are screened.
[0024]
(2) Screening for new antiepileptic drugs based on evaluation of anti-adhesion activity by SPARC protein
As described above, the SPARC protein has anti-cell adhesion activity to each cell including nerve cells, and this function is a basic function of the SPARC protein, and the spheronization activity is brought about by the anti-cell adhesion activity. . Therefore, a substance that inhibits these activities can be regarded as a substance that inhibits the function of the SPARC protein, and a substance that shows this activity inhibition may be an antiepileptic drug. Therefore, test drugs (including natural extracts, proteins, peptides, nucleic acids, other inorganic and organic compounds) are added to various types of cultured neurons or various types of primary cultured cells including hippocampus, and the culture Whether or not the anti-adhesion activity of the SPARC protein secreted from the cells is controlled can be screened for a novel antiepileptic drug using the degree of spheroidization of the cell morphology as an index.
[0025]
(3) Design of new antiepileptic drugs using transcriptome analysis and / or proteome analysis
By using comprehensive analysis methods such as transcriptome analysis or proteome analysis between the above-mentioned novel epilepsy model animals and normal animals, for example, differences in expressed gene profiles, expressed proteins, etc. are detected, and `` epileptic drug target for epilepsy '' Based on these findings, it becomes possible to search for and design novel antiepileptic drugs based on elucidation of “related factors”, physiological functions, molecular functions, and the like.
Furthermore, it goes without saying that the present invention contributes to the elucidation of the epileptogenesis mechanism or the control mechanism of the GABA sleeve system by analyzing a novel epilepsy animal model.
[0026]
Examples are shown below, and the SPARC fusion protein used in the examples follows the method developed by the present inventors (Japanese Patent No. 3012931). This method is shown below as a reference example.
[0027]
[Reference example]
(1) Preparation of SPARC fusion protein (recombinant Trx-SPARC) chimeric protein
a. Construction of SPARC fusion protein expression plasmid vector
The T vector was prepared according to the method of D. Marchuk et al. (Nucleic Acids Research, 19: 1154, 1990). That is, plasmid Bluescript SK (-) (10 μg / μl) (STARATAGENE) 1.0 μl, 10 × Buffer H (Takara Shuzo) 2.0 μl, EcoRV (10 U / μl) 2.0 μl, sterile water 15.0 μl Thus, the total amount was adjusted to 20.0 μl and reacted at 37 ° C. for 2 hours to completely cleave the plasmid. To the precipitate generated by ethanol precipitation, 10 × PCR buffer (PE Applied Systems) 4.0 μl, 10 mM dTTP 8.0 μl, Ampli Taq DNA polymerase (5 U / μl) (PE Applied Systems) 2.0 μl, sterile water 24.0 The total amount was adjusted to 40.0 μl by adding μl, and reacted at 70 ° C. for 2 hours. After purification by phenol chloroform extraction and ethanol precipitation, the resulting precipitate was dissolved in 20 μl of sterilized water to obtain a T vector solution. The T vector was stored at 4 ° C.
[0028]
b. Extraction of total RNA from mouse cerebral cortex
Total RNA was extracted according to the method of P. Chomczynski et al. (Analytical Biochemistry, 162: 156-159, 1987). That is, after removing the mouse cerebral cortex, it was frozen with liquid nitrogen and crushed with a hammer into a powder form. Dissolve 75 mg of this powder in 500 μl of guanidine solution (4 M guanidine thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sodium sarcosyl, 0.1 M mercaptoethanol), add 50 μl of 2 M sodium acetate solution (pH 4.0), DEPC water After adding 50 μl of saturated phenol (pH 4.0) and 100 μl of chloroform and stirring, the mixture was allowed to stand at 4 ° C. for 15 minutes. After centrifuging at 10,000 × g for 20 minutes, the aqueous layer was collected, and 600 μl of cooled isopropanol and 2 μl of glycogen (20 μg / ml) were added thereto and left at −20 ° C. for 1 hour. The precipitate formed by centrifugation at 16,000 × g for 10 minutes was completely dissolved in 500 μl of guanidine solution, and the above process was repeated. The precipitate formed by isopropanol precipitation was dissolved by adding 50 μl of DEPC water to obtain a total RNA solution. Finally, 0.3 to 254 μg of total RNA was obtained from mouse cerebral cortex.
[0029]
c. Cloning of mouse SPARC gene by RT-PCR
[Synthesis of cDNA]
B. Add 1.2 μl of total RNA obtained in the process (2.5 μg / μl) to 1.0 μl of random primer (100 ng / μl) and 2.8 μl of DEPC-treated sterilized water to make a total volume of 5.0 μl, and heat at 70 ° C. for 10 minutes. Denaturation treatment was performed. Add 5 μl buffer (GIBCO BRL) 2.0 μl, 0.1 M dithiothreitol 2.0 μl, 1.25 mM dNTP 8.0 μl, SUPERSCRIPTTM II Reverse Transcriptase (200 U / μl) 1.0 μl to this solution to make a total volume of 20.0 μl. And reacted at 42 ° C. for 2 hours. Furthermore, reverse transcriptase was completely inactivated by heat treatment at 70 ° C. for 15 minutes to obtain a single-stranded cDNA solution.
[0030]
[PCR reaction]
Next, 5.0 μl of the obtained single-stranded cDNA solution was added to 10 × PCR buffer (PE Applied Systems) 2.5 μl, Ampli Taq DNA polymerase (5 U / μl) (PE Applied Systems) 0.25 μl, 1.25 mM dNTP 8.0μl, 60ng / μl Primer 1 (5'-CCGAGAGTTCCCAGCATCAT-3 '); 20mer and Primer 2 (5'-TCAAACCAATTCACCAGTCT-3'); To prepare a reaction solution. Next, PCR using DNA Thermal cycler PJ2000 Model 480 (PE Applied Systems) (denaturation reaction; 94 ° C, 30 seconds, annealing reaction; 50 ° C, 45 seconds, synthesis reaction; 72 ° C, 90 seconds; 35 cycles) Subsequently, an extension reaction was performed at 72 ° C. for 10 minutes. An approximately 1750 bp PCR product was separated and purified by 1% agarose gel electrophoresis, and recovered from the gel using an ultra-free MC filter (Millipore). The recovered PCR product was subcloned into the T vector using DNA Ligation Kit Ver.2 (Takara Shuzo). The primer was prepared by chemically synthesizing an oligonucleotide sequence corresponding to a part of the mouse SPARC cDNA base sequence (JH McVey et. Al. Journal of Biochemistry 263, 11111-11116, 1988). Primers 1 and 2 are oligonucleotide sequences corresponding to SPARC cDNA base sequences 72-91 and 1801-1820, respectively.
[0031]
[Determination of cDNA sequence of PCR product]
The cDNA base sequence of the T vector (pBSK-SPARC vector) into which the PCR product was subcloned was determined by the cycle sequencing method. Specifically, 1.5 μg of the T vector purified by QIAGEN plasmid Minikit (Funakoshi) was used as a template, and a sequence sample was prepared using PRISM Ready Reaction Terminator Cycle Sequencing Kit (PE Applied Systems). PCR uses DNA Thermalcycler PJ2000 Model 480 (PE Applied Systems), denaturation reaction; 96 ° C, 30 seconds, annealing reaction; 50 ° C, 15 seconds, synthesis reaction; 60 ° C, 240 seconds; conditions under 25 cycles I went there. After removing unreacted PCR product by ethanol precipitation, the PCR product was dissolved in 4.0 μl of a denaturing solution (5 mg / ml blue dextran, 8.3 mM EDTA pH 8.0, 83.3% formamide) and heat-denatured at 90 ° C. for 2 minutes. It was. Immediately thereafter, the cDNA base sequence was analyzed using an ABI 373S sequencer (manufactured by PE Applied Systems), and it was confirmed that the PCR product had the mouse SPARC cDNA base sequence (72-1820).
[0032]
d. Subcloning into pGEX 4T-1 vector
Add 10 μl of the above pBSK-SPARC vector (6.25 μg / μl) to 10 μl 10 × buffer 4 (NEB) 10 μl, BamHI 2 μl, XhoI (12 U / μl) 2 μl, and sterilized water 76 μl to make a total volume of 100 μl. For 2 hours. An about 1800 bp BamHI-XhoI insert was separated and purified by 1% agarose gel electrophoresis, recovered from the gel using GENE CLEAN II kit (Funakoshi), and TE solution (Tris-HCl pH 8.0, 0.5 mM EDTA pH). 8.0) Dissolved in 48 μl. Next, add 42 μl of this BamHI-XhoI insert solution to 10 μl of 10 × Buffer 4 (NEB), 2 μl of BspHI (10 U / μl), 2 μl of SmaI (10 U / μl), and 44 μl of sterilized water to make a total amount. 100 μl was allowed to react at 37 ° C. for 2 hours. The approximately 1500 bp BspHI-SmaI insert was separated and purified by 1% agarose gel electrophoresis, recovered from the gel using GENE CLEAN II kit (Funakoshi), and dissolved in 48 μl of TE solution. This BspHI-SmaI insert fragment (960 ng) was blunt-ended using DNA Blunting Kit (Takara Shuzo) and dissolved in 10 μl of TE solution to obtain a blunt-ended insert fragment solution.
[0033]
On the other hand, add 2 μl of pGEX-4T1 vector (500 ng / μl) (Pharmacia Biotech) 2 μl of 10 × buffer 4 (NEB), 1 μl of SmaI (10 U / μl) and 15 μl of sterilized water to make a total volume of 20 μl. And reacted at 37 ° C. for 2 hours. After concentration by purification with phenol chloroform extraction and ethanol precipitation, add 5 μl of 10 × dephosphorylation buffer (Takara Shuzo), 1 μl of calf small intestine alkaline phosphatase (10 U / μl), and 44 μl of sterilized water to the resulting precipitate. Was subjected to dephosphorylation treatment by reacting at 37 ° C. for 15 minutes. After heat treatment at 52 ° C. for 15 minutes to inactivate the phosphatase, phenol-chloroform extraction and purification and concentration by ethanol precipitation were performed. The resulting precipitate was dissolved in 20 μl of TE solution to obtain a dephosphorylated pGEX-4T1 vector solution.
[0034]
Finally, 20 μl of dephosphorylated pGEX-4T1 vector solution and 10 μl of blunt-ended insert fragment solution were mixed and concentrated by ethanol precipitation, and the SmaI site of pGEX-4T1 vector was obtained using DNA Ligation Kit Ver.2 (Takara Shuzo). Was ligated with a BspHI-SmaI blunt-ended insert to obtain a pGEX-4T-SPARC vector. Note that the BspHI-SmaI blunt-ended insert was incorporated into the pGEX-4T1 vector as planned, indicating that the BamHI-XhoI insert of the pGEX-4T-SPARC vector was replaced by the pBluescript SK (-) vector (manufactured by STRATAGENE). This was confirmed by subcloning into the BamHI-XhoI site and cDNA sequence analysis.
[0035]
e. Construction of pET32a (+)-SPARC vector
Add 10 μl of Buffer 10 (Takara Shuzo) 10 μl, 2 μl of BamHI (10 U / μl), 2 μl of XhoI (12 U / μl), 46 μl of sterile water to 40 μl of the above pGEX-4T-SPARC vector (0.25 μg / μl) The total volume was adjusted to 100 μl and reacted at 37 ° C. for 2 hours. The BamHI-XhoI insert was separated and purified by 1% agarose gel electrophoresis, and the BamHI-XhoI insert was recovered from the gel using GENE CLEAN II kit (Funakoshi) and dissolved in 48 μl of TE solution. It was subcloned into the BamHI-XhoI site of pET32a (+) vector (Novagen). The finally obtained pET32a (+)-SPARC vector was subjected to cDNA nucleotide sequence analysis to confirm that the gene reading frame matched. The entire cDNA base sequence of the pET32a (+)-SPARC vector is shown in SEQ ID NO: 4, and the entire amino acid sequence of the SPARC fusion protein produced by this vector is shown in SEQ ID NO: 3 and FIG. An outline of the construction of the above pET32a (+)-SPARC vector is shown in FIG.
[0036]
f. Production of SPARC fusion proteins
Escherichia coli AD494 (DE3) was transformed with the pET32a (+)-SPARC vector. This Escherichia coli was cultured in LB solution containing 30 μg / ml kanamycin for 3 hours at 37 ° C. until OD560 = 0.6, followed by shaking culture at 20 ° C. for 6 hours in the presence of 1 mM IPTG. The cells are collected by centrifugation (6,000 × g, 30 minutes), suspended in 20 ml of buffer A (5 mM Imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9), subjected to ultrasonic treatment, and centrifuged. The soluble fraction was recovered by separation (30,000 × g, 30 minutes). After this soluble fraction was added to a His Bind Resin column (Novagnen), the adsorbed fraction was eluted with buffer B (1M Imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). The eluate was concentrated to about 1 ml, dialyzed overnight against buffer C (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% NP-40), and applied to a gel filtration column [Superdex 75 (Pharmacia)]. Added. Buffer D (10 mM Tris-HCl pH 7.4, 100 mM NaCl) is developed at a flow rate of 1 ml / min, fractionated every 5 ml, and a single peak (fraction 8-10) with a retention time of 40 minutes is recovered. Dialyzed overnight against buffer B. This sample was separated with 10% SDS-pAGE and then stained with CBB to confirm that the purified sample had a single band of 25 kDa and a purity of 98% or more (FIG. 3). The yield was about 1 mg per liter of LB culture.
[0037]
【Example】
[Example 1]
(1) Microbrain administration in the brain
Based on the previous report (Ikemoto, Iwata, Japanese Pharmacology Journal 118, 277-282, 2001). ddY mice (male, 7 weeks old) were intraperitoneally administered with 80 mg / kg of pentobarbital chloride and subjected to general anesthesia. After confirming that the mouse did not respond to the clapping sound, the mouse was fixed to the brain fixation device (Narimo Science Co., Ltd.), and the ophthalmic scissors were placed along the midline from the top of the head to the vicinity of the eye. The skin was incised. After exposing the entire cranium, the head was rubbed with a cotton swab to remove the bonded fabric, and the suture intersection was clarified. Bregma -1.80mm, Lateral ± 1 for the purpose of providing an insertion port of inner cannula (stainless steel pipe 0.2 ¢, Intermedical Co., Ltd.) in the skull after aligning the origin of the brain coordinate axis of the brain fixation device with the suture intersection ．Two holes with a diameter of about 1mm were drilled at a position of 5O mm using a dental electric drill, and the dura was cut so as not to damage the cerebral cortex. The inner cannula was carefully inserted at a constant speed into the stratum radiatum (Bregma -1.80 mm, Lat6ral ± 1.5 Omm, Ventral + 1.20 mm) of the hippocampal CA1 koto area on both sides. Trx-SPARC chimeric protein solution dissolved in physiological saline containing 0.04% NP-40 was administered at a rate of 0.1 μl / min for 5 minutes in a total amount of 2.5 to 10 pmoles in the brain.
[0038]
Immediately after completion of the microtrace administration in the brain, the inner cannula was swept out, and gelatin powder was applied to the surgical site to perform hemostasis treatment. Further, the canyula insertion site was occluded using resin Tocrily base first (Tokuso Co., Ltd.) for denture base rebase, and then the cut skin was adhered using Aron Alpha-jelly-like instant adhesive (Toagosei Chemical Co., Ltd.). After the skin was completely adhered, the mouse was removed from the brain fixing device, returned to the breeding cage and subjected to a recovery treatment. On the second day after surgery (after 48 hours), kainic acid monohydrate (Wako Pure Chemical Industries, Ltd.) was administered 20 mg / kg into the abdominal cavity in a single dose, and animal behavior was measured. The animal behavior was measured every 30 minutes for 90 minutes, including the number of convulsions and jumping, the number of drooling, and the number of wet dog shakes. The index of animal behavior was 1) the number of convulsions, 2) the convulsions score, and 3) the degree of convulsions (symptoms). The analytical data were statistically processed and tested for significance.
[0039]
The number of convulsions was not included in the count when transitioning to continuous convulsions within the measurement time. In addition, the degree of convulsions was observed over 4 hours after kainic acid administration to score and determine symptoms. For persistent convulsions, we defined “continuous convulsions” only when the convulsions persisted for more than 3 hours, and “transient convulsions” when the convulsions ended in less than 3 hours. Furthermore, the convulsions score and the degree of convulsions (symptoms) were classified as shown below.
[0040]
<Convulsions score>
1) Score 0 asymptomatic
2) Score 1 Transient convulsions (including drooling and wet dog shake)
3) Score 2 Transient convulsions (including persistent convulsions of less than 3 hours, including jumping)
4) Score 3 persistent convulsions (only persistent convulsions of 3 hours or more)
5) Score 4 death
Above 5 levels
<Degree of convulsions (symptoms)>
1) None
2) phasic (transient convulsions)
3) status (persistent convulsions)
4) Death
4 levels
[0041]
(2) Increased kainic acid sensitivity induced by intracerebral administration of Trx-SPAR protein
1) Elapsed time and kainic acid sensitivity after intracerebral administration of SPARC chimeric protein
Convulsions induced by acute kainic acid administration (20 mg / kg, intraperitoneal administration) after cerebral administration of 10 pmoles of Trx-SPARC chimeric protein to the hippocampal radiation layer of ddY mice (male, 7 weeks old) The effect on seizures was examined. The number of convulsions increased statistically significantly only in the Trx-SPARC protein-administered group on the second day after intracerebral micro-administration, and no effect was observed after 3 days after intra-cerebral micro-administration (Fig. 4). The convulsion score was statistically significantly increased only in the Trx-SPARC protein administration group on the second day after the intracerebral microadministration (FIG. 5). The above results indicate that the drug sensitivity to kainic acid is remarkably enhanced on the second day after administration of a trace amount of Trx-SPARC protein in the brain.
[0042]
2) Trace administration time of SPARC chimeric protein in the brain
The number of convulsions was significantly increased only in the Trx-SPARC protein administration group (FIG. 6).
This effect disappeared by heat treatment of the Trx-SPARC protein (FIG. 6).
The time course curve of the number of convulsions was the curve that descended to the maximum at 60 minutes in the vehicle administration group and the Trx-tag protein administration group, but the curve that increased only after 60 minutes in the Trx-SPARC protein administration group. (Fig. 7). In particular, the number of convulsions after 90 minutes markedly increased in the Trx-SPARC protein administration group (FIG. 7).
The spasm score also increased significantly in the condyles only in the Trx-SPARC protein administration group (FIG. 8).
[0043]
In addition, this effect disappeared in the heat treated Trx-SPARC protein brain administration group, and only convulsion scores and convulsions similar to those in the Vehicle brain administration group and Trx-tag protein brain administration group were observed (Fig. 8, 9).
The above results indicate that the enhancement of drug sensitivity to kainic acid is specifically induced by administration of a trace amount of Trx-SPARC protein in the brain.
[0044]
3) Trx-SPARC protein dose dependency
The number of convulsions increased depending on the micro dose of Trx-SPARC protein in the brain, and reached saturation with a micro dose of 7.5 pmoles or more of Trx-SPARC protein in the brain (FIG. 10). The time course curve of the number of convulsions moves upward depending on the micro dose of Trx-SPARC protein in the brain, and after 60 to 90 minutes, the brain dose-dependently dependent on the dose of Trx-SPARC protein. Differences were observed (Figure 11). On the other hand, the convulsions score also increased depending on the micro dose of Trx-SPARC protein in the brain, and reached saturation by micro administration of 7.5 pmoles or more of Trx-SPARC protein (FIGS. 12, 13). ). The above results indicate that the drug sensitivity to kainic acid is enhanced depending on the micro dose of Trx-SPARC protein in the brain.
[0045]
4） Kainic acid dose dependency
The number of convulsions increased in the Trx-SPARC protein administration group depending on the dose of kainic acid, and at any dose, it was statistically compared to the vehicle brain micro dose group and the Trx-tag brain micro dose group Markedly significant increase (FIG. 14). The time course curve of the number of convulsions moved upward depending on the kainic acid dose, and a significant difference depending on the kainic acid dose was observed 60 to 90 minutes later (FIG. 15). The convulsion score was statistically significant only in the trace dose group of Trx-SPARC protein in the brain when kainic acid at a dose of 20 mg / kg or less was administered, but statistically significant when kainic acid at 25 mg / kg was administered. Was not recognized (FIGS. 16 and 17). The above results indicate that the drug sensitivity to kainic acid induced by microinjection of Trx-SPARC protein in the brain is enhanced depending on the dose of kainic acid when doses of 20 mg / kg or less are administered. Show.
[0046]
【The invention's effect】
As is clear from the above explanation, the SPARC protein is related to the epileptogenesis mechanism due to the GABA-inhibiting nervous system abnormality, and the novel epilepsy model animal of the present invention in which the protein is present in an excessive amount in the brain is a conventional epilepsy model. The present invention has an onset mechanism not found in animals, and the present invention provides a basis for elucidating the onset mechanism of epilepsy, which is the basis for the development of new medicines and prevention methods for intractable epilepsy, or for the development of new drugs. It is extremely useful as a technology.
[0047]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the entire amino acid sequence of a SPARC fusion protein.
FIG. 2 shows the construction of pET32 (+)-SPARC vector.
FIG. 3 is a photograph showing the results of SDS-PAGE of a purified sample obtained in the production process of the SPARC fusion protein of Reference Example.
FIG. 4 is a graph showing the results of measuring changes over time in the number of convulsions when kainic acid-monohydrate was administered after intracerebral administration of SPARC fusion protein, physiological saline and thioredoxin (Trx), respectively. is there.
FIG. 5 is a graph showing the results of measuring changes over time in the convulsion score when kainic acid monohydrate was administered after intracerebral administration of SPARC fusion protein, physiological saline and thioredoxin (Trx), respectively.
FIG. 6: After administration of kainic acid monohydrate after administration of SPARC fusion protein, heat-treated SPARC fusion protein, thioredoxin (Trx-Tag) and vehicle alone in the brain and kainic acid monohydrate, respectively. It is a graph which shows each total number of convulsions for 90 minutes.
FIG. 7 shows the results of measuring changes over time in the number of convulsions when kainic acid monohydrate is administered after brain administration of SPARC fusion protein, heat-treated SPARC fusion protein, thioredoxin (Trx-Tag) and vehicle, respectively. It is a graph which shows.
FIG. 8: After administration of kainic acid monohydrate after administration of SPARC fusion protein, heat-treated SPARC fusion protein, thioredoxin (Trx-Tag) and vehicle alone in the brain and kainic acid monohydrate, respectively. It is a graph which shows each total seizure score for 90 minutes.
FIG. 9: After administration of kainic acid monohydrate after administration of SPARC fusion protein, heat-treated SPARC fusion protein, oredoxin (Trx-Tag) and vehicle, respectively, and kainic acid monohydrate. It is a graph which shows the ratio of each convulsions presented for 90 minutes.
FIG. 10 shows the dose of SPARC fusion protein and the number of convulsions exhibited 90 minutes after administration of kainic acid monohydrate when kainic acid monohydrate was administered after intracerebral administration of SPARC fusion protein. It is a graph which shows a relationship.
FIG. 11 is a graph showing the results of measuring changes over time in the number of convulsions for each dose of SPARC fusion protein when kainic acid monohydrate is administered after intracerebral administration of SPARC fusion protein.
FIG. 12 shows the relationship between the dose of SPARC fusion protein and the convulsion score exhibited 90 minutes after administration of kainic acid monohydrate when kainic acid monohydrate was administered after intracerebral administration of SPARC fusion protein. It is a graph to show.
FIG. 13 shows the proportion of each convulsions exhibited in 90 minutes after administration of kainic acid monohydrate after administration of SPARC fusion protein in the brain and then administration of kainic acid monohydrate. It is the graph measured for every dosage.
FIG. 14 shows the relationship between the dose of kainic acid monohydrate and the number of convulsions when kainic acid monohydrate is administered after intracerebral administration of SPARC fusion protein, thioredoxin (Trx-Tag) and vehicle, respectively. It is a graph which shows.
FIG. 15 is a graph showing the results of measuring changes over time in the number of convulsions for each dose of kainic acid monohydrate when kainic acid monohydrate was administered after intracerebral administration of SPARC fusion protein. is there.
FIG. 16 is a graph showing the relationship between the dose of kainic acid monohydrate and the convulsion score when kainic acid monohydrate is administered after brain administration of each SPARC fusion protein.
FIG. 17 shows the proportion of each convulsions exhibited in 90 minutes after administration of kainic acid monohydrate in the case where kainic acid monohydrate is administered after intracerebral administration of SPARC fusion protein. It is the graph measured for every dosage of the thing.

Claims (6)

An epilepsy model animal (excluding humans) , characterized in that the SPARC protein is administered into the brain. 2. The epilepsy model animal (excluding humans) according to claim 1, wherein the SPARC protein is a fusion protein with thioredoxin. A drug for producing epilepsy model animals (excluding humans) containing SPARC protein. 4. The drug for producing epilepsy model animals (excluding humans) according to claim 3, wherein the SPARC protein is a fusion protein with thioredoxin. A method for producing an epilepsy model animal (excluding humans), which comprises administering a SPARC protein in the brain. The method for producing an epileptic model animal (excluding humans) according to claim 5, wherein the SPARC protein is a fusion protein with thioredoxin.

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